Patent Application
20070259359
1. Field of the Invention
The present invention relates to detector systems for biomolecules using nanoelectronic sensor devices.
2. Description of Related Art
Because base sequences in polynucleotides encode genetic information, the ability to read these sequences has contributed to many advances in biotechnology. This work has identified many important sequences that are linked to medical conditions. For example, the BRCA gene is usually present in women who suffer from breast cancer. To take advantages of these linkages in medical testing, various techniques have been developed to scan tissue samples for the occurrence of specific important sequences. These techniques have shortcomings that make them expensive, slow, and complex, so that they are unlikely to be useful for routine medical testing.
These techniques universally rely on the tendency of polynucleotides to hybridize. 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. The result of this combination is double-stranded DNA (dsDNA), which can be processed and separated from ssDNA. Thus, to scan for a particular target sequence, 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.
Current methods have mainly focused on optical detection using fluorescence-labeled oligonucleotides with dyes, quantum dots or enhanced absorption of light by oligonucleotide-modified gold nanoparticles. For example, electrochemical detection of DNA hybridization may be performed using nanostructures as electrodes. However, electrochemical methods rely upon electrochemical behavior of the labels.
A first shortcoming arises because many methods of detecting this hybridization involve modification of the sample ssDNA before hybridization. Often, a fluorescent molecule is attached to the ssDNA. This molecule, known as a label, causes the ssDNA to be detected by optical instruments such as microscopes and spectrometers. Labeling is used to detect sample DNA after a hybridization step. If the target sequence is present in a labeled sample, the labeled ssDNA will be incorporated in labeled dsDNA, and the dsDNA will thus be detectable with optical instruments. Although the use of optical detection makes this approach convenient, the chemical reaction by which the DNA is labeled is expensive and time-consuming. A detection method which did not require labeling would significantly increase the usefulness of DNA scanning for routine medical tests.
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.
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. 2006-0040,286 entitled “Bio-Barcode Based Detection Of Target Analytes” and U.S. Pat. No. 6,750,016 entitled “Nanoparticles Having Oligonucleotides Attached Thereto And Uses Therefor”, each of which publication is incorporated by reference. However, these methods do not provide for an amplified detection response to a native target analyte (e.g., a non-PCR amplified analyte) and do not provide for electronic detection.
Certain work has explored amplified response to a native target analytes. See for example, H F Lee, Y Li, A W Wark and R M Corn, “Enzymatically Amplified SPR Imaging Detection of DNA by Exonuclease Ill Digestion of DNA Microarrays”, Analytical Chem., 77 5096-5100 (2005) and US Published App. 2005-0048,501 entitled “Method And Apparatus For Detection Or Identification Of DNA”, each of which publication is incorporated by reference. However, these methods do not provide a method of electronic detection.
Other biomolecules in addition to DNA are increasingly important in scientific understanding of biological processes and in practical applications such as medical diagnosis and treatment. Thus there is a need for detection of such biomolecules as proteins, polysaccharides, RNA and the like. Certain work has explored detection methods for such analytes as proteins. See for example, US Published App. 2006-0014,172 entitled “Aptamer-Nanoparticle Conjugates And Method Of Use For Target Analyte Detection”, which is incorporated by reference. However, these methods do not provide for an amplification of a target portion of a native target analyte and do not provide for electronic detection.
Thus, there is a clear need for a sensitive, fast and robust technique for detecting specific target biomolecules. Such a technique should operate without the use of PCR or other target analyte amplification methods and without the requirement for labeling of analyte species. Such a technique should provide of an amplified detection response, and should provide for electronic detection. In addition is desirable that such techniques provide for concentration and/or simplification or purification of an analyte sample, further increasing sensitivity and selectivity.
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.
Additional description and summary of devices and methods having aspects of the invention are described the following co-invented provisional and non provisional applications listed in the section above entitled “Cross-Reference To Related Applications”, each of which applications is incorporated by reference. The present application should be read together with the subject matter and descriptions of each of these applications. Among other things, these applications describe a number of alternative embodiments of nanoelectronic devices and methods for the label-free detection and identification of polynucleotides and other analyte species.
Nanoelectronic Detector Devices.
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.
Generally the nanostructure element and conductors are disposed adjacent to a supporting substrate, which 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. In a number of examples described herein, 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. In embodiments of nanosensor devices, the nanostructured element may comprise a collective structure which includes a plurality of nanostructures, such as SWNTs or other nanotubes arranged to form a collective structure. In a number of preferred embodiments of nanosensor devices, 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, U.S. patent application Ser. No. 10/177,929 (corresponding to WO2004-040,671); U.S. patent application Ser. No. 10/280,265; U.S. patent application Ser. No. 10/846,072; and L. Hu et al., Percolation in Transparent and Conducting Carbon Nanotube Networks, Nano Letters (2004), 4, 12, 2513-17, each of which applications and publication is incorporated herein by reference.
Properties of the nanostructure elements (e.g., nanotube network) may by measured using one or more contacts. 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. For example, 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.
Transistor embodiments. In some embodiments of the invention, 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. For example, the gate electrode is a conducting plane within the substrate beneath the silicon oxide. Alternatively, 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 U.S. patent application Ser. No. 10/656,898, filed Sep. 5, 2003 (Publication 2005-0279987) and Ser. No. 10/704,066, filed Nov. 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/Vg 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. To make a sensitive chemical sensors, a nanotube transistor has an on-off ratio preferably greater than 1.2, more preferably greater than 2, and most preferably greater than 10.
Recognition of Analytes. 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.
Polynucleotides Species. The invention provides an electronic sensor device with which to detect specific target sequences of polynucleotides. In certain embodiments, 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. In the particular examples described in detail, the nanostructured elements comprise carbon nanotubes, and more particularly, randomly oriented networks of carbon nanotubes. In these examples, the nanotubes are modified before sensing by the adsorption of ssDNA probe sequences. No labeling of the DNA is required. Further, the invention provides a method for using the sensor device.
As used herein, “DNA” means polynucleotides. Examples of 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. Accordingly, 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. Once a target sequence is specified, 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.
It should be noted that, with respect to all the described sensor embodiments, that 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. Alternatively or additionally, 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. For example, 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).
Through variations in stringency, it is possible to differentiate binding of strands with complete or incomplete complementary base pairs. Changes in electrical properties of the nanotubes in response to the stringency process allow discrimination of single base mismatches (SNP), among other things. One of ordinary skill in the art will be able to vary the hybridization conditions so as to tune the operation of certain embodiments of the sensors of the invention to obtain a selected degree of sensitivity to complete and less-than-complete hybridization of the target sequence.
For example, in an assay to discriminate between a DNA sample which is homozygous for a particular allele, on the one hand, and an otherwise comparable sample which is heterozygous for this allele, 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.
In the case of each of the sensor embodiments having aspects of the invention, these sensors may be constructed in arrays, e.g., arrays of transistor sensors functionalized for a plurality of different target DNA fragments. See U.S. application Ser. 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 U.S. patent application Ser. No. 60/901,538 filed Feb. 14, 2007 and Ser. No. 60/850,217 filed Oct. 6, 2006, each entitled “Electrochemical nanosensors for biomolecule detection”. Each of these applications is incorporated herein by reference.
Capacitance detector embodiments. In some embodiments of the invention, 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. See, for example, the detection methods and devices as described in co-invented U.S. patent application Ser. No. 11/588,845 filed Oct. 26, 2006 entitled “Anesthesia Monitor, Capacitance Nanosensors and Dynamic Sensor Sampling Method”, which claims priority to Ser. No. 60/850,217 filed Oct. 6, 2006; Ser. No. 60/773,138 filed Feb. 13, 2006; Ser. No. 60/748,834 filed Dec. 9, 2005 and Ser. No. 60/730,905 filed Oct. 27, 2005. Each of these applications is incorporated herein by reference.
Alternative detection methods, labels, markers and devices. Although the exemplary embodiments 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.
For example, 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. Likewise, 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.
One aspect of the invention 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. In certain embodiments having aspects of the invention, 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.
In a first embodiment having aspects of the invention, 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. For example, 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. In preparation of the probe assembly, 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.
In operation, 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). In certain embodiments (e.g. an magnetic bead or other substrate) immobilization of the substrate with bound analyte/probe complex permits purification or rinsing of the complex, thus simplifying the sample or lysis mixture. In a preferred embodiment, 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.
In a first embodiment of analyte-triggered release of reporter described below, 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′). It should be understood that 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. Still other alternatives are possible without departing from the spirit of the invention.
Alternative embodiments can employ 5′ 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.
In additional alternative embodiments other enzymes may be employed under selected conditions to achieve selective exonuclease activity. For example, 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.
In additional alternative embodiments, 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.
In additional alternative embodiments, 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.
Preferably, 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. Examples of 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, Calif. See also the separation methods and devices described in US Published Application 2005-0147,822 entitled “Process”; U.S. Pat. No. 5,512,439 entitled “Oligonucleotide-linked Magnetic Particles And Uses Thereof”; U.S. Pat. No. 6,994,971 entitled “Particle Analysis Assay For Biomolecular Quantification”; and U.S. Pat. No. 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.
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.
In one embodiment of a method of detecting an analyte polynucleotide in a sample, the method comprises:
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′ and 5′ terminal ends are reversed throughout the definition of the method, so that:
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:
In a first embodiment of amplifier method and apparatus described below, 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′). It should be understood that 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. Still other alternatives are possible without departing from the spirit of the invention.
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:
Duplex amplifier with companion. 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:
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. Thus, for example:
Note that 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.
Amplifier/Analyte Complex enzymatic degradation. In any one of these cases, 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. As degradation of the amplifier oligonucleotide continues, 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:
Thus, for each original analyte molecule, the above describe amplification process releases both the original un-degraded analyte polynucleotide molecule (with original Target A and Target B), two single-stranded synthetic targets derived from amplifier reagent (Synthetic Target A and Synthetic Target B), and any exonuclease enzyme which mediated the reaction.
Subsequent amplification of released targets. In the presence of additional pristine Amplifier A and Amplifier B and in the presence of exonuclease, each of these target sequences (synthetic or native analyte) 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:
Each “cycle” of the amplification produces (releases) both an original target and a corresponding new target, each of which can trigger additional amplification events. As a result, it may be seen that during the continued amplification process, and while the medium includes sufficient amplifier reagent and exonuclease, that the population of original analyte polynucleotide tends remains constant while the population of Synthetic Target A″ and Synthetic Target B″ tends to grow exponentially. Note the use of the term “cycle” in this context does not imply the particular cyclic variation of environmental conditions such as temperature that is typically employed in PCR, and the conditions of the medium may be selected and controlled so that both polynucleotide hybridization and enzymatic degradation activity are promoted simultaneously and continuously.
Multistage amplifiers. 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 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.
In a biomolecule detection method, the multistage amplification method may be used to detect analytes such as polynucleotides, proteins, polysaccharides, and other biomolecular species. In embodiments, the first probe assembly includes capture nucleotide sequence complementary to a target sequence of the template (e.g., template includes a DNA strand). In alternative embodiments, 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).
In an embodiment, 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:
For example, 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). Likewise, 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. In an embodiment, 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.
A more complete understanding of the devices and methods having aspects of the invention will be afforded to those skilled in the art, as well as a realization of additional advantages and objects thereof, by a consideration of the following detailed description of the preferred embodiment.
The following is a list which summarizes the drawings and figures herein:
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.
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.
FIGS. 1C(i) and 1C(ii) 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.
Note in this example 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. For example, 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.
In this example, the probe polynucleotide 34 is bound to substrate 32 adjacent a proximal 5′ terminal nucleotide, and comprises a nanocode sequence Y and a capture sequence X′, 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′ which in the assembled probe is hybridized to the nanocode sequence Y of the probe polynucleotide 34. Note that although
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.
It should be understood that, unless defined more distinctly for a specific purpose, the terms 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.
In this example, 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. In preparation of the probe assembly, 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.
FIGS. 1 D(i) and 1 D(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.
Where 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. In a detection system 20, there may advantageously be provided 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. 1F(i), 1F(ii), 1F(iii) 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. In this example, the non-protruding 3′ distal end of probe polynucleotide 34 is attacked and degraded progressively. In a preferred embodiment, specific activity of the exonuclease (added after optional rinsing) 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). It should be understood that 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. Likewise, 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.
As noted above, alternative embodiments may have probes configured to reverse the order of proximal and distal terminal strand ends (3′ for 5′) and employ exonucleases with a corresponding reversed activity (specific to non-protruding 5′ duplex ends). Likewise, in alternative embodiments, enzymes systems other than specific endonucleases and having multiple activity modes can be employed where non-specific activity is controlled or non-interfering. For example, various types of DNA polymerase may be employed in a media deprived of nucleotide tri-phosphates, so as to favor “proof-reading” activity at the expense of polymerase activity, thus functioning as a nuclease.
Note that the analyte molecule 33 released in the step of
Thus the accumulation of a comparatively high concentration of reporter oligonucleotides in the reaction buffer, which has optionally been previously purified or simplified by rinsing, permits a very low initial concentration of analyte in the sample to be electronically detected without significant cross reactivity by nanoelectronic detectors, as described herein.
As shown
Optionally, the 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.
In certain embodiments 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.
In some embodiments, 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.
Note that the term “amplification” is used herein in a somewhat different sense that the usage of the term in PCR. In an example of a PCR assay method, new copies of sequences may be created via polymerase activity, triggered by binding of primers to analyte in a sample. In the examples of methods and apparatus having aspects of the invention described herein, 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. The proliferation of released representative molecules in a reaction medium constitutes, in effect, an amplified signal representative of analyte presence.
“A” amplifier 51 comprises a synthetic oligonucleotide 52 having a sequence B′ (capture B′) 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. Alternatively, capping groups may be substituted (by know methods) for the “tail” sequence for this purpose.
The oligonucleotide 53 is bound to oligonucleotide 52 by hybridization of synthetic target A″ to capture A′. Note that “A” amplifier 51 is so designated because it can “reveal” a sequence (synthetic target A″) that is the same as or similar to the natural target sequence A of analyte 50.
Conversely, “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′ (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′. Note that “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.
Note the terms “natural” and “synthetic” are used in this context only to distinguish between the target analyte or “natural” polynucleotide putatively present in a sample, and the “synthetic” oligonucleotide provided in an amplifier reagent for purposes of carrying out an exemplary assay method. The target analyte or “native” polynucleotide of the sample often is, but need not be, from a natural source. The reagent amplifier oligonucleotides conveniently may be, but do not need to be, synthetically made.
Single target.
In a first phase, 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.
In a second phase, 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.
It may be seen that, since each target sequence that hybridizes with an additional amplifier group at each phase or step, the result is an exponential increase in released target presence. Table 1 shows the results of five phases or steps of this method.
Dual target. In contrast to
As described herein, 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.
Note the multi-analyte assay and matrix detector embodiment described below with respect to FIGS. 8A-B. The reporter purification method shown in
Exponential Non-PCR Target Amplification, Integrated Reporter Amplification
Initial immobilization.
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, and the B probe 61 includes capture sequence B′ which is complementary to target sequence B of analyte polynucleotide 50. Preferably, particularly where the sample comprises a very small quantity of analyte species 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. In the schematic illustration of
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). In certain embodiments, 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. For example, alternative arrangements may have distinct bead types (e.g., optionally employing bead segregation mechanisms), fixed plate or lumen zones, variable stringency conditions, distinct melting/denaturation properties, and the like. In certain embodiments substrate 71 may be integral with nano electronic detector elements (described further below) or may be disposed separate from the detector.
Purification.
Incubation.
Amplification.
In the example shown, 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
Reporter release.
In the example shown, 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
Note that the reporters 35 may be detected in the manner described with respect to FIGS. 1G-H (either via flow to an isolated nanoelectronic detector 40 or an integrated nanoelectronic detector communicating with the reaction region).
In certain alternative embodiments, substrate 71 may comprise one or more nanoelectronic detection elements, configured to directly detect the degradation of the probes 60, 61. For example, substrate 71 may comprise one or more regions of a nanoparticle, such as carbon nanotube elements communicating with electrical contacts. For example, 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.
In certain alternative embodiments, 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, counter electrode material, and the like or combinations of these). The applications incorporated by reference herein provide a number of examples of the preparation and operation of such devices. 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.
Exonuclease amplification.
In this alternative embodiment having aspects of the invention, 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′ end, so as to prevent degradation by the 3′ duplex-specific activity of exonuclease 37. In this manner the reaction of
In first alternative form, 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.
In a second alternative form, modified A probe 82 and modified B probe 84 have protective 3′ 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′ end).
Purification.
Probe denaturization.
One or more probe skeleton species 81′, 82′ 83′ and/or 84′ may remain attached to substrate 71 (in the example shown theses are single stranded oligonucletides). As in the example of
Reporter Probes With Internal Nuclease-resistant Blocking Groups.
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. Such blocking groups are known in the art, and may include, for example, analogs to natural nucleic acids, covalently bonded species.
Hybridize probes.
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′ vs. 5′ 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.
In certain embodiments, 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. In alternative embodiments, 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
Degrade probes.
Release reporter.
Following detachment of reporter 95, in certain embodiments, companion 92 is configured to remain attached to analyte 50. In alternative embodiments, 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
Purification.
Detection.
Multi-Analyte Assay With Multiple Reporter Types And A Matrix Detector Cell
Detection solution 103, in this example a purified reporter solution derived form the reaction mixture of
Note that although in the matrix cell embodiment 100 shown, 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.
Aptamer conformed to protect probe. As shown in
Aptamer bound to analyte, probe expose. As shown in
Probe degraded, reporter released. As shown in
Additional exemplary embodiments having aspects of the invention are described in Examples A through I set forth in the co-invented U.S. applications Ser. No. 11/318,354 filed Dec. 23, 2005 (see WO2006-071,895) and Ser. No. 11/212,026 filed Aug. 24, 2005 (see WO2006-024,023), each of which applications is incorporated by reference.
Multiple-stage Amplifiers (Power-law Amplification).
In the embodiments of
In the examples, only the initial stage of amplification is triggered directly by analyte presence, via hybridization with a pre-selected analyte target sequence A. Subsequent stage amplifiers are triggered by amplification products. The accumulation of the products of amplification (derived strands or reporter fragments) tends to follow a power-law relationship based on the number of successive amplifier stages (n=2,3, . . . ).
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.
For purposes of illustration of aspects of the invention, in the examples of
Two-stage amplification. For example,
Initial conditions. In the example illustrated in
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 121 to target sequence A of analyte strand 120. An exonuclease species (together with any necessary co-factors) is added or present in the reagent, so as to initiate degradation of capture strand 121 at its 3′ terminus.
In view 10c, enzymatic degradation of stage 1 is illustrated as complete, with the undegraded analyte strand 120 (original strand) and companion strand 122 (companion I or BC) released into solution. At this point (end of 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:
In view 10e, Amplifier II 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′ terminus. Simultaneously, additional Amplifier I is shown in duplex association with analyte strand 120 as described for stage 1.
In view 10f, 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 II or B) released into solution. At this point (end of step 2) there is one original strand and 3 derived (e.g., amplicon or reporter) strands in solution.
Additional steps (phases 1 and 2). View 10g 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:
In view 10h, Amplifier I is shown in duplex association with analyte strand 120 (stage 1) and Amplifier II 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.
In view 10i, enzymatic degradation is illustrated as complete, with the undegraded analyte strand 120, three of companion strand 122; and three of companion strand 124 (one from step 2 and two newly derived) released into solution. At this point (end of step 3) there is one original strand and 6 derived (e.g., amplicon or reporter) strands in solution.
Table 3 shows the results of six phases or steps of this method.
Three-stage amplification. In an alternative example, FIGS. 12A-C and 13 depict a three-stage (n=3) method embodiment, similar in many respect to the two-stage example shown in
Stage 1.
Stage 2.
Stage 3.
Table 4 shows the results of six phases or steps of this method.
Homologous sequences. With respect to the examples herein, and in particular the multi-stage examples of
For example, in
In other alternatives, homologous sequences in amplicon or reporter species (e.g., B′″ in strand 128 and B5 in strand 130) may have different labels, markers or other detection-enhancing properties, e.g., so as to permit distinct and separate detection (for example in quantitative amplification tracking), or detection by different devices or methods. In certain embodiments, multiple and/or quantitative detection schemes may be employed, e.g., to reduce false positives, to distinguish between similar analytes, and the like.
In this example, 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.
As described above several alternative embodiments of amplification and detection methods having aspect of the invention may be practiced employing a variety of nucleotide-active enzymes, including 3′-exonuclease, 5′-exonuclease, DNA polymerase (e.g., via proof-reading or nuclease activity), and the like. For example, T7 DNA polymerase has been demonstrated using methods of the invention. Similarly, 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.
Reporter strand 147 includes strand 143 which has a complementary sequence B′ 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.
As may be seen from 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-mer level in both channels 8 and 9, corresponding to the enzymatic digestion reaction mixture. One of ordinary skill in the art will readily appreciate that the 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.
As shown in
As shown in
As shown in
Having thus described a preferred embodiment and methods, it should be apparent to those skilled in the art that certain advantages have been achieved. It should also be appreciated that various modifications, adaptations, and alternative embodiments thereof may be made within the scope and spirit of the present invention.
This application claims priority pursuant to 35 USC. §119(e) to the following U.S. Provisional Applications, each of which applications are incorporated by reference: Ser. No. 60/901,538 filed Feb. 14, 2007 entitled “Electrochemical nanosensors for biomolecule detection”; Ser. No. 60/850,217 filed Oct. 6, 2006 entitled “Electrochemical nanosensors for biomolecule detection”; and Ser. No. 60/789,022 filed Apr. 4, 2006 entitled “Analyte amplification and reporters, and nanoelectronic detection of polynucleotides and other biomolecule”. This application is a continuation-in-part of and claims priority to U.S. patent application Ser. No. 11/318,354 filed Dec. 23, 2005, entitled “Nanoelectronic sensor devices for DNA detection and recognition of polynucleotide sequences” (equivalent published as WO2006-071,895), which claims priority to (among other applications) U.S. Provisional Applications No. 60/748,834, filed Dec. 9, 2005; Ser. No. 60/738,694 filed Nov. 21, 2005; Ser. No. 60/730,905, filed Oct. 27, 2005; Ser. No. 60/668,879, filed Apr. 5, 2005; Ser. No. 60/657,275 filed Feb. 28, 2005; and Ser. No. 60/639954, filed Dec. 28, 2004. This application is a continuation-in-part of and claims priority to U.S. patent application Ser. No. 11/212,026 filed Aug. 24, 2005, entitled “Nanotube sensor devices for DNA detection” (equivalent published as WO2006-024,023), which claims priority to (among other applications) U.S. Provisional Applications No. 60/629,604 filed Nov. 19, 2004; and Ser. No. 60/604,293 filed Aug. 24, 2004. This application is a continuation-in-part of and claims priority to U.S. patent application Ser. No. 11/588,845 filed Oct. 26, 2006 entitled “Anesthesia Monitor, Capacitance Nanosensors and Dynamic Sensor Sampling Method”, which in turn claims priority to the follow U.S. provisional applications No. 60/850,217 filed Oct. 6, 2006; Ser. No. 60/773,138 filed Feb. 13, 2006; Ser. No. 60/748,834 filed Dec. 9, 2005 and Ser. No. 60/730,905 filed Oct. 27, 2005. This application is a continuation-in-part of and claims priority to U.S. patent application Ser. No. 11/488,465 filed Jul. 18, 2006 entitled “Nanoelectronic Sensor With Integral Suspended Micro-Heater” (published as 2007-0045,756); which in turn claims priority to U.S. Provisional Application No. 60/700,953 filed Jul. 19, 2005. This application is related in subject matter to the following U.S. patent applications, each of which applications are incorporated by reference: Ser. No. 10/704,066 filed Nov. 7, 2003 entitled “Nanotube-Based Electronic Detection Of Biomolecules” (Publication 2004-0132,070); Ser. No. 10/388,701 filed Mar. 14, 2003 “Modification Of Selectivity For Sensing For Nanostructure Device Arrays” (U.S. Pat. No. 6,905,655); Ser. No. 10/345,783 filed Jan. 16, 2003, entitled “Electronic sensing of biological and chemical agents using functionalized nanostructures” (Publication 2003-0134,433); and Ser. No. 10/280,265 filed Oct. 26, 2002 entitled “Sensitivity Control For Nanotube Sensors” (U.S. Pat. No. 6,894,359); Ser. No. 10/177,929 filed Jun. 21, 2002 entitled “Dispersed Growth Of Nanotubes On A Substrate” (equivalent published as WO04-040,671).
| Number | Date | Country | |
|---|---|---|---|
| 60901538 | Feb 2007 | US | |
| 60850217 | Oct 2006 | US | |
| 60789022 | Apr 2006 | US | |
| 60748834 | Dec 2005 | US | |
| 60738694 | Nov 2005 | US | |
| 60730905 | Oct 2005 | US | |
| 60668879 | Apr 2005 | US | |
| 60657275 | Feb 2005 | US | |
| 60639954 | Dec 2004 | US | |
| 60629604 | Nov 2004 | US | |
| 60604293 | Aug 2004 | US | |
| 60850217 | Oct 2006 | US | |
| 60773138 | Feb 2006 | US | |
| 60748834 | Dec 2005 | US | |
| 60730905 | Oct 2005 | US | |
| 60700953 | Jul 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11318354 | Dec 2005 | US |
| Child | 11695401 | Apr 2007 | US |
| Parent | 11212026 | Aug 2005 | US |
| Child | 11695401 | Apr 2007 | US |
| Parent | 11588845 | Oct 2006 | US |
| Child | 11695401 | Apr 2007 | US |
| Parent | 11488456 | Jul 2006 | US |
| Child | 11695401 | Apr 2007 | US |
