EP2007379A2 - Detection nanoelectronique de biomolecules en employant l'amplification des analytes et des rapporteurs - Google Patents

Detection nanoelectronique de biomolecules en employant l'amplification des analytes et des rapporteurs

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Publication number
EP2007379A2
EP2007379A2 EP07754869A EP07754869A EP2007379A2 EP 2007379 A2 EP2007379 A2 EP 2007379A2 EP 07754869 A EP07754869 A EP 07754869A EP 07754869 A EP07754869 A EP 07754869A EP 2007379 A2 EP2007379 A2 EP 2007379A2
Authority
EP
European Patent Office
Prior art keywords
polynucleotide
probe
analyte
reporter
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07754869A
Other languages
German (de)
English (en)
Other versions
EP2007379A4 (fr
Inventor
Eugene Tu
Christian Valcke
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanomix Inc
Original Assignee
Nanomix Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US11/488,465 external-priority patent/US20070045756A1/en
Priority claimed from US11/588,845 external-priority patent/US20080021339A1/en
Application filed by Nanomix Inc filed Critical Nanomix Inc
Publication of EP2007379A2 publication Critical patent/EP2007379A2/fr
Publication of EP2007379A4 publication Critical patent/EP2007379A4/fr
Withdrawn legal-status Critical Current

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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
    • 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
    • C12Q1/682—Signal amplification
    • 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
    • C12Q1/6823—Release of bound markers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0645—Electrodes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5029—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures using swabs

Definitions

  • the present invention relates to detector systems for biomolecules using nanoelectronic sensor devices.
  • a strand of single-strand DNA (ssDNA) in solution readily combines with a complementary strand (cDNA) that contains an opposite base to pair with each base in the ssDNA.
  • cDNA complementary strand
  • dsDNA double-stranded DNA
  • an experimenter provides the appropriate cDNA as a probe sequence. If the target sequence is present in a sample, the target ssDNA will hybridize with the probe ssDNA to produce dsDNA, and this hybridization can be detected in some way.
  • a second problem results from the low sensitivity of traditional detection methods. Although some of these methods are sensitive to low concentrations of DNA, they require large absolute numbers of DNA molecules. In a medical application, only a few cells are usually available, and consequently only a few DNA molecules of the target sequence will be present in a sample. This problem has been ameliorated by the use of the polymerase chain reaction (PCR), which can amplify the quantity of target DNA a million-fold. Like labeling, PCR is a complex chemical reaction, which makes tests expensive and slow.
  • PCR polymerase chain reaction
  • Certain work has utilized oligonucleotides in systems for detection of target analytes, generally using optical or fluorescent detection methods. See for example, US Published App.
  • a salient objective of providing a method which avoids the necessity of labeling or amplification of analyte species and provides an electronic detection signal is to enable simplified, automated bio-detection capability conducive to fast and inexpensive point-of-care diagnostic applications, preferably that may be carried out non-laboratory clinical or home setting.
  • Nanostructures possess unique properties for sensor applications; in that they may be essentially one- dimensional so as to be extremely sensitive to electronic perturbations, are readily functionalized, and are compatible with many semiconducting manufacturing processes.
  • Embodiments having aspects of the invention employ nanostructures which have properties heavily influence by the atoms are on the surface, thus providing a basis for sensitive electronic detection.
  • Exemplary embodiments preferably include one or more carbon nanotubes, and more preferably one or more single-walled carbon nanotubes (SWNTs).
  • Alternative device embodiments generally include an element including at least one nanostructure (“nanostructure element”) whose electronic properties are highly sensitive to interaction with a target analyte.
  • One or more conducting elements may communicate with the nanostructure element to provide signal(s) for measurement of one or more device electronic properties which are influenced by the response of the nanostructure element to exposure to an analyte medium.
  • a supporting substrate typically includes at least a dielectric surface (or surface coating) to provide electrical isolation of device elements.
  • Substrates may be rigid or flexible, porous or non-porous, and may be generally planar or flat, or alternatively may have functional shapes, such as a tubular configuration, and may be of a number of alternative compositions, such as silicon oxide, silicon nitride, aluminum oxide, polyimide, and polycarbonate, and the like.
  • the substrate includes one or more layers, films or coatings comprising such materials as silicon oxide, SIO2, Si3N4, and the like, upon the surface of a silicon wafer or chip.
  • Nanotube network devices may comprise a collective structure which includes a plurality of nanostructures, such as SWNTs or other nanotubes arranged to form a collective structure.
  • the nanostructured element may advantageously comprise a random interconnected network of nanotubes ("nanotube network”) disposed on or adjacent a substrate, and communicating with at least one electrical lead.
  • Nanotube networks may be made by such methods as chemical vapor deposition (CVD) with traditional lithography, by solvent suspension deposition, vacuum deposition, and the like. See for example, US Patent Application No. 10/177,929 (corresponding to WO2004-040,671); US Patent Application No.
  • a contact includes a conducting element disposed such that the conducting element is in electrical communication with the nanostructure element, such as a nanotube network.
  • contacts may be disposed directly on a substrate surface, or alternatively may by disposed over a nanotube network.
  • Electric current flowing in the nanotube network may be measured by employing at least two contacts that are placed within the defined area of the nanotube network, such that each contact is in electrical communication with the network.
  • an additional conducting element referred to as a gate or counter electrode, is provided such that it is not in electrical communication with the nanostructured element (such as at least one nanotube), but such that there is an electrical capacitance between the gate electrode and the nanostructured element.
  • Exemplary devices comprise field-effect transistors where the channel of the transistor comprises the nanotube(s), and the device may be referred to as a nanotube field effect transistors or NTFET.
  • the gate electrode is a conducting plane within the substrate beneath the silicon oxide.
  • a gate or counter electrode may comprise a conductive layer disposed adjacent (e.g., under, above, beside), but electrically isolated from, the nanostructure element, such as a conductive polymeric material deposited on a flexible substrate. Resistance, impedance, transconductance or other properties of the nanotubes may be measured under the influence of a selected or variable gate voltage. Examples of such nanotube electronic devices are provided, among other places, in US patent applications No. 10/656,898, filed September 5, 2003 (Publication 2005-0279987) and No. 10/704,066, filed November 7, 2003 (Publication 2004-0132,070), both of which are incorporated herein, in their entirety, by reference.
  • a transistor device arrangement lends itself to measurement of the channel transconductance as a function of gate voltage (e.g., G ⁇ /g signal).
  • a transistor has a maximum conductance, which is the greatest conductance measured with the gate voltage in a range, and a minimum conductance, which is the least conductance measured with the gate voltage in a range.
  • a transistor has an on- off ratio, which is the ratio between the maximum conductance and the minimum conductance.
  • a nanotube transistor has an on-off ratio preferably greater than 1.2, more preferably greater than 2, and most preferably greater than 10.
  • Additional materials may be included in association with the nanostructure element (e.g., species or layers attached or absorbed upon one or more of the nanostructure element, the substrate, the conductor, and the like) to mediate the interaction of the device elements with the analyte medium, including target species, cross contaminants and the like.
  • Such materials may include one or more of recognition layers or molecular transducers (such as the ssDNA oligomer probes in the following examples), catalyst materials, passivation materials, inhibition materials, protective materials, filters, analyte attractors, concentrators, binding species, and the like.
  • Such materials and elements can function to improve selectivity, specificity and/or device service characteristics.
  • the invention provides an electronic sensor device with which to detect specific target sequences of polynucleotides.
  • the sensor comprises nanostructured elements, (for example single and/or multiwalled carbon nanotubes and/or interconnecting networks comprising such nanotubes) which interact with polynucleotides so as to act as sensing elements.
  • the nanostructured elements comprise carbon nanotubes, and more particularly, randomly oriented networks of carbon nanotubes.
  • the nanotubes are modified before sensing by the adsorption of ssDNA probe sequences. No labeling of the DNA is required.
  • the invention provides a method for using the sensor device.
  • DNA means polynucleotides.
  • polynucleotides include, but are not limited to, deoxyribonucleic acid, ribonucleic acid, messenger ribonucleic acid, transfer ribonucleic acid, and peptide nucleic acid.
  • the defining characteristics of polynucleotides are a chain of nucleic acids and a sequence of bases, each base chemically bonded to a nucleic acid and each base capable of pairing with an appropriate base on a matching sequence. Those skilled in the art will appreciate that other variations of polynucleotides may be produced which share these defining characteristics.
  • a “single- strand DNA”, referred to hereafter as “ssDNA”, may be a single strand of deoxyribonucleic acid, ribonucleic acid, or any other polynucleotide as described above.
  • a “double-strand DNA”, referred to hereafter as “dsDNA” or duplex polynucleotide, may be a double strand of any polynucleotide described above.
  • “Complimentary DNA”, referred to hereafter as “cDNA” may be any strand of a polynucleotide described above which is a single-strand sequence complimentary to an already referenced single-strand sequence.
  • the ssDNA in a particular sensor device may be selected to be cDNA for a particular target sequence.
  • the target sequence is the sequence of bases that the sensor device is intended to detect.
  • the cDNA for the target sequence is known as the probe sequence.
  • a quantity of DNA with the probe sequence must be obtained.
  • a variety of techniques are known for synthesizing DNA with specified sequences and for synthesizing DNA complementary to a given sequence. Those skilled in the art will have knowledge of these techniques. Further, appropriate cDNA or other polynucleotide to make a probe specific to a desired target sequence can generally be obtained from known commercial suppliers serving the biotechnology industry.
  • a sensor device may be used by exposing the nanotube network to a solution containing sample ssDNA.
  • the network should be exposed to the solution for a period of time long enough for hybridization to occur. This period of time depends on the concentration of the sample DNA, the quantity of the solution, the temperature of the room, the pH of the solution, and other variables. Those skilled in the art are familiar with the effect of these variables on DNA hybridization and are capable of choosing an appropriate period of time, solution composition, temperature and other conditions of hybridization without undue experimentation.
  • the occurrence, speed and specificity of polynucleotide hybridization depends on various conditions. In each of these hybridization schemes, the binding energy of the dsDNA can be challenged through stringency techniques. This can be done through temperature increases or buffer changes, for example sodium hydroxide.
  • Additional stringency controls may include various ionic constituents of the hybridization medium, such as sodium or magnesium ions.
  • a voltage may be applied to elements of the sensor (e.g., a nanotube network) before, during and/or after hybridization to influence polynucleotide behavior.
  • a polynucleotide such as cDNA has a phosphate-based backbone which typically is ionized in the hybridization medium so as to carry a localized negative charge.
  • Selectively charged sensor elements may be used to provide an attractive or repulsive stringency factor, for example, to destabilize a SNP-mismatched probe hybrid relative to a corresponding fully-matched probe hybrid (e.g., during incubation or during a rinse process).
  • the stringency of the hybridization conditions may be adjusted (e.g. by variation in temperature) so as to produce a distinctly different device measurement response between the homozygous and heterozygous samples.
  • these sensors may be constructed in arrays, e.g., arrays of transistor sensors functionalized for a plurality of different target DNA fragments. See US Application No. 10/388,701 entitled “Modification Of Selectivity For Sensing For Nanostructure Device Arrays” (publication 2003-0175,161), incorporated by reference herein.
  • Electrochemical detector embodiments In some embodiments of the invention, nanostructured elements, such as an electrode including a carbon nanotube network, may be employed to detect electrochemical interactions of analyte target species and/or reporters described herein, so as to permit measurement of presence or concentration of one or more analytes in a sample. See, for example, the detection methods and devices as described in co-invented US patent applications No. 60/901,538 filed February 14, 2007 and No. 60/850,217 filed October 6, 2006, each entitled “Electrochemical nanosensors for biomolecule detection”. Each of these applications is incorporated herein by reference. [036] Capacitance detector embodiments.
  • detectors including nanostructured capacitive elements may be employed to detect electrochemical interactions of analyte target species and/or reporters described herein, so as to permit measurement of presence or concentration of one or more analytes in a sample.
  • detection methods and devices as described in co-invented US patent application No. 11/588,845 filed October 26, 2006 entitled “Anesthesia Monitor, Capacitance Nanosensors and Dynamic Sensor Sampling Method", which claims priority to No. 60/850,217 filed October 6, 2006; No. 60/773,138 filed February 13, 2006; No. 60/748,834 filed December 9, 2005 and No. 60/730,905 filed October 27, 2005.
  • Alternative detection methods, labels, markers and devices are described in detail focus on reporters advantageously detectable by electronic signals independent of chemically or optically active labels or markers, alternative methodology is possible without departing from the spirit of the invention.
  • reporter fragments, species or amplicons derived by any of the amplification methods described herein may contain particular markers, labels or detection enhancers configured to effect any one of a number of different reporter or analyte detection methods known in the art, such as fluorescent markers, quenching groups, mass alteration, optical detection, spectroscopy, electrophoretic mobility alteration, receptor affinity alteration and the like.
  • reporter fragments, species or amplicons derived by any of the amplification methods of the invention may be configured to participate in secondary biomolecular reactions so as to have a detectable effect, e.g., by catalytic effects, or by triggering the vulnerability of a fluorescent probe to independent enzymatic attack or reconfiguration, or the like.
  • Non- PCR Reporter Amplification provides methods of electronically detecting a quantity of a reporter molecule, such as a specific DNA oligomer, following the amplified release of the reporter molecule in response to a much smaller quantity of target analyte.
  • enzymes having specific activity on polynucleotides may be employed to achieve a non-PCR amplification of a reporter moiety which is indicative of the presence of a selected analyte and which is electronically detectable by use of nanosensor having aspects of the invention.
  • Such embodiments may be described as having non-cyclical amplification in that the amplification process does not require a elaborate system of control for cyclically varying reaction conditions, as is the case in PCR for example.
  • a probe assembly comprises a substrate, such as a magnetic bead, a titration well, or the like, to which is bound one or more probe oligonucleotides.
  • a bead substrate may support a plurality of distinct and separately-functional probe oligonucleotides on the bead surface, so as to form a multi-probe assembly.
  • Each such probe oligonucleotide includes a nanocode sequence portion and a capture sequence portion. The nanocode sequence portion is selected to be complementary to a corresponding sequence on a reporter oligonucleotide.
  • the reporter oligonucleotide is hybridized, under suitable conditions, to the nanocode sequence.
  • the capture sequence portion is selected and configured to be complementary to a corresponding sequence on a target analyte polynucleotide (target sequence).
  • Probe oligonucleotide with capture sequence and nanocode may be manufactured to specification by known methods, and attached to substrates such as magnetic beads or other immobilization surface by known methods.
  • each target analyte polynucleotide is bound by a probe to form an analyte/probe complex (there may be an excess of probes, e.g. many probes per magnetic bead).
  • immobilization of the substrate with bound analyte/probe complex permits purification or rinsing of the complex, thus simplifying the sample or lysis mixture.
  • exonuclease (added after rinsing) has specific activity so as to degrade only capture sequence and nanocode of probe assembly portion of an analyte/probe complex, but not target analyte polynucleotide or reporter oligonucleotide (or pristine probes).
  • Exonuclease activity releases both target analyte polynucleotide and reporter oligonucleotide, so that: (a) analyte polynucleotide is free to react with additional pristine probes; and (b) reporter oligonucleotides accumulate in reaction buffer to a high concentration.
  • Accumulated (amplified) reporter oligonucleotides in simplified media may be electronically detected without significant cross reactivity by proprietary nanoelectronic detectors, as described herein.
  • the example employs an exonuclease having a specific activity for degradation (e.g. by hydrolysis of phosphate backbone bonds) of double stranded polynucleotides having a non-protruding 3' terminal end (e.g. blunt or recessed 3 1 ).
  • an exonuclease having a specific activity for degradation e.g. by hydrolysis of phosphate backbone bonds
  • double stranded polynucleotides having a non-protruding 3' terminal end e.g. blunt or recessed 3 1
  • alternative apparatus and methods may be configured to employ other exonuclease activity, such as a "mirror image" example, wherein the 3' and 5' configurations of polynucleotides and oligonucleotides are reversed, and the exonuclease has 5' end specificity.
  • Alternative embodiments can employ 5 1 reactive exonucleases, sticky-end exonulceases, etc; and can use other immobilization surfaces (polymer or Si surfaces, other bead types, etc.), cleavable linker between probe, media volume reduction, bead filtration, and the like.
  • enzymes may be employed under selected conditions to achieve selective exonuclease activity.
  • various types of DNA polymerase may be employed in a media deprived of nucleotides tri-phosphates, so as to favor "proof-reading" activity at the expense of polymerase activity, thus functioning as a nuclease.
  • the constituent elements of the probes may include polynucleotides with synthetic base analogs, such as locked nucleic acids (e.g., LNA Oligos or nucleic acids including a 2'-O, 4'-C methylene bridge, such as are available from Sigma-Aldrich Corp.). LNA oligomers may be used to block or limit exonuclease activity in selected portions of the probe molecules, and to control stability of hybridized duplexes.
  • LNA Oligos may be used to block or limit exonuclease activity in selected portions of the probe molecules, and to control stability of hybridized duplexes.
  • antibody composite probes and/or aptamer-based probes for protein, polysaccharide or other biomolecule target analytes may be employed to permit detection of non-polynucleotide analytes.
  • the exemplary detection methods and devices having aspects of the invention include concentration and simplification steps to increase sensor response, sensitivity and/or selectivity.
  • Probes may be bound or immobilized for separation or rinsing steps by attachment to any solid support or substrate suitable for binding the oligomers.
  • suitable substrate materials include, but are not limited to, glass, plastics, polyethylene, cellulose, polymethacrylate, latex, rubber, fluorocarbon resins , metals, and the like.
  • the substrate material may be configured as a slide, well or other enclosure, or alternatively as a particle, such as a microsphere or microbead. Paramagnetic coatings can render bead magnetically responsive.
  • Conjugating or complexing substances may be employed to bind oligonucleotides or other probe species to substrates, e.g. avidin or an avidin derivatives, suitable for binding biotin or biotin derivatives.
  • Magnetic bead probe conjugation and separation/immobilization techniques are well known and the constituents and accessories are commercially available. See for example, Invitrogen Corporation, http://www.invitrogen.com/ (formerly Dynal Biotech) of Carlsbad, California. See also the separation methods and devices described in US Published Application 2005-0147,822 entitled “Process”; US Patent 5,512,439 entitled “Oligonucleotide-linked Magnetic Particles And Uses Thereof; US Patent 6,994,971 entitled “Particle Analysis Assay For Biomolecular Quantification”; and US Patent 5,851,770 entitled “Detection Of Mismatches By Resolvase Cleavage Using A Magnetic Bead Support”; each of which publications are incorporated by reference.
  • Magnetic beads of sizes on the order of 1 micron or less are available, optionally pre-treated with binding constituents such as covalently bound streptavidin (e.g., for binding to avidin-treated oligonucleotides), bacterial wall proteins (e.g., for binding to antibodies), and the like.
  • binding constituents such as covalently bound streptavidin (e.g., for binding to avidin-treated oligonucleotides), bacterial wall proteins (e.g., for binding to antibodies), and the like.
  • Alternative separation/immobilization modalities for particulate probe substrates may be used, such as electrophoresis separation. Beads or particles having positive charged groups (e.g., amino) or negative charged groups (e.g., carboxylic acid) may be separated or immobilized by electric field forces. In other alternatives, particulate or bead substrates may be separated by filtration from a reaction medium.
  • the method comprises: (a) providing at least a first probe assembly, the first probe assembly comprising: ⁇ i) a substrate; (ii) at least one probe polynucleotide including a proximal 5'-terminal nucleotide and a distal 3'-terminal nucleotide, the probe polynucleotide bound adjacent the proximal ⁇ '-terminal nucleotide to the substrate, the probe polynucleotide comprising at least one nanocode nucleotide sequence portion, and a capture nucleotide sequence portion having a nucleotide sequence complementary to a corresponding target nucleotide sequence of the analyte polynucleotide; (iii) at least a first reporter polynucleotide having a nucleotide sequence complementary to the nanocode sequence of the probe polynucleo
  • the first probe/analyte complex including: (i) the first probe assembly; (ii) at least one analyte polynucleotide; and (iii) the analyte polynucleotide extending distally to form either of (1) a blunt distal 5'-terminal end or (2) a protruding distal 5'-terminal end;
  • An alternative embodiment of a method of detecting an analyte polynucleotide in a sample comprises the mirror-image method relative to that of the paragraph above, in which 3 1 and 5' terminal ends are reversed throughout the definition of the method, so that:
  • the substrate is conjugated adjacent a proximal 3' end of the probe polynucleotide;
  • the distal end of the first probe/analyte complex includes the analyte polynucleotide extending distally to form either of (1) a blunt distal 3'- terminal end or (2) a protruding distal 3'-terminal end;
  • the exonuclease has 5'-to-3' exo-deoxyribonuclease activity including specific binding to double-stranded DNA followed by selective hydrolysis of the 5 1 terminated strand of the DNA duplex;
  • the method includes maintaining conditions effective to allow hydrolysis of the distally 5' terminated strand of the probe/analyte complex to release the analyte polynucleotide and at least the first reporter polynucleotide;
  • the method includes determining the presence of the analyte polynucleotide in the sample by detecting the presence of the reporter polynucleotide released by the exonuclease 5'-to-3' hydrolysis. Exponential non-PCR target amplification.
  • One aspect of the invention provides methods of amplifying target analyte polynucleotide related species, so as to provide enhanced detection scope for rarified samples or minute quantities of sample analyte.
  • Alternative amplification methods and apparatus may include:
  • Homologous reporters Amplifying detectable species representative of target analyte polynucleotide presence in a sample by analyte-triggered release of corresponding synthetic target oligonucletides from amplifier reagent duplex species via exonuclease-mediated reactions, where the released synthetic target oligonucletides comprise detectable copies of portions of the target analyte polynucleotide sequence (homologous or quasi-homologous analog oligonucleotides); and/or (2) Non-homologous reporters.
  • the example employs an exonuclease having a specific activity for degradation (e.g. by hydrolysis of phosphate backbone bonds) of double stranded polynucleotides having a non-protruding 3' terminal end (e.g. blunt or recessed 3').
  • Dual target amplifiers In an embodiment having aspects of the invention, the sequences of two distinct portions of a single-stranded analyte polynucleotide are selected, conveniently designated Target A and Target B.
  • An amplifier reagent may be compounded so as to include at least two species of amplifier:
  • Amplifier "A” including an oligonucleotide which has a first capture sequence complementary to Target B (Capture B 1 ) and a second capture sequence complementary to Target A (Capture A') .
  • Amplifier "B” including an oligonucleotide which has a first capture sequence complementary to Target A (Capture A') and a second capture sequence complementary to Target B (Capture B') .
  • Each of the pristine Amplifier A and Amplifier B in the reagent comprises a duplex or double stranded form, in which the amplifier oligonucleotide is hybridized with a companion oligonucleotide configured to produce a duplex which lacks any non-protruding 3' end, so as to be protected from exonuclease activity in its pristine form.
  • the companion oligonucleotide includes a sequence (synthetic target) which mimics a corresponding target sequence of the analyte molecule , as follows:
  • the companion oligonucleotide of Amplifier "A” includes a synthetic target sequence the same or similar to Target A of the analyte (Synthetic Target A”), the Synthetic Target A” being hybridized in the pristine reagent to the second capture sequence Capture A'; and
  • the companion oligonucleotide of Amplifier "B” includes a synthetic target sequence the same or similar to Target B of the analyte (Synthetic Target B”), the Synthetic Target B" being hybridized in the pristine reagent to the second capture sequence Capture B'.
  • Amplifier hybridizing with target Each of Amplifier A and Amplifier B is configured (in the pristine duplex form) to expose a constituent capture sequence so that the capture sequence (under conditions effective to promote polynucleotide hybridization) may hybridize with the corresponding target sequence of the analyte polynucleotide, so as to form a corresponding Amplifier/Analyte Complex.
  • the capture sequence under conditions effective to promote polynucleotide hybridization
  • each Amplifier/Analyte Complex may include hybridization of either one or both of Amplifier A and Amplifier B with each analyte polynucleotide. The process need not proceed in synchronized form as the amplifiers types are configured to react in the describe process independently of each other. [059] Amplifier/Analyte Complex enzymatic degradation.
  • the selection of target sequence locations and/or configuration of the amplifier provides that the Amplifier/Analyte Complex (either having Amplifier A, an Amplifier B or both) includes an exposed non-protruding 3' end of each amplifier oligonucleotide, so that exonuclease present in the reagent (or added separately) may initiate degradation of the amplifier oligonucleotide.
  • the Amplifier/Analyte Complex either having Amplifier A, an Amplifier B or both
  • the Amplifier/Analyte Complex includes an exposed non-protruding 3' end of each amplifier oligonucleotide, so that exonuclease present in the reagent (or added separately) may initiate degradation of the amplifier oligonucleotide.
  • both capture sequences are removed, so as to release the target analyte from the amplifier oligonucleotide, and to release from the amplifier oligonucleotide the corresponding companion oligonucleotide having a synthetic target sequence, as follows:
  • each of these target sequences may participate in further processes of amplification as described above, because each amplifier is configured to hybridize (under conditions effective to promote polynucleotide hybridization) with a synthetic target with the same effect as with the native target: (1) the first capture sequence Capture B 1 of a pristine Amplifier A binds to
  • Synthetic Target B of the released companion oligonucleotide of a previously-degraded Amplifier B to form an all-synthetic Amplifier/Target
  • Synthetic Target A of the released companion oligonucleotide of a previously-degraded Amplifier A to form an all-synthetic Amplifier/Target
  • An embodiment having aspects of the invention includes a method of introducing a reporter species into a medium where the medium includes a biomolecular template species, the method comprising a non- PCR, template-triggered, enzyme-activated release of the reporter species from a probe assembly having a binding affinity for the template species.
  • the method including in any operative order the steps of:
  • the first probe assembly including: (i) at least a first probe strand having a capture nucleotide sequence which provides a selective binding affinity for a target portion of the template species; (ii) at least one first reporter species including a binding portion having a polynucleotide sequence configured to hybridize with a corresponding binding portion of the probe strand; (iii) wherein the first probe assembly includes at least one first probe strand and at least one first reporter species hybridized to comprise a polynucleotide duplex probe assembly; and (iv) wherein the duplex probe assembly is configured so as to have at least one enzyme-initiation site suited to promote the action of a selected enzyme having nuclease activity sufficient to degrade all or a portion of the probe strand so as to release the first reporter species from the probe assembly, the enzyme-initiation site being formed in the event that the capture nucleotide sequence binds with all or part of the selected target portion of the template species so as to form a template
  • step (c) contacting the medium with at least the selected enzyme under conditions effective to promote nuclease activity of the enzyme at an enzyme-initiation site, so that in the event that a first template-probe complex has been formed in step (b), the first probe strand is degraded so as to release the first reporter species from the probe assembly.
  • the amplification of this embodiment may be carried out so as to have multiple stages by additional steps whereby the first the first reporter species includes a template portion which is configured to act as a target template for at least one second non-PCR, template-triggered, enzyme-activated release of a second reporter species from a second probe assembly having a binding affinity for the template portion of the first reporter species. This may be extended to have a third or more stage in further reporter species are released.
  • the multistage amplification method may be used to detect analytes such as polynucleotides, proteins, polysaccharides, and other biomolecular species.
  • the first probe assembly includes capture nucleotide sequence complementary to a target sequence of the template (e.g., template includes a DNA strand).
  • the first probe assembly includes an aptamer as a capture nucleotide sequence, the aptamer having an affinity for a template target portion (e.g., template includes a polypeptide or polysaccharide target portion).
  • the biomolecular template species includes an analyte species in a sample
  • the first reporter species is configured to be directly or indirectly detectable when released from the probe assembly, the method further including the steps of:
  • the first reporter species is configured to be directly detectable when released from the first probe assembly, such as by including a detection portion having a detectable polynucleotide sequence or a detectable label group (single stage detection).
  • the first reporter species may indirectly detectable when released from the first probe assembly, via additional amplification stages in which further reporter species are detectable.
  • Hairpin probe assembly Further exemplary embodiments having aspects of the invention may eliminate separate capture and reporter portions of the probe assembly.
  • the first probe strand and the first reporter species comprise a co-linear polynucleotide strand, wherein portions of the co-linear polynucleotide strand are configured to self-hybridize when not in association with the target portion of the template species so as to be protected from degradation by the selected enzyme.
  • FIGS. 1A through 1H illustrate an exemplary embodiment of methods and apparatus having aspects of the invention of electronically detecting a quantity of a reporter molecule, such as a specific DNA oligomer, following the amplified release of the reporter molecule in response to a much smaller quantity of target analyte.
  • a reporter molecule such as a specific DNA oligomer
  • FIGS. 2A through 2H illustrate an exemplary embodiment of methods and apparatus having aspects of the invention for electronic detection which provide a non-linear or exponential analyte-responsive amplification.
  • FIGS. 3A and 3B summarize two examples of the process of amplification, both as shown in FIGURES 2A-H, one example employing two analyte targets (dual target) and an alternative example employing a single analyte target (single target).
  • FIG. 3C is a plot showing the data of Tables 1 and 2, depicting the increase or amplification of synthetic targets by the single target scheme and dual target scheme.
  • FIGS. 4A through 4B illustrate alternative apparatus and methods for reporter amplification and purification.
  • FIGS. 5A through 5E illustrate an alternative exemplary method and apparatus embodiment having aspects of the invention which provide exponential analyte- responsive amplification including a pre-amplification purification process.
  • FIGS. 6A through 6C illustrate one exemplary method and apparatus embodiment having aspects of the invention which provide exponential analyte-responsive amplification followed by removal of exonuclease from the media under analysis.
  • FIGS. 7A-F shows an example of a reporter probes having aspects of the invention and including with internal blocking groups which stop the processing of exonuclease at the sequence location of the blocking group.
  • FIGS. 8A and 8B illustrate one exemplary method and apparatus embodiment having aspects of the invention including a multi-analyte assay with multiple reporter types and a matrix detector cell.
  • FIGS. 8A and 8B illustrate one exemplary method and apparatus embodiment having aspects of the invention including a multi-analyte assay with multiple reporter types and a matrix detector cell.
  • FIGS. 9A to 9C illustrate one exemplary method having aspects of the invention employing a probe assembly including an aptamer portions for detection on biomolecules.
  • FIG. 10 comprises views 10a-10i which depict an exemplary embodiment of a two-stage method of target-initiated amplification so as to produce amplicons and/or reporter species.
  • FIG 11 is a plot showing exemplary data corresponding to the method of FIG. 10
  • FIGS. 12A-12C depict an exemplary embodiment of a three-stage method of target-initiated amplification so as to produce amplicons and/or reporter species.
  • FIG 13 is a plot showing exemplary data corresponding to the method of FIGS.
  • FIGS 14A-14C illustrate an example of single-target amplification method having aspects of the invention (See, e.g., FIG. 3A) utilizing fluorescent detection, wherein:
  • FIG. 14A is a diagram of the amplifier-analyte complex
  • FIG 14B is a photograph of a electrophoretic gel showing results and amplification product of the method
  • FIG 14B is a negative version of the photograph of FIG. 14B, which provides a more distinct indication of the data in a photo-reproducible image suitable for patent illustration.
  • FIGS 15A-15C illustrate an example of a "hairpin" type probe assembly.
  • the present invention provides a nanotube sensor device that detects a target DNA sequence.
  • the device requires no labeling of the target DNA and responds electronically to the presence of the target DNA. Exemplary embodiments are described below.
  • Analyte-Triggered Reporter Amplification [(mi FIGS 1A through 1H illustrate an exemplary embodiment of methods and apparatus having aspects of the invention of electronically detecting a quantity of a reporter molecule, such as a specific DNA oligomer, following the amplified release of the reporter molecule in response to a much smaller quantity of target analyte.
  • enzymes having specific activity on polynucleotides may be employed to achieve a non-PCR amplification of a reporter moiety which is indicative of the presence of a selected analyte and which is electronically detectable by use of nanosensor having aspects of the invention.
  • Such embodiments may be described as having non- cyclical amplification in that the amplification process does not require a elaborate system of control for cyclically varying reaction conditions, as is the case in PCR for example.
  • FIG. 1A shows a figurative example of sample collection and preparation step 10, depicting a sample applicator 11 (e.g., a throat swab or the like) inoculating a lysis buffer or media 12 in a container 13 to release cellular or viral material 14, which is in turn lysed to release sample polynucleotide material 15.
  • FIG. 1B shows a figurative example of sample processing in a fluidic system 20 comprising an applicator 21 (e.g., a pipette) applying lysed sample media to an inlet port 22, which communicates with a pattern of conduits 24 (e.g., of a microfluidic cartridge) connecting enclosure cells or vessels 23.
  • 1C(i) and 1C(H) show sample media 16 comprising polynucleotide material 15 as contained in vessel 23 containing one or more probe assemblies 30, each probe assembly comprising a substrate 32 attached to one or more capture probes 31.
  • the substrate 32 comprises a magnetic bead, but other particulate and non-particulate capture substrates (e.g., non-magnetic particles, fixed plates, enclosure walls, titration well, or the like) may be employed.
  • a bead substrate may support a plurality of distinct and separately- functional probes 31 on the bead surface, so as to form a bead-centered multi- probe assembly 30.
  • FIG. 1C(ii) shows detail of a probe assembly 30 and target analyte 33.
  • the target analyte is a single strand polynucleotide having a target nucleotide sequence X between 3' and 5' terminal strand ends.
  • Probe assembly 30 comprises a capture probe polynucleotide 34 bonded to substrate 32, for example by a biotin-streptavidin bond.
  • a probe such as probe 30 may be bound to a substrate by a complementary "sandwich” type oligonucleotide structure, for example the structure of complementary "sandwich” probe 79 described below with respect to FIG. 4B.
  • the probe polynucleotide 34 is bound to substrate 32 adjacent a proximal 5 1 terminal nucleotide, and comprises a nanocode sequence Y and a capture sequence X 1 , which is complementary to target sequence X on analyte polynucleotide 33.
  • the probe 31 also includes one or more reporter oligonucleotides 35 having a complementary sequence Y 1 which in the assembled probe is hybridized to the nanocode sequence Y of the probe polynucleotide 34. Note that although FIG. 1C(H) depicts the nanocode sequence Y as being proximal (relative to substrate 32) with respect to the capture sequence X', this need not be so.
  • Probe oligonucleotide 34 with selected capture and nanocode sequences as well as reporter oligonucleotide 35 may be manufactured to specification by known methods, such as by commercially available synthetic services. Likewise, biotinilated oligonucleotides are commercially available having designated base sequences, and substrates may be treated with streptavidin by know methods. Other suitable protocols for attaching polynucleotides to substrates are known, such as by use of cell surface ligands and the like. Reporters 35 may be hybridized to polynucleotide 34 under suitable conditions to complete probe assembly 30.
  • polynucleotide and oligonucleotide are generally used herein interchangeably. While the selected lengths of particular sequences is relevant to the hybridization properties of the molecules, the principals of the invention apply to a wide range of sequence lengths and molecular weights without departing from the spirit of the invention. Where the context or usage makes it clear that a polynucleotide or oligonucleotide is being referenced, such species may also be referred to as a "molecule” or “species”, or by molecule function, such as “analyte” or “reporter”, without loss of clarity.
  • each probe oligonucleotide includes a nanocode sequence portion and a capture sequence portion.
  • the nanocode sequence portion is selected to be complementary to a corresponding sequence on a reporter oligonucleotide.
  • the reporter oligonucleotide is hybridized, under suitable conditions, to the nanocode sequence.
  • the capture sequence portion is selected and configured to be complementary to a corresponding sequence on a target analyte polynucleotide (target sequence).
  • Probe oligonucleotide with capture sequence and nanocode may be manufactured to specification by known methods, and attached to substrates such as magnetic beads or other immobilization surface by known methods.
  • 1D(i) and 1D(ii) shows the vessel or cell 23 of fluidic system 20 in which target analyte polynucleotides 33 have hybridized with the probe nucleotides 34 of probes 31 to form one or more probe/analyte complexes 36, the probe/analyte complex 36 comprising the probe assembly 30 bound to the analyte polynucleotide 33 by hybridization of the target sequence X of the analyte 33 with the capture sequence X' of at least one probe polynucleotide 34.
  • FIG. 1D(ii) shows detail of a probe/analyte complex 36, depicting the target analyte 33 having a distal 5' end extending beyond and overlapping the distal 3' end of the probe polynucleotide 34.
  • FIG. 1E shows the one or more substrates 32 immobilized (or alternatively fixedly mounted) to permit one or more optional rinsing or washing steps in which sample medium 16 with un-reacted species 15 is removed and replaced by a reaction medium 17, leaving the one or more probe/analyte complexes 36 (and any unreacted probes or probe assemblies) in the vessel 23.
  • one or more substrates are magnetic beads immobilized by controllable magnet 18.
  • probe assembly 30 comprises a plurality of probes 31
  • a plurality of analytes 33 may bind to respective probes 31 of each such probe assembly 30.
  • probe assembly or assemblies 30 having in total an excess of probes 31 such that analyte 33 presented in a sample is efficiently bound by available capture probes 31 in the step of FIGS. 1D.
  • Rinsing/washing of the step of FIG. 1E may then advantageously remove non-target polynucleotides and other contaminants present in the initial sample or lysis mixture, so as to minimize the likelihood or extent of non-specific binding and cross reactivity during detection, loss] FIGS.
  • 1F(i), 1F(H) 1 1 R(Ui) and 1G(i) show the treatment of the probe/analyte complexes 36 with a exonuclease 37, in this example the exonuclease 37 having a specific activity to degrade (e.g., hydrolysis of phosphate bonds) duplex hybridized polynucleotides strands having a blunt or recessed (non-protruding) 3' end exposed.
  • the non-protruding 3' distal end of probe polynucleotide 34 is attacked and degraded progressively.
  • specific activity of the exonuclease acts to degrade only capture sequence and nanocode of probe assembly portion of an analyte/probe complex, but not target analyte polynucleotide 33 or reporter oligonucleotide 35 (or any pristine or unreacted probes 31).
  • nucleases as with other enzymes, have variable processivity depending on type and environment, and what for convenience is illustrated as a continuous process of degradation may be discontinuous or episodic as nuclease diffuses on or off the substrate oligonucleotide.
  • nucleases may have both primary and secondary activity (such as endonuclease activity), and one of ordinary skill in the art will be able to adjust concentrations and conditions to favor desired exonuclease activity.
  • alternative embodiments may have probes configured to reverse the order of proximal and distal terminal strand ends (3 1 for 5 1 ) and employ exonucleases with a corresponding reversed activity (specific to non-protruding 5' duplex ends).
  • enzymes systems other than specific endonucleases and having multiple activity modes can be employed where non-specific activity is controlled or non-interfering.
  • FIG. 1 F(iii) shows the degradation of probe molecule 34 proceeding so as to release the analyte molecule 33 by removal of capture sequence X'.
  • FIG. 1G(i) shows the completed degradation of probe molecule 34 so as to release the reporter molecule 35 by removal of nanocode sequence Y.
  • 1F(iii) is free to diffuse within medium 17 and hybridize with any remaining unreacted ("pristine") probes 31 to form additional probe/analyte complexes 36.
  • Such subsequently formed complexes 36 are in turn likewise vulnerable to exonuclease degradation, so as to release additional reporters 35.
  • the provision of an excess of probes 31 permits an advantageous amplification process whereby each analyte molecule 33 is permitted to generate the sequential release of a plurality of reporter molecules from the analyte-stimulated degradation of a corresponding plurality of probes 31.
  • FIG. 1G(ii) shows the flow of accumulated reporters 35 to an adjacent detector cell 25 having one or more nanoelectronic sensors 40 as described herein (see incorporated patent applications and further sensor embodiments described below).
  • Sensor 40 has a sensitivity for reporter probe 35, for example by having one or more detector probe 41 comprising a oligonucleotide complementary to at least a portion of the sequence of reporter 35.
  • the detector 40 may be included in the reaction cell 23.
  • reporter 35 results in a specific detectable change in the electronic properties detector 40 so as to permit the determination of the presences of reporter 35 by operation of measurement circuitry 42: a. reporters hybridize with detector probes. b. optional step: reaction buffer may be replaced by measurement buffer (e.g., to inhibit nuclease activity at detector). c. signal acquired (e.g., transistor . or capacitive properties, etc.) d. reporters detected.
  • reaction medium 17 may be removed (e.g., replaced with a measurement buffer 18) following detector probe hybridization, prior to signal acquisition by circuitry 42. Exponential non-PCR amplification.
  • FIGS. 2A through 2H illustrate an exemplary embodiment of methods and apparatus having aspects of the invention (among a number of alternative methods having aspects in common) for electronic detection which provide a nonlinear or exponential analyte-responsive amplification (additional non-sample targets are made available or "revealed” in response to analyte presence in sample, e.g., so as to participate in stimulating reporter release).
  • a method such as in FIGS. 2A-H may be employed independently or in combination with methods such as exemplified by FIGS. 1A-H, which may be characterized as providing linear analyte-responsive amplification (e.g., only target analyte present in initial sample participates in stimulating reporter release).
  • an initial analyte-responsive enzyme-mediated reaction releases "synthetic" (i.e., pre-prepared natural or synthesized oligonucleotide not present in sample) oligonucleotide targets which participate in additional enzyme-mediated reactions which in turn result in the release of further synthetic targets.
  • the synthetic oligonucleotide targets are provided in a duplex form configured to be protected from exonuclease activity in the absence of analyte polynucleotide in the sample.
  • the "synthetic" targets may mimic natural sequences of the target analyte, and in alternative embodiments, the synthetic targets may be entirely non-natural, or combinations of natural and non-natural sequences.
  • the non-linear or exponential proliferation of such targets in the reaction medium may be employed to stimulate the release of reporter probes, such as by the methods shown in FIGS. 1A-H.
  • amplification is used herein in a somewhat different sense that the usage of the term in PCR.
  • new copies of sequences may be created via polymerase activity, triggered by binding of primers to analyte in a sample.
  • copies of selected target sequences are pre-synthesized, and pre-compounded as amplifier groups, for example, as a reagent material
  • the release of pre-synthetic targets is specifically analyte-triggered upon use of the reagent material in an assay, so that the synthetic targets are representative of the presence of analyte species in the sample.
  • FIG. 2A shows the sample medium including a target analyte polynucleotide 50 comprising a target sequence A (target A), and also a target sequence B (target B).
  • the sample is shown treated with a mixture including exonuclease 37, "A" amplifier 51 and "B" amplifier 54.
  • the sample medium may be subjected to optional purification steps prior to amplifier treatment, as described herein.
  • the exonuclease 37 may be the same or similar enzyme as in the example of FIG.
  • a amplifier 51 comprises a synthetic oligonucleotide 52 having a sequence B 1 (capture B 1 ) which is complementary to the natural target sequence B (target B), and also having a sequence A' (capture A') which is complementary to the target sequence A (target A).
  • A amplifier 51 further comprises a second oligonucleotide 53 having a sequence A" (synthetic target A") which is complementary to the natural target sequence A (target A).
  • the second oligonucleotide 53 has a "tail" sequence at its 3' terminal end so as to configure oligonucleotide 53 so that it has a protruding 3' "sticky end” (i.e., does not have a blunt or recessed 3' end) so that it does not form a point of attack for the 3' specific exonuclease 37.
  • capping groups may be substituted (by know methods) for the "tail” sequence for this purpose.
  • "B" amplifier 54 comprises a synthetic oligonucleotide 55 having a sequence A' (capture A') which is complementary to the natural target sequence A (target A), and also having a sequence B 1 (capture B') which is complementary to the target sequence B (target B).
  • "B” amplifier 54 further comprises a second oligonucleotide 56 having a sequence B" (synthetic target B") which is complementary to the natural target sequence B (target B).
  • the second oligonucleotide 56 has a "tail" sequence (or other capping group) at its 3' terminal end.
  • the oligonucleotide 56 is bound to oligonucleotide 55 by hybridization of synthetic target B" to capture B 1 .
  • B amplifier 54 is so designated because it can “reveal” a sequence (synthetic target B") that is the same as or similar to the natural target sequence B of analyte 50.
  • synthetic target B sequence that is the same as or similar to the natural target sequence B of analyte 50.
  • FIG. 2B shows the elements of FIGURE 2A, in which the "A" amplifier 51 and “B” amplifier 54 have reacted so as to hybridize with the target B and target A sequences respectively of analyte polynucleotide 50, so as to form a analyte/amplifier complex 57.
  • both the "A” and “B” amplifiers 51, 54 are illustrated as having reacted simultaneously with a single analyte molecule 50, this need not be the case, as the amplification process described herein related to each amplifier may proceed independent of the other amplifier. For example, each amplifier may bind to a separate analyte molecule.
  • FiG. 2C shows analyte/amplifier complex 57 of FIG. 2B, in which the non- protruding 3' terminal ends of the oligonucleotide 52 of "A" amplifier 51 and the comparable non-protruding 3' terminal ends of the oligonucleotide 55 of "B" amplifier 54 have begun to be degraded by action of a pair of exonuclease 37 (not that the action of the enzyme at both sites need not be simultaneous).
  • FIG. 2D shows analyte/amplifier complex 57 of FIG. 2C, in which the activity of the exonuclease 37 has proceeded so as to remove the capture B 1 sequence of oligonucleotide 52 and the capture A 1 sequence of oligonucleotide 55, so as to release the native analyte polynucleotide 50.
  • FIG. 2E shows analyte/amplifier complex 57 of FIG.
  • FIG. 2F shows the elements of FIG. 2E, in which both the released native analyte polynucleotide 50 and the released synthetic targets A and B (oligonucleotides 53 and 56 with their corresponding targets A" and B" respectively) have reacted with additional "A" amplifier 51 and "B” amplifier 54, so as to form additional hybridized duplex forms: and additional amplifier/analyte complex 57, a synthetic A target/amplifier complex 58, and a synthetic B target/amplifier complex 59.
  • FIG- 2G shows the elements of FIG. 2F, in which further activity of exonuclease 37 has begun degrading the exposed non-protruding 3' ends of complexes 57, 58 and 59 in the manner shown in FIGS. 2D-2E above, so as to release additional synthetic targets A and B, as well as the native analyte 50 and the previously released synthetic targets A and B.
  • FIG. 2H shows the elements of FIG. 2G (original accumulated native analyte 50 and accumulated synthetic targets A and B being treated with capture/reporter probe assemblies generally similar to those described with respect to the methods exemplified by FIGS. 1A-H.
  • FIGS. 3A and 3B summarize two examples of the process of amplification, both as shown in FIGS. 2A-H (employing two analyte targets) and an alternative example employing a single analyte target.
  • FIG. 3A schematically illustrates the outcome of an amplification method using a single analyte capture sequence to trigger the release of synthetic targets.
  • Analyte 50 has a single selected target sequence portion A (target A).
  • Amplifier 75 is similar to amplifier 51 shown in FIGURE 2A, except that both capture sequences are complementary to target A.
  • a single analyte 50 reacts with a single amplifier 75 to produce an analyte/amplifier complex 76.
  • Exonuclease 37 degrades complex 76 to release analyte 50 and synthetic A target 53. Thus 2 targets are released.
  • both analyte 50 and synthetic A target 53 react with two additional amplifiers 75 to produce an analyte/amplifier complex 76 as well as a synthetic A target/amplifier complex 77.
  • Exonuclease 37 degrades both complex 76 and complex 77 to release analyte 50 and three synthetic A targets 53. Thus 4 targets are released.
  • FIG. 3B schematically illustrates the outcome of an amplification method using a more than one analyte capture sequence to trigger the release of synthetic targets.
  • the method is the same as illustrated in FIGURES 2A-H, but other alternatives are possible.
  • Target A and Target B need not be selected to be in the sample analyte polynucleotide, but may be selected to be portions of co-analytes, for example, where the amplified target response is triggered by the simultaneous presence of both co-analytes in a sample.
  • analyte 50 includes two selected target sequence portions that can react with two distinct amplifiers 51, 54 to produce complex 57. Exonuclease reaction then degrades the amplifiers to release both analyte 50 (two targets) and two distinct synthetic targets 53, 56. In the subsequent phase both synthetic and analyte targets react with additional amplifiers 53, 56 to produce both synthetic target/amplifier complex and an analyte target/amplifier complex.
  • Exonuclease reaction then degrades the amplifiers to release both analyte 50 (two targets) and six distinct synthetic targets 53, 56. It can be seen that each phase results in a doubling of released target sequences, so that the amplification is likewise exponential. Table 2 shows the results of five phases or steps of this method.
  • FIG. 3C is a plot showing the data of Tables 1 and 2, depicting the increase or amplification of synthetic targets by the single target scheme and dual target scheme.
  • FIGS. 4A through 4B illustrate alternative apparatus and methods for reporter amplification and purification.
  • FIGS. 4A shows generally the elements illustrated in FIG. 2H, with modified reporter probes 61,62.
  • the reporter probes are not bound to a substantial substrate, and have a capping ground 78 on the 3' terminal end of the reporter oligonucleotide 35.
  • Two alternatives are shown, one in which the capping group is present at the terminus of the complementary reporter sequence (35") and one in which the capping group is present at the terminus of short tail sequence.
  • Capping groups can be any of a number of suitable species known in the art which resist exonuclease attachment and/or processing, so as to protect the 3' end from degradation, e.g., nucleotide analogs, covalently bound species and the like.
  • FIGS. 4B shown an alternative example of a method and apparatus having aspects of the invention for purifying released reporters 35' and 35" prior to nanoelectronic detection, so as to simplify the detection environment and improve sensitivity and selectivity (e.g., "noise reduction").
  • the removal of high molecular weight polynucleotides prior to detector operation may be advantageous.
  • the substrate may advantageously comprise a magnetic bead or one of the other alternative substrates describe herein.
  • One or a plurality of "sandwich" probes 79 comprising an oligonucleotide having a sequence complementary to at least a portion of reporter 35' or 35" is bound to substrate 32 as described with respect FIGS. 1A-H, such as by a biotin/streptavidin bond.
  • the reporter 35' is shown hybridized by the short tail region to probe 79.
  • the reporter 35' is shown hybridized by at least a portion of the reporter sequence which is complementary to the nanocode of probes 60,61.
  • the substrate-bound reporter 35', 35" may be rinsed or washed to remove the enzymatic reaction media with unbound species, and replaced by a buffer, which may be optimized for nanoelectronic detector operation (for example, by magnetic immobilization of beads).
  • the probe 79 may have a nucleotide sequence selected to permit stable reporter attachment during washing, and to permit subsequent convenient denaturization to release reporters for detection.
  • 4B may conveniently employ "sandwich" probes 79 complementary to a common sequence portion of the reporter 35, where other portions of the reporter sequence are specific to one of a plurality of analyte types. What ever reporter types are released in the assay reaction may then be collectively purified prior to matrix detector contact.
  • FIGS. 5A through 5E illustrate one exemplary method and apparatus embodiment having aspects of the invention (among a number of alternative methods having aspects in common) which provide exponential analyte- responsive amplification including a pre-amplification purification process, having a number of aspects in common with the methods and apparatus shown in FIGS. 2A-2H, and share common reference numerals in many cases.
  • Methods and apparatus such as depicted in FIGS. 5A-5E may be employed independently or in combination with other methods described herein, in particular with the method and apparatus, such as is exemplified by FIGS. 1A-1H.
  • FIG. 5A shows the sample medium including a target analyte polynucleotide 50 comprising a target sequence A (target A), and also a target sequence B (target B), and including a variety of sample contaminants 63 (e.g., non-analyte polynucleotides, interfering enzymes, undesired chemical constituents, excess diluent, and the like).
  • the sample is contacted, under conditions sufficient to effect polynucleotide hybridization, to an analysis system 70 comprising a substrate 71 and a plurality of capture probes 60, 61 attached to the substrate, for example by biotin-streptavidin bonding 62.
  • the probes comprise "A" probe 60 and "B" probe 61 (which may be essentially the same as probe 60, 61 in FIGS. 2A-H).
  • the A probe 60 includes capture sequence A' which is complementary to target sequence A of analyte polynucleotide 50
  • the B probe 61 includes capture sequence B' which is complementary to target sequence B of analyte polynucleotide 50.
  • system 70 comprises a sufficient plurality of A probes 60 and/or B probe 61 to bind a substantial fraction of the molecules of analyte 50 that may be present in the sample, so as to maximize sensitivity to rarified samples.
  • all analyte molecules 50 are bound, either to a probe 60 or a probe 61. Excess probes 60, 61 are functional for steps described below.
  • spacial separation of probe types avoids aglutination/cross-linking while leaving about half of the analyte
  • Substrate 71 may be particulate (e.g., one or a plurality of beads) or non- particulate (e.g., a well, plate, belt or the like).
  • substrate 71 is configured so as to reduce potential for multiply bound analyte polynucleotides 50 (an analyte hybridized at both A target and B target sequences to corresponding probes 60 and 61. In the example shown this is represented by separate right and left hand zones providing distinct regions for the one or more of A probe 60 and B probe 61, but many alternatives are possible.
  • substrate 71 may be integral with nano electronic detector elements (described further below) or may be disposed separate from the detector. 10127J Purification.
  • FIG. 5B shows the elements of FIG. 5A, in which a rinsing and/or washing step is carried out to remove excess sample medium and contaminants 63, retaining the analyte 50 bound to substrates by probes 60, 61 (immobilization/separation techniques are used to retain any particulate substrates, if present).
  • FIG. 5C shows the elements of FIG. 5B, in which the sample medium has been replace with a buffer including exoriuclease 37 (e.g., having activity as in FIGS. 2A-H) and amplifier duplex oligonucleotides (may be essentially the same as "A" amplifier 51 and "B" amplifier 54 in FIG. 2A).
  • exoriuclease 37 e.g., having activity as in FIGS. 2A-H
  • amplifier duplex oligonucleotides may be essentially the same as "A" amplifier 51 and "B” amplifier 54 in FIG. 2A.
  • synthetic A targets 53 and synthetic B targets 56 hybridize with excess of the plurality of pristine A probes 60 and B probes 61 provided in the step of FIG. 5A (alternatively, additional probes may be provided in the step of FIGURE 5D).
  • This reaction has produced a plurality of synthetic target/probe complexes in the manner depicted in FIG. 2H.
  • an excess of amplifiers 51 , 54 are provided to permit exonuclease- mediated amplification to continue so as to produce sufficient synthetic A and B targets to saturate a plurality of probes 60 and 61 respectively via hybrid complex formation.
  • FIG. 5E shows the elements of FIG.
  • exonuclease 37 has reacted with the synthetic target/probe complexes as well as the native analyte/probe complexes in the manner depicted in FIG. 2H, so as to degrade the probes 60 and 61 and release of reporter oligonucleotides 35 (as well as target species) into the buffer medium.
  • the exonuclease degrades all probes to release reporters for detection.
  • the only substantial population of polynucleotides present are the native analyte and those species released in responsive to analyte presence (synthetic targets and reporters), and thus cross reactivity is minimized or avoided.
  • Reporters can be optimized (e.g., in size or composition) for detector response. Note that the reaction of FIG. 5E may generally proceed simultaneously with the reaction shown in FIG. 5D, where the conditions permit. Alternatively, stringency controls, probe segregation, and the like may be used to isolate these reaction steps.
  • substrate 71 may comprise one or more nanoelectronic detection elements, configured to directly detect the degradation of the probes 60, 61.
  • substrate 71 may comprise one or more regions of a nanoparticle, such as carbon nanotube elements communicating with electrical contacts.
  • changes in the properties one or more nanotubes due to probe degradation are detectable as described herein, and as described in Examples A-I of the incorporated patent applications.
  • substrate 71 comprises one or more regions comprising an interlocking network of nanoparticles, such as carbon nanotubes, contacted by one or more electrodes, (e.g., at least one spaced-apart pair of source/drain electrodes).
  • the nanoparticle network may be supported by wafer-like substrate structure (e.g., silicon, SiO2, Si3N4, PET 1 counter electrode material, and the like or combinations of these).
  • wafer-like substrate structure e.g., silicon, SiO2, Si3N4, PET 1 counter electrode material, and the like or combinations of these.
  • the degradation of probes 60, 61 (in some cases leaving residual ligand moieties 62') produces at least one change in the electrical, mechanical and/or electrochemical environment of the nanoparticle elements which is detectable by suitable circuitry (not shown), such as a change in capacitance of a nanoparticle relative to a counter electrode, a change in transistor characteristics under the influence of a gate electrode, and the like properties- Amplification With Subsequent Enzyme Removal.
  • FIGS. 6A through 6C illustrate one exemplary method and apparatus embodiment having aspects of the invention (among a number of alternative methods having aspects in common) which provide exponential analyte- responsive amplification followed by removal of exonuclease from the media under analysis.
  • the method and apparatus illustrated have a number of aspects in common with the methods and apparatus shown in FIGS. 2A-H and 5A-E, and share common reference numerals in many cases.
  • FIG. 6A shows the sample medium including a target analyte polynucleotide 50 comprising a target sequence A (target A), and also a target sequence B (target B).
  • the sample may be processed in a manner generally the same or similar to the steps depicted in FIGS. 5A-D, so as to react analyte 50 (under conditions suitable for hybridization and enzymatic activity) with the A amplifier 51 , B amplifier 54 and exonuclease 37, so as to release a plurality of synthetic A targets 53 and synthetic B targets 56 into the buffer medium.
  • exonuclease-mediated amplification is carried out so as to contact one or more modified A probes 81 and/or 82, and one or more modified B probes 83 and/or 84.
  • the modified probes have protective 3' tails which eliminate the non-protruding (e.g. blunt or recessed) 3 1 end, so as to prevent degradation by the 3 1 duplex- specific activity of exonuclease 37. In this manner the reaction of FIG. 5E is prevented.
  • excess amplifier has permitted excess synthetic A and B targets to saturate the modified probes 81-84.
  • modified A probe 81 and modified B probe 83 have protective 3' tails which are non-complementary with an adjacent portion of the bound synthetic A and B target respectively, so that (in the probe/target hybrid complex) the protective tail has a single stranded 3' terminal portion.
  • modified A probe 82 and modified B probe 84 have protective 3 1 tails which are complementary with an adjacent portion of the bound synthetic A and B target respectively, so that (in the probe/target hybrid complex) the protective tail has a duplex or double-stranded 3' terminal portion, the 3' terminal portion protruding beyond the adjacent 5' terminal end of the respective synthetic target oligonucleotide (sticky 3 1 end).
  • FIG. 6B shows the elements of FIG. 6A, in which a rinse and/or wash step is carried out following probe/target complex formation (e.g., rinse exonuclease buffer from substrate-bound probe/target complex with a replacement buffer), so as to remove excess reagent material (and any remaining sample material) including exonuclease 37 from the medium.
  • probe/target complex formation e.g., rinse exonuclease buffer from substrate-bound probe/target complex with a replacement buffer
  • the replaced medium may be optimized for detector operation (and need not be suitable for enzymatic activity) as in the step illustrated in FIG. 1G.
  • FIG. 6C shows the substrate 71 and probe/target elements of FIG. 6B, in which denaturization step has been carried out to denature the probe/target complex (e.g., by use of heat, electric field, stringency controls) so as to release reporter molecules 35 and/or synthetic target molecules 53, 56.
  • the denaturization process may be either total or selective.
  • a detector may be optimized to detect reporter 35, target 53, target 56, or any combination of these (alternative probes may optionally omit reporter 35).
  • the substrate 71 may comprise a nanoelectronic detector such as is described herein (direct substrate device detection), configured to detect the denaturization status directly.
  • direct substrate device detection may include detecting the difference between the denaturization event (e.g. comparison of signals before, during and after denaturization) involving pristine probes 81-84 (no analyte 50 present in sample) and the denaturization event involving probes/target complexes (created in response to the presence of analyte 50).
  • both such direct substrate device detection combined with remote nanodetector e.g. as in FIG. 1H may be carried out.
  • FIGS. 7A-F shows an example of a reporter probes having aspects of the invention and including with internal blocking groups which stop the processing of exonuclease at the sequence location of the blocking group.
  • blocking groups are known in the art, and may include, for example, analogs to natural nucleic acids, covalently bonded species.
  • FIG. 7A shows an analyte polynucleotide 50 which has been treated with a probe 90 so as to hybridize probe 90 to a target sequence A of the analyte 50 so as to form an analyte/probe complex.
  • the probe 90 includes a linked reporter oligonucleotide 91 and a companion oligonucleotide 92.
  • the linked reporter oligonucleotide 91 includes a proximal capture sequence portion 94 complementary to at least a portion of target A of analyte 50, and includes a distal reporter sequence portion 95 complementary to at least a portion of companion oligonucleotide 92
  • the capture sequence 94 and reporter sequence 95 are linked by an intervening resistant link group 93 (proximal and distal in this example are arbitrarily described with 3' as proximal, 5' as distal, it being understood that alternative embodiments may have 5' exonuclease activity and a "mirror image" oligonucleotide structure with reversal of the 3 1 vs. 5 1 sense).
  • the companion oligonucleotide 92 includes a proximal anchor sequence 96 complementary to at least a portion of the reporter sequence 95, and a distal capture sequence 97 complementary to at least a portion of analyte 50.
  • an assembled probe 90 including a hybridized duplex of linked reporter 91 and companion 92 may be provided, e.g., in a reagent solution, and reacted with a sample including analyte 50.
  • a single-stranded linked reporter 91 and a single stranded companion 92 may be provided, and each contacted with analyte 50, so as to hybridize in situ with each other and with analyte 50, forming an analyte/probe complex as shown in FIG. 7A.
  • FIG. 7B shows the elements of FIG. 7, further including a exonuclease 37 having activity to degrade duplex polynucleotide with non-protruding 3 1 end. The exonuclease has attached to and begun degrading the proximal portion of linked reporter 91 comprising capture sequence 94.
  • FIG. 7C shows the elements of FIG. 7B, the exonuclease 37 having continued to degrade linked reporter 91 so as to remove the portion of linked reporter 91 proximal to resistant link 93.
  • FIG. 7D shows the elements of FIG. 7C, the exonuclease 37 having detached form the probe analyte complex upon reaching resistant link 93, reporter seq. 35 remaining intact.
  • Reporter seq. 95 is shown denatured and detached form companion 92.
  • the conditions of the reaction medium temperature, pH, ionic composition, and the like
  • the length and nucleotide composition of reporter seq. 35 and the corresponding complementary portion of companion anchor sequence 96 may be selected so that the duplex of the remaining reporter seq. 95 is generally unstable (as duplex form) when the proximal portion of oligonucleotide 91 is degraded.
  • conditions may be controlled following enzymatic degradation to promote denaturization of this reporter 95 duplex.
  • companion 92 is configured to remain attached to analyte 50.
  • companion 92 may also be denatured and detach from analyte 50. In either case, the companion 92 may be recycled by binding with additional pristine linked reporter oligonucleotide 91 in the media, so as to form an additional probe/analyte complex, as shown in FIG. 7A.
  • FIG. 7E shows an optional purification step in which one or more complementary "sandwich" probes 79 are provided, having one or more probe oligonucleotides attached to a substrate, each having a sequence complementary to reporter sequence 95, e.g., in the manner described with respect to FIGURE 4B. Binding of reporter 95 to probe 79, followed by rinsing/washing of substrate 32, enables the sample medium to be highly simplified upon release (via denaturization) of reporter 95.
  • FIG. 7F illustrates the detection of reporters 95 by a nanoelectronic sensor 42 having aspect so the invention, as described with respect to FIG. 1G-H.
  • Detector 42 has one or more detector probes 98 attached, with can hybridize via complementary sequences with reporter 95, so as to produce a detectable change in sensor properties. Removal or deactivation of exonuclease 37, such as by optional purification step of 7E, removes constraints on probe/reporter complex configuration in that a distal (with respect to sensor) non-protruding 3' end is not degraded (rapid detection by sensor 42 also may also obviates this constraint).
  • probe 98 may be configured (as shown) to have a either a distal protruding 3 1 end.
  • Alternative detector probe 99 has a distal 5 1 end. Note that in the example shown, resistant link 93 is at or adjacent to the 3' end of reporter 95, so as to resist endonuclease activity in the duplex probe/reporter complex. Multi-Analyte Assay With Multiple Reporter Types And A Matrix Detector Cell
  • FIGS. 8A and 8B illustrate one exemplary method and apparatus embodiment having aspects of the invention including a multi-analyte assay with multiple reporter types and a matrix detector cell.
  • the method includes providing a plurality of probe types configured to amplify and provide detection species (e.g., reporters) corresponding to a plurality of different analytes.
  • FIG. 8A is a flow diagram depicting a multi-analyte assay with multiple reporter probe types, and optionally having purification steps and multiple amplifier types.
  • a reagent mixture is compounded having both reporter probes and amplifiers corresponding to putative analytes 1 , 2, 3 and 4 respectively.
  • a sample medium containing only analytes 2 and 4 is contacted to the reagent mixture and exonuclease 37, under conditions effective to promote hybridization and enzyme activity. Following amplification and reporter release reactions as described above, reporter (as well as synthetic targets) are released corresponding to analytes 1 and 2. Reporter probes and amplifiers corresponding to analytes 1 and 3 remain unreacted. Note that the reporter probes and amplifiers (and rinsing or purification) may be any of the embodiments described herein, such as with respect to FIGS. 1-7, or similar embodiments without departing from the spirit of the invention.
  • FIG. 8B illustrates detector cell 100 having enclosure walls 101 and one or more ports 102, and a matrix of sensors, each having a distinct detector sensitivity corresponding to a different analyte reporter molecule (four are shown, sensors 42a, b, c and d). Note that the sensors may include any of the sensor embodiments described herein and in the incorporated patent applications.
  • each sensor 42 includes a substrate (e.g., a common substrate 106 having a dielectric layer 107 is shown, such as a silicon wafer and SiO2 surface layer, alternatively a polymer sheet, or the like), a nanostructure element 104 disposed adjacent the substrate (in this example an interconnected CNT network) and at lease one contact 105 in electrical communication with the nanostructure element 104 (an interdigitated pair of source-drain electrodes are shown.
  • a substrate material such as a doped Si wafer may serve as a common gate electrode for the matrix.
  • Nanostructure element 104 in each sensor is sensitized with a different recognition material specific to one of the putative analytes of the assay, in this example a oligonucleotide detector probe complementary to a selected one of the reporter oligonucleotides corresponding to one of analytes 1 , 2, 3 and 4 respectively.
  • Detection solution 103 in this example a purified reporter solution derived form the reaction mixture of FIG. 8A, is contacted to the sensors 42 in cell 100.
  • Suitable measurement circuitry (not shown) detects changes in the properties of those ones of sensor 42a-d in which hybridization with a corresponding reporter occurs.
  • the sensors 42 are configured to detect a distinct reporter molecule, alternative embodiments are possible without departing from the spirit of the invention, such a having sensors including probes sensitive to synthetic targets or analyte molecules. Aptamer-Reporter Complex.
  • FIGS. 9A to 9C illustrate one exemplary method having aspects of the invention employing a probe assembly including an aptamer portions for detection of analytes, including biomolecules.
  • the aptamer/base sequence composite is selected so that there is a conformal change upon binding of the aptamer 112 to analyte 111.
  • an Aptamer-Reporter Complex is immobilized to a substrate, such as a bead, e.g., by methods described above.
  • probe assembly 110 includes a reporter 113 in a duplex form bound to base sequence 114.
  • the probe 110 can generally similar to any of the examples described herein.
  • the probe 110 is generally similar to that described with respect to FIGS. 7A-F, in which the base sequence 114 acts in the same manner as the analyte 50.
  • the base sequence is connected to an aptamer sequence 112, the aptamer having a specific binding capacity for a target analyte 111.
  • the analyte may be any one of a number of substances having a specific affinity or reactivity with an aptamer or similar polynucleotide construct, e.g., a globular protein, a polysaccharide, or the like.
  • [0i6i] Aptamer conformed to protect probe. As shown in FIGURE 9A, when the aptamer 112 is not bound to analyte 111, the conformation is protective of the probe 110, in that the endonuclease 37 is prevented from substantially degrading the probe.
  • Nanoelectronic sensors for polynucleotide detection EXAMPLES A through I.
  • FIGS. 10-13 depict exemplary embodiments having aspects of the invention which provide for analyte-responsive enzyme-mediated amplification in a manner similar in a number of respects and operative schemes as the embodiments described above with respect to FIGS. 2 and 3, and wherein a multistage series of amplifier reagent species is employed.
  • the product of the analyte-responsive enzyme reaction of an initial stage amplifier forms the substrate for the amplification of a second-stage amplifier, so as to produce a second amplification product upon subsequent enzyme action. Additional stages may be include.
  • Detector devices employing such multiple-stage amplification schemes may detect any or all of such amplification products (reporters), so as to permit measurement of the presence or concentration of a biomolecule analyte in a sample. Such detection may employ any of the nanoelectronic devices described herein.
  • the oligonucleotide complexes and enzymes are configured for degradation by a 3'-exonuclease selective for a initiation on a duplex DNA having a non-protruding 3 1 terminus on one strand, with degradation proceeding in 3' to 5' direction along the non-protruding strand.
  • a 3'-exonuclease selective for a initiation on a duplex DNA having a non-protruding 3 1 terminus on one strand
  • degradation proceeding in 3' to 5' direction along the non-protruding strand.
  • other enzyme types may be employed, such as a 5' exonuclease, DNA polymerase, and the like, with amplifier reagents species configured accordingly.
  • an initial analyte- responsive enzyme-mediated reaction releases "synthetic" (i.e., pre- prepared/selected natural or synthesized oligonucleotides not present in sample) targets which participate in additional enzyme-mediated reactions which in turn result in the release of further synthetic strands or fragments.
  • the synthetic oligonucleotides are provided in a duplex form configured to be protected from exonuclease activity in the absence of analyte polynucleotide in the sample. [oi7i] Initial conditions. In the example illustrated in Fig.
  • the first view portion, view 10a shows the initial state of sample measurement in which: a. Analyte polynucleotide strand 120, having a pre-selected sequence A is exposed, under conditions sufficient to effect polynucleotide hybridization, to a reagent/buffer having at least Amplifier I; b. Amplifier I comprises a duplex structure which includes a capture strand 121 and a companion strand 122; c. Capture strand 121 comprises (in 3' to 5' order) a capture sequence A'
  • Companion strand 122 comprises (in 3' to 5 1 order) two companion sequences C and B 1 which are complementary to and hybridized to (all or a portion of) the companion sequences C and B' respectively of capture strand 121, so as to leave the capture sequence A' of strand 121 exposed.
  • Stage 1 In view 10b, Amplifier I is shown in duplex association with analyte strand 120 by means of hybridization of the capture sequence A' of strand
  • stage 1 enzymatic degradation of stage 1 is illustrated as complete, with the undegraded analyte strand 120 (original strand) and companion strand
  • step 1 there is one original strand and one derived (e.g., reporter) strand in solution.
  • Stage 2 View 10d illustrates the beginning of stage 2 of the method, in which the products of stage 1 (stands 120 and 122) are shown exposed in the reagent/buffer medium to additional Amplifier I and also to Amplifier II: a.
  • Amplifier Il comprises a duplex structure includes a capture strand 123 and a companion strand 122; b.
  • Capture strand 123 comprises (in 3' to 5' order) a capture sequence C" (complementary to all or a portion of sequence C of companion strand
  • Companion strand 124 comprises companion sequence B 1 ", which is complementary to and hybridized to (all or a portion of) the companion sequence B" of capture strand 123, so as to leave the capture sequence C" of strand 123 exposed.
  • Amplifier Il is shown in duplex association with companion strand 122 by means of hybridization of the capture sequence C" of strand 123 to sequence C of companion strand 122.
  • An exonuclease species (together with any necessary co-factors) is added or present in the reagent, so as to initiate degradation of capture strand 123 at its 3 1 terminus.
  • additional Amplifier I is shown in duplex association with analyte strand 120 as described for stage 1.
  • stage 2 enzymatic degradation of stage 2 is illustrated as complete, with the undegraded analyte strand 120 (original strand), two of companion strand 122 (first derived and second derived); and companion strand 124 (companion Il or B) released into solution.
  • the undegraded analyte strand 120 original strand
  • two of companion strand 122 first derived and second derived
  • companion strand 124 complement Il or B
  • FIG. 1Og illustrates the beginning of subsequent step 3 of the method, in which the products of both stage 1 (strands 120 and 122) and stage 2 (stand 124) are shown exposed in the reagent/buffer medium to additional Amplifier I and Amplifier II: [0178] In view 10h, Amplifier I is shown in duplex association with analyte strand 120 (stage 1) and Amplifier Il is shown in duplex association with companion strand 122 (stage 2). Exonuclease initiates degradation of capture strand 121 of the analyte duplex and capture strand 123 of the each of the companion I duplexes.
  • FIG. 11 is a plot which shows the data of table 3 with respect to a two- stage amplification.
  • FIGS. 12A-C and 12A-C Three-stage amplification.
  • FIGS. 12A-C and 12A-C Three-stage amplification.
  • FIGS. 12A shows three portions connected by arrows depicting the progressive activity of Amplifier #1 , in which: a. Amplifier #1 comprises a duplex structure which includes a capture strand
  • Capture strand 125 comprises (in 3 1 to 5' order) a capture sequence A 1
  • Companion strand 126 comprises (in 3' to 5" order) three companion sequences D, C and B, which are complementary to and hybridized to (all or a portion of) the companion sequences D 1 , C and B' respectively of capture strand 125, so as to leave the capture sequence A' of strand 125 exposed; d.
  • Analyte polynucleotide strand 120 having a pre-selected sequence A, is exposed, under conditions sufficient to effect polynucleotide hybridization, to a reagent/buffer having at least Amplifier #1, so as to bind and produce a duplex Analyte-Amplifier #1 complex; e. An exonuclease species (together with any necessary co-factors) is added or present in the reagent, so as to initiate degradation of capture strand 125 at its 3' terminus; and f. enzymatic degradation continues to completion, so as to release the undegraded analyte strand 120 and companion strand 126 (companion #1 or BCD) into solution.
  • FIGS. 12B shows three portions connected by arrows depicting the progressive activity of Amplifier #2, in which a. Amplifier #2 comprises a duplex structure which includes a capture strand
  • Capture strand 127 comprises (in 3' to 5' order) a capture sequence C"
  • Companion strand 128 comprises (in 3' to 5' order) two companion sequences B'" and D'", which are complementary to and hybridized to (all or a portion of) the companion sequences B" and D" respectively of capture strand 127, so as to leave the capture sequence C" of strand 127 exposed; d.
  • Companion #1 (strand 126) is exposed, under conditions sufficient to effect polynucleotide hybridization, to a reagent/buffer having at least Amplifier #2, so as to bind and produce a duplex Companion #1 -Amplifier #2 complex; e.
  • exonuclease species (together with any necessary co-factors) is added or present in the reagent, so as to initiate degradation of capture strand 127 at its 3' terminus; and f. enzymatic degradation continues to completion, so as to release the undegraded Companion #1 (126) and companion strand 128 (companion
  • FIGS. 12C shows three portions connected by arrows depicting the progressive activity of Amplifier #3, in which a. Amplifier #3 comprises a duplex structure which includes a capture strand 129 and a companion strand 130; b. Capture strand 129 comprises (in 3' to 5' order) a capture sequence D 4 (complementary to all or a portion of sequence C" of strand 128), and an additional sequence B 4 ; c. Companion strand 130 comprises a companion sequence B 5 , which is complementary to and hybridized to (all or a portion of) the companion sequence B 4 of capture strand 129, so as to leave the capture sequence D 4 of strand 129 exposed; d.
  • Amplifier #3 comprises a duplex structure which includes a capture strand 129 and a companion strand 130
  • Capture strand 129 comprises (in 3' to 5' order) a capture sequence D 4 (complementary to all or a portion of sequence C" of strand 128), and an additional sequence B
  • Companion #2 (strand 128) is exposed, under conditions sufficient to effect polynucleotide hybridization, to a reagent/buffer having at least Amplifier #3, so as to bind and produce a duplex Companion #2-Amplifier #3 complex; e. An exonuclease species (together with any necessary co-factors) is added or present in the reagent, so as to initiate degradation of capture strand 129 at its 3' terminus; and f. enzymatic degradation continues to completion, so as to release the undegraded Companion #2 (126) and companion strand 130 (companion #2 or B) into solution.
  • a reagent/buffer having at least Amplifier #3 so as to bind and produce a duplex Companion #2-Amplifier #3 complex
  • An exonuclease species (together with any necessary co-factors) is added or present in the reagent, so as to initiate degradation of capture strand 129 at its 3' terminus; and f.
  • Table 4 shows the results of six phases or steps of this method.
  • FIG. 13 is a plot which shows the data of table 4 with respect to a three- stage amplification.
  • homologous sequences With respect to the examples herein, and in particular the multi-stage examples of FIGS. 10-13, it should be understood that the separate designation of homologous sequences which correspond to a sequences present in more than one amplifier reagent species, does not imply that such homologous sequences are identical in nucleotide length or sequence, or in overall chemical composition. Such homologous sequences may be identical, or they may differ in a number of respects. 101891 For example, in FIGS.
  • sequence D" 1 of strand 128 may be seen to hybridize with sequence D" (in Amplifier #2, strand 127) and also hybridize with sequence D 4 (in Amplifier #3-Companion #2 complex, , strand 129).
  • sequence D" and D 4 may be configured to have different degrees of completeness of hybridization or may have different denaturalization or annealing stringency properties.
  • sequences D" and D 4 may be configured to have different lengths, terminal groups, intermediate groups, non-natural nucleotides, or the like, e.g., so to regulate the initiation, termination or specificity or blocking of enzymatic activity, as the case may be. I0i90
  • homologous sequences in amplicon or reporter species e.g., B 1 " in strand 128 and B5 in strand 130
  • multiple and/or quantitative detection schemes may be employed, e.g., to reduce false positives, to distinguish between similar analytes, and the like.
  • FIGS 14A-14C illustrate an example of single-target amplification method having aspects of the invention (See, e.g., FlG. 3A) utilizing fluorescent detection, through one step of the method.
  • the amplifier capture probe sequence is configured to form an analyte-amplifier complex whereby the capture stand has a terminal non- protruding 5' end, subject to degradation by a 5'»3 * exonuclease.
  • the enzyme used in this example is a T7 polymerase having 5'»3' exonuclease activity.
  • nucleotide-active enzymes including 3'-exonuclease, 5'-exonuclease, DNA polymerase (e.g., via proof-reading or nuclease activity), and the like.
  • DNA polymerase e.g., via proof-reading or nuclease activity
  • T7 DNA polymerase has been demonstrated using methods of the invention.
  • alternative amplifier systems having aspects of the invention may utilize the activity of endonucleases when configured to form an analyte- amplifier complex with a suitable enzyme initiation site, without departing from the spirit of the invention. (01941 FIG.
  • 14A is a diagram showing the components of the analyte and amplifier system 140, including analyte 141 having a target sequence A (in this example, having about 54 bases), capture strand 142 (in this example, having about 102 bases) and having a complementary sequence A 1 and a extended sequence B, and reporter strand 147 (in this example, having about 19 bases).
  • the of amplifier-analyte complex 145 in this example has about 121 base pairs.
  • Reporter strand 147 includes strand 143 which has a complementary sequence B f which binds to extended sequence B' of strand 142, and in this example includes also a FAM fluorescent group 144, attached to strand 143 by conventional practice, to permit convenient detection by optical methods.
  • FIGS. 14B-14C show the results of electorphoretic gel separation of various components of the reactions of the method, where FIG 14B is a positive photograph of a electrophoretic gel following separation, and where FIG 14C is a negative version of the photograph of FIG. 14B, which provides a more distinct indication of the data in a photo-reproducible gray-scale image suitable for patent illustration.
  • various combinations of reaction or reagent components were tested for comparison purposes that need not necessarily be present in a useful reagent system.
  • the gel was configured to have 10 parallel channels, in which the buffer in each channel is described in the list below: 1 - Oligomer calibration ladder of units of about 10 base pairs (bp), the "bright line” component corresponding to 100 bp.
  • FIGS. 14B-C the enzymatic digestion product, reporter 147, is not visible in channels 7 corresponding to the complex without enzyme, but is readily apparent at the 19-meir level in both channels 8 and 9, corresponding to the enzymatic digestion reaction mixture.
  • concentration of enzyme and other reaction conditions may be optimized without undue experimentation of facilitate convenient, sensitive and selective detection of such an analyte.
  • Hairpin probe assembly Further exemplary embodiments having aspects of the invention may eliminate separate capture and reporter portions of the probe assembly.
  • FIGS 15A-15C illustrate an example of a "hairpin" type probe assembly.
  • the probe assembly comprises a co-linear polynucleotide strand 152 which includes a capture portion A' (complementary to the target A of analyte strand 151) and a reporter portion, which may have one or more labels or other detection enhancement 154 (e.g., a fluorescent marker group) disposed on or adjacent any convenient place on the strand 152.
  • a marker 154 is shown in a mid-portion of stand 152, but it may be disposed alternatively, such as adjacent the 3' end.
  • strand 152 includes at least one addition portion A" which has a degree of hybridization affinity such that portions A 1 and A" tend to self-hybridize (e.g., to form a hairpin-like configuration) when not in association with the target portion A of the template species 151 so as to be protected from degradation by the selected enzyme.
  • the hairpin form of strand 152 has a protruding 5' end, thus being protected from an enzyme 146 (e.g. an endonuclease) requiring a duplex having a non-protruding 5' terminus for initiation of activity.

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Abstract

La présente invention concerne des procédés de détection de biomolécules, y compris des procédés d'amplification d'espèces d'analytes cibles et de rapporteurs cibles par l'action déclenchée par l'analyte d'une enzyme telle qu'une nucléase, une polymérase ou une enzyme similaire. Les espèces cibles amplifiées (par exemple des produits d'amplification et des rapporteurs) sont détectables à l'aide de plusieurs modes de réalisation de capteurs nanoélectroniques selon des aspects de l'invention et, à titre d'alternative, par des procédés conventionnels de détection des biomolécules.
EP07754869A 2006-04-04 2007-04-03 Detection nanoelectronique de biomolecules en employant l'amplification des analytes et des rapporteurs Withdrawn EP2007379A4 (fr)

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US11/488,465 US20070045756A1 (en) 2002-09-04 2006-07-18 Nanoelectronic sensor with integral suspended micro-heater
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US11/588,845 US20080021339A1 (en) 2005-10-27 2006-10-26 Anesthesia monitor, capacitance nanosensors and dynamic sensor sampling method
US90153807P 2007-02-14 2007-02-14
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