WO2009093813A2 - Methods for electrically detecting interactions between biomolecules using scanning tunneling microscope - Google Patents

Methods for electrically detecting interactions between biomolecules using scanning tunneling microscope Download PDF

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Publication number
WO2009093813A2
WO2009093813A2 PCT/KR2008/007655 KR2008007655W WO2009093813A2 WO 2009093813 A2 WO2009093813 A2 WO 2009093813A2 KR 2008007655 W KR2008007655 W KR 2008007655W WO 2009093813 A2 WO2009093813 A2 WO 2009093813A2
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biomolecule
biomolecules
nanoparticle
stm
interaction
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PCT/KR2008/007655
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French (fr)
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WO2009093813A3 (en
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Jeong-Woo Choi
Byung-Keun Oh
Woochang Lee
Da-Yeon Kang
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Industry-University Cooperation Foundation Sogang University
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Publication of WO2009093813A3 publication Critical patent/WO2009093813A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q60/00—Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
    • G01Q60/10—STM [Scanning Tunnelling Microscopy] or apparatus therefor, e.g. STM probes
    • G01Q60/14—STP [Scanning Tunnelling Potentiometry]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01Q—SCANNING-PROBE TECHNIQUES OR APPARATUS; APPLICATIONS OF SCANNING-PROBE TECHNIQUES, e.g. SCANNING PROBE MICROSCOPY [SPM]
    • G01Q60/00—Particular types of SPM [Scanning Probe Microscopy] or microscopes; Essential components thereof
    • G01Q60/10—STM [Scanning Tunnelling Microscopy] or apparatus therefor, e.g. STM probes
    • G01Q60/12—STS [Scanning Tunnelling Spectroscopy]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J37/00—Discharge tubes with provision for introducing objects or material to be exposed to the discharge, e.g. for the purpose of examination or processing thereof
    • H01J37/26—Electron or ion microscopes; Electron or ion diffraction tubes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y35/00—Methods or apparatus for measurement or analysis of nanostructures

Definitions

  • the present invention relates to a method for electrically detecting interactions between biomolecules using scanning tunneling microscope.
  • Hybrid of Au nanoparticle-bio/organic material has been studied for the investigation of molecular switch, material property and other electronic devices, and has still been enlarged its applications such as the measurement of biological binding events (Gittins et a/., 2000; Csaki et a/., 2001; Xiao et a/., 2003).
  • the small label size, the unique optical and electrical properties of Au nanoparticles, and easiness of signal enhancement through silver staining make them effective tools for the detection of biological binding events.
  • biosensors developed up to date by a number of researchers, they are described as follows.
  • SRP Surface Plasmon resonance
  • LSPR Localized surface plasmon resonance
  • Piezoelectric biosensor also allows easy miniaturization and has defect that sensitivity is decreased to the level of almost 1/10 compared with SRP.
  • Nano-FET biosensor has some disadvantages in the sense that response is significantly dependent on non-specificity and the properties of measured substance and that detection distance from surface is far.
  • Amperomet biosensor has low selectivity due to difficulty of common utilization and Impedence biosensor represents drawbacks like low reproductivity and non-specific response irrespective of its easy miniaturization and high sensitivity. Therefore, the biosensor equipped with a new analysis system to overcome defects of biosensors described above has been needed for a nano-size protein chip integrated analysis system.
  • the present inventors have made intensive researches to develop a method for electrically detecting interactions or bindings between biomolecules in a high throughput manner. As results, we have discovered that the interactions or bindings between biomolecules were detected in a more convenient manner and with higher sensitivity using a scanning tunneling microscope (STM).
  • STM scanning tunneling microscope
  • STM scanning tunneling microscope
  • a method for electrically detecting an interaction between biomolecules using a scanning tunneling microscope comprising the steps of:
  • the present inventors have made intensive researches to develop a method for electrically detecting interactions or bindings between biomolecules in a high throughput manner. As results, we have discovered that the interactions or bindings between biomolecules were detected in a more convenient manner and with higher sensitivity using a scanning tunneling microscope (STM).
  • STM scanning tunneling microscope
  • the biomolecule to be detected in this invention includes, but not limited to, an biomolecule to specifically combine with a counterpart.
  • the first and second biomolecules are
  • DNA, RNA, protein or carbohydrate more preferably, DNA, RNA or protein, much more preferably, DNA or protein and most preferably, protein.
  • the first biomolecule is antibodies (polyclonal or monoclonal).
  • the first and second biomolecules are specifically bound to each other in case that the former is an antibody as a kind of protein and the latter is an antigen as a kind of protein.
  • the first and second biomolecules are specifically bound to each other in case that the former is a protein receptor and the latter is a protein ligand.
  • the first and second biomolecules are specifically bound to each other in case that the former is DNA and the latter is an DNA-binding protein.
  • the present method permits to specifically detect the interactions or bindings between biomolecules with higher sensitivity.
  • the first biomolecule is immobilized on the metal substrate.
  • the metal substrate to be used in this invention includes, but is not limited to, metal substrates with electrical conductivity and is used in any one known to those skilled in the art.
  • the shape, size and chemical composition of surface of the metal substrate to be used in this invention are not particularly restricted.
  • metal substrate refers to all substrates composed of metal, metal oxide and alloy.
  • the metal substrate to be used in this invention includes gold, silver, bronze, white gold, aluminium, metals alloy ⁇ e.g., alloy of gold and copper) or metal oxide substrates.
  • the metal substrate includes the substrates metal is coated on their surface as well as metal substrates.
  • the metal substrate to be used in this invention is a gold (Au) substrate.
  • the metal "gold substrate” refers to all substrates which gold is coated on their surface.
  • To immobilize the first biomolecule on the metal substrate may be carried out by various methods known to those skilled in the art.
  • the first biomolecule is a protein containing an additive thiol group (-SH) and is immobilized on the surface of metal therethrough.
  • -SH additive thiol group
  • Proteins will combine to a cysteine residue through its R group.
  • the present invention endows wild type protein to contain an additive thiol group.
  • the addition of thiol group is performed according to two different methods.
  • the addition of thiol group is carried out by applying a reducing agent ⁇ e.g., 2-mercaptoethylamine) to wild type protein containing sulfide or disulfide bond.
  • a reducing agent e.g., 2-mercaptoethylamine
  • the addition of thiol group is carried out by further introducing a cysteine residue into N- or C-terminal end of protein through a genetic engineering technique.
  • the first biomolecule is antibody.
  • the antibodies become fragmentation. These antibodies were designated as "fragmented antibody” in this specification.
  • substrate preferably gold substrate was annealed at high temperature and washed with piranha solution.
  • the proteins with additive thiol groups were dispersed on the surface of gold substrate and kept to stand on the substrate for forming self-assembled monolayer (SAM), generating the substrate in which proteins were immobilized.
  • SAM self-assembled monolayer
  • To immobilize proteins on the metal substrate through additive thiol groups has some advantages of: (i) direct immobilizing proteins on the substrate via the molecule in proteins without help of other linkers,
  • the interactions or bindings between the first and second biomolecules are induced by applying the second biomolecule to the first biomolecule immobilized on the metal substrate.
  • the second biomolecule includes DNA, RNA, protein or carbohydrate, more preferably, DNA, RNA or protein, much more preferably, DNA or protein and most preferably, protein.
  • the second biomolecule is antigen where the first biomolecule is antibody.
  • the second biomolecule includes isolated or other substance-mixed forms.
  • the present method may be used in qualitative or quantitative analysis to examine whether target substances is involved in unknown samples. Therefore, the second biomolecule may be provided in a form of analyte or sample.
  • the second biomolecule is bound to the first biomolecule where the second biomolecule of target is present in the analytes or samples.
  • the present method after step (b), further induces the interaction between the second biomolecule and a third biomolecule by applying the third biolmolecule with affinity to the second biomolecule.
  • the second biomolecule is antigen
  • other antigen specifically bound to it may be used as a third biomolecule.
  • the third biomolecule is conjugated to an metal nanoparticle with electrical conductivity.
  • the electrical conductive metal nanoparticle bound to the third biomolecule is not particularly limited.
  • the metal nanoparticle includes gold, silver, bronze, white gold, aluminium, metal alloys ⁇ e.g., alloy of gold and copper) or metal oxide substrates.
  • the metal nanoparticle used in this invention is gold (Au) nanoparticle.
  • the third biomolecule is a protein containing additive thiol groups (-SH) and immobilized on the surface of metal nanoparticle therethrough.
  • the metal substrate is scanned by STM to measure an electric signal, whereby the occurrence of the interaction between the first and the second biomolecules is determined.
  • the basic principle of the present detection method is set forth as follows: Au nanoparticle-antibody conjugate with affinity to target molecules ⁇ i.e., the second biomolecule) exhibits a peak-like pulse in scanning the substrate containing Au nanoparticle-antibody conjugate by STM tip (or probe) in fixed current-mode and tunneling current also exhibits the peak-like pulse in a position most adjacent between STM tips (or probe).
  • the frequency of peak-like pulse is dependent on the surface density of Au nanoparticle-antibody conjugate. Therefore, it is possible to quantitatively analyze target molecules ⁇ i.e., the second biomolecule).
  • the density of Au nanoparticle-antibody conjugate may be analogized using STM images and the density of Au nanoparticle-antibody conjugate and frequency of current peak may be analyzed by observing the periodicity and strength change of current peak obtained from total scanning area.
  • the present invention includes the following steps: (a) immobilizing antibodies (first biomolecule) on gold substrate; (b) applying antigens (second biomolecule) to the gold substrate to induce the interaction between the first biomolecule and a second biomolecule; (c) applying antibodies (third biomolecule) with affinity to the second biomolecule to the gold substrate to induce the interaction between the second and third biomolecules; and (d) scanning the metal substrate using STM to measure an electric signal, whereby the occurrence of the interaction between the first and the second biomolecules is determined.
  • This invention is carried out by protein array (or protein chip) as a whole.
  • the first biomolecule is immobilized on the substrate of protein chip and the second and third biomolecules are added to protein chip.
  • Fig. 1 schematically represents a diagram of electrical detection system of protein based on STM.
  • Panal B shows topography and tunneling current profile of dispersed gold nanoparticles on the surface.
  • Fig. 2 schematically represents a monolayer preparation process of the present invention for electrically measuring currents using proteins and nanoparticles.
  • Fig. 3 schematically represents topographical STM images of Au nanoparticle layer immobilized on 1,8-ODT SAM with different particle concentration; a) 0.01%, b) 0.0001%, c) 0.0001%.
  • Fig. 3 represents power spectra of the current profiles acquired in the corresponding STM images (including bare gold).
  • Fig. 4 represents a schematic diagram (panel a) of immunoassay based on antibody fragment, I-V characteristics of bare Au (circle), thin film of antibody fragment (square) (panel b) and of immunocomplex (diamond) with its corresponding image (paenl c).
  • Fig. 5 shows regression curves of the power spectra with respect to the applied target (HSA) concentration. The regression curve in the lowest region is that of bare Au.
  • Fig. 6 represents a schematic diagram of the present assay system (panel a), and a micrograph of fabricated assay system after silver enhancement.
  • HSA human serum albumin
  • chromium and gold surface was sequentially fabricated with a thickness of 2 nm and 43 nm by DC magnetron sputtering (Kim et a/., 1999).
  • the sputtered Au substrate was cleaned, comprising the steps of: immersing in (i) the piranha solution (a mixture of 70:30 (v/v) of sulphric acid and hydrogen peroxide) for 5 min, (ii) ethanol solution for 30 min and (iii) deionized water for 2 hrs (Oh et a/., 2004; Lee et a/., 2005). All solutions were prepared with Millipore (MiIIi-Q) water followed by distillation.
  • nanoparticles observed in the prepared nanostructure was additionally fabricated because a blocking procedure which inhibited non-specific binding during immobilization was not carried out.
  • gold nanoparticle was self-assembled and immobilized on 1,8-ODT monolayer according to total surface morphology of STM represented in Fig. 3 and the surface density of gold substrate was changed by concentrations of nanoparticle introduced into SAM.
  • the regression curves of periodogram were obtained from the current profiles for the display of STM topography (Fig. 5). Each regression curve corresponds to a specific concentration of target analyte. Because the surface density of the Au nanoparticle depends on the amount of antibody-antigen binding events in the measurement system of this invention, the present inventors could control the surface density of Au nanoparticle through the regulation of the antigen (HSA) concentration applied to the surface.
  • HSA antigen
  • the pattern of monoclonal antibody fragment against HSA was fabricated with micro-contact printing method. After HSA and Au nanoparticle-antibody conjugate was added on surface according to the procedure.
  • the specific binding event of Au nanoparticle complex on the patterned surface was visualized by silver enhancement (Fig. 6).
  • the sandwich type binding event was observed on the patterned antibody fragment without nonspecific binding.
  • the estimated periodograms could be regressed with logarithmic curve, which showed monotonic decrease of dimensionless intensity. At an arbitrary point less than p/4 period, correlation of the dimensionless intensity could be observed with its corresponding concentration of target analytes.
  • the present invention demonstrates that the current profiles obtained from STM can be utilized for the quantitative measurement in protein array. Since the tunneling current in constant current-mode is dependent on the tip-to-sample separation, localized Au nanoparticles cause this separation to be close. It ultimately made a pulse-like peak occurred in STM current profile. The frequency of the change in tip-to-sample separation was related with the surface density of Au nanoparticle immobilized on surface. Characterization of the obtained current profile was performed by means of periodogram analysis. As the interval between immobilized nanoparticles would be decreased according the increase of their surface density, the dimensionless intensity at short period regime was significantly changed with respect to the amount of Au nanoparticle. The method of this invention was successfully extended to the measurement of antibody-antigen binding events.
  • the present detection system makes it possible to measure DNA, RNA, and other kinds of antigen, and allows multiple detections in a single chip.
  • the conception of measurement of the present invention can be carried out regardless of the spot size fabricated for protein array and allows ultrahigh sensitivity by signal enhancement through silver staining of Au nanoparticle.

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Abstract

The present invention relates to a method for electrically detecting an interaction between biomolecules using a scanning tunneling microscope (STM), comprising the steps of: (a) immobilizing a first biomolecule on a metal substrate; (b) applying a second biomolecule to the metal substrate to induce the interaction between the first biomolecule and the second biomolecule; and (c) scanning the metal substrate using STM to measure an electric signal, whereby the occurrence of the interaction between the first and the second biomolecules is determined. The present STM-based electrical detection method enables to detect analytes having much lower concentrations {e.g., 100 fg/mL). In addition, the method of the present invention allows multiple detections in a single measurement system {e.g., single protein chip). On the other hand, the present invention permits to successfully analyze a nano-size array of which detection is impossible in a conventional method based on the fluorescence and allows ultrahigh sensitivity by signal enhancement through silver staining of Au nanoparticles.

Description

METHODS FOR ELECTRICALLY DETECTING INTERACTIONS BETWEEN BIOMOLECULES USING SCANNING TUNNELING MICROSCOPE
BACKGROUND OF THE INVENTION FIELD OF THE INVENTION
The present invention relates to a method for electrically detecting interactions between biomolecules using scanning tunneling microscope.
BACKGROUND OF TECHNIQUE Electrical detection of biological binding events such as DNA hybridization, proteinprotein interaction, and small molecules has been introduced as an alternative emerging technology of conventional colorimetric and fluorescence-based method (Park et a/., 2002; Lasseter et a/., 2004; Rawlett et a/., 2002). Owing to its advantages of immediate response, cost effectiveness, and integration with semiconductor technology, the technological demand is anticipated to be still expanded. The advancement of these electrical detection technologies has been started from metal nanoparticle-biomolecule complex, nanowires, and conductive single-walled carbon nanotubes (SWNT) (Willner et a/., 2002; Chen et a/., 2004; Bunimovich et a/., 2006; Zheng et a/., 2005). Typical configuration of the proposed electrical detections is to create electrical circuit with two electrodes (Syvanen et a/., 2002; Tsai et a/., 2006). The readout can be performed with electrical resistance of the embedded conductive materials placed to gaps between electrodes. However, the existing electrical detection technology of DNA/protein binding events is restrictive for multiple detections because the configuration of device is designed in plane. Therefore, the major challenge of the electrical detection is to develop method that fulfills multiple detections.
Hybrid of Au nanoparticle-bio/organic material has been studied for the investigation of molecular switch, material property and other electronic devices, and has still been enlarged its applications such as the measurement of biological binding events (Gittins et a/., 2000; Csaki et a/., 2001; Xiao et a/., 2003). The small label size, the unique optical and electrical properties of Au nanoparticles, and easiness of signal enhancement through silver staining make them effective tools for the detection of biological binding events. Compared with the merits and defects of biosensors developed up to date by a number of researchers, they are described as follows. Surface Plasmon resonance (SRP) biosensor has various advantages such as reproductivity of measurement, minimalization of non-specific response, wide measurement range and potential of multi-component measurement, while has problem which miniaturization is difficult. Localized surface plasmon resonance (LSPR) biosensor allows easy miniaturization compared to SRP but has defect that detection distance from surface is near. Piezoelectric biosensor also allows easy miniaturization and has defect that sensitivity is decreased to the level of almost 1/10 compared with SRP. Nano-FET biosensor has some disadvantages in the sense that response is significantly dependent on non-specificity and the properties of measured substance and that detection distance from surface is far. On the other hand, Amperomet biosensor has low selectivity due to difficulty of common utilization and Impedence biosensor represents drawbacks like low reproductivity and non-specific response irrespective of its easy miniaturization and high sensitivity. Therefore, the biosensor equipped with a new analysis system to overcome defects of biosensors described above has been needed for a nano-size protein chip integrated analysis system.
Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains. DETAILED DESCRIPTION OF THE INVENTION
The present inventors have made intensive researches to develop a method for electrically detecting interactions or bindings between biomolecules in a high throughput manner. As results, we have discovered that the interactions or bindings between biomolecules were detected in a more convenient manner and with higher sensitivity using a scanning tunneling microscope (STM).
Accordingly, it is an object of this invention to provide a method for electrically detecting an interaction between biomolecules using a scanning tunneling microscope (STM).
Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.
In one aspect of this invention, there is provided a method for electrically detecting an interaction between biomolecules using a scanning tunneling microscope (STM), comprising the steps of:
(a) immobilizing a first biomolecule on a metal substrate;
(b) applying a second biomolecule to the metal substrate to induce the interaction between the first biomolecule and the second biomolecule; and
(c) scanning the metal substrate using STM to measure an electric signal, whereby the occurrence of the interaction between the first and the second biomolecules is determined.
The present inventors have made intensive researches to develop a method for electrically detecting interactions or bindings between biomolecules in a high throughput manner. As results, we have discovered that the interactions or bindings between biomolecules were detected in a more convenient manner and with higher sensitivity using a scanning tunneling microscope (STM).
The biomolecule to be detected in this invention includes, but not limited to, an biomolecule to specifically combine with a counterpart. According to a preferable embodiment, the first and second biomolecules are
DNA, RNA, protein or carbohydrate, more preferably, DNA, RNA or protein, much more preferably, DNA or protein and most preferably, protein.
According to a preferable embodiment, the first biomolecule is antibodies (polyclonal or monoclonal). For instance, the first and second biomolecules are specifically bound to each other in case that the former is an antibody as a kind of protein and the latter is an antigen as a kind of protein. In addition, the first and second biomolecules are specifically bound to each other in case that the former is a protein receptor and the latter is a protein ligand. In other example, the first and second biomolecules are specifically bound to each other in case that the former is DNA and the latter is an DNA-binding protein.
The present method permits to specifically detect the interactions or bindings between biomolecules with higher sensitivity.
According to the method of this invention, the first biomolecule is immobilized on the metal substrate.
The metal substrate to be used in this invention includes, but is not limited to, metal substrates with electrical conductivity and is used in any one known to those skilled in the art.
In addition, the shape, size and chemical composition of surface of the metal substrate to be used in this invention are not particularly restricted.
The term "metal substrate" refers to all substrates composed of metal, metal oxide and alloy. For example, the metal substrate to be used in this invention includes gold, silver, bronze, white gold, aluminium, metals alloy {e.g., alloy of gold and copper) or metal oxide substrates.
The metal substrate includes the substrates metal is coated on their surface as well as metal substrates. Most preferably, the metal substrate to be used in this invention is a gold (Au) substrate.
The metal "gold substrate" refers to all substrates which gold is coated on their surface.
To immobilize the first biomolecule on the metal substrate may be carried out by various methods known to those skilled in the art.
According to a preferable embodiment, the first biomolecule is a protein containing an additive thiol group (-SH) and is immobilized on the surface of metal therethrough.
Proteins will combine to a cysteine residue through its R group. According to a preferable embodiment, the present invention endows wild type protein to contain an additive thiol group. In general, the addition of thiol group is performed according to two different methods.
According to one method, the addition of thiol group is carried out by applying a reducing agent {e.g., 2-mercaptoethylamine) to wild type protein containing sulfide or disulfide bond.
In the other method, the addition of thiol group is carried out by further introducing a cysteine residue into N- or C-terminal end of protein through a genetic engineering technique.
It is preferable to perform former method where the first biomolecule is antibody. In treating the reducing agent to introduce additive thiol groups into the first biomolecule, the antibodies become fragmentation. These antibodies were designated as "fragmented antibody" in this specification. The illustrative Examples to be immobilized through these additive thiol groups follow as: substrate, preferably gold substrate was annealed at high temperature and washed with piranha solution. The proteins with additive thiol groups were dispersed on the surface of gold substrate and kept to stand on the substrate for forming self-assembled monolayer (SAM), generating the substrate in which proteins were immobilized. To immobilize proteins on the metal substrate through additive thiol groups has some advantages of: (i) direct immobilizing proteins on the substrate via the molecule in proteins without help of other linkers,
(ii) maximizing protein immobilization under predetermined conditions and (iii) immobilizing protein on the substrate in a correct orientation.
And then, the interactions or bindings between the first and second biomolecules are induced by applying the second biomolecule to the first biomolecule immobilized on the metal substrate.
According to a preferable embodiment, the second biomolecule includes DNA, RNA, protein or carbohydrate, more preferably, DNA, RNA or protein, much more preferably, DNA or protein and most preferably, protein.
The second biomolecule is antigen where the first biomolecule is antibody.
The second biomolecule includes isolated or other substance-mixed forms.
The present method may be used in qualitative or quantitative analysis to examine whether target substances is involved in unknown samples. Therefore, the second biomolecule may be provided in a form of analyte or sample.
The second biomolecule is bound to the first biomolecule where the second biomolecule of target is present in the analytes or samples.
According to a preferable embodiment, the present method, after step (b), further induces the interaction between the second biomolecule and a third biomolecule by applying the third biolmolecule with affinity to the second biomolecule. For example, where the second biomolecule is antigen, other antigen specifically bound to it may be used as a third biomolecule.
According to a preferable embodiment, the third biomolecule is conjugated to an metal nanoparticle with electrical conductivity. The electrical conductive metal nanoparticle bound to the third biomolecule is not particularly limited. Preferably, the metal nanoparticle includes gold, silver, bronze, white gold, aluminium, metal alloys {e.g., alloy of gold and copper) or metal oxide substrates. Most preferably, the metal nanoparticle used in this invention is gold (Au) nanoparticle. To combine the third biomolecule to the metal nanoparticle is carried out by any one known to those skilled in the art. According to a preferable embodiment, the third biomolecule is a protein containing additive thiol groups (-SH) and immobilized on the surface of metal nanoparticle therethrough.
After completing the reaction between biomolecules described above, the metal substrate is scanned by STM to measure an electric signal, whereby the occurrence of the interaction between the first and the second biomolecules is determined.
As the illustrative examples of the present method using the third biomolecule conjugated with Au nanoparticle, the basic principle of the present detection method is set forth as follows: Au nanoparticle-antibody conjugate with affinity to target molecules {i.e., the second biomolecule) exhibits a peak-like pulse in scanning the substrate containing Au nanoparticle-antibody conjugate by STM tip (or probe) in fixed current-mode and tunneling current also exhibits the peak-like pulse in a position most adjacent between STM tips (or probe). The frequency of peak-like pulse is dependent on the surface density of Au nanoparticle-antibody conjugate. Therefore, it is possible to quantitatively analyze target molecules {i.e., the second biomolecule). Additionally, the density of Au nanoparticle-antibody conjugate may be analogized using STM images and the density of Au nanoparticle-antibody conjugate and frequency of current peak may be analyzed by observing the periodicity and strength change of current peak obtained from total scanning area. According to the most preferable embodiment, the present invention includes the following steps: (a) immobilizing antibodies (first biomolecule) on gold substrate; (b) applying antigens (second biomolecule) to the gold substrate to induce the interaction between the first biomolecule and a second biomolecule; (c) applying antibodies (third biomolecule) with affinity to the second biomolecule to the gold substrate to induce the interaction between the second and third biomolecules; and (d) scanning the metal substrate using STM to measure an electric signal, whereby the occurrence of the interaction between the first and the second biomolecules is determined.
This invention is carried out by protein array (or protein chip) as a whole. In detail, the first biomolecule is immobilized on the substrate of protein chip and the second and third biomolecules are added to protein chip.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 schematically represents a diagram of electrical detection system of protein based on STM. Panal B shows topography and tunneling current profile of dispersed gold nanoparticles on the surface.
Fig. 2 schematically represents a monolayer preparation process of the present invention for electrically measuring currents using proteins and nanoparticles. Fig. 3 schematically represents topographical STM images of Au nanoparticle layer immobilized on 1,8-ODT SAM with different particle concentration; a) 0.01%, b) 0.0001%, c) 0.0001%. Fig. 3 represents power spectra of the current profiles acquired in the corresponding STM images (including bare gold). Fig. 4 represents a schematic diagram (panel a) of immunoassay based on antibody fragment, I-V characteristics of bare Au (circle), thin film of antibody fragment (square) (panel b) and of immunocomplex (diamond) with its corresponding image (paenl c). Fig. 5 shows regression curves of the power spectra with respect to the applied target (HSA) concentration. The regression curve in the lowest region is that of bare Au.
Fig. 6 represents a schematic diagram of the present assay system (panel a), and a micrograph of fabricated assay system after silver enhancement.
The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.
EXAMPLES Experimental Materials and Methods
Monoclonal and polyclonal antibody against human serum albumin (HSA) was purchased from Sigma-Aldrich (St. Louis, MO, USA). 2-mercaptoethylamine (MEA) and gold nanoparticle with a mean diameter of 5 nm was also purchased from Sigma-Aldrich.
Preparation of Substrate
On the cover glass with a thickness of 0.1 mm purchased from Superior (Germany), chromium and gold surface was sequentially fabricated with a thickness of 2 nm and 43 nm by DC magnetron sputtering (Kim et a/., 1999). The sputtered Au substrate was cleaned, comprising the steps of: immersing in (i) the piranha solution (a mixture of 70:30 (v/v) of sulphric acid and hydrogen peroxide) for 5 min, (ii) ethanol solution for 30 min and (iii) deionized water for 2 hrs (Oh et a/., 2004; Lee et a/., 2005). All solutions were prepared with Millipore (MiIIi-Q) water followed by distillation.
Fabrication and Measurement of Nanopartide-Antibody Conjugate
To carry out the oriented immobilization of antibody, gold-thiol interaction was utilized for both the conjugation of nanoparticle-antibody complex and the biosurface fabrication. 2-mercaptoethylamine (2-MEA) was applied to antibody solution for the fragmentation of immunoglobulin G (IgG) molecules (polyclonal antibody). After incubation for 90 min at 37°C, the residual 2-MEA was removed through the dialysis membrane with molecular cut-off membrane against phosphate buffered saline (PBS)-ethylenediaminetetraacetic acid (EDTA) buffer (pH 7.4) which is PBS containing 5 mM of EDTA. The above solution, Au nanoparticle and distilled water were mixed at a ratio of 3:6: 1, respectively and then incubated for 2 hrs at 4°C. For the stabilization of Au nanoparticle-antibody conjugate, 5% casein was added in the mixed solution and it was incubated for 1 hr. The prepared Au nanoparticle-antibody conjugate was centrifuged at 34,000 rpm for 1 hr at 40C using ultracentrifuge. And then, the sediments corresponding to Au nanoparticle-antibody complexes were recovered and resuspended in PBS. The fabrication of conjugates was confirmed by UV-visible spectroscopy.
Protein Introduction and Fabrication of Self-Assembled Monolayer 2-MEA was applied to antibody solution for the fragmentation of HSA molecules (monoclonal antibody). After incubation for 90 min at 370C, the monoclonal antibodies were fragmented. Cysteine residues were exposed on the surface of the antibodies due to fragmentation. Au-coated substrate was incorporated in the solution of fragmented antibody for 12 hrs, generating self- assembled monolayer of antibody on the substrate. The antibodies were linked to the substrate through their exposed cysteine residues. The fragmented antibodies were immobilized on gold substrate with orientation in a constant pattern.
Antigen Introduction The antibody which was not fixed on the substrate was washed with
PBS/EDTA buffer and then the substrate was incorporated in HAS solution of predetermined concentration for 6 hrs.
Introduction of Nanoparticle-antibody (polyclonal) Conjugate The antigen which was not fixed on the substrate was washed with
PBS/EDTA buffer and then the substrate was incorporated for 6 hrs in nanoparticle-antibody (polyclonal) conjugate solution described above.
Measurement and Assay The surface topography and current file of protein array was obtained by commercially available scanning probe microscopy (SPM, XE-100, Park Systems, Korea). Image acquisition was carried out under the condition of Iset = 0.5 nA in the applied voltage of above 0.5 V.
Results and discussion
For the proof-of-principle of the suggested electrical detection, a simple model system was investigated with Au nanoparticle and self-assembled dithiolated organic molecule (1,8-octanedithiol, 1,8-0DT). Au nanoparticle with 5 nm of diameter was immobilized on the 1,8-ODT monolayer. Localized binding event of Au nanoparticle was observed as shown in Figs. 3 (a) to 3 (c). The surface density of the Au nanoparticle immobilized on 1,8-ODT self-assembled monolayer (SAM) was proportional to the added concentration of Au nanoparticle. In concentration of over a critical limit, it can ultimately form a thin film. The current profile obtained for the display of STM topography was utilized as input variables for periodogram analysis.
The size distribution of nanoparticles observed in the prepared nanostructure was additionally fabricated because a blocking procedure which inhibited non-specific binding during immobilization was not carried out. However, it could be appreciated that gold nanoparticle was self-assembled and immobilized on 1,8-ODT monolayer according to total surface morphology of STM represented in Fig. 3 and the surface density of gold substrate was changed by concentrations of nanoparticle introduced into SAM.
This principle can be extended to sandwich type immunoassay for quantitative measurement (Fig. 4). In this case, Au nanoparticle can be placed on the bound antigen which is also localized on the surface. Experiment results shows that the Au nanoparticle-antibody conjugates was bound to the antigen-antibody cluster in a localized fashion on Au surface (Fig. 4 (b)). The existence of Au nanoparticle on surface can be observed with STM. Current-voltage (I-V) characteristics of the localized Au nanoparticle-antibody conjugate (Fig. 4 (c)) shows that the point where Au nanoparticle is located has high electrical resistance as well as topology difference. This local difference of electrical property can lead to pulse-like peaks of tunneling currents while STM tip is scanning on surface. Therefore, the analysis method of the present invention can be identically employed for the measurement of antibody-antigen binding events.
In order to verify the potential for semi-quantitative measurement of target molecules, the regression curves of periodogram were obtained from the current profiles for the display of STM topography (Fig. 5). Each regression curve corresponds to a specific concentration of target analyte. Because the surface density of the Au nanoparticle depends on the amount of antibody-antigen binding events in the measurement system of this invention, the present inventors could control the surface density of Au nanoparticle through the regulation of the antigen (HSA) concentration applied to the surface. For the detection of HSA on protein array, the pattern of monoclonal antibody fragment against HSA was fabricated with micro-contact printing method. After HSA and Au nanoparticle-antibody conjugate was added on surface according to the procedure. The specific binding event of Au nanoparticle complex on the patterned surface was visualized by silver enhancement (Fig. 6). The sandwich type binding event was observed on the patterned antibody fragment without nonspecific binding. The estimated periodograms could be regressed with logarithmic curve, which showed monotonic decrease of dimensionless intensity. At an arbitrary point less than p/4 period, correlation of the dimensionless intensity could be observed with its corresponding concentration of target analytes.
Compared with the periodogram taken from the sample less than 100 fg/mL of HSA, the obtained periodogram was not so significant that it could not be distinguished between them. From these experimental results, 100 fg/mL of HSA could be also successfully detected and the potential of electrical detection technique based on STM on protein array could be remarkably proposed.
Conclusion
The present invention demonstrates that the current profiles obtained from STM can be utilized for the quantitative measurement in protein array. Since the tunneling current in constant current-mode is dependent on the tip-to-sample separation, localized Au nanoparticles cause this separation to be close. It ultimately made a pulse-like peak occurred in STM current profile. The frequency of the change in tip-to-sample separation was related with the surface density of Au nanoparticle immobilized on surface. Characterization of the obtained current profile was performed by means of periodogram analysis. As the interval between immobilized nanoparticles would be decreased according the increase of their surface density, the dimensionless intensity at short period regime was significantly changed with respect to the amount of Au nanoparticle. The method of this invention was successfully extended to the measurement of antibody-antigen binding events. So far as the molecular dimension for detection is not much large as to interrupt the tip-to-sample interactions, the present detection system makes it possible to measure DNA, RNA, and other kinds of antigen, and allows multiple detections in a single chip. In addition, the conception of measurement of the present invention can be carried out regardless of the spot size fabricated for protein array and allows ultrahigh sensitivity by signal enhancement through silver staining of Au nanoparticle.
Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in this art, and the scope of this invention is to be determined by appended claims and their equivalents.
References
Bunimovich,, Y. L., Chin, Y. S., Yeo, W.-S., Amori, M., Kwong, G., Heath, J. R., J.
Am. Chem. Soc, 128: 16323-16331 (2006).
Chen, R. 1, Choi, H. C, Bangsaruntip, S., Yenilmez, E., Tang, X., Wang, Q., Chang,
Y.-L, Dai, H., J. Am. Chem. Soc, 126: 1563-1568 (2004). Csaki, A., Moller, R., Straube,W., Kohler, J. M., Fritzsche,W., Nucleic Acids Res.,
29: e81 (2001).
Gittins,D. L, Bethell, D., Schiffrin, D. J., Nichols, R. J., Nature, 408: 67-69 (2000).
Jin, Y., Kang, X., Song, Y., Zhang, B., Cheng, G., Dong, S., Anal. Chem., 73: 2843-
2849 (2001). Karyakin, A. A., Presnova,G. V., Rubtsova, M. Y., Egorov, A. M., Anal. Chem., 72:
3805 (2000).
Kim, M. H., Park, T. S., Yoon, E., Lee, D. S. Park, D. Y., Woo, Y. J., Chun, D. L, Ha,
J., J. Mater. Res., 14: 1255 (1999). Kossek, S., Padeste, C, Tiefenauer, L. X., Siegenthaler, H., Biosens. Bioelec, 13:
31-43 (1998).
Lasseter, T. L., Cai, W., Hamers, R. J., Analyst, 129: 3-8 (2004).
Lee, W., Oh, B. K., Lee., W. H., Choi, J. W., Colloids and Surf., B: Biointerfaces, 40: 143 (2005).
Oh, B. K., Kim,Y. K., Park, K. W., Lee, W. H., Choi, J. W., Biosens. Bioelectron., 19:
1497 (2004).
Park, SJ., Taton, T. A., Mirkin, C. A., Science, 295: 1503-1506 (2002).
Rawlett, A. M., Hopson,T. J., Nagahara, L. A., Tsui, R. K., Ramachandran, G. K., Lindsay, S. M., Appl. Phys. Lett, 81: 3043-3045 (2002).
Syvanen, A.-C, Soderlund. H., Nat. Biotechnol., 20: 349-340 (2002).
Tsai, C-Y., Chang, T.-L, Kuo, L-S., Chen, P.-H., Appl. Phys. Lett., 89: 203902
(2006)
Willner, L, Willner, B., Pure Appl. Chem., 74: 1773-1783 (2002). Xiao, Y., Patolsky, F., Katz, E., Hainfeld, J. F., Willner, I., Science, 299: 1877-1881
(2003).
Zheng, G., Patolski, F., Cui, Y., Wang, W. U., Lieber, C. M., Nat. Biotechnol., 23:
1294-1301 (2005).

Claims

What is claimed is:
1. A method for electrically detecting an interaction between biomolecules using a scanning tunneling microscope (STM), comprising the steps of:
(a) immobilizing a first biomolecule on a metal substrate; (b) applying a second biomolecule to the metal substrate to induce the interaction between the first biomolecule and the second biomolecule; and (c) scanning the metal substrate using STM to measure an electric signal, whereby the occurrence of the interaction between the first and the second biomolecules is determined.
2. The method according to claim 1, wherein the first and second biomolecules comprise DNA, RNA, protein or carbohydrate.
3. The method according to claim 1, wherein the metal substrate is a gold substrate.
4. The method according to claim 1, wherein the first and second biomolecules are proteins.
5. The method according to claim 4, wherein the first biomolecule is an antibody.
6. The method according to claim 4 or 5, wherein the protein or antibody further comprises a thiol group (-SH) and is immobilized on the metal surface therethrough.
7. The method according to claim 1, wherein the second biomolecule is an antigen.
8. The method according to claim 1, wherein the method further, after step (b), comprises the step of inducing the interaction between the second biomolecule and a third biomolecule by applying the third biolmolecule with affinity to the second biomolecule.
9. The method according to claim 8, wherein the third biomolecule is linked to a metal nanoparticle.
10. The method according to claim 9, wherein the metal nanoparticle is a gold nanoparticle.
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