Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals – Carrier is inorganic
Reexamination Certificate
2000-11-16
2004-01-13
Geperley, Mary E. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
Carrier is inorganic
C435S006120, C435S181000, C436S823000, C530S391100, C530S402000, C530S408000, C530S409000, C530S410000
Reexamination Certificate
active
06677163
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the field of modified surfaces, more particularly to silicon surfaces modified with functionalised molecules.
BACKGROUND OF THE INVENTION
The covalent attachment of organic monolayers to semiconductor surfaces provides a route to passivation and a method to incorporate chemical and biochemical function into solid-state devices to produce bio-sensors or bio-sensor arrays.
Particularly sought after are methods for immobilising bio-molecules with specific binding functions, such as proteins or DNA, on silicon surfaces.
Several methods have been proposed for the preparation of organic layers on silicon surfaces, attached through Si—C and Si—O—C bonds. The relative susceptibility to hydrolysis of the Si—O—C bond is a disadvantage of this type of linkage for applications in which an aqueous environment is encountered.
Hydrogen terminated silicon is interesting as a substrate for immobilisation of organic molecules. Chidsey et al. demonstrated that the Si(111)H surface formed by etching with fluoride may be hydrosilylated by immersion in a neat alkene followed by irradiation
1
. Similar work is disclosed in U.S. Pat. No. 5,429,708 (to Linford et al., issued Jul. 4, 1995). The layers formed are extremely durable.
Bateman et al. have reacted hydrogen-terminated porous silicon surfaces thermally with 1-octene, 1-octyne, 1-undecene and vinyl ferrocene, by refluxing in toluene at 110-180° C. The surface-attached ferrocene moiety may be observed by cyclic voltammetry
2
. Boukherroub et al. use a modification of the method of Buriak et al. to achieve the modification of porous silicon by etching the silicon surface with ammonium fluoride to form a hydrogen-terminated Si(111) surface, followed by hydrosilylation with an alkene in the presence of a Lewis acid catalyst (AlEtCl
2
)
3,4
.
Sieval et al. report the thermal reaction of alkenes terminated with ester groups, with an Si(100) hydrogen terminated surface
5
. The ester groups can be hydrolysed to release a carboxylic acid-modified surface, or reduced with LiAlH
4
to produce an alcohol-modified surface. Re-esterification of either of these surfaces by refluxing with an alcohol or carboxylic acid, respectively, in the presence of acid catalyst is possible. The required conditions are unfortunately too harsh to be compatible with most biological molecules.
Hydrogen-terminated Si(100) and Si(111) modified through Si—O linkages have been reported by Zhu et al. A silicon substrate is etched with NH
4
F, to produced a hydrogen-terminated surface. Exposure of the surface to gaseous chlorine results in a Cl-capped surface, which is subsequently reacted with dodecanol or octadecanol to produce C
12
and C
18
chains, respectively, immobilised on the Si surface by Si—O bonds
6
. Zhu et al. has reported a similar procedure, wherein a Cl-capped Si(100) surface is exposed to amine vapour, to produce amines attached to the silicon surface via two N—Si bonds
7
. They have reported the use of an analogous procedure to attach molecules to a clean Si(100) surface and a porous silicon surface via Si—N bonds
8
.
Lewis et al. formed Si(111)—C linkages using a two-step method, whereby an Si(111) surface is first chlorinated, and then reacted with an alkyl Grignard or alkyl lithium reagent
9
. Boukherroub et al. have demonstrated that similar modification can be achieved by direct reaction of alkyl magnesium bromides with an Si(111) surface
10
.
The photochemical hydrosilylation of aldehydes with an Si(111)—H surface, to form an Si(111)—OCH
2
R surface has been reported by Effenberger et al
11
.
Sailor et al. disclose modification of porous silicon with BSA or protein A via a complicated 8-step method. The linkage to the silicon surface is through an Si—O—Si linker on the oxidised (i.e. SiO
2
) surface. Binding of IgG to surface bound protein A is measured by observing the shift in effective optical thickness using interferometry
12
.
Strother et al. report the modification of Si(111) with non-covalently attached DNA. A hydrogen-terminated Si(111) surface is reacted with an &ohgr;-undecylenic acid methyl or trifluoroethyl ester by UV irradiation of a thin film of the ester applied to the surface. The ester functionality is hydrolysed by treatment with potassium t-butoxide in DMSO, to yield a carboxylic acid-modified surface. Polylysine is added, becoming immobilised on the surface through ionic interactions between the polylysine amino groups and the surface carboxylate groups. Thiol-modified DNA is then linked to the polylysine via the bi-functional linker sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate
13
.
There remains to be found a simple, versatile method for durable covalent attachment of organic molecules to hydrogen-terminated silicon surfaces, under conditions that are compatible with bio-molecules.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a method for immobilising molecules on a hydrogen-terminated silicon surface.
It is another object of the invention to provide a silicon surface with an immobilised layer which layer is suitable to be further reacted to attach a bio-molecule.
It is a further object of the invention to provide a silicon surface modified with a bio-molecule.
In a first aspect the invention provides a method for immobilising a desired molecule on a silicon substrate, the method comprising the steps:
(A) providing an Si—H surface on the silicon substrate; and
(B) attaching the desired molecule to the Si—H surface via a covalent bond.
In a second aspect, the invention provides a method for providing a coupling group on a silicon substrate, the method comprising the steps:
(A) providing an Si—H surface on the silicon substrate;
(B) reacting the Si—H surface with a linker-molecule possessing at least one anchor functionality capable of reacting with the Si—H surface to form an Si—C or Si—O linkage, and further possessing at least one coupling group and/or protected coupling group which does not react with the Si—H surface; and
(C) removing unreacted linker-molecule.
In a third aspect, the invention provides a method for immobilising a desired molecule on a silicon substrate, the method comprising the steps:
(A) providing an Si—H surface on the silicon substrate;
(B) reacting the Si—H surface with a linker-molecule possessing at least one anchor functionality capable of reacting with the Si—H surface to form an Si—C or Si—O linkage, and further possessing at least one coupling group and/or protected coupling group which does not react with the Si—H surface;
(C) removing unreacted linker-molecule;
(D) if a protected coupling group is present, deprotecting the protected coupling group; and
(E) reacting the coupling group with the desired molecule.
In a fourth aspect, the invention provides a silicon substrate bearing immobilised coupling groups attached to the silicon substrate by Si—C bonds or Si—O bonds.
In a fifth aspect, the invention provides a silicon substrate bearing a bio-molecule attached to the silicon substrate covalently via Si—C bonds or Si—O bonds.
DETAILED DESCRIPTION OF THE INVENTION
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patent: 5773308 (1998-06-01), Conrad et al.
patent: 5843767 (1998-12-01), Beattie
Boukherroub, Rabah, et al.: “Insights into the Formation Mechanisms of Si-OR Monolayers from the Thermal Reactions of Alcohols and Aldehydes with Si(111)H1”. National Research Council of Canada, Ottawa.
Lu, Wuyuan et al.: “Comparative Total Syntheses of Turkey Ovomucoid Third Domain by Both Stepwise Solid Phase Peptide Synthesis and Native Chemical Ligation.” J. Am. Chem. Soc. 1996, 118, 8518-8523.
Stewart, Michael P.: “Chemical and Biological Applications of Porous Silicon Technology.” Advanced Materials, 2000, 12, No. 12.
Xia, Younan et al.: “Soft Lithography”. Agnew, Chem. Int. Ed. 1998, 37, 550-575.
Buriak, Jillian M. et al.: “Lewis Acid Mediated Functionalization of Porous Silicon wi
Boukherroub Rabah
Wayner Danial D. M.
Wojtyk James
Geperley Mary E.
National Research Council of Canada
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