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WO1992015709A1 - Technique immunologique de microscopie par sonde a balayage - Google Patents

Technique immunologique de microscopie par sonde a balayage Download PDF

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Publication number
WO1992015709A1
WO1992015709A1 PCT/US1992/001572 US9201572W WO9215709A1 WO 1992015709 A1 WO1992015709 A1 WO 1992015709A1 US 9201572 W US9201572 W US 9201572W WO 9215709 A1 WO9215709 A1 WO 9215709A1
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WO
WIPO (PCT)
Prior art keywords
analyte
test piece
scanning
test
amount
Prior art date
Application number
PCT/US1992/001572
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English (en)
Inventor
Stephen D. Stroupe
Original Assignee
Abbott Laboratories
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Publication of WO1992015709A1 publication Critical patent/WO1992015709A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • 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
    • 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
    • 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

  • This invention relates generally to the detection of analytes, and more particularly, relates to the detection of analytes by utilizing scanning probe microscopy.
  • STM and AFM instruments are commercially available from several sources, such as Digital Instruments, Inc. (Santa Barbara, CA 93117), TopoMetrix (Pasadena, CA 91106), and EG&G Princeton Applied ⁇ Research (Princeton, NJ 08543).
  • the probe is electrical.
  • the sensor At each X, Y coordinate position, the sensor, a very fine wire drawn to an atomically sharp tip, is maintained a constant distance above the surface by a piezoelectric ceramic driver.
  • a constant potential is maintained between the tip and the surface to be examined; hence, the requirement that the substrate be conductive.
  • Conductive substrates such as Highly Oriented Pyrolytic Graphite (HOPG) (available from Digital Instruments, Santa Barbara CA) and gold have been used. Crystals also can be examined, since they are usually conductive.
  • HOPG Highly Oriented Pyrolytic Graphite
  • Crystals also can be examined, since they are usually conductive.
  • the Z coordinate at a constant distance above the surface is recorded using the tunneling current from the tip to the surface in a feedback circuit to keep the tunneling current constant, and hence, the distance from the tip to surface constant.
  • the "map" resulting from such measurements consists of the height, Z, of the surface at various X, Y coordinates. For conductive substrates, this method allows the resolution of atoms in crystalline latices (Hansma et al., 1988, supra).
  • STM can be used to image samples immersed in an aqueous solution.
  • S. M. Lindsay et al, Science.244: 1063 - 1064 (1989) electrophoretically deposited double-stranded DNA onto a gold surface and observed the double helix conformation of the macromolecule in an aqueous environment.
  • the use of STM in ultra high vacuum has allowed the imaging of DNA at the atomic level with DNA dried onto HOPG, as reported by R. J. Driscoll et al, Nature. 346: 294 - 296 (1990).
  • Atomic Force Microscopy is usually performed by observing the deflection of a microscopic spring as a probe is lowered to the surface of the material to be examined.
  • the spring ends in a fine tip and its deflection by contact with the surface can be observed by monitoring a beam of laser light reflected from a mirrored surface on the back of the probe.
  • AFM does not require a conductive substrate, and further it does not require that the material to be imaged be conductive.
  • the former allows non-conductive materials such as plastic, glass, and silicon which are compatible with various biochemical and biomedical processes to be used.
  • the latter allows the direct detection of various kinds of biological molecules without the necessity of coating them with a conductive layer.
  • Probes other than electron tunneling or atomic force can be used to explore the surfaces of various substrates.
  • atomic scale friction can be measured in a Scanning Force Microscope, as well as surface "hardness” or “softness”.
  • Electrostatic interactions also provide another probe of a surface; the sensor is essentially an AFM probe, but with a charge (positive or negative) imparted to its surface. Electrostatic repulsion or attraction is measured by deflection of the probe as it approaches the surface giving a charge map of the surface.
  • the present invention provides a first method for determining the presence and/or amount of an analyte in a test sample, which method comprises contacting a test sample with a test piece to which an analyte specific substance has been attached for a time and under conditions sufficient to allow binding of the analyte to the test piece, and scanning the test piece by scanning probe microscopy to determine the presence or absence of the analyte.
  • Quantitation of the amount of analyte present in the test sample can be accomplished by counting the number of analyte molecules in one or more fields of the test piece and determining the concentration of analyte present in the test sample either by reference to previously measured standards or mathematically.
  • Scanning is performed by monitoring the response of a microsensor as it is systematically scanned across " a surface of the test piece in raster fashion to define an X, Y plane such as to control the vertical displacement of the microsensor thereby making a map of a function of the surface of the test piece at various X, Y coordinates. Scanning may be performed either by a scanning tunnelling microscope or an atomic force microscope.
  • the analyte specific substance is attached to the test piece by adsorption or by covalent binding.
  • the preferred solid phases of the test piece used for adsorption attachment are plastic or metal.
  • the preferred solid phases of the test piece used for covalent attachment are derivatized plastic, metal, glass and silicon.
  • the analyte specific substance can be an antibody, an antigen, an antigen analog, DNA, RNA, a lectin, a hapten, avidin, biotin, folate binding protein, or intrinsic factor.
  • the present invention also provides a second method of determining the presence of an analyte which may be present in a test sample, which method comprises (a) mixing the test sample with a known .amount of its specific binding partner to form a mixture; (b) applying the mixture to the test piece to which the analyte or an analyte analog has been attached, for a time and under conditions sufficient for a reaction to occur; and (c) scanning the test piece by scanning probe microscopy to determine the presence and/or amount of the specific binding partner, wherein the presence and amount of the specific binding partner bound is inversely proportional to the amount of analyte present in the test sample.
  • Quantitation of the amount of analyte present in the test sample can be accomplished by counting the number of specific binding partner molecules in one or more fields of the test piece and determining the concentration of analyte present in the test sample either by reference to previously measured standards or mathematically. Scanning is performed by monitoring the response of a microsensor as it is systematically scanned across a surface of the test piece in raster fashion to define an X, Y plane such as to control the vertical displacement of the microsensor thereby making a map of a function of the surface of the test piece at various X, Y coordinates.
  • the analyte specific substance is attached to the test piece by adsorption or by covalent binding.
  • the preferred solid phases of the test piece used for adsorption are plastic or metal.
  • the preferred solid phases of the test piece used for covalent attachment are derivatized plastic, metal, glass and silicon.
  • the analyte specific substance can be an antibody, an antigen, an antigen analog, a hapten, DNA, RNA, a lectin, avidin, biotin, folate binding protein or intrinsic factor.
  • test kits useful for determining the presence of an analyte suspected of being present in a test sample by scanning probe microscopy, comprising a test piece having bound thereto an analyte specific substance, an analyte, or an analyte analog.
  • the analyte specific substance, analyte or analyte analog preferably is covalently attached to the test piece of the test kit.
  • the test piece of the test kit preferably comprises a solid phase.the solid phase being selected from the group consisting of derivatized plastic, glass, silicon and metal.
  • the test piece of the test kit most preferably comprises a silicon wafer.
  • FIG IA is an SPM image of an amine-activated silicon test piece.
  • FIG. IB is a graph of the profile of the surface of an amine- activated silicon test piece wherein height (in .angstroms [A]) of the surface of the test piece is plotted against surface length (in nanometers [nm]).
  • FIG. 2A is an SPM image of an activated silicon test piece to which an analyte specific substance (anti-Hepatitis B surface antigen monoclonal antibody) has been attached.
  • an analyte specific substance anti-Hepatitis B surface antigen monoclonal antibody
  • FIG. 2B is a graph of the profile of the surface of an amine- activated silicon test piece to which an analyte specific substance (anti- Hepatitis B surface antigen monoclonal antibody) has been attached, wherein height (in angstroms [A]) of the surface of the test piece is plotted against surface length (in nanometers [nm]).
  • analyte specific substance anti- Hepatitis B surface antigen monoclonal antibody
  • FIG. 3A is an SPM image of an activated silicon test pieces to which an analyte specific substance (anti-Hepatitis B surface antigen monoclonal antibody) has been attached, after reaction with an analyte (r Hepatitis B surface antigen).
  • FIG. 3B is a graph of the profile of the surface of an amine- activated silicon test piece to which an analyte specific substance (anti- Hepatitis B surface antigen monoclonal antibody) has been attached, after reaction with an analyte (r Hepatitis B surface antigen), wherein height (in nanometers [nm]) of the surface of the test piece is plotted against surface length (in nanometers [nm]).
  • FIG. 4 is an SPM image of an amine-activated silicon test piece to which an analyte specific substance (anti-Hepatitis B surface antigen monoclonal antibody) has been attached, after reaction with an analyte (r Hepatitis B surface antigen).
  • analyte specific substance anti-Hepatitis B surface antigen monoclonal antibody
  • the present invention provides a means for specifically binding various analytes to a surface and a molecule by molecule determination of the presence or absence of the analyte in question using Scanning Probe Microscopy techniques.
  • the present invention thus provides the application of a powerful new analytical tool to the problem of determining the presence or absence of specific molecules.
  • the molecules are typically, though not limited to, molecules having biological properties such as antibodies, antigens, drugs, and the like.
  • the molecules can be nucleic acids such as DNA and RNA. Or, supramolecular structures consisting of assemblages of macromolecules such as virus coat protein particles, viruses, bacteria, membranes or membrane fragments, cells or fragments of cells can be the subject of the current invention.
  • the determination of the presence of the molecule of interest also can result in the quantitation of the amount of analyte present, if desired.
  • the analytical tool used in this invention is termed Scanning Probe
  • Microscopy and consists of a related family of techniques which can determine the properties of a surface at the molecular and even atomic level.
  • a micro-sensor is rapidly and systematically scanned across a surface in (for example) the X direction.
  • the sensor is advanced a small distance in the Y direction, .and another trace is made back in the X direction, thereby defining an X, Y plane.
  • the response of the sensor is monitored as it is rastered (i.e., systematically scanned) across the surface.
  • the sensor response is utilized in a feedback loop to control the Z position of the sensor.
  • a complete map is made of a function of the surface at various X, Y coordinates.
  • the microsensor detects current flowing across the gap between the surface and the microsensor. The current flow is used in a feedback loop to control the height of the senor above the surface so as to keep the current flow at a preselected value.
  • the microsensor physically contacts the surface, and the displacement of the sensor is used in a feedback loop to control the height of the sensor above the surface.
  • the present invention provides an immunoassay which utilizes specific binding members.
  • a "specific binding member,” as used herein, is a member of a specific binding pair. That is, two different molecules where one of the molecules through chemical or physical means specifically binds to the second molecule. Therefore, in addition to antigen and antibody specific binding pairs of common immunoassays, other specific binding pairs can include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, effector and receptor molecules, cofactors and enzymes, enzyme inhibitors and enzymes, and the like. Furthermore, specific binding pairs can include members that are analogs of the original specific binding members, for example, an analyte-analog.
  • Immunoreactive specific binding members include antigens, antigen fragments, antibodies and antibody fragments, both monoclonal and polyclonal, and complexes thereof, including those formed by recombinant DNA molecules.
  • hapten refers to a partial antigen or non-protein binding member which is capable of binding to an antibody, but which is not capable of eliciting antibody formation unless coupled to a carrier protein.
  • analyte is the substance to be detected which may be present in the test sample.
  • the analyte can be any substance for which there exists a naturally occurring specific binding member (such as, an antibody), or for which a specific binding member can be prepared.
  • an analyte is a substance that can bind to one or more specific binding members in an assay.
  • “Analyte” also includes any antigenic substances, haptens, antibodies, and combinations thereof.
  • the analyte can be detected by means of naturally occurring specific binding partners (pairs) such as the use of intrinsic factor protein as a member of a specific binding pair for the determination of Vitamin B12, the use of folate-binding protein to determine folic acid, or the use of a lectin as a member of a specific binding pair for the determination of a carbohydrate.
  • the analyte can include a protein, a peptide, an amino acid, a hormone, a steroid, a vitamin, a drug including those administered for therapeutic purposes as well as those administered for illicit purposes, a bacterium, a virus, and metabolites of or antibodies to any of the above substances.
  • EMIT Multiplied Immunoassay Technique
  • Supra-molecular structures frequently have been determined by binding specific binding agents such as antibodies with a measurable signal generating compound (measurable detectable label) to surface markers and observing the measurable bound label after removal of excess reagent.
  • specific binding agents such as antibodies with a measurable signal generating compound (measurable detectable label)
  • features internal to the structure could be stained allowing observation of the particle. Specificity in the latter case can be imparted by immunologically trapping the structure using specific binding agents such as antibodies against surface markers.
  • supramolecular analytes such as cells, malignant cells, blood cells, sperm, and microorganisms including bacteria, viruses, parasites, rickettsia, fungi, mycobacterium, mycoplasma, may be analyzed by the method of this invention.
  • Test samples which may be tested by the methods of the invention includes both liquid and solid substances of biological and non-biological origin.
  • Liquid and/or solid samples include those of biological origin, for example, human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, biological fluids such as cell culture supernatants, tissue specimens and cell specimens, and the like. Tissue specimens and cell specimens do not need to be fixed in order to be assayed by the method of the invention.
  • liquid and solid non-biological specimens including soil samples, water samples, oil samples, and the like.
  • a test piece comprising a solid phase may be used according to the method of the invention.
  • a "solid phase”, as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction.
  • the solid phase can be chosen for its intrinsic ability to attract and immobilize the capture reagent.
  • the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent.
  • the additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent.
  • the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid phase and which has the ability to immobilize the capture reagent through a specific binding reaction.
  • the receptor molecule enables the indirect binding of the capture reagent to a solid phase material before the performance of the assay or during the performance of the assay.
  • the solid phase thus can be a plastic, derivatized plastic, metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, .and other configurations known to those of ordinary skill in the art.
  • test piece comprising a solid phase also can comprise any suitable porous material.
  • porous is meant that the material is one through which the test sample can easily pass.
  • the solid phase can include NucleoporeTM membranes (available from VWR Scientific,
  • the solid phase can also comprise polymeric or glass beads, microparticles, tubes, sheets, plates, slides, wells, tapes, test tubes, or the like.
  • surface probes other than Electron Tunnelling or Atomic Force, such as atomic friction, scanning magnetic, scanning thermal or other scanning micromechanical instruments, when used in a scanning mode.
  • one member of a specific binding partner is attached to a surface suitable for scanning.
  • the attachment of the analyte specific substance may be by adsorption to a test piece which comprises a solid phase of a plastic or metal surface, following methods known to those of ordinary skill in the art.
  • covalent attachment of a specific binding partner (analyte specific substance) to a test piece which test piece comprises a solid phase of derivatized plastic, metal, silicon, or glass may be utilized.
  • Covalent attachment methods are known to those skilled in the art and include a variety of means to irreversibly link specific binding partners to the test piece.
  • the surface must be activated prior to attaching the specific bindmg partner.
  • Activated silane compounds such as triethoxy amino propyl silane (available from Sigma Chemical Co., St. Louis, MO), triethoxy vinyl ' silane (Aldrich Chemical Co., Milwaukee, WI), and (3-mercapto-propyl)- trimethoxy silane (Sigma Chemical Co., St. Louis, MO) can be used to introduce reactive groups such as amino-, vinyl, and thiol, respectively.
  • Such activated surfaces can be used to link the binding partner directly (in the cases of amino or thiol) or the activated surface can be further reacted with linkers such as glutaraldehyde, bis (succinimidyl) suberate, SPPD 9 succinimidyl 3-[2-pyridyldithio] propionate), SMCC (succinimidyl-
  • the vinyl group can be oxidized to provide a means for covalent attachment. It also can be used as an anchor for the polymerization of various polymers such as poly acrylic acid, which can provide multiple attachment points for specific binding partners.
  • the amino surface can be reacted with oxidized dextrans of various molecular weights to provide hydrophilic linkers of different size and capacity.
  • oxidizable dextrans examples include Dextran T-40 (molecular weight 40,000 daltons), Dextran T-110 (molecular weight 110,000 daltons), Dextran T-500 (molecular weight 500,000 daltons), Dextran T-2M (molecular weight 2,000,000 daltons) (all of which are available from Pharmacia,
  • the surface may be further treated with materials such as serum, proteins, or other blocking agents to minimize non-specific binding.
  • the surface also may be scanned either at the site of manufacture or point of use to verify its suitability for assay purposes. The scanning process is not anticipated to alter the specific binding properties of the test piece.
  • test pieces contemplated include surfaces of glass, plastic, metal and silicon.
  • the test piece comprises a silicon wafer.
  • the wafer can be of varying thickness. Therefore, silicon wafers from about 10 to about 30 mils can be used as the test piece.
  • the silicon wafer will be of a thickness of about 18 to about 22 mils.
  • the silicon wafer is sliced from a large crystal and can have an orientation of (100) or (111).
  • a silicon wafer such as described will be a polished, monocrystalline silicon slice.
  • the test piece to which an analyte specific substance has been attached is contacted with a test sample for an adequate period of time under conditions sufficient to .allow the binding of the analyte specific binding partner, if present in the test sample, to the analyte specific substance.
  • the test piece is scanned by scanning probe microscopy methods described herein to determine the presence or absence of the analyte.
  • a micro-sensor is rapidly and systematically scanned across a surface in (for example) the X direction. Upon the completion of one pass, the sensor is advanced in a small direction in the Y direction, and another trace is made back in the X direction.
  • the response of the sensor is monitored as it is rastered (i.e., systematically scanned) across the surface.
  • the sensor response is utilized in a feedback loop to control the Z position of the sensor. In this fashion, a complete map is made of a function of the surface at various X, Y coordinates.
  • the number of molecules in a particular field is counted and, either by reference to previously measured standards or mathematically, the concentration in the sample is determined.
  • Multiple fields on the same test piece may be examined to extend the sensitivity and accuracy of the method.
  • the test piece Before the sample is scanned, the test piece may be washed and dried or washed and scanned wet. Optionally, the test piece may be scanned with the sample still in place. The probe will sense only those molecules tightly bound at the surface of the test piece; therefore, weakly bound molecules will not interfere. It can be anticipated that the probe itself will sweep the surface clean of lightly bound interfering molecules.
  • the sample containing the analyte may be mixed with a known amount of its specific binding partner and the mixture applied to the test piece to which the analyte or an analyte .analog has been attached.
  • This amount of specific binding partner can be determined prior to beginning the assay.
  • This so-formed mixture is incubated for a time and under cond tions sufi ⁇ icient for a binding reaction to occur. Again, upon completion of the reaction, the test piece is scanned as previously described for the presence and amount of the specific binding partner.
  • the presence and amount of the specific binding partner bound will be inversely proportional to the amount of analyte in the sample.
  • the sensitivity and accuracy of the measurement process may be enhanced by scanning multiple fields of the same test piece.
  • the appropriate specific binding partner is a polynucleic acid complementary to one or more target sequences suspected of being present in the test sample.
  • Such methods are well known in the art. See, for example, Falkow et al., U.S. Patent No. 4, 358,535 and Ranki et al., U.S. Patent No. 4, 486,539, both of which are incorporated herein by reference Attachment of the capture sequence onto the test piece may be achieved covalently as, for example, described by Stabinsky, U.S. Patent No.
  • the capture sequence may be derivatized with a hapten, which in turn may be bound to the surface of the test piece through an antibody or the like as described by Snitman et al., published as International Patent Publication WO 86/07387, which is incorporated herein by reference.
  • the hapten derivatized capture sequence may be mixed with the sample prior to exposure to the test piece or it may be pre-bound to the antibody activated test piece.
  • the antibody may be attached as described above.
  • the target sequence Prior to its exposure to the test piece, the target sequence may be amplified by use of the polymerase chain reaction (PCR) or ligase chain reaction (LCR) such as to lessen the number of fields which need to be scanned to determine tiie presence or absence of analyte.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • U. S. Patents No.4,683,195 and 4,683,202 teach a method of amplifying DNA sequences by using PCR. Both of these patents are incorporated herein by reference. Briefly, in PCR, two complementary polynucleotide strands are amplified by treating the strands with two oligonucleotide primers such that an extension product of each primer is synthesized which is complementary to each nucleic acid strand. The primers are selected such that the extension product of one primer forms a template for the synthesis of an extension product from the other primer once the extension product of the one primer is separated from the template.
  • a chain reaction is maintained by a cycle of denaturing the primer extension products from their templates, treating the single- stranded molecule generated with the same primers to re-anneal, and allowing the primers to form further extension products.
  • the cycle is repeated for any many times as it takes to increase the target nucleic acid segments to a concentration where they can be detected.
  • the amplified target sequence can be detected by denaturing the double-stranded products formed by PCR, and treating those products with one or more reporter probes which hybridize with the extension products.
  • the Ligase Chain Reaction amplifies sections of polynucleic acid by copying the section of polynucleic acid, and copying the copies of that section of polynucleic acid, many times over.
  • the nucleic acid of the sample is provided either as single stranded or as double-stranded polynucleic acid which is denatured to separate the strands.
  • Four probes are utilized: the first two probes (A and B) are the so-called primary probes, and the second two probes (A * and B') are the so-called secondary probes.
  • the first probe (A) is a single strand capable of hybridizing to a first segment of the primary strand of the target nucleotide sequence.
  • the second probe (b) is capable of hybridizing to a second segment of the primary strand of the target nucleotide sequence.
  • the 5' end of the first segment of the primary strand of the target is positioned relative to the 3' end of the second segment of the primary strand of the target to enable joining of the 3' end of the first probe to the 5' end of the second probe, when the probes are hybridized to the primary strand of the target nucleotide sequence.
  • the third probe (A') is capable of hybridizing to the first probe
  • the fourth probe (B') is capable of hybridizing to the second probe (B).
  • the hybridized probes are ligated to form reorganized fused probe sequences. Then, the polynucleic acid in the sample is denatured to separate ligated probes from sample polynucleic acid.
  • Successive cycles wherein the ligated probes and target polynucleic acid undergo the above-described process are performed to increase the amount of detectable polynucleic acid in the sample.
  • the amount of cycles performed is dependent upon the sequence used and the sensitivity required of the test. Usually, the cycle can be repeated from 15 to 60 times.
  • the sensitivity of SPM immunoassays may be enhanced by use of standard immunological methods appropriately adapted to the type of probe used.
  • a second antibody labelled with materials such as colloidal metals, colloidal non- metals, small latex particles, charged polymers, liposomes, fixed cells, and the like can be used for enhanced detection of the analyte.
  • the specific binding ligand used in limited amounts may be appropriately labelled.
  • a complementary, non-overlapping, label sequence can be used for polynucleic acid analytes.
  • long sequences of double stranded DNA, linear or circular, may be used to enhance the sensitivity of the method.
  • a high degree of specificity can be imparted to a Scanning Probe Microscope Assay. That is, if an antibody, monoclonal or polyclonal, with very low cross-reactivity, is used to bind analyte to the surface of the test piece, it is anticipated that by proper combination of washings and probe manipulations a high degree of specificity will be exhibited by the assay. That is, only the desired analyte will be specifically immobilized on the surface of the test piece and so the detection of any material bound incremental to the specific binding partner originally attached to the surface will constitute a positive binding event. Additional specificity can be built into the assay if an additional requirement for a binding event to be scored as a positive result is used. Since the scanning probe microscope affords information on the size, shape and other characteristics can be used to provide additional specificity requirements for the assay result to be regarded as positive. For example, in an assay for Carcino-Embryonic .Antigen
  • CEA Cerebral astyrene-maleic anhydride
  • SP.MIA Scanning Probe Microscopy Immunoassay
  • stringency conditions will be used which limit the non-specific binding of target to capture probe.
  • Commonly used stringency conditions include high temperature, denaturing surfactants, organic solvents, high ionic strength buffers and the like. If a capture probe is used, it must be of sufficient length to allow efficient binding under conditions of high stringency. It is anticipated that there will be limited information available from the size and shape of the captured target sequence relevant to establishing sufficiency conditions to allow additional specificity in the assay. However, as pointed out by Driscoll et al. (supra). SPM allows atomic resolution of double stranded nucleic acids, thereby allowing verification of the "fit" of the target to capture sequence as well as confirmation that the sequence is that which is expected .
  • means of accelerating the binding reaction will provide shorter assay time. Temperature changes wherein the temperature used to affix the analyte specific substance to the test piece, as well as, the use of polyethylene glycol, ultrasonic exposure and other means, each may contribute either alone or in combination to the acceleration of the reaction. Reactions of macromolecular and supramolecular analytes can be accelerated by conducting the reaction in an ultracentrifuge wherein the analyte is sedimented onto the surface of the test piece at high gravitational force. Non-bound material can be removed by washing, and the specifically bound material analyzed by SPM. These accelerating means are contemplated to be within the scope of the present invention.
  • the concentration in the sample is determined.
  • Example 2 Coating of Test Pieces With Antibody A solution of murine monoclonal antibody directed against Hepatitis B Surface Antigen (anti-HBsAg) (IgGl subtype) (available from Abbott Laboratories, Abbott Park, IL in the commercially available kit AUZYME® MONO) was prepared in 2-[N-morpholino] ethane sulfonic acid (MES) (Sigma Chemical Co., St. Louis, MO) buffer (50mM, pH 7.0) at a concentration of 400 ⁇ ⁇ g/.mL.
  • MES 2-[N-morpholino] ethane sulfonic acid
  • a solution of l-ethyl-3-(3- dimethylaminopropryl) carbodiimide (EDAC) (.Aldrich Chemical Co., Milwaukee, WI) also was prepared in 50 mM MES, pH 7.0, at a concentration of 500 ⁇ g/mL. Equal volumes of the two reagents then were mixed to form a solution and immediately, 50 ⁇ L of the solution were applied to each silicon wafer, which had been activated following the procedure described in Example 1. The samples were allowed to react at room temperature for about 15 minutes and then were rinsed with distilled water and blown dry with a nitrogen stream as described in Example 1. The dry, highly reflective surface showed no visible residue.
  • EDAC l-ethyl-3-(3- dimethylaminopropryl) carbodiimide
  • Example 3 Reaction With rHBsAg Test pieces with anti-HBsAg covalently attached and prepared as described in Example 2, were covered with 50 ⁇ L of a solution containing 200 ⁇ g/mL of recombinant Hepatitis B Surface .Antigen subtype Ay
  • rHBsAg (available from Abbott Laboratories, Abbott Park, IL in the commercially available .kit Abbott Mx® AUSAB®) in 50 mM MES buffer pH 7.0, and allowed to react for approximately 20 minutes at room temperature. After reaction, the test pieces were washed with distilled water and dried with a stream of nitrogen gas as described in Example 1.
  • a test piece carried through the sequences described in Examples 1 and 2 was measured in a Rudolph Ellipsometer (model Auto EL, available from Rudolph Research, Flanders, NJ). Using a refractive index of 1.46, a film thickness of 37.7 Angstroms was obtained. Then, a test piece carried through the sequences described in Examples 1, 2, and 3 was measured in the Rudolph Ellipsometer. Again using a refractive index of 1.46, a thickness of 65.1 Angstroms was obtained. The thickness increase of 27.4 Angstroms was due to binding of the rHBsAg to the antibody coated surface of the test piece.
  • Example 5 Atomic Force Microscopy Examination of Test Pieces An Atomic Force Microscope, built by TopoMetrix (Pasadena, CA) was used to scan the surfaces of the various test pieces produced according to Examples 1, 2 and 3 detailed hereinbelow in points a through d , as follows. A force of approximately 6 nN (nanoNewtons) was applied by the sensor which scanned across the surface at 500 nm/s (nanometers per second) in the X direction. Upon the completion of one pass, the sensor was advanced in a small direction in the Y direction, and another trace was made back in the X direction. The response of the sensor was monitored as it was rastered (i.e., systematically scanned) across the surface. The sensor response was utilized in a feedback loop to control the Z position of the sensor. In this fashion, a complete map was made of a function of the surface at various X, Y coordinates. Two hundred or four hundred line images were obtained.
  • nN nanoNewtons
  • Example 2 A test piece prepared as described in Example 1 was scanned.
  • FIG. IA is an SPM image of the surface of the activated silicon test piece.
  • the image shows a smooth, featureless surface.
  • FIG. IB which traces the apparent elevation of the probe above the surface across the X, Y plane at three different lines (1, 2 and 3)
  • the traces approximate a smooth catenary.
  • the apparent increase in thickness at the edges of the trace are thought to be artifact.
  • FIG.2A is an SPM image of the silicon test piece coated with an analyte specific substance (antibody). Referring to FIG. 2A, the image shows a smooth, essentially featureless surface. Three different lines (1, 2 and 3) were traced across the X, Y plane. FIG. 2B , which traces the apparent elevation of the probe across the X, Y plane at three different lines (1, 2 and 3) above the surface, the traces approximate a smooth catenary. The apparent increase in thickness at the edges of the traces are thought to be artifact.
  • d A test piece prepared according to Example 3 was scanned and multiple fields were examined. FIG.
  • FIG. 3A is an SPM image of a test piece coated with analyte-specific substance (antibody directed against Hepatitis B Surface .Antigen [anti-HBsAg]) reacted with analyte (recombinant Hepatitis B Surface Antigen [rHBsAg]).
  • the figure shows multiple antigen particles immobilized on the surface of the test piece.
  • One major cluster of particles is seen approximately in the center with other particles in contact with another distributed around the image.
  • Three different lines (1, 2 .and 3) were traced across the X, Y plane.
  • FIG. 3B gives three traces across the field.
  • the upper trace (1) shows essentially constant elevation above the surface, while traces 2 and 3 traverse the large grouping of antigen particles. The artifact at the edges of the trace are still present.
  • FIG. 4 which provides a different projection of a field, shows the image of multiple globular objects, crowded together, others presenting as isolated hemispheres when not in contact with other
  • the method of the invention described herein can be used to assay for analytes which may be present in a test sample. It will be appreciated by those skilled in the art that many of the concepts of the present invention are equally applicable to other types of binding assays.
  • the embodiments described and presented herein are intended as examples rather than as limitations. Thus, the description of the invention is not intended to limit the invention to the particular embodiments disclosed, but it is intended to encompass all equivalents and subject matter within the spirit .and scope of the invention as described and contemplated above, and as set forth in the following claims.

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Abstract

Procédés et kits d'analyse servant à détecter la présence d'un analyte dans un spécimen, procédant molécule par molécule, au moyen de la microscopie par sonde à balayage, dans lesquels le spécimen susceptible de contenir l'analyte recherché est exposé à un élément d'analyse auquel on a fixé une substance spécifique à l'analyte et on analyse ledit élément au moyen de la microscopie à sonde d'analyse pour déterminer la présence ou l'absence de l'analyte.
PCT/US1992/001572 1991-02-28 1992-02-28 Technique immunologique de microscopie par sonde a balayage WO1992015709A1 (fr)

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WO1997049989A3 (fr) * 1996-06-25 1998-02-12 Interactiva Biotechnologie Gmb Ensembles a vaste specificite et affinite: approche qualitative de la discrimination d'echantillons complexes
EP0829722A3 (fr) * 1996-08-02 1998-09-16 Basf Aktiengesellschaft Méthode pour le criblage des agents actifs utilisant la microscopie à forces atomiques
WO2003008941A3 (fr) * 2001-07-17 2003-05-30 Bioforce Nanosciences Inc Detection de liaison moleculaire combinee au moyen de la microscopie a force atomique et de la spectrometrie de masse
WO2004027095A1 (fr) * 2002-09-20 2004-04-01 Intel Corporation Regulation de l'alignement de nanocodes a barres codant des informations specifiques pour lecture par microscope-sonde a balayage (spm)
US6897015B2 (en) 2000-03-07 2005-05-24 Bioforce Nanosciences, Inc. Device and method of use for detection and characterization of pathogens and biological materials
US6998228B2 (en) 1999-05-21 2006-02-14 Bioforce Nanosciences, Inc. Method and apparatus for solid state molecular analysis
US7008769B2 (en) 2000-08-15 2006-03-07 Bioforce Nanosciences, Inc. Nanoscale molecular arrayer
EP1546983A4 (fr) * 2002-09-20 2006-03-15 Intel Corp Alignement oriente de codes a barre nanometriques codant une information specifique pour la lecture par microscopie electronique a balayage (spm)
US7042488B2 (en) 2001-09-27 2006-05-09 Fujinon Corporation Electronic endoscope for highlighting blood vessel
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WO2007011405A3 (fr) * 2004-10-29 2008-01-17 Bioforce Nanosciences Inc Dispositif et procede a utiliser pour la detection et la caracterisation de micro-organismes et de microparticules
US7344832B2 (en) 2003-01-02 2008-03-18 Bioforce Nanosciences, Inc. Method and apparatus for molecular analysis in small sample volumes

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Cited By (18)

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AU729960B2 (en) * 1996-06-25 2001-02-15 Michael Mecklenburg Broad specificity affinity arrays: a qualitative approach to complex sample discrimination
WO1997049989A3 (fr) * 1996-06-25 1998-02-12 Interactiva Biotechnologie Gmb Ensembles a vaste specificite et affinite: approche qualitative de la discrimination d'echantillons complexes
US6872522B1 (en) 1996-06-25 2005-03-29 Michael Mecklenburg Broad specificity affinity arrays: a qualitative approach to complex sample discrimination
EP0829722A3 (fr) * 1996-08-02 1998-09-16 Basf Aktiengesellschaft Méthode pour le criblage des agents actifs utilisant la microscopie à forces atomiques
US6998228B2 (en) 1999-05-21 2006-02-14 Bioforce Nanosciences, Inc. Method and apparatus for solid state molecular analysis
US6897015B2 (en) 2000-03-07 2005-05-24 Bioforce Nanosciences, Inc. Device and method of use for detection and characterization of pathogens and biological materials
US7008769B2 (en) 2000-08-15 2006-03-07 Bioforce Nanosciences, Inc. Nanoscale molecular arrayer
US7060448B2 (en) 2000-10-10 2006-06-13 Bioforce Nanosciences, Inc. Evaluating binding affinities by force stratification and force panning
WO2003008941A3 (fr) * 2001-07-17 2003-05-30 Bioforce Nanosciences Inc Detection de liaison moleculaire combinee au moyen de la microscopie a force atomique et de la spectrometrie de masse
US7042488B2 (en) 2001-09-27 2006-05-09 Fujinon Corporation Electronic endoscope for highlighting blood vessel
WO2004027095A1 (fr) * 2002-09-20 2004-04-01 Intel Corporation Regulation de l'alignement de nanocodes a barres codant des informations specifiques pour lecture par microscope-sonde a balayage (spm)
EP1546983A4 (fr) * 2002-09-20 2006-03-15 Intel Corp Alignement oriente de codes a barre nanometriques codant une information specifique pour la lecture par microscopie electronique a balayage (spm)
US7361821B2 (en) 2002-09-20 2008-04-22 Intel Corporation Controlled alignment of nanobarcodes encoding specific information for scanning probe microscopy (SPM) reading
US7476786B2 (en) 2002-09-20 2009-01-13 Intel Corporation Controlled alignment of nano-barcodes encoding specific information for scanning probe microscopy (SPM) reading
US7531726B2 (en) 2002-09-20 2009-05-12 Intel Corporation Controlled alignment of nanobarcodes encoding specific information for scanning probe microscopy (SPM) reading
US7705222B2 (en) 2002-09-20 2010-04-27 Intel Corporation Controlled alignment of nano-barcodes encoding specific information for scanning probe microscopy (SPM)
US7344832B2 (en) 2003-01-02 2008-03-18 Bioforce Nanosciences, Inc. Method and apparatus for molecular analysis in small sample volumes
WO2007011405A3 (fr) * 2004-10-29 2008-01-17 Bioforce Nanosciences Inc Dispositif et procede a utiliser pour la detection et la caracterisation de micro-organismes et de microparticules

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EP0570535A1 (fr) 1993-11-24
AU1584292A (en) 1992-10-06
CA2100683A1 (fr) 1992-08-29

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