Note: Descriptions are shown in the official language in which they were submitted.
~13~9!~ 6
RAN 4090/24$
The present invention is concerned with novel silanes and
their application for the silanization of dielectric materials and
s for anchoring biologically active compounds. Dielectric materials
equipped with silane layers are used as solid phases in analytic
methods (Methods in Enzymology 44 ( 1976), 134). The new silanes
are preferably used for coating signal transformers, e.g. an
optical waveguide in sensor analysis.
w
Optical biosensors consist, for example, of a recognition
element and an optical signal transformer (Trends in Biotechnol.
2 (1984), 59); (Opt. L_ett. 9 (1984) 137); (Sensors and Actuators A,
25 (1990) 185); (Sensors and Actuators B, 6 (1992) 122); (Proc.
~ Biosensors 92, extended abstracts 1992, pp. 339 and 347). The
task of the recognition element consists in selectively binding or
converting the analyte. This task is accomplished by immobilizing
biological recognition molecules (e.g., antibodies, antigens,
ligands, ssDNA) on the surface of a signal transformer. In general,
~o for this purpose the surface of the signal transformer (e.g. the
surface of a dielectric waveguide) is provided with an organic
carrier layer to which the recognition molecules are bound cova-
lently. Organic carrier layers with an ordered, compact molecule
arrangement, as described in European Patent Application EPA-
~ 596421, have proved particularly well-adapted in this context.
These recognition molecules can be used both in their naturally
occurring and isolated form, as well as in their chemically or
biologically produced form.
ao These types of biosensors can be employed to determine
analyte concentrations, e.g. in human and animal diagnostics, in
environmental analyses and in food analyses or in the field of
biochemical research for quantifying intermolecular interactions
of biologically active substances (e.g. antibody-antigen inter-
~ actions, receptor-ligand interactions, DNA-protein interactions,
etc. ).
Hu/So 23.8.94
2~339~6
2
For example, the organic carrier layer is constructed by
treating the waveguide surface with silanes of general formula I.
(R~R2R3)Si-Y-X
- Si(R~ R2R3) represents a coupling group to the wave-
guiding layer. R~~ R2 and R3 signify alkyl, alkoxy or halogen,
with at least one being halogen or alkoxy.
- Y is a spacer group and, as such, can be, for example, an
alkylene chain, a fluoroalkylene chain or an oligooxyalkylene
chain.
- X is a chemically reactive group by means of which bio-
logical recognition molecules can be bound' to the organic
carrier layer. Known reactive groups are, for example,
carboxylic acid halides (-COHaI), olefins (-CH=CH2), nitrites
(-CN), thiocyanates (-SCN), thioacetates (-SCOCH3).
The dielectric waveguides coated with aforementioned
silanes are hydrolysis-unstable when the reactive group X is e.g. a
carboxylic acid halide. When, for example, the reactive group X is
an olefin, then the olefin has to be modified and activated in a
follow-up step for the subsequent immobilization of biological
recognition molecules. This follow-up treatment leads to
hydrolysis-stable organic carrier layers with reactive groups.
Such subsequently formed reactive groups are, for example,
epoxides, N-hydroxysuccinimide-activated carboxylic acids,
ao thiols and the like. In general, it is very expensive to produce
such reactive groups quantitatively at a surface.
Accordingly, the object of invention is to provide silanes
which can be bound to dielectric materials, with the silanes
as already being provided with hydrolysis-stable, reactive X groups
which permit the direct immobilization of an organic or bio-
logical recognition molecule on the surface without an additional
activation step.
CA 02133946 2001-11-06
3
This object is achieved by silanes of general formula I
(R~R.2R3)Si-Y-X I
wherein R~, R2 and R3 signify halogen, Y signifies an alkylene chain
[-CH2-(CH2)n-CH2-], a fluoroalkylene chain [-CHz-(CF2)~-CH2-],
[-CH2,-(CF2)~-CF2-] with n = 1-20 or an oligooxyalkylene chain
-[(CH2)~'-O-(CH2)n"]m- with n', n"= 2-6 and m = 2-6 and X signifies
0
an epoxide or an anhydride of a dicarboxylic acid with 4-5 C atoms.
The Si(R~ RZR3) group represents a coupling group to the
wave-guiding layer. In the scope of the present invention the term
"halogen" signifies chlorine or bromine. Chlorine is preferred. By
~o using trichlorosilanes as organic carrier layers on dielectric
waveguides it is possible to produce very stable, compact and
ordered sensor surfaces with good optical properties.
The term "alkyl" signifies in the scope of the present
25 invention straight or branched alkyl chains with 1-6 C atoms such
as, for example, methyl, ethyl, n-propyl, isopropyl, butyl, tert.-
butyl, pentyl, hexyl and the like.
The term "alkoxy" signifies in the scope of the present
3o invention groups in which the alkyl group has the foregoing
significance.
The term "aryl" signifies an unsubstituted or substituted
phenyl or naphthyl group or the like. An alkyl group is, for
a~ example, a suitable substituent.
The X group is a hydrolysis-stable, reactive group which is
used for the anchoring of additional organic molecules or for the
CA 02133946 2001-11-06
4
anchoring of biomolecules after application of the silane to the
surface.
The novel silanes according to the invention have the
s following advantages:
The reactive groups X of the silanes are hydrolysis-stable,
but sufficiently reactive for the immobilization of an organic or
biological recognition molecule to take place without further
w activation of the silane layer.
The silanes are suitable for the direct coating of dielectric
materials, in particular of dielectric waveguides which are
preferably made of Zr02, Hf02, Ta205, Si02, A1203, or Ti02.
The silanes can be applied to the surface of dielectric
materials not only from solution, but also from the gas phase.
The hydrolysis stability of the reactive groups gives rise to
2o better stability during storage of silane layers and permits their
direct application in an aqueous medium.
Short-chain silanes having, for example, an alkylene chain
[-CH2-(CH2)~-CH2-] with n = 1-3 are also suitable for silaniz-
~s ations, but long-chain analogues are preferred for the construct-
ion of oriented, compact layers as are used, for example, in bio-
sensors.
A further object of the present invention is to provide
ao dielectric waveguides provided with oriented, compact organic
carrier layers.
This object is achieved by a dielectric waveguide to whose
surface is bound an organic carrier layer, which is constructed
~ from silanes as described above and which consists of sub-
units of the general formula
CA 02133946 2001-11-06
Si-Y-X II
wherein Y signifies an alkylene chain [-CHz-(CH2)n-CH2-], a
fluoroalkylene chain [-CHZ-(CF2)~,-CH2-], [-CH2-(CFZ)n-CF2-]
with n = 6-20 or an oligooxyalkylene chain -[(CH2)n~-0-
(CH2)n"]m- with n', n'" = 2-6 and m = 2-6 and X is an epoxide,
an anhydride of a dic:arboxylic acid with 4-5 C atoms.
m
The silane layers can be made up of pure alkylene chains,
fluoroalkylene chains or oiigooxyalkylene chains or a combination
of alkylene chains and fluoroalkylene chains or a combination of
alkylene chains and oligooxyalkylene chains.
Due to the long spacer group Y, self-aligning mono-layers
are formed during silanization. These layers are compact, exhibit
a high degree of order with respect to reactive groups X and they
do not affect the optical properties of the waveguide.
m
A further object of the invention is concerned with the
application of the novel silanes of formula I according to the
invention for coating dielectric materials, in particular
dielectric waveguides which are preferably made of Zr02, Hf02,
~~ Ta205 or Ti02. If necessary, another thin layer (d < 20 nm) of a
silanizable material (Si02, A1203 etc.) can first be applied to the
actual waveguide. This supplementary organic layer is used for
coupling additional organic molecules or for coupling biologically
active molecules.
m
After coating dielectric waveguides ~~~rith the novel silanes
according to the invention additional molecules can be coupled to
reactive group X, as described in the European Patent Application
EPA-596421, so as to form an ordered layer consisting of sub-
~ units according to general formula Ila
X133946
6
Si-Y-Z IIa
with the Si atom being bound directly to the solid phase, e.g. of a
dielectric waveguide.
w
In this case Z signifies:
- hydroxyl, carboxyl, amine, methyl, alkyl, fluoroalkyl
groups;
- derivatives of hydrophilic, short-chain molecules such as
oligovinyl alcohols, oligoacrylic acids, oligoethylene
glycols;
- derivatives of mono- or oligo-saccharides with 1-7 sugar
units;
- derivatives of carboxyglycosides;
~o - derivatives of aminoglycosides such as fradiomycin,
kanamycin, streptomycin, xylostasin, butirosin, chitosan;
- derivatives of hydrogel-forming groups of natural or
synthetic origin such as dextran, agarose, alginic acid,
2s starch, cellulose and derivatives of such polysaccharides,
or hydrophilic synthetic polymers such as polyvinyl
alcohols, polyacrylic acids, polyethylene glycols and
derivatives of such polymers;
30 - a recognition molecule, e.g. antibodies, antigens, ligands,
ssDNA;
- a group having the above definition of Z to which a
recognition molecule is bound.
as
~I339~6
7
The following Examples illustrate methods for the
production and application of the novel compounds.
I. Production of trichlorosilanes which carry an epoxide
s as the reactive group.
Examl to a 1
Production of 7,8-epoxy-octyl-trichlorosilane
w
0.05-0.2 g of H2PtCl6 was stirred in 20 ml of dry tetra-
hydrofuran. 5 ml (7 g) of trichlorosilane were added to the yellow
solution. 5 g of 1,7-octadiene monoxide. were carefully added
dropwise to the orange suspension obtained. Then, the mixture
was stirred for 5 hours at room temperature and subsequently for
about 15 hours at 50~C. The reaction mass was concentrated in a
water jet vacuum and distilled at 180~C. 7.1 g of a colourless oil
were obtained. The compound was characterized by elementary
analysis. This showed:
Calculated: C = 36.72; H = 5.78; CI = 40.65.
Found: C = 36.12; H = 6.28; CI = 40.19.
Example 2
Production of 9,10-epoxy-decyl-tricholorosilane
0.05-0.2 g of H2PtClg was stirred in 20 ml of dry tetra-
3o hydrofuran. 5 ml (7 g) of trichlorosilane were added to the yellow
solution. 6 g of 9,10-epoxydecene (decadiene monoxide) were
carefully added dropwise to the orange suspension obtained. Then,
the mixture was stirred for 5 hours at room temperature and
subsequently for about 1 S hours at SO~C. The reaction mass was
concentrated in a water jet vacuum and distilled at 200~C. About
8 g of a colourless oil were obtained. The compound was char-
acterized by elementary analysis. This showed:
z~~3~~s
8
Calculated: C = 41.46; H = 6.61; CI = 36.71.
Found: C = 41.92; H = 6.86; CI = 36.17.
s Cxami la a 3
Production of 13,14-epoxy-tetradecyl-tricholorosilane
0.05-0.2 g of H2PtCl6 was stirred in 20 ml of dry tetra-
w hydrofuran. 5 ml (7 g) of trichlorosilane were added to the yellow
solution. 8 g of 13,14-epoxytetradecene were carefully added
dropwise to the orange suspension obtained. Then, the mixture
was stirred for 5 hours at room temperature and subsequently for
about 15 hours at 50~C. The reaction mass was concentrated in a
~s water-jet vacuum and distilled at 230~C. About 10 g of a colour-
less oil were obtained. The compound was characterized by
elementary analysis. This showed:
Calculated: C = 48.63; H = 7.87; CI = 30.76.
~o
Found: C = 48.01; H = 7.45; CI = 31.19.
I1. Production of trichlorosilanes carrying an isothio-
cyanate as the reactive group.
8xamal,~ 4
Production of 3-(trichlorosilyl)propyl-isothiocyanate
30 0.1 g of H2PtClg was suspended in 5 ml of trichlorosilane.
ml of allyl-isothiocyanate (allyl mustard oil) were added drop-
wise to the suspension. Then, the mixture was stirred for about
hours at room temperature. The reaction mass was distilled
under a water jet vacuum at 130~C in a bulb tube. 8.6 g of a
colourless, oil were obtained. The compound was characterized by
elementary analysis. This showed:
z~33~~s
9
Calculated: C = 20.48; H = 2.58; N = S = 13.67; CI = 45.34.
Found: C = 21.01; H = 2.77; N = 6.43; S = 14.26;
CI = 44.92.
III. Production of triethoxysilanes carrying an isothio-
cyanate as the reactive group.
Exam Ip a 5
w
Production of 3-(triethoxysilyl)propyl-isothiocyanate
0.1 g of H2PtCl6 was suspended in 5 ml of triethoxysilane.
5 ml of allyl-isothiocyanate (allyl mustard oil) were added drop-
~5 wise to the suspension. Then, the mixture was stirred for about
hours at room temperature. The reaction mass was distilled in
a water jet-vacuum at 140~C in a bulb tube. 8.2 g of a colourless
oil were obtained. The compound was characterized by elementary
analysis. This showed:
~o
Calculated: C = 45.60; H = 8.04; N = 5.32; S = 12.17.
Found: C = 45.91; H = 7.71; N = 5.69; S = 12.64.
~5 IV. Production of trichlorosilanes carrying an acid anhydride
as the reactive group.
ao Production of .2-(11-trichlorosilyl-undecenyl)-succinic
anhydride
0.01 g of H2PtClg was stirred in 10 ml of dry tetrahydro-
furan. 0.3 ml (0.35 g) of trichlorosilane was added to the yellow
solution. 0.3 g of 2-(10-undecenyl)-succinic anhydride was
carefully added dropwise to the orange suspension obtained. Then,
. the mixture was stirred for 5 hours at room temperature and sub-
sequently for about 1 S hours at 50~C. The reactio~~ mass was
21~3~t~6
concentrated in a water-jet vacuum and distilled at 230~C. About
0.4 g of a colourless oil was obtained. The compound was char-
acterized by elementary analysis. This showed:
s Calculated: C = 46.46; H = 6.50; CI = 27.43.
Found: C = 47.01; H = 6.92; CI = 26.83.
V. Production of the starting materials.
w
Exam Ip a 7
Production of 2-(10-undecenyl)-succinic anhydride
(Grignard reaction)
0.1 mol of 10-undecenyl-magnesium halide was added drop-
wise at -78~C to a suspension of 30 g of malefic anhydride and
10 g of copper I iodide in 100 ml of dry tetrahydrofuran. The
reaction mass was heated to room temperature and subsequently
2o stirred for about 15 hours. The reaction mass was concentrated
and the residue was taken up in 100 ml of diethyl ether con-
taining 2% water and stirred for a further 5 hours. The resulting
suspension was filtered and the residue was washed with dry
ether. The filtrate was concentrated and distilled in a water jet
2s vacuum at 230~C in a bulb tube. 9.2 g of 2-(10-undecenyl)succinic
anhydride were obtained. The compound was characterized by
elementary analysis. This showed:
Calculated: C = 71.39; H = 9.59.
so
Found: C = 71.12; H = 9.64.
Examiole 8
Production of 2-(10-undecenyl)-succinic anhydride from 2-
(10-undecenyl)-succinic acid
X233946
11
2.7 g ofi 2-(10-undecenyl)-succinic acid were stirred with
ml of acetic anhydride at 100~C and subsequently concentrated
and distilled in a water-jet vacuum at 230~C. 2.5 g of 2-(10-
undecenyl)-succinic anhydride were obtained.
5
Exam~l~ 9
Production of 2-(10-undecenyl)-succinic acid firom methyl
2-(10-undecenyl)-succinate or ethyl 2-(10-undecenyl)-succinate
w
2.8 g of methyl 2-(10-undecenyl)-succinate or 3.0 g of ethyl
2-(10-undecenyl)-succinate were stirred with cone. sulphuric
acid for 1 hour at room temperature. 2.4 g of 2-(10-undecenyl)-
succinic anhydride were obtained after extraction with methylene
~5 chloride.
Exam Ip a 10
Production of methyl 2-(10-undecenyl)-succinate
1.76 g of methyl succinate were converted in dry tetra-
hydrofuran using 6 mmol of Li diisopropylamide (LDA) into the
enolate and then reacted with 10-undecenyl iodide. The reaction
mass was treated with 1 ml of methanol and subsequently with
25 10 ml of water and extracted 3 times with 20 ml of diethyl
ether. The organic phases were dried, concentrated and distilled
under a water jet vacuum at 200~C in a bulb tube. 534 mg of
colourless oil were obtained. The compound was characterized by
elementary analysis. This showed:
so
Calculated: C = 68.42; H = 10.13.
Found: C = 68.61; H = 9.92.
a5 Exam Ip a 1 1
Production of 10-undecenyl iodide
~~p~~~~s
12
0.27 mol of 10-undecenyl tosylate was reacted with 250 g
of sodium iodide and 4.4 g of tetrabutylammonium oxide as the
phase transfer catalyst for about 15 hours under reflux. The
reaction mass was extracted 3 times with 400 ml of hexane. The
s organic phases were combined, decolorized with sodium
bisulphite and dried with sodium sulphate. Concentration and
vacuum distillation followed. B.p. (0.3 mbar) 100~C. 72.3 g of a
colourless oil were obtained. The compound was characterized by
elementary analysis. This showed:
w
Calculated: C = 47.15; H = 7.55; I = 45.29.
Found: C = 47.42; H = 7.72; I = 45.01.
VI. Application of densely packed, organic mono-layers to
Ti02 surfaces, e.g. from the gas phase to the wave-guiding layer
of an optical signal transformer.
Exam I_p a 12
Formation of an organic mono-layer on Ti02 surfaces by
treatment with CI3Si-(CH2)~ 2-CH-CJ-12 in a CVD process (chemical
vapour deposition). 0
2s A reaction vessel, which can be operated under a pressure
of 10-5 mbar and in which the probe to be coated can be heated to
temperatures between 30-100~C, was provided for the deposition
of silanes of formula I from the gas phase. This reaction vessel
was connected to an evacuatable and heatable supply vessel in
ao which the compound used for coating can be placed (alternatively,
the apparatus can also be epuipped with several of such supply
vessels).
For the coating, the optical signal transformer was placed
as in the reaction vessel. After introducing the silane Cl3Si-
(CH2)~2- \-CH2 into the supply vessel, the supply vessel and the
0
za~~~~6
13
reaction chamber were brought to a working pressure of 10-5
mbar. The probe to be coated was heated to 100oC. After warming
the silane placed in the supply vessel to 50~C the surface was
treated for 1 hour with reagent from the gas phase. Subsequently
s the flow of reagent was stopped and the probe was subjected to
post-treatment in a vacuum at 150~C for 15 min.
The detection of organic mono-layers on surfaces is
effected by XPS (X-ray photoelectron spectroscopy) and contact
~o angle measurements).
All silanes of general formula I can be applied to a surface
from the gas phase by an analogous procedure.
VII. Application of densely packed, organic mono-layers to
Ti02 surfaces, e.g. to the wave-guiding layer of an optical signal
transformer using a (CI3Si-(CH2)~ 2-CH-CH2) solution.
y
~o A 0.5% (v/v) solution of (CI3Si-(CH2)~2-CH-CH2) in CCI4
0
was placed in a reaction vessel under an inert gas atmosphere.
The surface to be coated was brought into contact with this
solution for 25 min. under an inert gas. After this treatment the
2s surface was cleaned with CCI4, ethanol and water.
All silanes of general formula I can be applied to a surface
from the liquid phase by an analogous procedure.
