WO2016160877A1

WO2016160877A1 - Non-enzymatic nucleic acid detection using an oligonucleotide linker with a large cell gap

Info

Publication number: WO2016160877A1
Application number: PCT/US2016/024835
Authority: WO
Inventors: Peter Condon; Uma Mahesh Babu
Original assignee: Rapid Pathogen Screening, Inc.
Priority date: 2015-03-31
Filing date: 2016-03-30
Publication date: 2016-10-06

Abstract

Each electrode pair in a nucleic acid detection apparatus includes a first electrode, a second electrode, an insulator between the first electrode and the second electrode, and at least one bridge between the first electrode and the second electrode across the insulator. The insulator includes a channel providing a gap between the first electrode and second electrode. The bridge includes at least one nucleic acid strand having a sequence complementary to a target nucleic acid sequence. In some embodiments, the bridge includes a first nucleic acid strand permanently attached to the first electrode, a conductive linker permanently attached to the first nucleic acid, and a second nucleic acid strand permanently attached to the conductive linker and to the second electrode. In some embodiments, the bridge is made of non-nucleic acid conducting material upon which strands of nucleic acids are permanently bound as tethers along the length of the bridge.

Description

NON-ENZYMATIC NUCLEIC ACID DETECTION USING AN OLIGONUCLEOTIDE

LINKER WITH A LARGE CELL GAP

REFERENCE TO RELATED APPLICATIONS

This application claims one or more inventions which were disclosed in

Provisional Application Number 62/141,012, filed March 31, 2015, entitled "NON- ENZYMATIC NUCLEIC ACID DETECTION USING AN OLIGONUCLEOTIDE LINKER WITH A LARGE CELL GAP". The benefit under 35 USC § 119(e) of the United States provisional application is hereby claimed, and the aforementioned application is hereby incorporated herein by reference. BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The invention pertains to the field of nucleic acid detection. More particularly, the invention pertains to nucleic acid detection by measuring the electrical conductivity between two electrodes. DESCRIPTION OF RELATED ART

U.S. Pat. App. Pub. No. 2004/0048241, entitled "Methods for Attaching

Molecules", by Freeman et al., published March 11, 2004, and hereby incorporated herein by reference, discloses a method of electrically coupling an electrode pair in a plurality of electrode pairs in a biosensor with a macromolecule. The electrode pair includes a first electrode and a second electrode. A first portion of the macromolecule is derivatized with a first reactive group and a second portion of the macromolecule is derivatized with a second reactive group. The first reactive group is masked with a first electrolabile masking group and the second reactive group is masked with a second electrolabile masking group. A first voltage is applied at the first electrode in the electrode pair under conditions that are sufficient to unmask the first reactive group. The unmasked first reactive group binds to the first electrode thereby linking the macromolecule to the first electrode. A second voltage is applied at the second electrode in the electrode pair under conditions that are

{00651183.DOCX 7} sufficient to unmask the second reactive group. The unmasked second reactive group binds to the second electrode, thereby electrically coupling the electrode pair in the biosensor with the macromolecule.

U.S. Pat. App. Pub. No. 2004/0023253, entitled "Device Structure for Closely Spaced Electrodes", by Kunwar et al., published February 5, 2004, and hereby incorporated by reference herein, discloses a biosensor with a plurality of devices on a substrate. Each device occupies a different region on the substrate. Each device includes a first electrically conducting material, a spacer, and a second electrically conducting material. The first electrically conducting material is overlaid on a first portion of the different region on the substrate occupied by a device and the spacer is overlaid on a second portion of the different region on the substrate that is occupied by the device. The first electrically conducting material and the spacer abut each other. The second electrically conducting material is overlaid on a portion of the spacer.

U.S. Pat. App. Pub. No. 2004/0248282, entitled "Electronic Detection of

Biological Molecules using Thin Layers", by Pisharody et al., published December 9, 2004, and hereby incorporated by reference herein, discloses sensors that facilitate the detection of an analyte. In general, the biosensors utilize a binding agent (e.g.

biomolecule) to specifically bind to one or more target analytes. The biomolecule spans a gap between two electrodes. Binding of the target analyte changes conductivity of the sensor, thereby facilitating ready detection of the binding event and thus detection and/or quantitation of the bound analyte.

U.S. Patent No. 6,824,974, entitled "Electronic Detection of Biological Molecules using Thin Layers", issued November 30, 2014 to Pisharody et al., and hereby incorporated by reference herein, discloses sensors that facilitate the detection of analytes. The sensors use a binding agent (e.g. biomolecule) to specifically bind to one or more target analytes. The biomolecule spans a gap between two electrodes. The gap is generally less than 30 nanometers. Binding of the target analyte changes the conductivity of the sensor, which facilitates detection of the binding and thus detection and/or quantitation of the analyte.

{00651183.DOCX 7} U.S. Patent No. 8,078,048, entitled "Bridged Element for Detection of a Target Substance", issued December 13, 2011 to Albert et al., and hereby incorporated by reference herein, discloses detecting physical changes resulting from an association between a template molecule and a target molecule by monitoring changes in the template molecule. Changes may include a change in a physical dimension or stiffness of the template molecule, a change in electrical conductivity of the template molecule, or a change in the energy required to dissociate the target molecule and the template molecule. The magnitude of the change indicates the specific identity of the target molecule. An electrical field is used to dissociate deoxyribonucleic acid (DNA) from the cell. Some examples include 150-200 base pair segments of DNA as DNA bridge templates.

SUMMARY OF THE INVENTION

A nucleic acid detection apparatus includes electrode pairs. Each electrode pair includes a first electrode, a second electrode, an insulator between the first electrode and the second electrode, and at least one bridge between the first electrode and the second electrode across the insulator. The insulator includes a channel providing a gap between the first electrode and the second electrode. In some embodiments, the gap is at least 70 nanometers in length. The bridge includes at least one nucleic acid strand having a sequence complementary to a target nucleic acid sequence. In some embodiments, the bridge includes a first nucleic acid strand permanently attached to the first electrode, a conductive linker permanently attached to the first nucleic acid, and a second nucleic acid strand permanently attached to the conductive linker and to the second electrode. In some embodiments, the bridge is made of non- nucleic acid conducting material upon which strands of nucleic acids are permanently bound as tethers along the length of the bridge.

A nucleic acid detection apparatus includes a plurality of electrode pairs, each electrode pair including a first electrode, a second electrode and an insulator between the first electrode and the second electrode. The insulator includes a channel providing a gap between the first electrode and the second electrode. The nucleic acid detection apparatus also includes at least one bridge between the first electrode and the second electrode extending across the insulator. The bridge includes a first nucleic acid strand permanently attached to the first electrode at a first end of the first nucleic acid strand, a conductive linker having a first end and a second end, where the first end of the conductive linker is

{00651183.DOCX 7} permanently attached to a second end of the first nucleic acid strand, and a second nucleic acid strand permanently attached at a first end to the second end of the conductive linker.

By contouring the electrodes in some embodiments, the width of the gap can vary. In some embodiments, the bridge includes a first nucleic acid strand permanently attached to the first electrode, a conductive linker permanently attached to the first nucleic acid strand, and a second nucleic acid strand permanently attached to the conductive linker and to the second electrode.

A method of non-enzymatic nucleic acid detection for a presence of at least one target nucleic acid in a sample includes the step of measuring a first electrical conductivity between at least one electrode pair, the electrode pair including a first electrode, a second electrode, an insulator between the first electrode and the second electrode, and at least one bridge between the first electrode and the second electrode extending across the insulator. The insulator includes a channel providing a gap between the first electrode and the second electrode, where the gap is either less than approximately 50 nanometers or greater than approximately 100 nanometers. The bridge includes a first nucleic acid strand having a sequence complementary to a first target nucleic acid sequence, where the first nucleic acid strand is permanently attached to the first electrode at a first end of the first nucleic acid strand, a conductive linker having a first end and a second end, where the first end of the conductive linker is permanently attached to a second end of the first nucleic acid strand, and a second nucleic acid strand having a sequence complementary to a second target nucleic acid sequence permanently attached at a first end to the second end of the conductive linker. The method also includes the steps of exposing the electrode pair to the sample, measuring a second electrical conductivity between the electrode pair, and quantifying a difference between the first electrical conductivity and the second electrical conductivity to determine the presence of the first target nucleic acid sequence and the second target nucleic acid sequence in the sample.

An alternative embodiment of a nucleic acid detection apparatus includes electrode pairs, where each electrode pair includes a first electrode, a second electrode and an insulator between the first electrode and the second electrode. The insulator includes a channel providing a gap between the first electrode and the second electrode. The nucleic acid detection apparatus also includes at least one bridge between the first electrode and

{00651183.DOCX 7} the second electrode extending across the insulator. The bridge includes a first conductive linker permanently attached to the first electrode at a first end of the first conductive linker, a nucleic acid strand having a first end and a second end and a second conductive linker. The first end of the nucleic acid strand is permanently attached to a second end of the first conductive linker and the second conductive linker is permanently attached at a first end to the second end of the nucleic acid strand.

Another alternative embodiment of a nucleic acid detection apparatus includes electrode pairs, wherein each electrode pair includes a first electrode, a second electrode, and an insulator between the first electrode and the second electrode. The insulator includes a channel providing a gap between the first electrode and the second electrode. The nucleic acid detection apparatus also includes at least one bridge between the first electrode and the second electrode extending across the insulator. The bridge includes at least one conductive linker having a length, a first end and a second end. The conductive linker is made of non-nucleic acid material. The first end of the conductive linker is attached to the first electrode and the second end of the conductive linker is attached to the second electrode. At least one nucleic acid strand is permanently attached to the conductive linker along the length of the conductive linker.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig- 1A shows an electrode design from the prior art with a constant gap between the electrodes such that the circuit accommodates a specific length of nucleic acid.

Fig. IB shows a micrograph of the electrode design of Fig. 1 A.

Fig- 2 shows schematically two electrodes with a gap bridged by a nucleic acid. Fig. 3A shows schematically two electrodes with a gap and a conductive linker with

nucleic acids attached at either end. Fig. 3B shows schematically the components of Fig. 3A with the conductive linker and nucleic acids bridging the gap. Fig. 3C shows schematically two electrodes with a gap and a bridge including two

conductive linkers and a single nucleic acid strand.

{00651183.DOCX 7} Fig. 3D shows schematically two electrodes with a gap and a bridge including two conductive linkers and three nucleic acids.

Fig. 4 shows schematically two electrodes with a gap and a bridge formed by a conductive linker, with nucleic acid strands attached to the conductive linker along the length of the conductive linker.

Fig. 5 shows a portion of a chip with a bridge.

Fig. 6 shows a micrograph of the bridge of Fig. 5 including two electrodes with a tapered gap between the two electrodes.

DETAILED DESCRIPTION OF THE INVENTION

The above-described prior art technology has been used with DNA to bridge a fixed gap in a circuit and to detect DNA in a sample when it hybridizes. In some embodiments, electrical circuitry used to apply a current and detect an electrical conductance across the gap of a chip in an apparatus of the present invention is similar to the electrical circuitry disclosed in U.S. Patent No. 6,824,974.

"Nucleic acid strand", as defined herein, refers to a single strand of nucleic acid.

When a single stranded DNA molecule bridges a circuit gap and hybridizes to a target sequence in a sample, the double stranded DNA conducts charge better than the single stranded section. However, not all DNA sequences conduct charge well. Lewis et al. ("Distance-Dependent Electron Transfer in DNA Hairpins", Science, 277, 1997, pp. 673-676, herein incorporated by reference) found that sequential deoxyadenosine- deoxythymidine base pairs quenched charge transport. Therefore, designing a tool to specifically recognize a DNA fragment in a sample requires finding a unique section of DNA that is specific to the target sequence but still conducts charge well when hybridized to its complement.

Referring to Figs. 1A and IB, a prior art bridged element analyte recognition biosensor is shown (Bridger Technologies, Inc., Bozeman, Montana). The chip device includes an upper electrode 10 and a lower electrode 12 separated by a gap. The gap is bridged by a nucleic acid 14, which is permanently bound at one end to the upper

{00651183.DOCX 7} electrode 10 and at the other end to the lower electrode 12. The micrograph in Fig. IB shows preferred shapes for the first electrode 10 and the second electrode 12 with a permanently bound nucleic acid receptor bridging the gap.

A prior art device was evaluated for its ability to identify bacterial samples that had been blinded. DNA from seven organisms was grown in culture and purified. The seven organisms were S. dysgalactiae, E. coli, K. pneumonia, S. enterica, P. aeruginosa, P. vulgaris and H. sapiens. Chips were prepared to detect S. dysgalactiae and S. enterica. The DNA from the other organisms was used as a negative control. Evaluation was conducted in two ways: 1) Purified DNA was serially diluted and tested until negative results were obtained, and

2) Cultured bacteria were serially diluted and tested until negative results were obtained.

The procedure was similar to Southern blotting or in-situ hybridization in which denatured DNA is allowed to hybridize with labeled probe. In this case, the process started with placing a sample into a lysis/extraction solution. No amplification or purification step was needed. The chip included 200 base pair (bp) segments of DNA chemically bridging two electrodes in a prior art device similar to that disclosed in U.S. Patent No. 6,824,974. When a voltage potential is applied across the electrodes, single stranded DNA conducts electricity very poorly. Once the sample is introduced to the chip, if the single-stranded DNA hybridizes with complementary DNA, the conductivity is dramatically increased. The DNA coupled with the electrode is referred to herein as a receptor.

The sample, either culture or purified DNA, was diluted into a buffer containing detergents, buffering capacity, and formamide. The buffer denatures genomic DNA into shorter fragments. Formamide is added to stabilize the DNA in a single stranded state as is typically done in Southern blots. This process occurs within seconds. As soon as the sample was diluted, it was introduced into a cavity containing the receptors. The denatured DNA was allowed to anneal with the receptors within the device. The annealing is based on standard Watson-crick base pairing. The annealing/hybridization is facilitated by

{00651183.DOCX 7} applying a voltage potential across the receptor. This process also occurs within seconds. The process is specific because large pieces (200 bp) of DNA are annealed.

If annealing occurs due to complementary DNA being present, current flow through the receptor increases significantly. If no annealing occurs because no complementary DNA is present, the current does not change. On the device,

approximately 224 receptors were present. A threshold current was selected for specificity and the hybridization was considered to have occurred if the current through the receptor exceeded that threshold. The data is reported as the percent of receptors that indicated an increase in conductivity over the threshold value. Tables 1 and 2, respectively, show the results of the evaluation of Salmonella chip bacterial culture sensitivity and specificity.

Table 1: Salmonella Test Sensitivity

As shown in Table 2, when purified DNA from other organisms was used as a !gative control, none of the receptors had a current that exceeded the threshold value.

{00651183.DOCX 7} Table 2: Salmonella Test Specificity

Evaluation of the current through receptors for the negative samples indicated an average current of 0.0005 μΑ. The average current through the receptors mixed with positive sample at the level of detection was 0.22204 μΑ. Comparison of these currents over 2,240 observations for the positive sample and 2,240 observations for the negative samples, using a student's t-test, produced a p-value of 0.00001, indicating that the chance that these two populations differ due to a random event is extremely unlikely. A cut off for current of the average value of the current through the negative samples plus three standard deviations would be 0.007 μΑ. The average positive current value is 31 times greater than this value, so the separation between negative and positive samples is quite significant.

In addition to evaluating purified DNA, bacterial culture was evaluated. The S. enterica culture was found to be positive with no additional sample processing, so at least in the case of salmonella, the buffer was capable of lysing the cells to expose the DNA to the hybridization conditions. E. coli culture did not produce a positive result nor did it inhibit a positive result from the S. enterica culture (see Table 3). It is not known precisely what concentration of DNA was contained in the culture. The preferred or optimal lysis conditions may vary depending on the nature of the culture or sample.

{00651183.DOCX 7} Table 3: Percent Occupancy as a Function of Culture Dilution for Negative Samples

A similar evaluation with S. dysgalactiae produced the results shown in Tables 4 and 5, which are consistent with the S. enterica results.

Table 4: Streptococcus Test Sensitivity

{00651183.DOCX 7} K. pneumoniae 50,000,000 0

S. enterica 50,000,000 0

S. enterica 25,000,000 0

P. aeruginosa 50,000,000 0

P. vulgaris 50,000,000 0

H. sapiens 250,476 0

The test provides discrimination between positive and negative samples within a 2 minute time frame. The test was able to discriminate between positive and negative bacteria grown in culture as well as purified DNA. Mixed samples, such as, for example, E. coli plus S. enterica, did not inhibit a positive response. No cross reaction was observed with human DNA, which presumably would be the most likely contaminant. Of all of the negative samples evaluated, not a single current above the threshold was observed with more than 2,000 observations. The test has sensitivity levels rivaling PCR. This combination of sensitivity, specificity, and speed to result is an unequaled combination. However, given that the gap is a constant width, the number of base pairs in the length of DNA must be constant. While this prior art system has been found to work well if there is a unique sequence in the target sequence that is exact length needed to bridge the gap between the electrodes that conducts charge well in a hybridized form, not all target sequences have such a long unique sequence that conducts well. This has been found to be severely limiting for nucleic acid detection in some organisms.

Therefore, the embodiments described herein teach a novel circuit that allows varying widths of DNA to be used in the same circuit.

A nucleic acid detection apparatus includes electrode pairs. Each electrode pair includes a first electrode, a second electrode, an insulator between the first electrode and the second electrode, and at least one bridge between the first electrode and the second electrode across the insulator. The insulator includes a channel providing a gap between the first electrode and the second electrode. The bridge includes at least one nucleic acid strand having a sequence complementary to a target nucleic acid sequence. By contouring

{00651183.DOCX 7} the electrodes, the width of the gap can vary. In some embodiments, the bridge includes a first nucleic acid strand permanently attached to the first electrode, a conductive linker permanently attached to the first nucleic acid, and a second nucleic acid strand permanently attached to the conductive linker and to the second electrode.

Apparatus and methods of the present invention preferably provide a clinical benefit of point-of-care nucleic acid testing with accurate, immediate results to the physician rather than a several day lag for test results. In some embodiments, an unpurified sample is run over a chip and in less than two minutes one to fifty copies of a target DNA sequence in a 50 microliter volume is detected. In some embodiments, the apparatus and method determine whether there is the presence of at least one specific pathogen in the test sample. In some embodiments, the apparatus and method provide a cancer screening for at least one specific nucleic acid sequence associated with at least one cancer.

In some embodiments, the apparatus uses a chip with a circuit technology similar to in situ hybridization.

Referring to Fig. 2, a first electrode 20 and a second electrode 22 are separated by a gap 26. A nucleic acid strand 24 bridges the gap 26 and is permanently bound at one end to the first electrode 20 and at the other end to the second electrode 22.

In some embodiments, the Non Enzymatic Nucleic Acid (NENA) detection described herein uses a conductive linker with oligonucleotides at either end in combination with a pair of electrodes having a large cell gap between electrodes. In preferred embodiments, a disposable chip uses large cell gaps in combination with a fragment with two DNA sequences at each end connected to a conductive linker. In other embodiments, different configurations and numbers of the nucleic acid strands and conductive linkers form a bridge across the cell gap between the electrodes.

In some embodiments, the bridge includes only one strand of nucleic acid permanently attached between the first and the second electrode using two conductive linkers.

{00651183.DOCX 7} In some embodiments, the bridge between the two electrodes is made of non- nucleic acid material to which, at various points all along the bridge, strands of nucleic acids are attached. The nucleic acid strands protrude from the length of the bridge. The length, the number and the multiplicity of these strands of nucleic acids may be varied. The difference in the basal level of conductivity of the bridge with these strands and that from the measured conductivity when the target nucleic acid hybridizes with them is the basis of detection of the target nucleic acid.

The conductive linker may be any material able to conduct electricity and able to permanently attach to nucleic acids on the two ends of the conductive linker. The two DNA sequences may be the same or different from each other. Some preferred conductive linker materials include, but are not limited to, gold, a single-walled carbon nanotube (SWNT), and polysilicon. In some embodiments, the linker material is gold and the linkage is an alkanethiol. In other embodiments, the linker material is graphitic and the linkage is a pyrene.

In some embodiments, the conductive material and the linkage between the conductive material and the nucleic acid are a conductive material and a linkage disclosed by Hihath et al. ["Study of Single-Nucleotide Polymorphisms by Means of Electrical Conductance Measurements", PNAS 102, pp. 16979-16983 (2005)], Muren et al.

["Solution, Surface, and Single Molecule Platforms for the Study of DNA-mediated Charge Transport", Phys. Chem. Chem. Phys. 14, pp. 13754-13771 (2012)], Guo et al. ["Conductivity of a Single DNA Duplex Bridging a Carbon Nanotube Gap", Nat.

Nanotechol. 3, pp. 163-167 (2008)], Lee [U.S. Pat. App. Pub. No. 2005/0136419, entitled "Method and Apparatus for Nanogap Device and Array" and published June 23, 2005], or Guo et al. [U.S. Pat. App. Pub. No. 2011/0275062, entitled "Systems and Methods for Integrating a Single DNA Molecule into a Molecular Electronic Device" and published November 10, 2011], all of which are hereby incorporated by reference herein.

In some embodiments, a naphthalimide (NI) derivative, a metallointercalator, or an organic probe is used to enhance the electrical conductivity of the nucleic acid [see, for example, Muren et al.]. For example, the cell gap can be about 2 micrometers and the conductive linker about 1.9 micrometers such that the strands of DNA would be about 50 nanometers on each side. An advantage of this approach is that DNA sequences within a

{00651183.DOCX 7} range of lengths may be used as long as the total length of the combined oligo-linker-oligo is about the same as the length of the cell gap. In other words, shorter DNA fragments may be used with longer conductive linkers. Alternatively, in some embodiments, the cell gap and conductive linker are long enough that the system is able to tolerate some variation in the length of the oligonucleotides at each end.

Referring to Figs. 3A and 3B, a first electrode 30 and a second electrode 32 are separated by a gap 40. A conductive linker 34 has a first nucleic acid strand 36 permanently attached at one end of the conductive linker 34 and a second nucleic acid strand 38 permanently attached at the other end of the conductive linker 34. Preferably, the gap 40 is 2 μιη in length, the conductive linker 34 is 1.9 μιη in length, and the first and second nucleic acid strands 36, 38 are each 50 nm in length.

The nucleic acid strands are preferably attached to the conductive linker, which may be done outside the presence of the chip, prior to being attached to the first and second electrodes. In Fig. 3B, the other end of the first nucleic acid strand 36 is permanently bound to the first electrode 30 and the other end of the second nucleic acid strand 38 is permanently bound to the second electrode 32. The first nucleic acid 36- conductive linker 34-second nucleic acid 38 complex bridges the gap 40 between first electrode 30 and the second electrode 32.

Different configurations of conductive linkers and nucleic acid strands are alternatively possible. For example, in one alternative embodiment, shown in Fig. 3C, two conductive linkers 34, 35 are permanently attached to the electrodes 30, 32 respectively. A single nucleic acid strand 37 is permanently attached to each of the two conductive linkers. The two conductive linkers, 34, 35 and the nucleic acid strand 37 form the bridge across the gap 40. In other embodiments, additional conductive linkers and nucleic acid strands may be used to test for three or more target nucleic acid sequences. One example, showing a first nucleic acid strand 36, a first conductive linker 34, a second nucleic acid strand 38, a second conductive linker 35 and a third nucleic acid strand 37, can be seen in Fig. 3D.

Additional nucleic acid strands and conductive linkers may be added to increase the number of target sequences being tested for. In some of these embodiments, the

{00651183.DOCX 7} nucleic acid strands (as shown in Figs. 3A, 3B and 3D) or the conductive linkers (as shown in Fig. 3C) are permanently attached to the first electrode and the second electrode. In other embodiments, a nucleic acid strand is permanently attached to the first electrode and a conductive linker is permanently attached to the second electrode.

In still other embodiments, the bridge across the gap includes only conductive linkers and one end of the nucleic acid strands are connected to the conductive linkers at locations along the length of the conductive linkers at an angle with respect to the conductive linkers. In these embodiments, the entire bridge is made of non-nucleic acid material upon which at strategic points throughout the length of the bridge are strands of nucleic acids are permanently attached or tethered, for example, similarly to flags on a long flag pole. One example of this embodiment is shown in Fig. 4. Fig. 4 shows a single conductive linker 34 that bridges the gap 40 between the first electrode 30 and the second electrode 32. There are multiple nucleic acid strands 41 extending or protruding from the conductive linker 34 along a length of the conductive linker 34. The difference in the basal level of conductivity of the bridge with these strands 41 and that from the measured conductivity when the target nucleic acid hybridizes with them is the basis of detection of the target nucleic acid. While multiple nucleic acid strands 41 are shown in Fig. 4, one or more nucleic strands 41 may be included in this embodiment.

In some embodiments, the gap between the two electrodes is tapered to provide a range of gap distances between a pair of electrodes rather than having a uniform gap distance between the pair of electrodes. The tapered gap allows flexibility in the length of the DNA hybridization fragments loaded onto the chip.

Fig. 5 shows a chip 51 with a bridge 56 (for example, the first nucleic acid 36- conductive-linker 34-second nucleic acid 38 complex bridge shown in Figs. 3A and B or the first nucleic acid 36-first conductive linker 34-third nucleic acid 37- second conductive linker 35-second nucleic acid 38 complex bridge shown in Fig. 4) between electrodes 50 and 52. Fig. 6 shows a micrograph of a resulting pair of electrodes 60 and 62 intended to be fabricated with a 30 nm to 100 nm taper 70 between the electrodes 60 and 62. An actual measurement of the fabricated electrode pair determined the taper 70 to be 31 nm to 102 nm, which is well within the tolerance range for the gap distance. The tapered gap 70 in Fig. 6 was formed between two gold electrodes 60 and 62. In the example micrograph

{00651183.DOCX 7} shown in Fig. 6, 80 nm gold conductor electrodes 60 and 62 were used. An 18 nm thick gold bridge 80 is also shown in Fig. 6.

In some embodiments, a method of Non Enzymatic Nucleic Acid (NENA) detection includes the following procedural steps. The sample is diluted, the system is purged, the sample is injected, and the electrical conductivity is read. The sample dilution step preferably takes about 15 seconds, the system purge step preferably takes about 20 seconds, the sample injection step preferably takes about 5 seconds, and the electrical reading preferably takes about 15 seconds. This gives a total time for the procedure of less than one minute. Some advantages of the NENA detection methods and devices described herein are that they are an advance over real-time polymerase chain reaction (PCR), they may qualify for Clinical Laboratory Improvement Amendments (CLIA) waivability, they have a lower cost per test compared to PCR, the sample preparation can be relatively impure, and their detection is analogous to ion selective electrode (ISE) analysis. Other benefits include PCR-like sensitivity, only minutes from sample to result, no interference from blood or proteins, no requirement for isolation of DNA from the rest of a sample, no enzymatic activity involved (no enzymatic inhibition), no temperature control required, and many nucleic acid sequences per test. Each chip can include multiple circuits that can be bridged with redundant or distinct nucleic acid tests. For example, if a chip was loaded with 20 nucleic acid sequences of the same sequence, for 10 different sets of the sequences, the chip would detect up to 20 copies of DNA for 10 unique sequences. One of those sets of sequences could be a control sequence that is included in the extraction or sample buffer such that the hybridization of the control sequence provides a control that the device is working properly. A comparison of attributes of NENA detection with PCR is shown in Table 6:

Table 6: Test Attributes

{00651183.DOCX 7} polymerase inhibitors because of built in controls

Quality of DNA Highly dependent Purity is irrelevant

Interferences Susceptible to various None observed yet

interferences

Ease of Use Multi-step Simple, CLIA waivable

Point of Care Remote from patient Point of care test

location

Multiplexing Yes Yes

Temperature Thermocycling required Ambient temperature

Multiple analytes may be simultaneously detected in one sample. In some embodiments, the number of analytes detected simultaneously in one sample is in the range of 10 to 50. Unlike PCR, NENA detection does not require labile enzymes or time- consuming thermal cycling amplification, non-enzymatic.

In preferred methods of non-enzymatic nucleic acid detection, a sample with target analytes is placed into a handheld portable test device. The device contains complimentary nucleotide sequences (~100-mer) to the target analytes. The sequences are attached at each end to a conductive linker and to each electrode. The device monitors the change in charges across the complimentary nucleotide sequence and indicates the present or lack of the target analytes in the sample.

In some embodiments, the conductive linker materials are gold surfaces. In some embodiments, the conductive linker materials are polymers. For example, Polyaniline, Polypyrole, Polyethylenedioxythiophene, Polythiophenes, Polycarbazole, Polyacetylene, and Poly(p-phenylene vinylene) are all inherently conductive polymers. The production method of these polymers can have an impact on the dimensions of the polymer chain that is produced. In theory, enhanced electrical conductivity is derived from enhanced molecular order. The molecular order is frequently associated with the radius of the polymer chain since controlled, ordered growth tends to result in smaller diameter chains and unregulated growth tends to create molecular "excursions" which result in larger diametered, lower ordered chains. Diameters of polymer chains range from the low nm

{00651183.DOCX 7} range to the multiple micron range. Controlled polymer chain production has produced polymers with conductivity values of 100,000 S/CM. This is the level of conductivity associated with copper wire.

In some embodiments, composites of conjugated polymers (CPs), co-polymers (COPs) and Carbon Nanotubes (CNs) can also be used as conductive linkers. Doping CNs with CPs or COPs can imbue the CNs with greater conductive properties. Some authors have described these composites as having synergistic combinations of attributes including conductivity.

Oligonucleotides can be synthesized with a large number of functional groups. These modifications include reactive moieties such as carboxyl groups, free amines, maleimides or chemical groups consistent with click chemistry.

Selection of a conductive polymer can facilitate chemical cross linking by standard techniques employing agents such as (l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) 1EDC or N-Hydroxysuccinimide (NHS). Additionally, the synthesis of the polymer can be done under conditions that incorporate active groups consistent with click chemistry such as PEDOT (poly(3,4-ethylenedioxythiophene) polystyrene sulfonate).

A 34 nm long monolayer of double- stranded DNA of just 100 base pairs on a gold surface is capable of carrying a low current, which is another small step towards the ultimate goal of nanoelectronics but also holds promise for exquisite DNA sensors [see, for example, Slinker et al., "DNA Charge Transport over 34 nm", Nature Chem. 3, pp. 230-235 (2011), hereby incorporated by reference herein]. A 100-mer formed through piece- wise synthesis may be attached to a gold surface at one end and may have a probe attached at the other. A length of 100 base pairs is close to the persistent length of DNA strands in aqueous solution and is therefore a natural length with which to work. A single base pair mismatch in the DNA reduces the flow of electrons significantly, which might be exploited in sensors.

In some embodiments, methods of the present invention are applied to viruses, but lysis of virus is different from the lysis of bacteria. Lysis using the methods described herein is not especially efficient and optimization of that process would improve the sensitivity of the methods. Additionally, many of the target pathogens encode their genetic

{00651183.DOCX 7} information in ribonucleic acid (RNA), so preferred methods of the present invention are preferably adaptable to RNA detection.

Apparatus and methods detect highly homologous DNA. Any mismatch decreases current flow through the DNA. The location of the mismatch, the type of mismatch, and the stringency of the hybridization conditions determines the extent to which current flow is changed. It is therefore possible to detect known genetic mutations based on these current flow changes. The apparatus and methods have been successfully tested with both bacteria and viruses as well as with human cells, which are relatively simple to lyse. No reaction inhibitors have been found to date, including a 100-fold molar excess of human DNA, whole blood, proteins, cell debris, house dust, drywall particulates, oil, and gasoline.

In preferred embodiments, the sample chip is a one-use sample chip that is inexpensive to produce and disposable, which eliminates cross contamination issues. The apparatus preferably includes a dedicated reader capable of reading multiple test types. Each test may be for a single nucleic acid sequence or a panel of sequences. The method and apparatus preferably permit use of nucleic acid hybridization fragments of various sizes.

One feature of using conductive linker materials is that two or more DNA fragments can be connected with the conductive linker materials. Those pieces of DNA could be identical or distinct. If the pieces of DNA are distinct, they may be also be the same or different lengths. For example, if the only suitable sections of DNA for conductivity were not unique to the organism being detected, but a combination of smaller sections of DNA were unique and conductive, the DNA could be different at each end of the conductive linker material. For example, a segment of 100bps could be added to each end where each sequence of lOObp shared significant homology with multiple organisms, but the combination of both lOObp sequences is unique to the organism of interest. Then the hybridization of only one section of the DNA attached to the conductive linker material would not provide the increased conductivity that would result in a positive detection result. In addition, two sections of conductive linker material could be added with an additional section of DNA connected between the two sections of conductive linker material providing for three or more unique sections of DNA that could hybridize.

{00651183.DOCX 7} Shorter sections of DNA might be particularly useful for detections of single nucleotide polymorphisms (SNP). For example, longer sections of DNA might identify the organism or specific gene in question, but shorter sections at the point mutation that hybridize differentially to various SNPs could be used to identify specific strains or mutations.

All above-mentioned references are hereby incorporated by reference herein.

Accordingly, it is to be understood that the embodiments of the invention herein described are merely illustrative of the application of the principles of the invention. Reference herein to details of the illustrated embodiments is not intended to limit the scope of the claims, which themselves recite those features regarded as essential to the invention.

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Claims

What is claimed is:

1. A nucleic acid detection apparatus comprising: electrode pairs, each electrode pair comprising: a first electrode; a second electrode; an insulator between the first electrode and the second electrode, the insulator comprising a channel providing a gap between the first electrode and the second electrode; and at least one bridge between the first electrode and the second electrode extending across the insulator, the bridge comprising: a first nucleic acid strand permanently attached to the first electrode at a first end of the first nucleic acid strand; a first conductive linker having a first end and a second end, the first end of the conductive linker being permanently attached to a second end of the first nucleic acid strand; and a second nucleic acid strand permanently attached at a first end to the second end of the conductive linker.

2. The nucleic acid detection apparatus of claim 1, wherein the conductive linker is made of a material selected from the group consisting of gold, a single-walled carbon nanotube, Polyaniline, Polypyrole, Polyethylenedioxythiophene, Polythiophenes,

Polycarbazole, Poly acetylene, and Poly(p-phenylene vinylene) and polysilicon.

3. The nucleic acid detection apparatus of claim 1, wherein the first nucleic acid strand has a different sequence from the second nucleic acid strand.

4. The nucleic acid detection apparatus of claim 1, wherein the first nucleic acid strand and the second nucleic acid strand have an identical sequence.

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5. The nucleic acid detection apparatus of claim 1, wherein the gap has a uniform length.

6. The nucleic acid detection apparatus of claim 1 , wherein the gap has a tapered length.

7. The nucleic acid detection apparatus of claim 6, wherein the gap has an approximately

30 nm to 100 nm taper.

8. The nucleic acid detection apparatus of claim 1, wherein the second nucleic acid strand is permanently attached at a second end to the second electrode.

9. The nucleic acid detection apparatus of claim 1 , wherein the bridge further comprises a third nucleic acid strand and a second conductive linker having a first end and a second end, wherein the first end of the second conductive linker is permanently attached to a second end of the second nucleic acid strand, the second end of the second conductive linker is permanently attached to a first end of the third nucleic acid strand, and the third nucleic acid strand is permanently attached at a second end to the second electrode.

10. The nucleic acid detection apparatus of claim 1, wherein the gap is less than 50

nanometers.

11. The nucleic acid detection apparatus of claim 1, wherein the gap is greater than 100 nanometers.

12. The nucleic acid detection apparatus of claim 1, wherein the first nucleic acid strand has approximately 100 base pairs.

13. The nucleic acid detection apparatus of claim 1, wherein the second nucleic acid strand has approximately 100 base pairs.

14. A method of non-enzymatic nucleic acid detection for a presence of at least one target nucleic acid in a sample comprising: a) measuring a first electrical conductivity between at least one electrode pair, the electrode pair comprising: a first electrode;

{00651183.DOCX 7} a second electrode; an insulator between the first electrode and the second electrode, the insulator comprising a channel providing a gap between the first electrode and the second electrode, wherein the gap varies between 50 nanometers or less and at least 100 nanometers; and at least one bridge between the first electrode and the second electrode extending across the insulator, the bridge comprising: a first nucleic acid strand having a sequence complementary to a first target nucleic acid sequence, wherein the first nucleic acid strand is permanently attached to the first electrode at a first end of the first nucleic acid strand, a conductive linker having a first end and a second end, the first end of the conductive linker being permanently attached to a second end of the first nucleic acid strand; and a second nucleic acid strand having a sequence complementary to a second target nucleic acid sequence permanently attached at a first end to the second end of the conductive linker; b) exposing the electrode pair to the sample; c) measuring a second electrical conductivity between the electrode pair; and d) quantifying a difference between the first electrical conductivity and the second electrical conductivity to determine the presence of the first target nucleic acid sequence and the second target nucleic acid sequence in the sample.

15. The method of claim 14, wherein the second nucleic acid stand is permanently

attached at a second end to the second electrode.

16. The method of claim 14, wherein the bridge further comprises a third nucleic acid strand and a second conductive linker having a first end and a second end, wherein the first end of the second conductive linker is permanently attached to a second

{00651183.DOCX 7} end of the second nucleic acid strand, the second end of the second conductive linker is permanently attached to a first end of the third nucleic acid strand, and the third nucleic acid strand is permanently attached at a second end to the second electrode.

17. The method of claim 14, wherein the conductive linker is made of a material selected from the group consisting of gold, a single-walled carbon nanotube, Polyaniline, Polypyrole, Polyethylenedioxythiophene, Polythiophenes, Polycarbazole,

Polyacetylene, and Poly(p-phenylene vinylene) and polysilicon.

18. The method of claim 14, wherein the first nucleic acid strand has a different sequence from the second nucleic acid strand.

19. The method of claim 14, wherein the first nucleic acid strand and the second nucleic acid strand have an identical sequence.

20. The method of claim 14, wherein the gap has a uniform length.

21. The method of claim 14, wherein the gap has a tapered length.

22. The method of claim 21, wherein the gap has an approximately 30 nm to 100 nm taper.

23. The method of claim 14, wherein the first nucleic acid strand has approximately 100 base pairs.

24. The method of claim 14, wherein the second nucleic acid strand has approximately

100 base pairs.

25. A nucleic acid detection apparatus comprising: electrode pairs, each electrode pair comprising: a first electrode; a second electrode;

{00651183.DOCX 7} an insulator between the first electrode and the second electrode, the insulator comprising a channel providing a gap between the first electrode and the second electrode; and at least one bridge between the first electrode and the second

electrode extending across the insulator, the bridge comprising: a first conductive linker permanently attached to the first

electrode at a first end of the first conductive linker; a first nucleic acid strand having a first end and a second end, the first end of the nucleic acid strand being permanently

attached to a second end of the first conductive linker;

and a second conductive linker permanently attached at a first end to the second end of the first nucleic acid strand.

26. The nucleic acid detection apparatus of claim 25, wherein the conductive linkers are made of a material selected from the group consisting of gold, a single-walled carbon nanotube, Polyaniline, Polypyrole, Polyethylenedioxythiophene, Polythiophenes, Polycarbazole, Polyacetylene, and Poly(p-phenylene vinylene) and polysilicon.

27. The nucleic acid detection apparatus of claim 25, wherein the gap has a uniform length.

28. The nucleic acid detection apparatus of claim 25, wherein the gap has a tapered length.

29. The nucleic acid detection apparatus of claim 28, wherein the gap has an

approximately 30 nm to 100 nm taper.

30. The nucleic acid detection apparatus of claim 25, wherein the second conductive linker is permanently attached at a second end to the second electrode.

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31. The nucleic acid detection apparatus of claim 25, wherein the bridge further comprises a third conductive linker and a second nucleic acid strand having a first end and a second end, wherein the first end of the second nucleic acid strand is permanently attached to a second end of the second conductive linker, the second end of the second nucleic acid strand is permanently attached to a first end of the third conductive linker, and the third conductive linker is permanently attached at a second end to the second electrode.

32. The nucleic acid detection apparatus of claim 25, wherein the gap is less than 50

nanometers.

33. The nucleic acid detection apparatus of claim 25, wherein the gap is greater than 100 nanometers.

34. A nucleic acid detection apparatus comprising: electrode pairs, each electrode pair comprising: a first electrode; a second electrode; an insulator between the first electrode and the second electrode, the insulator comprising a channel providing a gap between the first electrode and the second electrode; and at least one bridge between the first electrode and the second

electrode extending across the insulator, the bridge comprising at least one conductive linker having a length,

a first end and a second end, wherein the conductive linker is made of non-nucleic acid material, the first end

of the conductive linker is attached to the first electrode

and the second end of the conductive linker is attached to

the second electrode; and

{00651183.DOCX 7} at least one nucleic acid strand permanently attached to the conductive

linker along the length of the conductive linker.

35. The nucleic acid detection apparatus of claim 34, comprising a plurality of nucleic acid strands permanently attached to the conductive linker along the length of the conductive linker.

36. The nucleic acid detection apparatus of claim 35, wherein the plurality of nucleic acid strands do not have identical sequences.

37. The nucleic acid detection apparatus of claim 35, wherein the plurality of nucleic acid strands have identical sequences.

38. The nucleic acid detection apparatus of claim 35, wherein the conductive linker is made of a material selected from the group consisting of gold, a single-walled carbon nanotube, Polyaniline, Polypyrole, Polyethylenedioxythiophene,

Polythiophenes, Polycarbazole, Polyacetylene, and Poly(p-phenylene vinylene) and polysilicon.

39. The nucleic acid detection apparatus of claim 35, wherein the gap has a uniform

length.

40. The nucleic acid detection apparatus of claim 35, wherein the gap is less than 50

nanometers.

41. The nucleic acid detection apparatus of claim 35, wherein the gap is greater than 100 nanometers.

42. The nucleic acid detection apparatus of claim 35, wherein the nucleic acid strands each have approximately 100 base pairs.

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