WO2010135800A1

WO2010135800A1 - Inhibiting oxidation and immobilizing biomolecules on a hydrogen-terminated crystalline silicon substrate

Info

Publication number: WO2010135800A1
Application number: PCT/CA2009/000713
Authority: WO
Inventors: Trevor K. Mischki; Gregory P. Lopinski; Danial D.M. Wayner
Original assignee: National Research Council Of Canada
Priority date: 2009-05-27
Filing date: 2009-05-27
Publication date: 2010-12-02

Abstract

A silicon substrate for use in biosensing applications is provided, having a protective layer made of a hydrophobic dendrimer monolayer linked to a hydrophilic layer presenting biomolecule probes. The hydrophobic monolayer inhibits oxidation of the substrate surface while the biomolecule probes optimize recognition and capture of biomolecules. A process for establishing the functionalized silicon substrate and a method of making the protective layer is also provided.

Description

INHIBITING OXIDATION AND IMMOBILIZING BIOMOLECULES ON A

HYDROGEN-TERMINATED CRYSTALLINE

SILICON SUBSTRATE

Field of the Invention

This application relates to modification of silicon substrates for use in biosensing applications.

Background of the Invention

Hydrogen terminated silicon surfaces are particularly useful platforms for field-effect based biosensing applications. Additionally, the lack of an insulating oxide layer allows direct access to the electronic levels of the semiconductor. However, in the presence of oxygen and water, the hydrogen terminated surfaces tend to oxidize, introducing electrically active defects, and ultimately ionizable hydroxyl groups, which diminish biosensing abilities. This is particularly true in aqueous environments, which are common in biosensing applications. These limitations have hindered the development of sensing and biomolecular electronic applications on silicon substrate surfaces. Particularly sought after are methods for immobilising biomolecules with specific binding functions, such as proteins or DNA, on silicon surfaces, while also preventing oxidation of the hydrogen terminated sites.

A number of methods have been proposed for the preparation of organic layers on silicon surfaces. U.S. Pat. No. 6,677,163 (Boukherroub et al., issued Jan. 13, 2004) and U.S. Publ. No. 2004/0096893 (Boukherroub et al., published May 20, 2004) disclose a method of immobilizing a biomolecule on a hydrogen terminated silicon surface by covalently attaching a bifunctional organic compound to the silicon surface through hydrosilylation and then activating the carboxy group of the organic compound by converting it to an active ester, which can then be reacted with a primary amino group of a biomolecule, to thereby capture it. However, the density of this organic layer is not always sufficient to successfully inhibit oxidation. Chidsey et al. demonstrated that the Si(Hl)H surface formed by etching with fluoride may be hydrosilylated by immersion in a neat alkene followed by irradiation. Similar work is disclosed in U.S. Pat. No. 5,429,708 (Linford et al., issued JuI. 4, 1995).

Sieval et al. report the thermal reaction of alkenes terminated with ester groups, with an Si(IOO) hydrogen terminated surface. The ester groups can be hydrolysed to release a carboxylic acid-modified surface, or reduced with LiAIH₄ 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 silicon modified through Si-O linkages have been reported by Zhu et al. A silicon substrate is etched with NH₄F, to produce 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 C₁₂ and Ci₈ chains, respectively, immobilised on the Si surface by Si-O bonds. It is therefore desirable to find a versatile method of forming a protective layer on hydrogen-terminated silicon surfaces, which is dense enough to inhibit oxidation of the surface, particularly in aqueous buffers. At the same time, it is of value to maintain and enhance biomolecule capture performance of the silicon substrate.

Summary of the Invention

In a first aspect of the invention, a method is provided for inhibiting oxidation and immobilizing biomolecules on a hydrogen-terminated silicon substrate. The method comprises first functionalizing a surface of the silicon substrate to initiate growth of a hydrophobic dendrimer monolayer. Next, the hydrophobic dendrimer monolayer is linked to a hydrophilic layer bearing biomolecule probes to capture biomolecules.

In a further aspect, the invention provides a method of inhibiting oxidation and immobilizing biomolecules on a hydrogen-terminated silicon substrate. The method comprises functionalizing a surface of the hydrogen-terminated silicon substrate with a linear hydrocarbon possessing an ester to initiate growth of a hydrophobic dendrimer layer. Next, the hydrophobic dendrimer layer is expanded via reaction with a Grignard reagent possessing terminal functional groups that serve as linkage points to a hydrophilic layer. Finally the terminal functional groups are modified to present biomolecule probes. The hydrophobic dendrimer monolayer serves to inhibit oxidization of the silicon substrate and the biomolecule probes recognize and capture biomolecules. In a further still aspect, a method is provided for providing a protective layer on a hydrogen-terminated silicon substrate to inhibit oxidation and immobilize biomolecules. The method comprises first functionalizing a surface of the hydrogen-terminated silicon substrate to promote growth of a hydrophobic dendrimer monolayer to inhibit oxidation. Next, the hydrophobic dendrimer monolayer is linked to a hydrophilic layer bearing biomolecule probes to capture biomolecules. A further method is provided for providing a protective layer on a hydrogen-terminated silicon substrate to inhibit oxidation and capture biomolecules. The method comprises functionalizing the hydrogen- terminated silicon substrate surface with a linear hydrocarbon possessing an ester to initiate growth of a hydrophobic dendrimer layer. Next, the hydrophobic dendrimer layer is expanded via reaction with a Grignard reagent possessing terminal functional groups that serve as linkage point to a hydrophilic layer. Finally, the terminal functional groups are modified to present biomolecule probes. The hydrophobic dendrimer monolayer serves to inhibit oxidization of the silicon substrate and the biomolecule probes immobilize biomolecules.

The invention also provides a functionalized silicon substrate for biosensing applications comprising a hydrogen-terminated silicon substrate and a protective a monolayer of hydrophobic dendrimer formed on the surface of the silicon substrate. The hydrophobic dendrimer layer is chemically linked to a hydrophilic layer bearing biomolecule probes. The hydrophobic dendrimer monolayer inhibits oxidation of the silicon substrate and the biomolecule probes capture biomolecules.

The invention further provides a protective multi-component layer formed on a hydrogen-terminated silicon substrate. The layer comprises a hydrophobic dendrimer monolayer to inhibit oxidation of the silicon substrate and a hydrophilic layer bearing biomolecule probes chemically linked to the hydrophobic dendrimer monolayer, to capture biomolecules. Finally, the invention provides the use of an organic dendrimer monolayer to protect a surface of a silicon substrate from oxidation by attaching to more than 80% of surface silicon atoms on the silicon substrate.

Description of the Drawings The invention will now be described in further detail with reference to the following drawings, in which:

Figure 1 illustrates the components of the present monolayers on a silicon substrate;

Figure 2 is a process diagram illustrating one example of the process of the present invention, specifically showing an alcohol terminated monolayer activated using hexamethylene diisocyanate;

Figure 3 is a drawing illustrating a number of possible routes to activate an alcohol terminated monolayer derived from Grignard reagents terminated with protected alcohols for reaction with a nudeophile such as an amine, thiol or alcohol;

Figure 4 is a drawing illustrating a number of possible routes to functionalize an alkene terminated monolayer created with Grignard reagents terminated with alkenes;

Figure 5 is a process diagram illustrating a process of the present invention using the alcohol-isocyanate route;

Figure 6 is an FTIR spectra graph showing preparation of functionalized UDA surface;

Figure 7 is an FTIR spectra graph showing preparation of branched alkene monolayer ; Figure 8 is an FTIR spectra graph showing preparation of biotin presenting monolayer via metathesis route;

Figure 9 is an FTIR spectra graph showing preparation of modified surface via oxidation route;

Figure 10 is an FTIR spectra graph of a biotin surface prepared via succinic anhydride route; Figure 11 is a graph of XPS data for biotin terminated silicon surface prepared via Scheme 7 of the

Example;

Figure 12 is a graph of XPS data for biotin terminated silicon surface prepared via Scheme 7 after immersion in water for 18 hours;

Figure 13 is a graph of XPS data for biotin terminated silicon surface prepared via Scheme 8 of the Example;

Figure 14 is a graph of XPS data for biotin terminated silicon surface prepared via Scheme 8 after immersion in water for 18 hours; and

Figure 15 shows three graphs of data relating to biotin terminated surface prepared via Scheme 11: A surface potential map (A); as prepared (B) after exposure to Streptavidin and (C) fluorescence image of the sample.

Detailed Description of the Preferred Embodiments

In optimizing silicon substrates used for biosensing applications, it is very important to both inhibit oxidation of the hydrogen-terminated silicon surface and to maintain biomolecule recognition, capture or immobilization on the substrate surface. Although attempts have been made in the past to functionalize the silicon surface, the resulting monolayers have never been dense enough to fully protect the silicon substrate from oxidation, particularly in aqueous buffers. Furthermore, biomolecule capture and immobilization is often diminished.

Clare et al demonstrate that on extended exposure to aqueous environments, monolayers on crystalline silicon are removed due to extensive oxidation. Optical sensors measure changes in refractive index at the interface, if the monolayer is chemically unstable and is lost due to exposure to measurement conditions, it will manifest itself as baseline drift, thereby limiting sensitivity.

Electrical sensors are sensitive to charge at the interface, and are also affected by both chemical instability of the monolayer and as the silicon is oxidized electrical defects are introduced. Thus, in any application, inhibiting the oxidation of the substrate will stabilize the physical-chemical properties of the material. The attachment of organic monolayers to semiconductor surfaces provides a method to incorporate chemical and biochemical function into solid-state devices to produce bio- sensors or bio-sensor arrays.

The present invention provides a method of functionalizing the silicon substrate surface, which creates an organic monolayer that is both sufficiently dense to prevent oxidation and also presents biomolecule probes that can capture biomolecules. For the purposes of the present invention, biomolecules refer to targets or analytes in solution, which may include but are not limited to: oligo-nucleotides of various lengths, peptides, proteins, peptides and cells.

The monolayer of the present invention has a unique branched structure, specifically in the form of a first generation dendrimer. The branches of the monolayer are established using a Grignard reaction. Solution phase dendrimer chemistry is well known in the art and would be familiar to a person skilled in the art. In the present process, the silicon substrate surface is functionalized with an ω-alkene bearing functional groups which can act as the starting point for the growth of a hydrophobic dendrimer. The hydrophobic layer prevents water from reaching the silicon-carbon interface and acts to stabilize the electrical properties of the surface. Once this layer is sufficiently dense to passivate the silicon substrate, reactive groups are established that link it to a hydrophilic layer that may bear biomolecule probes. Optionally, biomolecule probes may be added to the hydrophilic layer in a stepwise manner. The nature of the interface between the substrate and the biomolecules depend on the application: capture of proteins, oligo-nucleotides of various lengths, or cells. Figure 1 illustrates the components of the present protective layer on a hydrogen-terminated silicon substrate. The use of an organic layer in the form of a dendrimer presents a number of desirable properties. The coverage of Si-H groups with standard alkyl chains is limited by the mismatch in diameter between the surface silicon atoms (12.8 A) and the alkyl chains (15.3 A), as only 83 % of the Si-H sites can be capped by an alkyl chain. This coverage of Si-H sites represents 35 - 60 % of a closed packed hydrocarbon monolayer. Effectively this means that 40 - 65 % of the silicon surface remains unreacted and is available for attack by water or oxygen.

By contrast, theoretical studies suggest methyl terminated layers, such as those of dendrimer monolayers, are capable of reaction at more than 80% of Si-H sites, thereby providing a denser, more complete coverage of the silicon substrate surface. Most importantly, the use of a branching organic monolayer, such as dendrimer monolayers, has been found to successfully separate the requirements for monolayer density for protective reasons, from the constraints of the tight silicon crystal lattice spacing. The layering structure of the present invention thus favourably applies dendrimer chemistry to separate monolayer density requirements from the constraints imposed by the silicon lattice spacing. The process of the present invention progresses in several steps and employs multifunctional and bifunctional organic molecules to progressively add components to form the resultant monolayer. The present process can be generally outlined as first providing a hydrogen-terminated silicon surface and reacting this surface with a linear hydrocarbon possessing an ester. Subsequently, the ester is reacted with a Grignard reagent terminated in a functional group. The terminal functional group is then modified for coupling via a number of different possible processes. Figure 2 illustrates one example of the present process. In the first step, the silicon substrate surface is preferably functionalized with an ω-alkene bearing functional groups which can act as the starting point for the growth of a hydrophobic dendrimer. Next, the hydrophobic dendrimer layer expanded via reaction with a Grignard reagent possessing a terminal function group that serve as a linkage point to a hydrophilic layer. The terminal functional group can be any group that can undergo standard organic chemistry transformations to form a covalent bond. The hydrophilic layer serves to inhibit non-specific bonding of biomolecules to ensure that only target biomolecules are recognized and captured by the silicon substrate. This is particularly important for protein-capture applications. Examples of suitable hydrophilic layers of the present invention include, but are not limited to ethylene glycol or dextran based oligomers and polymers.

The hydrophilic layer in turn bonds to biomolecule probes. The bonding of biomolecule probes can happen in one or two steps. In a general embodiment, the hydrophilic layer will have functional groups available for installation of the biomolecule probes. The inventors have experimented with covalently attaching biotin as a biomolecule probe to an oligoethylene glycol amine. In other cases, the inventors have used a two step process of attaching the oligoethylene glycol to the surface then spotting and covalently binding many different biomolecule probes to the surface. In other cases, such as DNA, the DNA may optionally be directly reacted with the terminal functional groups and fill in the background with a blocking group such as oligoethylene glycols or some other molecule. Most commonly, the biomolecules have been attached by covalent bonds linking one layer to the next. The Grignard reaction of esters is a preferred for a number of reasons. The Grignard reaction is thermodynamically favourable, therefore ensuring that the reaction proceeds. Furthermore, Grignard reagents react directly with Si-H surfaces thereby filling any holes in the monolayer. Finally methyl terminated monolayers prepared via this process have shown good stabilization of the electrical properties. The terminal functional group of the Grignard reagents of the present invention can be any reactive groups compatible with the Grignard reagents and capable of functionalizing the ester surface. Preferred terminal groups include, but are not limited to protected alkyne, protected alcohol, protected amines, protected carboxylic acid, protected aldehydes, alkenes and azides. An example of a preferred protected alkyne is trimethyl silyl. An example of a preferred protected alcohol is trimethyl silyl ether. Examples of preferred protected amines are disilyl protecting groups. Examples of preferred protected carboxylic acids are orthoesters. Examples of preferred protected aldehydes are acetals. Silyl based protecting groups are most preferred because the deprotection is mediated by hydrofluoric acid which is compatible with crystalline silicon but will remove any hydrocarbon from oxide regions of the surface ensuring probe molecules are located exclusively on the sensing region of a device. Figure 3 illustrates a number of possible routes to activate an alcohol terminated monolayer derived from Grignard reagents terminated with protected alcohols for reaction with a nucleophile such as an amine, thiol or alcohol. Figure 4 illustrates a number of possible routes to functionalize an alkene terminated monolayer derived from Grignard reagents terminated with alkenes.

The terminal functional group of the Grignard reagent is further modified for coupling. This can involve a number of different processes. These can include epoxidation, metathesis, deprotection or activation. Epoxidation and metatheses are preferably carried out with alkene terminal functional groups. Deprotection or activation processes are preferably carried out with orthoesters, protected aldehydes or protected alcohols as terminal functional groups. The modification of the terminal functional groups allows for bonding of biomolecule probes, also known as biological probe molecules, which act to capture and immobilize particular biomolecules for biosensing applications. The hydrophilic layer may present molecular recognition elements in the form of biomolecule probes such as oligo-nucleotides of various lengths, small molecules, peptides, sugars, oligosaccharides, proteins or protein fragments.

The modification process chosen depends on the type of biomolecules that are to be captured. The biomolecule probes interact with their target biomolecules by an affinity interaction. Silicon surfaces with a high density of functional groups are important for protein attachment and immobilization because the interface between substrate and protein must be hydrophilic in order to inhibit non-specific binding. This hydrophilicity can be achieved by, for example, addition of ethylene glycol containing molecules. For immobilization of proteins a preferred higher density monolayer can be achieved through deprotection, preferably using alcohol-isocyanate. Also preferably, alcohol terminal functional groups can be modified by activation by hexamethylene diisocyanate to capture and immobilize proteins. Figure 5 illustrates the process using the alcohol-isocyanate activation to modify the silicon surface. Surfaces that present lower densities of reactive groups are compatible for DNA detection. The density of reactive groups can be lower for DNA applications because the mechanism for non-specific binding does not exist, although, the density must be sufficient to ensure a signal is measurable. In this case, biomolecules probes act to recognize a complementary strand of DNA through the molecular recognition. The present inventors have found that for immobilization of DNA, either metathesis or oxidation of an alkene terminated branched surface is preferred as it provides a suitable density for capture. Generally speaking preferred routes for electrical sensing applications result in no ionizable groups remaining in the monolayer. Thus, carbonates, carbamates, esters or ethers for example, are more preferred over acids or amines. Optionally, there is also the possibility that if the hydrophilic layer does not present probes, it may be useful as a coating to impart desired interfacial properties for applications such as implants or as a substrate for cell or tissue growth.

The monolayers of the present invention have been found to work well with a number of types of silicon substrate surfaces, including but not limited to hydrogen terminated silicon (111) and (100), hydrogen terminated silicon on insulator and any structures derived from it. The inventors also predict application of the present invention to functionalize Si (110), hydrogen terminated silicon nanowires and nanoparticles.

Examples The creation of a suitable organic monolayer for silicon surfaces was tested and various properties of the resulting surfaces have been measured, confirming the absence of significant oxidation or growth of electrically active defects due to biomolecule attachment. The following examples serve to illustrate aspects of the present invention, without limiting the scope thereof:

Surface Characterization Techniques

Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR). ATR-FTIR spectra were recorded using a Nicolet MAGNA-IR 860 spectrometer at 4cm^"1 resolution. The ATR crystals were mounted in a purged sample chamber with the light focused normal to one of the 45° bevels. Background spectra were obtained using an oxidized surface. X-Ray Photoelectron Spectroscopy (XPS). XPS spectra were recorded on a PHI 5500 instrument, using monobromated Al Ka (1486eV) radiation with detection on the surface normal. The pressure during analysis was ~ 5xlO^"8 Torr. Spectra were fitted with Gaussian profiles using standard procedures. The positions of all peaks were normalized to C Is at 285.0 eV.

Surface Photovoltage Measurements (SPV). A scanning Kelvin probe (KP Technology Ltd, Wick, Scotland) was used to measure the SPV, which provides a direct measure of band-bending (the difference in surface potential in the dark and under illumination). A white light fiber optic source was used to illuminate the surface; the light was determined to be of sufficient intensity to saturate the photovoltage. A motorized stage was used to scan the sample allowing measurement of the SPV at 16 spatially distinct points to be collected per sample.

Fluorescence Measurements. Silicon wafers were affixed to a glass slide and scanned in a BioRAD VersArray Chip Reader with 5 μm resolution using 635 and 532 nm excitation.

Materials

10-Undecylenic acid 98% (UDA) was purchased from Aldrich and distilled in a Kugelrohr apparatus under vacuum at ~160°C. Ethyl-undecylenate 97% (UDE) and 11-bromoundecene were purchased from Aldrich and distilled under vacuum at 134°C. N-hydroxysuccinimide (NHS), N-ethyl-N'-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC), trimethyl silyl chloride (TIvIS-CI), 11-bromo- undecan-1-ol, Grubbs 2^nd Generation catalyst, 4-pentenoic acid, thionyl chloride and other synthetic reagents were purchased from Aldrich at >95% purity and used as received. Unless otherwise specified, solvents were obtained from a PureSolv purification system. ATR silicon crystals (25 x 4.5 x 1 mm³) were purchased from Harrick and the silicon wafers from Virginia Semiconductor. Cleaning and etching solutions were clean-room grade. Sulfuric acid, 96% (H₂SO₄), and ammonium fluoride, 40% (NH₄F) were purchased from J. T. Baker, hydrochloric acid, 30% (HCI) and ammonium hydroxide, 30% (NH₄OH) from Olin Microelectronic Materials, hydrogen peroxide, 30% (H₂O₂) from Anachemica, and hydrofluoric acid, 48% (HF) from Arch. MiIIiQ water (18 MΩ) was used for all experiments. Silicon shards and ATR elements were cleaned after reactions in a Soxhlet Extractor under argon with continuously refluxing 1,1,1-trichloroethane (TCE) purchased from Aldrich.

Synthesis

I)PPh₃, THF

²)H₂O H₂N^^°^^-^/^^/0H

Scheme 1 - Synthesis of EG3NH2 (3)

2-[2-(2-azidoethoxy)ethoxy]ethanol (2). 2-[2-(2-chloroethoxy)ethoxy]ethanol (1) (1Og, 59.3mmol) was dissolved in 95% ethanol (4OmL) in a 25OmL round bottom flask equipped with a magnetic stir bar. To this was added Kl (3g, lδmmol) and sodium azide (5g, 78mmol) the reaction was then heated at 8O⁰C over night. The solution was allowed to cool to room temperature, filtered and concentrated to afford a clear yellow oil. The crude was purified by silica gel column chromatography to afford a clear oil (7.3g, 71% yield). ¹H NMR (400 MHz, CDC)₃) δ ppm 3.73 (t, 7=4.88 Hz, 2 H), 3.68 (m, 6 H), 3.61 (t, 7=4.88 Hz, 2 H), 3.40 (t, 7=4.89 Hz, 2 H), 2.64 (s, 1 H). 2-[2-(2-aminoethoxy)ethoxy]ethanol (3). 2-[2-(2-azidoethoxy)ethoxy]ethanol (2) (2g, 11.4mmol) was dissolved in dry tetrahydrofuran (4OmL) in a 25OmL round bottom flask, to this was added triphenylphosphine (3.3g, 12.6mmol). The reaction was sealed under a nitrogen balloon and stirred at room temperature over night. The reaction was opened and water (821μL, 45.6mmol) was added and stirred for four hours. The reaction was concentrated, suspended in water (2OmL) and washed with toluene (5 x 2OmL). The water was concentrated to a clear yellow oil which was dissolved in dichloromethane, dried with MgSO₄, filtered concentrated and flashed eluting with 10-25% methanol : chloroform : 1-2.5% NH₄OH to yield a pale yellow oil (1.5g, 88% yield). ES-MS: calc 149.1, found MH⁺ 150.1.

BoC₂O₁ TEA THF

Scheme 2 - Synthesis of 5 tert-butyl 3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy}propylcarbamate (5). 3-{2-[2-(3- aminopropoxy)ethoxy]ethoxy}-l-propanamine (4) (8.8mL, 40mmol) was dissolved in dichloromethane (15OmL) in a 25OmL round bottom flask. To this was added di-teitbutyl dicarbonate (2.2g, lOmmol) in four portions over 20 minutes. The reaction was stirred overnight, concentrated and purified by silica gel column chromatography eluting with 20% methanol : chloroform : 2% NH₄OH to yield a pale yellow oil (2.2g, 68% yield). ¹H NMR (400 MHz, CDCI₃) δ ppm 5.15 (s, 1 H), 3.63 (m, 4 H), 3.56 (m, 8 H), 3.21 (m, 2 H), 2.82 (t, J=6.66Hz, 2 H), 2.12 (s, 2 H), 1.74 ( p, J=6.44, Hz, 4 H), 1.42 (s, 9 H).

Scheme 3 - Synthesis of Biotin NHS (7)

(+)-Biotin N-hydroxysuccinimide ester (7). (D)-biotin (6) (l.Olg, 4.14mmol) was suspended in dry chloroform (6mL), a drop of N,N-dimethylformamide was added and the reaction sealed under nitrogen. Thionyl chloride (4mL, 55mmol) was added drop-wise and the reaction stirred at room temperature for 1 hour. The solvent and thionyl chloride were evaporated under reduced pressure and the residue taken up in dry chloroform (1OmL) this was drop-wise added to a stirring suspension of N- hydroxysuccinimide (952mg, 8.28mmol) and triethylamine (1.45mL, 10.3mmol) in chloroform (3OmL) under nitrogen. The reaction was stirred 3 hours then concentrated and the residue purified by silica gel column chromatography eluting with 10% methanol : chloroform (l.lg, 77% yield). ¹H NMR (400 MHz, DMSO-d6) δ ppm 6.43 (s, 1 H), 6.37 (s, 1 H), 4.31 (s, 1 H), 4.15 (s, 1 H), 3.11 (dd_; J=6.97, 11.46 Hz, 1 H), 2.84 (s, 5 H), 2.68 (t, J=7.35 Hz, 2 H), 2.59 (d, J=12.43 Hz, 1 H), 1.65 (m, 3 H), 1.48 ( m, 3 H).

Boc biotin (8). tert-butyl 3-{2-[2-(3-aminopropoxy)ethoxy]ethoxy} propylcarbamate (5) (l.lg, 3.43mmol) and triethylamine (482μL, 3.43mmol) were dissolved in dichloromethane (1OmL) and added to a stirring suspension of (+)-Biotin N-hydroxysuccinimide ester (7) in dichloromethane (1OmL) the reaction was stirred at room temperature over night. It was then concentrated and purified by silica gel column chromatography eluting with 1-5% methanol : chloroform (1.2g_; 93% yield) as a pale yellow oil. ¹H NMR (400 MHz, CDCI₃G] ppm 6.59 (s, 1 H), 6.25 (s, 1 H), 5.46 (s, 1 H), 5.06 (s, 1 H), 4.52 (dd, 7-5.01, 7.58 Hz, 1 H), 4.33 (dd, 7=5.08, 7.15 Hz, 1 H), 3.61 (s, 12 H), 3.36 (dd, 7=6.00, 12.08 Hz, 2 H), 3.23 (dd, 7=5.83, 11.83 Hz, 2 H), 3.16 (dt, 7=4.78, 7.30 Hz, 1 H), 2.92 (dd, 7=4.92, 12.81 Hz, 1 H), 2.76 (d, 7=12.77 Hz, 1 H), 2.21 (t, 7=7.47 Hz, 2 H), 1.73 (s, 8 H), 1.47 (m, 11 H). Amino biotin (9). Amino boc (8) (1.2g, 2.12mmol) was dissolved in dichloromethane (1OmL) and trifluoroacetic acid (2mL) and stirred at room temperature for 2 hours. The reaction was concentrated then dissolved in methanol (2OmL) added potassium carbonate powder and stirred for 5 minutes then filtered and concentrated. The pale yellow oil was taken up in dichloromethane, dried with MgSO₄ filtered and concentrated to give a pale yellow oil (830 mg, 88% yield). ¹H NMR (400 MHz, CDCI₃) ppm 7.04 (s, 1 H), 6.62 (s, 1 H), 4.50 (dd, J=4.86, 7.26 Hz, 1 H), 4.31 (dd, J=4.50, 7.47 Hz, 1 H), 4.11 (s, 1 H), 3.58 (m, 14 H), 3.32 (m, 1 H), 3.14 (dd, J=7.20, 11.69 Hz, 1 H), 2.86 (m, 2 H), 2.74 (d, J=12.72 Hz, 1 H), 2.47 (m, 1 H), 2.20 (t, J=7.05 Hz, 1 H), 1.72 ( m, 8 H), 1.43 (td, J=8.17, 16.33 Hz, 2 H). ES-MS: calc 446.3, found M H⁺ 447.3.

10 11

Scheme 4 - Preparation of 11

Compound 11 (11). 4-pentenoic acid (10) (5g, 50mmol) was drop-wise added to a solution of thionyl chloride (1OmL) under nitrogen and stirred at room temperature for 2 hours. The solution was concentrated under reduced pressure and dissolved in methylene chloride (5OmL), which was added over 10 minutes to NHS (11.5g, lOOmmol) and triethylamine (2OmL, 150mmol) dissolved in methylene chloride (10OmL) under nitrogen. After 2 hours the solution was concentrated suspended in diethyl ether, filtered, concentrated and purified by silica gel column chromatography eluting with 5% hexanes : chloroform to afford 5.5g as a clear oil which solidified on standing (36 % yield). ¹H NMR (400 MHz, CDCI₃) ppm 5.85 (m, 1 H), 5.12 (m, 2 H), 2.84 (s, 4 H), 2.72 (t, J=7.43, 7.43 Hz, 2 H), 2.50 (td, J=6.88, 6.88, 13.76 Hz, 2 H). Me₃SiCl,

12 13

Scheme 5 - Synthesis of 11-bromo-l-trimethylsilγloxγundecane (13) 11-bromo-l-trimethylsilyloxyundecane (13). 11-bromoundecan-l-ol (3.83g, 15.2mmol) was dissolved in tetrahydrofuran (10OmL), triethylamine (2.4mL, 17.2mmol) was added and the reaction sealed under nitrogen. Trimethylsilyl chloride (2.02mL, 15.8mmol) was added dropwise and the reaction stirred at room temperature over night. The solution was filtered, concentrated, taken up in hexanes, filtered a second time then concentrated and purified by silica gel column chromatography eluting with 5% ethyl acetate : hexanes to afford 4.13g (84% yield) as a clear oil. ¹H NMR (400 MHz, CDCj₃ppm 3.58 (t, J=6.73, 6.73 Hz, 2 H), 3.42 (t, 7=6.87, 6.87 Hz, 2 H), 1.87 (m, 1 H), 1.53 (dd, J=6.75, 13.52 Hz, 2 H), 1.43 (dd, J=6.97, 14.35 Hz, 2 H), 1.30 (m, 14 H), 0.13 (s, 9 H).

1) MePhSO₂Cl, Et₃N, THF

16

Scheme 6 - Synthesis of 1-amino hexaethyleneglycol (16)

1-azido hexaethyleneglycol (15). Hexaethylene glycol (14) (5g, 17.71 mmol) was dissolved in diethylether : methylene chloride 6OmL : 1OmL, triethylamine (2.75mL, 19.5 mmol) was added followed by p-toluenesulfonyl chloride (3.38g, 17.71 mmol) and stirred at room temperature over night. The solvent was evaporated and the residue taken up in ethyl acetate filtered and concentrated. The residue was dissolved in ethyl alcohol (4OmL), sodium azide (1.73g, 26.6mmol) was added and the reaction heated at 80 "C overnight. The reaction was concentrated and then purified by silica gel column chromatography eluting with 10% methanol : ethyl acetate l.lg (21% yield) as a clear oil. ¹H NMR (400 MHz, CDCI₃) ppm 3.68 (m, 2 H), 3.63 (m, 18 H), 3.57 (m, 2 H), 3.37 (s, 2 H), 2.92 (s, 1 H).

1-amino hexaethyleneglycol (16). 1-azido hexaethyleneglycol (15) (550mg, Ulmmol) and triphylphosphine (565mg, 2.15mmol) were dissolved in tetrahydrofuran (1OmL) and sealed under a nitrogen atmosphere. The reaction was stirred for 4 hours and then MiIIi-Q water (ImL) was added and the reaction stirred a further Ih. The solvent was evaporated under reduced pressure and the residue suspended in water (1OmL), and extracted with toluene (4 x 1OmL). The aqueous was isolated evaporated to dryness, taken up in methylene chloride, dried (MgSO₄) and concentrated to afford 465mg (96% yield) as a clear pale yellow oil. ¹H NMR (400 MHz, CDCI₃) ppm 3.62 (m, 2 H), 3.55 (m, 16 H), 3.50 (m, 2 H), 3.44 (t, -/=5.19Hz, 2 H), 2.77 (t, J=5.19Hz, 2 H), 2.74 (s, 3 H)

ES-MS: calc 281.35, found MH⁺ 282.3.

Surface Preparation

Cleaning and Hydrogen termination. Single sided silicon crystals were used once and discarded; ATR elements were cleaned and reused. All crystals were cleaned with piranha solution (3:1 H₂SO₄, 96% : H₂O₂, 30%) at 120⁰C for 30 minutes, then rinsed with MiIIi-Q water. (Warning: Piranha solutions should be handled with care and kept isolated from organic materials). ATR elements were then cleaned with 4:1:1 MiIIi-Q water : NH₄OH, 30% : H₂O₂, 30% at 80⁰C for 15 minutes, rinsed with MiIIi-Q water and heated in 4:1:1 MiIIi-Q water : HCI, 30% : H₂O₂, 30% at 80⁰C for 15 minutes then rinsed with MiIIi-Q water. Samples were hydrogen terminated by etching in degassed ammonium fluoride for 15 minutes followed by a brief rinse in degassed MiIIi-Q water.

Scheme 7 - Preparation of unbranched biotin surface

UDA monolayer formation. Freshly Distilled UDA was transferred to a Pyrex Schenk tube and degassed with argon for 10 minutes. The silicon samples were added and degassed a further 5 minutes then placed in a Rayonette photoreactor for 3 hours. After the reaction the samples were removed and washed with either continuously refluxing 1,1,1-trichloroethane in a soxhlet or with copious volumes of chloroform and ethanol at room temperature. The FTIR spectrum shows the presence of methylene stretches at 2924 and 2954 cm^"1, the carbonyl stretch of the acid group is observed at 1714 cm^"1.

NHS Ester Formation. EDC (lOmg / mL, 52mM) and NHS (6mg/mL, 52mM) were dissolved in MiIIi-Q water and the silicon samples immersed for 1 hour. The samples were removed rinsed with MiIIi-Q water, and ethanol then blown dry with a stream of nitrogen. The FTIR spectrum indicates methelene stretches at 2924 and 2953 cm^"1, imide peaks at 1816 and 1784 cm^"1 and ester carbonyl at 1743 cm^"1.

Amide formation -ATR element. A NHS activated silicon ATR element was immersed in a solution of compound 3 (20 mM) in pH 8.0 carbonate buffer (0.2 M) for 3 hours. The sample was washed with MiIIi- Q water, ethanol and dried under a stream of nitrogen. The FTIR spectrum indicates methelene stretches at 2923 and 2953 cm^"1, acid carbonyl at 1716 cm^"1, and amide I and Il bands at 1647 and 1548 cm^"1 respectively. The observed carboxylic acid is the result of a hydrolysis reaction competing with amide formation. Amide formation - Silicon wafer. A NHS activated silicon wafer manually spotted with 2 μL drops of compound 9 (20 mM) in pH 8.0 carbonate buffer (0.2 M) and placed in a humid chamber for 1 hour. The sample was rinsed in MiIIi-Q water and the entire surface covered in compound 3 (20 mM) in pH 8.0 carbonate buffer (0.2 M) for 3 hours. The sample was washed with MiIIi-Q water, ethanol and dried under a stream of nitrogen.

UndecyienylMgBr

Compound 9 Carbonate pH 8.0

Scheme 8 - Metathesis route

UDE monolayer formation. Freshly Distilled UDA was transferred to a Pyrex Schenk tube and degassed with argon for 10 minutes. The silicon samples were added and degassed a further 5 minutes then placed in a Rayonette photoreactor for 3 hours. After the reaction the samples were removed and washed with either continuously refluxing 1,1,1-trichloroethane in a soxhlet or with copious volumes of chloroform and ethanol at room temperature. The FTIR spectrum, as seen in Figure 6, shows the presence of a methyl stretch at 2982 cm^"1, methylene stretches at 2922 and 2952 cm^'1, the carbonyl stretch of the ester group is observed at 1741 cm^"1. Grignard reaction - Alkenβ Terminated Monolayers. Magnesium turnings (350mg, 14.6mmol) were added to a round bottom flask and sealed under an atmosphere of nitrogen with a condenser. Dry tetrahydrofuran (9mL) was added via syringe and the reaction heated to 80 °C. The magnesium was activated by 1,2-dibromoethane (200 μL) for 15 minutes and then 11-bromo-l-undecene (2.8mL, 13 mmol) was added via syringe. The reaction was heated a further 1 hour at 80 "C. Just prior to introduction to the reaction vessel the silicon sample can be exposed to 2% HF solution for 2 minutes in order to remove any silicon oxide which may have formed during the hydrosilation reaction. The silicon sample was sealed under an atmosphere of nitrogen in a clean, dry test tube with a silicone septum and the Grignard reagent was introduced via syringe. The sample was allowed to react at 80 °C for 90 minutes. At this point the Grignard reaction was stopped by successive dilution with dry tetrahydrofuran ensuring the sample remained covered in solvent. After 3 exchanges 2 mL of glacial acetic acid was introduced to quench remaining Grignard reagent. The sample was washed with glacial acetic acid, MiIIi-Q water, ethanol and dried under a stream of nitrogen. The FTIR spectrum, as seen in Figure 7, shows the presence of an alkene C-H stretch at 2082 cm^'1, methylene stretches at 2924 and 2952 cm^"1, the remaining carbonyl stretch of the ester group is observed at 1738 cm^"1 and ketone from the partially reacted ester at 1718 cm^"1.

Metathesis reaction. Compound 11 (100 mg, 0.51 mmol) was dissolved in dry methylene chloride 5mL in a 25 mL sealed tube to this was added an alkene terminated silicon and the mixture degassed with bubbling argon for 5 minutes. To this was added Grubbes 2^nd generation catalyst (22mg, 5 mol%) and the sealed tube closed under an argon atmosphere. The reaction was heated at 50 °C over night, then opened and the sample washed with methylene chloride, ethyl alcohol and blown dry under a stream of nitrogen. The FTIR spectrum shows the methylene stretches at 2924 and 2952 cm^"1, imide peaks at 1818 and 1787 cm^"1, the ester carbonyl stretch is observed at 1743 cm^"1, it has increased due to the presence of the N-hydroxysuccinimide ester and ketone from the partially reacted ester at 1718 cm^"1. Amide formation -ATR element. A NHS activated silicon ATR element was immersed in a solution of compound 3 (20 mM) in pH 8.0 carbonate buffer (0.2 M) for 3 hours. The sample was washed with MiIIi- Q water, ethanol and dried under a stream of nitrogen. The FTIR spectrum indicates methelene stretches at 2924 and 2952 cm^"1, the ester carbonyl stretch is observed at 1738 cm^"1, ketone from the partially reacted ester and potentially acid from hydrolyzed N-hydroxysuccinimide esters at 1714 cm^"1, and amide I and Il bands at 1655 and 1547 cm^"1 respectively.

Scheme 9 - Alkene oxidation route

Permanganate - Periodate Oxidation. An oxidizing solution consisting of 0.5 mM KMnO₄, 19.5 mM NaIO₄, and 1.8 mM K₂CO₃, at a pH of 7.5 was prepared in MiIIi-Q water. The silicon sample was immersed in this solution for 30 minutes and then washed with 0.3 M NaHSO₃, MiIIi-Q water, 0.1 N HCI, MiIIi-Q water, ethanol and blown dry under a stream of nitrogen. The FTIR spectrum, as seen in Figure 8, indicates loss of alkene signals, methelene stretches at 2925 and 2953 cm^"1, remaining ester carbonyl at 1743 cm^"1, and ketone and carboxylic acid stretches overlapping at 1718 cm^"1.

NHS Ester Formation. EDC (10 mg / mL, 52mM) and NHS (6mg/mL, 52mM) were dissolved in MiIIi-Q water and the silicon samples immersed for 1 hour. The samples were removed rinsed with MiIIi-Q water, and ethanol then blown dry with a stream of nitrogen. The FTIR spectrum indicates methelene stretches at 2924 and 2952 cm^"1, imide peaks at 1813 and 1785 cm^"1 and ester carbonyl at 1738 cm^"1 ketone at 1718 cm^"1.

Amide formation -ATR element. A NHS activated silicon ATR element was immersed in a solution of compound 3 (20 mM) in pH 8.0 carbonate buffer (0.2 M) for 3 hours. The sample was washed with MiIIi- Q water, ethanol and dried under a stream of nitrogen. The FTIR spectrum indicates methelene stretches at 2924 and 2952 cm^"1, remaining ethyl ester carbonyl at 1743 cm^"1 hydrolyzed acid and ketone carbonyl at 1720 cm^"1, and amide I and Il bands at 1648 and 1542 cm^"1 respectively. The observed carboxylic acid is the result of a hydrolysis reaction competing with amide formation.

Amide formation - Silicon wafer. A NHS activated silicon wafer manually spotted with 2 μL drops of compound 9 (20 mM) in pH 8.0 carbonate buffer (0.2 M) and placed in a humid chamber for 1 hour. The sample was rinsed in MiIIi-Q water and the entire surface covered in compound 3 (20 mM) in pH 8.0 carbonate buffer (0.2 M) for 3 hours. The sample was washed with MiIIi-Q water, ethanol and dried under a stream of nitrogen.

Scheme 10 - Preparation of Alcohol Terminated Monolayer

Grignard reaction -Alcohol Terminated Monolayers. Magnesium turnings (340 mg, 14 mmol) were added to a round bottom flask and sealed under an atmosphere of nitrogen with a condenser. Dry tetrahydrofuran (10 mL) was added via syringe and the reaction heated to 80 °C. The magnesium was activated by 1,2-dibromoethane (200 μL) for 15 minutes and then 11-bromo-l- trimethylsilyloxyundecane (3.1 g, 9.6 mmol) was added via syringe. The reaction was heated a further 1 hour at 80⁰C. Just prior to introduction to the reaction vessel the silicon sample can be exposed to 2% HF solution for 2 minutes in order to remove any silicon oxide which may have formed during the hydrosilation reaction. The silicon sample was sealed under an atmosphere of nitrogen in a clean, dry test tube with a silicone septum and the Grignard reagent was introduced via syringe. The sample was allowed to react at 80⁰C for 90 minutes. At this point the Grignard reaction was stopped by successive dilution with dry tetrahydrofuran ensuring the sample remained covered in solvent. After 3 exchanges 2 mL of glacial acetic acid was introduced to quench remaining Grignard reagent. The sample was washed with glacial acetic acid, MiIIi-Q water, ethanol and dried under a stream of nitrogen. The FTIR spectrum, as seen in Figure 9, shows the presence of methylene stretches at 2924 and 2952 cm^"1, the remaining carbonyl stretch of the ester group is observed at 1737 cm ^α and ketone from the partially reacted ester at 1716 cm^":

Scheme 11 - Isocγate Route to Biotin Surface

lsocyanate Terminated Monolayer. An alcohol terminated silicon sample was introduced into an argon purged solution of neet hexamethylene diisocyanate, sealed under nitrogen and heated at 60⁰C for 90 minutes. The sample was removed washed with methylene chloride and dried under a stream of nitrogen. The FTIR spectrum shows the presence of methylene stretches at 2925 and 2952 cm^"1, isocyanate carbonyl at 2272 cm^"1, and carbonate stretches at 1699 cm^"1 and 1537 cm ¹.

Carbamate Formation - ATR Element. The isocyanate surface was immersed a solution of compound 9 (20 mM) in pH 8.0 carbonate buffer (0.2 M) for 1 hours. It was washed with water, ethanol and methylene chloride then dried under a stream of nitrogen. The FTIR spectrum indicates a broad NH stretch at 3346 cm^"1 from the carbamate and urea functional groups, methylene stretches at 2924 and 2954 cm^"1, carbonate and urea carbonyl stretching at 1699, 1647and 1548 cm^"1.

Carbamate formation - Silicon wafer. A NHS activated silicon wafer manually spotted with 2 μL drops of compound 9 (20 mM) in pH 8.0 carbonate buffer (0.2 M) and placed in a humid chamber for 1 hour. The sample was rinsed in MiIIi-Q water and the entire surface covered in compound 3 (20 mM) in pH 8.0 carbonate buffer (0.2 M) for 1 hours. The sample was washed with MiIIi-Q water, ethanol and dried under a stream of nitrogen.

Scheme 12 - Biotin Surface via Succinic acid route

Carboxylic Acid Terminated Monolayer. An alcohol terminated silicon sample was introduced into an argon purged solution of 1 M succinic anhydride, 0.05M N,N-dimethylaminopyridine in dimethylformaide, sealed under nitrogen and heated at 90⁰C for 18 hours. The sample was removed washed with dimethylformamide, ethanol, and dried under a stream of nitrogen. The FTIR spectrum, as seen in Figure 10, shows the presence of methylene stretches at 2924 and 2952 cm^"1, ester carbonyls (product plus unreacted ethyl ester) at 1737 cm^'1, and a ketone shoulder at 1716 cm^"1.

NHS Ester Formation. EDC (10 mg / mL, 52mM) and NHS (6mg/mL, 52mM) were dissolved in MiIIi-Q water and the silicon samples immersed for 1 hour. The samples were removed rinsed with MiIIi-Q water, and ethanol then blown dry with a stream of nitrogen. The FTIR spectrum indicates methelene stretches at 2924 and 2953 cm^"1, imide stretches at 1814 and 1781 cm^"1, ester carbonyls at 1740 cm^"1, and a ketone shoulder at 1717 cm^"1.

Amide formation -ATR element. A NHS activated silicon ATR element was immersed in a solution of compound 3 (20 mM) in pH 8.0 carbonate buffer (0.2 M) for 3 hours. The sample was washed with MiIIi- Q water, ethanol and dried under a stream of nitrogen. The FTIR, spectrum indicates methelene stretches at 2920 and 2950 cm^"1, ester carbonyls at 1735 cm^"1, a ketone at 1717 cm^"1, and amide bands at 1656 and 1568 cm^"1.

Example of Branched surface stability

A biotin terminated silicon surface was prepared according to Scheme 7. The sample was analyzed by X- Ray photoelectron spectroscopy before, as seen in Figure 11, and after, as seen in Figure 12, exposure to water for 18 hours. The inset shows an expansion of the region corresponding to oxidized silicon. The relative peak area of the oxidized silicon peak compared with the unshifted main silicon peaks is reported in Table 1 along with surface photovoltage data as a measure of the stability of the electrical properties of the interface.

A biotin terminated silicon surface was prepared according to Scheme 8. The sample was analyzed by X- Ray photoelectron spectroscopy before, as seen in Figure 13, and after, as seen in Figure 14, exposure to water for 18 hours. The inset shows an expansion of the region corresponding to oxidized silicon. The relative peak area of the oxidized silicon peak compared with the unshifted main silicon peaks is reported in Table 1 with surface photovoltage data as a measure of the stability of the electrical properties of the interface.

Table 1 - Oxide Peak area and Photovoltage Data for Biotin terminated silicon surfaces

These data confirm that functional branched monolayers stabilize the silicon surface chemically and electrically.

Example of Protein recognition by electrical detection

A biotin terminated silicon sample was prepared according to Scheme 11. The isocyanate surface was reacted with 2 x 2 μL drops of 20 mM Compound 9 in pH 8.0 carbonate buffer (0.2 M) for 1 hour then the entire surface was covered in a 20 mM solution of Compound 16 in pH 8.0 carbonate buffer (0.2 M) for 1 hour. The surface was washed with water and ethanol dried under a stream of nitrogen and scanned with a Kelvin Probe. This surface was then exposed to a 0.1 mg/mL solution of Cy-3 labelled Streptavidin in pH 7.4 phosphate buffered saline for 1 hour. The sample was washed 3 x 1 minute with 0.1 % Tween-20 in pH 7.4 phosphate buffered saline followed by MiIIi-Q water then dried under a stream of nitrogen. The surface potential was measured by Kelvin Probe and the fluorescent protein imaged using a BioRad chip scanner. The results are shown in Figure 15. These measurements indicate substantial changes in the surface potential which correlate with a fluorescence image and the protein is selectively captured in the biotin terminated regions.

This detailed description of the compositions and methods is used to illustrate certain embodiments of the present invention. It will be apparent to a person skilled in the art that various modifications can be made in the present composition and methods and that various alternate embodiments can be utilized without departing from the scope of the present application, which is limited only by the appended claims.

Claims

ClaimsWe claim:

1. A method of inhibiting oxidation and immobilizing biomolecules on a hydrogen-terminated silicon substrate comprising: a. functionalizing a surface of the silicon substrate to initiate growth of a hydrophobic dendrimer monolayer; and b. linking to the hydrophobic dendrimer monolayer a hydrophilic layer presenting biomolecule probes to capture biomolecules.

2. The method of claim 1, wherein the surface of the hydrogen-terminated silicon substrate is functionalized with a linear hydrocarbon possessing an ester.

3. The method of claim 1, wherein the hydrophobic dendrimer monolayer is grown by reaction with Grignard reagents having terminal functional groups, said terminal functional groups serving as linkage points to the hydrophilic layer.

4. The method of claim 3, wherein the biomolecule probes are bonded to the hydrophilic layer by modifying the terminal functional groups.

5. The method of claim 4, wherein the biomolecule probes are bonded to the hydrophilic layer stepwise, in a one or two step process.

6. The method of claim 2, wherein the hydrogen terminated silicon substrate is functionalized by hydrosilylation of a terminal C-C double bond of the ester of the linear hydrocarbon.

7. The method of claim 3, wherein the Grignard reagent is terminated in a protected alkyne, protected alcohol, protected amine, protected carboxylic acid, protected aldehyde, alkene or azide.

8. The method of claim 4, wherein the terminal functional groups are modified by a process selected from epoxidation, metathesis, deprotection or activation.

9. The method of claim 4, wherein protected carboxylic acid, protected aldehyde or protected alcohol terminal functional groups are modified by metathesis to capture and immobilize DNA.

10. The method of claim 4, wherein alkene terminal functional groups are modified by activation by alcohol-isocyanate to capture and immobilize proteins.

11. The method of claim I₁ wherein the hydrophilic layer is ethylene glycol, a dextran-based oligomer or a polymer.

12. The method of claim 1, wherein the biomolecule probes are oligo-nucleotides of various lengths, small molecules, peptides, sugars, oligo-saccharides, proteins or protein fragments.

13. A method of inhibiting oxidation and immobilizing biomolecules on a hydrogen-terminated silicon substrate, the method comprising: a. functionalizing a surface of the hydrogen-terminated silicon substrate with a linear hydrocarbon possessing an ester to initiate growth of a hydrophobic dendrimer layer; b. growing the hydrophobic dendrimer layer by reacting the ester with a Grignard reagent possessing terminal functional groups, said terminal function groups serving as linkage points to a hydrophilic layer; and c. modifying the terminal functional groups to present biomolecule probes wherein the hydrophobic dendrimer monolayer inhibits oxidization of the silicon substrate, the hydrophilic layer serves to inhibit non-specific bonding and the biomolecule probes recognize and capture biomolecules.

14. A method for providing a protective layer on a hydrogen-terminated silicon substrate to inhibit oxidation and capture biomolecules, comprising: a. functionalizing a surface of the hydrogen-terminated silicon substrate to initiate growth of a hydrophobic dendrimer monolayer to inhibit oxidation; and b. linking the hydrophobic dendrimer monolayer to a hydrophilic layer presenting biomolecule probes to capture biomolecules.

15. The method of claim 14, wherein the surface of the hydrogen-terminated silicon substrate is functionalized with a linear hydrocarbon possessing an ester.

16. The method of claim 14, wherein the hydrophobic dendrimer monolayer is grown by reaction with Grignard reagents having terminal functional groups, said terminal functional groups serving as linkage points to the hydrophilic layer.

17. The method of claim 16, wherein the biomolecule probes are bonded to the hydrophilic layer by modifying the terminal functional group.

18. The method of claim 17, wherein the biomolecule probes are bonded to the hydrophilic layer stepwise, in a one or two step process.

19. The method of claim 16, wherein the hydrogen terminated silicon substrate is functionalized by hydrosilylation of a terminal C-C double bond of the ester of the linear hydrocarbon.

20. The method of claim 16, wherein the Grignard reagent is terminated in a protected alkyne, protected alcohol, protected amine, protected carboxylic acid, protected aldehyde, alkene or azide.

21. The method of claim 17, wherein the terminal functional groups are modified by a process selected from epoxidation, metathesis, deprotection or activation.

22. The method of claim 17, wherein protected carboxylic acid, protected aldehyde or protected alcohol terminal functional groups are modified by metathesis, to capture and immobilize DNA.

23. The method of claim 17, wherein alcohol terminal functional groups are modified by activation by hexamethylene diisocyanate to capture and immobilize proteins.

24. The method of claim 14, wherein the hydrophilic layer is ethylene glycol, a dextran-based oligomer or a polymer.

25. The method of claim 14, wherein the biomolecule probes are oligo-nucleotides of various lengths, small molecules, peptides, sugars, oligosaccharides, proteins or protein fragments.

26. A method for providing a protective layer on a hydrogen-terminated silicon substrate to inhibit oxidation and capture biomolecules, comprising: a. functionalizing the hydrogen-terminated silicon substrate surface with a linear hydrocarbon possessing an ester to form a hydrophobic dendrimer monolayer; b. growing the hydrophobic dendrimer layer by reacting the ester with a Grignard reagent possessing terminal functional groups, said terminal function groups serving as linkage points to a hydrophilic layer; and c. modifying the terminal functional group to present biomolecule probes wherein the hydrophobic dendrimer monolayer inhibits oxidization of the silicon substrate, the hydrophilic layer serves to inhibit non-specific bonding and the biomolecule probes capture biomolecules.

27. A functionalized silicon substrate for biosensing applications, comprising: a. a hydrogen-terminated silicon substrate; and b. a protective a monolayer of hydrophobic dendrimers formed on a surface of the silicon substrate and chemically linked to a hydrophilic layer presenting biomolecule probes, wherein the hydrophobic dendrimer monolayer inhibits oxidation of the silicon substrate and the biomolecule probes capture biomolecules.

28. The functionalized silicon substrate of claim 27, wherein the hydrophilic layer is ethylene glycol, a dextran-based oligomer or a polymer.

29. The functionalized silicon substrate of claim 27, wherein the biomolecule probes are oligo- nucleotides of various lengths, small molecules, peptides, sugars, oligo-saccha rides, proteins or protein fragments.

30. A protective multi-component layer formed on a hydrogen-terminated silicon substrate comprising a hydrophobic dendrimer monolayer to inhibit oxidation of the silicon substrate and a hydrophilic layer bearing biomolecule probes chemically linked to the hydrophobic dendrimer monolayer, to capture biomolecules.

31. The protective layer of claim 30, wherein the hydrophilic layer is ethylene glycol, a dextran-based oligomer or a polymer.

32. The protective layer of claim 30, wherein the biomolecule probes are oligo-nucleotides of various lengths, small molecules, peptides, sugars, oligo-saccharides, proteins or protein fragments.

33. The use of an organic dendrimer monolayer to protect a surface of a silicon substrate from oxidation by attaching to more than 80% of surface silicon atoms on the silicon substrate.