Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
2 ~
E~L~
The present invention is concerned with optical biosensors
and with a methad for their production; in particular, it is
concerned with methods and compounds for appiying biologically
recognizing elements to TiO2 wave guides which are used for
these novel optical sensors.
According to definition, a biosensor is a device consisting
of a transducer and a biologically recognizing element (Trends in
Biotechnol. 2 (1984), 59). Such biosensors can be used to deter-
mine anaiyte concentrations e.g. in human and veterinary diag-
nostics, in environmental analysis and in the analysis of food or
in biochemical r~search for the quantification of intermolecular
interactions of biologically active substances (e.g. antibody-
antigen interaction, receptor-ligand interaction, DNA-protein
interaction ctc.).
The function of th~ biologically recognizin~ element of a
biosensor is to r~cognize an analyte molecule in solution and to ~ -
bind to it (so-called affinity sensor) or ~o catalytically modify it
(so-called enzymatic, metabolic sensor). The change in the
biologically recognizing element which accompanies this is
recognizecl by tha transducer (si~nal transform~r), which is in
close contact with the element, and is converted into a process-
able si~nal.
One class of such transducers detects alterations in the
optical properties of the biologically recognizing element (e.g.
absorption, refraction) by means of an optical surface wave,
which is conducted in a wave-guiding layer along the interface of
the transducer/recognizing element. This class of transducers
can be divided into two groups on the basis of different wave-
guiding layer structures. A first group embraces transducers in
which this wave-guiding structure is the interface between a
metal and a dielectric. The wave which is conducted at this
interface is the surface piasmone (Sensor and Actuators 4 (1983)
299). The second ~roup embrac~s transducers having diel.qctric
wave-guiding layers. The optioal waves which are conducted are
wave guide modes (Opt. Lett. 9 (1g8~) 137; Sensors and Actuators
A, 25 (1990) 185; Sensors and Actuatnrs B, 6 (19~ 122; Proc.
Biosensors 92, extended abstracts, pp 339 & pp 347).
The present invention is concerned with biosensors which
are based on dielectric wave guides. The basic principle of such
transciucers can be expiained on the basis of the field distribution
of the modes which are conducted in such wave guides. The
electriG field of the conducted mode is not limited solely to the
geometric dimensions of the wave guicie, but has so-called
evanescent ccmponents, i.e. the field distribution of the
conducted modes dies away exponentially in the media adjacent
to the wave guide (e.g. in the substrate or in the superstrate in
which the wave-guiding layer is situated). Changes in the optical
properties of the substrate or superstrate adjacent to the wave
guide within the ~ransmission range of the evanescent field
influerlc~ the propagation of these modes and can be detec~ed by
suitabl~ measuring instruments. When th~ sup~rstrate contains
the biologically recogniziny element within the transmission
range of the evanPscent field, then the changes in th~ opticai
properties of this element which accompany the bindin~ or
mcdification can be detected by this optical, surface-sensitive
method and calibrated in terms of an anallyte concentration.
It is known from the lit~rature that the hi~her are the
surfac~ specificity and ~he surface sensitivity of the method,
then the sma11er is the effective density of the wave-guiding
layer (monomode wave guide) and the higher is the jump in the
refraetive index at the interface of the wave guide/substrate and
wave guide/superstrate~ Having regard to its high refractive
index, TiO2 is therefore especially suitable as a materiai for such
wave guides and it has been shown recently (Proc. Materials res.
Soc. Spring i\/ieeting, San Francisco, 1992) that by using PICVD
technoio~y wave-guiding films can be produced from this
rnateriai with a refractive index of 2.45 for sensorics.
"~
, . . - - . , , ,,. : :
... , ; ,
3 ~ ~3~7~
The present invention is concerned with the coating of such
TiO2 wave guides with biologically recognizing elements, there
being obtained biosensors of high sensitivity and specificity for
an analyte molecule. The basis for these recognizing elements is
that a so-called recognizing molecule (e.g. antibody, membrane
r~ceptor, ssDNA sampl~ etc) comes into play for a selective
recognition and binding (and/or modification~ of an analyte
moleculc (e.g. antigen, ligand, active substance, ssDNA, etc).
These recognizing molecules can be used no~ only in their
naturally occurring and isolatable form, but also in a chemically
or biotechnolo~ically prepared form.
The object of the present invention is to provide an optical
biosensor and a method for its production, ~he biosensor
consisting of a TiO2 wave ~uide and an or~anic carrier layer with
the receptor molecules bonded thereon and this carrier layer
satisfying the requirements of optical biosensorics, i.e.
- its layer thickness is not greater than the transmis-
sion range of the evanescent field of the mode
conduct~d into the wav~ guide;
- its construction shows optical homogenicity with
respect to the light conducted into the wave guide;
- it shows ch~mical resistance to media with which
it comes into contact (sera, fermentation solutions,
etc);
- the recognizing molecules are anchored to it such
that their natural activity is maintained;
- the recognizing molecules are anchored to it such ~
that they are no~ lost by dissociation in contact with ::
the sample.
The object of the invention is to provide corresponding
recognizing ~lements on the novel TiO2 wave guides appropriate
~"":.," ;,: .," ,~ ~, " ,~
to the different analyte molecules and, respectively, recognizing
receptor moiecules.
In accordance with the invention the object is achieved by
providing an optical biosensor consisting of a dieleetric wave
~uide and an organic carrier layer to which receptor molecules
are bonded, wherein the organic oarrier layer and the receptor
molecules bonded thereon form an ordered monomolecular layer
and the carrier layer consists of molecules of general formula I
/ Si-Y- Z
and wherein the molecules of the carrier layer are bonded to a
TiO2 waYe guide directly via th~ Si atom or, if desired, are bonded
to a TiO2 wav0 guide via an intermediate laysr.
Examples of optical biosensors and a process for the .
production of the biosensors in accordance with the invention
will be described hereinafter.
For tho construction of the organic carrier layers having the
r~quired prop0rti~s, the TiO2 wav~ guid~ surface is firstly
provided with a homogeneous organic suppl~mentary layer. The
cornpounds used for the derivatization of the TiO2 surface are
silanes o~ general formula ll
(R1 R2R3)Si-Y-X 11
wherein
-Si(R1R2R3~ represents a coupling group to the TiV2 layer
and R1, R2, R3 can be alkyl, alkoxy ar halogen, but at least one of
these residues is either alkoxy or halogen,
-Y is a spacer group and as such represents either an
alkylene chain -CH2-(CH2)n-CH2-, a fluoroalkylene chain
-Ctl2~(CF2~n~CH2~ or -CH2-(CF2)n CF2- with n - 1-30, an
oli~oethylane chain ~[tCH~)n~~O-(CH2)nl]m~ with n', n" = 2-6 and
3 ~
m= 2-6 or a cornbination of alkylene, fiuoroalkylene or
oligoalkyiene glycol and
-X is either hydrogen or fluorine or a chemically reactive
group which is compatible with -Si(R1R~R3) such as e.g.
carboxylio acid halide ~-COHal), olefin (-CH=CH2), nitrile (-CN),
thiocyanate [-SCN) and thioacetate (-SCOCH3) or, when R1,R2,R3=
alkoxy, also amine (-NH2).
After the addition of the compounds to the TiO2 surface,
these groups can also be converted by suitable subsequent
chemical treatments into groups which are not compatible with
-Si(R1R2R3) such as e.g. into a7ide and further into amiJle, or
nitrile into amine, or halogen into thiocyanate and further into
thiol, or thioacetate into thioi or olefin into epoxide, diol, halide,
dihalide or carboxylic acid etc.
Other molecules can be coupled to the original group X or to
the group X subsequently treated as just described to give an
organic carrier layer to which the r~ceptor molecul~s are bond~d. -::
An optical biosensor is obtained in which the organic
carrier layer forms ordered monomolecular layers and consists of
molacules of general formula l.
Si -Y- Z
In ~his formula Z signifies the groups~
- hydroxyl, carboxyl, amine, methyl, alkyl, fluoroalkyl
groups;
- dorivatives oli hydrophilic short-chain molecules such
as oligovinyl alcohols, oligoacrylic aoids, oligo-
ethylene glycols;
- derivative of mono- or oligo-saccharides with 1~7 :: ~
sugar units; : -
,: ~ . . . , , ' . , : , ' ,
6 2 ~
- carboxyglycosid~ derivatives,
- aminoglycoside derivatives suoh as fradiomycin,
kanamycin, straptomycin, xylostatin, butirosin,
ohitcsan
- derivative of hydro3el-forming groups of natural or
synth~tic origin such as dextran, agarose, alginic acid,
starch, cellulose and deriva$ives of such polysac-
charides or hydrophilic polym*rs such as polyvinyl
aicohols, polyacrylic acid, polyethylene glycols and -:
derivatives of such polymers.
It has surprisingly been found th~ the compounds of
formula 11 are outstandingly suitable for applying to TiO2
monomolecular, densely packed, ordered organie films having the
quality required for optical sensorics.
For low-molecular representatives of the cornpounds of
formula 11 this coating is pre~crably carrie~d out frcm the gas
phase (chemical vapor deposition (CVD) method). For high-mole- :;~
cular compounds this coating can also be carried out from the
liquid phase. In order, however, in the coating of the TiO~ wave
guide to achievs a homogenicity which is sufficient for use in
optiGs, the soivent used must be coordinated with thc compounds
of formula 11.
The biologically recognizing eiemcnts in biosensorics are
3snerally constructed from an organic carrier layer which is
covalently linked with ths substrate (transducer surf~ce) and to
which biologically recognizing moiecules are absorbativeiy or,
preferably, covalently bondQd. As will be evident ~rom the
following, in biosensorics the specifk: construction of a bio-
logically recognizing element is on th~ one hand closely linked
with the type of analyte molecule to be detected and thus with
the nature of the receptor molecule which is usecl for the
detection and is on ths other hand, however, also dependent on the
;" " . ~ s, "~ ; , "
2 ~ o ~ ~ !3 ~
probiem position to be resolved with a particular biosensor for a
receptor/analyte molecule pair.
The biologically recognizing elements, which are claimed
here for use in optical biosensorics in combination with TiO2
wave guides, are divided into two main classes A and B, with eaoh
of these main classes being divided into two sub-classes A1, A2
and B1, B2. The criterium relevant for the assignment of a
reco~nition element to one of the main classes relates to the
arrangemant of th~ receptor moiecules on the sensor surface. In
the first class (A) the receptor molecules are ordered
approximately in one plane (two dimensional arrangement~ on the
surface of the optical transducer. Such a ~No dimenslonal
arrangement of the receptor molecules results only when the
dimension of the organic carrier layer perpendicular to the
transducer surface is not substantialiy greater than the mo!e-
cular size of the reoeptor molecule bonded to this carrier layer.
In reoognizing elements of class B the imrnobilized receptors
have a three dimensional arrangement. This three dimensional
arrangement can only be realized with a carrier layer having a
thickness which is substantially greater than the moleeular
dimensions of the receptor molecula and which proves to be
parmeabl~ for the receptor molecule. This type of carrier iayer
can be denoted as a porous, three dim~nsional matrix. The
criterion for the assignment to one of the sub-classes (A1, B1 or,
respectively, A2, B2) relates to the manner in which the receptor
molecules are irnmobilized on the carrier layer; the differenti-
ation being a non-directed ~A1, B1) and a directed (A2, B23 mode
of immobilization, the term immobilization being used not oniy
for an absorptive but also for a covalent bonding to the organic
carriar layer. Non-directed immobilization of a receptor
molecule to the organic carrier layer signifies that in the bonding
of the receptor molecule to the organic carrier layer no regard is -
had to particular structural features of the receptor molecule, i.e.
the immobilization takes place at any position on the surface of
the receptor molecule. Directed immobilization signifies that in
the immobilization of the receptor molecule regard is had to the
analyte-recognizing domains and for the immobilization those
,. ~, . . . .
f~
structural elements are chosen which are well separated
spatially from the analyte-recognizing domains.
As mentioned earlier, each of these different typas of
biologically recognizing elements has its specific suitability for
us~ in different fields or investigational areas of bioanalytics.
This will be substantiated using some exampies:
A three dimensional matrix permits the immobilizatian of a
larger number of receptor molecules per surface unit. Since the
total number of receptor molecules per surface unit in the case of
directed biosensors detsrmines th~ steepness of th~ sensor curve
and consequently the analytical capabili~y in the concentration
range relevant to the sensor, a sensor which is equipped with a
three dimensional recognizin~ element is accordingly preferred
for an exact determination of an analyte concentration (e.g. in
diagnostic use). A three dimensional matrix can, however, also bs
of disadvan~age when the analyte molecule to be detected
possesses several repetitive epitopes. The binding of this
analyte molecule to several receptor molecules in ~he outer
regions of the element then leads to a cross-linking of the carrier
layer, whereby the access to free binding sites on the inside of
the recognizing element is impeded for subsequent analyte
molecules. The disadvantage of a three dirnensional arrangement
oiF r~ceptor molecules is also obvious in a quantitative represent-
ation of the kinetics of the binding process between a receptor
molecule and an analyte molecule. The time-dependent sensor
response observed in such an inv~stigation using a three dimen-
siona5 matrix can also be marked by the hindered diffusion of the
analyte molecule in this matrix.
In an analogou~ manner, advantages and disadvantages of a
directed or non-directed immobilization can be demonstrated in
different bioanalytical investigations. For the detection of an
antigen in immunodiagnostics it is without doubt important to
irnmobilize the antibody used for the detection on the surface
such that the antigen-recognizing domains are not influenced by
the immobilization, e.g. over the Fc part which is well separated
2 ~
from the antigsn-recognizing domains. When, however, a
biosensor is used to test for the presence of an ensemble of
polyclonal antibodies a~ainst one and the same antigen, then it is
convenient to irnmobilize the antigen in a non-directed manner,
since thereby all epitopes of the antigen are equally available for
recognition by the antibody in solution.
As a further embodiment of the invention it ensues
accordingly that the TiO2 surfaces for the construction of the
biologically recognizing elements of type A1, A2, B1 and B2 must
be provided with organic carrier layers which permit the directed -~
or non-directed immobilization of receptor molecules in a two or
three dimansional arrangement.
In addition to the aforementioned requirements relating to
the dimensions and permeability of a carrier layer, which is
providsd for the two a,r three dimensional arrangement of
receptor molecules, these organic carrier layers for the
immobilization of the receptor molecul~s must also satisfy
chemieal and, respectively, physicochemical requirements and
must have
a) reactive groups by means of which receptor molecules
can be covalently anchored to/in the two/three dimensional
carrier layer
and
b) functional groups or molecules and/or molecular
associations which permit an efficient concentration of receptor
molecules to/in this two/three dimensional matrix before the
covalent anchoring, so that the immobilization of receptor
molecules can be carried out from dilute solutions.
ad a): A large number of reactive groups which can be used
for such a covalent immobilization are known from the literature.
A differentiation is made between groups which are per se
chemically active and which can enter into a bonding with
1 0
functional groups on the receptor molecule, such as e.g. amino,
hydrazina or hydrazide ~roups on the carrier layer, which can
react with aldehy~e groups on the receptor moiecule, and vice
versa, aotivated disulphide bonds on the carrier layer which react
with free thiol ~roups on the receptor moiecule, c~rboxylic acid
halides or activated carboxylic acid esters on the carrier layer
which react with amino groups on the surface of the receptor
molccule etc, and groups which react with functional groups on
the receptor molecule after an in situ activation (chemical or
photochemical activation~, such as e.g. aziridins or phenylazide
which are converted by a photochemical activation into reactive
carbene or nitrene.
ad b) such a concentrating property can be conferred to a
carrier layer in various ways, e.g. by ionic ~roups by means of
which receptor molecules of opposite total charge are
concentrated on the basis of a Couloumb interaction with the
ionic groups of the carrier layer in an approximately non-directed
manner,
or by molecular associations which confer a hydrophobic
character to a carrier layer such that receptor molecules havin~
hydrophobic domains are concentrated via these domains at these
surfaces in a directed manner,
or by metal complexes having non-saturated coordination
spheres which are saturated by particular functionai groups or
domains of a receptor molecule and therPby a directed concen-
tra~ion on the carrier layer is effected,
or by molecules having the capacity of a molecular
recognition ~e.g. protein A, protein G, str~ptavklin, antibodies
against particular epitopes of a recognition molecule etc), which
have a high affinity to particular ~omains of a recognition mole-
cule and which concentrate a receptor molecule as a result of
this affinity to/in tha carrier layer in a directed manner.
i ..... .. . ~ . ~ , .
8 ~
It has surprisingly been found that in the coating in
accordance with the invention of planar ~iO2 wave guides there ~-
are obtained organic layers which have an analogous construction
and a comparable arrangement to s)rganic monolayers which are
applied using the compounds of formula ll to materials such as
silicone, silicone oxide and aluminium oxide (Advanced Materials
2 (1990) 573; Langmuir 8 (1992) 947) or to organic monolayers
which can be applied using functionalized thioalkanes to gold
surfaces (Langmuir 6 (1990) 87). This class of organic layers is
denoted by the term "self assembled monolayer" in the technical
literature. Under the described conditions there are obtained
organic monolayers on the TiO2 surfaces in which the oompounds
of formula ll are covalently bonded with ~he TiO~ via the terminal
Si atom and the spaeer group Y having the reactive group X stands
clear of the surface. The compounds of formula ll bond to the
TiO2 layers via the reactive group (R1R2R3)-Si- in that at least
one of the groups (R1, R2, R3) reacts with free hydroxyl groups on
the surFaees. In order to obtain a dense packing and resistant
layers, it is accordingly important to pre-treat the TiO2 surface
in a suitable manner SQ that a high density of hydroxyl functions
re~ults on the surface. The layers obtained on the TiO2 surfaces
with the compounds of formula ll have been found to be stable in
organic solven~s and in aqueous m~dia with a 9>pH>1. In basic
aqueous media pH>10 their stability deereases, namely with
decreasing number of reactive groups R at the terminal Si atom of
the compounds of formula ll.
With referenoe to their adhesion to the TiO2 surface, these
monoiayers are also stable towards reduction a~ents such as BH3
or LiAlH4 and, respectively, towards oxidation agents such as
aqueous permanganate solution or perchlorate solution.
These monolayers of organic compounds covalently bonded
h the surface of the TiO2 can be used directly as two
dimensional carrier layers when the reactive groups are those
which react with functional groups on receptor molecules (e.g.
acid halide, epoxide, aldehyde, hydrazide). Otherwise, thess
groups must be modified in a suitable manner (e.g. olefin into
, ~, - ~ , . . .
12 21g~7~
carboxylic acid, halide, epoxide, or halide into azide and further
into amine, or thiocyanate into thiol~ and/or activated (~.~. car-
boxylic acid into activated ester). Such procedures for the
rnodification or activation of functional groups on surfaces are
known from the literature (e.g. IEEE Transactions on Biomedical
En~incering 35 (~988), 466; Analytica Chimica Acta 229 (1990)
169; Analytica Chimica Acta 228 (1990) 107; Biosensors and
Bioelectronics 7 (1991) 207, Langmuir 6 (1990), 1621). A further
possibility of modification comprises adding heterobifunctional
photoreagents (e.g. compounds having phenylazido or aziridino
groups as photoreactive groups and an activated carboxylic acid
as chemically reactive ~roups) via the ch~mically reactive groups
on the functional gruups X to give a surface to which, during
exposure to light, reccptor molacules can be immobilized on the
surFace (Journal of Photochemistry and Photobiology, B:Biology 7
(1 990) 277).
A variant of the monofunctional carrier layer described
above, which leads to recognizing elements of type A, comprises
producing a surface having different functional groups using
compounds of formula ll so that a multifunctional organic mono-
layer is obtained. Such a mixed layer can be prodllced by using a
mixture of compounds of formula ll for the coating of the TiO2
surface or by a subsequent chemical modification in which the
chemically reactive groups X on the surface are only transformed
partially into a group X'. Such a mixed layer can then carry
chemically reactive (such as e.g. activated carboxylic acid) or
activatable (such as e.g. a7iridine or phenyla~ide) groups and at
the same time also functional groups, molecules and/or molecular
associations which permit the aforementioned concentration of
receptor molecules for the immobilization.
For example, an organic monolayer which carries hydroxyl
groups as functional groups X can be produced in a first coating
step. This is carried out in a simple manner by treating the TiO2
surface with a compound of formula ll which carries a double
bond as a functionai group X. This double bond is converted into a
diol group by treating this surface with peracids (e.g. chloroper- -
- - , ,
,
; ,' , , ,
,,. ~ -
13 2 ~
benzoic acid and subsequent treatment with acidic-aqueous
solutions of pH = 3). Upon treatment of this surface with
compounds of formula ll in which the spacer grcup is a perfluoro-
alkane chain and ~he functional group is a fluorine atom there
results a surface which bonds receptor molecules preferably over
hydrophobic domains. The thereby resulting recognizing element
has the characteristic properties of an element of type A2
(directed immobilization of receptor molecules in a two dimen-
sional arrangement). It has e.g. surprisingly been found that
membrane proteins concentrated and anchored on such surfaces
preferably bond to this surface via the transmembrane part and
thus preserve approximately 100% of their natural activity.
An alternative modification of such organic monolayers
comprises th~ addition of biomolecules which recognize and bond
specific, non-analyte binding domains of the receptor molecules
to be immobilized. A monolayer of protein A can be immobilized
e.g. on an organic monoiayer via activated carboxylic aoid groups
For the concentration of antibodies under suitable buffer
conditions the antibodies ar~ adsorbed via their F~ part on protein
A. By unspecific coabsorption of molecules, which carry photo-
ac~ivatable groups (e.g. BSA modified with phenylazido
compounds), these adsorbed antibodies can subsequently b~
anchored to the surface in a light-inducecl reaction and there
again results a recognizing element of typ2 A2.
In a preferred modification of such bifunctional carrier
layers, which leads to recognizing elements of type A1, the
r~activ~ groups of the organic monolayer are used to anchor low-
molecular (MW > 1500) hydrophilic molecules to this surface.
Preferrad representatives of these short-chain, hydrophilic
compounds are low-molecular polymers such as oligovinyl
alcohols, oligoacrylic acids and oligoacrylic acid derivatives,
oiigoethylene ~Iycols and low-molecular natural compounds such
as monosaccharides, oligosaccharides having 2-7 sugar units, or
carboxyglycosides or aminoglycosides (such as e.g. fradiomycin,
kanarnycin, streptomycin, xylostasin, butirosin, chitosan etc). By ~:
an addition of such low-molecular compnunds there are obtained
21 !~7~3
14
carrier layers which still retain their two dimensional character,
but simultaneously have a high degree of biocompatibility. It has
surprisingly been found that such low-molecular, hydrophilic
compounds are outstandingly suitable for the ccnstruction of two
dimensional carrier layers to which receptor molecules can be
concentrated and anchored from dilute solutions when these
compounds are equipped with ionic groups (e.g. carboxylate) and
with reactive ~roups X' (e.g. chemicaliy reactive groups such as
aldehyde, epoxide, activated ester etc or photochemically
reactive groups such as aziridine or phenylazide). In this manner
there is ~hen obtained e.g. a recogni~ing element olF type A1.
A suitable modification of such hydrophilic surfaces occurs
especially readily when the aforementioned amino~lycosides are
used. These aminoglycosides, most of which have antibiotic
activity, are generally synthesized from 1-5 sugar units which
are derivatized with one or more amino groups. One of these
aminc groups can be used to immobilize the aminoglycoside on the
organic monolayer described above when this monolayer carries
suitable reactive groups ~e.g. carboxylic acid halide, activated
carboxylic acid, aldehyde). The remaining amino groups can be
modified in such a manner that the carrier layer subsequently has
the mentioned bifunctionality (concentration, bonding). In a
simple procedure, succinic anhydride can be added e.g. to the
amino groups. Some of tha thereby resulting free carboxylic acid
functions can be modified for the chemical bonding by conversion
into the N-hydroxysuccinimide derivative (alternatively, photo-
chemically active groups can also be added), while the non-
modified free carboxylic acid functions can be used in the form of
a carboxylate in order to corlcentrate receptor molecules haYing a
positive total charge on the carrier layer. It has surprisingly
been found that the receptor molecules (recognizing element of
type A1) irnmobilized on such a carrier layer in a non-directed
mann~r g~nerally exhibit a very much higher binding activity for
the analyte molecule than receptor rnolecules which are
immobilized directly on a carrier layer prepared with a compound
of formula ll.
. .
,
15 ~a~7~3~
Low-molecular, hydrophilic compounds such as mono^ and
oligo~accharides, which in their na~ive form carry neither
reactive groups for the directed immobilization nor functional
groups, molecules or molecular associations for the concen-
tration, can be modified in a suitable manner, with such a modifi-
cation being generally effected more efficiently when it is not
camed out on the compound already anchored to the surface.
A typical procedure will be demonstrated using dextran
1500 (7glucose sub-units~ by way of example. This dextran can
be ~ctivated in solution (e.~. DMSO) by converting the hydroxyl
groups into hydroxylate groups effective for a methylcarboxyl-
ation. Since this activation takes place using NaH in a very basic
medium, it oan not be carried out directly on ~he solid phase
having regard to the aforementioned instability of the organic
monolayers on TiO2. Tho externally methylcarboxylated dextran
1500 can, however, be anchored very simply to an organic mono-
layer which carries ~ 9. epoxide groups, there being obtained two
dimensional carrier la3~ers having a high concentration of
oarboxyl groups which can be used in an analogous manner for the
construction of recognizing elernents of type A1 such as the
organic monolayers modified with amino~lycosides and succinic
acid.
Such carrier layers constructed using these low-molecular,
hydrophilic compounds oan also be modified further to carrier
layers in order to achieve a concentration in a directed manner
(recognizing elemen~s of type A2).
For example, the carboxyl groups introduced via amino-
glycosides or oligosaccharides can be used to anchor bio-
molecules (e.g. protein A, protein G, streptavidine etc) to the
surfaces, which have a hi~h affinity to one domain of the receptor
moleoule which is to be immobilized subsequently.
In addi~ion to use as two dimensional carrier layers for
receptor molecules, these aforementioned organic layers are also
16 2~q~3
suitable as a basis for the construction of three dimensional
carrier layers which lead to the recngnizing elements of type B.
The convsrsion of the two dimensional carrier layer into a
three dimensional carrier layer is carried out by the addition of
long-chain synthetic or natural hydrophilic polymers which are
capable of forming a porous, three dimensienal matrix in the
nature of a hydrogel of the surface of the transducer. Typical
representatives of suitable natural polymers are e.g. polysac-
charides such as dextran, a~arose, alginic acid, starch, cellulose
or derivatives of such polysaccharides such as methylcar-
boxylated derivatives or synthetic, hydrophiiic polymers such as
polyvinyl alcohol, polyacrylic acid, polyethylene glycol.
It is important that these long-chain polymers such as the
low-molacular, hydrophilic compounds are a3so provided with
reactive groups X which permit an anchoring of receptor mole-
cules to thes0 three dimensional carrier layers. In a preferred
embodiment this carrier layer carries, moreover, ionic groups or
directing molecules and/or molecular associations which permit
the concentration of receptor molecules in a non-directed (type
IB1) or directed (type B2) manner.
For example, dextran 500000 can be methylcarboxyiated and
subsequently anchored to an organic monolayer which is modified
with epoxide groups. There is thus obtained an about 100 nm
thick, porous carrier layer havin~ carboxylic acid groups of which
sokme, as carboxylate, permit the concentration of biomolecules
having a positive total charge and the remainder, in activated
form, can be used for the subsequent covalent anchoring.
. .
If desired, the biologically recognizing element in accord-
ance with the invention can be applied to the optical TiO2 wave
guide via a thin intermediate layer (d ~ 20 nm) of SiO2 or A12O3.
The following Examples illustrate the invention in more
detail:
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1 7
1. Application of densely packed, organic monolayers to
a TiO2 wave guide.
1.1. Formation of an organic monolayer on TiO2 surfaces by
treatment ~ith Cl(CH3)2Si-(CH2)11-COCI in a CVD proc~ss:
For the application of the compounds of formula li from the
gas phase, a reaction vesssl is prepared which can operate at a
pressure of 10-5 mbar and in which the sample to be coated can
bs brought to temperaturcs between 30-100C. This reaction
vessel is attached to an evacuatable, heatable supply vessel in
which the compound used for the coating can be placed ~if
desired, the apparatus can also be equipped with several such
supply vessels).
For the coating, the substrate is introduced into the ~
reaction vessel. After introducing ~he silane Cl(CH3)2Si-~CH2)1 1 -
COCI into the supply vessel, the supply v~ssel and reaction
oharnber are brought to an operating pressure of 10-5 mbar. The
sample to be coat~d is heated to 100C. After heating the reagen~ :
in the supply vessel to 50C,i the surface is treated for 1 h. with :~ -
reag0nt from the gas phase. Subsequently, the reagent flow is ~:
stopped and the samplc is treated in a vacuum at 150C for
15 min.
(The detection of an organic monolayer on the surface is
carried out by XPS and contact angle measurements).
1.2. Formation of an organic monolayer on TiO2 surfaces by
treatment with Cl(CH3)2Si-(CH~6~ H=CH2 in a CVD process:
The procedure described under 1.1. is used for the coating of
the surface.
1.3. Formation of an organic rnonolay~r on TiO2 surfaces by
treatment with (Cl 13O)3Si-(CH2)3-NH2 in a CVD process: :~
2 ~ 7 3 ~
1 8
The coating takes place using the corresponding compound
according to the procedure described under 1.1.
1.4. Formation of an organic monolayer on TiO2 by treat-
rnent with a solution of Cl(CH3)2Si-(CH2)11-COCI.
A 0.5% (vlv) solution of C:l(CH3)2Si-(CH2)11-COCI in CCI4 is
placed in a reaction vessel under an inert gas atmosphere. The
surface to be coated is brought into contact with this solution for
25 min. under an inert gas. After this treatment, the surface is
washed with CC4, ethanol and water.
1.5. Formation of an organic monolay~r on TiO2 by treat-
ment with a solution of Cl3Si-(CH2)6-CH=Ctl2: ~ :
A 0.5% solution (v/v) of C:13Si-(CH2)6-CH=CH2 in hexadecane -:
is prepared in a reaction vessel under an inert ~as atmosphere.
The surface to be coated is brought into contact with this
solution for 5 min. und~r an inert gas. After this treatment, the
surface is washed with hexadecane, hexane and ethanol.
1.6. Formation of an organic monolayer on TiO2 by treat-
m~nt with a solution of Cl(C313)2Si-(CH2)7-(CH2-O-CH2)2-CH2-O-
CH3:
The coating is carried out accordin~ to the procedure
described under 1.4.
1.7. Formation of an organic monolayer on TiO2 by treating
the surface with a solution of Cl3Si-(CH2)gBr:
The co~ting is carri~d out according to the procedure
describecl under 1.4.
2. Modification of functional ~roups on organic mono~
layers on TiO2 prepared according to the procedure described
under 1.
.:
19
2.1. Conversion of olefins into epoxides:
A TiO2 surface treated according to paragraph 1.2. or 1.5. is
contacted at 4C for 24 h. with a solution of 3-ehloroperbenzoic
acid ~0.06M) in die~hyl ether (abs.). The surface is subsequently
washed with diethyi ether, ethanol and water (4C).
2.2. Conversion of epoxides into diols:
The surface having epoxide functions prepared und~r 2.1. is
treated with an aquaous solution of pH Y 2.5 at 80C for 1 h. and .::
subsequently washed with H2O.
2.3. Conversion of olefins into carboxylic acids: ::
The TiO2 surfaces treated according to paragraph 1.2. or 1.5.
are brought into contact with an a~ueous solution of potassium
permanganate (0.1M) and NalC)4 (0.1M) for 20 min. Subsequently,
the surface is washed with 0.1M aqueous hlaHSO3, with ethanol
and wat~r.
2.4. Conversion of halides into azides: :
A TiO2 surface treated according to paragraph 1.7. is
brought into contaet with a solution of NaN3 (6 mg/mi) in DMF
(abs.) for 15 h. and subsequently washed with DMF and water.
2.5. Conversion of azides into amines:
The TiC:)2 surFace havin~ N3 groups prepared according to 2.4.
is brou~ht into contact with a solution of SnCI2 in absolute
methanol for 4 h. The surface is subsequently washed with
methanol and water. ::
2.6. Activatioll of carboxylic acids with ethyl chloro~
formate and N-hydroxysuccinimide: ~:
' " ', '.
~o ~ 5
The surface having COOH groups prepared according to para-
graph 2.3. is brought into contact with a 2.5~/o solutiorl (v/v) of
ethyl chloroformate in CH2CI2/pyridine (100/2.5) for 1 h.
Subsequently, the surface is contacted with a solution of N-
hydroxysuccinimide (0.5M) in pyridine. There are thus obtained N-
hydroxysuccinimide-activated carboxylic acid functions to which
molecules having amino groups can be added directly.
3. Modification of organic monolayers prepared according
to th~ procedure described under 2. with low-molecular, hydro-
philic compounds.
3.1. Addition of fradiomycin to a TiO2 surface provided
with an organic monolayer:
:
The surface having activated carboxylic acid functions
prepared according to paragraph 2.6. is brought into contact wi~h
a solution of fradiomycin (20 mM in PBS; pH = 7.2) for 1 h. It is
subsequently washed with H2
3.2. In situ modification of immobilized fradiomycin:
a) In~rodu~tion of carboxylic acid functions: The amino
groups of the fradiomycin immobilized on the surface according
to paragraph 2.1. are quantitatively reacted by contact with a
solution (1% w/w) of succinic anhydride in pyridine. There is
thus produoed a hydrophilic carrier layer which has a high density
of available acid functions.
b) Introduction of activated disulphid2 bonds: 3-(2-
pyridinyl)dithispropionate can be coupled to the amino ~roups of
tha fradiomycin irnmobilized on the surface according to para-
graph 3.1 by contact with an ethanolio solution (2 mM) of N-
succinimidyi 3-(2-pyridinyl)dithiopropionate. The dithiopyridinyl
group can bc used for the planned immobilization of molecules
(e.g. Fab' fragments of IgG molecules) on the carrier layer via free
~hio functions. The unreacted amino groups in this procedure can
". .. . . . .. . . .
21 ~ l~8~
be used according to the procedure described under a) in order to
immobilize carboxylic acid functions on the surface. ~-
c) Introduction of photoactivatable phenylazido ~roups:
ô-(4'-azido-2'-nitrophenylamino)hexanoate can be immobilized on
the amino groups of the fradiomycin immobilized on the surface
according to paragraph 3.1. by contact with an aqueous (10%
DMSO) solution (2 mM) of N-succinimidyl 6-(4'-azido-2'-
nitrophenylamino)hexanoate. Other reactive amino functions can
be used according ~o the procedure described under a) in order
simultaneously to modify the carrier layer with carboxylic acid
groups.
4. Construction of a porous carrier layer on the organic
monolayers for the production of three dimensional recognizing
elements:
4.1. Methylcarboxylation of dextran 500000:
75 rnl of dry DMSO are added to 7.5 9 of NaH. The concen- -
tration of thus~obtained DMSO anions is determined by titration.
0.2equivalent (based on glucose sub-units) of dextran 500000 is
dissolved in 150 ml of dry DMSO and the solution is mixed with
the DMSO anions. The mixture is stirred at room temperature for
4 h. and added to a two-fold excess (bas~d an glucose sub-units)
of bromoacetic acid. The solution is stirred for 1~ h.
Subsequently, th~ dextran is precipitated with acetone, filtered
off, dissolved in 20 ml of water and dialyzed against water for
24 h. Af~er Iyophilization, the amount of methylcarboxylated
glucose sub-units is determined by titration (about 1 carboxyl
grouplS glucos~ sub-units).
~ æ Immobilization of methylcarboxylated dextran 500000
on organic monolayers~
The immobilization of methylcarboxylated dextran starts
from organic monolayers which are constructed accordin~ to
paragraph 1.7 on TiO2 surfaces and which have been modified
--- Z 2 ~ 3
according to paragraphs ~.4. and 2.5. The amino groups of these
or~anic monolay~rs are reacted with ~pichlorohydrin solution
(1 ml of epichlorohydrin in 10 ml of NaC)H (0.4~A)/10 ml of
diglyme). After wa~hing with ethanol and water, it is treated
with a solution of methylcarboxyla~ed dextran (0.3 9 of dextran
in aqueous NaOH solution (0.01M NaOH~) for 24 h. The surface is
subsequently washed well with watcr at 50C:.
5. Preparation of recognizing ~lements of type A1, A2,
B1 and B2 on the basis of the carrier layers ref~rred to in parts
1 -4 .
5.1. Preparation of reeo~nizing elements of type A1 having
immobiliz~d IFNa (interferon a) as the receptor molecule:
For the immobilization of IFNoc, a carrier layer is prepared
which has been modified with succinic anhydrid~ ~coating of TiO2 :
according to 1.1., addition of fradiomycin a~cording to 3.1., modi-
fication of the fradiomycin according to 3.2.a.). This surface is
treated for 5 min. with an aqueous solution of 1~1-(3-dimethyl-
aminopropyl~-N'-ethylcarbodiimide (~Om~/ml) and N-hydroxy-
succinimide (3 mg/ml). After washing with acet~te buffar
(0.01M; pH = 5.5), it is incubated with a solution of inter~eron
(0.9 ~lg/ml) for 20 min. The surfacs concentration of IFNc~
achieved using this procedure is 0.36 ng/mm2 after washing with
acetate buffer and 0.01M H(~
5.2. Preparation of a recognizing elem~nt on TiO2 accord-
ing to type A2 with Gpllb-llla (glycoprotein llb-llla) as the
receptor molecule: :~
~: .
The immobilization of the Gpllb-llla s~arts from a TiO2
layer which is modified with a diol-containing surfa~e :
(preparation according to paragraph 1.5., 2.1. and 2.2.~. This u: ~:
surface is treated with a 0.5% solution of l H, 1 H,2H,2H-perfluoro- :
octyldimethylchlorosilane in CC!4. The thus-obtained strongly
hydrophobic surFace is brought into contact with an aqueous
solution of Gplib-llla (0.~ ~/ml) ~0.1M Tris; pH = 7.2) for
23 2~7~
20 min. The su~ace concentration of Gpllb-illa achieved using
this procedure is 1.5 n3/mm2 after washing with buff2r solution.
5.3. Preparation of a recognizing element of TiO2 accord-
ing to type B1 havin~ TnFoc (tumor necrosis factor a) as the
receptor molecule:
A TiO2 surface modified with methylcarboxyiated dextran
according to paragraph 1.5., 4.2. is us0d for the immobilizatian of
TNFa. This surface is tr0ated for 5 min. with an aqueous solution
of N-~3-dimethylaminopropyl)~ ethylcarbodiimide (20mg/ml) :
and N-hydroxysuccinimida (3 mg/ml). After washing with acetate
buffer (O.OlM, pH = 5.5), it is incubated with a solution of TNFa in
acetate buffer 1 ~,lg/ml for 10 min. Accordin~ to this procedure .
and after washing with acetat~ buffQr, PBS buffsr (O.lN: pH - 7.2)
and ethanolamine (1M, pH = 8.5~, about 2 n~/mm2 of TNFa are
covalently immobilized.
5.4. Preparation of a recognizing element on TiO2
according to type E~2 with antibodies as receptor molecules:
Tha directed immobilization of the antibodies is effected on
a dextran carrier layer which has bcen preparsd according to
paragraph 1.5., 4.2. For the directed imrnobilizatiorl, frse
aldehyde functions are produced on the carbohydrate residues of
the antibodies by oxidation according to known procedur~s. The
dextran layer on the TiO2 wave ~uide is treated for 5 min. with
an aqueous solution of N-(3-dimethylarninopropyl)-N'-ethylcarbo-
diimida (20mg/ml) and N-hydroxysuccinimide (3 mg/ml). After
washing with water, the activated carboxylic aeid functions o
the surfac~ are eonverted into hydrazides by contact with an
aqueous solution of hydrazine rncnohydrochloride (1 mM). This
surface is brought into contact with a solution of the
oxidatively-treated antibodies (1 llg/ml in acetate buffer (0.01M,
pH = 5.5)) for 20 min. The surface is washed with PBS ~û.1M; pHI
= 7.2) and an aqueous solution of ethanolamine (1M; pH = 8.5).
This procedure leads to a directed immobilization of about
5 ng/mm2 of antibodies on the dextran carrier layer.
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