WO2003083437A2

WO2003083437A2 - Method and apparatus for identifying molecular species on a conductive surface

Info

Publication number: WO2003083437A2
Application number: PCT/US2003/008813
Authority: WO
Inventors: Iii Louis C. Brousseau
Original assignee: Quantum Logic Devices, Inc.
Priority date: 2002-03-22
Filing date: 2003-03-21
Publication date: 2003-10-09
Also published as: AU2003215025A1; AU2003215025A8; WO2003083437A3

Abstract

A nano-probe for performing single electron molecular orbital tunneling spectroscopy (SEMOTS) is disclosed. Unlike the conventional probes, the nano-probe is capable of obtaining highly resolved spectra of single molecule electronic structures on conductive surfaces. The increase in spectral resolution allows the unique spectrum of adsorbed molecules to be collected into a database and subsequently used to identify adsorbates on surface on unknown samples. The nano-probe can be utilized for genetic sequencing of single molecules of deoxyribose nucleic acid and other oligonucleotides based on the differences in orbital energy spectra of the nucleotide bases.

Description

METHOD AND APPARATUS FOR IDENTIFYING MOLECULAR SPECIES

ON A CONDUCTIVE SURFACE

RELATED PATENT APPLICATIONS

The present patent application claims priority is related to copending application U.S. Serial No. 60/366,840 filed on March 22, 2002.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to tunneling spectroscopy in general, and in particular to scanning tunneling microscopy. Still more particularly, the present invention relates to an improved method and apparatus for identifying molecular species on a conductive surface utilizing scanning tunneling microscopy.

2. Description of the Related Art

It is a common belief that dramatic improvements in human health can be made possible by the understanding of the ordering of the billions base pairs contained in deoxyribose nucleic acid (DNA). Thus, sequencing human genome is one of the major scientific goals in the United States as well as the rest of the world today. Incidentally, the completion of the Human Genome project has fueled a great deal of speculation on how such knowledge may impact medicine. M.any agree that genetic- based drugs and medicine (also known as pharmacogenomics) can enhance physicians' ability to cure human illness. It is anticipated that genetic-based technology will impact many aspects of health care — from gene-based identification of new chemical entities' for drug development to smarter selections of candidates for clinical trials. The greatest impact of genetic-based technology is expected to be in its use as a point-of-care genotyping tool to enable the diagnosis and treatment of disease on an individual patient basis. However, such paradigm shift in health care requires a rapid, cost-effective, accurate, and non-complex technique to perform genetic profiling and screening of patients.

A number of research groups, including those at Lawrence Livermore National Laboratory, have attempted, with limited success, to use scanning probe microscopes, such as scanning tunneling microscopes (STMs) and atomic force microscopes (AFMs), to sequence DNA by resolving individual nucleotide bases in tunneling images. STM of DNA structure has been performed in air, buffer, and vacuum, but is limited from sequencing due to lack of chemical identification capabilities. Attempts to overcome such limitation have been restricted to inferred sequencing based on the capture of a probe sequence, measured by height differences, or attached nanoparticle labels. However, such at sequencing based on strand topology have not been very successful.

Today, the demands on the sequencing technology are even greater than those of the Genome Project because many scientists now also want to understand the DNA of different animals, plants, and micro-organisms as quickly as possible. Consequently, it would be desirable to provide an improved method and apparatus for identifying molecular species effectively and efficiently.

SUMMARY OF THE INVENTION

In accordance with a preferred embodiment of the present invention, a scanning tunneling microscope includes a conducting nano-probe, a drive means, a variable bias voltage source, a servo control means and a display means. The drive means moves the nano-probe relative to a test sample in two dimensions. The variable bias voltage source supplies a variable potential to the nano-probe with respect to the test sample. The servo control means varies a distance between the nano-probe and the test sample such that a tunneling current flowing between the nano-probe and the test sample is kept constant. The display means displays a scanning tunneling microscope image based upon an output signal in relation to a scanning position of the nano-probe.

The nano-probe has a conducting metallic probe body with a sharp tip. A nanoparticle is attached to the sharp tip.

All objects, features, and advantages of the present invention will become apparent in the following detailed written description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention itself, as well as a preferred mode of use, further objects, and advantages thereof, will best be understood by reference to the following detailed description of an illustrative embodiment when read in conjunction with the accompanying drawings, wherein:

Figure 1 is a block diagram of a scanning tunneling microscope having a nano-probe, in accordance with a preferred embodiment of the invention;

Figures 2a-2e are a graphical illustration of the fabrication of a nano- probe used in the scanning tunneling microscope from Figure 1, in accordance with a preferred embodiment of the present invention;

Figures 3a-3d are topographical and current images for an 1-octanethiol monolayer intercalated with ferrocene-terminated 1-decanethiol (Ferrocene-thiol);

Figures 4a-4c ares tunneling spectroscopy data taken over a single ferrocene-thiol molecule;

Figures 5a-5b are topographical images of gold and CdS nanoparticles adsorbed to a gold electrode surface;

Figures 6a-6d are I verses V spectroscopy data over a single nanoparticle of gold and CdS;

Figure 7 is the current (left) and topographical (right) image of thymine adsorbed to a gold electrode surface;

Figure 8 is the current (left) and topographical (right) image of guanine adsorbed to a gold electrode surface; Figures 9a-9b are I verses N spectra taken with a nano-probe over a single molecule of thymine and guanine, respectively;

Figure 10 is I verses N and dl/dN verses N spectra taken over a single molecule of guanine with a conventional probe; and

Figure 11 is a comparison plot of the spectrum of single molecules of thymine (lower current traces) and guanine (higher current traces) taken with a nano-probe.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

L SCANNING TUNNELING MICROSCOPE

Referring now to the drawings and in particular to Figure 1, there is depicted a block diagram of a scanning tunneling microscope (STM) having a nano- probe, in accordance with a preferred embodiment of the invention. As shown, an STM 10 includes a three-dimensional drive unit 11 having a conducting nano-probe 12 attached to the front end of drive unit 11. Drive unit 11 preferably includes a three-layer piezoelectric unit. A variable-bias voltage source 13 supplies a bias voltage between nano-probe 12 and a test sample 14. A servo control circuit 15 detects the tunneling current flowing between nano-probe 12 and test sample 14 and produces a control signal for controlling a z drive (not shown) of drive unit 11 to control the distance between nano-probe 12 and test sample 14 such that the tunneling current is maintained at a predetermined constant value. In other words, nano-probe 12 is moved slowly across the surface of test sample 14, and raised and lowered (or nearer and farther when placed horizontally) so as to keep the control signal constant. The control signal for controlling the z drive represents the topography of the surface of test sample 14, and such control signal is sent to a memory 22 via an analog-to-digital converter (ADC) 16. In addition, a central processing unit (CPU) 21 causes an XY drive signal generator 17 to produce a signal for driving nano-probe 12 along the x-y plane that is generally parallel to the surface of test sample 14.

STM 10 is also equipped with a keyboard 23, a display 24 and a pointing device, such as a mouse 25, all connected to a system bus 20. Variable-bias voltage source 13 provides a negative or positive potential of a few volts to nano-probe 12 with respect to test sample 14. Servo control circuit 15 ensures that the tunneling current is maintained at approximately 1 nA. CPU 21 causes XY drive signal generator 17 to produce an XY scanning signal that is sent to the XY drive portion of drive unit 11. In turn, test sample 14 is scanned by nano-probe 12 in two dimensions. A z-axis control signal produced in response to the scan is sent to memory 22, where the signal is stored. The signal can be read from memory 22 to generate a computer- generated contour map (i.e.. STM image) of the surface of test sample 14. The contour map can be displayed on display 24 or printed via a printer (not shown). The above- mentioned scanning tunneling microscopy technique is capable of resolving individual molecules on test sample 14, but requires conductive materials for image formation.

IL NANO-PROBE FABRICATION

With reference now to Figures 2a-2e, there are a graphical illustration of the fabrication of nano-probe 12, in accordance with a preferred embodiment of the present invention. Initially, a metal probe 31 is made by either cutting or etching a metal wire, as shown in Figure 2a. The metal wire can be made of 80% platinum/20% iridiurn, tungsten, gold, or similar type of materials. For the intended applications to be described infra, metal probe 31 is preferably made by electrochemical etching in order to yield a high aspect ratio taper to an extremely narrow tip 32 having a radius of less than 50 nm, as depicted in Figure 2b. Metal probe 31 is then coated with an insulation layer 33 of no more than 10 nm thick, as shown in Figure 2c. Insulation layer 33 can be made of wax, polymer, glue, oxide, ceramic or other self-assembled molecular materials. The application of insulation layer 33 can be performed by any well-known coating techniques designed for that specific one of the above-mentioned materials, including solution and vapor phase deposition or electrochemical growth.

Next, a nanoscopic sized pore or nanopore 34 is opened in insulation layer 33 in order to expose tip 32 of metal probe 31, as depicted in Figure 2d. Nanopore 34 preferably has a diameter of approximately 10 nm. The opening procedure can be performed by various well-known methods, depending on the composition of insulation layer 33. For wax, polymer, or self- assembled molecular materials that have characterized melting or desorption properties, tip 32 may be exposed by applying an appropriate voltage potential to tip 32 and a grounded counter electrode in close proximity to tip 32 to allow electrons flow from tip 32 to the counter electrode such that a current is produced. The effect of such current is to heat the coating in the immediate vicinity of tip 32, causing the coating to melt or desorb until tip 32 is exposed. In the case of the melting process, the cooler regions of material not in the immediate vicinity of tip 32 attracts the melted material by capillary action on account of its lower surface free energy, and the melted material then re-solidifies through loss of heat to the cooled regions of the material and/or environment. As such, only the portion of tip 32 that is conducting current is exposed. In the case of desorption process, the molecules of insulation layer 33 are electrochemically released from the surface of metal probe 31 and are removed by diffusion or other kinetic processes. By careful controlling the applied voltage potential, only the molecules at the sharpest part of tip 32 receive sufficient energy to desorb because the electric field is concentrated at tip 32.

Similarly, for a ceramic or oxide coating, the field concentration enables an electrochemical etching process to proceed only at the sharpest part of tip 32. Such process has been well characterized for aluminum/aluminum oxide electrodes, but it is believed that such process is also applicable to other metals/oxides/ceramics. Also, concentrated phosphoric or sulfuric acid can be applied to, for example, aluminum or silicon oxide, for etching pores into the oxide with a calibration of approximately 1 nm of diameter per volt applied. One skilled in the relevant art will recognize that other etchants and metals are possible.

After nanopore 34 has been formed to expose the metal surface of tip 32, a quantum dot 35 is placed in nanopore 34 to attach to the apex of tip 32 to form nano-probe 12. Quantum dot 35 is preferably metallic, however, other non-metallic may also be used. The size of quantum dot 35 is preferably 5 nm or less. Quantum dot 35 may be a chemically synthesized n-moparticle that is attached by an appropriate linking chemistry, or fabricated in situ via electrochemical, solution or vapor phase methods that are well-known in the art.

If a chemically synthesized nanoparticle is being used as quantum dot 35, the attachment linker may be a functionalized self-assembled monolayer for generating a separation distance between quantum dot 35 and tip 32. The functionalized self-assembled monolayer can be in the form of HS-(CH₂)_n-SH, where 4<n<8. For example, a gold nanoparticle can be immobilized by using a thiol terminated molecule. It is understood by those skilled in the art that for other compositions of nanoparticle, other linker chemistries will be appropriate, such as isonitriles for platinum, carboxylic acids for oxides, etc.

If quantum dot 35 is made in situ, it is desirable to construct a spacer layer between quantum dot 35 and tip 32 such that there is a separation distance between quantum dot 35 and tip 32. The spacer layer is preferably made an insulative material. As a result, moderately strong electronic coupling of the Fermi level of tip 32 and the pseudo-Fermi level of quantum dot 35 can be achieved by adjustment of the separation distance (i.e., the thickness of the space layer).

The tunneling current from nano-probe 12 (i.e., metal probe 31 and quantum dot 35) to a test sample is related to the spatial cross-section of the electron beam, as follows:

I oc _e ^2R

where I is the tunneling current, r is the distance away from the central axis of the tunneling current, R is the radius of quantum dot 35, and k is an empirical constant. It is understood that by reducing the radius of quantum dot 35 (i.e., nanoprobe 12), the width of the electron beam is also reduced, providing better spatial resolution of the device.

IIL MOLECULAR IDENTIFICATIONS USING NANO-PROBE

Nano-probe 12 can be placed in an STM, such as STM 10 from Figure 1, for performing single electron molecular orbital tunneling spectroscopy (SEMOTS) investigation of single molecule on selected surfaces. Although operational procedures for nano-probe 12 are identical to those for standard metal probes with regard to imaging and spectroscopy, significant differences in the molecular orbital spectra of adsorbed molecules, including resolution of molecular orbital fine structure and single-electron tunneling effects, will become apparent later.

As a preferred embodiment of the present invention, nano-probe 12 and SEMOT technique can be specifically used to obtain the base pair sequences of single molecules of double and single stranded oligonucleotides by spectral mapping. Samples of DNA and other oligonucleotides (generically known as polynucleic acids or PNAs) can be acquire by many methods that are well-known in the art. Once acquired, the molecules of a PNA can be deposited on a conductive surface in a chain-extended formation using methods that are also well-known in the art. It is not necessary for the PNA sample to be in the chain-extended formation, although overlapping portions of the molecule may present challenges in the process of obtaining clear resolution of adjacent bases.

An exemplary method for preforming direct sequencing of molecules of a PNA sample on a conductive surface is described as follows. Initially, a solution of PNA sample is translated across a surface that has some affinity for the PNA, and viscous flow of the solution forces the molecular chains of the PNA to align parallel to the direction of the viscous flow. After the PNA sample has been prepared, the molecules of the PNA sample can be imaged with an STM having a nano-probe, such as STM 10 from Figure 1, and the identified molecules can be sequenced by obtaining the SEMOT spectra of the identified molecules at different positions along the axis of the molecular chain. Ideally, the different positions should be spaced by no more than 0.1 nm because the approximate spacing of the bases is 0.3 nm. The obtained SEMOT spectra are compared to a reference library to perform peak matching The peak matching process facilitates the identification of each base in the sequence.

As an alternative embodiment of the present invention, nano-probe 12 is held at a constant distance (for proper tunneling) from a counter electrode and the PNA molecule is moved past the tip of nano-probe 12 in an aligned manner. The alignment should be orthogonal to the tunneling axis between the tip of nano-probe 12 and the counter electrode. Such arrangement allows higher sequencing rates, and may be manifested in a microfluidic channel.

In order to demonstrate the advantages of the SEMOT technique of the present invention over those of the conventional tunneling spectroscopies, the tunneling spectra obtained by the SEMOT technique of the present invention are compared to the tunneling spectra collected with conventional STM probes using several test cases.

A. Single ferrocene terminated decanethiol (Fc-thiol)

The electrochemistry of self assembled mono-layer of ferrocene terminated thiols has been well studied. First, a platinum/iridium probe was coated with an insulating layer of polyethylene glue, which was thinned at the tip of the platinum/iridium probe by carefully approaching the tip of the platinum/iridium probe with a hot soldering iron. As the glue melted, the glue was pulled away from the tip of the platinum/iridium probe by surface tension. The tip of the platinum/iridium probe was then placed into a STM, such as STM 10 from Figure 1. A gold electrode substrate is connected to a potential of 1 volt and a current 1.5 nanoamperes. When the tip neared the surface and tunneling current began to flow, localized melting occurs at the apex, fully exposing the bare metal of the platinum/iridium probe. A 5 nm gold nanoparticle quantum dot was then attached by sequential incubation of the platinum/ iridium probe in an ethanol/water (95/5) solution of hexane dithiol, followed by rinsing and re-immersion into an aqueous solution of nanoparticles. The nanoscopic hole in the glue layer ensures that only a small number (approximately one to five) nanoparticles are bound to the tip of the platinum/iridium probe.

Next, a sample having isolated ferrocene terminated decanethiols (Fc-thiol) was prepared by partial substitution of the molecules into an octane thiol monolayer. That was achieved by initially preparing a 1 cm² gold on glass slide with an octane thiol self-assembled mono-layer (SAM) by incubation in a bath of 1 mM concentration (in ethanol) for 30 minutes. The sample was then rinsed copiously with ethanol and immersed in a bath containing a 10 mM solution of Fc-thiol for 24 hours. The sample was rinsed with ethanol and stored under fresh absolute ethanol until it is ready to be used. Prior to examination, the sample was blown dry in a stream of argon gas. All of the above-mentioned steps were performed at ambient conditions.

The corresponding topographic and current images are shown in Figure 3. The line in the left panel of Figure 3 indicates two ferrocene groups present on top of the octane thiol SAM. The measured cross sectional height of the molecules (topographic and current, respectively, Figure 3b and 3d) corresponds to the calculated difference between the 8-carbon .and 10-carbon chains lengths. As can be observed in the current image, tunneling is enhanced over these two features of the film.

The tunneling spectra for a conventional STM probe is shown in Figure 4a (I vs. V) and 4b (dl/dN vs. N). The spectra from the conventional STM probe shows symmetric current enhancement at both positive and negative bias, which appear as broad peaks in the dl/dN plot (numerical differentiation). These features indicate the enhancement of tunneling due to interaction between the electronic states of the STM probe and the Fc-Thiol. This is in remarkable contrasts for the single-electron I/N spectrum shown in panel b of Figure 4c. A dominant sharp peak is seen at +1.06 N, which corresponds to the electron tunneling through only the lowest occupied molecular orbital (LUMO) of ferrocene. The improved spectral resolution is a consequence of the tunneling electrons having a narrower energy distribution due to tunneling from the quantum dot.

The superior energetic resolution of the quantum dot probe can be understood according to the uncertainty relation, which states that the product of spatial distribution and momentum distribution must equal approximately one. In the conventional STM tip, where tunneling is believed to occur through a single apex atom of spatial distribution Δx » 2 A, the momentum resolution would be approximately Δp « 0.5 A^"1. For tunneling through the quantum dot where Δx « 25 A, the resolution of the energy distribution is then Δp « 0.04 A^"1. An equation has been developed to relate an observed conductance peaks to the standard reduction potentials with good predictive ability:

A V= aV= _M- Φ_SCE -E₀ ± δ (2)

where Φ_M and Φ_SCE are the work function of a metal and saturated calomel electrode reference electrode, αV is the energy shift of the molecular state due to the applied bias, and δ is a small correction factor for the difference between the vacuum level and the potential at the edge of the electrode double layer. Typically, α is between 0 and 1, and relates to the asymmetry of the drop in potential across the molecule. The amount of the applied potential that is dropped across the molecule is a function of the coupling to the substrate and the distance of a tip over the molecule.

Thus, the above-mentioned peak value compares very well with an estimate value of 1.04 V from equation (2), taking into account the work functions of the gold surface (Φ_M = 5.5V), the measured reduction potential for such a molecular surface (E₀ = 0.19V) and the work function of the Ag/Cl reference electrode used in that work (Φ_AgC1 = 4.65V).

B. Single gold and CdS nanocrystals

Nanoparticles of gold or CdS were prepared by known solution phase routes that provide size limited crystallites through the use of reverse micelles. The nanoparticles were then dispersed onto a gold electrode that had been coated with a 1,6-hexanedithiol anchoring layer that formed chemical bonds to both the gold and the CdS particles. The preparation of the nano-probe was performed as previously described. Topographical images show the particles adsorbed to the surface, and cross sections permit measurement of their size, as shown in Figure 5a (Au) and Figure 5b (CdS). Several particles were selected for investigation by tunneling spectroscopy with conventional STM probe and nano-probe. The differences in spectra are illustrated in Figures 6a-6d. For the conventional STM probe, only Coulombic current steps are seen in the I/N spectra for both the gold and CdS nanoparticles, which appear as peaks in the dl/dV plot. For the nano-probe, however, sharp peaks corresponding to the estimated HOMO-LUMO gap are visible even in the I/V spectrum of CdS (Figure 6d). Results are tablated and compared to the expected bandgap for three different particles in Table I. In addition, fine structure is observed in each band. For the gold nanoparticles (Figure 6c), the observed Coulombic gap (flat portion through the middle of the spectrum) is broadened as expected for the series addition of tunneling barriers. The spectra obtained at different tip-particle distances also show the expected progression from Coulombic staircases (at low current/large distance) to ohmic response (at high current/small distance). This demonstrates the ability of the SEMOT technique and the nano-probe to discriminate between nanoparticle adsorbates that otherwise look the same to the STM images from a conventional STM probe.

C. Single molecules of guanine and thymine

Gold substrates were prepared with surfaces that provide hydrogen-bond stabilization of guanine and thymine adsorbates. For guanine, a mono-layer of mercaptoethylamine was grown by incubation in a 2 mM ethanolic solution for 1 hour. Likewise for thymine adsorption, a monolayer of mercatopropionic acid was generated by incubation in a 2 mM ethanolic solution. Solutions of the nucleobases were then prepared in distilled water. The solubility of guanine is very low, so a saturated solution was prepared. Thymine was prepared at a concentration of 2 mM. The above-mentioned solutions were filtered with a 0.22 micron syringe membrane filter and drops were cast on the corresponding substrates and allowed to dry slowly. Nano- probes were prepared as described previously and STM images were obtained from both samples.

Figures 7 and 8 shows the current and topographic images of the thymine and guanine layers, respectively. Although mobility of the molecules made collection of high-resolution images difficult, several individual molecules were located, as indicated by the circle in the right panel of Figure 7 for thymine, .and as can be seen in the bottom half of Figure 8 for guanine. Evidence of the presence of thymine and guanine is inferred from the height of the features relative to the surface below, and for the guanine image, by comparative reference to the underlying gold lattice visible between molecules. Spectra collected over these features with the nano-probe show sharp peaks in the positive half of the I/N trace, as shown for thymine in Figure 9a and guanine, in Figure 9b. Figure 10a and 10b show I verses N and dl/dN verses N plots for the guanine sample taken with a conventional STM probe, that show no peaks, only the enhanced tunneling at the extreme of the bias sweep. The measured resonances are listed in Table I, and the overlaid spectra for guanine and thymine are presented in Figure 11.

Table I

Calculations of the expected resonance positions are different than the value suggested in known reference for guanine, although the thymine energies are remarkably close. The guanine spectra obtained with a conventional STM probe shows the enhanced tunneling occurring at the same voltage, suggesting that the features are real. Differences are expected due to the additional tunneling barrier and the proportionally larger drop of the applied bias across the outside junctions of the three junction arrangement, as well as the correction for the applied bias used in the other experiments. Despite some overlap of resonances, however, the comparison plot illustrates that the spectra are distinct enough to make a distinction between the two. Based upon the very different reduction potentials of the bases as determined by selective electrochemical experiments, as well as gas phase-ionization and electron on the line shapes and line widths of the preliminary data, it is anticipated that the SEMOT technique will be able to resolve them sufficiently to allow direct sequencing of oligonucleotides, including DNA single molecules.

As has been described, the present invention provides an improved method and apparatus for identifying molecular species on a conductive surface utilizing scanning tunneling microscopy. With the present invention, an enhanced resolution of the tunneling spectra allows a specific assignment of energy level structure for individual molecules sufficient to facilitate the identification and discrimination of similar species. The present invention also allows for the collection and compilation of such orbital spectra into a reference database for the identification and discrimination of energy level structure for individual molecules.

While the invention has been particularly shown and described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

Claims

What is claimed is:

1. A scanning tunneling microscope comprising:

a conducting nano-probe;

a drive means for moving said nano-probe relative to a test sample in two dimensions;

a variable bias voltage source for supplying a variable potential to said nano-probe with respect to said test sample;

a servo control means for varying a distance between said nano-probe and said test sample so that a tunneling current flowing between said nanoprobe and said test sample is kept constant; and

a display means for displaying a scanning tunneling microscope image based upon an output signal in relation to a scanning position of said nanoprobe.

2. The scanning tunneling microscope of Claim 1, wherein said nano-probe includes a nanoparticle placed at a tip of said nano-probe.

3. The scanning tunneling microscope of Claim 2, wherein said nanoparticle is 5 nm or less in diameter.

4. The scanning tunneling microscope of Claim 1, wherein said nano-probe is 50 nm or less in radius.

e for use in a scanning tunneling microscope, said probe comprising:

a conducting metallic probe body having a sharp tip; and

a nanoparticle attached to said sharp tip.

The probe of Claim 5, wherein said nanoparticle is less than 5 nm in diameter.

7. The probe of Claim 5, wherein said sharp tip is less than 50 nm in radius.

8. A method for performing sequencing on oligonucleotides, said method comprising:

depositing an oligonucleotide sample on a surface in a chain-extended conformation;

imaging said oligonucleotide sample with a scanning tunneling microscope (STM);

obtaining tunneling spectra of the identified sample at various positions along said oligonucleotide sample; and

comparing said obtained tunneling spectra of said oligonucleotide sample with a plurality of spectrum stored within a reference library to identify each base within the sequence of said oligonucleotide sample.

9. The method of Claim 8, wherein said oligonucleotide sample is single stranded or double stranded.

10. The method of Claim 8, wherein said oligonucleotide sample is a deoxyribose nucleic acid (DNA) molecule.

11. The method of Claim 8, wherein said oligonucleotide is a polynucleic acid molecule.

12. The method of Claim 8, wherein said tunneling spectra are single electron molecular orbital tunneling (SEMOT) spectra.

13. The method of Claim 8, wherein said positions are no more than 0.1 nm apart.

4. A method for performing sequencing oligonucleotides, said method comprising:

positioning a nano-probe at a fixed and appropriate distance from a counter electrode;

moving an oligonucleotide sample past a tip of said nano-probe in an aligned position orthogonal with a tunneling axis between said tip of said nanoprobe and said counter electrode;

imaging said oligonucleotide sample with a scanning-tunneling microscope (STM) having said nano-probe;

obtaining tunneling spectra of said oligonucleotide sample at various positions along said tunneling axis; and

15. The method of Claim 14, wherein said oligonucleotide sample is single stranded or double stranded.

16. The method of Claim 14, wherein said oligonucleotide sample is a deoxyribose nucleic acid (DNA) molecule.

17. The method of Claim 14, wherein said oligonucleotide is a polynucleic acid molecule.

18. The method of Claim 14, wherein said tunneling spectra are single electron molecular orbital tunneling (SEMOT) spectra.

19. The method of Claim 14, wherein said positions are no more than 0.1 nm apart.