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US005942397 A 

[11] Patent Number: 5,942,397 

[45] Date of Patent: Aug. 24, 1999 

United States Patent [19] 

Tarlov et ai. 

[54] 	 SURFACE IMMOBILIZATION OF 

BIOPOLYMERS 

[76] 	 Inventors: Michael J. Tarlov; Tonya M. Herne; 

Keith H. McKenney, all of 

NIST-Office of Tech. Development, 

Bldg. 820, Room 213, Gaithersburg, 

Md. 20899-0001 

[21] 	 Appl. No.: 08/988,338 

[22] 	 Filed: Dec. 10, 1997 

Related U.S. Application Data 

[60] 	 Provisional application No. 60/033,042, Dec. 11, 1996. 

[51] 	 Int. CI.6 ....................................................... C12Q 1/68 


[52] 	 U.S. CI. ................................................ 435/6; 536/24.1 


[58] 	 Field of Search ................................. 435/6; 536/24.1 


[56] References Cited 

U.S. PATENT DOCUMENTS 

5,302,509 4/1994 Cheeseman ................................. 435/6 


5,445,934 8/1995 Fodor et al. ................................ 435/6 


5,472,881 12/1995 Beebe et al. .............................. 436/94 


5,514,501 5/1996 Tarlov ......................................... 430/5 


5,622,826 4/1997 Varma ......................................... 435/6 


OTHER PUBLICATIONS 

Leavitt et aI., "Angle-Dependent X-ray Photoelectron 

Spectroscopy and Atomic Force Microscopy of SulfurModified DNA on Au(111)" in J. Phys. Chern., (1994), 98, 

8742-8746. 

Lee et aI., "Direct Measurement of the Forces Between 

Complementary Strands of DNA" in Science, (1994), 266, 

771-773. 

Okahata et aI., "Hybridization of Nucleic Acids Immobilized 

on a Quartz Crystal Microbalance" in J. Arner. Chern. Soc., 

(1992), 114,8299-8300. 

Primary Exarniner~cott W. Houtteman 

Attorney, Agent, or Firrn-Jagtiani & Associates 

[57] ABSTRACT 

In one embodiment, the present invention provides a 

biopolymer-containing monolayer comprising: thiolderivatized biopolymers and organic thiols bound to a metal 

substrate. In another embodiment, the present invention 

provides a method for forming this biopolymer-containing 

monolayer. Preferably, the biopolymers are single-stranded 

DNA probes. 

22 Claims, 6 Drawing Sheets 

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u.s. Patent Aug. 24, 1999 Sheet 1 of 6 5,942,397 

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5.8x105 

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405 400 395 390 

Binding Energy (eV) 

Fig. 1 
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u.s. Patent Aug. 24, 1999 Sheet 2 of 6 5,942,397 

Fig. 2 
u.s. Patent Aug. 24, 1999 Sheet 3 of 6 5,942,397 

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u.s. Patent Aug. 24, 1999 Sheet 5 of 6 5,942,397 

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Fig. 5 
u.s. Patent Aug. 24, 1999 Sheet 6 of 6 5,942,397 

TABLE 1. Data obtained from radio-images ofHS-ssDNAfMCH monolayers exposed to 

32P-radiolabeled complement. 

Sample Time in HS-ssDNA Time in MCH 
 Intensity' 32 p radiolabeled DNAb 

(min) (min) 
 Counts/cm2 (molecules/cm2) 

Bare Au 00 00 
 6836 ± 83 3 1 (±0.03) x 1012 


Ie 00 
 60 
 64±8 1010 
 2.9 (±04) x 

2 

3 

4 

025 


I 0 


5.0 

I 


I 

I 


60 


60 


60 


773±28 

4018±64 

8818±94 

3 5 (±0.1) X lOll 


1012
 I 8 (±O 03) X 


10 12
 4.0 (±0.04) X 


5 10.0 60 
 7064±84 1012
 32 (±004) X 


6 32.5 60 
 7627±87 10 12
 34 (±0.04) X 


7 60.0 
 60 
 8491±92 1 012
 3 8 (±O. 04) X 


8 1200 
 60 
 12627±112 1012
 57 (±005) X 


9 240.0 
 60 
 11418±107 1012
 5.2 (±004) X 


10 13130 
 60 
 9764±99 1012
 44 (±004) X 


"The intensity is obtained by totaling the number of counts in a circle of area 0 11 cm2 

bCalculated by comparing the number of photostimulated luminescence counts measured on the 

gold substrates to a radio-image obtained of a spot of 32p radio labeled DNA of known 

concentration. 

cMCR control sample 

Fig. 6 

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5,942,397 


1 

SURFACE IMMOBILIZATION OF 


BIOPOLYMERS 


RELATED APPLICATIONS 

The present application is based on U.S. Provisional 

Patent Application No. 60/033,042 filed Dec. 11, 1996, the 

entire disclosure and contents of which is hereby incorporated by reference. 

BACKGROUND OF THE INVENTION 

1. Field of the Invention 

The present invention relates generally to biopolymer 

probes immobilized on the surface of a substrate. 

2. Description of the Prior Art 

New methods for genetic screening and sequencing are 

based on the hybridization of surface immobilized singlestranded oligonucleotides, or DNA probes. Thiolderivatized, single-stranded DNA has been used to model 

hybridization reactions at surfaces. For example, Lee et aI. 

have described covalently attaching thiolated DNA to 

derivatized silane layers on silica for use in atomic force 

microscopy studies (Lee et aI., "Direct Measurement" in 

Science, (1994), 266, 771-773). Okahata et aI. have prepared a monolayer consisting of thiol-derivatized DNA 

10-mers on gold, and measured the mass changes during 

hybridization using a quartz crystal microbalance (Okahata 

et aI., "Hybridization of Nucleic Acids Immobilized on a 

Quartz Crystal Microbalance" in J. Arner. Chern. Soc., 

(1992),114,8299-8300). Leavitt et aI. describe modifying 

the phosphate group of the nucleotide backbone with a 

sulfur group which is adsorbed directly on the surface 

(Leavitt et aI., "Angle-Dependent X-ray Photoelectron 

Spectroscopy and Atomic Force Microscopy of SulfurModified DNA on Au(111)" in J. Phys. Chern., (1994), 98, 

8742-8746). 

A problem with all of these prior methods is that is that the 

DNA can interact strongly with the gold surface, resulting in 

probes with low hybridization activity. In addition, the prior 

methods do not permit facile control of the surface coverage 

of DNA probes. 

SUMMARY OF THE INVENTION 

The surface-confined biopolymers of the present invention differ from prior oligonucleotide probes in that the 

surface coverage of the thiol-derivatized biopolymers of the 

present invention can be precisely controlled by creating 

mixed mono layers of thiol-derivatized probes and a diluent 

thiol such as mercaptohexanoI. Because it has been discovered that controlling the surface coverage of thiolderivatized DNA on a gold surface can maximize the 

hybridization efficiency of surface-bound probes, the present 

invention is more useful for specific hybridization of singlestranded DNA and other nucleotides than prior immobilization strategies. 

In one embodiment, the present invention provides a 

biopolymer-containing monolayer comprising thiolderivatized biopolymers and organic thiols bound to a metal 

substrate. Preferably, the biopolymers are oligonucleotides 

and are most preferably single-stranded DNA probes. 

In another embodiment, the present invention provides a 

method for forming a biopolymer-containing monolayer 

comprising the steps of: applying thiol-derivatized biopolymers to a metal substrate to bind the thiol-derivatized 

biopolymers thereto; and applying an organic thiol to the 

substrate to bind the organic thiol to the substrate, to remove 

2 

nonspecifically bound biopolymers and to passivate the 

surface, preventing adsorption of target biopolymers from 

solution. In addition, passivation of the surface with a 

diluent thiol increases the biological activity of the immobilized biopolymers. Preferably, the biopolymer is an oligonucleotide and most preferably is single-stranded DNA. 

Particularly with respect to oligonucleotides, the present 

invention has advantages over prior immobilization strategies in that precise control can be achieved over the coverage of the thiol-derivatized, single-stranded oligonucleotides on metal surfaces. Also, the surface-immobilized 

oligonucleotide retains high activity and specificity for 

hybridization with the complementary sequence as a result 

of posttreatment with the diluent thioI. In addition, very 

little, if any, oligonucleotide from solution adsorbs nonspecifically on the two component monolayer. 

Other objects and features of the present invention will be 

apparent from the following detailed description of the 

preferred embodiment. 

BRIEF DESCRIPTION OF THE DRAWINGS 

The invention will be described in conjunction with the 

accompanying drawings, in which: 

FIG. 1 is a graph of XPS N Is data obtained from 1.0,uM 

HS-ssDNA in pure water and in 1.0 M in KH2P04 ; 

FIG. 2 is a graph of normalized XPS N Is peak areas 

plotted as a function of ionic strength of the 1.0 ,uM 

HS-ssDNA solution; 

FIG. 3 shows the XPS N Is spectra of thiol-derivatized 

and non-thiol derivatized ssDNA before (solid lines) and 

after (triangles) exposure to MCH; 

FIG. 4A is a schematic diagram of a HS-ssDNA/MCH 

monolayer before posttreatment with MCH; 

FIG. 4B is a schematic diagram of a HS-ssDNA/MCH 

monolayer after posttreatment with MCH; 

FIG. 5 is a graph of normalized N Is peak areas plotted 

as a function of sample exposure time to HS-ssDNA; 

FIG. 6 is a table (Table 1) which contains data obtained 

from radioimages of HS-ssDNA/MCH monolayers exposed 

to 32P-radiolabeled complement. 

DETAILED DESCRIPTION OF THE 


PREFERRED EMBODIMENT 


Definitions 


For the purposes of the present invention, the term "oligonucleotide" refers to any oligonucleotide, including 

double and single-stranded DNA, RNA, PNAs (peptide 

nucleic acids) and any sequence of nucleic acids, either 

natural or synthetic, derivatized or underivatized. 

For the purposes of the present invention, the term "HSssDNA" refers to a thiol-derivatized single-stranded DNA 

probe. 

For the purposes of the present invention, the term 

"biopolymer" refers to any oligonucleotide, polypeptide, 

polysaccharide, and derivatives thereof. 

For the purposes of the present invention, the term 

"organic linker" refers broadly to an a organic chain which 

links a thiol group to a biopolymer. The organic chain may 

be saturated or unsaturated, may include aryl groups, may 

include alkyl, alkenyl, or aryl side chains, and may include 

one or more functional groups such as ether groups, hydroxy 

groups, carboxy groups, halide groups, etc. 

Description 

The probes of the present invention can be formed using 

any biopolymer which can be derivatized with a thiol and 
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5,942,397 


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bound to a metal substrate through the sulfur atom of the 

thiol. The probes of the present invention are preferably 

formed from oligonucleotides. Most preferably the probes of 

the present invention are formed from single-stranded DNA 

(ssDNA) which have been reacted with a thiol to form 

thiol-derivatized DNA (HS-ssDNA). 

The hybridization activity of surface-bound HS-ssDNAis 

dependent on surface coverage. One method of varying the 

surface coverage is to control the ionic strength of the 

HS-ssDNA solution. Another method to control coverage is 

to vary the immersion time in the HS-ssDNA solution. A 

primary advantage of the surface-bound probes of the 

present invention is that nonspecifically adsorbed DNA is 

largely removed from the surface. Thus, the majority of 

surface bound probes are accessible for specific hybridization with complementary oligonucleotides and can discriminate between complementary and non-complementary target 

molecules. Moreover, the surface-bound probes of the 

present invention are stable, and can survive temperature 

cycling and exposure to air. 

The probes of the present invention are preferably bound 

to a gold substrate. However, preferred metal substrates also 

include other noble metals, such as: silver, copper, platinum, 

palladium, ruthenium, and iridium to which thiols readily 

attach through the sulfur atom. Alloys of these metals may 

also be used as a substrate in the present invention. 

The diluent thiol used in the present invention serves two 

roles: it removes nonspecifically bound HS-ssDNA, and it 

prevents nonspecific adsorption of oligonucleotides from 

solution. Specifically, the hydroxyl group functionality of 

the diluent thiol prevents nonspecific adsorption of DNA 

from solution. It is possible that thiols including functional 

groups other than a hydroxyl group may also be effective in 

preventing nonspecific adsorption of DNA from solution. 

Therefore, although hydroxy-terminated organic thiols such 

as mercaptohexanol are preferred thiols, it is contemplated 

that other suitable thiols that prevent nonspecific adsorption 

may be used for the purposes of the present invention. 

Furthermore, it is contemplated that diluent thiols having 

both a shorter and longer chain length than mercaptohexanol 

could be used for the purposes of the invention. Such diluent 

thiols may include thiols having saturated or unsaturated 

carbon chains, having aryl groups as part of the carbon 

chain, having side chains, having ether linkages, etc. 

The biopolymer is preferably derivatized so that an 

organic linker, such as a hexamethylene linker (-(CH2)6)' 

acts as a spacer group to separate the biopolymer from the 

thiol (-SH) group. When the biopolymer is ssDNA, the 

presence of this organic linker/spacer group in the 

HS-ssDNA allows all of the nucleotides of the ssDNA to be 

available for hybridization. However, the present invention 

also contemplates that the biopolymer, such as ssDNA, 

could be directly bound to the thiol group. In this situation, 

some of the ssDNA nucleotides would be used as the spacer 

group with the balance available for hybridization. 

Although HS-ssDNA is a preferred thiol-derivatized oligonucleotide for the present invention, it is contemplated 

that oligonucleotide monolayers could be formed using 

thiol-derivatized RNA, PNAs and other modified and 

un-modified oligonucleotides which are used for hybridization reactions. 

The invention will now be described by way of example. 

The following example is illustrative and is not meant to 

limit the scope of the invention which is set forth by the 

appointed claims. 

EXAMPLE 

Single-crystal (100) silicon wafers were used as substrates 

in the preparation of evaporated Au films. The Au thin films 

were prepared by thermal evaporation of 200 nm ofAu over 

a 10 nm Cr adhesion layer. The Au substrates were cleaned 

in piranha solution (70% H2S04 : 30% H20 2) before exposure to the sample solutions. 

The thiolated single-stranded DNA, abbreviated 

HS-ssDNA25, is a 25-base oligonucleotide with the following sequence: 5'-HS-(CH2kCAC GAC GTT GTA AAA 

CGA CGG CCA G-3'. The complementary single-stranded 

DNA is a 25-mer with the sequence 5'-CTG GCC GTC GTT 

TTA CAA CGT CGT G-3', abbreviated ssDNA-C25. The 

non-complementary control has the same sequence as the 

immobilized probe, 5'-CAC GAC GTT GTA AAA CGA 

CGG CCA G-3', without the HS-(CH2)6 attachment. 

HS-ssDNA25 surfaces were prepared by placing piranhacleaned Au in KH2P04 buffer solutions of DNA. The 

KH2P04 solution concentration was 1.0 M unless otherwise 

stated. Mixed monolayer surfaces containing HS-ssDNA25 

and mercaptohexanol (MCH) were prepared by immersing 

the clean gold substrate in a 1.0 ,uM solution of 

HS-ssDNA25 for 15 seconds to 22 hours, followed by a one 

hour exposure of the sample to an aqueous solution of 1.0 

mM MCH. Before analysis or hybridization, each sample 

was rinsed thoroughly with deionized water. FIG. 4Ashows, 

in schematic form, the HS-ssDNA/MCH monolayer before 

posttreatment with MCH and FIG. 4B shows, in schematic 

form, the HS-ssDNA/MCH monolayer after posttreatment 

with MCH. 

Hybridization of the HS-ssDNA25 immobilized on gold 

was measured using 32p radiolabeling. Complementary and 

non-complementary DNA oligonucleotides were radio labeled with 32p using T4 polynucleotide kinase and gamma 

32p ATP (30000 Ci/mmole). Hybridization was done at 24°0 

C. for 90 minutes in TE-1M NaCI (10 mM Tris-HCI, 1 mM 

EDTA, 1 M NaCI). After hybridization, samples were rinsed 

in 1 mL of TE eight times, ten seconds each rinse. Samples 

were air-dried before imaging. Hybridization of the surface 

bound probe with its complement was monitored by obtaining radio-images using a photostimulatable storage imaging 

plate system. The imaging instrument is capable of 100,uM 

spatial resolution. 

The amount of HS-ssDNA25 adsorbed on the surface was 

monitored by measuring the XPS N 1s signal. The inventors 

have found that the presence of the N 1s peak in the XPS 

data is a reliable indication that DNA is adsorbed on the 

surface. Bare gold samples exposed to air or buffer solutions 

containing no DNA exhibit no-XPS-detectable nitrogen. 

Therefore, any observed N is signal originated exclusively 

from the nitrogen containing purine and pyrimidine bases of 

DNA. Furthermore, the relative amounts of adsorbed DNA 

for different samples was determined by comparison of N 1s 

peak areas. Shown in FIG. 1 are XPS data obtained from 

samples immersed in 1.0,uM HS-ssDNA25 solutions, prepared either in pure water or in 1.0 M KH2P04 buffer. For 

the HS-ssDNA25 solution prepared in pure water, essentially no N 1s peak is observed, indicating that HS-ssDNA 

dissolved in water does not adsorb on gold. By contrast, a 

relatively intense N 1s peak is observed when a sample is 

exposed to an identical HS-ssDNAsequence prepared in 1.0 

M KH2P04 buffer. To explore further the role of ionic 

strength on adsorption of HS-ssDNA, XPS data were 

obtained from a series of samples exposed to 1.0 ,uM 

HS-ssDNA25 solutions prepared in different concentrations 

of KH2P04 buffer. Normalized N 1s peak areas obtained 

from this series of solutions plotted as a function of KH2PO4 

concentration are displayed in FIG. 2. The XPS N 1s peak 

area for buffer concentrations of 2.7xlO-4 to 1.0 M KH2P04 

grows five-fold as the buffer concentration is increased, 

evidence that ionic strength plays a critical role in adsorption 

of DNA. 
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5,942,397 


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After determining from these XPS studies that the surface 

coverage obtained for the HS-ssDNAin 1.0 M KH2P04 was 

maximized, an attempt was made to hybridize the surfacebound probe with its radiolabeled complement, ssDNAC25. Hybridization was measured by obtaining radio images 

of the HS-ssDNA25 sample after exposing it to the radiolabeled complement. No signal from the radiolabel was 

measured, indicating that hybridization did not occur. It is 

believed that hybridization on this surface is inhibited due to 

steric and electrostatic factors. The complement cannot 

access the surface-bound HS-ssDNA25, as the molecules on 

the surface are too tightly packed. The dense packing of 

these charged groups on the surface may also prohibit the 

approach and binding of another similarly charged DNA 

molecule. 

To determine whether HS-ssDNA25 is adsorbed on the 

gold surface "specifically" through the sulfur atom or "nonspecifically" through the nitrogen-containing nucleotide side 

chains interacting directly with the surface, or some other 

functionality of the DNA, XPS data from the thiolderivatized DNA (HSssDNA25 ) and the non-thiol derivatized DNA (ssDNA25 ) were obtained and compared. The 

XPS N 1s data for HS-ssDNA and the ssDNA are shown in 

FIG. 3. The N 1s peak area of the non-thiol derivatized DNA 

is approximately 50 to 60% of the intensity measured for 

adsorbed HS-ssDNA, indicating that more DNA is adsorbed 

when the molecule is thiol-derivatized. There is a significant 

amount of DNA adsorption for the non-derivatized 

oligonucleotide, suggesting that DNA will interact with and 

adsorb on the surface when no thiol group is present. The 

non-derivatized DNA is adsorbed strongly on the surface; 

heating the gold surface exposed to non-thiol-derivatized 

ssDNA25 to 75° C. did not cause desorption of ssDNA25. 

None intensity of the N 1s signal for HS-ssDNA is evidence 

that the strong thiol-gold interaction drives the adsorption of 

HS-ssDNA25 to higher coverages, compared to the nonthiolated DNA. 

As a means of measuring how tightly the HS-ssDNA25 

and non-thiolated ssDNA25 mono layers are bound to the 

surface, both HS-ssDNA25 (thiolated) and ssDNA (nonthiolated) surfaces were exposed to another thiol, mercaptohexanol (MCH), for one hour. The XPS N 1s data obtained 

after posttreatment with MCH for both HS-ssDNA and 

ssDNA samples are shown in the scatter plots in FIG. 3, 

along with the XPS data obtained before MCH exposure. It 

is clear that both HS-ssDNA25 and non-thiolated ssDNA25 

coverages are altered by posttreatment with MCH. The N 1s 

peak obtained from the HS-ssDNA25 sample is slightly less 

intense than that observed before exposure to MCH, indicating that a small amount of HS-ssDNA has been removed 

or displaced form the surface. A much more dramatic 

difference between the "before" and "after" XPS data for 

non-thiolated ssDNA25 is observed. Essentially all of the 

non-thiolated DNA is removed from the surface, presumably 

displaced by MCH. The significance of this result is threefold: First, it is clear that the HS-ssDNA25 is adsorbed 

through the sulfur atom, as the HS-ssDNA25 is not displaced by MCH posttreatment, but the non-thiolated 

ssDNA25 is almost completely removed. Second, posttreatment with MCH results in removal of nonspecifically bound, 

single-stranded DNA. Third, the HS-ssDNA25 molecules 

remaining on the surface after MCH treatment are raised off 

the surface, tethered by the sulfur group. 

Mixed HS-ssDNNMCH monolayers of varying coverage 

were formed by a two step process. First, clean bare gold 

was immersed in a 1.0,uM HS-ssDNA25 solution in 1.0 M 

KH2P04 for a specific amount of time (referred to here as 

6 

"exposure time"), followed by rinsing with water. Second, 

the HS-ssDNA25-treated surface was exposed to a 1.0 mM 

aqueous solution of MCH for exactly one hour. 

M CH was used for three reasons: First, and, perhaps, most 

importantly, nonspecific binding of DNA on a selfassembled monolayer (SAM) of MCH does not occur. That 

is, if a pure MCH monolayer is formed, DNAwill not adsorb 

on the hydroxy-terminated surface of the MCH monolayer. 

Second, MCH is soluble in aqueous solutions. Third, the 6 

carbon chain of MCH is the same length as the methylene 

spacer in HS-ssDNA. 

A series of HS-ssDNA25/M CH surfaces were prepared by 

the above method, with exposure times ranging from 1 

minute to 21.9 hours. The relative amount of HS-ssDNA on 

the surface was determined by measuring the XPS N 1s peak 

areas. Shown in FIG. 5 are the normalized N 1s peak areas 

plotted as a function of time in the HS-ssDNA solution. The 

N 1s peak intensity is observed to increase monotonically 

with exposure time. The amount of HS-ssDNA25 on the 

surface appears to reach a maximum at 240 minutes; the 

sample that had been exposed to HS-ssDNA25 for 21.9 

hours does not have more DNA on the surface than the 

sample exposed for 240 minutes. 

In order to measure the optimal HS-ssDNA25 coverage 

for hybridization, a series of surfaces with varying 

HS-ssDNA25 coverages in an HS-ssDNA25/MCH mixed 

monolayer were exposed to 32P-radiolabeled complement. 

Each surface was exposed to a separate hybridization solution containing the radiolabeled complement for 90 minutes 

and then rinsed in TE buffer. The results from this experiment are summarized in Table 1 of FIG. 6, along with results 

obtained from exposing both a bare gold sample and a pure 

MCH monolayer to the radio labeled complement. The bare 

gold and pure MCH samples served as controls which 

monitored nonspecific adsorption. All of these surfaces were 

subjected to the same hybridization protocol with the 32p 

radio labeled complement, ssDNA-C25. In addition, a second set of identically prepared surfaces was exposed to a 

different radio labeled oligonucleotide that was not complementary to the surface bound probe. This set of samples was 

used to estimate any nonspecific binding of the radio labeled 

probe to the surface as well as nonspecific hybridization 

between mismatched oligonucleotides. 

Hybridization or adsorption of the complement on the 

HS-ssDNA25-coated substrates and the control samples was 

uniform, without any indication of bare patches or clumping 

of radiolabeled oligonucleotides. The control sample of the 

pure MCH monolayer shows negligible adsorption of the 

radio labeled target. By contrast, the bare gold substrate has 

a significant amount of the radiolabeled complement 

adsorbed. For the HS-ssDNA25/MCH coated surfaces, 

hybridization is evidenced by the binding of the radio labeled 

probe to the substrate. As the HS-ssDNAcoverage increases, 

more of the radio labeled probe is observed on the surfaces. 

Hybridization was greatest for sample 8, which corresponds 

to an exposure time of 120 minutes. It is interesting to note 

that the most hybridization is not observed for the highest 

HS-ssDNA25 coverage samples (samples 9 and 10). In fact, 

for exposure times longer than 120 minutes, a decrease in 

hybridization efficiency is observed (defined for the purposes of the present invention as the product of the total 

number of surface bound probes and the percentage of 

probes undergoing hybridization), as evidenced by the 

decrease in the amount of radio labeled probe measured at 

the surface. It is believed that the decrease in the number of 

duplexes formed on the surface for higher coverage samples 

(samples 9 and 10) is due to steric and electrostatic hin­
5,942,397 


7 	 8 


drance arising from the more tightly packed DNA 

monolayer, as described earlier. 

It is clear from the hybridization experiment that the 

HS-ssDNA25 appears to be uniformly distributed on the 

surfaces, and that the optimal surface coverage for hybrid­ 5 

ization for the samples examined is that obtained for the 120 

minute HS-ssDNAexposure time sample. The hybridization 

efficiency on this surface can be estimated by comparing the 

numher of counts in a known volume of radiolaheled DNA 

that has been spotted on the paper and exposed to the 10 

imaging plate. It appears that there are 5.7 (±0.05)x1012 

molecules/cm2 of the radiolabeled probe bound to sample 8. 

From independent measurement of the HS-ssDNA coverage 

using surface plasmon resonance, the hybridization efficiency is estimated to be close to 100%. 15 

Although the present invention has been fully described in 

conjunction with the preferred embodiment thereof with 

reference to the accompanying drawings, it is to be understood that various changes and modifications may be apparent to those skilled in the art. Such changes and modifica­ 20 

tions are to be understood as included within the scope of the 

present invention as defined by the appended claims, unless 

they depart therefrom. 

What is claimed is: 

1. A biopolymer-containing monolayer comprising: 
 25 

a metal substrate; 


a plurality of thiol-derivatized oligonucleotides bound to 


said substrate by a sulfur-substrate linkage, said thiolderivatized oligonucleotides having the formula: 30 

HS-X-Y 

where 

X is either nothing or an organic linker, and 35 

Y comprises an oligonucleotide terminally bound to X 

where X is an organic linker or terminally bound to S 

where X is nothing; and 

a 	plurality of organic thiols bound to said substrate, 

wherein said plurality of bound thiol-derivatized boli­ 40 

gonucleotides and said plurality of bound organic thiols 

comprise a mixed monolayer. 

2. The biopolymer-containing monolayer of claim 1, 

wherein said thiol-derivatized oligonucleotide comprise 

thiol-derivatized single-stranded DNA. 45 

3. The biopolymer-containing monolayer of claim 1, 

wherein said thiol-derivatized oligonucleotides comprise 

RNA. 

4. The biopolymer-containing monolayer of claim 1, 

wherein said thiol-derivatized oligonucleotides comprise 50 

PNA. 

5. The biopolymer-containing monolayer of claim 1, 

wherein Y is a nucleotide sequence of at least six nucleotides. 

6. The biopolymer of claim 1, wherein X comprises an 55 

organic linker. 

7. The biopolymer-containing monolayer of claim 1, 

wherein said metal substrate comprises a metal selected 

from the group consisting of the metals gold, silver, 

platinum, palladium, ruthenium and iridium and alloys of 60 

said metals. 

8. The biopolymer-containing monolayer of claim 1, 

wherein said substrate comprises gold. 

9. The biopolymer-containing monolayer of claim 1, 

wherein said organic thiol includes a terminal hydroxy 

group. 

10. The biopolymer-containing monolayer of claim 1, 

wherein X comprises a hexamethylene linker and said 

organic thiol compound comprises mercaptohexanol. 

11. A method for forming a biopolymer-containing monolayer comprising the steps of: 

applying 	a solution comprising thiol-derivatized oligonucleotides to a substrate comprising a metal to bind 

said thiol-derivatized oligonucleotides thereto by a 

sulfur-substrate linkage, the thiol-derivatized oligonucleotides having the formula: 

HS-X-Y 

where 

X is either nothing or an organic linker; and 

Y comprises an oligonucleotide terminally bound to X 

where X is an organic linker or terminally bound S 

where X is nothing; and 

applying a solution comprising an organic thiol to displace nonspecifically adsorbed oligonucleotides and to 

prevent nonspecific adsorption of oligonucleotides. 

12. The method of claim 11, further comprising rinsing 

the substrate to which the thiol-derivatized oligonucleotides 

and the thiols are bound with water to remove compounds 

not bound to the substrate. 

13. The method of claim 11, wherein said thiolderivatized oligonucleotides comprise thiol-derivatized 

single-stranded DNA. 

14. The method of claim 11, wherein said thiolderivatized oligonucleotides comprise RNA. 

15. The method of claim 11, wherein said thiolderivatized oligonucleotides comprise PNA. 

16. The method of claim 11, wherein Y is a nucleotide 

sequence of at least six nucleotides. 

17. The method of claim 11, wherein the organic thiol is 

applied to the substrate by immersing the substrate in an 

aqueous solution of the organic thiol, thereby removing 

nonspecifically adsorbed thiol-derivatized oligonucleotides 

and passivating the surface against nonspecific adsorption of 

oligonucleotides in solution. 

18. The method of claim 11, wherein X comprises an 

organic linker. 

19. The method of claim 11, wherein the substrate comprises a metal selected from the group consisting of the 

metals gold, silver, platinum, palladium, iridium and ruthenium and alloys of said metals. 

20. The method of claim 11, wherein the substrate comprises gold. 

21. The method of claim 11, wherein the organic thiol 

includes a terminal hydroxy group. 

22. The method of claim 11, wherein X comprises a 

hexamethylene linker and said organic thiol comprises mercaptohexanol. 

* * * * *