FUNCTIONALIZED NANOTUBES

NANOTUBES FONCTIONNALISES

English Abstract

Graphitic nanotubes, which include tubular fullerenes (commonly called
'buckytubes") and fibrils, which are functionalized by
chemical substitution or by adsorption of functional moieties. More
specifically the invention relates to graphitic nanotubes which are
uniformly or non-uniformly substituted with chemical moieties or upon which
certain cyclic compounds are adsorbed and to complex
structures comprised of such functionalized nanotubes linked to one another.
The invention also relates to methods for introducing functional
groups onto the surface of such nanotubes. The invention further relates to
uses for functionalized nanotubes.

French Abstract

L'invention concerne les nanotubes graphitiques, incluant les fullerènes tubulaires (souvent appelés "buckytubes" en anglais) et les fibrilles, qui sont fonctionnalisés par substitution chimique ou par adsorption de fractions fonctionnelles. L'invention est plus particulièrement applicable aux nanotubes graphitiques qui sont uniformément ou non uniformément remplacés par des fractions chimiques ou sur lesquels certains composés cycliques sont adsorbés. La présente invention s'applique également aux structures complexes comprenant de tels nanotubes fonctionnalisés reliés entre eux. L'invention concerne aussi les méthodes permettant d'introduire des groupes fonctionnels dans la surface de tels nanotubes. L'invention se rapporte en outre aux utilisations des nanotubes fonctionnalisés.

Claims

91
CLAIMS:

1. A composition of matter made of carbon atoms,
hydrogen atoms and groups R;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube having a
length to diameter ratio of greater than 5 and a diameter of
less than 0.5 micron;

numbers of the hydrogen atoms and the groups R are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;

each of R is the same and is:

(a) R'CHOH, where R' is hydrogen, alkyl, aryl,
cycloalkyl, aralkyl, cycloaryl or poly(alkylether);

(b) COOR' or SR', where R is cycloaryl or
poly(alkylether); or

(c) SiR'3, Si~OR'~y R' 3-y, Si~O-SiR'2~OR' or AlR'2,
where R' is hydrogen, cycloaryl or poly(alkylether); and
y is an integer of 1 to 3.

2. A composition of matter made of carbon atoms,
hydrogen atoms and groups R;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic fibril being
substantially free of pyrolytically deposited carbon, a
projection of the graphite layers on the fibrils extends for
a distance of at least two fibril diameters;



92

numbers of the hydrogen atoms and the groups R are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;

each of R is the same and is:

(a) R'CHOH, where R' is hydrogen, alkyl, aryl,
cycloalkyl, aralkyl, cycloaryl or poly(alkylether);

(b) COOR' or SR', where R is cycloaryl or
poly(alkylether); or

(c) SiR'3, Si~-OR~y R' 3-y, Si~O-SiR'2~OR' or Al R'2,
where R' is hydrogen, cycloaryl or poly(alkylether); and
y is an integer of 1 to 3.

3. A composition of matter made of carbon atoms,
hydrogen atoms and groups R;

wherein the carbon atoms are surface atoms of a
fishbone fibril;

numbers of the hydrogen atoms and the groups R are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;

each of R is the same and is:

(a) R'CHOH, where R' is hydrogen, alkyl, aryl,
cycloalkyl, aralkyl, cycloaryl or poly(alkylether);

(b) COOR' or SR', where R is cycloaryl or
poly(alkylether); or

(c) SiR'3, Si~OR'~ y R'3-y, Si~O-SiR'2~OR' or A1R'2,
where R' is hydrogen, cycloaryl or poly(alkylether); and
y is an integer of 1 to 3.



93

4. A composition of matter made of carbon atoms,
hydrogen atoms and groups R;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube having a
length to diameter ratio of greater than 5 and a diameter of
less than 0.5 micron;

numbers of the hydrogen atoms and the groups R are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;

each of R is:

(a) the same and is:
(i) COOH;

(ii) R'CHOH, where R' is hydrogen, alkyl, aryl,
cycloalkyl, aralkyl, cycloaryl or poly(alkylether);

(iii) COOR' or SR', where R is cycloaryl or
poly(alkylether); or

(iv) SiR'3, Si~OR'~y R'3-y, Si~O-SiR' 2~OR' or A1R'2,
where R' is hydrogen, cycloaryl or poly(alkylether) and y is
an integer of 1 to 3; or

(b) different and is selected from SO3H, COOH, NH2,
OH, R'CHOH, CHO, CN, COCl, halide, COSH, SH, COOR', SR',
SiR'3, Si~OR'~ y R'3-y, Si~O-SiR'2~OR', R", Li, AlR'2, Hg-X, TlZ2
and Mg-X,

y is an integer of 1 to 3,

R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl,
cycloaryl or poly(alkylether),



94

R" is a fluoroalkyl, fluoroaryl, fluorocycloalkyl
or fluoroaralkyl,

X is a halide,

Z is carboxylate or trifluoroacetate,

and further provided that where each of R is an
oxygen-containing group COOH is not present.

5. A composition of matter made of carbon atoms,
hydrogen atoms and groups R;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic fibril being
substantially free of pyrolytically deposited carbon, a
projection of the graphite layers on the fibrils extends for
a distance of at least two fibril diameters;

numbers of the hydrogen atoms and the groups R are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;

each of R is:

(a) the same and is:
(i) COOH;

(ii) R'CHOH, where R' is hydrogen, alkyl, aryl,
cycloalkyl, aralkyl, cycloaryl or poly(alkylether);

(iii) COOR' or SR', where R is cycloaryl or
poly(alkylether); or

(iv) SiR'3, Si~OR'~y R' 3-y, Si~O-SiR' 2~OR' or AlR'2,
where R' is hydrogen, cycloaryl or poly(alkylether) and y is
an integer of 1 to 3; or



95

(b) different and is selected from SO3H, COOH, NH2,
OH, R'CHOH, CHO, CN, COCl, halide, COSH, SH, COOR', SR',
SiR'3, Si~OR'~y R' 3-y, Si~O-SiR'2~OR', R", Li, AlR'2, Hg-X, TlZ2
and Mg-X,

y is an integer of 1 to 3,

R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl,
cycloaryl or poly(alkylether),

R" is a fluoroalkyl, fluoroaryl, fluorocycloalkyl
or fluoroaralkyl,

X is a halide,

Z is carboxylate or trifluoroacetate,

and further provided that where each of R is an
oxygen-containing group COOH is not present.

6. A composition of matter made of carbon atoms,
hydrogen atoms and groups R;

wherein the carbon atoms are surface atoms of a
fishbone fibril;

numbers of the hydrogen atoms and the groups R are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;

each of R is:

(a) the same and is:
(i) COOH;

(ii) R'CHOH, where R' is hydrogen, alkyl, aryl,
cycloalkyl, aralkyl, cycloaryl or poly(alkylether);



96

(iii) COOR' or SR', where R is cycloaryl or
poly(alkylether); or

(iv) SiR'3, Si~OR'~y R' 3-y, Si~O-SiR'2`OR' or AlR'2,
where R' is hydrogen, cycloaryl or poly(alkylether) and y is
an integer of 1 to 3; or

(b) different and is selected from SO3H, COOH, NH2,
OH, R'CHOH, CHO, CN, COCl, halide, COSH, SH, COOR', SR',
SiR'3, Si~OR'~y R' 3-y, Si~O-SiR'2~OR', R", Li, A1R'2, Hg-X, TlZ2
and Mg-X,

y is an integer of 1 to 3,

R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl,
cycloaryl or poly(alkylether),

R" is a fluoroalkyl, fluoroaryl, fluorocycloalkyl
or fluoroaralkyl,

X is a halide,

Z is carboxylate or trifluoroacetate,

and further provided that where each of R is an
oxygen-containing group COOH is not present.

7. A composition of matter made of carbon atoms,
hydrogen atoms and groups A;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube having a
length to diameter ratio of greater than 5 and a diameter of
less than 0.1 micron;

numbers of the hydrogen atoms and the groups A are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;



97
each of A is:

Image where Y is an appropriate functional
group of a protein, a peptide, an amino acid, an enzyme, an
antibody, an oligonucleotide, a nucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH,

R'-N(R')2, R'SH, R'CHO, R'CN, R'X, R'SiR'3, R'-N+(R')3X-,

R'Si~OR'~y R'3-y, R'-R", R'-N-CO, R'Si~O-SiR'2~OR', (C2H4O~w H,
~C3H6O~w H, +C2H4O)w-R', (C3H6O)w-R', R'Image, where

R' is hydrogen, alkyl, aryl, cycloaryl, aralkyl or
cycloaryl;

(b) OY, NHY, Image N=Y or C=Y, where
Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image,
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloalkyl; or

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'Si~O-SiR'2~OR', R'Si~OR'~y R'3-y, R'-R", R'-N-CO,
~C2H4O)w-R', (C3H6O)w-R' and R', where

R' is hydrogen or cycloaryl; or

(c) Image or -CR'2-OY, where Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloalkyl; or



98


(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,

R'SiR'3, R'-Si~OR'~y R'3-y, R'Si~O-SiR'2~OR', R'-R", R'-N-CO,
(C2H4O~w H, ~C3H6O~w H, -~C2H4O)w-R', (C3H6O)w-R' and R' or is an
appropriate functional group of a protein, a peptide, an
enzyme, an antibody, an oligonucleotide, a nucleotide, an
antigen, an enzyme substrate, an enzyme inhibitor or a
transition state analog of an enzyme substrate, where R' is
hydrogen or cycloaryl;

y is an integer of 1 to 3;

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide; and

w is an integer greater than one and less than 200.
8. The composition according to claim 7 wherein

A is Image

R' is H, and

Y is an amino acid selected from the group
consisting of lysine, serine, threonine, tyrosine, aspartic
acid and glutamic acid.

9. A composition of matter made of carbon atoms,
hydrogen atoms and groups A;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic fibril being
substantially free of pyrolytically deposited carbon, a
projection of the graphite layers on the fibrils extends for
a distance of at least two fibril diameters;




99


numbers of the hydrogen atoms and the groups A are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;

each of A is:
(a) Image where Y is an appropriate functional

group of a protein, a peptide, an amino acid, an enzyme, an
antibody, an oligonucleotide, a nucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH,

R'-N(R')2, R'SH, R'CHO, R'CN, R'X, R'SiR'3, R'-N+(R')3X-,

R'Si~OR'~y R'3-y, R'-R", R'-N-CO, R'Si~O-SiR'2~OR', (C2H4O~w H,
~C3H6O~w H, ~C2H4O)w-R', (C3H6O)w-R', R' and Image where

R' is hydrogen, alkyl, aryl, cycloaryl, aralkyl or
cycloaryl;

(b) OY, NHY, Image N=Y or C=Y, where
Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloalkyl; or

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'Si~O-SiR'2~OR', R'Si~OR'~y R'3-y, R'-R", R'-N-CO,
~C2H4O)w-R', (C3H6O)w-R' and R', where

R' is hydrogen or cycloaryl; or
(c) Image or -CR'2-OY, where Y is:

(i) an appropriate functional group of an amino



100


acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloalkyl; or

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'-Si~OR'~y R'3-y, R'Si~O-SiR'2~OR', R'-R", R'-N-CO,
(C2H4O~w H, ~C3H6O~w H, ~C2H4O)w-R', (C3H6O)w-R' and R' or is an
appropriate functional group of a protein, a peptide, an
enzyme, an antibody, an oligonucleotide, a nucleotide, an
antigen, an enzyme substrate, an enzyme inhibitor or a
transition state analog of an enzyme substrate, where R' is
hydrogen or cycloaryl;

y is an integer of 1 to 3;

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide; and

w is an integer greater than one and less than 200.
10. The composition according to claim 9, wherein

A is Image
R' is H, and

Y is an amino acid selected from the group
consisting of lysine, serine, threonine, tyrosine, aspartic
acid and glutamic acid.

11. A composition of matter made of carbon atoms,
hydrogen atoms and groups A;

wherein the carbon atoms are surface atoms of a
fishbone fibril;



101


numbers of the hydrogen atoms and the groups A are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;

each of A is:
(a) Image where Y is an appropriate functional
group of a protein, a peptide, an amino acid, an enzyme, an
antibody, an oligonucleotide, a nucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH,

R'-N (R')2, R'SH, R'CHO, R'CN, R'X, R'SiR'3, R'-N+(R')3X-,

R'Si~OR'~y R'3-y, R'-R", R'-N-CO, R'Si~O-SiR'2~OR', (C2H4O)w H,
~C3H6O~w H, ~C2H4O)w-R', (C3H6O)w-R', R' and Image where
R' is hydrogen, alkyl, aryl, cycloaryl, aralkyl or
cycloaryl;

(b) OY, NHY, Image N=Y or C=Y, where
Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloalkyl; or

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'Si~O-SiR'2~OR', R'Si~OR'~y R'3-y, R'-R", R'-N-CO,
~C2H4O)w-R', (C3H6O)w-R' and R', where

R' is hydrogen or cycloaryl; or
(c) Image or -CR'2-OY, where Y is:

(i) an appropriate functional group of an amino



102


acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloalkyl; or

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'-Si~OR'~y R'3-y, R'Si~O-SiR'2~OR', R'-R", R'-N-CO,
(C2H4O~w H, ~C3H6O~w H, ~C2H4O)w-R', (C3H6O)w-R' and R' or is an
appropriate functional group of a protein, a peptide, an
enzyme, an antibody, an oligonucleotide, a nucleotide, an
antigen, an enzyme substrate, an enzyme inhibitor or a
transition state analog of an enzyme substrate, where R' is
hydrogen or cycloaryl;

y is an integer of 1 to 3;

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide; and

w is an integer greater than one and less than 200.
12. The composition according to claim 11, wherein

A is Image
R' is H, and

Y is an amino acid selected from the group
consisting of lysine, serine, threonine, tyrosine, aspartic
acid and glutamic acid.

13. A composition of matter made of carbon atoms,
hydrogen atoms and groups R'-A;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube having a



103


length to diameter ratio of greater than 5 and a diameter of
less than 0.5 micron;

numbers of the hydrogen atoms and the groups R'-A
are less than 0.1 and less than 0.5, respectively, of that
of the carbon atoms;

each of R' is:

(a) cycloaryl or poly(alkylether), where
A is selected from:

OY, NHY, Image -CR'2-OY, N=Y,
Image and C=Y;

Y is an appropriate functional group of a protein,
a peptide, an amino acid, an enzyme, an antibody, a
nucleotide, an oligonucleotide, an antigen, an enzyme
substrate, an enzyme inhibitor or a transition state analog
of an enzyme substrate or is selected from R'-OH, R'-N(R')2,
R'SH, R'CHO, R'CN, R'X, R'N+(R')3X-, R'SiR'3, R'Si~OR'~y R'3-y,
R'Si~O-SiR'2~OR', R'-R", R'-N-CO, (C2H4O~w H, ~C3H6O~w H,

~C2H4O)w-R', (C3H6O)w-R', R' and Image or

(b) each of R' is alkyl, aryl, cycloalkyl or
aralkyl, where A is:

(j) Image where Y is an appropriate functional
group of a protein, a peptide, an amino acid, an enzyme, an
antibody, a nucleotide, an oligonucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH,

R'-N(R')2, R'SH, R'CHO, R'CN, R'X, R'N+(R')3X-, R'SiR'3,

R'Si~OR'~y R'3-y, R'Si~O-SiR'2~OR', R'-R", R'-N-CO, (C2H4O~w H,



104


~C3H6O~w H, ~C2H4O~w R', (C3H6O)w-R', R' and Image or
(ii) OY, Image CR'2-OY, N=Y

or C=Y, where Y is an appropriate functional group of an
amino acid or is selected from R'-N(R')2, R'N+(R')3X-

and Image

y is an integer of 1 to 3;

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide; and

w is an integer greater than one and less than 200.
14. The composition according to claim 13, wherein

A is Image
R' is H, and

Y is an amino acid selected from the group
consisting of lysine, serine, threonine, tyrosine, aspartic
acid and glutamic acid.

15. A composition of matter made of carbon atoms,
hydrogen atoms and groups R'-A;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic fibril being
substantially free of pyrolytically deposited carbon, a
projection of the graphite layers on the fibrils extends for
a distance of at least two fibril diameters;



105

numbers of the hydrogen atoms and the groups R'-A
are less than 0.1 and less than 0.5, respectively, of that
of the carbon atoms;

each of R' is:

(a) cycloaryl or poly(alkylether), where
A is selected from:

OY, NHY, Image -CR'2-OY, N=Y,
Image
and C=Y;

Y is an appropriate functional group of a protein,
a peptide, an amino acid, an enzyme, an antibody, a
nucleotide, an oligonucleotide, an antigen, an enzyme
substrate, an enzyme inhibitor or a transition state analog
of an enzyme substrate or is selected from R'-OH, R'-N(R')2,
R'SH, R'CHO, R'CN, R'X, R'N+(R')3X, R'SiR'3, R'Si~R'~y R'3-y,
R'Si~O-SiR'2~OR', R'-R", R'-N-CO, (C2H4O~w H, ~C3H6O~w H,

~C2H4O)w-R', (C3H6O)w-R', R' and Image ; or

(b) each of R' is alkyl, aryl, cycloalkyl or
aralkyl, where A is:

(i) Image, where Y is an appropriate functional
group of a protein, a peptide, an amino acid, an enzyme, an
antibody, a nucleotide, an oligonucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH,
R'-N(R')2, R'SH, R'CHO, R'CN, R'X, R'N+(R')3X, R'SiR'3,

R'Si~OR'~y R'3-y, R'Si~O-SiR'2~OR', R'-R", R'-N-CO, (C2H4O~w H,
~C3H6O~w H, ~C2H4O~w R', (C3H60)w-R', R' and Image ; or




106



(ii) OY, NHY, Image CR'2-0Y, N=Y
or C=Y, where Y is an appropriate functional group of an
amino acid or is selected from R'-N(R')2, R'N+(R')3X-

and Image ;

y is an integer of 1 to 3;

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide; and

w is an integer greater than one and less than 200.
16. The composition according to claim 15 wherein:

A is Image,
R' is H, and

Y is an amino acid selected from the group
consisting of lysine, serine, threonine, tyrosine, aspartic
acid and glutamic acid.

17. A composition of matter made of carbon atoms,
hydrogen atoms and groups R'-A;

wherein the carbon atoms are surface atoms of a
fishbone fibril;

numbers of the hydrogen atoms and the groups R'-A
are less than 0.1 and less than 0.5, respectively, of that
of the carbon atoms;

each of R' is:

(a) cycloaryl or poly(alkylether), where



107

A is selected from:

OY, NHY, Image -CR'2-OY, N=Y,
Image and C=Y;

Y is an appropriate functional group of a protein,
a peptide, an amino acid, an enzyme, an antibody, a
nucleotide, an oligonucleotide, an antigen, an enzyme
substrate, an enzyme inhibitor or a transition state analog
of an enzyme substrate or is selected from R'-OH, R'-N(R')2,
R'SH, R'CHO, R'CN, R'X, R'N+(R')3X , R'SiR'3, R'Si~OR'~y R'3-y,
R'Si~O-SiR'2~OR', R'-R", R'-N-CO, (C2H4O~w H, ~C3H6O~w H,

~C2H4O)w-R', (C3H6O)w-R', R' and Image ; or

(b) each of R' is alkyl, aryl, cycloalkyl or
aralkyl, where A is:
(i) Image , where Y is an appropriate functional
group of a protein, a peptide, an amino acid, an enzyme, an
antibody, a nucleotide, an oligonucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH,

R'-N(R')2, R'SH, R'CHO, R'CN, R'X, R'N+(R')3X-, R'SiR'3,

R'Si~OR'~y R' 3-y, R' Si~O-SiR'2~OR', R'-R", R'-N-CO, (C2H4O~w H,
~C3H6O~w H, ~C2H9O~w R',(C3H6O)w -R', R' and Image ; or
(ii) OY, NHY, Image CR'2-OY, N=Y
or C=Y, where Y is an appropriate functional group of an
amino acid or is selected from R'-N(R')2, R'N+(R')3X-

and Image;

y is an integer of 1 to 3;



108

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide; and

w is an integer greater than one and less than 200.
18. A composition of matter made of carbon atoms,
hydrogen atoms and groups X'-A a;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube having a
length to diameter ratio of greater than 5 and a diameter of
less than 0.5 micron;

numbers of the hydrogen atoms and the groups X'-A a
are less than 0.1 and less than 0.5, respectively, of that
of the carbon atoms;

a is an integer of 1 to 9;
each of A is:

(a) Image , where Y is an appropriate functional
group of a protein, a peptide, an amino acid, an enzyme, an
antibody, a nucleotide, an oligonucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH, R'-
N(R')2, R'SH, R'CHO, R'CN, R'X, R' N+(R')3X-, R'SiR'3,
R'Si~OR'~y R'3-y, R'Si~O-SiR'2~OR', R'-R", R'-N-CO, (C2H9O~w H,
~C3H6O~w H, ~C2H4O)w-R', (C3H6O)w-R', R' and Image , where R'
is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl;

(b) OY, NHY, Image N=Y or C=Y, where Y
is:

(i) an appropriate functional group of an amino



109

acid or is selected from R'-N(R')2, R'N+(R')3X- and Image

where R' is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl;
or

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'Si~OR'~y R'3-y, R'Si~O-SiR'2~OR', R' -R", R'-N-CO,
~C2H4O)w-R', (C3H6O)w-R' and R', where R' is cycloaryl; or

(c) Image or -CR'2-OY, where Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl;
or

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'Si~OR'~y R'3-y, R'S~O-SiR'2~OR', R'-R", R'-N-CO,
(C2H4O~w H, ~C3H6O~w H, ~C2H4O~w-R',(C3H6O)w-R' and R', or is an
appropriate functional group of a protein, a peptide, an
enzyme, an antibody, a nucleotide, an oligonucleotide, an
antigen, an enzyme substrate, an enzyme inhibitor or a
transition state analog of an enzyme substrate, where R' is
cycloaryl;

y is an integer from 1 to 3;

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide;

X' is a polynuclear aromatic, polyheteronuclear
aromatic or metallopolyheteronuclear aromatic moiety; and

w is an integer greater than one and less than 200.



110

19. A composition of matter made of carbon atoms,
hydrogen atoms and groups X'-A a;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic fibril being
substantially free of pyrolytically deposited carbon, the
projection of the graphite layers on said fibrils extends
for a distance of at least two fibril diameters;

numbers of the hydrogen atoms and the groups X'-A a
are less than 0.1 and less than 0.5, respectively, of that
of the carbon atoms;

a is an integer of 1 to 9;
each of A is:

(a) Image , where Y is an appropriate functional
group of a protein, a peptide, an amino acid, an enzyme, an
antibody, a nucleotide, an oligonucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH, R'-
N(R')2, R'SH, R'CHO, R'CN, R'X, R'N+ (R')3X-, R'SiR'3,
R'Si~OR'~y R'3-y, R'Si~O-SiR'2~OR', R'-R", R'-N-CO, (C2H4O~w H,
~C3H6O~w H, ~C2H4O)w-R',(C3H6O)w-R', R' and Image ; where R'
is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl;

(b) OY, NHY, Image N=Y or C=Y, where Y
is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N (R')2, R'N+(R')3X- and Image
where R' is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl;
or



111

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'Si~OR'~y R'3-y, R'Si~O-SiR'2~OR', R'-R", R'-N-CO,
~C2H4O)w-R', (C3H6O)w-R'and R', where R' is cycloaryl; or
(c)Image or -CR'2-OY, where Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R'is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl;
or

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'Si~OR'~y R'3-y, R'Si~O-SiR'2~OR', R'-R", R'-N-CO,
(C2H4O~w H, ~C3H6O~w H, ~C2H4O)w-R',(C3H60)w-R' and R', or is an
appropriate functional group of a protein, a peptide, an
enzyme, an antibody, a nucleotide, an oligonucleotide, an
antigen, an enzyme substrate, an enzyme inhibitor or a
transition state analog of an enzyme substrate, where R' is
cycloaryl;

y is an integer from 1 to 3;

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide;

X' is a polynuclear aromatic, polyheteronuclear
aromatic or metallopolyheteronuclear aromatic moiety; and

w is an integer greater than one and less than 200.
20. A composition of matter made of carbon atoms,
hydrogen atoms and groups X'-A a;

wherein the carbon atoms are surface atoms of a
fishbone fibril;



112

numbers of the hydrogen atoms and the groups X'-A a
are less than 0.1 and less than 0.5, respectively, of that
of the carbon atoms;

a is an integer of 1 to 9;
each of A is:
(a)Image , where Y is an appropriate functional
group of a protein, a peptide, an amino acid, an enzyme, an
antibody, a nucleotide, an oligonucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH,

R'-N(R')2, R'SH, R'CHO, R'CN, R'X, R'N+(R')3X-, R'SiR'3,

R'Si~OR'~y R'3-y, R'Si~O-SiR'2~OR', R'-R", R'-N-CO, (C2H4O~w H,
~C3H6O~w H, ~C2H4O)w-R', (C3H6O)w-R', R'and Image , where R'
is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl;

(b) OY, NHY, Image N=Y or C=Y, where Y
is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'N+(R')3X- and Image
where R'is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl;
or

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'Si~OR'~y R'3-y, R'Si~O-SiR'2~OR', R'-R", R'-N-CO,
~C2H4O)w-R', (C3H6O)w-R' and R', where R' is cycloaryl; or

(c) Image or -CR'2-OY, where Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image ,



113

where R' is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl;
or

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'Si~OR'~y R'3-y, R'Si~O-SiR'2~OR', R'-R", R'-N-CO,
(C2H4O~w H, ~C3H6O~w H, ~C2H4O)w-R', (C3H6O)w-R' and R', or is an
appropriate functional group of a protein, a peptide, an
enzyme, an antibody, a nucleotide, an oligonucleotide, an
antigen, an enzyme substrate, an enzyme inhibitor or a
transition state analog of an enzyme substrate, where R' is
cycloaryl;

y is an integer from 1 to 3;

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide;

X' is a polynuclear aromatic, polyheteronuclear
aromatic or metallopolyheteronuclear aromatic moiety; and

w is an integer greater than one and less than 200.
21. A method of forming a composition of matter made of
carbon atoms, hydrogen atoms and groups CH(R')OH;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube;

numbers of the hydrogen atoms and the groups
CH(R')OH are less than 0.1 and less than 0.5, respectively,
of that of the carbon atoms;

R'is a hydrogen, alkyl, aryl, cycloalkyl, aralkyl,
cycloaryl, or poly(alkylether),



114


which method comprises: reacting the surface
carbons with a compound having the formula R'CH2OH in the
presence of a free radical initiator under conditions
sufficient to form functionalized nanotubes having the group
CH(R')OH.

22. The method according to claim 21 wherein the free
radical initiator is benzoyl peroxide.

23. A method of forming a composition of matter made
of carbon atoms, hydrogen atoms and groups A;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube;

numbers of the hydrogen atoms and the groups A are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;

each of A is:

(a) Image where Y is an appropriate functional
group of a protein, a peptide, an amino acid, an enzyme, an
antibody, an oligonucleotide, a nucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH,

R'-N(R')2, R'SH, R'CHO, R'CN, R'X, R'SiR'3, R'-N+(R')3X-,

R'-R", R'-N-CO, (C2H4O~w H, ~C3H6O~w H, ~C2H4O)w-R', (C3H6O)w-R',
R' and Image where R' is hydrogen, alkyl, aryl,
cycloalkyl, aralkyl or cycloaryl;

(b) OY, NHY, Image N=Y or C=Y, where
Y is:

(i) an appropriate functional group of an amino



115


acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloaryl; or

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'-R", R'-N-CO, ~C2H4O)w-R', (C3H6O)w-R' and R', where
R' is hydrogen or cycloaryl; or

(c) or -CR'2-OY, where Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloaryl; or

(ii) an appropriate functional group of a protein,
a peptide, an enzyme, an antibody, an oligonucleotide, a
nucleotide, an antigen, an enzyme substrate, an enzyme
inhibitor or a transition state analog of an enzyme
substrate or is selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'-R", R'-N-CO, (C2H4O~w H, ~C3H6O~w H, ~C2H4O)w-R',
(C3H6O)w-R' and R', where R' is hydrogen or cycloalkyl;

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide;

Z is carboxylate or trifluoroacetate; and

w is an integer greater than one and less than 200,
which method comprises:

(a) reacting the surface carbons with at least one
appropriate reagent under conditions sufficient to form



116


substituted nanotubes having groups R wherein each of R is
the same and is selected from SO3H, COOH, NH2, OH, CH(R')OH,
CHO, CN, COCl, halide, COSH, SH, COOR', SR', SiR'3,

Si~OR'~y R'3-y, Si~O-SiR'2~OR', R", Li, AlR'2, Hg-X, TlZ2 and
Mg-X, and y is an integer of 1 to 3; and

(b) reacting the substituted nanotubes with at
least one appropriate reagent under conditions sufficient to
form functionalized nanotubes having the groups A.

24. A method of forming a composition of matter made
of carbon atoms, hydrogen atoms and groups A;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube having a
length to diameter ratio of greater than 5 and a diameter of
less than 0.1 micron;

numbers of the hydrogen atoms and the groups A are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;

each of A is:
(a) Image where Y is an appropriate functional
group of a protein, a peptide, an amino acid, an enzyme, an
antibody, an oligonucleotide, a nucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH,

R'-N(R')2, R'SH, R'CHO, R'CN, R'X, R'SiR'3, R'-N+(R')3X-,

R'-R", R'-N-CO, (C2H4O~w H, ~C3H6O~w H, ~C2H4O)w-R', (C3H6O)w-R',
R' and Image where R' is hydrogen, alkyl, aryl,
cycloalkyl, aralkyl or cycloaryl;
(b) OY, NHY, Image N=Y or C=Y, where

Y is:



117


(i) an appropriate functional group of an amino

acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloaryl; or

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'-R", R'-N-CO, ~C2H4O)w-R', (C3H6O)w-R' and R', where
R' is hydrogen or cycloaryl; or

(c) Image where Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloaryl; or

(ii) an appropriate functional group of a protein,
a peptide, an enzyme, an antibody, an oligonucleotide, a
nucleotide, an antigen, an enzyme substrate, an enzyme
inhibitor or a transition state analog of an enzyme
substrate or is selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'-R", R'-N-CO, (C2H4O~w H, 4C3H6O~w H, ~C2H4O)w-R',
(C3H6O)w-R' and R', where R' is hydrogen or cycloalkyl;

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide;

Z is carboxylate or trifluoroacetate; and

w is an integer greater than one and less than 200,
which method comprises:



118


(a) reacting the surface carbons with at least one
appropriate reagent under conditions sufficient to form
substituted nanotubes having groups R wherein each of R is
the same and is selected from SO3H, COOH, NH2, OH, CH(R')OH,
CHO, CN, COCl, halide, COSH, SH, COOR', SR', SiR'3,

Si~OR'-}y R'3-y, Si~O-SiR'2~OR', R", Li, AlR'2, Hg-X, TlZ2 and
Mg-X, and y is an integer of 1 to 3; and

(b) reacting the substituted nanotubes with at
least one appropriate reagent under conditions sufficient to
form functionalized nanotubes having the groups A.

25. A method of forming a composition of matter made
of carbon atoms, hydrogen atoms and groups A;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube being
substantially free of pyrolytically deposited carbon;

numbers of the hydrogen atoms and the groups A are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;

each of A is:
(a) Image where Y is an appropriate functional
group of a protein, a peptide, an amino acid, an enzyme, an
antibody, an oligonucleotide, a nucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH,

R'-N(R')2, R'SH, R'CHO, R'CN, R'X, R'SiR'3, R'-N+(R')3X-,

R'-R", R'-N-CO, (C2H4O~w H, ~C3H6O~w H, ~C2H4O)w-R', (C3H6~)w-R',



119

R' and Image , where R' is hydrogen, alkyl, aryl,
cycloalkyl, aralkyl or cycloaryl;

(b) OY, NHY, Image N=Y or C=Y, where
Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloaryl; or

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'-R", R'-N-CO, ~C2H4O)w-R', (C3H6O)w-R' and R', where
R' is hydrogen or cycloaryl; or

Image
(c) or -CR'2-OY, where Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloaryl; or

(ii) an appropriate functional group of a protein,
a peptide, an enzyme, an antibody, an oligonucleotide, a
nucleotide, an antigen, an enzyme substrate, an enzyme
inhibitor or a transition state analog of an enzyme
substrate or is selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'-R", R'-N-CO, (C2H4O}w H, ~C3H6O~w H, ~C2H4O)w-R',
(C3H6O)w-R' and R', where R' is hydrogen or cycloalkyl;

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide;



120

Z is carboxylate or trifluoroacetate; and

w is an integer greater than one and less than 200,
which method comprises:

(a) reacting the surface carbons with at least one
appropriate reagent under conditions sufficient to form
substituted nanotubes having groups R wherein each of R is
the same and is selected from SO3H, COOH, NH2, OH, CH(R')OH,
CHO, CN, COCl, halide, COSH, SH, COOR', SR', SiR'3,
Si~OR'~y R' 3-y, Si~O-SiR'2~OR', R", Li, AlR'2, Hg-X, TlZ2 and
Mg-X, and y is an integer of 1 to 3; and

(b) reacting the substituted nanotubes with at
least one appropriate reagent under conditions sufficient to
form functionalized nanotubes having the groups A.

26. A method of forming a composition of matter made
of carbon atoms, hydrogen atoms and groups A;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube;

numbers of the hydrogen atoms and the groups A are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;

each of A is:

(a) Image where Y is an appropriate functional
group of a protein, a peptide, an amino acid, an enzyme, an
antibody, an oligonucleotide, a nucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH,

R'-N(R')2, R'SH, R'CHO, R'CN, R'X, R'SiR'3, R'-N+(R')3X-,



121



R'-R", R'-N-CO, (C2H4O~w H, ~C3H6O~w H, ~C2H40)w-R', (C3H6O)w-R',
R' and Image where R' is hydrogen, alkyl, aryl,
cycloalkyl, aralkyl or cycloaryl;

(b) OY, NHY, Image N=Y or C=Y, where
Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloaryl; or

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'-R", R'-N-CO, ~C2H4O)w-R', (C3H6O)w-R' and R', where
R' is hydrogen or cycloaryl; or

Image
(c) or -CR'2-OY, where Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloaryl; or

(ii) an appropriate functional group of a protein,
a peptide, an enzyme, an antibody, an oligonucleotide, a
nucleotide, an antigen, an enzyme substrate, an enzyme
inhibitor or a transition state analog of an enzyme
substrate or is selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'-R", R'-N-CO, (C2H4O~w H, ~C3H6O~w H, ~C2H4O~w-R',
(C3H6O)w-R' and R', where R' is hydrogen or cycloalkyl;

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;



122

X is a halide;

Z is carboxylate or trifluoroacetate; and

w is an integer greater than one and less than 200,
which method comprises:

reacting substituted nanotubes made of carbon
atoms, hydrogen atoms and groups R with at least one
appropriate reagent under conditions sufficient to form
functionalized nanotubes having groups A, where in the
substituted nanotubes, each of R is the same and is selected
from SO3H, COOH, NH2, OH, CH(R')OH, CHO, CN, COCl, halide,
COSH, SH, COOR', SR', SiR'3, Si~OR'~y R'3-y, Si~O-SiR'2~OR',
R", Li, AlR'2, Hg-X, TlZ2 and Mg-X, and y is an integer of 1
to 3, and numbers of the hydrogen atoms and the groups R are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms.

27. A method of forming a composition of matter made
of carbon atoms, hydrogen atoms and groups A;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube having a
length to diameter ratio of greater than 5 and a diameter of
less than 0.1 micron;

numbers of the hydrogen atoms and the groups A are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;

each of A is:
(a) Image where Y is an appropriate functional

group of a protein, a peptide, an amino acid, an enzyme, an
antibody, an oligonucleotide, a nucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state



123

analog of an enzyme substrate or is selected from R'-OH,
R'-N(R')2, R'SH, R'CHO, R'CN, R'X, R'SiR'3, R'-N+(R')3X-,

R'-R", R'-N-CO, (C2H4O~w H, ~C3H6O~w H, ~C2H4O)w-R', (C3H6O)w-R',
R' and Image where R' is hydrogen, alkyl, aryl,
cycloalkyl, aralkyl or cycloaryl;

(b) OY, NHY, Image N=Y or C=Y, where
Y is:

i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloaryl; or

(ii) selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'-R", R'-N-CO, ~C2H4O)w-R', (C3H6O)w-R' and R', where
R' is hydrogen or cycloaryl; or
(c) Image or -CR'2-OY, where Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloaryl; or

(ii) an appropriate functional group of a protein,
a peptide, an enzyme, an antibody, an oligonucleotide, a
nucleotide, an antigen, an enzyme substrate, an enzyme
inhibitor or a transition state analog of an enzyme
substrate or is selected from R'-OH, R'SH, R'CHO, R'CN, R'X,
R'SiR'3, R'-R", R'-N-CO, (C2H4O~w H, ~C3H6O~w H, ~C2H4O~w-R',
(C3H6O)w-R' and R', where R' is hydrogen or cycloalkyl;



124

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide;

Z is carboxylate or trifluoroacetate; and

w is an integer greater than one and less than 200,
which method comprises:

reacting substituted nanotubes made of carbon
atoms, hydrogen atoms and groups R with at least one
appropriate reagent under conditions sufficient to form
functionalized nanotubes having groups A, where in the
substituted nanotubes, each of R is the same and is selected
from SO3H, COOH, NH2, OH, CH(R')OH, CHO, CN, COCl, halide,
COSH, SH, COOR', SR', SiR'3, Si~OR~y R'3-y, Si~O-SiR'2~OR',

R", Li, AlR'2, Hg-X, TlZ2 and Mg-X, and y is an integer of 1
to 3, and numbers of the hydrogen atoms and the groups R are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms.

28. A method of forming a composition of matter made
of carbon atoms, hydrogen atoms and groups A;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube being
substantially free of pyrolytically deposited carbon;

numbers of the hydrogen atoms and the groups A are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;

each of A is:
(a) Image



125


where Y is an appropriate functional group of a
protein, a peptide, an amino acid, an enzyme, an antibody,
an oligonucleotide, a nucleotide, an antigen, an enzyme
substrate, an enzyme inhibitor or a transition state analog
of an enzyme substrate or is selected from R'-OH, R'-N(R')2,
R'SH, R'CHO, R'CN, R'X, R'SiR'3, R'-N+(R')3X-, R'-R", R'-N-CO,
(C2H9O~w H, ~C3H6O~w H, ~C2H4O~w-R', (C3H6O)w-R', R' and Image
where R' is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl;

(b) OY, NHY, Image N=Y or C=Y, where
Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- or Image
where R' is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl;
or
(ii) is selected from R'-OH, R'SH, R'CHO, R'CN,

R'X, R'SiR'3, R'-R", R'-N-CO, ~C2H4O)w-R', (C3H6O)w-R' and R',
where R' is cycloaryl; or

(c) Image or -CR'2-OY, where Y is:

(i) an appropriate functional group of an amino
acid or is selected from R'-N(R')2, R'-N+(R')3X- and Image
where R' is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl;

(ii) an appropriate functional group of a protein,
a peptide, an enzyme, an antibody, an oligonucleotide, a
nucleotide, an antigen, an enzyme substrate, an enzyme
inhibitor or a transition state analog of an enzyme
substrate or is selected from R'-OH, R'SH, R'CHO, R'CN, R'X,



126


R'SiR'3, R'-R", R'-N-CO, (C2H4O~w H, ~C3H6O~w H, ~C2H4O)w-R',
(C3H6O)w-R'and R', where R' is cycloaryl;

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide;

Z is carboxylate or trifluoroacetate; and

w is an integer greater than one and less than 200,
which method comprises reacting substituted
nanotubes made of carbon atoms, hydrogen atoms and groups R
with at least one appropriate reagent under conditions
sufficient to form functionalized nanotubes having groups A,
wherein the substituted nanotubes, each of R is the same and
is selected from SO3H, COOH, NH2, OH, CH('R)OH, CHO, CN,

COCl, halide, COSH, SH, COOR', SR', SiR'3, Si~OR'~y R'3-y,
Si~O-SiR'2~OR', R", Li, AlR'2, Hg-X, TlZ2 and Mg-X, and y is
an integer of 1 to 3, and numbers of hydrogen atoms and the
groups R are less than 0.1 and less than 0.5, respectively,
of that of the carbon atoms.

29. A method of forming a composition of matter made
of carbon atoms, hydrogen atoms and groups R'-A;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube;

numbers of the hydrogen atoms and the groups R'-A
are less than 0.1 and less than 0.5, respectively, of that
of the carbon atoms;

R' is:

(a) cycloaryl or poly(alkylether), where



127


each of A is selected from

Image
Y is an appropriate functional group of a protein,
a peptide, an amino acid, an enzyme, an antibody, an
oligonucleotide, a nucleotide, an antigen, an enzyme
substrate, an enzyme inhibitor or a transition state analog
of an enzyme substrate or is selected from R'-OH, R'-NH2,
R'SH, R'CHO, R'CN, R'X, R'SiR'3, R'-R", R'-N-CO, (C2H4O~w H,
~C3H6O~w H, ~C2H4O~w-R', (C3H6O)w-R', R'and Image or

(b) alkyl, aryl, cycloalkyl or aralkyl, where A
is:
(i) Image where Y is an appropriate functional
group of a protein, a peptide, an amino acid, an enzyme, an
antibody, an oligonucleotide, a nucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH,
R'-NH2, R'SH, R'CHO, R'CN, R'X, R'SiR'3, R'-R", R'-N-CO,
(C2H4O~w H, ~C3H6O~w H, ~C2H4O~w-R', (C3H6O)w-R', R' and Image
or
(ii) OY, Image -CR'2-OY, N=Y
or C=Y, where Y is an appropriate functional group of an
amino acid or is Image

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide; and



128


Z is carboxylate or trifluoroacetate,
which method comprises:

(a) deoxygenating the graphitic nanotubes under
conditions sufficient to form deoxygenated nanotubes;

(b) reacting the deoxygenated nanotubes with at
least one appropriate activated olefin to form substituted
nanotubes having groups R'-R where each of R is selected
from SO3H, COOH, NH2, OH, CHO, CN, COCl, halide, COSH, SH,
COOR', SR', SiR'3, Si~OR'~y R'3-y, Si~O-SiR'2~OR', R", Li,
AlR'2, Hg-X, TlZ2 and Mg-X, and y is an integer of 1 to 3;
and

(c) reacting the substituted nanotubes having the
groups R'-R with at least one appropriate reagent under
conditions sufficient to form functionalized nanotubes
having the groups R'-A.

30. A method of forming a composition of matter made
of carbon atoms, hydrogen atoms and groups X'-R a;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube;

numbers of the carbon atoms and the groups X'-R a
are less than 0.1 and less than 0.5 of that of the carbon
atoms, respectively;

a is zero or an integer of 1 to 9;
each of R is:

(a) CH(R')OH, where R' is alkyl, aryl, cycloalkyl,
aralkyl or cycloaryl; or



129


(b) selected from COOR', SR', SiR'3, Si~OR'~y R'3-y,
Si~O-SiR'2~OR' and AlR'2, where R' is cycloaryl;
y is an integer of 1 to 3; and

X' is a polynuclear aromatic, polyheteronuclear
aromatic or metallopolyheteronuclear aromatic moiety,
which method comprises adsorbing at least one

appropriate macrocyclic compound onto a surface of the
graphitic nanotube under conditions sufficient to form a
functionalized nanotube having the groups X'-R a.

31. A method of forming a composition of matter made
of carbon atoms, hydrogen atoms and groups X'-A a;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube;

numbers of the hydrogen atoms and the groups X'-A a
are less than 0.1 and less than 0.5, respectively, of that
of the carbon atoms;

a is an integer of 1 to 9;
each of A is:

(a) Image where Y is an appropriate functional
group of a protein, a peptide, an amino acid, an enzyme, an
antibody, an oligonucleotide, a nucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH,
R'-NH2, R'SH, R'CHO, R'CN, R'X, R'SiR'3, R'-R", R'-N-CO,
~C2H4O~w H, ~C3H6O~w H, ~C2H4O~w-R', (C3H6O)w-R', R' and Image
where R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl or
cycloaryl;



130


(b) OY, Image N=Y or C=Y, where Y
is:

(i) an appropriate functional group of an amino
acid or is Image where R' is hydrogen, alkyl, aryl,
cycloalkyl, aralkyl or cycloaryl; or

(ii) selected from R'-OH, R'-NH2, R'SH, R'CHO,
R'CN, R'X, R'SiR'3, R'-R", R'-N-CO, ~2H4O)w-R', (C3H6O)w-R'
and R', where R' is hydrogen or cycloaryl; or

(c) Image or -CR'2-OY, where Y is:

(i) an appropriate functional group of an amino
acid or is Image where R' is hydrogen, alkyl, aryl,
cycloalkyl, aralkyl or cycloaryl;

(ii) an appropriate functional group of a protein,
a peptide, an enzyme, an antibody, an oligonucleotide, a
nucleotide, an antigen, an enzyme substrate, an enzyme
inhibitor or a transition state analog of an enzyme
substrate or is selected from R'-OH, R'-NH2, R'SH, R'CHO,
R'CN, R'X, R'SiR'3, R'-R", R'-N-CO, (C2H4O~w H, ~C3H6O~w H,
~C2H4O)w-R', (C3H6O)w-R' and R', where R' is hydrogen or
cycloaryl;

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide;

X' is a polynuclear aromatic, polyheteronuclear
aromatic or metallopolyheteronuclear aromatic moiety;



131


Z is carboxylate or trifluoroacetate; and

w is an integer greater than one and less than 200,
which method comprises:

(a) adsorbing at least one appropriate macrocyclic
compound onto a surface of the graphitic nanotube under
conditions sufficient to form a substituted nanotube having
groups X'-R a, where each of R is selected from SO3H, COOH,
NH2, OH, CHO, CN, COCl, halide, COSH, SH, COOR', SR', SiR'3,
Si~OR'~y R'3-y, Si~O-SiR'2~OR', R", Li, AlR'2, Hg-X, TlZ2 and
Mg-X, and y is an integer of 1 to 3; and

(b) reacting the substituted nanotubes with at
least one appropriate reagent under conditions sufficient to
form a functionalized nanotube having the groups X'-A a.

32. A method of forming a composition of matter made
of carbon atoms, hydrogen atoms and groups X'-A a;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube;

numbers of the hydrogen atoms and the groups X'-A a
are less than 0.1 and less than 0.5, respectively, of that
of the carbon atoms;

a is an integer of 1 to 9;
each of A is:

(a) Image where Y is an appropriate functional
group of a protein, a peptide, an amino acid, an enzyme, an
antibody, an oligonucleotide, a nucleotide, an antigen, an
enzyme substrate, an enzyme inhibitor or a transition state
analog of an enzyme substrate or is selected from R'-OH,
R'-NH2, R'SH, R'CHO, R'CN, R'X, R'SiR'3, R'-R", R'-N-CO,



132


(C2H4O~w H, ~C3H6O~w H, ~C2H4O)w-R', (C3H6O)w-R', R' and Image
where R' is alkyl, aryl, cycloalkyl, aralkyl or cycloaryl;

(b) OY, Image N=Y or C=Y, where Y
is:

(i) an appropriate functional group of an amino
acid or is Image where R' is alkyl, aryl, cycloalkyl,
aralkyl or cycloaryl; or

(ii) selected from R'-OH, R'-NH2, R'SH, R'CHO,
R'CN, R'X, R'SiR'3, R'-R", R'-N-CO, ~C2H4O)w-R', (C3H6O)w-R'
and R', where R' is cycloaryl; or

(c) Image or -CR'2-OY, where Y is:

(i) an appropriate functional group of an amino
acid or is Image where R' is alkyl, aryl, cycloalkyl,
aralkyl or cycloaryl;

(ii) an appropriate functional group of a protein,
a peptide, an enzyme, an antibody, an oligonucleotide, a
nucleotide, an antigen, an enzyme substrate, an enzyme
inhibitor or a transition state analog of an enzyme
substrate or is selected from R'-OH, R'-NH2, R'SH, R'CHO,
R'CN, R'X, R'SiR'3, R'-R", R'-N-CO, (C2H4O~w H, ~C3H6O~w H,
~C2H4O)w-R', (C3H6O)w-R' and R', where R' is cycloaryl;

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide;

X' is a polynuclear aromatic, polyheteronuclear
aromatic or metallopolyheteronuclear aromatic moiety;



133


Z is carboxylate or trifluoroacetate; and

w is an integer greater than one and less than 200,
which method comprises reacting a substituted
nanotube with at least one appropriate reagent under
conditions sufficient to form functionalized nanotubes
having the groups X'-A a,

wherein the substituted nanotubes are made of
carbon atoms, hydrogen atoms and groups X'-R a;

numbers of the hydrogen atoms and the groups X'-R a
are less than 0.1 and less than 0.5 of the carbon atoms,
respectively;

each of R is selected from SO3H, COOH, NH2, OH,
CHO, CN, COCl, halide, COSH, SH, COOR', SR', SiR'3,

Si~OR'~y R'3-y, Si~O-SiR'2~OR', R", Li, AlR'2r Hg-X, TlZ2 and
Mg-X, and y is an integer of 1 to 3.

33. A method for forming a composition of matter made
of carbon atoms, hydrogen atoms and groups Image

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube;

numbers of the hydrogen atoms and the groups
Image are less than 0.1 and less than 0.5,
respectfully, of that of the carbon atoms;

R' is alkyl, aryl, cycloalkyl or cycloaryl,
which methods comprises:

reacting the surface carbons with at least one
appropriate reagent under conditions sufficient to form
functionalized nanotubes having groups COOH; and


134
reacting the functionalized nanotubes with a
compound having two or more amino groups under conditions
sufficient to form the functionalized nanotubes having the
group Image

34. A method of forming a composition of matter made
of carbon atoms, hydrogen atoms and groups R;

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube;

numbers of the hydrogen atoms and the groups R are
less than 0.1 and less than 0.5, respectively, of that of
the carbon atoms;

each of R is the same and is selected from SO3H,
COOH, NH2, OH, CH(R')OH, CHO, CN, COC1, halide, COSH, SH,
COOR', SR', SiR' 3, , Si+OR' +-y R' 3-y', Si~-O-SiR'2~OR' , R", Li,
AlR' 2, Hg-X, T1Z2 and Mg-X;

y is an integer of 1 to 3;

R' is hydrogen, alkyl, aryl, cycloalkyl, aralkyl
or cycloaryl,

R" is fluoroalkyl, fluoroaryl, fluorocycloalkyl or
fluoroaralkyl;

X is a halide; and

Z is carboxylate or trifluoroacetate,
comprising the step of reacting the surface
carbons with at least one enzyme capable of accepting the
nanotube as a substrate and of performing a chemical
reaction to form functionalized nanotubes having the
group R, in aqueous suspension under conditions acceptable
for the at least one enzyme to carry out the reaction.


135
35. The method according to claim 34, wherein the
group R is -OH and the enzyme is a cytochrome p450 enzyme or
a peroxidase.

36. A method for forming a composition of matter made
of carbon atoms, hydrogen atoms and groups NH2,

wherein the carbon atoms are surface carbons of a
substantially cylindrical, graphitic nanotube,

numbers of the hydrogen atoms and the groups NH2
are less than 0.1 and less than 0.5, respectively, of that
of the carbon atoms;

which method comprises:

reacting the surface carbons with nitric acid and
sulfuric acid to form nitrated nanotubes; and

reducing the nitrated nanotubes to form
functionalized nanotubes having the group NH2.

37. A method of uniformly substituting a surface of
carbon nanotubes with a functional group which comprises
contacting carbon nanotubes with a reactant capable of
uniformly substituting a functional group onto the surface
of the carbon nanotubes, wherein the reactant is nickel (II)
phthalocyaninetetrasulfonic acid (tetrasodium salt) or
1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine.
38. A surface-modified carbon nanotube made by a
method which comprises contacting a carbon nanotube with a
reactant for substituting a functional group onto a surface
of the carbon nanotube, wherein the reactant is nickel (II)
phthalocyaninetetrasulfonic acid (tetrasodium salt) or
1,4,8,11,15,18,22,25-octabutoxy-29H,31H-phthalocyanine.


136
39. A method for linking a protein to a nanotube which
comprises:

contacting a nanotube bearing an NHS ester group
with a protein under conditions sufficient to form a
covalent bond between the NHS ester and an amine group of
the protein.

40. An electrode comprising functionalized nanotubes
wherein the electrode is a porous flow through electrode.
41. The electrode according to claim 40, wherein the
functionalized nanotubes are phthalocyanine substituted
nanotubes.

42. A method for separating a solute of interest from
a sample which comprises:

exposing substituted nanotubes to a fraction
containing the solute of interest under conditions
sufficient for the solute of interest to bind a substance
being capable of binding the solute of interest and being
immobilized on the substituted nanotubes, wherein the
substituted nanotubes are obtained by:

physically or chemically modifying surface carbons
of graphitic nanotubes with at least one appropriate reagent
under conditions sufficient to form functionalized
nanotubes; and

immobilizing the substance capable of binding the
solute of interest on the functionalized nanotubes.

43. The method according to claim 42 wherein the
solute of interest is a protein.

44. The method according to claim 43, which further
comprises recovering the functionalized nanotubes.


137
45. The method according to claim 42, 43 or 44,
wherein the functionalized nanotubes are in a form of a
porous mat.

46. The method according to claim 42, 43 or 44,
wherein the functionalized nanotubes are in a form of a
packed column.

47. The method according to any one of
claims 42 to 46, wherein the binding is reversible.
48. The method according to any one of
claims 42 to 46, wherein the binding is an ionic
interaction.

49. The method according to any one of

claims 42 to 46, wherein the binding is a hydrophobic
interaction.

50. The method according to any one of

claims 42 to 46, wherein the binding is through specific
molecular recognition.

51. A polymer bead which comprises a bead with a
diameter of less than 25µ to which is linked a plurality of
functionalized nanotubes.

52. The polymer bead according to claim 51, wherein
the bead is magnetic.

53. A method for catalyzing a reaction wherein at
least one reactant is converted to at least one product
which comprises:

contacting functionalized nanotubes having a
biocatalyst capable of catalyzing the reaction immobilized
thereon, with the reactant under conditions sufficient for
the reactant to be converted to the product, wherein the



138


biocatalyst-immobilized functionalized nanotubes are
obtained by:

physically or chemically modifying surface carbons
of graphitic nanotubes with at least one appropriate reagent
under conditions sufficient to form functionalized
nanotubes; and

immobilizing the biocatalyst capable of catalyzing
a reaction on the functionalized nanotubes.

54. The method according to claim 53, which further
comprises recovering the biocatalyst-immobilized
functionalized nanotubes after the reaction is complete.
55. The method according to claim 53 or 54, wherein
the biocatalyst-immobilized functionalized nanotubes are a
porous mat.

56. The method according to claim 53 or 54, wherein
the functionalized nanotubes are a packed column.

57. A method for synthesizing a peptide which
comprises:

attaching a terminal amino acid of the peptide to
a nanotube via a reversible linker; and

then adding one or more amino acids to the
terminal amino acid.

58. The method according to claim 57, wherein the
linker is 4-(hydroxymethyl)phenoxyacetic acid.

Description

1 i
CA 02247820 2007-06-15
51096-13

1
FIINCTIONALIZED NANOTUBEB

FIELD OF THE INVENTION
The invention relates broadly to graphitic
nanotubes, which includes tubular fullerenes (commonly
called "buckytubes") and fibrils, which are
functionalized by chemical substitution or by adsorption
of functional moieties. More specifically the invention
relates to graphitic nanotubes which are uniformly or
non-uniformly substituted with chemical moieties or upon
which certain cyclic compounds are adsorbed and to
complex structures comprised of such functionalized
fibrils linked to one another. The invention also
relates to methods of introducing functional groups onto
the surface of such fibrils.
BACKaROUND OF THE INVENTION
This invention lies in the field of submicron
graphitic fibrils, sometimes called vapor grown carbon
fibers. Carbon fibrils are vermicular carbon deposits
having diameters less than 1.O , preferably less than
0.5Fc, and even more preferably less than 0.24. They
exist in a variety of forms and have been prepared
through the catalytic decomposition of various carbon-
containing gases at metal surfaces. Such vermicular
carbon deposits have been observed almost since the
advent of electron microscopy. A good early survey and
reference is found in Baker and Harris, Chemistry and
Physics of Carbon, Walker and Thrower ed., Vol. 14, 1978,
p. 83. See also,
Rodriguez, N., J. Mater. Research, Vol. 8, p. 3233
(1993).
In 1976, Endo et al. (see Obelin, A. and Endo,
M., J. of Crystal Growth, Vol. 32 (1976), pp. 335-349)


CA 02247820 2007-06-15
51096-13

2
elucidated the basic
mechanism by which such carbon fibrils grow. There were
seen to originate from a metal catalyst particle, which,
in the presence of a hydrocarbon containing gas, becomes
supersaturated in carbon. A cylindrical ordered
graphitic core is extruded which immediately, according
to Endo et al., becomes coated with an outer layer of
pyrolytically deposited graphite. These fibrils with a
pyrolytic overcoat. typically have diameters in excess of
0.1 , more typically 0.2 to 0.5Et.
In 1983, Tennent, U.S. Patent No. 4,663,230,
succeeded in growing
cylindrical ordered graphite cores, uncontaminated with
pyrolytic carbon. Thus, the Tennent invention provided
access to smaller diameter fibrils, typically 35 to 700 A
(0.0035 to 0.070 ) and to an ordered, "as grown"
graphitic surface. Fibrillar carbons of less perfect
structure, but also without a pyrolytic carbon outer
layer have also been grown.
The fibrils, buckytubes and nanofibers that are
functionalized in this application are distinguishable
from continuous carbon fibers commercially available as
reinforcement materials. In contrast to fibrils, which
have, desirably large, but unavoidably finite aspect
ratios, continuous carbon fibers have aspect ratios (L/D)
of at least 104 and often 106 or more. The diameter of
continuous fibers is also far larger than that of
fibrils, being always >1.o and typically 5 to 7 .
Continuous carbon fibers are made by the
pyrolysis of organic precursor fibers, usually rayon,
polyacrylonitrile (PAN) and pitch. Thus, they may
include heteroatoms within their structure. The
graphitic nature of "as made" continuous carbon fibers
varies, but they may be subjected to a subsequent
graphitization step. Differences in degree of
graphitization, orientation and crystallinity of graphite
planes, if they are present, the potential presence of


CA 02247820 2007-06-15
51096-13

3
heteroatoms and even the absolute difference in substrate
diameter make experience with continuous fibers poor
predictors of nanofiber chemistry.
Tennent, U.S. Patent No. 4,663,230 describes
carbon fibrils that are free of a continuous thermal
carbon overcoat and have multiple graphitic outer layers
that are substantially parallel to the fibril axis. As
such they may be characterized as having their c-axes,
the axes which are perpendicular to the tangents of the
curved layers of graphite, substantially perpendicular to
their cylindrical axes. They generally have diameters no
greater than 0.1 and length to diameter ratios of at
least 5. Desirably they are substantially free of a
continuous thermal carbon overcoat, i.e., pyrolytically
deposited carbon resulting from thermal cracking of the
gas feed used to prepare them.
Tennent, et al., US Patent No. 5,171,560,
describes carbon
fibrils free of thermal overcoat and having graphitic
layers substantially parallel to the .fibril axes such
that the projection of the layers on the fibril axes
extends for a distance of at least two fibril diameters.
Typically, such fibrils are substantially cylindrical,
graphitic nanotubes of substantially constant diameter
and comprise cylindrical graphitic sheets whose c-axes
are substantially perpendicular to their cylindrical
axis. They are substantially free of pyrolytically
deposited carbon, have a diameter less than 0.1 and a
length to diameter ratio of greater than 5. These
fibrils are of primary interest in the invention.
Further details regarding the formation of
carbon fibril aggregates may be found in the disclosure
of Snyder et al., U.S. Patent No. 5,707,919 and PCT
Publication No. WO 89/07163 ("Carbon Fibrils"), and Moy
et al., U.S. Patent No. 5,456,897 and PCT Publication


CA 02247820 2007-06-15
51096-13

4
No. WO 91/05089 ("Fibril Aggregates and Method of Making
Same"), all of which are assigned to the same assignee as
the present application.
Moy et al., U.S. Patent No. 6,143,689 describes
fibrils prepared as aggregates having various macroscopic
morphologies (as determined by scanning electron
microscopy) in which they are randomly entangled with
each other to form entangled balls of fibrils resembling
bird nests ("BN"); or as aggregates consisting of bundles
of straight to slightly bent or kinked carbon fibrils
having substantially the same relative orientation, and
having the appearance of combed yarn ("CY") e.g., the
longitudinal axis of each fibril (despite individual
bends or kinks) extends in the same direction as that of
the surrounding fibrils in the bundles; or as aggregates
consisting of straight to slightly bent or kinked fibrils
which are loosely entangled with each other to form an
"open net" ("ON") structure. In open net structures the
degree of fibril entanglement is greater than observed in
the combed yarn aggregates (in which the individual
fibrils have substantially the same relative orientation)
but less than that of bird nests. CY and ON aggregates
are more readily dispersed than BN making them useful in
composite fabrication where uniform properties throughout
the structure are desired.
When the projection of the graphitic layers on
the fibril axis extends for a distance of less than two
fibril diameters, the carbon planes of the graphitic
nanofiber, in cross section, take on a herring bone
appearance. These are termed fishbone fibrils. Geus,
U.S. Patent No. 4,855,091
provides a procedure for preparation of
fishbone fibrils substantially free of a pyrolytic
overcoat. These fibrils are also useful in the practice
of the invention.


CA 02247820 2007-06-15
51096-13

Carbon nanotubes of a morphology similar to the
catalytically grown fibrils described above have been
grown in a high temperature carbon arc (Iijima, Nature
354 56 1991). It is now generally accepted (Weaver,
5 Science 265 1994) that these arc-grown nanofibers have
the same morphology as the earlier catalytically grown
fibrils of Tennent. Arc grown carbon nanofibers are also
useful in the invention.
McCarthy et al., U.S. Patent No. 5,965,470
describes processes for oxidizing the surface
of carbon fibrils that include contacting the fibrils
with an oxidizing agent that includes sulfuric acid
(H2SO4) and potassium chlorate (KC103) under reaction
conditions (e.g., time, temperature, and pressure)
sufficient to oxidize the surface of the fibril. The
fibrils oxidized according to the processes of McCarthy,
et al. are non-uniformly oxidized, that is, the carbon
atoms are substituted with a mixture of carboxyl,
aldehyde, ketone, phenolic and other carbonyl groups.
Fibrils have also been oxidized non-uniformly
by treatment with nitric acid. International Publication
WO 95/07316 discloses the formation of oxidized
fibrils containing a mixture of functional groups.
Hoogenvaad, M.S., et al. ("Metal Catalysts supported on a
Novel Carbon Support", Presented at Sixth International
Conference on Scientific Basis for the Preparation of
Heterogeneous Catalysts, Brussels, Belgium, September
1994) also found it beneficial in the preparation of
fibril-supported precious metals to first oxidize the
fibril surface with nitric acid. Such pretreatment with
acid is a standard step in the preparation of carbon-
supported noble metal catalysts, where, given the usual
sources of such carbon, it serves as much to clean the
surface of undesirable materials as to functionalize it.
In published work, McCarthy and Bening (Polymer
Preprints ACS Div. of Polymer Chem. 30 (1)420(1990))


CA 02247820 2007-06-15
51096-13

6
prepared derivatives of oxidized fibrils in order to
demonstrate that the surface comprised a variety of
oxidized groups. The compounds they prepared,
phenyihydrazones, haloaromaticesters, thallous salts,
etc., were selected because of their analytical utility,
being, for example, brightly colored, or exhibiting some
other strong and easily identified and differentiated
signal. These compounds were not isolated and are,
unlike the derivatives described herein, of no practical
significance.
While many uses have been found for carbon
fibrils and aggregates of carbon fibrils, as described in
the patents and patent applications referred to above,
many different and important uses may be developed if the
fibril surfaces are functionalized. Functionalization,
either uniformly or non-uniformly, permits interaction of
the functionalized fibrils with various substrates to
form unique compositions of matter with unique properties
and permits fibril structures to be created based on
linkages between the functional sites on the f ibrils'
surf aces .
OBJECTS OF THE INVENTION
It is therefore a primary object of this
invention to provide functionalized fibrils, i.e. fibrils
whose surfaces are uniformly or non-uniformly modified so
as to have a functional chemical moiety associated
therewith.


CA 02247820 2007-06-15
51096-13

7
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a graphical representation of an
assay of BSA binding to plain fibrils, carboxy fibrils,
and PEG-modified fibrils.
Fig. 2 is a graphical representation of an
assay of B-lactoglobulin binding to carboxy fibrils and
PEG-modified fibrils prepared by two different methods.
Fig. 3 is a graphical representation of the
elution profile of bovine serum albumin (BSA) on a
tertiary amine fibril column.
Fig. 4 is a graphical representation of the
elution profile of BSA on a quaternary amine fibril
column.
Fig. 5 is the reaction sequence for the
preparation of lysine-based dendrimeric fibrils.


CA 02247820 2007-06-15
51096-13

8
Fig. 6 is a graphical representation of cyclic
voltammograms demonstrating the use of iron
phthalocyanine modified fibrils in a flow cell.
Fig. 7 is the reaction sequence for the
preparation of bifunctional fibrils by the addition of
NE-(tert-butoxycarbonyl)-L-lysine.
Fig. 8 is a graphical representation of the
results of the synthesis of ethyl butyrate using fibril-
immobilized lipass.
Fig. 9 is a graphical representation of the
results of separation of alkaline phosphatase (AP) from a
mixture of AP and P-galactosidase (BG) using AP
inhibitor-modified fibrils.
Fig. 10 is a graphical representation of the
results of separation of BG from a mixture of AP and BG
using BG-modified fibrils.
DETAILED DESCRIPTION OF THE INVENTION
The invention is directed to compositions which
broadly have the formula
[ CnHLJ-Rm
where n is an integer, L is a number less than
0.1n, m is a number less than 0.5n,
each of R is the same and is selected from
SO3H., COOH, NHZ, OH, R'CHOH, CHO, CN, COCI, halide, COSH,
SH, COOR', SR', SiR' 3, Si-fOR'}yR' 3_Y, Si-{-O-SiR' 2}OR' , R",
Li, AlR'2, Hg-X, T1Z2 and Mg-X,
y is an integer equal to or less than 3,
R' is hydrogen, alkyl, aryl, cycloalkyl, or
aralkyl, cycloaryl, or poly(alkylether),

R" is fluoroalkyl, fluoroaryl,
fluorocycloalkyl, fluoroaralkyl or cycloaryl,
X is halide, and

Z is carboxylate or trifluoroacetate.


CA 02247820 2007-06-15
51096-13

8a
The compositions may be expressed alternatively as
those made of carbon atoms, hydrogen atoms and groups R, in
which number of the hydrogen atoms and the groups R are less
than 0.1 and less than 0.5 of that of the carbon atoms,

respectively.

The carbon atoms, C, are surface carbons of a
substantially cylindrical, graphitic nanotube of


CA 02247820 1998-08-28

WO 97/32571 PCT/US97/03553
9
substantially constant diameter. The nanotubes include
those having a length to diameter ratio of greater than 5
and a diameter of less than 0.5 , preferably less than
0.1 . The nanotubes can also be substantially
cylindrical, graphitic nanotubes which are substantially
free of pyrolytically deposited carbon, more preferably
those characterized by having a projection of the
graphite layers on the fibril axis which extends for a
distance of at least two fibril diameters and/or those
having cylindrical graphitic sheets whose c-axes are
substantially perpendicular to their cylindrical axis.
These compositions are uniform in that each of R is the
same.
Non-uniformly substituted nanotubes are also
prepared. These include compositions of the formula
[ CnHL~Rm
where n, L, m, R and the nanotube itself are as defined
above, provided that each of R does not contain oxygen,
or, if each of R is an oxygen-containing group COOH is
not present.
Functionalized nanotubes having the formula
[CnHL+ Rm
where n, L, m, R and R' have the same meaning as above
and the carbon atoms are surface carbon atoms of a
fishbone fibril having a length to diameter ratio greater
than 5, are also included within the invention. These
may be uniformly or non-uniformly substituted.
Preferably, the nanotubes are free of thermal overcoat
and have diameters less than 0.5 .
Also included in the invention are
functionalized nanotubes having the formula
[CnHL}[R-R]m
where n, L, m, R' and R have the same meaning as above.
The carbon atoms, Cn, are surface carbons of a
substantially cylindrical, graphitic nanotube of
substantially constant diameter. The nanotubes have a
length to diameter ratio of greater than 5 and a diameter


CA 02247820 1998-08-28

WO 97/32571 PCT/US97/03553
of less than 0.5g, preferably less than 0.1 . The
nanotubes may be nanotubes which are substantially free
of pyrolytically deposited carbon. More preferably, the
nanotubes are those in which the projection of the
5 graphite layers on the fibril axes extends for a distance
of at least two fibril diameters and/or those having
cylindrical graphitic sheets whose c-axes are
substantially perpendicular to their cylindrical axis.
In both uniformly and non-uniformly substituted
10 nanotubes, the surface atoms Cn are reacted. Most carbon
atoms in the surface layer of a graphitic fibril, as in
graphite, are basal plane carbons. Basal plane carbons
are relatively inert to chemical attack. At defect
sites, where, for example, the graphitic plane fails to
extend fully around the fibril, there are carbon atoms
analogous to the edge carbon atoms of a graphite plane
(See Urry, Elementary Equilibrium Chemistry of Carbon,
Wiley, New York 1989.) for a discussion of edge and basal
plane carbons).
At defect sites, edge or basal plane carbons of
lower, interior layers of the nanotube may be exposed.
The term surface carbon includes all the carbons, basal
plane and edge, of the outermost layer of the nanotube,
as well as carbons, both basal plane and/or edge, of
lower layers that may be exposed at defect sites of the
outermost layer. The edge carbons are reactive and must
contain some heteroatom or group to satisfy carbon
valency.
The substituted nanotubes described above may
advantageously be further functionalized. Such
compositions include compositions of the formula

[CnHL+ Am
where the carbons are surface carbons of a nanotube, n, L
and m are as described above,
A is selected from
0 0 0 0 0
II II II I! ~)


CA 02247820 1998-08-28

WO 97/32571 PCT/US97/03553
11
OY, NHY, C-OY, C-NR'Y, C-SY, C-Y, -CR'2-OY, N=Y, -NHCY or
C=Y,
Y is an appropriate functional group of a
protein, a peptide, an amino acid, an enzyme, an
antibody, a nucleotide, an oligonucleotide, an antigen,
or an enzyme substrate, enzyme inhibitor or the
transition state analog of an enzyme substrate or is
selected from R'-OH, R'-NR'2, R'SH, R'CHO, R'CN, R'X,
R'N{'(R')3X-, R'SiR'3, R'Si-fOR'3-yR'3_y, R'Si-fO-SiR'2-}OR',
r


CA 02247820 1998-08-28

WO 97/32571 PCT/US97/03553
12
R I-R" , R I-N-CO , ( C2H4,0-)-WH , -(-C3H60}WH , +C2H40 ) w R I,
(C3H60)w R'.

0 R' A' -N

0
and w is an integer greater than one and less
than 200.
The carbon atoms, Cn, are surface carbons of a
substantially cylindrical, graphitic nanotube of
substantially constant diameter. The nanotubes include
those having a length to diameter ratio of greater than 5
and a diameter of less than O.l , preferably less than
0.05/.c. The nanotubes can also be substantially
cylindrical, graphitic nanotubes which are substantially
free of pyrolytically deposited carbon. More preferably
they are characterized by having a projection of the
graphite layers on the fibril axes which extends for a
distance of at least two fibril diameters and/or they are
comprised of cylindrical graphitic sheets whose c-axes
are substantially perpendicular to their cylindrical
axes. Preferably, the nanotubes are free of thermal
overcoat and have diameters less than 0.5 .
The functional nanotubes of structure
[CnHL-j-[R-R]m
may also be functionalized to produce compositions having
the formula
LCnHI,-1-[ R-A] m
where n, L, m, RI and A are as defined above. The carbon
atoms, Cn, are surface carbons of a substantially
cylindrical, graphitic nanotube of substantially constant
diameter. The nanotubes include those having a length to
diameter ratio of greater than 5 and a diameter of less
than 0.5 , preferably less than 0.1 . The nanotubes can


CA 02247820 1998-08-28

WO 97/32571 PCT/US97/03553
13
also be substantially cylindrical, graphitic nanotubes
which are substantially free of pyrolytically deposited
carbon. More preferably they are characterized by having
a projection of the graphite layers on the fibril axes
which extends for a distance of at least two fibril
diameters and/or by having cylindrical graphitic sheets
whose c-axes are substantially perpendicular to their
cylindrical axis. Preferably, the nanotubes are free of
thermal overcoat and have diameters less than 0.5 .
The compositions of the invention also include
nanotubes upon which certain cyclic compounds are
adsorbed. These include compositions of matter of the
formula
jCnHL-3-jX-Rajm
where n is an integer, L is a number less than O.in, m is
less than 0.5n, a is zero or a number less than 10, X is
a polynuclear aromatic, polyheteronuclear aromatic or
metallopolyheteronuclear aromatic moiety and R is as
recited above. The carbon atoms, Cn, are surface carbons
of a substantially cylindrical, graphitic nanotube of
substantially constant diameter. The nanotubes include
those having a length to diameter ratio of greater than 5
and a diameter of less than 0.5 , preferably less than
0.1/.t. The nanotubes can also be substantially
cylindrical, graphitic nanotubes which are substantially
free of pyrolytically deposited carbon and more
preferably those characterized by having a projection of
the graphite layers on said fibril axes which extend for
a distance of at least two fibril diameters and/or those
having cylindrical graphitic sheets whose c-axes are
substantially perpendicular to their cylindrical axes.
Preferably, the nanotubes are free of thermal overcoat
and have diameters less than 0.5 .
Preferred cyclic compounds are planar
macrocycles as described on p. 76 of Cotton and
Wilkinson, Advanced Organic Chemistry. More preferred


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
14
cyclic compounds for adsorption are porphyrins and
phthalocyanines.
The adsorbed cyclic compounds may be
functionalized. Such compositions include compounds of
the formula
[CnHi,-3-CX-AaI m
where m, n, L, a, X and A are as defined above and the
carbons are surface carbons of a substantially
cylindrical graphitic nanotube as described above.
The carbon fibrils functionalized as described
above may be incorporated in a matrix. Preferably, the
matrix is an organic polymer (e.g., a thermoset resin
such as epoxy, bismaleimide, polyamide, or polyester
resin; a thermoplastic resin; a reaction injection molded
resin; or an elastomer such as natural rubber, styrene-
butadiene rubber, or cis-1,4-polybutadiene); an inorganic
polymer (e.g., a polymeric inorganic oxide such as
glass), a metal (e.g., lead or copper), or a ceramic
material (e.g., Portland cement). Beads may be formed
from the matrix into which the fibrils have been
incorporated. Alternately, functionalized fibrils can be
attached to the outer surface of functionalized beads.
Without being bound to a particular theory, the
functionalized fibrils are better dispersed into polymer
systems because the modified surface properties are more
compatible with the polymer, or, because the modified
functional groups (particularly hydroxyl or amine groups)
are bonded directly to the polymer as terminal groups.
In this way, polymer systems such as polycarbonates,
polyurethanes, polyesters or polyamides/imides bond
directly to the fibrils making the fibrils easier to
disperse with improved adherence.
The invention is also in methods of introducing -
functional groups onto the surface of carbon fibrils by
contacting carbon fibrils with a strong oxidizing agent
for a period of time sufficient to oxidize the surface of
said fibrils and further contacting said fibrils with a


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
reactant suitable for adding a functional group to the
oxidized surface. In a preferred embodiment of the
invention, the oxidizing agent is comprised of a solution
of an alkali metal chlorate in a strong acid. In other
5 embodiments of the invention the alkali metal chlorate is
sodium chlorate or potassium chlorate. In preferred
embodiments the strong acid used is sulfuric acid.
Periods of time sufficient for oxidation are from about
0.5 hours to about 24 hours.
10 In a further preferred embodiment, a
composition having the formula [CnHL}J-CH(R')OH]m, wherein
n, L, R' and m are as defined above, is formed by
reacting R'CH2OH with the surface carbons of a nanotube
in the presence of a free radical initiator such as
15 benzoyl peroxide.
The invention is also in a method for linking
proteins to nanotubes modified by an NHS ester, by
forming a covalent bond between the NHS ester and the
amino group of the protein.
The invention is also in methods for producing
a network of carbon fibrils comprising contacting carbon
fibrils with an oxidizing agent for a period of time
sufficient to oxidize the surface of the carbon fibrils,
contacting the surface-oxidized carbon fibrils with
reactant suitable for adding a functional group to the
surface of the carbon fibrils, and further contacting the
surface-functionalized fibrils with a cross-linking agent
effective for producing a network of carbon fibrils. A
preferred cross-linking agent is a polyol, polyamine or
polycarboxylic acid.
Functionalized fibrils also are useful for
preparing rigid networks of fibrils. A well-dispersed,
three-dimensional network of acid-functionalized fibrils
may, for example, be stabilized by cross-linking the acid
groups (inter-fibril) with polyols or polyamines to form
a rigid network.


CA 02247820 1998-08-28

WO 97/32571 PCT/US97/03553
16
The invention also includes three-dimensional
networks formed by linking functionalized fibrils of the
invention. These complexes include at least two functionalized fibrils linked
by one or more linkers

comprising a direct bond or chemical moiety. These
networks comprise porous media of remarkably uniform
equivalent pore size. They are useful as adsorbents,
catalyst supports and separation media.
Although the interstices between these fibrils
are irregular in both size and shape, they can be thought
of as pores and characterized by the methods used to
characterize porous media. The size of the interstices
in such networks can be controlled by the concentration
and level of dispersion of fibrils, and the concentration
and chain lengths of the cross-linking agents. Such
materials can act as structured catalyst supports and may
be tailored to exclude or include molecules of a certain
size. Aside from conventional industrial catalysis, they
have special applications as large pore supports for
biocatalysts.
The rigid networks can also serve as the
backbone in biomimetic systems for molecular recognition.
Such systems have been described in US Patent No.
5,110,833 and International Patent Publication No.
W093/19844. The appropriate choices for cross-linkers
and complexing agents allow for stabilization of specific
molecular frameworks.
MlETHODS OF FUNCTIONALIZING NANOTUBES
The uniformly functionalized fibrils of the
invention can be directly prepared by sulfonation,
electrophilic addition to deoxygenated fibril surfaces or
metallation. When arc grown nanofibers are used, they
may require extensive purification prior to
functionalization. Ebbesen et al. (Nature 367 519
(1994)) give a procedure for such purification.
Preferably, the carbon fibrils are processed
prior to contacting them with the functionalizing agent.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
17
Such processing may include dispersing the fibrils in a
solvent. In some instances the carbon fibrils may then
be filtered and dried prior to further contact.
1. SIILFONATION
Background techniques are described in March,
J.P., Advanced Organic Chemistry, 3rd Ed. Wiley, New York
1985; House, H., Modern Synthetic Reactions, 2nd Ed.,
Benjamin/Cummings, Menlo Park, CA 1972.
Activated C-H (including aromatic C-H) bonds
can be sulfonated using fuming sulfuric acid (oleum),
which is a solution of conc. sulfuric acid containing up
to 20% SO3. The conventional method is via liquid phase
at T-80 C using oleum; however, activated C-H bonds can
also be sulfonated using SO3 in inert, aprotic solvents,
or SO3 in the vapor phase. The reaction is:
-C-H + S03 ----> -C-SO3H
Over-reaction results in formation of sulfones, according
to the reaction:
2 -C-H + SO3 ----> -C-S02-C- + H20
EXAMPLE 1
Activation of C-H Bonds Using Sulfuric Acid
Reactions were carried out in the gas phase and
in solution without any significant difference in
results. The vapor phase reaction was carried out in a
horizontal quartz tube reactor heated by a Lindberg
furnace. A multi-neck flask containing 20% SO3 in conc.
H2SO4 fitted with gas inlet/outlet tubes was.used as the
SO3 source.
A weighed sample of fibrils (BN or CC) in a
porcelain boat was placed in the 1" tube fitted with a
gas inlet; the outlet was connected to a conc. H2SO4
bubbler trap. Argon was flushed through the reactor for
20 min to remove all air, and the sample was heated to
300 C for 1 hour to remove residual moisture. After
drying, the temperature was adjusted to reaction
temperature under argon.


CA 02247820 1998-08-28
WO 97/32571 PCTIUS97/03553
18
When the desired temperature was stabilized,
the SO3 source was connected to the reactor tube and an
argon stream was used to carry S03 vapors into the quartz
tube reactor. Reaction was carried out for the desired
time at the desired temperature, after which the reactor
was cooled under flowing argon. The fibrils were then
dried at 90 C at 5" Hg vacuum to obtain the dry weight
gain. Sulfonic acid (-SO3H) content was determined by
reaction with 0.100N NaOH and back-titration with 0.100N
HC1 using pH 6.0 as the end point.
The liquid phase reaction was carried out in
conc. sulfuric acid containing 20% SO3 in a multi-neck
100 cc flask fitted with a thermometer/temperature
controller and a magnetic stirrer. A fibril slurry in
conc. H2SO4 (50) was placed in the flask. The oleum
solution (20 cc) was preheated to -60 C before addition
to the reactor. After reaction, the acid slurry was
poured onto cracked ice, and diluted immediately with 1 1
DI water. The solids were filtered and washed
exhaustively with DI water until there was no change in
pH of the wash effluent. Fibrils were dried at 100 C at
5"' Hg vacuum. Due to transfer losses on filtration,
accurate weight gains could not be obtained. Results are
listed in Table 1.
TABLE I
Summary of Reactions

SAMPLE FIBRIL DRY Wt SO3H CONC
EX= RUN REACT Wt.q TYPE T C TIME GAIN meq/g
1A 118-60A Vap 0.20 CY 110 15 m 9.3% 0.50
1B 118-61A Vap 0.20 BN 100 30 m 8.5% 0.31
1C 118-61B Vap 0.20 BN 65 15 m 4.2% 0.45
1D 118-56A Liq 1.2 CY 50 10 m 0.33


CA 02247820 1998-08-28
WO 97/32571 PCTIUS97/03553
19
1E 118-56B Liq 1.0 CY 25 20 m 0.40

There was no significant difference in sulfonic
acid content by reaction in the vapor phase or liquid
phase. There was a temperature effect. Higher
temperature of reaction (vapor phase) gives higher
amounts of sulfones. In 118-61B, the 4.2% wt gain agreed
with the sulfonic acid content (theoretical was 0.51
meq/g). Runs 60A and 61A had too high a wt gain to be
accounted for solely by sulfonic acid content. It was
therefore assumed that appreciable amounts of sulfones
were also made.
2. ADDITIONS TO OXIDE-FREE FIBRIL SURFACES
Background techniques are described in Urry,
G., Elementary Equ.i.Zibrium Chemistry of Carbon, Wiley,
New York 1989.
The surface carbons in fibrils behave like
graphite, i.e., they are arranged in hexagonal sheets
containing both basal plane and edge carbons. While
basal plane carbons are relatively inert to chemical
attack, edge carbons are reactive and must contain some
heteroatom or group to satisfy carbon valency. Fibrils
also have surface defect sites which are basically edge
carbons and contain heteroatoms or groups.
The most common heteroatoms attached to surface
carbons of fibrils are hydrogen, the predominant gaseous
component during manufacture; oxygen, due to its high
reactivity and because traces of it are very difficult to
avoid; and H20, which is always present due to the
catalyst. Pyrolysis at -1000 C in a vacuum will
deoxygenate the surface in a complex reaction with
unknown mechanism, but with known stoichiometry. The
products are CO and CO2, in a 2:1 ratio. The resulting
fibril surface contains radicals in a CZ-C4 alignment
which are very reactive to activated olefins. The
surface is stable in a vacuum or in the presence of an
inert gas, but retains its high reactivity until exposed


CA 02247820 2007-06-15
51096-13

to a reactive gas. Thus, fibrils can be pyrolized at
--1000 C in vacuum or inert atmosphere, cooled under these
same conditions and reacted with an appropriate molecule
at lower temperature to give a stable functional group.
5 Typical examples are:
1000 C
Fibril-O -------> Reactive Fibril Surface (RFS) + 2
CO + CO2
followed by:
10 1000 C
RFS + CH2=CHCOX -------> Fibril-R'COX X=-OH,-Cl,-NH2,-
H

RFS + Maleic anhydride --------> Fibril-R'(COOH)2
RFS + Cyanogen -------> Fibril-CN

15 RFS + CH2=CH-CH2X -------> Fibril-R'CH2X X=-NH2,-OH, -
Halogen,

RFS + H20 -------> Fibril=O (quinoidal)

RFS + CH2=CHCHO -------> Fibril-R'CHO (aldehydic)
RFS + CH2=CH-CN -------> Fibril-R'CN

20 where R' is a hydrocarbon radical (alkyl, cycloalkyl,
etc.)

EXAMPLE 2
Preparation of Functionalized Fibrils by Reacting
Acrylic Acid with Oxide-Free Fibril Surfaces
One gram of BN fibrils in a porcelain boat is
placed in a horizontal 1" quartz tube fitted with a
thermocouple and situated in a Lindberg*tube furnace.
The ends are fitted with a gas inlet/outlets. The tube
is purged with dry, deoxygenated argon for 10 minutes,
after which the temperature of the furnace is raised to
300 C and held for 30 minutes. Thereafter, under a
continued flow of argon, the temperature is raised in
100 C increments to 1000 C, and held there for 16 hours.
At the end of that time, the tube is cooled to room

* Trade-mark


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
21
temperature (RT) under flowing argon. The flow of argon
is then shunted to pass through a multi-neck flask
containing neat purified acrylic acid at 50 C and fitted
with gas inlet/outlets. The flow of acrylic acid/argon
vapors is continued at RT for 6 hours. At the end of
that time, residual unreacted acrylic acid is removed,
first by purging with argon, then by vacuum drying at
100 C at <511 vacuum. The carboxylic acid content is
determined by reaction with excess 0.100N NaOH and back-
titrating with 0.100N HCl to an endpoint at pH 7.5.
EXAMPLE 3
Preparation of Funationalized Fibrils by Reacting
Acrylic Acid with Oxide-Free Fibril Surfaces
The procedure is repeated in a similar manner
to the above procedure, except that the pyrolysis and
cool-down are carried out at 10-4 Torr vacuum. Purified
acrylic acid vapors are diluted with argon as in the
previous procedure.
EXAMPLE 4
Preparation of Functionalized Fibrils by Reacting
Maleic Acid with Oxide-Free Fibril Surfaces
The procedure is repeated as in Ex. 2, except
that the reactant at RT is purified maleic anhydride
(MAN) which is fed to the reactor by passing argon gas
through a molten MAN bath at 80 C.
EXAMPLE 5
Preparation of Functionalized Fibrils by Reacting
Acryloyl Chloride with Oxide-Free Fibril Surfaces
The procedure is repeated as in Ex. 2, except
that the reactant at RT is purified acryloyl chloride,
which is fed to the reactor by passing argon over neat
acryloyl chloride at 25 C. Acid chloride content is
determined by reaction with excess 0.100N NaOH and back-
titration with 0.100N HC1.
Pyrolysis of fibrils in vacuum deoxygenates the
fibril surface. In a TGA apparatus, pyrolysis at 1000 C
either in vacuum or in a purified Ar flow gives an


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
22
average wt loss of 3% for 3 samples of BN fibrils. Gas
chromatographic analyses detected only CO and C02, in
-2:1 ratio, respectively. The resulting surface is very
reactive and activated olefins such as acrylic acid,
acryloyl chloride, acrylamide, acrolein, maleic
anhydride, allyl amine, allyl alcohol or allyl halides
will react even at room temperature to form clean
products containing only that functionality bonded to the
activated olefin. Thus, surfaces containing only
carboxylic acids are available by reaction with acrylic
acid or maleic anhydride; surf only acid chloride by
reaction with acryloyl chloride; only aldehyde from
acrolein; only hydroxyl from allyl alcohol; only amine
from allyl amine, and only halide from allyl halide.
3. METALLATION
Background techniques are given in March,
Advanced Orqanic Chemistry, 3rd ed., p 545.
Aromatic C-H bonds can be metallated with a
variety of organometallic reagents to produce carbon-
metal bonds (C-M). M is usually Li, Be, Mg, Al, or Tl;
however, other metals can also be used. The simplest
reaction is by direct displacement of hydrogen in
activated aromatics:
1. Fibril-H + R-Li ------> Fibril-Li + RH
The reaction may require additionally, a strong
base, such as potassium t-butoxide or chelating diamines.
Aprotic solvents are necessary (paraffins, benzene).
2. Fibril-H + A1R3 ------> Fibril-AlR2 + RH
3. Fibril-H + Tl(TFA)3 ----> Fibril-Tl(TFA)2 +
HTFA
TFA=Trifluoroacetate HTFA=Trifluoroacetic
acid
The metallated derivatives are examples of .
primary singly-functionalized fibrils. However, they can
be reacted further to give other primary singly-
functionalized fibrils. Some reactions can be carried


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
23
out sequentially in the same apparatus without isolation
of intermediates.
4. Fibril-M + 02 --------> Fibril-OH + MO M= Li,
Al
H+
Fibril-M + S --------> Fibril-SH + M+
Fibril-M + X2 --------> Fibril-X + MX
X=Halogen
catalyst
Fibril-M + CH3ONH2.HC1 --------> Fibril-NH2 +
MOCH3
ether

catalyst
Fibril-Tl(TFA)2 + NaOH --------> Fibril-OH
catalyst
Fibril-Tl(TFA)2 + NH3OH --------> Fibril-NH2 +
HTFA

Fibril-Tl(TFA)2 + aq. KCN ----> Fibril-CN +
T1TFA +KTFA

Fibril-CN + H2 ----------------> Fibril-CH2-NH2
EXAMPLE 6
, Preparation of Fibril-Li
One gram of CC fibrils is placed in a porcelain
boat and inserted into a 1" quartz tube reactor which is
enclosed in a Lindberg tube furnace. The ends of the
tube are fitted with gas inlet/outlets. Under continuous


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
24
flow of H21 the fibrils are heated to 700 C for 2 hours
to convert any surface oxygenates to C-H bonds. The
reactor is then cooled to RT under flowing H2.
The hydrogenated fibrils are transferred with
dry, de-oxygenated heptane (with LiAlH4) to a 1 liter
multi-neck round bottom flask equipped with a purified
argon purging system to remove all air and maintain an
inert atmosphere, a condenser, a magnetic stirrer and
rubber septum through which liquids can be added by a
syringe. Under an argon atmosphere, a 2% solution
containing 5 mmol butyllithium in heptane is added by
syringe and the slurry stirred under gentle reflux for 4
hours. At the end of that time, the fibrils are
separated by gravity filtration in an argon atmosphere
glove box and washed several times on the filter with
dry, deoxygenated heptane. Fibrils are transferred to a
50 cc r.b. flask fitted with a stopcock and dried under
10-4 torr vacuum at 50 C. The lithium concentration is
determined by reaction of a sample of fibrils with excess
0.100N HC1 in DI water and back-titration with 0.100N
NaOH to an endpoint at pH 5Ø
EXAMPLE 7
Preparation of Fibril-Tl(TFA)Z
One gram of CC fibrils are hydrogenated as in
Ex. 5 and loaded into the multi-neck flask with HTFA
which has been degassed by repeated purging with dry
argon. A 5% solution of 5 mmol Tl(TFA)3 in HTFA is added
to the flask through the rubber septum and the slurry is
stirred at gentle reflux for 6 hours. After reaction,
the fibrils are collected and dried as in Ex. 1.
EXAMPLE 8
Preparation of Fibril-OH
(Oxygenated Derivative Containing Only OH
Funationali.zati.on)
One half g of lithiated fibrils prepared in ~
Ex. 6 are transferred with dry, deoxygenated heptane in
an argon-atmosphere glove bag to a 50 cc single neck


CA 02247820 1998-08-28
WO 97/32571 PCTlUS97/03553
flask fitted with a stopcock and magnetic stirring bar.
The flask is removed from the glove bag and stirred on a
magnetic stirrer. The stopcock is then opened to the air
and the slurry stirred for 24 hours. At the end of that
5 time, the fibrils are separated by filtration and washed
with aqueous MeOH, and dried at 50 C at 5" vacuum. The
concentration of OH groups is determined by reaction with
a standardized solution of acetic anhydride in dioxane
(0.252 M) at 80 C to convert the OH groups to acetate
10 esters, in so doing, releasing 1 equivalent of acetic
acid/mole of anhydride reacted. The total acid content,
free acetic acid and unreacted acetic anhydride, is
determined by titration with 0.100N NaOH to an endpoint
at pH 7.5.
15 EXAMPLE 9
Preparation of Fibril-NH2
One gram of thallated fibrils is prepared as in
Ex. 7. The fibrils are slurried in dioxane and 0.5 g
triphenyl phosphine dissolved in dioxane is added. The
20 slurry is stirred at 50 C for several minutes, followed
by addition at 50 C of gaseous ammonia for 30 min. The
fibrils are then separated by filtration, washed in
dioxane, then DI water and dried at 80 C at 5" vacuum.
The amine concentration is determined by reaction with
25 excess acetic anhydride and back-titration of free acetic
acid and unreacted anhydride with 0.100N NaOH.
4. DERIVATIZED POLYNUCLEAR AROMATIC, POLYHETERODTUCLEAR
AROMATIC AND PLANAR MACROCYCLIC COMPOUNDS
The graphitic surfaces of fibrils allow for
physical adsorption of aromatic compounds. The
attraction is through van der Waals forces. These forces
are considerable between multi-ring heteronuclear
aromatic compounds and the basal plane carbons of
graphitic surfaces. Desorption may occur under
conditions where competitive surface adsorption is
possible or where the adsorbate has high solubility.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
26
For example, it has been found that fibrils can
be functionalized by the adsorption of phthalocyanine
derivatives. These phthalocyanine derivative fibrils can
then be used as solid supports for protein
immobilization. Different chemical groups can be
introduced on the fibril surface simply by choosing
different derivatives of phthalocyanine.
The use of phthalocyanine derivative fibrils
for protein immobilization has significant advantages
over the prior art methods of protein immobilization. In
particular, it is simpler than covalent modifications.
In addition, the phthalocyanine derivative fibrils have
high surface area and are stable in almost any kind of
solvent over a wide range of temperature and pH.
EXAMPLE 10
Adsorption of Porphyrins and Phthalocyanines onto Fibrils
The preferred compounds for physical adsorption
on fibrils are derivatized porphyrins or phthalocyanines
which are known to adsorb strongly on graphite or carbon
blacks. several compounds are available, e.g., a
tetracarboxylic acid porphyrin, cobalt (II)
phthalocyanine or dilithium phthalocyanine. The latter
two can be derivatized to a carboxylic acid form.
Dilithium ghthalocyanine
In general, the two Li' ions are displaced from
the phthalocyanine (Pc) group by most metal (particularly
multi-valent) complexes. Therefore, displacement of the
Li+ ions with a metal ion bonded with non-labile ligands
is a method of putting stable functional groups onto
fibril surfaces. Nearly all transition metal complexes
will displace Li+ from Pc to form a stable, non-labile
chelate. The point is then to couple this metal with a
suitable ligand. }


CA 02247820 1998-08-28
WO 97/32571 PCTIUS97/03553
27
Cobalt (II) Phthalocyanine
Cobalt (II) complexes are particularly suited
for this. Co++ ion can be substituted for the two Li+
ions to form a very stable chelate. The Co+{ ion can then
be coordinated to a ligand such as nicotinic acid, which
contains a pyridine ring with a pendant carboxylic acid
group and which is known to bond preferentially to the
pyridine group. In the presence of excess nicotinic
acid, Co(II)Pc can be electrochemically oxidized to
Co(III)Pc, forming a non-labile complex with the pyridine
moiety of nicotinic acid. Thus, the free carboxylic acid
group of the nicotinic acid ligand is firmly attached to
the fibril surface.
Other suitable ligands are the aminopyridines
or ethylenediamine (pendant NH2)1 mercaptopyridine (SH),
or other polyfunctional ligands containing either an
amino- or pyridyl- moiety on one end, and any desirable
function on the other.
- -
The--loading capacity of the porphyrin or
phthalocyanines can be determined by decoloration of
solutions when they are added incrementally. The deep
colors of the solutions (deep pink for the
tetracarboxylic acid porphyrin in MeOH, dark blue-green
for the Co(II) or the dilithium phthalocyanine in acetone
or pyridine) are discharged as the molecules are removed
by adsorption onto the black surface of the fibrils.
Loading capacities were estimated by this
method and the footprints of the derivatives were
calculated from their approximate measurements (-140 sq.
Angstroms). For an average surface area for fibrils of
250 m2/g, maximum loading will be -0.3 mmol/g.
The tetracarboxylic acid porphyrin was analyzed
by titration. The integrity of the adsorption was tested
by color release in aqueous systems at ambient and
elevated temperatures.
The fibril slurries were initially mixed
(Waring blender) and stirred during loading. Some of the


CA 02247820 1998-08-28

WO 97/32571 PCT/US97/03553
28
slurries were ultra-sounded after color was no longer
discharged, but with no effect.

After loading, Runs 169-11, -12, -14 and -19-1 (see Table II) were washed in
the same solvent to remove

occluded pigment. All gave a continuous faint tint in
the wash effluent, so it was difficult to determine the
saturation point precisely. Runs 168-18 and -19-2 used
the calculated amounts of pigment for loading and were
washed only very lightly after loading.
The tetracarboxylic acid porphyrin (from
acetone) and the Co phthalocyanine (from pyridine) were
loaded onto fibrils for further characterization (Runs
169-18 and -19-2, respectively).
Analysis of Tetracarboxylic Acid Porphyrin
Addition of excess base (pH 11-12) caused an
immediate pink coloration in the titrating slurry. While
this did not interfere with the titration, it showed that
at high pH, porphyrin desorbed. The carboxylic acid
concentration was determined by back titration of excess
NaOH using Ph 7.5 as end-point. The titration gave a
loading of 1.10 meq/g of acid, equivalent to 0.275 meq/g
porphyrin.
Analysis of Cobalt or Dilithium Phthalocyanine
The concentrations of these adsorbates were
estimated from decoloration experiments only. The point
where the blue-green tint did not fade after 30 min was
taken as the saturation-point.
A number of substituted polynuclear aromatic or
polyheteronuclear aromatic compounds were adsorbed on
fibril surfaces. For adhesion, the number of aromatic
rings should be greater than two per rings/pendant
functional group. Thus, substituted anthracenes,
phenanthrenes, etc., containing three fused rings, or
polyfunctional derivatives containing four or more fused
rings can be used in place of the porphyrin or
,
phthalocayanine derivatives. Likewise, substituted
aromatic heterocycles such as the quinolines, or multiply


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
29
substituted heteroaromatics containing four or more rings
can be used.
Table II summarizes the results of the loading
experiments for the three porphyrin/phthalocyanine
derivatives.

TABLE II

Summary of Adsorption Runs

Wgt. Loading meq/g
Ea. RUN Adsorbate Fib,a Solv. a/a Form Titration
10A 169-11 TCAPorph 19.6 mg Acet 0.18g/g Acid na

lOB 169-12 TCAPorph 33.3 mg H20 0.11 Na Salt na
lOC 169-14 DiLiPhth 119.0 mg Acet 0.170 Li na

lOD 169-19-1 CoPhth 250.0 mg Pyr 0.187 Co 0.335(cal)
10E 169-18 TCAPorph 1.00 g Acet 0.205 Acid 1.10(T)
10F 169-19-2 CoPhth 1.40 g Pyr 0.172 Co 0.303(cal)

TCAPorph=Tetracarboxylic Acid Porphyrin
(cal)=calculated
DiLiPhth=Dilithium Phthalocyanine (T)=Titration
CoPhth=Cobalt(II) Phthalocyanine

The following Examples 11 and 12 illustrate
methods for the adsorption of two different
phthalocyanine derivatives on carbon nanotubes.
EXAMPLE 11
Fibrils Functionalized by Adsorption
of Nickel (II) Phthalocyaninetetrasulfonic Acid
Two milligrams of Nickel (II) phthalocyanine-
tetrasulfonic acid (tetrasodium salt) was mixed with 4.2
milligrams of plain fibrils in one milliliter of dH2O.
The mixture was sonicated for 50 minutes and rotated at
room temperature overnight.


CA 02247820 1998-08-28

WO 97/32571 PCT/US97/03553
The fibrils were washed with 3 x 1 ml of dH2O,
3 x 1 ml of MeOH, and 3 x 1 ml of CH2C12 and dried under
vacuum.
Thermolysin was immobilized on these
5 phthalocyanine derivative fibrils by adsorption. 0.5 mg
of fibrils were suspended in 250 l of dH2O and sonicated
for 20 minutes. The supernatant was discarded and the
fibrils were suspended in 250 /sl of 0.05 M Tris (pH=8.0)
and mixed with 250 l of 0.6 mM thermolysin solution made
10 in the same buffer. The mixture was rotated at room
temperature for 2 hours and stored at 4 C overnight. The
fibrils were then washed three times with 1 ml of 25 mM
Tris (pH=8) and suspended in 250 l of buffer containing
mM Tris and ZOmM CaC12 at pH 7.5.
15 The amount of thermolysin on these fibrils was
determined by measuring the enzyme activity of the
fibrils. Thermolysin can react with substrate FAGLA (N-
(3-[2-furyl]acryloyl)-gly-leuamide) and produce a
compound that causes absorbance decrease at 345 nm with
20 extinction coefficient of -310 MlcYri 1. The assay buffer
condition for this reaction was 40mM Tris, lOmM CaC12 and
1.75 M NaCl at pH 7.5. The reaction was performed in 1
ml cuvette by mixing 5 l of FAGLA stock solution (25.5
mM in 30% DMF in dH2O) and 10 g of thermolysin fibrils in
25 1 ml of assay buffer. The absorbance decrease at 345 nm
was monitored by time scan over 10 minutes. The enzyme
activity (EtM/min) was then calculated from the initial
slope using the extinction coefficient -310 M-lcin 1. The
amount of active thermolysin per gram of fibril was 0.61
30 moles.
EXAMPLE 12
Fibrils Functionalised by Adsorption of
1,4,8,11,15,18,22,25-Octabutoxy-29H,31H-phthalocyanine
Three milligrams of 1,4,8,11,15,22,25-octabutoxy-

35 29H,31H-phthalocyanine and 5.3 mg of plain fibrils were mixed in 1 ml of
CHC13. The mixture was sonicated for 50

minutes and rotated at room temperature overnight.


CA 02247820 1998-08-28
WO 97/32571 PCT/iJS97/03553
31
The fibrils were washed with 3 x 1 ml of CH2C12
and dried under vacuum.
Thermolysin was immobilized on these
phthalocyanine derivative fibrils by adsorption according
to the method of Example 34. The amount of active
thermolysin per gram of fibrils was 0.70 moles.
EXAMPLE 13
Aspartame Precursor Synthesis Using
Phthalocyanine Derivative Fibrils
With Thermolysin Immobilized Thereon

Phthalocyanine derivative fibrils on which
thermolysin has been immobilized can be used to catalyze
the synthesis of a precursor of the artificial sweetener
aspartame. The reaction is carried out by mixing 80 mM
L-Z-Asp and 220 mM L-PheOMe in ethyl acetate with 10 M
fibril immobilized thermolysin. The product Z-Asp-PheOMe
is monitored by HPLC to determine the yield.

5. CHLORATE OR NITRIC ACID OXIDATION
Literature on the oxidation of graphite by
strong oxidants such as potassium chlorate in conc.
sulfuric acid or nitric acid, includes R.N. Smith,
Ouarterly Review 13, 287 (1959); M.J.D. Low, Chem. Rev.
60, 267 (1960)). Generally, edge carbons (including
defect sites) are attacked to give mixtures of carboxylic
acids, phenols and other oxygenated groups. The
mechanism is complex involving radical reactions.
EXAMPLE 14
Preparation of Carboxylic Acid-Funationalized
Fibrils Using Chlorate
The sample of CC fibrils was slurried in conc.
H2SO4 by mixing with a spatula and then transferred to a
reactor flask fitted with gas inlet/outlets and an
overhead stirrer. With stirring and under a slow flow of
argon, the charge of NaC103 was added in portions at RT


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
32
over the duration of the run. Chlorine vapors were
generated during the entire course of the run and were
swept out of the reactor into a aqueous NaOH trap. At
the end of the run, the fibril slurry was poured over
cracked ice and vacuum filtered. The filter cake was
then transferred to a Soxhlet thimble and washed in a Soxhlet extractor with
DI water, exchanging fresh water

every several hours. Washing was continued until a
sample of fibrils, when added to fresh DI water, did not
change the pH of the water. The fibrils were then
separated by filtration and dried at 100 C at 5" vacuum
overnight.
The carboxylic acid content was determined by
reacting a sample with excess 0.100N NaOH and back-
titrating with 0.100n HC1 to an endpoint at pH 7.5. The
results are listed in the Table.
TABLE III

Summary of Direct Oxidati.on Runs
Components,a Time
Rec Acid,
Ex. RUN # Fibrils NaClO3 cc HaSO4 hours Wash Ph Wat
mecr/cr
11A 168-30 10.0 8.68 450 24 5.7
10.0 0.78

11B 168-36 12.0 13.9 600 24 5.9
13.7 0.75


CA 02247820 1998-08-28
WO 97/32571 PCTRJS97/03553
33
EXAMPLE 15

Preparation of Carboxylic Acid-Functionalized
Fibrils Using Nitric Acid
A weighed sample of fibrils was slurried with
nitric acid of the appropriate strength in a bound bottom
multi-neck indented reactor flask equipped with an
overhead stirrer and a water condenser. With constant
stirring, the temperature was adjusted and the reaction
carried out for the specified time. Brown fumes were
liberated shortly after the temperature exceeded 70 C,
regardless of acid strength. After the reaction, the
slurry was poured onto cracked ice and diluted with DI
water. The slurry was filtered and excess acid removed
by washing in a Soxhlet extractor, replacing the
reservoir with fresh DI water every several hours, until
a slurried sample gave no change in Ph from DI water.
The fibrils were dried at 100 C at 5" vacuum overnight.
A weighed portion of fibrils was reacted with standard
0.100 N NaOH and the carboxylic acid content determined
by back-titration with 0.100 N HC1. Surface oxygen
content was determined by XPS. Dispersibility in water
was tested at 0.1 wt% by mixing in a Waring Blender at
high for 2 min. Results are summarized in Table 4.
TASLE IV

Summary of Direct Oxidation Runs
COMPONENTS
Gms. cc Acid Temp. Wgt. COOH ESCA, at%
Disp
Ex. Fibrils Acid Conc. C Time Loss mecr/a C 0
BZO

12A 1(BN) 300 70% RT 24 hr 0 <0.1 98 2
P

12B 1(BN) 300 15 rflx 48 <5% <0.1 not analyzed
P


CA 02247820 1998-08-28

WO 97/32571 PCT/US97/03553
34
12C 20(BN) 1.0 1 70 rflx 7 25% 0.8 not analyzed
G

12D 48(BN) 1.0 1 70 rflx 7 20% 0.9 not analyzed
G

P=Poor; G=Good
6. AMINO FUNCTIONALIZATION OF FIBRILS
Amino groups can be introduced directly onto
graphitic fibrils by treating the fibrils with nitric
acid and sulfuric acid to get nitrated fibrils, then
reducing the nitrated form with a reducing agent such as
sodium dithionite to get amino-functionalized fibrils
according to the following formula:
Fib gN03f HESO4 Fib-NO NaSZ04
z Fib-NH2
The
resulting fibrils have many utilities, including the
immobilization of proteins (e.g., enzymes and
antibodies), and affinity and ion exchange
chromatography.
EXAMPLE 16
Preparation of Amino-Punctionalized Fibrils Using Nitric
Acid
To a cooled suspension (0 C) of fibrils (70 mg)
in water (1.6 ml) and acetic acid (0.8 ml) was added
nitric acid (0.4 ml) in a dropwise manner. The reaction
mixture was stirred for 15 minutes at 0 C and stirred for
further 1 hour at room temperature. A mixture of
sulfuric acid (0.4 ml) and hydrochloric acid (0.4 ml) was
added slowly and stirred for 1 hour at room temperature.
The reaction was stopped and centrifuged. The aqueous
layer was removed and the fibrils washed with water (X5).
The residue was treated with 10% sodium hydroxide (X3),
and washed with water (X5) to furnish nitrated fibrils.
To a suspension of nitrated fibrils in water (3
ml) and ammonium hydroxide (2 ml) was added sodium


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
dithionite (200 mg) in three portions at O C. The
reaction mixture was stirred for 5 minutes at room
temperature and refluxed for 1 hour at 100 C. The
reaction was stopped, cooled to O C and the pH adjusted
5 with acetic acid (pH 4). After standing overnight at
room temperature, the suspension was filtered, washed
with water (X1O), methanol (X5) and dried in vaccuo to
give amino fibrils.
To test the amino functionalized fibrils, the
10 fibrils were coupled with horseradish peroxidaese. The
HRP-coupled amino fibrils were then extensively dialyzed.
Following dialysis, the fibrils were washed 15 times over
the following week. The enzyme-modified fibrils were
assayed as follows:

H202 + ABTS (clear) gRP . 2H20 + product (green)

The results indicated that HRP coupled with Fib-NH2
showed good enzyme activity which was retained over a
period of one week.
7. ATTACHMENT OF TERMINAL ALCOHOLS USING A FREE
RADICAL INITIATOR
The high degree of stability of carbon
nanotubes, while allowing them to be used in harsh
environments, makes them difficult to activate for
further modification. Previous methods have involved the
use of harsh oxidants and acids. It has now been
surprisingly found that terminal alcohols can be attached
to carbon nanotubes using a free radical initiator such
as benzoyl peroxide (BPO). Carbon nanotubes are added to
an alcohol having the formula RCH2OH, wherein R is
hydrogen, alkyl, aryl, cycloalkyl, aralkyl, cycloaryl, or
poly(alkylether) along with a free radical initiator and
heated to from about 60 C to about 90 C. Preferred
alcohols include ethanol and methanol. When sufficient
time has elapsed for all of the free radical initiator to


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
36
decompose, the reaction mixture is filtered and the
carbon nanotube material is washed and dried, yielding
modified nanotubes of the formula Nanotube-CH(R)OH. This
method can also be used to couple bifuncti.onal alcohols.
This allows one end to be linked to the carbon nanotube
and the other to be used for the indirect linkage of
another material to the surface.
EXAMPLE 17
Preparation of Alcohol Functionalized Nanotubes
Using Benzoyl Peroxide

0.277 grams of carbon nanotubes were dispersed
in MeOH using a probe sonicator. 0.126 grams of BPO were
added at RT and the temperature was increased to 60 C and
an additional 0.128 grams of BPO were added. After an
additional 45 minutes at 60 C, a final BPO charge of
0.129 grams was added and the mixture was kept at 60 C
for an additional 30 minutes. The product was filtered
onto a membrane and washed several times with MeOH and
EtOH and dried in an oven at 90 C. The yield was 0.285
grams. ESCA analysis showed an oxygen content of 2.5
atomic percent compared with 0.74% for a control sample
refluxed in MeOH without BPO.
_EXAMPLE 18
Modification of Carbon Nanotubes with
Poly(ethylene Glycol) Using Benzoyl Peroxide
0.1 grams of carbon nanotubes, 0.5 grams BPO
and 10 grams poly(ethyleneglycol), avg. mol. wt. 1000
(PEG-1000) were mixed together at room temperature. The
mixture was heated to 90 C to melt the PEG and the
mixture was left to react at 90 C overnight. The entire
mixture was then filtered and washed to remove the excess
PEG and was then dried. The resultant material can be
used either as is, or it can be further modified by
attaching materials of interest to the free end of the
PEG.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
37
EXAMPLE 19
Use of Carbon Nanotubes Modified With PEG
to Reduce Nonspecific Binding

Non-specific binding to high surface area
carbon material is ubiquitous. It has been found that
attaching hydrophilic oligomers such as PEG to carbon
nanotubes can reduce non-specific binding. Further, it
has been found that by attaching one end of chain-like
molecules such as PEG to the surface of the nanotubes the
free end can contain a functional group that can be used
for attachment of other materials of interest while still
retaining the properties of the PEG (or other material)
layer to reduce non-specific binding.
Reduction of Non-specific Binding of Bovine Serum Albumen
with PEG-modified Fibrils

Stock dispersions of unmodified fibrils,
ch.Corate oXidizec.d- fibrils and PEG mod].fied fibrils at 0.1
mg/ml in 50 mM potassium phosphate buffer at pH 7.0 were
prepared by dispersing 1.0 mg of each in 10 mis of buffer
with sonication. 2 mis of 2-fold serial dilutions of
each were placed in each of 9 polypropylene tubes. 100
l of a 0.2 mg/mi solution of bovine serum albumin (BSA)
in the same buffer was added to each tube and to three
buffer blanks. Three buffer tubes without protein were
also prepared. All tubes were mixed on a vortex mixer
and allowed to incubate for 30 minutes with 30 seconds of
vortexing every 10 minutes. All tubes were centrifuged
to separate the fibrils and 1 ml aliquots of the
supernatant were transferred to new tubes and analyzed
for total protein content using a Micro BCA protein assay
(Pierce). The level of protein remaining in the
supernatant was an indirect measure of the amount that
had been non-specifically bound to the fibrils. All the
BSA remained in the supernatant for the PEG modified


CA 02247820 1998-08-28

WO 97/32571 PCT/US97/03553
38
fibrils while there was nearly complete binding to the
unmodified or chlorate oxidized fibrils (see Fig. 1).
Comparison of Reduction of Non-soecific Binding by PEG-
modified Fibrils Prepared Using Benzoyl Peroxide and by
NHS Ester Coupling Stock dispersions of chlorate
oxidized fibrils, fibrils modified with PEG using benzoyl
peroxide and chlorate oxidized fibrils modified with PEG
by NHS ester coupling were prepared at 1.0 mg/ml in 50 mM
potassium phosphate buffer, pH 7.0 with sonication. 2
mis of 3-fold serial dilutions of each were placed in
each of 7 polypropylene tubes. 100 1 of a 0.2 mg/ml
solution of (3-lactoglobulin (/3LG) in the same buffer was
added to each tube and to 3 buffer blanks. Three buffer
tubes without protein were also prepared. All tubes were
mixed on a vortex mixer and allowed to incubate for 60
minutes with 30 seconds of vortexing every 10 minutes.
All tubes were centrifuged to separate the fibrils and 1
ml aliquots of the supernatant were transferred to new
tubes and analyzed for total protein content using a
Micro BCA protein assay (Pierce). The level of protein
remaining in the supernatant was an indirect measure of
the amount that had been non-specifically bound to the
protein (see Fig. 2). For each of the tubes the PLG
remained in the supernatant for the fibrils modified with
PEG via the NHS ester route signifying no non-specific
binding. The fibrils modified with PEG via the BPO route
exhibited only slight (approx. 10%) binding of the PLG at
the highest fibril level of 1.0 mg/ml and no significant
binding at lower levels. In contrast, there was nearly
complete binding to the chlorate oxidized fibrils at
fibril levels of 0.1 mg/ml and above and substantial
binding down to 0.01 mg/ml of these fibrils.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
39
8. SECONDARY DERIVATIVES OF FUNCTIONALIZED NANOTUBES

Carboxvlic Acid-functionalized Nanotubes

The number of secondary derivatives which can
be prepared from just carboxylic acid is essentially
limitless. Alcohols or amines are easily linked to acid
to give stable esters or amides. If the alcohol or amine
is part of a di- or bifunctional poly-functional
molecule, then linkage through the 0- or NH- leaves the
other functionalities as pendant groups. Typical
examples of secondary reagents are:

PENDANT
GENERAL FORMULA GROUP EXAMPLES

HO-R, R=alkyl, aralkyl, R- Methanol, phenol, tri-
aryl, fluoroethanol, fluorocarbon, OH-terminated
polymer, SiR'3 Polyester, silanols

H2N-R R=same as above R- Amines, anilines,
fluorinated amines,
silylamines, amine
terminated polyamides,
proteins
Cl-SiR3 SiR3- Chlorosilanes

HO-R-OH, R=alkyl, HO- Ethyleneglycol, PEG, Penta-
aralkyl, CH2O- erythritol, bis-Phenol A
H2N-R-NH2, R=alkyl, H2N- Ethylenediamine, polyethyl-
aralkyl eneamines


CA 02247820 1998-08-28
WO 97/32571 PC'T/US97/03553
X-R-Y, R=alkyl, etc; Y- Polyamine amides,
X=OH or NH2; Y=SH, CN, Mercaptoethanol
C=O, CHO, alkene,
5 alkyne, aromatic,
heterocycles

The reactions can be carried out using any of
the methods developed for esterifying or aminating
10 carboxylic acids with alcohols or amines. Of these, the
methods of H.A. Staab, Angew. Chem. Internat. Edit., (1),
351 (1962) using N,N'-carbonyl diimidazole (CDI) as the
acylating agent for esters or amides, and of G.W.
Anderson, et al., J. Amer. Chem. Soc. 86, 1839 (1964),
15 using N-hydroxysuccinimide (NHS) to activate carboxylic
acids for amidation were used.
EXAMPLE 20
Preparation of Secondary Derivatives of Functionalized
Fibrils
20 N. NI-Carbonvl Diimidazole
Clean, dry, aprotic solvents (e.g., toluene or
dioxane) are required for this procedure.
Stoichiometric amounts of reagents are sufficient. For
esters, the carboxylic acid compound is reacted in an
25 inert atmosphere (argon) in toluene with a stoichiometric
amount of CDI dissolved in toluene at R.T. for 2 hours.
During this time, CO2 is evolved. After two hours, the
alcohol is added along with catalytic amounts of Na
ethoxide and the reaction continued at 80 C for 4 hr.
30 For normal alcohols, the yields are quantitative. The
reactions are:

1. R-COOH + Im-CO-Im ----> R-CO-Im + HIm + C02, Im=Imidazolide,

35 HIm=Imidazole

NaOEt


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
41
2. R-CO-Im + R'OH ------->R-CO-OR' + HIm

Amidation of amines occurs uncatalyzed at RT.
The first step in the procedure is the same. After
evolution of C02, a stoichiometric amount of amine is
added at RT and reacted for 1-2 hours. The reaction is
quantitative. The reaction is:

3. R-CO-Im + R'NHZ ------> R-CO-NHR + HIm
Silylation
Trialkylsilylchiorides or trialkylsilanols
react immediately with acidic H according to:
R-COOH + Cl-SiR'3 ------> R-CO-SiR'3 + HCl
Small amounts of Diaza-1,1,1-bicyclooctane
(DABCO) are used as catalysts. Suitable solvents are
dioxane and toluene.
Sulfonic Acid-Functionalized Fibrils
Ary1 sulfonic acids, as prepared in Example 1,
can be further reacted to yield secondary derivatives.
Sulfonic acids can be reduced to mercaptans by LiA1H4 or
the combination of triphenyl phosphine and iodine (March,
J.P., p. 1107). They can also be converted to sulfonate
esters by reaction with dialkyl ethers, i.e., Fibril--
So3H + R-O-R ----> Fibril-SO2OR + ROH
N-Hydroxysuccinimide
Activation of carboxylic acids for amidation
with primary amines occurs through the N-
hydroxysuccinamyl ester; carbodiimide is used to tie up
the water released as a substituted urea. The NHS ester
is then converted at RT to the amide by reaction with
primary amine. The reactions are:

1. R-COOH + NHS + carbodimide-----> R-CONHS +
Subst. Urea
2. R-CONHS + R'NH2 ------> R-CO-NHR'


CA 02247820 1998-08-28
WO 97/32571 PCT/CTS97/03553
42
This method is particularly useful for the
covalent attachment of protein to graphitic fibrils via
the free NH2 on the protein's side chain. Examples of
proteins which can be immobilized on fibrils by this
method include trypsin, streptavidin and avidin. The
streptavidin (or avidin) fibrils provide a solid carrier for any biotinylated
substance

EXAMPLE 21

Covalent Attachment of Proteins to Fibrils via NHS Ester
To demonstrate that protein can be covalently
linked to fibrils via NHS ester, streptavidin, avidin and
trypsin were attached to fibrils as follows.
0.5 mg of NHS-ester fibrils were washed with 5
mM sodium phosphate buffer (pH 7.1) and the supernatant
was discarded. 200 l streptavidin solution (1.5 mg in
the same buffer) was added to the fibrils and the mixture
was rotated at room temperature for 5.5 hours. The
fibrils were then washed with 1 ml of following buffers
in sequence: 5 mM sodium phosphate (pH 7.1), PBS (0.1 M
sodium phosphate, 0.15 M NaCl, pH 7.4), ORIGENTM assay
buffer (IGEN, Inc., Gaithersburg, MD) and PBS. The
streptavidin fibrils were stored in PBS buffer for
further use.
2.25 mg NHS-ester fibrils were sonicated in 500
l of 5 mM sodium phosphate buffer (pH 7.1) for 40
minutes and the supernatant was discarded. The fibrils
were suspended in 500 l of 5 mM sodium phosphate buffer
(pH 7.1) and 300 l of avidin solution made in the same
buffer containing 2 mg avidin (Sigma, A-9390) was added
The mixture were rotated at room temperature for two
hours, stored at 4 C overnight and rotated at room
temperature for another hour. The fibrils were washed with 1 ml of 5 mM sodium
phosphate buffer (pH 7.1) four

times and PBS buffer twice. The avidin fibrils were
suspended in 200 ,ul PBS buffer for storage.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
43
Trypsin fibrils were prepared by mixing 1.1 mg
NHS-ester fibrils (treated as in avidin fibrils) and 200
l of 1.06 mM trypsin solution made in 5 mM sodium
phosphate buffer
(pH 7.1) and rotating at room temperature for 6.5 hours.
The trypsin fibrils were then washed by 1 ml of 5 mM
sodium phosphate buffer (pH 7.1) three times and
suspended in 400 l of the same buffer for storage.
EXAMPLE 22
Measurement of Enzyme Activity of Trypsin on Fibrils
Trypsin can react with substrate L-BAPNA (Na-
benzoyl-L-arginine p-nitroanilide) and release a colored
compound that absorbs light at 410 nm. The assay buffer
for this reaction was 0.05 M Tris, 0.02 M CaC121 pH 8.2.
The reaction was performed in 1 ml cuvette by mixing 5 l
of L-BAPNA stock solution (50 mM in 37% DMSO in H20) and
10-25 gg of trypsin fibrils in a 1 ml of assay buffer.
The absorbance increase at 410 nm was monitored over 10
minutes. The enzyme activity ( M/min) was then
calculated from the initial slope.
For covalently bound trypsin fibrils, the activity
was 5.24 M/min per 13 g fibrils. This result can be
converted to the amount of active trypsin on fibrils by
dividing the activity of a known concentration of trypsin
solution, which was measured to be 46 M/min per 1 M
trypsin under the same assay conditions. Therefore the
amount of active trypsin per gram of fibrils was 8.3
gmoles (or 195 mg).
EXAMPLE 23
Carbon Nanotubes with Surface Thiols
0.112 gms of amino carbon nanotubes (CN)
prepared by modification with ethylenediamine as
described in Example 27 (below) were suspended in 20 mis
of pH 8.0 0.05 M potassium phosphate buffer containing 50
mM EDTA. The suspension was sonicated with a Branson 450


CA 02247820 1998-08-28

WO 97/32571 PCT/US97/03553
44
Watt probe sonicator for 5 minutes to disperse the CN.
The resulting suspension was quite thick. Argon was
bubbled though the suspension for 30 minutes with
stirring. 50 mgs of 2-iminothiolane=HCl was added and
the mixture was allowed to react for 70 minutes with
continued stirring under argon. The resulting material
was filtered onto a polycarbonate membrane filter, washed
2X with buffer, iX with DI water and 2X with absolute
EtOH, all under an argon blanket. The thiol modified CN
were placed in a vacuum desiccator and pumped on
overnight. Final weight = 0.118 gms, 55% conversion,
based on weight gain.
A 10 mg sample of thiolated nanotubes was
suspended in 10 mis. of DI water with sonication and
filtered onto 0.45 m nylon membrane to form a felt-like
mat. The mat section was stored in a vacuum desiccator
prior to analysis by ESCA which showed 0.46% sulfur and
1.69% nitrogen, confirming successful conversion to
thiol-modified CN.
EXAMPLE 24
Attachment of Thiol-modified Carbon Nanotubes to Gold
Surfaces
Gold foil (Alfa/Aesar), 2 cm x 0.8 cm, was
cleaned with a solution of 1 part 30% H202 and 3 parts
concentrated H2SO4 for 10 minutes and rinsed with DI
water. The foil piece was connected to an Au wire lead
and cycled electrochemically between -0.35 V vs. Ag/AgCl
and 1.45 V vs. Ag/AgCl in,1 M H2SO4 at 50 mv/sec until the
cyclic voltammograms were unchanged, approx. 10 minutes.
It was then rinsed with DI water and dried. The large
piece was cut into four strips 0.5 cm x 0.8 cm.
10 mis of absolute EtOH, deoxygenated by argon
purging for 30 min., was placed in each of two glass vials. In one vial was
suspended 16 mgs of thiol-

modified CN (CN/SH) and 2 Au pieces and in the other vial
1 piece of Au and 10 mgs of the ethylene diamine modified
CN used to make the thiol derivative. All manipulations


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
were carried out in an Ar filled glove bag. The vials
were sealed under Ar and placed in a chilled ultrasonic
bath for 1 hour. The sealed vials were left at RT for 72
hours. The Au samples were removed from the vials,
5 rinsed 3X with EtOH, air dried and placed in protective
vials.
The Au foil samples exposed to the
CN/ethylenediamine and CN/SH were examined by scanning
electron microscopy (SEM) to detect the presence or
10 absence of CN on the surface. Examination at 40,000X
revealed the presence of CN distributed over the surface
exposed to CN/SH but no CN were observed on the Au foil
sample exposed to CN/ethylenediamine.
EXAMPLE 25
15 Preparation of Maleimide Fibrils From Amino Fibrils
Amino fibrils were prepared according to
Example 13. The amino fibrils (62.2 mg) were then
sonicated in sodium phosphate buffer (5 ml, 5 mM at pH
7.2). Sulfosuccinmidyl-4-(N-maleimidomethyl)cyclohexane-
20 1-carboxylate (SMCC; 28.8 mg, 0.66 mmols; Pierce, Cat.
No.22360) was added to the fibril suspension. The
reaction mixture was stirred overnight at room
temperature. The fibrils were washed with water and
methanol, and the product fibrils were dried under
25 vacuum. Antibody immobilization on the product confirmed
the presence of maleimide fibrils. Other maleimides with
different linkers (e.g., sulfo-SMCC, succinimidyl 4-[p-
maleimidophenyl]butyrate [SMPB], sulfo-SMPB, m-
maleimidobenzyl-N-hydroxysuccinimide ester [MBS], sulfo-
30 MBS etc.) fibrils can be made through the same method.
The resulting maleimide fibrils can be used as
a solid support for the covalent immobilization of
proteins, e.g. antibodies and enzymes. Antibodies were
covalently immobilized on malemide activated fibrils.
35 The capacity of antibody was 1.84 milligrams per gram of
fibrils when amino fibrils obtained from
nitration/reduction method (Example 13) were used and


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
46
0.875 milligrams per gram of fibrils when amino fibrils
derivatized from carboxyl fibrils were used.
EXAMPLE 26

Preparation of Ester/Alcohol Derivatives
from Carboxylic Acid-Functionalised Fibrils
The carboxylic acid functionalized fibrils were
prepared as in Example 14. The carboxylic acid content
was 0.75 meq/g. Fibrils were reacted with a
stoichiometric amount of CDI in an inert atmosphere with
toluene as solvent at R.T. until CO2 evolution ceased.
Thereafter, the slurry was reacted at 80 C with a 10-
fold molar excess of polyethyleneglycol (MW 600) and a
small amount of NaOEt as catalyst. After two hours
reaction, the fibrils were separated by filtration,
washed with toluene and dried at l00 C.
EXAMPLE 27

Preparation of Amide/Ami.ne Derivatives
from Carboxylic Acid-Functionalized Fibrils (177-041-1)
0.242 g of chlorate-oxidized fibrils (0.62
meq/g) was suspended in 20 ml anhydrous dioxane with
stirring in a 100 ml RB flask fitted with a serum
stopper. A 20-fold molar excess of N-hydroxysuccinimide
(0.299 g) was added and allowed to dissolve. This was
followed by addition of 20-fold molar excess of i-ethyl-
3-(3-dimethylaminopropyl)carbodiimide (EDAC) (0.510 g),
and stirring was continued for 2 hr at RT. At the end of
this period stirring was stopped, and the supernatant
aspirated and the solids were washed with anhydrous
dioxane and MeOH and filtered on a 0.45 micron
polysulfone membrane. The solids were washed with
additional MeOH on the filter membrane and vacuum-dried
until no further weight reduction was observed. Yield of
NHS-activated oxidized fibrils was 100% based on the 6%
weight gain observed.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
47
100 l ethylenediamine (en) was added to 10 ml
0.2 M NaHCO3 buffer. An equivalent volume of acetic acid
(HOAc) was added to maintain the pH near S. NHS-
activated oxidized fibrils (0.310 g) was added with
vigorous stirring and reacted for 1 hr. An additional
300 l of en and 300 l HOAc was added for an additional
min. The solution was filtered on 0.45 micron
polysulfone membrane and washed successively with NaHCO3
buffer, 1t HC1, DI water and EtOH. The solids were dried
10 under vacuo overnight. The HC1 salt was converted back
to the free amine by reaction with NaOH (177-046-1) for
further analysis and reactions.
ESCA was carried out to quantify the amount of
N present on the aminated fibrils (GF/NH2). ESCA
analysis of 177-046-1 showed 0.90 at% N (177-059). To
further assess how much of this N is present as both
accessible and reactive amine groups, a derivative was
made by the gas phase reaction with
pentafluorobenzaldehyde to produce the corresponding
Schiff Base linkages with available primary amine groups.
ESCA analysis still showed the 0.91 at% N, as expected,
and 1.68 at%F. This translates into a 0.34 at% of N
present as reactive primary amine on the aminated fibrils
(5 F per pentafluorobenzaldehyde molecule). A level of
0.45 at% N would be expected assuming complete reaction
with the free ends of each N. The observed level
indicates a very high yield from the reaction of N with
NHS-activated fibril and confirms the reactivity of the
available free amine groups.
At the level of 0.34 at% N present as free
amine calculated from the ESCA data, there would be
almost complete coverage of the fibrils by the free amine
groups allowing coupling of other materials.
Carboxyl fibrils were also converted to amino
fibrils using mono-protected 1,6-diaminohexane (a six-
carbon linker), rather than ethylenediamine (a two-carbon
linker).


CA 02247820 1998-08-28

WO 97/32571 PCTIUS97/03553
48
BXAMPLE 28
Preparation of Amine Derivatives from
Carboxylic Acid Functionalized Fibrils

Carboxyl groups on fibrils can be modified by
reacting the carboxyl groups with one amino group of a
compound having two or more amino groups (at least one of
which is unprotected by groups such as t-Boc or CBZ).
The fibrils so generated are amide derivatives in which
the amide carbonyl is derived from the fibril carboxyl
group and the amide nitrogen is substituted with a group
(such as an alkyl group) containing one or more primary
amines. The amino groups are then available for use or
further modification.
One gram of carbon fibrils was placed in a dry
scintered glass filter tunnel, the outlet of which was
tightly stoppered with a rubber serum septum, and
anhydrous dichloromethane was added to cover. N-
Methylmorpholine (758 L, 7 mmol) was added, the
suspension was mixed with the aid of a spatula. Then
isobutyl chloroformate (915 L, 7 mmol) was added, and
the suspension mixed periodically for one hour. The
mixture was protected from atmospheric moisture by a
cover of Parafilm as much as was practical.
Meanwhile, N-boc-1,6-diaminohexane
hydrochloride (1.94 g, 7.7 mmol) was partitioned between
dichloromethane (10 mL) and 1 M NaOH (10 mL). The lower,
organic phase was dried over anhydrous potassium
carbonate and filtered through a disposable Pasteur
pipette containing a cotton plug, and N-methylmorpholine
(758 L, 7 mmol) was added.
The serum septum was removed from the filter
funnel, the reagents were removed from the fibrils by
vacuum filtration, and the fibrils were washed with
anhydrous dichloromethane. The serum septum was
replaced, and the mixture of N-methylmorpholine and
monoprotected diaminohexane was added to the fibrils.


CA 02247820 1998-08-28

WO 97/32571 PCT1US97/03553
49
The mixture was stirred periodically for one hour. Then,
the reagents were removed by filtration, and the fibrils
were washed successively with dichioromethane, methanol,
water, methanol, and dichloromethane.
A 50% mixture of trifluoric acid and
dichioromethane was added to the fibrils and the mixture
stirred periodically for 20 minutes. The solvents were
removed by filtration, and the fibrils were washed
successively with dichloromethane, methanol, water, 0.1 M
NaOH, and water.
To demonstrate the efficacy of the method, a
small sample of amino fibrils were reacted with
"activated" horseradish peroxidase (HRP; 5 mg, Pierce)
which was modified to specifically react with amino
groups. The fibrils were washed repeatedly for several
days (by suspension, rotation, and centrifugation in an
Eppendorf tube) while kept cold. After approximately two
weeks of washing, the enzyme was assayed with H202/ABTS in
glycine buffer, pH 4.4. A green color appeared in the
solution within 10 minutes indicating the presence of
enzyme. Control fibrils (COOH fibrils treated with
activated HRP and washed for the same period of time)
showed little if any catalytic activity.
EXAMPLE 29
Preparation of Silyl Derivative
from Carboxylic Acid-Functionalized Fibrils
Acid functionalized fibrils prepared as in
Example 14 were slurried in dioxane in an inert
atmosphere. With stirring, a stoichiometric amount of
chlorotriethyl silane was added and reacted for 0.5 hr,
after which several drops of a 5% solution of DABCO in
dioxane was added. The system was reacted for an
additional hour, after which the fibrils were collected
by filtration and washed in dioxane. The fibrils were
dried at 100 C in 5" vacuum overnight.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
Table 5 summarizes the secondary derivative
preparations. The products were analyzed by ESCA for C,
0, N, Si and F surface contents.
TABLE V
5
Bummary of Secondary Derivative Preparations
ESCA ANALYSIS, ATOM %
REACTANT PENDANT GROUP S C N 0 Si F
As Grown ---- -- 98.5 -- 1.5 -- --
Chlorate -COOH, C=O, C-OH -- 92.4 -- 7.6 -- --
Oxidized

H2N-C2H4-NH2 -CONHC2H4NH2 -- 99.10 0.90 -- -- --
-CONHCZH4N=OC6F5 -- 97.41 0.91 -- -- 1.68
EXAMPLE 30
Preparation of Silyl Derivative
from Carboxylic Acid-Functionalized Fibrils
Acid functionalized fibrils prepared as in
Example 14 are slurried in dioxane in an inert
atmosphere. With stirring, a stoichiometric amount of
chlorotriethyl silane is added and reacted for 0.5 hr,
after which several drops of a 5% solution of DABCO in
dioxane is added. The system is reacted for an
additional hour, after which the fibrils are collected by
filtration and washed in dioxane. The fibrils are dried
at 100 C in 5" vacuum overnight.
Table VI summarizes the secondary derivative
preparations. Products are analyzed by ESCA. The
analysis confirms the incorporation of the desired
pendant groups. The products are analyzed by ESCA for C,
0, N, Si and F surface contents.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
51
TABLE VI

Summary of Secondary Derivative Preparations
ESCA ANALYSIS, ATOM %
REACTANT PENDANT GROUP S C N 0 Si F
CF3CH20H -COOCH2CF3 NOT ANALYZED

PolyEG-600 -CO-(OC2H40-)H NOT ANALYZED
HO-CZH4 SH -COOCZH4SH

Cl-SiEt3 -COSiEt3

EXAMPLE 31
Preparation of Tertiary and
Quaternary Amine Derivatives from
Carboxylic Acid Functionalized Fibrils

Tertiary and quaternary amine functional groups
can be attached to the surface of carbon nanotubes via an
amide or ester bond via a carboxyl group on the nanotube
and either an amine or hydroxyl group of the tertiary or
quaternary amine precursor. Such tertiary or quaternary
amine fibrils are useful as chromatographic matrices for
the separation of biomolecules. The tertiary or
quaternary amine fibrils can be fabricated into disk-
shaped mats or mixed with conventional chromatographic
media (such as agarose) for separation purposes.
preparation of triethylethanolamine iodide precursor
In a 100 ml round bottom flask, 10 g N,N-
diethylethanolamine (85.3 mmole) was mixed with 10 ml
anhydrous methanol. A mixture of 20 g ethyl iodide
(127.95 mmole) and 10 ml anhydrous methanol was then
added dropwise using a pipette. The reaction mixture was
refluxed for 30 minutes. White crystalline product
formed when the reaction mixture was allowed to cool to
room temperature. The white solid product was collected


CA 02247820 1998-08-28

WO 97/32571 PCT/US97/03553
52
by filtration and washed with anhydrous methanol. The
product was further dried overnight in a desiccator under
vacuum. Product (10.3 g, 37.7 mmole) was obtained in a
yield of 33%.
Preparation of quaternarv amine functionalized araphite
,
fibriis
In a vacuum dried 25 ml Wheaton disposable
scintillation vial, 100 mg dry carboxyl fibril (about 0.7
mmole COOH per gram of fibrils) was mixed with 2 ml
anhydrous dimethylformamide and the mixture was sonicated
for 60 seconds. Two more milliliters of
dimethylformamide, 39 mg dimethyl-aminopyridine (0.316
mmole), and 50 gl diisopropylcarbodiimide (0.316 mmole)
were added to the reaction vial. The reaction mixture
was stirred for one hour at room temperature, then 88 mg
triethylethanolamine iodide (0.316 mmole) was added to
the vial and the reaction was allowed to go overnight.
The resulting fibrils were washed three times with 20 ml
dimethylformamide, three times with 20 ml methylene
chloride, three times with 20 ml methanol and finally
three times with de-ionized water. The product was dried
under vacuum. Results from an elemental analysis of
nitrogen showed that about 50% of the carboxyl groups on
the fibril had reacted with the primary amino group in
the quaternary amine moiety.
EXAMPLE 32
Chromatography of Bovine
Serum Albumin (BSA) on Tertiary
Amine Functionalized Graphite Fibrils.
An aqueous slurry containing 60 mg 2-
diethylamino ethylamine modified carboxyl fibrils and 180
mg Sephadex G-25 superfine resin (Pharmacia, Uppsala,
Sweden) was allowed to stand overnight at room
temperature to ensure full hydration of the solid
support. The slurry was packed into a 1 cm x 3.5 cm
column. The column was equilibrated with 5 mM sodium


CA 02247820 2007-06-15
51096-13

53
phosphate buffer (pH 7.3) at a flow rate of 0.2 ml/min.
BSA (0.6 mg in 0.1 ml de-ionized water) was loaded on the
column. The column was eluted with 5 mM sodium phosphate
at a.flow rate of 0.2 ml/min and 0.6m1 fractions were
collected. The elution profile was monitored using a UV-
visible detector, and is shown in Fig 3. Once the
detector indicated that no more protein was eluting from
the column, bound BSA was eluted by adding 1 M KC1 in 5
mM sodium phosphate (pH 7.3). The presence.of the
lo protein in each fraction was identified by micro BCA
assay (Pierce, Rockford, Il).
EXAMPLE 33
Chromatography of Bovine Serum
Albumin (BSA) on Quaternary Amine
Functionalized Graphite Fibrils.
An aqueous slurry containing 100 mg
2-triethylammonioethanol modified carboxyl fibril and
300 mg Sephadex*G-25 superfine resin was allowed to stand
overnight at room temperature. The resulting slurry was
used to pack a 1 cm diameter column. The column was
equilibrated with 5 mM sodium phosphate buffer (pH 7.3)
at a flow rate of 0.1-0.6 ml/min. BSA (2.7 mg in 0.2 ml
de-ionized water) was loaded on the column. The column
was eluted with 5 mM sodium phosphate at a flow rate of
0.2 ml/min and 0.6 ml fractions were collected. The
elution profile was monitored using a UV-visible detector
(Fig. 4). Once the detector indicated that protein was
no longer being eluted with 5 mM sodium phosphate buffer,
the solvent was changed to 1 M KC1 in 5 mM sodium
phosphate (pH 7.3). The presence of the protein in each
fraction was identified by micro BCA assay (Pierce,
Rockf ord , I 1) .
9. ENZYMATIC FIINCTIONALIZATION OF GRAPHITIC CARBON
Biocatalysts can be used to introduce
functional groups onto the surface of graphitic carbon,
especially carbon nanotubes. Until now, graphitic carbon
*Trade-mark


CA 02247820 2007-06-15
51096-13

54
has been modified by purely chemical means (see e.g.,
WO 97/32571; Canadian Patent Application No. 2,207,282).
These chemical methods have drawbacks of: (1)
harshness of conditions (use of extreme temperatures,
extreme acidity or toxic chemicals), and (2) lack of
specificity (e.g., oxidation can introduce COOH, COH, and
CHO groups). Aqueous suspensions of solid graphitic
carbon (such as carbon fibrils; Hyperion, Inc.) are made
containing one or more enzymes that are capable of
accepting the graphitic carbon as a substrate and
performing a chemical reaction resulting in chemically-
modified graphitic carbon. The aqueous suspension is
maintained at conditions acceptable for the enzyme(s) to
carry out the reaction (temperature, pH, salt
concentration, etc.) for a time sufficient for the
enzyme(s) to catalytically modify the surface of the
graphitic carbon. During the reaction, the suspension is
continually mixed to allow the enzyme(s) access to the
surface of the graphitic carbon. Following a reaction
time acceptable for the reaction to proceed to a
satisfactory degree, the enzyme is removed from the
carbon by f iltration washing.
To date two types of enzymes have been used:
cytochrome p450 enzymes and peroxidase enzymes. In both
cases, the types of enzymes have been well-studied, they
accept aromatic type substrates, and their optimal
reaction conditions have been worked out. Both enzyme
types introduce hydroxyl groups into their substrates and
may introduce hydroxyl groups into graphitic carbon.
Besides enzymes, other biocatalysts such as ribozymes and
catalytic antibodies, or non-biological mimics of
enzymes, could be designed to catalytically functionalize
carbon nanotubes.


CA 02247820 1998-08-28

WO 97/32571 PCTIUS97/03553
EXAMPLE 34
Enzymatic Functionalization Using Rat Liver Microsomes
Cytochrome p450 enzymes are generally believed
to function in the liver as detoxifying agents (F. Peter
5 Guengerich, American Scientist, 81, 440-447 and F. Peter
Guengerich, J. Biol. Chem., 266, 10019-10022). They
hydroxylate foreign compounds such as polyaromatic toxic
compounds. Hydroxylation allows these compounds to
become water soluble so that they can be eliminated from
10 the body via the urine. There are many different
cytochrome p450 enzymes in the liver, each with different
substrate specificities. These broad range of
specificities is believed to be important because of the
wide range of environmental toxins whose detoxification
15 is required. Although individual cytochrome p450s are
commercially available, no information is available
regarding whether any of these would accept carbon
nanotubes as a substrate. Because of this uncertainty,
we decided to initially incubate carbon nanotubes with a
20 rat liver extract which contained many different
cytochrome p450s.
Two rats ("experimental" rats) were
administered phenobarbital (ig/L, pH 7.0) in their
drinking water for one week to induce expression of
25 cytochrome p450 enzymes. Two other rats ("control" rats)
were given water without phenobarbital. The rats were
then sacrificed and cytochrome p450-containing microsomes
were prepared from their livers by standard procedures
(see for example, Methods in Enzymology, Vol. 206).
30 The microsomes were mixed with carhnn nannt-õhps
(fibrils) to allow the cytochrome p450s to react with the
graphitic carbon. In these experiments, 5 mg of fibrils
(both "plain" or nonfunctionalized and "COOH" or oxidized
fibrils) were mixed with microsomes (both experimental
35 and control microsomes) in a buffered solution containing
0.1 M Tris, 1.0 mM NADPH, 0.01% NaN3, 10 mM glucose-6-
phosphate, glucose-6-phosphate dehydrogenase (1 unit/mL),


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
56
pH 7.4. NADPH was included as a co-substrate for
cytochrome p450s and glucose-6-phosphate, glucose-6-
phosphate dehydrogenase were added to regenerate NADPH
from NADP+ (if NADP+ is generated by cytochrome p450s).
The mixtures were rotated at room temperature for about
1.5 days in microcentrifuge tubes. Following the
incubation, the fibrils were washed extensively in
deionized water, 1 M HC1, 1 M NaOH, 0.05% Triton X-100,
0.05% Tween, methanol, and 1 M NaCl. Following washing,
microBCA assay for proteins (Pierce) showed that fibrils
seemed to still have protein associated with them
(although no protein was detected in the wash solution).
To determine whether hydroxyl groups had been
introduced onto the fibril surfaces, the fibrils were
reacted with N-FMOC-isoleucine. The different batches of
fibrils (control and experimental) (1.5 mg each) were
reacted with 333 microliters of a solution of dry DMF
containing 4.45 mg/mL FMOC-isoleucine, 1.54 mg/mL
dimethylaminopyridine (DMAP) and 2.6 mg/mL 1,3-
dicyclohexylcarbodiimide (DCC). Following reaction for
two days (while being continuously rotated), the fibrils
were washed with DMF, piperidine, methanol, water, DMF,
methanol, methylene chloride (600 microliters of each).
This wash sequence was repeated three times. Fibrils
were sent to Gaibraith Laboratories (Knoxville, TN) for
amino acid analysis for isoleucine present. The results
were equivocal because many other amino acids were seen
in addition to isoleucine, indicating that proteins,
peptides, and amino acids present in the rat liver
microsomal extracts had not completely washed away from
the fibrils. Thus, because of technical difficulties in
washing and analysis it could not be determined whether
or not cytochrome p450's had functionalized the fibrils. .


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
57
EXAMPLE 35
Fibril Functionalization Using
Commercially-Available Recombinant Cytochrome p450
Enzymes
To avoid the impurities associated with using
rat liver microsomes as a source of cytochrome p450s,
individual cytochrome p450 enzymes were purchased
(GENTEST, Woburn, MA). Because cytochrome p450 enzymes
are only active in association with membranes, these
enzymes are supplied as microsomal preparations. Using a
reaction procedure similar to that described above, we
tested the following cytochrome p450s: CYP1A1 (cat.#
M111b), CYP1A2 (cat.# M103c), CYP2B6 (cat.# 110a), CYP3A4
(with reductase, cat.# 107r). MgC12 (0.67 mg/mL) was
also included in the reaction solution. In this
experiment, fibrils were washed with the aid of a Soxhlet
apparatus.
Analysis of introduced hydroxyl groups was
carried out by reaction of cytochrome p450-reacted,
washed fibrils with the colored reagent 3,5-
dinitrobenzoic acid (DNBA). Coupling was carried out as
described above for N-FMOC-isoleucine. Following
reaction with DNBA, the fibrils were washed with DMF and
residual (covalently attached) DNBA was hydrolyzed using
either 6 M HC1 or 46 units/mL pig liver esterase (Sigma).
Analysis of liberated DNBA was carried out by HPLC
analysis of the supernatant surrounding the fibrils
following hydrolytic treatment. HPLC analysis of
liberated DNBA was carried out on a Waters HPLC system
equipped with a Vydac C18 reversed phase analytical
column (cat.# 218TP54) and a linear gradient from
deionized water containing 0.1% TFA (solvent A) to
acetonitrile containing 0.1% TFA (solvent B).


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
58
EXAMPLE 36
Functionalisation of Fibrils 1Tsing Peroxidase
Literature descriptions of peroxidase substrate
specificities indicated that carbon nanotubes may be
substrates for these enzymes (J.S. Dorick et al.,
Biochemistrv (1986), 25, 2946-2951; D.R. Buhier et al.,
Arch. Biochem. Biophys. (1961) 92, 424-437; H.S. Mason,
Advances in Enzvmology, (1957) 19, 79; G.D. Nordblom et
al., Arch. Biochem. Biophvs. (1976) 175, 524-533). To
determine whether peroxidase (hydrogen peroxidase, Type
II, Sigma) could introduce hydroxyl groups onto the
surface of fibrils, fibrils (11 mg) were mixed in a
solution containing 50 mM sodium acetate (1.25 mL, pH
5.0), horseradish peroxidase (200 nM), and
dihydroxyfumaric acid (15 mg) was added 5 mg at a time
for the first 3 hours of the reaction. The reaction was
carried out for a total of 5 hours at 40 C with
intermittent bubbling of gaseous oxygen. Following the
reaction, the fibrils were washed with water, 1 N NaOH,
methanol, and methylene chloride (200 mL of each). A
control reaction was carried out using peroxidase that
had been heat inactivated (100 C for 5 minutes).
For analysis of the extent of peroxidase-
catalyzed fibril hydroxylation, fibrils were reacted with
t-butyldimethylsilyl chloride (Aldrich) in dry DMF in the
presence of imidazole. Following washing of the fibrils,
the fibrils were sent to Robertson Microlit Laboratories,
Inc (Madison, NJ) for elemental analysis of silicon
incorporated into the fibrils. The results of the
analysis were equivocal for the presence of silicon on
the surface of the fibrils. It is believed that silicon
from glassware used in the experiment was present in
small chips in the fibrils submitted for elemental
analysis. This resulted in a high level of silicon in
both experimental and control samples. The conclusion of
the experiment is that peroxidase may have introduced
hydroxyl groups into the fibrils but technical


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
59
difficulties precluded us from determining the presence
of any introduced hydroxyl groups.
10. NANOTUBES FUNCTIONALIZED BY ELECTROPHILIC
ADDITION TO OXYGEN-FREE FIBRIL SURFACES
OR BY METALLIZATION
The primary products obtainable by addition of
activated electrophiles to oxygen-free fibril surfaces
have pendant -COOH, -COC1, -CN, -CH2NH2, -CH2OH, -CH2-
Halogen, or HC=O. These can be converted to secondary
derivatives by the following:
Fibril-COOH -----> see above.
Fibril-COC1 (acid chloride) + HO-R-Y ----> F-COO-R-Y
(Sec. 4/5)
Fibril-COC1 + NH2-R-Y -------> F-CONH-R-Y
Fibril-CN + H2 -----> F-CH2-NH2
Fibril-CH2NH2 + HOOC-R-Y ----> F-CH2NHC0-R-Y
Fibril-CH2NH2 + O=CR-R'Y ----> F-CH2N=CR-R'-Y
Fibril-CH2OH + O(COR-Y)2 ----> F-CH2OCOR-Y
Fibril-CH2OH + HOOC-R-Y -----> F-CH2OCOR-Y
Fibril-CH2-Halogen + Y- ----> F-CH2-Y + X- Y- = NCO-, -
OR-
Fibril-C=O + H2N-R-Y -----> F-C=N-R-Y

11. DENDRIMERIC NANOTUBES
The concentration of functional groups on the
surface of nanotubes can be increased by modifying the
nanotubes with a series of generations of a
polyfunctional reagent that results in the number of the
specific functional groups increasing with each
generation to form a dendrimer-like structure. The
resulting dendrimeric nanotubes are particularly useful
as a solid support upon which to covalently immobilize
proteins, because they increase the density of protein
immobilized on the nanotube surface. The present
invention demonstrates that high densities of a specific
chemical functionality can be imparted to the surface of


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
high surface area particulate carbon, which has been
difficult with previous high surface area carbons.
EXAMPLE 37

5 Preparation of Lysine-Based Dendrimers
The reaction sequence is shown in Fig. 5.
To a suspension of amino fibrils (90 mg) in
sodium bicarbonate (5 ml, 0.2 M, pH 8.6) was added a
solution of Nc,NE-di-t-boc-L-lysine N-hydroxysuccinimide
10 ester (120 mg, 0.27 mmol) in diosane (5 ml). The
reaction mixture was stirred overnight at room
temperature. The tert-butoxycarbonyl protected lysine
fibrils were extensively washed with water, methanol and
methylene chloride and dried under vacuum. The tert-
15 butoxycarbonyl protected lysine fibrils were then treated
with trifloroacetic acid (5 ml) in methylene chloride (5
ml) for 2 hours at room temperature. The product amino
lysine fibrils were extensively washed with methylene
chloride, methanol and water and dried under vacuum.
20 Preparation of the second and the third generation lysine
fibrils followed the same procedure. The amino acid
analysis data showed that the first generation lysine
fibrils contained 0.6 mols lysine per gram of fibrils,
the second generation lysine fibrils contained 1.8 mols
25 per gram of fibrils, and the third generation lysine had
3.6 mols lysine per gram of fibrils.
Carboxyl dendrimeric fibrils can be prepared by the
same method by using aspartic or glutamic acid with
carboxyl fibrils.
30 EXAPSPLE 38
Preparation of Carboxylate-Terminated Dendrimers
Carboxylate terminated dendrimers with a carbon
nanotube (CN) core are produced by successive, sequential
couplings of aminobuty-nitrilotriacetic acid (NTA) and
35 beginning with the NHS ester of chlorate oxidized carbon
nanotubes.


CA 02247820 2007-06-15
51096-13

61
Preparation of NTA
NTA was prepared according to the method of
Hochuli (E. Hochuli, H. Dobeli, and A. Schacher, J.
Chromatoaraphv. 411, 177-184 (1987)).
Preparation of CNf NHS
CN/NHS were prepared according to the method of
Example 20.
PreQaration of CN/NTA
0.4 g of NTA=HC1 was dissolved in 25 mis of
0.2M NaHCO31 pH 8.1. 1M NaOH was added to bring the pH
back up to 7.8. 0.5 g of CNJNHS was added, the mixture
was sonicated to disperse the CN and the resultant slurry
was left to react for 30 minutes with stirring. The
slurry was filtered onto a 0.45 m nylon membrane and
washed 2X with pH 8.1 carbonate buffer and 2X with DI
water on filter. The modified CN were twice resuspended
in 25 mis of MeOH with sonication, filtered to a solid
cake and finally dried in a vacuum desiccator.
Preparation of CN/NTA/NTA
CN/NTA was first converted to the NHS active
ester. 0.396 grams of CN/NTA was dried in an oven at
90 C for 30 minutes and then placed in a 100 ml RB flask
with 30 mis of anhydrous dioxane and purged with argon.
0.4 g of N-hydroxysuccinimide added with stirring
followed by 0.67 grams of EDC with continued stirring for
an additional hour. The CN tended to agglomerate
together during this time. The dioxane was decanted off
and the solids were washed 2X with 20 mis of anhydrous
dioxane. The solids were washed with 20 mis of anhydrous
MeOH during which the agglomerates broke up. The solids
were filtered onto a 0.45Am nylon membrane, resuspended
in MeOH, filtered and washed on the filter with MeOH.
0.2 g of NTA added to a 50 ml flask and
dissolved with 10 drops of 1M NaOH. 20 mis of 0.2M
NaHCO3 at pH 8.1, was added and then all of the
CN/NTA/NHS was added and the solution lightly sonicated


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
62
with a probe sonicator. The mixture was left to react
for 2.5 hours at room temperature. The modified CN were
filtered onto a 0.45 m nylon membrane, washed 2X with
carbonate buffer, resuspended in DI water with
sonication, filtered and washed with DI water. They were
then placed in vacuum desiccator to dry.
Preparation of CN/NTA/NTA/NTA
An additional level of NTA was added by
following the procedure described above.
Preparation of CN/NTA/NTA/NTA/NTA
An additional level of NTA was added by
following the procedure described above.
Samples (approx. 10 mg) of each of the four
generation of NTA addition were suspended in 10 mis of DI
water with sonication and filtered onto 0.45 /.cm nylon
membranes to form felt-like mats. The mat sections were
stored in a vacuum desiccator and analyzed by ESCA for
nitrogen (N) to indicate relative amounts of NTA. The
results are shown in the table below.
Material ~ N by ESCA
CN/NTA 0
CN/NTA/NTA 1.45
CN/NTA/NTA/NTA 1.87
CN/NTA/NTA/NTA/NTA 2.20
The ESCA results verify incorporation of
increasing amounts with each successive generation.
EXAMPLE 39
Carbon Nanotube Dendrimers as Protein supports
The density of protein immobilized on carbon
nanotubes can be greatly increased by using fibrils
derivatized to bear dendrimers. Horseradish peroxidase
(HRP) has been immobilized on dendrimeric nanotubes
according to the following method:
Plain fibrils (0.49 mg), amino fibrils (0.32
mg), first generation lysine fibrils (0.82 mg), second
generation lysine fibrils and third generation lysine
fibrils were sonicated with sodium bicarbonate conjugate


CA 02247820 1998-08-28
WO 97/32571 PCT/US97103553
63
buffer (600 l, 0.1 M, containing 0.9% NaCl) for 15
minutes at room temperature. Then they were incubated
with HRP solution in sodium bicarbonate conjugate buffer
(490 ml, enzyme stock solution of 5.6 mg/ml) for 19 hours
at room temperature. The HRP immobilized fibrils were
washed with the following buffer (1 ml): 10 mM NaHCO3
buffer containing 0.9% NaCl at pH 9.5 (1X washing buffer)
seven times, 0.1% Triton X-100 in iX washing buffer five
times, 50% ethylene glycol in IX washing buffer three
times. The activity of HRP was assayed with hydrogen
peroxide solution (10 l, 10 mM stock solution) and
2,2-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid
diammonium salt (ABTS, 3 l, mM stock solution) in
glycine assay buffer (50 mM, pH 4.4) at 414 nm. The
results are shown in the following table:

Fibrils nmol HRP/gram fibrils
plain Fib 3.82
Fib-NH2 8.58
Fib-NH-Lys 28.09
Fib-NH-Lys(Lys)2 28.30
Fib-NH-Lys(Lys)4 46.28

12. BIFUNCTIONAL FIBRILS

It has been found that more than one type of
functional group (e.g. a carboxyl group and an amino
group) can be introduced onto a fibril simultaneously by
reacting a functionalized nanotube, e.g. a carboxy
nanotube, with an amino acid. Such bifunctional fibrils
can be used to immobilize multiple molecules,
particularly in 1:1 stoichiometries and in close
proximity.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
64
ERAMPLE 40
Preparation of Bifunctional Fibrils
by Addition of Lysine
5gvnthesis of Na-CBZ-L-lysine Benzvl Ester
The reaction sequence is shown in Fig. 7. NE-
(tert-butoxycarbonyl)-L-lysine (2 g, 8.12 mmol) was
dissolved in methanol (40 ml) and water (40 ml), and the
pH was adjusted to 8 with triethylamine. A solution of
N-(benzyloxycarbonyl-oxy)succinimide in dioxane (2.4 g,
9.7 mmol in 20 ml) was added to the above mixture and the
pH was maintained at 8-9 with triethylamine. The
reaction mixture was stirred overnight. The solvent was
removed by rotary evaporation to obtain crude Na-CBZ-NE-
(tert-butoxycarbonyl)-L-lysine. Na-CBZ-NE-(tert-
butoxycarbonyl)-L-lysine was treated with 0.2 M calcium
carbonate (4 ml) and the aqueous layer was removed to
obtain a white solid. The solid was resuspended in N,N-
dimethylformamide(40 ml) and benzyl bromide (1.16 ml).
The reaction mixture was stirred overnight at room
temperature. The reaction mixture was worked up with
ethyl acetate and water, and the organic layer was dried
over magnesium sulphate. The solvent was removed to
obtain crude Na-CBZ-NE- (tert-butoxycarbonyl) -L-lysine
benzyl ester which was purified by silica gel
chromatography using 25% hexane in ethyl acetate as a
solvent. To Na-CBZ-NE- (tert-butoxycarbonyl) -L-lysine
benzyl ester (1 g, 2.2 mmol) in methylene chloride (10
ml) was added trifluoroacetic acid at 0 C. The reaction
mixture was stirred for 10 minutes at 0 C, then stirred
for further 2.5 hr at room temperature. The solvent was
removed and the crude product was obtained. Pure Na-
CBZ-L-lysine benzyl ester was obtained by silica gel
chromatography.


CA 02247820 1998-08-28
WO 97/32571 PCTlUS97/03553
Synthesis of N -CBZ-L-lysine Benzyl Ester Fibrils
To a suspension of carboxyl fibrils (300 mg) in
methylene chloride (18 ml) was added a solution of Nq-CBZ-L-
lysine benzyl ester (148 mg, 0.32 mmol in 20 ml methylene
5 chloride and 176 l triethylamine). HOBT (43.3 mg, 0.32
mmol) and EDC (61.3 mg, 0.32 mmol) were then added. The
reaction mixture was stirred overnight at room
temperature to obtain the crude product. The product
fibrils were extensively washed with methanol, methylene
10 chloride, and water, then dried under vacuum.
Synthesis of Bifunctional Fibrils Fib-Lys(COOH)NH2
To Na-CBZ-L-lysine benzyl ester fibrils (113
mg) in methanol (4 ml) was added sodium hydroxide (1 N, 4
ml) and the reaction mixture was stirred overnight. The
15 product Na-CBZ-L-lysine fibrils was extensively washed
with water and methanol and the fibrils were dried under
vacuum. To a suspension of Na-CBZ-L-lysine fibrils (50
mg) in acetonitrile (4 ml) was added trimethyl silyl
iodide (1 ml).- The miXture- was stirred f"or 3hours at
20 40 C. The final bifunctional fibrils were extensively
washed with water, methanol, 0.5 N sodium hydroxide,
acetonitrile and methylene chloride. Amino acid analysis
showed 0.3 mols lysine per gram of fibrils.
Hydroxyl and carboxyl (or amino) bifunctional
25 fibrils can be made by a similar method to that described
here by using serine, threonine, or tyrosine. Thiolated
and carboxyl (or amino) bifunctional fibrils can be made
using cysteine. Carboxyl and amino bifunctional fibrils
can be made using aspartic or glutamic acid.
USES FOR FUNCTIONALIZED NANOTUBES
Functionalized graphitic nanotubes are useful
as solid supports in many biotechnology applications due
to their high porosity, chemical and thermal stability
and high surface area. They have been found to be
compatible with harsh chemical and thermal treatments and
very amenable to chemical functionalization.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
66
For example, an enzyme can be covalently
immobilized on a modified nanotube while retaining its
biological activity. In addition, nanotubes are also
suitable for use as affinity chromatographic supports in
biomolecular separations. For example, enzyme inhibitors
have been prepared on nanotubes in multi-step syntheses
such that the immobilized inhibitors were accessible to
macromolecules, and reversible specific biological
recognition occurred between proteins and modified
fibrils.
The hydrophobicity of the nanotube surface is
not enough to immobilize high densities of proteins by
adsorption. To increase the hydrophobicity of the
nanotube surface and to expand the hydrophobic
environment from two dimensions to three dimensions,
alkyl chains of varying lengths have been coupled to the
nanotube surface. Proteins that have been immobilized on
alkyl nanotubes by adsorption include trypsin, alkaline
phosphatase, lipase and avidin. The enzyme activities of
these immobilized proteins are comparable with those of
the free enzymes, proven by the catalytic efficiencies
toward the hydrolysis of their substrates in aqueous
solutions.
In addition, phenyl-alkyl nanotubes, which are
alkyl nanotubes with the addition of a phenyl group on
the end of the alkyl chain, have also been prepared.
This modification introduced an aromatic structure that
interacts with the amino acids phenylalanine, tyrosine,
and tryptophan in proteins through v-v interactions. The
adsorption of alkaline phosphatase and lipase on phenyl-
alkyl nanotubes was comparable to the adsorption on C8-
alkyl nanotubes.
Functionalized fibrils have also been found to
be useful as solid supports for protein synthesis.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
67
1. FUNCTIONALIZED NANOTUBES AS SOLID SUPPORTS FOR ENZYMES
E}CAMPLE 41
Enzyme Immobilization by Adsorption
Preparation of Alkyl Fibrils
Alkyl fibrils were prepared by reacting 10mg of
carboxyl fibrils, which contained approximately 0.007
mmoles of
-COOH group (10 mg fibrils x 0.7 mmoles-COOH/mg of
fibrils = 0.007 mmoles), with 0.14 mmoles of alkylamines
in 1.5 ml DMF
(N,N-dimethylformamide) using 0.14 mmoles of EDC (1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 0.14
mmoles of DMAP (4-dimethylaminopyridine). The chemical
reaction is as follows:
Fibril-COOH + NH2(CH2)nCH2R (R=H or OH) ----->
Fibril-CONH(CH2)nCH2R
Several different alkyl fibrils with different lengths of
alkyl chains (n = 5, 7, 9, 17; R=OH only for n=5) were
prepared by this procedure. After the reaction was
stirred at ambient temperature overnight, fibrils were
washed rigorously with 3 x 25 ml CH2C12, 3 x 25 ml MeOH,
and 3 x 25 ml dH2O. Elemental analysis of the nitrogen
content in the fibrils showed that the yields of the
reaction were 65-100%.
Adsorption of Enzymes
The enzymes lipase, trypsin, alkaline
phosphatase and avidin were immobilized on the alkyl
fibrils of this example by adsorption. The alkyl fibrils
and enzyme were mixed at room temperature for three to
four hours, followed by washing two to four times with
5mM sodium phosphate (pH 7.1). Alkaline phosphatase was
immobilized on C8-fibrils and CsOH-fibrils; trypsin on C6-
, Cg-, CZO- and C18-fibrils, lipase on C6OH-, Cg-, Clo- and
C18-fibrils, and avidin on C8-fibrils. The results are
shown in the following table:


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
68

Enze umol fibr.i.l I_.mg/g fibril
lipase - 6.8 816
trypsin 1.7 40

alkaline phosphatase 0.66 56
avidin not determined

The kinetic properties of the immobilized
enzymes were found to be comparable to those of the free
enzymes, as shown in
the following table:
--: .
,;::. ....
Enz e M k s"a 3c M.", s- .:
lipase 40 x 10-6 0.040 0.99 x 103
lipase-Fibrils 36 x 10-6 0.048 1.34 x 103

trypsin 1.2 X 10'3 4.8 4.17 x 103
trypsin-Fibrils 7.9 x 10-3 19.1 2.43 x i03
substrate: lipase 1, 2-O-dilauryl-rac-glycero-3-
glutaric acid resorufin ester
trypsin N-benzoyl-L-arginine-p-
nitroanilide
BXAMPLE 42
Esterification Catalyzed by Fibril-Lipase
(Synthesis of Ethyl Butyrate)
Lipase was immobilized on C8-alkyl fibrils
according to the procedure of Example 41. The lipase
fibrils were washed first by dioxane, then a mixture of
dioxane and heptane, and finally heptane in order to
disperse the fibrils in heptane. To synthesize ethyl
butyrate (a food additive which provides pineapple-banana
flavor), ethanol (0.4M) and butyric acid (0.25M) were
mixed in heptane with 6.2 gm fibril-immobilized lipase. The reaction mixture
was stirred at room temperature.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
69
The yield was 60% in 7 hours, which was determined by
measuring ethanol concentration in the reaction mixture
using an established method. The reaction and results
are shown in Fig. S.
EXAMPLE 43
Immobilization of Alkaline Phosphatase
on Phenyl-akyl Fibrils
Preparation of Phenyl-Alkyl Fibrils
Phenyl-alkyl fibrils were prepared by two
different reactions. Reaction 1 mixed 20 mg carboxyl
fibrils (containing approximately 0.014 mmoles of -COOH
group) with 0.28 mmoles of 4-phenylbutylamine, 0.28
mmoles EDC and 0.28 mmoles DMAP (4-dimethylaminopyridine)
in 1.5 ml of DMF (N,N-dimethylformamide). Reaction 2
mixed 20 mg carboxyl fibrils with 0.28 mmoles of 6-
phenyl-l-hexanol, 0.28 mmoles DCC (1,3-
dicyclohexylcarbodiimide) and 0.28 mmoles DMAP in 1.5 ml
of DMF. The reactions were performed at room temperature
with stirring overnight. The fibrils were then washed
rigorously with 3 x 25 ml CH2C12, 3 x 25 ml MeOH, and 3 x
ml dH2O.
Preparation of Alkaline Phosphatase-Immobi.lized Fibrils
0.5 mg of phenyl-alkyl fibrils were suspended
in 400 Al of 0.05 M Tris (pH 8.6) and sonicated for 20
25 minutes. To these fibrils 150 l of alkaline phosphatase
solution (1.67 mg/ml in 5 mM sodium phosphate buffer, pH
7.0) were added and the mixture was rotated at room
temperature for 2 hours and stored at 4 C overnight. The
fibrils were then washed with 600 l of 5 mM sodium
phosphate buffer (pH 7.1) twice and suspended in 200 l
of the same buffer.
Ouantitation of Specifically Immobilized Alkali.ne-
Phosphatase by Measurement of Catalytic Activity
Alkaline phosphatase reacts with substrate p-
nitrophenyl phosphate and releases a color compound that
absorbs light at 405 nm with extinction coefficient of
18,200 M-lcm 1. The assay buffer condition for this


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
reaction was 10 mM Tris, 1 mM MgC12 and 0.1 mM ZnCl21 pH =
8.4. The reaction was performed in 1 ml cuvette by
mixing 5 l of p-nitrophenyl phosphate stock solution
(0.5 M in 33% DMSO in assay buffer) and 13 gg of alkaline
5 phosphatase fibrils in 1 ml of assay buffer. The
absorbance increase of 405 nm was monitored by time scan
over 0 minutes. The enzyme activity (/iM/min) was then
calculated from the initial slope using the extinction
coefficient 18200 M-lcui 1.
10 For alkaline phosphatase adsorbed on phenyl
fibrils from reaction 1, the activity was 6.95 ,uM/min per
13 g fibrils. For alkaline phosphatase adsorbed on
phenyl fibrils from reaction 2, the activity was 2.58
uM/min per 13 g fibrils. These results were converted
15 to 0.63 moles (or 54 mg) and 0.23 moles (or 20 mg)
active alkaline phosphatase per gram of fibrils,
respectively, by dividing the activity of a known
concentration of alkaline phosphatase solution, which was
measured to be 879.8 M/min per 1 M alkaline phosphatase
20 under the same assay condition.
EXAMPLE 44
Immobilization of Lipase on Phenyl Alkyl Fibrils
Preparation of Lipase-imimobilized Fibrils
0.5 mg of phenyl-alkyl fibrils were suspended
25 in 50 l of 5 mM sodium phosphate buffer (pH 7.1) and
sonicated for 20 minutes. To these fibrils 350 l of
lipase solution (0.2 mM in 5 mM sodium phosphate buffer,
pH 7.1) were added and the mixture was rotated at room
temperature for 5 hours and stored at 4 C overnight. The
30 fibrils were then washed with 600 l of 5 mM sodium
phosphate buffer (pH 7.1) three times and suspended in
200 l of the same buffer.


CA 02247820 1998-08-28
WO 97/32571 PCTIUS97/03553
71
Ouantitation of Specifically Immobilized Lipase by
Measurement of Catalytic Activity
Lipase can react with the substrate 1,2-o-
dilauryl-rac-glycero-3-glutaric acid-resorufin ester
(Boehringer Mannheim, 1179943) and produce a color
compound that absorbs light at 572 nm with extinction
coefficient of 60,000 M-Icm 1. The assay buffer condition
for this reaction was 0.1 M KH2PO41 pH = 6.8. The
reaction was performed in 1 ml cuvette by mixing 5 l of
substrate stock solution (7.6 mM in 50% dioxane in
Thesit) and 13 g of alkaline phosphatase fibrils in 1 ml
of assay buffer. The absorbance increase at 572 nm was
monitored by time scan over 10 minutes. The enzyme
activity ( M/min) was then calculated from the initial
slope using the extinction coefficient 60,000 M-lcm 1.
For lipase adsorbed on phenylalkyl fibrils from
reaction 1 of Example 43, the activity was 0.078 M/min
per 13 g fibrils. For lipase adsorbed on phenylalkyl
fibrils from reaction 2 of Example 43, the activity was
0.054 M/min per 13 E,cg fibrils. These results were
converted to 4.7 moles (or 564 mg) and 3.3 moles (or
396 mg) active lipase per gram of fibrils, respectively,
by dividing the activity of a known concentration of
lipase solution, which was measured to be 1.3 M/min per
1 M lipase under the same assay condition.
#,;XAMPIaE 45
Immobilization of Horseradish Peroxidase (HRP)
on Amino Alkyl-modified Fibrils

Preparation of Carboxvla.c Acid-Functionalized Fibrils
(Carboxyl Fibrils)

A 10.0 g sample of graphitic fibrils was
slurried in 450 mL concentrated H2SO4 by mixing with a
spatula, then transferred to a reactor flask fitted with
inlet/outlets and an overhead stirrer. With stirring and
under a slow flow of argon, a charge of 8.68 g of NaC103


CA 02247820 2007-06-15
51096-13

72
was added in portions at room temperature over a 24 hour
period. Chlorine vapors, which were generated during the
entire course of the run, were swept out of the reactor
into an aqueous NaOH trap. At the end of the run, the
fibril slurry was poured over cracked ice and vacuum
filtered. The filter cake was then transferred to a
Soxhlet thimble and washed in a Soxhlet extractor with
deionized water, exchanging fresh water every several
hours. Washing continued until a sample of fibrils, when
added to fresh deionized water, did not change the pH of
the water. The carboxylated fibrils were then recovered
by filtration and dried overnight at 100 C and 5" vacuum.
The yield was 10.0 g.
Preparation of HRP-Immobilized Fibrils
Amino fibrils made from 1,6-diaminohexane using
the method of Example 27 (1.2 mg) were added to
conjugation buffer (0.1 M NaHCO3, 0.9% NaCl, pH 9.5) and
the suspension was sonicated for 20 minutes. The fibrils
were then washed twice with conjugation buffer in an
Eppendorf tube and suspended 430 L conjugation buffer.
A 50- L aliquot of the suspension (0.14 mg fibrils) was
mixed with 4.0 mg activated HRP (Pierce, Rockford, IL)
dissolved in 50 L deionized water and the resulting
suspension was rotated overnight at 4 C. The HRP-
conjugated fibrils were washed extensively in an
Eppendorf centrifuge tube with a combination of the
following solutions; conjugation buffer, washing buffer
(20 mM KH2PO4, 0.45% NaCl, pH 6.2), washing buffer
containing 0.03-0.1% Triton*X-100, and washing buffer
containing 50% ethylene glycol. As a control, identical
manipulations with activated HRP were carried out with
plain (non-derivatized) fibrils, which indicated that the
attachment of HRP to amino fibrils was indeed a specific
covalent linkage.

* Trade-mark


CA 02247820 2007-06-15
51096-13

73
4uantitation of Specifically Immobilized HRP by
Measur ment of Catalvtic Activity

Extensive washing removed the majority of non-
specifically bound enzyme. Immobilized active HRP was
quantitated by substrate turnover using H202 and the
chromogenic substrate 2,2'-azino-bis(3-
ethylbenzthiazoline-6-sulfonic acid), diammonium salt
(ABTS). Catalytic.activity of HRP was
spectrophotometrically monitored at 414 nm using 100 M
H202 and 30 M ABTS as substrates. The total amount of
enzyme bound to amino fibrils in these preliminary
studies was 0.0230 mol HRP/g fibrils. By comparison,
control (plain fibrils) nonspecifically bound 0.0048 mol
HRP/g fibrils. By subtraction, the amount of covalently
(specifically attached) HRP was 0.0182 pmol/g fibrils.
EXAMPLE 46
Affinity Chromatographic separation of Alkaline
Phosphatase (AP) and B-Galactosidase (BG) on Pibrils
Bearing Immobilized Enzyme anhibitors

Premaration of Alkaline Phosnhatase Inhibitor Fibrils
Preparation of AP-inhibitor modified fibrils
was based on the method of Brenna et al. (1975), Biochem
J., 151:291-296.
Carboxylated fibrils were used to prepare NHS ester
fibrils as described in Example 50 above. NHS ester
fibrils (114 mg) were suspended in 4 mL acetone and 10
equivalents (based on the estimation of 0.7 meq NHS ester
per gram of fibrils) of tyramine were added. Dry
triethylamine (10 equiv.) was added and the mixture was
stirred for 3 hours at room temperature. The tyraminyl
fibrils were washed under vacuum in a scintered glass
funnel first with acetone, then extensively with
deionized water..
4-(p-Aminophenylazo)-phenylarsonic acid (66 mg)
was suspended in 4 mL of 1 N HC1. The suspension was


CA 02247820 2007-06-15
51096-13

74
cooled to 4 C and mixed slowly with 0.36 mL of 0.5 M
NaNO2. After 15 minutes, the arsonic acid/NaN02 mixture
was added to the tyraminyl fibrils, which were suspended
in 10 mL of 0.1 M NaCO3 (pH 10.0). The reaction mixture
(pH -_ 10) was stirred overnight at 4 C. The fibrils were
then treated with successive washes of 0.1 M Na2CO3 (pH
10.0), 8 M guanidine HC1, 25 mM NaOH, and water until the
effluent became clear. Atomic absorption analysis of
arsenic in the AP-inhibitor fibrils was carried out by
Galbraith Laboratories (Knoxville, TN). AP-inhibitor
fibrils which contain sidechains containing one atom of
arsenic were found by atomic absorption analysis to have
any arsenic content of 0.4%. This indicates that roughly
10t of the estimated initial COOH groups were converted
to AP-inhibitors in this multi-step synthesis. Based on
the surface area of fibrils, this means that there would
be one inhibitor molecule (enzyme binding site) for every
5o0A2 of surface area.
Preparation of !3-Galactosidase-Inhibitor Fibrils
p-Amino-phenyl-B-D-thiogalactoside (TPEG)
derivatized fibrils were prepared based on the method of
Uliman, (1984) Gene, 29:27-31. To 8 mg of carboxylated
fibrils in 0.2 mL deionized water was added 2.24 mg TPEG.
The pH of the suspension was adjusted to 4.0 with 0.1 M
HC1 and 15 mg EDAC was added. The mixture was stirred
for 3 hours at pH 4.0 and room temperature. The reaction
was stopped by rapid centrifugation in an Eppendorf*tube
and removal of the liquid. The 8-galactosidase-inhibitor
fibrils were washed five times by repeated resuspension
in deionized water and centrifugation.
Affinity Separations
Mixtures of alkaline phosphatase (AP), from E.
coli, Type III; Sigma Chemical Co., St. Louis, MO) and B-
galactosidase (BG) (from E. coli; Calbiochem, La Jolla,
CA) were separated batchwise on either AP-inhibitor
fibrils or BG-inhibitor fibrils in Eppendorf*
*Trade-mark


CA 02247820 1998-08-28

WO 97/32571 PCT/US97/03553
microcentrifuge tubes. For affinity separations, 1.0 mL
solutions of loading buffer (20 mM Tris, 10 mM MgCl, 1.6
M NaCl, 10 mM cysteine, pH 7.4) containing both AP
(generally approximately 10 units) and BG (generally
5 approximately 280 units) were added to 0.8-1.0 mg of
either AP-or BG-inhibitor fibrils. The resulting
suspensions were gently vortexed, then rotated at room
temperature for 2 hours. Following enzyme binding, the
fibrils were sedimented by brief centrifugation in a
10 tabletop centrifuge and the liquid phase containing
unbound enzyme was withdrawn and saved for enzyme assay.
Washes (7 x 1.0 mL) with loading buffer were carried out
by repeated buffer addition, gentle vortexing, 15-minute
rotation, brief centrifugation, and solvent withdrawal
15 with a Pasteur pipette. After the seventh wash, the same
manipulations were repeatedly carried out (5 x 1.0 mL)
with the appropriate elution buffer for either f3G-
inhibitor fibrils (100 mM sodium borate, 10 mM cysteine,
10 mM cysteine, pH 10.0) or AP-inhibitor fibrils (40 mM
20 NaHPO4, 10 mM Tris, 1.0 mM MgC121 0.1 mM ZnC121 pH 8.4).
All fractions (unbound enzyme, washes, and
elutions) were assayed for both AP and BG activity.
Alkaline phosphatase activity was determined by
spectrophotometrically monitoring the rate of hydrolysis
25 of 500 M p-nitro-phenylphosphate (PNPP) at 410
nm(ee=18,000 M-lcin 1). Alkaline phosphatase activity
measurements were carried out in 10 mM Tris, 1.0 mM
MgC12, and 0.1 mM ZnC12 at pH 8.4. B-Galactosidase was
assayed by spectrophotometrically monitoring the enzyme's
30 ability to hydrolyze 2-nitro-galacto-B-D-pyranoside
(ONPG). Initial rates of !3-galactosidase-catalyzed
hydrolysis of 5.0 mM ONPG were measured at 405 nm
(ee=3500 M-lcm 1) in 10 mM Tris, 10 mM MgC121 1.6 M NaCl,
10 mM cysteine, pH 7.4.
35 For both AP-inhibitor and BG-inhibitor fibrils,
a mixture of AP and BG were added. To facilitate
determinations of specific binding capacities, the


CA 02247820 1998-08-28
WO 97/32571 PCT/US97103553
76
concentrations of added enzymes were in large excess of
the immobilized inhibitor concentrations. For AP-
inhibitor fibrils, 0.550 mol AP/g fibrils was bound (as
opposed to non-specific binding of 0.020 mol BG/g
fibrils). For BG-inhibitor fibrils, the capacity was
determined to be 0.093 mol BG/g fibrils (in contrast
with non-specific binding of 0.012 mol AP/g fibrils).
The results of the affinity chromatography experiments
are shown in Figs. 9 and 10. AP-inhibitor fibrils did
not appreciably bind BG, but bound AP, which specifically
eluted when 40 mM phosphate, a competing inhibitor, was
added to the buffer (Fig. 9). Fibrils derivatized with
BG did not bind substantial amounts of AP, but bound BG,
which specifically eluted when the pH was raised to
weaken the enzyme-inhibitor association (Fig. 10). These
results show that inhibitors were successfully covalently
attached to the fibrils, that the immobilized inhibitors
were accessible to large molecules, that the inhibitors
were available for specific enzyme binding, and when
specifically eluted, that the enzymes remained active.
In Fig. 10, there appears to be continued leaching of BG
from BG-inhibitor fibrils. This may be a result of a
natural weak enzyme-inhibitor affinity rather than a
shortcoming of the fibrils because the same phenomenon is
not seen in Fig. 9 with AP-inhibitor fibrils.
2. FUNCTIONALIZED NANOTUBES AS SOLID SUPPORTS FOR
ANTIBODIES
It has been found that antibodies can be
immobilized on functionalized nanotubes, and that such
antibody nanotubes have unique advantages for many
applications due to their high surface area per weight,
electrical conductivity, and chemical and physical
stability. For example, antibody nanotubes can be used
as affinity reagents for molecular separations. Antibody
nanotubes are also useful for analytical applications, ,
including diagnostic immunoassays such as ECL-based
immunoassays.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
77
Antibodies can be immobilized either by
covalent binding or non-covalent adsorption. Covalent
immobilization was accomplished by various methods;
including reductive amination of antibody carbohydrate
groups, NHS ester activation of carboxylated fibrils (see
Example 27, supra), and reaction of thiolated or
maleimido fibrils with reduced or maleimido-modified
antibodies (see Examples 23 and 25 supra).
The best method for attaching antibodies to
nanotubes will depend on the application they are to be
used in. For separations applications, the preferred
method may be non-covalent adsorption because the
capacity of protein binding seems to be the highest for
this method. For methods involving ECL, where the
electrical conductivity of the fibrils may be important,
covalent methods may be preferred (the alkyl appendages
are weak electrical conductors and can be expected to
insulate the fibrils). Reductive amination may be the
best way to covalently attach antibodies to fibrils
because, by using this method, the antibodies are
correctly oriented so that their binding sites are
pointing outward (away from the fibrils).
3. ADDITION OF NAD{ TO FUNCTIONALIZED NANOTUBES
It has been found that cofactors such as NAD+
can be added to and used as a solid support for
biospecific affinity chromatography of proteins that bind
to enzyme cofactors. For example, NAD+ fibrils have been
used as a solid support for the purification of
dehydrogenases. The main advantage of using fibrils is
their large amount of accessible surface area. An
affinity matrix with high surface area is desirable
because of the high potential capacity. The fibrils may
either be a loose dispersion or fixed into a column or
mat.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
78
EXAMPLE 47
Affinity Chromatographic Separation of
Dehydrogenases on NAD+ Fibrils

Preparation of NAD+ Fibrils
Fibrils were oxidized to introduce carboxyl
groups according to Examples 14 and 15. To the
suspension of fibrils (31 mg) in sodium bicarbonate
solution (3m1, 0.2 M, pH 8.6) was added N6-
[aminohexyl]carbamoylmethyl)-nicotinamide adenine
dinucleotide lithium salt solution (25 mg from Sigma in 5
ml sodium bicarbonate solution). The reaction mixture
was stirred overnight at room temperature. The product
fibrils were extensively washed with water, N,N-
dimethylformamide, and methanol. The elemental analysis
data showed that the product fibrils contained 130 mmols
of NAD molecules per gram of fibrils by nitrogen analysis
and 147 mmols of NAD molecules per gram of fibrils by
phosphorus analysis. other NAD+ analogs having linkers
terminating in an amino group can be used to prepare NAD+
fibrils.
Affinity Separation
The NAD+ immobilized fibrils (0.26 mg) and
plain fibrils (0.37 mg) were sonicated with 0.1%
polyethylene glycol (PEG, MW 1000) in sodium phosphate (1
ml, 0.1 M, at pH 7.1) for 30 minutes at 40 C, then
incubated for 30 minutes at 40 C. The fibril suspension
was centrifuged and the supernatant were removed. The
fibrils were incubated with the mixture of L-lactate
dehydrogenase (LDH) in 0.1% PEG (1000) sodium phosphate
buffer (250 l, the ratio of the LDH solution and the
0.1% PEG buffer was 1:1) for 90 minutes at 4 C. Then the
mixtures were equilibrated for 30 minutes at room
temperature. After the incubation of the fibrils with
LDH, the fibrils were washed with 0.1% PEG (1000) in ~
sodium phosphate buffer (5 X 1000 l) and every washing
took 15 minutes with rotation. The LDH was eluted with a


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
79
mM solution of NADH in 0.1% PEG (1000) sodium phosphate
buffer (5 mM 3X1000 E.cl). During each elution wash the
fibrils were rotated for 15 minutes. The LDH activity in
the eluents was assayed by measuring the absorbance
5 change at 340 nm during reduction of pyruvate. The assay
mixture contained 0.1% PEG (1000) in sodium phosphate
buffer (980 l), pyruvate (3.3 l, 100 mM stock
solution), and each elution fraction (16.7 l). The
enzyme reaction is shown below:
LDH
pyruvate + NADH ------> Lactate dehydrogenase +
NAD+

The results showed that the capacity of LDH on
the NAD+ immobilized fibrils was 484 nmols per gram of
fibrils and the capacity of LDH on the plain fibrils
(control) was 3.68 nmols per gram of fibrils. The
nonspecific binding of LDH was 5.6%.
4. FUNCTIONALIZED NANOTUBES AS
SOLID SUPPORTS FOR PROTEIN SYNTHESIS
NXW4PLE 48
Use of Funationalized Fibrils as Solid
Support for Peptide Synthesis
To a mixture of amino fibrils (400 mg) and a 4-
(hydroxymethyl)-phenoxyacetic acid suspension (255 mg,
1.4 mmol) in methylene chloride (20 ml) were added 1-
ethyl-3-(3-
dimethylaminopropyl) carbodiimide (EDC, 268 mg, 1.40
mmol) and 1-hydroxybenzotriazole hydrate (HOBT, 189 mg,
1.4mmol). The reaction mixture was stirred overnight at
room temperature under argon gas. The product fibrils
were extensively washed with methylene chloride, methanol
and water, then dried under vacuum to get fibrils. To


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
the suspension of fibrils in N,N-dimethylforrnamide (DMF,
2 ml) and methylene chloride (8 ml) were added N-(9-
fluorenylmethoxycarbonyl)-O-butyl-L-serine (215 mg, 0.56
mmol), 1,3-dicyclohexylcarbodiimide (DCC, 115 mg, 0.56
5 mmol) and 4 dimethylaminopyridine (DMAP, 3.4 mg, 0.028
mmol). The reaction mixture was stirred overnight at
room temperature and the product fibrils were treated
with 20% piperidine in DMF (5 X 40 ml, each time soaked 1
min.). The product fibrils were then extensively washed
10 with DMF, water, sodium hydroxide (1N), methanol and
methylene chloride. The product Fib-Handle-Ser(O+)-COOH
(ninhydrin test was positive) was dried under vacuum.
For synthesis of dipeptide, the same procedure was used
to add arginine. The amino acid analysis data of Fib-
15 Handle-Ser(O+)-Arg(NE-2,2,5,7,8-pentamethylchroman-6-
sulfonyl) shows that it contains 6.5 gmol serine per gram
fibrils and 7.6 mol arginine per gram fibrils. Any
other peptide can be made by the same method.
5. BIOTINYLATED FIBRILS AND
20 BIOTINYLATED ALRYL FIBRILS
It has been found that fibril surfaces can be
functionalized by biotinylation or by both alkylation and
biotinylation. The fibrils containing such modifications
can then bind any streptavidin conjugated substances such
25 as streptavidin beads and streptavidin enzymes.
Fibrils offer great advantages as solid
carriers because of their high surface area. Beads,
which can be made strongly magnetic, are extremely useful
in separation assays. The biotinylated fibrils described
30 herein combine the advantages of both the fibrils and the
beads. The biotinylated alkyl fibrils are an extension
of the same concept but exhibit the additional protein
adsorption property of alkyl fibrils.
The streptavidin- and biotin-coated fibrils can
35 be used in diagnostics and can be used as capture agents
for assays such as electrochemiluminescence assays.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
81
A novel feature of this invention is the
combination of two solid carriers on one fibril to create
a bifunctional fibril. Moreover, the disclosed process
increases the surface area for beads and magnifies fibril
magnetization.
EXAMPLE 49
Preparation of Biotinylated Fibrils
Biotinylated fibrils were prepared by mixing
2.4 mg of amino fibrils prepared as described in Example
16 and 9 mg of NHS ester long chain biotin in buffer 0.2
M NaHCO3 at a pH of 8.15. The mixture was rotated at
room temperature for four hours and washed with the same
buffer twice.
EXAMPLE 50
Preparation of Biotinylated Alkyl Fibrils
Biotinylated alkyl fibrils were prepared by a
two step reaction. First, 4.25 mg of bifunctional
fibrils (containing both amino and carboxyl) and 25 mg of
NHS ester long chain biotin were mixed. The fibrils were
washed and dried under vacuum.
The second reaction was carried out by mixing 4
mg of biotinylated bifunctional fibrils with 11 mg of EDC
(1-ethyl-3-3-dimethylaminopropyl)carbodiimide), 7.5 mg of
DMAP (4-dimethylaminopyridine) and 10 l of NH2(CH2)7CH3
in 0.5 ml of DMF. The mixture was stirred at room
temperature overnight. The final biotinylated alkyl
fibrils were washed by CH2C121 MeOH, and dHaO.
BXAMPLE 51
Biotinylated Fibrils
as a Solid Support in Assays
Biotinylated fibrils can be used in assays
involving formats that require streptavidin-biotin or
avidin-biotin interactions. Biotinylated fibrils could,
for example, be further derivatized with streptavidin.
Biotin covalently linked to fibrils (see Example 50)
could form strong non-covalent binding interactions with
streptavidin. Because streptavidin is a tetrameric


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
82
protein with four equivalent binding sites, streptavidin
bound to biotinylated fibrils would almost certainly have
unoccupied binding sites to which additional biotinylated
reagents could bind. Thus, biotinylated fibrils would be
converted to streptavidin-coated fibrils.
There are a number of analytical tests that
could be performed with such fibril-biotin-streptavidin
(FBS) supports. For example, a biotinylated anti-analyte
antibody could be captured on the FBS support (either
before or after the antibody has complexed to an
analyte). Assays using biotinylated anti-analyte
antibodies are well established. Such assays include
competitive assays where the analyte of interest competes
with a labeled analyte for binding to the anti-analyte
antibody. Free (unbound) analyte and free (unbound)
labeled analyte can be washed from the fibril immobilized
antibody. The washing step depends on the fibrils being
physically separated from the solution phase by common
practices involving centrifugation, filtration, or by
attraction to a magnet.
Besides a competition assay, a sandwich type
immunoassay could be carried out on FBS supports.
sandwich immunoassays are well known in the field of
diagnostics. Such assays involve an analyte being bound
simultaneously by two antibodies; a first "primary"
antibody which is captured on a solid surface by for
example being labeled with biotin, and a secondary"
antibody which is not captured by a solid surface but is
labeled with a reporter group. Such a sandwich assay
could be carried out using fibrils as a solid capture
support whereby the fibrils are captured as described in
the previous paragraph. Hence, in such an assay, the
fibril would have covalently linked to it biotin, which
would be bound to streptavidin, which would in turn be
bound to a biotinylated primary antibody, which would be
bound to analyte (if present), which would be bound to a
labeled secondary antibody.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
83
Similarly, DNA probe assays could be carried
out using FBS supports. Biotinylated single stranded DNA
can be bound to FBS supports and competitive
hybridization can occur between complementary single
stranded analyte DNA molecules and complementary labeled
oligonucleotides.
Another type of biotinylated fibrils,
biotinylated alkylated fibrils, can be used in
immunoassays and DNA probe assays. As described in
Example 51, bifunctional fibrils can be modified by
covalent attachment of biotin to one type of functional
group and alkyl chains to the other type of functional
group. The resultant alkylated, biotinylated fibrils can
be used both in specific association with streptavidin or
avidin (via biotin) and also for adsorption of proteins
(via the alkyl chains).
Alkyl fibrils could be used in conjunction with
other solid supports, such as streptavidin-coated
magnetic beads. One advantage of fibrils over such beads
is that they have a much higher surface area (per unit
weight). Thus, if fibrils could be attached to the
outside surface of the magnetic beads, this would
dramatically improve the surface area and hence the
binding capacity of the beads. It is envisioned that
alkylated, biotinylated fibrils could be mixed with
streptavidin-coated beads resulting in high affinity
streptavidin(bead)-biotin(fibril) interactions and hence
fibril-coated beads with an extremely high surface area.
Because alkyl fibrils can bind proteins by adsorption,
the fibril-coated beads could be further derivatized with
adsorbed proteins including streptavidin and antibodies.
As described above, streptavidin or antibody coated
fibrils can be_used in immunoassays and DNA probe assays.
Thus, fibril-coated beads could improve the properties of
the beads by dramatically increasing their surface area
such that fewer beads would be required in a given assay
to give the same result.


CA 02247820 1998-08-28

WO 97/32571 PCT/US97/03553
84
6. 3-DIMENSIONA7, STRUCTURES
The oxidized fibrils are more easily dispersed
in aqueous media than unoxidized fibrils. Stable, porous
3-dimensional structures with meso- and macropores (pores
>2 nm) are very useful as catalysts or chromatography
supports. Since fibrils can be dispersed on an
individualized basis, a well-dispersed sample which is
stabilized by cross-links allows one to construct such a
support. Functionalized fibrils are ideal for this
application since they are easily dispersed in aqueous or
polar media and the functionality provides cross-link
points. Additionally, the functionality provides points
to support the catalytic or chromatographic sites. The
end result is a rigid, 3-dimensional structure with its
total surface area accessible with functional sites on
which to support the active agent.
Typical applications for these supports in
catalysis include their use as a highly porous support
for metal catalysts laid down by impregnation, e.g.,
precious metal hydrogenation catalysts. Moreover, the
ability to anchor molecular catalysts by tether to the
support via the functionality combined with the very high
porosity of the structure allows one to carry out
homogeneous reactions in a heterogeneous manner. The
tethered molecular catalyst is essentially dangling in a
continuous liquid phase, similar to a homogeneous
reactor, in which it can make use of the advantages in
selectivities and rates that go along with homogeneous
reactions. However, being tethered to the solid support
allows easy separation and recovery of the active, and in
many cases, very expensive catalyst.
These stable, rigid structures also permits
carrying out heretofore very difficult reactions, such as
asymmetric syntheses or affinity chromatography by
attaching a suitable enantiomeric catalyst or selective ~
substrate to the support. Derivatization through
Metallo-Pc or Metallo-porphyrin complexes also allows for


CA 02247820 1998-08-28

WO 97/32571 PCT/US97/03553
retrieval of the ligand bonded to the metal ion, and
furthermore, any molecule which is bonded to the ligand
through the secondary derivatives. For example, in the
case where the 3-dimensional structure of functionalized
5 fibrils is an electrode, or part of an electrode, and the
functionalization has resulted from adsorption of
Co(II)Pc, electrochemical oxidation of Co(II) to Co(III)
in the presence of nicotinic acid will produce a non-
labile Co(III)-pyridyl complex with a carboxylic acid as
10 the pendent group. Attaching a suitable antigen,
antibody, catalytic antibody, or other site-specific
trapping agent will permit selective separations of
molecules (affinity chromatography) which are otherwise
very difficult to achieve. After washing the electrode
15 to remove occluded material, the Co(III) complex
containing the target molecule can be electrochemically
reduced to recover the labile Co(II) complex. The ligand
on Co(II) containing the target molecule can then be
recovered by mass action substitution of the labile
20 Co(II) ligand, thereby effecting a separation and
recovery of molecules which are otherwise very difficult
or expensive to perform (e.g., chiral drugs).
Previously, it was believed that the pores
within the functionalized carbon fibril mats were too
25 small to allow significant flow and thus would not be
useful as flow through electrodes. There were also
problems associated with the use of particulate carbon or
other carbon based materials (such as Reticulated
Vitreous Carbon (RVC)) as electrode materials. For
30 example, the porous electrode materials could not be
formed in situ, packed too densely and formed voids or
channels, were subject to dimensional instability during
changes in solvent and flow conditions, and were unable
to form very thin electrodes. The use of functionalized
35 carbon fibrils as electrodes in a flow cell solved such
problems.


CA 02247820 1998-08-28
WO 97/32571 PCTIUS97/03553
86
The functionalized carbon fibrils used as
electrodes in a flow cell can be modified by surface
treatment with electroactive agents. The fibrils can
also be modified with non-electroactive materials that
may serve a catalytic or electrocatalytic function or
serve to inhibit unwanted reactions or adsorption of
materials from the flowing stream.
These flow through electrodes are useful in
separation techniques such as electrochromatography,
electrochemically modulated affinity chromatography,
electrosynthesis or electrochemically modulated ion
exchange chromatography. They can also be used in
diagnostic devices that separate and/or analyze material
trapped on the carbon fibril mat.
Composite mats composed of functionalized
carbon fibrils and other fibers or particulates can also
be used. These fibers or particulates can be added to
the suspension to alter the final porosity or
conductivity of the carbon fibril mat.
EXAMPLE 52
Use of Iron Phthalocyanine Functionalized
Fibrils as Electrodes in a Flow Cell
Graphitic fibrils were modified by adsorbing
Iron(III)phthalocyanine-bis-pyridine (FePc-2Py) (Aldrich
41,016-0). 0.403 grams of fibrils and 0.130 grams of
FePc-2Py were added to 150 mis of absolute EtOH and
sonicated with a 450 Watt Branson probe sonicator for 5
min. The resulting slurry was filtered onto a 0.45 m
MSI nylon filter in a 47 mm Millipore membrane vacuum
filter manifold, rinsed with water and dried in a vacuum
oven overnight at 35 C. The final weight was 0.528
grams, indicating substantial adsorption. A
spectrophotometric analysis of the filtrate accounted for
the remaining FeP-2Py.
5 mgs of the FePc-2Py modified fibrils were
dispersed in 10 mls of DI water with sonication. The


CA 02247820 1998-08-28
WO 97/32571 PCTIUS97/03553
87
dispersion was deposited onto a piece of 200 mesh
stainless steel (SS) woven screen held in a 25 mm
membrane filter manifold and allowed to dry at room
temperature. A 0.5 inch diameter disk of the SS screen
supported fibril mat was cut using an arch punch.
A electrochemical flow cell was constructed
from a 13 mm, plastic, Swinney type membrane filter
holder by placing a 13 mm diameter disk of gold mesh (400
mesh, Ladd Industries) on top of the membrane support and
making electrical contact to the screen with a platinum
wire, insulated with Teflon heat shrink tubing that was
fed through the wall of the filter holder for external
connection as the working electrode of a three electrode
potentiostat circuit. The gold mesh was fixed in place
with a minimal amount of epoxy around the outer edge. A
strip of gold foil was fashioned into a ring and placed
in the bottom, down stream section of the filter holder
and connected with an insulated Pt wire lead for
connection as the counter electrode of a three electrode
potentiostat circuit. A ring of 0.5mm diameter silver
wire, electrochemically oxidized in 1M HC1, was placed in
the top section of the filter holder with an insulated
lead for connection as the reference electrode.
The 0.5 inch diameter disk of FePc-2Py modified
CN was placed in the flow cell, which was then connected
to the appropriate leads of an EG&G PAR 273 potentiostat.
The flow cell was connected to a Sage syringe pump filled
with 0.1M KC1 in 0.1M potassium phosphate buffer at pH
7Ø Cyclic voltammograms (CVs) were recorded under no
flow (static) and flow (0.4 mls/min.) at a potential scan
rate of 20 mv/sec. (see Fig. 6). The CVs were nearly
identical with and without flow and showed two
persistent, reversible oxidation and reduction waves
consistent with surface confined FePc-2Py. The
persistence of the redox peaks under fluid flow
conditions demonstrates that the FePc-2Py is strongly
bound to the carbon fibrils and that the use of iron


CA 02247820 1998-08-28
WO 97/32571 PCT/US97l03553
88
phthalocyanine modified fibrils function well as a flow
through electrode material.
Another example of 3-dimensional structures are
fibril-ceramic composites.
EXAMPLE 53
Preparation of Alumina-Fibril composites (185-02-01)
One g of nitric acid oxidized fibrils (185-01-
02) was highly dispersed in 100 cc DI water using and U/S
disintegrator. The fibril slurry was heated to 90 C and
a solution of 0.04 mol aluminum tributoxide dissolved in
cc propanol was slowly added. Reflux was continued
for 4 hr, after which the condenser was removed to drive
out the alcohol. After 30 min the condenser was put back
and the slurry refluxed at 100 C overnight. A black sol
15 with uniform appearance was obtained. The sol was cooled
to RT and after one week, a black gel with a smooth
surface was formed. The gel was heated at 300 C in air
for 12 hr.
The alumina-fibril composites were examined by
20 SEM. Micrographs of cracked surfaces showed a
homogeneous dispersion of fibrils in the gel.
EXAMPLE 54
Preparation of Silica-Fibril Composites (173-85-03)
Two g of nitric acid oxidized fibrils (173-83-
03) were highly dispersed on 200 cc ethanol using
ultrasonification. A solution of 0.1 mol
tetraethoxysilane dissolved in 50 cc ethanol was slowly
added to the slurry at RT, followed by 3 cc conc. HCL.
The mixture was heated to 85 C and maintained at that
temperature until the volume was reduced to 100 cc. The
mixture was cooled and set aside until it formed a black
solid gel. The gel was heated at 300 C in air.
The silica-fibril composites were examined by
SEM. Micrographs of cracked surfaces showed a
homogeneous dispersion of fibrils in the gel.


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
89
Similar preparations with other ceramics, such
as zirconia, titania, rare earth oxides as well as
ternary oxides can be prepared.
7. INCORPORATION OF GRAPHITIC NANOTIIBEB
ONTO POLYMER BEADS
Polymer beads, especially magnetic polymer
beads containing an Fe304 core, such as those manufactured
by Dynal and others, have many uses in diagnostics.
These beads suffer, however, from having a low surface
area compared to that available from nanotubes.
Functionalized fibrils can be incorporated onto the
surface of beads, which allows the polymer/fibril
composites to be used as solid supports for separations
or analytical application (e.g., electrochemiluminescence
assays, enzyme immobilization).
EXAMPLE 55
Attachment of Functionalized Fibrils to Functionaiized
Beads
7.5 mg of magnetic tosyl-activated Dynabeads M-450
(30 mg/ml) beads (Dynal, Oslo, Norway) were washed three
times with 0.1 M sodium phosphate buffer, pH 7.5. Then
0.9 ml of 0.1 M sodium phosphate buffer, pH 8.4 was added
to the beads and 0.1 ml of amine fibrils were added. The
mixture was allowed to rotate for 16-24 hours at room
temperature.
When viewed under the microscope clumps of fibrils
with beads on the surface of the fibrils were evident.
As illustrated by the foregoing description and
examples, the invention has application in the
formulation of a wide variety of functionalized nanotubes
and uses therefor.
The terms and expressions which have been
employed are used as terms of description and not of
limitations, and there is no intention in the use of such
terms or expressions of excluding any equivalents of the
features shown and described as portions thereof, its


CA 02247820 1998-08-28
WO 97/32571 PCT/US97/03553
being recognized that various modifications are possible
within the scope of the invention.