Patent Publication Number: US-7582422-B2

Title: Modified carbon and germanium surfaces

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional of U.S. application Ser. No. 09/659,442, filed Sep. 8, 2000 (now U.S. Pat. No. 6,569,979, issued May 27, 2003), the entirety of which is incorporated herein by reference. 
    
    
     U.S. GOVERNMENT SUPPORT 
     This invention was made with United States government support awarded by the following agency: NSF Grant No. 9613799. The United States has certain rights in this invention. 
    
    
     FIELD OF THE INVENTION 
     The invention is directed to modified carbon, silicon, and germanium surfaces (including modified surfaces of alloys containing any combination of carbon, silicon and/or germanium). Specifically, the invention is directed to unoxidized carbon, silicon, and germanium surfaces modified to have bonded thereto a linker moiety capable of anchoring various other chemical moieties to the unoxidized surface. These modified surfaces are suitable for the fabrication of surface-bound chemicals, including surfaces of bound nucleic acids and nucleic acid arrays comprising two or more such surfaces. 
     DESCRIPTION OF THE PRIOR ART 
     Surfaces suitable for the immobilization of nucleic acids have become an increasingly important biological tool in recent years. Arrays of nucleic acid molecules, either as double-stranded segments or as short, single-stranded oligonucleotides, have been utilized for drug development, genetic sequencing, medical diagnostics, nucleic acid-ligand binding studies and DNA computing. 1-21  The principal advantages of using surface-bound oligonucleotides over those in solution include ease of purification, conservation of material and reagents, reduction of interference between oligonucleotides, and facilitated sample handling. 12  Previously explored surfaces for immobilization of nucleic acids include latex beads, 5  polystyrene, 1  carbon electrodes, 22-25  gold, 17,23,26-28  and oxidized silicon or glass. 3,8,11,29-32    
     The surface chemistries involved with these substrates, however, do not possess many of the desired characteristics of an ideal surface. These ideal surface characteristics include: surface flatness and homogeneity; control of surface properties; thermal and chemical stability; reproducibility; and amenability to nucleic acid immobilization and biochemical manipulation. More recently, the desire to use immobilized nucleic acid as a biosensor in integrated circuits 33  would require an ideal surface to be amenable to integration into a microelectronics format. Thus, the limitations of the prior art surfaces and attachment chemistries indicate a need for alternative substrates that more closely resemble the ideal. As described herein, unoxidized crystalline carbon, silicon, and germanium, allotropes thereof, and alloys thereof offer advantages as substrates for immobilization of nucleic acids and other chemicals having similar or analogous reactivity because of their high purity, highly organized and defined crystalline structure and robustness. Silicon is particularly preferred due to its ubiquitous use in the microelectronics industry. 
     However, native silicon surfaces, for example, react with air under ambient conditions to form a thin surface layer of silicon oxide. This oxidized silicon surface is chemically similar to glass and suffers from some of the same drawbacks, namely inhomogeneity and variability in the relative number of Si—O—Si and Si—OH linkages. This inhomogeneity and the concomitant chemical variability it engenders can lead to difficulties in the reproducibility and homogeneity of the subsequent chemical-modified surfaces, particularly as the silane chemistry generally employed to couple to such surfaces is itself prone to stability problems and difficult-to-control polymerization processes. 34,35    
     Chemical pathways for direct functionalization of silicon substrates without an oxide layer have opened up new possibilities for highly-controlled nucleic acid attachment. These new attachment methods provide modified silicon surfaces through direct carbon-silicon bonds, 19,36-42  and have resulted in methyl-, chlorine-, ester-, or acid-terminated substrates. 36,39,40,43,44  Although Wagner et al. 19  describe using an N-hydroxysuccinimidyl ester-terminated silicon surface to immobilize a 1752 bp double-stranded section of DNA, this reference does not disclose a systematic, reproducible method to produce chemically-modified silicon surfaces in general, nor nucleic acid-modified silicon surfaces in particular. 
     Reactions of ω-alkenes with silicon surfaces have been described in the prior art, either mediated by diacyl peroxides, 36  through UV irradiation, 19,41  or by direct thermal activation. 39  The ω-alkenes used have contained a variety of functional groups, including esters, acids, 39,44  and chlorides. 36    
     SUMMARY OF THE INVENTION 
     The invention is directed to a method of creating modified carbon, silicon, and germanium surfaces comprising reacting a straight, branched, or cyclic alkene having a substitutent thereon with an unoxidized carbon, silicon, or germanium substrate to yield a modified substrate having bonded directly thereto, in the absence of any intervening oxygen atoms, substituted alkyl moieties. 
     A preferred embodiment of the invention is directed to a method of creating modified surfaces on unoxidized carbon, silicon, and germanium surfaces, the method comprising first reacting an amino-, carboxy, or thiol-modified and protected alkene with an unoxidized carbon, silicon, or germanium substrate to yield a surface of protected, amino-, carboxy, or thiol-modified alkane molecules covalently bonded to the substrate in the absence of any intervening oxygen molecules. Then the modified alkane molecules of the first step are deprotected, thereby yielding a surface of unprotected modified alkane molecules bonded to the substrate. Next, the deprotected modified alkane molecules are reacted with a crosslinker, whereby the crosslinker is attached to the modified alkane molecules. Lastly, molecules such as nucleic acids or proteins are attached to the crosslinker of the previous step, thereby yielding a surface of molecules immobilized on the substrate. 
     The invention is also directed to modified substrate produced according to these methods. 
     Similarly, another embodiment of the invention is drawn to a modified carbon, silicon or germanium substrate comprising a substrate selected from the group consisting of unoxidized, hydrogen-terminated carbon, silicon, and germanium and a layer of substituted alkane molecules bonded directly to the substrate, in the absence of any intervening oxygen molecules, the substituted alkane molecules having attached thereto a substituent selected from the group consisting of an amino-containing substituent, a carboxy-containing substituent, and a thiol-containing substituent. 
     In the preferred embodiment of the method, UV radiation mediates the reaction of a protected ω-unsaturated aminoalkene, hydroxyalkene, carboxyalkene, or thioalkene (the most preferred being 10-aminodec-1-ene protected with t-BOC) with hydrogen-terminated silicon (001). Removal of the protecting group yields an aminoalkane-modified, hydroxyalkane-modified, carboxyalkane, orthioalkane-modified silicon surface. These reactive terminal groups (amino, hydroxy, carboxy, thio) can then be coupled to modified oligonucleotides or other biomolecules using a heterobifunctional crosslinker, preferably SSMCC, thereby permitting the preparation of biomolecule arrays on an otherwise inert substrate. 
     A primary advantage of the present invention is that it enables the formation of highly ordered biomolecule arrays of defined surface density, including oligonucleotide arrays, on an inert, inorganic, unoxidized substrate. 
     Another distinct advantage of the present invention is that the surface density of biomolecules in the array can easily be controlled and manipulated. Control of surface density is provided in two fashions: In the first approach, binary mixtures of the protected ω-unsaturated aminoalkene and dodecene are utilized in an initial UV-mediated coupling reaction. Here, a linear relationship was found between the mole fraction of ω-unsaturated aminoalkene in the binary mixture and the density of oligonucleotide hybridization sites. In the second approach, only a portion of the protecting groups is removed from the surface by limiting the time allowed for the deprotection reaction, thereby limiting the number of available binding sites for oligonucleotide attachment. 
     Another advantage of the invention is the stability of the arrays. Oligonucleotide-modified surfaces formed utilizing the present invention are extremely stable and perform well in DNA hybridization assays. These surfaces provide an alternative to gold or glass for surface immobilization of oligonucleotides in DNA arrays as well as a route for the coupling of biomolecular recognition elements to semiconductor materials. 
     By permitting control of the density of the biomolecules within the array, the invention described herein also permits control of the hydrophobicity and concomitant wetting characteristics of the functionalized silicon surfaces, an important aspect in preparing reproducible and well-defined biomolecule arrays. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 : Reaction scheme for immobilization of DNA on an amine-modified silicon (001) wafer. A layer of t-BOC-protected 10-aminodec-1-ene was bound to the surface by applying a thin layer of the protected amine to the wafer and exposing it to ultraviolet light from a low pressure mercury vapor lamp. Deprotection was accomplished by treating the surface with 25% TFA in CH 2 Cl 2  for 1 hour followed by a 5 minute rinse in 10% NH 4 OH. The cross-linker SSMCC was added to the surface, whereby it bound to the free amines through an N-hydroxysuccinimidyl ester. Thiol DNA was then bound to the maleimide groups of the SSMCC. 
         FIGS. 2   a  through  2   f : X-ray photoelectron spectra of the t-BOC-protected 10-aminodec-1-ene ( FIG. 2   a ). The nitrogen spectrum shows a single peak at 400.5 eV. After treatment in 25% TFA for 1 hour ( FIG. 2   b ), two peaks are recorded. The NH 3   +  and the primary amine have been assigned the peaks at 401.9 and 400.2 eV, respectively. A surface that has been treated with 25% TFA followed by a five-minute rinse in 10% NH 4 OH shows only one peak at 400.3 eV ( FIG. 2   c ). This indicates the NH 3   +  nitrogens have been completely deprotonated. The carbon spectrum of the t-BOC-protected amine shows a strong peak at 285 eV ( FIG. 2   d ) assigned to the bulk carbons, as well as a peak at 289.7 eV assigned to the carbonyl carbon of the t-BOC group. The carbonyl peak is reduced by approximately 70% after deprotection with TFA ( FIG. 2   e ) and NH 4 OH ( FIG. 2   f ). The shoulder at 287.2 eV has been attributed to the t-butyl carbon. 
         FIGS. 3   a  through  3   c : Images of DNA-modified silicon (001) obtained using a Molecular Dynamics “FluorImager”-brand fluorescent imaging device. Two different oligonucleotides are attached to the surface in two spots approximately 2 mm in diameter. Hybridization with the fluorescent complement of the upper spot shows the expected image ( FIG. 3   a ). Denaturation and hybridization to the lower spot also shows the expected image ( FIG. 3   b ). Denaturation and hybridization with both complements allows both spots to be visualized ( FIG. 3   c ). 
         FIG. 4 : Control of oligonucleotide density using binary mixtures of 10-aminodec-1-ene and dodecene. Mixtures containing different percentages of t-BOC-protected 10-aminodec-1-ene and dodecene were attached to the surfaces using UV light. Deprotection and addition of SSMCC and thiol DNA followed by hybridization with the fluorescent complement allowed for quantification of the signal. A linear relationship is observed between the fluorescence signal obtained and the percent of amine attached to the surface. (y=0.0081x+0.1664; R 2 =0.9045) 
         FIG. 5 : Control of oligonucleotide density demonstrated by exposing surfaces to deprotection conditions for differing lengths of time. Surfaces were deprotected, then modified with SSMCC and thiol DNA. Hybridizing with the fluorescent complement followed by imaging on a “FluorImager”-brand imaging device allowed for quantification of the signal. Exposure to TFA rapidly deprotects the t-BOC-modified 10-aminodec-1-ene molecules to yield surface amines. 
         FIG. 6 : Stability of silicon surfaces covalently modified with fluorescently tagged oligonucleotides. DNA-modified silicon surfaces were prepared using 3′ thiol DNA with fluorescein on the 5′ end. The surfaces were stored overnight in HB then scanned with a “FluorImager”-brand imaging device. Hourly scans recorded the surface fluorescence intensity. The observed fluorescence signal is stable throughout the experiment. (y=−0.0041x+0.9395). 
         FIG. 7 : Stability of surfaces during 15 hybridization and denaturation cycles. The measured fluorescence signal from both the hybridized DNA and the fluorescein tagged DNA decreases by approximately 1% with each hybridization cycle. The surfaces were also imaged after each denaturation step to demonstrate the complete removal of the fluorescent complement. No fluorescence signal from the complement was observed after denaturation. (hybridized DNA signal, y=−0.0097x+1.0059; fluorescein tagged DNA, y=−0.01 13x+1.0241) 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Abbreviations: 
     The following abbreviations are used throughout the specification. 
     “Alkane(s)”=C 2 -C 24  linear, branched, or cyclic alkane, and, where appropriate, corresponding alkyl groups. 
     “Alkene(s)”=C 2 -C 24  linear, branched, or cyclic alkene, mono- or poly-unsaturated, cis or trans or a combination thereof, and, where appropriate, corresponding alkenyl groups. 
     “Biomolecule”=any molecule found in biological material, expressly including, but not limited to nucleic acids, proteins, peptides, antibodies, enzymes, cell-wall components such as phospholipids, etc., and modified and synthetic forms thereof, such as labeled biomolecules and recombinant biomolecules. 
     “Crosslinker”=Any chemical crosslinking reagent having two reactive moieties available for forming intermolecular bonds; the two reactive moieties may be the same or different. 
     “Fmoc”=9-fluorenylmethoxycarbonyl. 
     “HB”=Hybridization buffer; synonymous with 2×SSPE/0.2% SDS buffer (20 mM NaH 2 PO 4 , 300 mM NaCl, 2 mM EDTA, and 7 mM sodium dodecyl sulfate, pH 7.4.) or any buffer of equivalent functionality. 
     “Heterobifunctional crosslinker”=Any chemical crosslinking reagent having two reactive moieties available for forming intermolecular bonds wherein the two reactive moieties are different from each other. 
     “Modified alkene” and “ω-Modified alkene”=A“modified alkene” is an alkene modified by the addition of a chemically-reactive moiety such as an amino, hydroxy, carboxy, or thiol moiety. The reactive moiety may be located anywhere in the alkene with the exception of an alkenyl carbon. An “ω-modified alkene” is an alkene wherein the reactive moiety is situated on the terminal carbon of the alkene chain. 
     “Nucleic acid”=deoxyribonucleic acids (DNA), ribonucleic acids (RNA), and peptide nucleic acids from any source, and modified forms thereof, including, without limitation, labeled (radioactive, fluorescent, etc.) nucleic acids, and nucleic acids modified to include a binding moiety such as a thiol group or a biotin tag. “Nucleic acid” includes both DNA and RNA oligonucleotides comprised of the standard bases A, C, T, G, and U, and modified bases thereof, such as mono- or dimethylated bases, dideoxy bases, etc. 
     “Protecting group”=a chemical moiety capable of selective addition to and removal from a reactive site to allow manipulation of a chemical entity at sites other than the reactive site. A host of amine and hydroxyl protecting groups are known in the art. tBOC and Fmoc are examples of protecting groups. 
     “SSMCC”=sulfosuccinimidyl 4-N-maleimidomethyl)-cyclohexane-1-carboxylate. 
     “tBOC”=tert butoxycarbonyl, a protecting group 
     “TFA”=trifluoroacetic acid. 
     “UV”=ultraviolet 
     Reaction Chamber: 
     In the most preferred embodiment of the method described herein, an ω-modified and protected alkene is reacted with a carbon, silicon, or germanium substrate to yield an array of protected, ω-modified alkane molecules covalently bonded to the substrate. It is most preferred that this reaction be photoinitiated by contacting the alkene with the substrate and then exposing the two to a suitable wavelength of UV light, for example 254 nm. For this purpose, any reaction chamber designed for conducting photoinitiated chemical reactions may be utilized. Such reaction chambers are well known to the art and can be fabricated or purchased from many commercial suppliers. A suitable reaction chamber for use in the present invention is described in detail in Example 4. 
     Because the initial reaction of the inventive method is photoinitiated, by controlling where the light falls upon the substrate/alkene reactants, the points of attachment can be photopatterned. UV photopatterning is preferred to create a patterned surface, although the means to create the patterned surface is not critical so long as the method reliably yields the desired pattern. For example, microcontact printing methods can also be used to yield a patterned surface. Using UV patterning, the surface is exposed to the UV radiation through a suitably UV-opaque mask that prevents reaction from occurring at the shielded locations. 
     After photopatterning, the surface of the substrate is gently rinsed to remove any remaining reactants. 
     Photopatterning is, of course, optional. The entire substrate may be exposed to the UV radiation to yield a substrate uniformly coated with covalently attached ω-modified alkanes. Note that the photoreactive moiety is the double bond of the alkene, which is consumed in the reaction to yield an C—Si covalent attachment in the case of a silicon substrate. 
     ω-Modified Alkenes: 
     An alkene having a reactive moiety, and preferably a terminal reactive moiety, is utilized in the present reaction to provide a means to attach molecules to the substrate surface. The reactive moiety may be situated anywhere within the modified alkene, except on an alkenyl carbon. If the reactive moiety is attached at a terminus of the alkene, the alkene is defined herein as an ω-modified alkene. It is much preferred that the alkene be between 3 and 24 carbons long and have a single unsaturation at or near a terminal carbon of the alkene. The alkene may have more than one unsaturation, and may be linear, branched, or cyclic. 
     The modification to the alkene may be the addition of any moiety which enables further covalent linkages to be made to the alkene. As noted in the definitions, the modification need not necessarily be located at the physical terminus of the alkene, although that is much preferred. Such modifications include, without limitation, the addition of an amine-containing group, a carboxyl-containing group, or a thiol-containing group to or near the co carbon of the alkene chain. 
     Because the modification may itself be reactive, it may need to be protected during the first reaction of the alkene with the substrate. Not all reactive modifications need to be protected, however. For example, many carboxy-containing groups will not be adversely affected by the chemistry used to attach the alkene to the substrate, and therefore do not need to be protected. In general, protecting the reactive modification on the alkene can be done with any type of protecting group suitable for the reactive modification chosen. 50  In short, the choice of a protecting group used is dependent in large measure upon the nature of the modification made to the alkene. If the modification is the addition of a carboxyl group, there may be no need for a protecting group at all, or a carboxy protecting group would be used. Likewise, if the modification is the addition of an amino or thiol group, an amino or thiol protecting group, respectively, would be used. Numerous such protecting groups, for any number of reactive moieties, such as amine, carboxy, and thiol functionalities, are known to the art. Many are quite hydrophobic. For amino-terminated alkenes, t-BOC is most preferred. t-BOC is a common acid labile protecting group. 50  Other acid-labile protecting groups are known to the art. 
     An illustrative example of a photoinitiated reaction of an amino-modified, t-BOC-protected alkene with a silicon substrate is shown in  FIG. 1 . Here, the protected, amino-modified alkene is contacted with the substrate and exposed to UV light. This causes the terminal double bonds of the alkene to form covalent bonds to the unoxidized silicon substrate. 
     Once the protected alkene has been reacted with the substrate, the protecting groups (if any) are removed. Removal can be accomplished chemically and depends in large measure upon the type of protecting group chosen. Such methods of protecting group removal are well known to those in the art. For the t-BOC protecting group, treatment with TFA removes the protecting group to yield a reactive primary amine group. 
     The preferred modified alkene for use in the invention is an ω-amino-C 3 -C 24 -alkene, having a single α unsaturation, a straight-chain or cyclic alkene being preferred over a branched alkene; the most preferred ω-modified alkene is 10-aminodec-1-ene. As noted above, the preferred protecting group for amine-modified alkenes is t-BOC. 
     Attaching Molecules To The Surface-Bound Alkane: 
     Once the substrate surface has been modified to contain a monolayer of reactive moieties in the form of modified alkanes bound to the surface of the substrate, molecules can then be bound to the modification by any means known to the art. The molecules can be attached directly to the modification or a crosslinker can be interposed between the modification of the surface-bound alkane and the molecule to be attached. 
     Where the modification to the alkene is either a carboxy or amino moiety, well characterized chemistries, widely used in the solid-phase synthesis of polypeptides, can be utilized to link proteins or other carboxy- or amino-group bearing biomolecules directly to the ω-modification of the surface-bound alkane. 
     Briefly, in solid phase reactions, reactive moieties in the incoming molecule to be bound to the substrate are protected, with the exception of one or more moieties which will serve as the attachment point. In certain instances, the moiety residing at the desired attachment point must be activated to induce a reaction between the incoming molecule and the modification of the surface-bound alkane. For example, by activating a molecule bearing a carboxy group, such as by reacting with dicyclohexylcarbodiimide, the incoming molecule will bind to an exposed amino group at the activated carboxy moiety. Whereas in peptide synthesis, the process is repeated over and over to add additional amino acids, here, only a single coupling reaction is employed to link the incoming molecule onto alkane-covered surface. 
     It is more preferred, however, to utilize a crosslinker, and more preferred still to use a heterobifunctional linker to provide a link between the molecule and the alkane-covered substrate. The most preferred linker is SSMCC. SSMCC contains an amine-reactive N-hydroxysuccinimide ester as well as a thiol-reactive maleimide moiety. This bifunctionality makes SSMCC the preferred linker due to its ability to bind nucleic acids to the alkane-modified substrate. Such a link is shown in the last reaction step depicted in  FIG. 1 . 
     Other suitable crosslinking moieties are known for other types of modifications. For example, diamines, dialdehydes, dicarboxylic acids, amino acids, and the like can be used as crosslinking agents. 
     To attach nucleic acids to the modified, substrate-bound alkanes, the deprotected surface is contacted with SSMCC for a time sufficient to allow the N-hydroxysuccinimide ester portion of the SSMCC to react with the amino group of the bound alkane. The substrate is gently rinsed to remove unreacted SSMCC. The SSMCC-modified substrate is then reacted with thiol-modified nucleic acid to form nucleic acid arrays on a silicon substrate. The nucleic acid-modified surfaces are then rinsed with distilled water and stored at 37° C. in HB to remove non-specifically bound strands of nucleic acid. 
     To ensure the quality of the initial reaction with the modified alkene, the reaction with the crosslinker (if any), or the reaction with an incoming molecule such as a nucleic acid, each reaction step may be monitored using PM-FTIRRAS, XPS, contact angle measurements, etc. 
     X-Ray Photoelectron (XPS) Measurements: 
     X-ray photoelectron spectroscopy (XPS) was performed on protected and deprotected surfaces to measure the extent of deprotection. X-ray photoelectron spectra of-BOC-protected 10-aminodec-1-ene bound to a silicon substrate are shown in  FIG. 2   a . (See Examples 2, 3, and 5.) The nitrogen spectrum shows a single peak at 400.5 eV. After treatment in 25% TFA for 1 hour ( FIG. 2   b ), two peaks are recorded. The NH 3   +  and the primary amine have been assigned the peaks at 401.9 and 400.2 eV, respectively. A surface that has been treated with 25% TFA followed by a five-minute rinse in 10% NH 4 OH shows only one peak at 400.3 eV ( FIG. 2   c ). This indicates the NH 3   +  nitrogens have been completely deprotonated. The carbon spectrum of the t-BOC-protected amine shows a strong peak at 285 eV ( FIG. 2   d ) assigned to the bulk carbons, as well as a peak at 289.7 eV assigned to the carbonyl carbon of the t-BOC group. The carbonyl peak is reduced by approximately 70% after deprotection with TFA ( FIG. 2   e ) and NH 4 OH ( FIG. 2   f ). The shoulder at 287.2 eV has been attributed to the t-butyl carbon. 
     Regrettably, the nitrogen I s peak of the t-BOC protected surface is nearly identical to that of the nitrogen peak on the primary amine surface (see  FIGS. 2   a  and  2   c ). XPS of the amine salt, taken before rinsing in ammonium hydroxide, clearly shows two nitrogen peaks ( FIG. 2   b ). A peak at 401.9 eV binding energy is thought to represent the NH 3   +  while a peak at 400.2 eV is assigned to the primary amine nitrogens. It is interesting to note that the nitrogens do not become completely protonated after exposure to the TFA solution. After rinsing in ammonium hydroxide a single peak at 400.3 eV appears ( FIG. 2   c ). The carbon 1s spectrum of the protected surface shows a peak at 289.7 eV assigned to the carbonyl carbon of the t-BOC group ( FIG. 2   d ). This peak is dramatically reduced using the deprotection conditions, indicating that most of the t-BOC groups have been removed ( FIG. 2   e  and  2   f ). Peak fitting using IGOR indicates approximately 70% of the t-BOC groups have been removed. It should be noted, however, that areas of these smaller peaks could only be estimated due to the large amount of error associated with the fitting parameters. Optimizing the deprotection conditions through increasing the temperature, TFA concentration, and time of reaction yielded similar results. 
     Contact Angle Measurements: 
     Contact angles were measured for the surface at various stages of deprotection. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 SURFACE 
                 CONTACT ANGLE θ 
               
               
                   
                   
               
             
            
               
                   
                 t-BOC-protected amine 
                 78.1° 
               
               
                   
                 TFA-treated 
                 55.4° 
               
               
                   
                 TFA &amp; NH 4 OH-treated 
                 76.8° 
               
               
                   
                   
               
            
           
         
       
     
     The t-BOC protected amine was the most hydrophobic. Upon exposure to 25% trifluoroacetic acid for one hour the contact angles became smaller, consistent with the behavior expected from the hydrophilic surface associated with the protonated amine salt. Rinsing the surface further in 10% ammonium hydroxide removed the salt and produced the primary amine surface, which was evidenced by an increase in the contact angle measurements 
     Specific Hybridization: 
     As described in the Examples, amine-modified surfaces were employed as substrates for the preparation of small DNA arrays, which were characterized with respect to their performance in DNA hybridization experiments. See  FIGS. 3   a - 3   c . Two different types of oligonucleotides were immobilized on an unoxidized silicon (001) surface (approximately 1 cm×1 cm) in approximately 2 mm diameter spots. A solution of fluorescently labeled oligonucleotide complementary to one of the immobilized oligonucleotides was placed on the surface and allowed to hybridize.  FIG. 3   a  shows the image obtained after hybridization and washing. A single spot appears, indicating successful hybridization to the selected oligonucleotide. Imaging following denaturation and hybridization with the alternate fluorescent oligonucleotide shows the second individual spot ( FIG. 3   b ). A final denaturation and subsequent hybridization with both fluorescently tagged complements, followed by imaging revealed both spots, as expected ( FIG. 3   c ). 
     Control of Surface Density of Attachment Sites: 
     Control of the density of surface attachment sites is a major advantage of the present invention because it allows for control of the density of bound biomolecules such as oligonucleotides. For example, while some applications may require densely packed DNA strands, others may require greater spacing between bound oligonucleotides. Control of the surface density of attachment points on the substrate surface was demonstrated in two different ways; 1) by using mixtures of a protected amino-modified alkene and dodecene during the attachment step; and 2) by controlling the exposure time of the t-BOC protected amine surface to TFA, thereby limiting the extent of deprotection of the amine-modified surface. 
     Control of the number of surface reactive amine sites was demonstrated using mixtures of t-BOC-protected 10-aminodec-1-ene and dodecene. Dodecene, because it lacks an ω reactive moiety, is unreactive once attached to the silicon surface. Mixtures containing increasing fractions of t-BOC-protected 10-aminodec-1-ene were applied to the surfaces and allowed to react under the UV lamp. The surfaces were then treated as described herein with TFA (to deprotect), SSMCC (a crosslinker), and DNA. Hybridization with the fluorescent complement and subsequent quantification of the fluorescence image was done on the surfaces. The plot shown in  FIG. 4  shows a linear relationship between the measured fluorescence signal and the fraction of 10-aminodec-1-ene. This finding clearly demonstrates that the density of surface amine groups can be readily controlled, which in turn allows for control of the ultimate surface density of bound oligonucleotides or other biomolecules available for hybridization or further study. 
     An alternate method of controlling the density of surface attachment sites involves controlling the exposure time of the initially protected, ω-modified surface to deprotection conditions. To demonstrate this approach, four silicon surfaces were prepared with t-BOC protected 10-aminodec-1-ene. The surfaces were then exposed to 25% TFA for 0, 2, 15, and 60 minutes. The surfaces were then modified with SSMCC and 5′-thiol-DNA and hybridized with fluorescent complement.  FIG. 5  is a plot of the fluorescence signals of each of these surfaces. The amount of fluorescence measured depends on the time of exposure to the deprotecting agent. Deprotection occurs rapidly up to about 15 minutes of exposure time, at which point most of the t-BOC groups have been removed. Continuing the deprotection for 60 minutes yields slightly more free amines available for reaction. 
     Stability: 
     An important parameter of the DNA-modified silicon surfaces is their stability to the conditions employed for biochemical procedures such as nucleic acid hybridization and enzymatic manipulation. Two experiments were performed to evaluate the stability of the DNA-modified surfaces. The stability of the bonds linking a DNA oligonucleotide covalently to the surface was evaluated by means of a fluorescent tag covalently attached to the oligonucleotide (SEQ. ID. NO:3); the surface was incubated in HB overnight and the surface fluorescence was measured repeatedly (see  FIG. 6 ). No significant decrease in the fluorescence intensity was observed, indicating that the DNA-surface linkage was stable under the conditions examined. 
     In a second experiment, a surface modified with fluorescein labeled DNA (SEQ. ID. NO:3) in one area and non-labeled DNA (SEQ. ID. NO:1) in another was hybridized repeatedly with the fluorescent complement of SEQ. ID. NO:1. The measured fluorescence intensity of SEQ. ID. NO:3 was typically two thirds that of SEQ. ID. NO:1 hybridized to its fluorescent complement. The normalized data show both signals decay at essentially identical rates ( FIG. 7 ). The fluorescence intensity after 15 cycles decays to approximately 85% of the initial value. This corresponds to a loss of approximately 1% per cycle. This stability is superior to previously published results using a non-covalent surface attachment chemistry. 44  These two findings demonstrate that unoxidized silicon surfaces modified according to the invention exhibit excellent stability under the storage and hybridization conditions employed. 
     EXAMPLES 
     The following Examples are included solely to provide a more complete understanding of the invention disclosed and claimed herein. The Examples do not limit the scope or generality of the attached claims in any fashion. 
     Example 1 
     Synthesis of Oligonucleotides 
     All oligonucleotides were synthesized by the University of Wisconsin Biotechnology Center, Madison, Wis. Two types of oligonucleotides were employed for the Examples which follow: oligonucleotides to be attached to the surface, and oligonucleotides employed for hybridization to the surface. Oligonucleotides to be attached to the surface were thiol-modified at either the 5′ end using the reagent 5′-Thiol-Modifier C6 (HSC6, Glen Research, 22825 Davis Dr., Sterling, Va. 28164, www.glenres.com), or at the 3′ end using the reagent 3′-Thiol-Modifier C3 S-S CPG (C3SH, Glen Research); each of these oligonucleotides were 31 nt in length, comprised of a 15-mer dT spacer sequence at the 5′ end, and a specific 16-mer sequence at the 3′ end. The three 31-mer sequences employed were 
     
       
         
           
               
               
               
            
               
                 (SEQ. ID. NO:1) 
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 5′-HSC6-T 15 AA CGA TCG AGC TGC AA3′, 
                   
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                 (SEQ. ID. NO:2) 
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 5′-HSC6-T 15 AA CGA TGC AGG AGC AA3′, and 
                   
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                 (SEQ ID NO:3) 
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 5′-FAM-T 15 AA CGA TCG AGC TGC AA-C 3 SH3′. 
                   
               
            
           
         
       
     
     In SEQ. ID. NO:3, “FAM” refers to a fluorescein dye incorporated into the oligonucleotide during synthesis using the reagent 6-FAM phosphoramidite (Perkin-Elmer Biosystems, Foster, Calif.). Oligonucleotides employed for hybridization to their surface-bound complements were 16 nt in length and complementary to the 3′ 16 nt of either SEQ. ID. NO:1 or SEQ. ID. NO:2. These oligonucleotides were also modified with FAM on the 5′ end. The thiol-modified oligonucleotides were deprotected according to guidelines from Glen Research, 45,46  and purified by reversed-phase HPLC with a binary gradient elution. The 16 nt complements were purified in the same manner. Other reagents were obtained from Aldrich Chemical Company, Milwaukee, Wis. unless otherwise noted and are as follows: 1-dodecene, 10-undecenoyl chloride, sodium azide, trifluoroacetic acid (TFA), di-tert-butyl dicarbonate (Acros Organics, Janssens Pharmaceuticalaan 3A, 2440 Geel, Belgium, www.acros.com), sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SSMCC) (Pierce Chemical, Rockford, Ill.), silicon (001) wafers (Virginia Semiconductor, Fredricksburg, Va., www.VirginiaSemi.com). Ultra pure water used for rinsing the wafers was obtained from a Millipore system (Marlborough, Mass.). 
     Example 2 
     Synthesis of 10-Aminodec-1-ene 
     The 10-aminodec-1-ene used to modify the silicon surfaces was prepared using a variant of the Curtius reaction. 47  A solution of 40.5 g of 10-undecenoyl chloride and tetrabutylammonium bromide (200 mg) was prepared in 300 mL of dichloromethane and cooled in an ice bath. Sodium azide (15.6 g) was dissolved in 50 mL of water and added to the mixture. This mixture was stirred continuously for 2 hours in an ice bath. The organic layer was removed, washed twice with 50 mL of water, dried over anhydrous magnesium sulfate for 36 hours and filtered. Trifluoroacetic acid (20.3 mL) was slowly added with stirring and the mixture was refluxed for 6 hours. The resultant mixture containing the trifluoroacetamide was cooled and washed twice with 50 mL of saturated aqueous sodium bicarbonate, then dried over magnesium sulfate and filtered. The solvent was removed by rotary evaporation to yield an oil which was vacuum distilled to produce the pure trifluoroacetamide. Nuclear magnetic resonance confirmed the identity of the trifluoroacetamide. Cleavage of a 10 mL portion of the trifluoroacetamide was accomplished by refluxing for 3 hours in 500 mL of a 7% potassium carbonate water:methanol (2:5) solution. 48  The free amine was extracted twice with 100 mL of ether, which was again dried over magnesium sulfate and filtered. Removal of the solvent by rotary evaporation and vacuum distillation yielded the desired product. Nuclear confirmed its identity as the pure amine. 
     Example 3 
     Synthesis of t-BOC-Protected Amine 
     t-BOC protected 10-aminodec-1-ene was prepared by standard methods. 49  A portion of the purified amine (5.11 g) was dissolved in 60 mL of chloroform that was added to a solution of 3 g NaHCO 3  in 50 mL of water. Sodium chloride (6.45 g) was added along with 7.18 g of di-tert-butyl dicarbonate dissolved in a few milliliters of chloroform. This mixture was refluxed for 90 min and extracted twice with 50 mL of ether. The collected organic extracts were dried over magnesium sulfate, filtered and the ether removed by rotary evaporation. The t-BOC-protected product was purified by vacuum distillation, and its identity was confirmed by NMR. Treating a 10 μL aliquot of this product with 1 mL of 25% TFA in methylene chloride for 1 hour at room temperature recovered the free amine as demonstrated by thin layer chromatography. 
     Example 4 
     Reaction Chamber 
     The chamber used for the UV mediated reaction consisted of a cylindrical aluminum base 11.5 cm in diameter and 5 cm tall. The interior of the base was milled to create a chamber 9 cm in diameter and 4 cm deep. A thin Teflon disk was placed in the bottom of this chamber. Inlet and outlet ports were added to allow for the continual passage of nitrogen gas. A Teflon lid 11.5 cm in diameter and 3.5 cm tall with an 8 cm diameter section removed from the middle was manufactured to fit over the aluminum base. The 8 cm diameter hole was milled to secure a quartz window. When assembled and in use, the lid was secured and a UV lamp was placed over the chamber. The UV lamp was purchased from UV Products (part number CPQ-7446) and consisted of a low-pressure mercury vapor ozone-producing quartz grid lamp mounted on an aluminum reflector. The intensity of the lamp at 15 cm was 2000 microwatts/cm 2  (254 nm). 
     Example 5 
     Preparation of DNA Modified Surface 
     Silicon wafers were prepared for modification as previously described. 19,41,44  The wafers were briefly exposed to a 2% solution of hydrofluoric acid in water to hydrogen terminate the surface atoms. The wafers were then immediately placed in the reaction chamber, covered with 10-30 μL of the t-BOC-protected 10-aminodec-1-ene, and exposed to ultraviolet light from the UV lamp for 2 hours. The volume of the t-BOC-protected 10-aminodec-1-ene used was enough to just cover the surface and varied with the size of the wafers. The thickness of the layer covering the wafer was not directly measured or controlled but was typically less than 1 mm. The modified surfaces were then subjected to 25% TFA in methylene chloride followed by a 3 minute rinse in 10% NH 4 OH to remove the t-BOC protecting group and form the primary amine-terminated surface. These surfaces were placed in a 50 μL droplet containing the heterobifunctional crosslinker SSMCC (1.5 mM in 100 mM triethanolamine buffer, pH 7) for 20 minutes. SSMCC contains an amine reactive N-hydroxysuccinimide ester as well as a thiol reactive maleimide moiety. The maleimide activated surfaces were reacted overnight with 0.4-0.8 μL droplets of approximately 1 mM thiol-modified DNA to form the DNA arrays. The DNA modified surfaces were then rinsed with distilled water and stored at 37° C. for 1 hour in 2×SSPE/0.2% SDS buffer (20 mM NaH 2 PO 4 , 300 mM NaCl, 2 mM EDTA, and 7 mM sodium dodecyl sulfate, pH 7.4, designated herein as “HB”) to remove non-specifically bound strands. 
     Example 6 
     Hybridization and Denaturation 
     Procedures utilized to hybridize fluorescent complements to the surface-bound oligonucleotides have been described in detail elsewhere. 28,44  Briefly, the DNA-modified silicon surfaces were removed from HB and rinsed with distilled water then exposed to 5-10 μl of 2 μM 5′-fluorescein labeled complement in HB, and allowed to hybridize for 20 minutes at room temperature in a humid chamber. The hybridized surfaces were then rinsed twice for five minutes in HB to remove any unhybridized complement. The hybridized DNA modified surfaces were then placed facedown in a droplet of HB on a Molecular Dynamics FluorImager 575 tray and scanned. This allowed visualization and quantitation of the fluorescent areas on the silicon surfaces. Denaturation was accomplished by placing the surfaces in an 8.3 M urea solution at room temperature for 5 minutes followed by a water rinse The surfaces were then rescanned with the FluorImager to ensure that complete denaturation had been accomplished. Subsequent hybridizations, if required, could be repeated using the same procedure. Alternatively, for the number density experiment, denaturation was accomplished by placing the hybridized surfaces in a small volume of water at 90° C. for 15 minutes. This denatured the duplex and allowed the fluorescent complements to be collected for quantitation.(28,44) 
     Example 7 
     Control of DNA-Modified Amine Surface 
     A series of Examples were conducted to characterize the behavior of the bound oligonucleotides and to control the density of the free amines. Hybridization specificity was tested by immobilizing two different kinds of oligonucleotides (SEQ. ID. NO:1 and SEQ. ID. NO:2) onto the surface, followed by selective hybridization to either of the fluorescent complements. The density of free amines on the surface can be controlled by reaction of the hydrogen terminated silicon surface with mixtures of t-BOC protected amine and dodecene. Alternatively, the t-BOC protected amine surfaces were partially deprotected by exposure to 25% trifluoroacetic acid for a variety of times. 
     Example 8 
     Surface Density Measurements 
     The number of molecules hybridized to the surface was determined using a previously published procedure.(28,44) Oligonucleotides were immobilized on the surface and their fluorescent complements were allowed to hybridize as before. The surface was imaged and the area of fluorescence was measured. The surface was then placed in 800 μL of 90° C. water in a microfuge tube for 15 minutes to completely denature the duplexes. The surface was then rinsed twice with 200 μL water, which were quantitatively eluted into the original 800 μL. The surfaces were rescanned to ensure complete denaturation had occurred. The volume of water containing the complements was then reduced to about 10 μL by vacuum centrifugation and loaded on one lane of a polyacrylamide gel, while known amounts of fluorescent oligonucleotide standard were loaded in the other lanes. After electrophoresis the gel was imaged and the amount of fluorescent oligonucleotide eluted from the surface was calculated by reference to a standard curve developed from the known samples. Using this method, the density of hybridized molecules was found to be approximately 2.3×10 12  molecules/cm 2 . 
     Example 9 
     Stability Measurements 
     Stability of this surface chemistry was demonstrated both with direct monitoring of immobilized fluorescent oligonucleotides and through repeated hybridizations. For direct monitoring, oligonucleotides labeled with fluorescein on their 5′ ends and a thiol group on their 3′ ends (SEQ. ID. NO:3) were covalently attached to the activated surface. After covalent attachment was allowed to occur, the surface was stored overnight in HB at 27° C. The fluorescence intensity of the surface was then measured by scanning on the FluorImager, after which the surface was returned to the buffer for 1 hour at 27° C. The surface was then rescanned and the fluorescence measured. Subsequent scans were performed every hour for a total of 10 hours while the surface was stored in the buffer at 27° C. Exposure to light was kept to a minimum throughout this experiment. 
     The stability of the surface with respect to hybridization conditions was also tested. Fluorescein labeled SEQ. ID. NO:3 as well as the non-fluorescent SEQ. ID. NO:1 were covalently attached to two different areas of the surface. The DNA modified surface was exposed to the fluorescently tagged complement of SEQ. ID. NO:1 for 20 minutes and hybridization was allowed to occur. The hybridized surface was then scanned on the FluorImager and the fluorescence signals of both DNA modified areas were quantitatively measured. The hybridized surface was placed in 8.3M urea to denature the duplex strands then rescanned to ensure the fluorescent complements were removed. The surface was subjected to 15 such cycles of hybridization, scanning, denaturing, rescanning and rehybridization (see results). 
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