[0001] This application claims the benefit under 35 U.S.C. § 119(e) to

U.S. Provisional Application No. 62/267,529, filed December 15, 2015, the entire contents of which are incorporated herein by reference.

BACKGROUND

[0002] Viral load or viral titer is a measure of the severity of a viral infection, and can be calculated from the amount of virus in a body fluid. Tracking viral load can be used for monitoring the course of viral infections, monitoring a therapy during viral infections, and can also help to determine if someone is or was recently infected. Indeed, viral load itself is an important indicator for initiation of HIV or HCV treatments. Viral load testing can be categorized into three types, (1) nucleic acid amplification-based tests (NATs or NAATs), some of which are commercially available in the United States with Food and Drug Administration (FDA) approval, or on the market in the European Economic Area (EEA) with the CE-mark; (2) "homebrew" or in-house NATs; and (3) non-nucleic acid-based tests.

[0003] The current standard for viral load measurement uses a multi-step, laborious process based on polymerase chain reaction (PCR). Briefly, RNA is extracted from a body fluid sample and processed to be ready for quantitative amplification. The process includes, for instance, reverse transcription and basal level amplification. Finally, quantitative amplification is performed in a standardized qPCR equipment. For example, HIV RNA levels are measured by the amount of used tagged-HIV primers and used to calculate the amount of virus. Roche and Abbott have commercialized this type of tests, e.g. , Amplicor™ A modified version of the test, which is not as widely used, is a bDNA (branched DNA) method. The bDNA method measures the level of HIV in infected cells. This test has been commercialized by Bayer. A still more recently developed method, based on a modification of the previously described methods, is NASBA (nucleic acid sequence based

amplification). NASBA (manufactured by bioMerieux) amplifies viral proteins to derive a count. [0004] All of these methods suffer from similar problems, including the length of time required to carry out the methods, the requirement for extensive human labor, and the lack of immediate results. Moreover, the current methods cannot be applied in any resource-limited settings, and blood samples typically must be processed and shipped to a certified reference laboratory for analysis. These problems impact patient care. For example, a patient does not receive viral load results immediately, and often must return to the doctor's office or health care facility days later, delaying diagnosis and/or treatment. Along the same lines, the physician or health care provider cannot readily know whether the patient has been exposed to or infected with the virus or whether a current treatment is effective such as may be indicated by a decreased viral load, or whether a current treatment is not effective such as may be indicated by an increased viral load. These delays can have a significant clinical impact where, for example, a given course of therapy is time-sensitive, such as postexposure phrophylaxis that is indicated within hours of HIV exposure, or where patients cannot easily return to the doctor's office or health care facility for follow-up. This is a particular problem in countries with limited resources, and impedes the implementation of available treatments.

[0005] Thus, there is a need for improved methods detecting and quantitating target polynucleotides in a physiological samples.

SUMMARY

[0006] Described herein are devices and methods for detecting and quantitating a target polynucleotide in a sample. In one embodiment, provided is a probe for a target nucleotide sequence comprising a carbon nanotube labeled with a probe nucleotide sequence complementary to the target sequence, wherein the probe nucleotide sequence comprises a plurality of guanine nucleotide bases.

[0007] In some aspects, the probe nucleotide sequence is bound via a biotin-avidin or biotin-streptavidin linkage to a surface of the carbon nanotube. In some aspects, the probe nucleotide sequence is conjugated to a biotin moiety that is bound to an avidin or streptavidin moiety on the surface of the carbon nanotube.

[0008] In some aspects, the target nucleotide sequence is a viral DNA or RNA. In some aspects, the viral DNA or RNA is from HIV, a hepatitis virus or an influenza virus. In some aspects, the viral DNA or RNA is from HIV. In some aspects, the target nucleotide sequence is a bacterial DNA or RNA. In some aspects, the bacterial DNA or RNA is from tuberculosis, E. coli, or Chlamydia. In some aspects, the target nucleotide sequence is a nucleotide sequence specific to a tumor cell. In some aspects, the nucleotide sequence contains a mutation specific to the tumor cell or is overexpressed by the tumor cell (such as in comparison to a normal cell). In some aspects, the target nucleotide sequence is an HIV DNA or RNA sequence. In some aspects, the probe nucleotide sequence comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-3. In some aspects, the target nucleotide sequence is a fungal DNA or RNA.

[0009] Also provided, in one embodiment, is an electrode comprising a plurality of probes as provided in the present disclosure. In some aspects, the electrode is a graphite electrode or a reticulated vitreous carbon foam.

[0010] In a still further embodiment, provided is a device comprising a housing containing an electrode as described herein, connected to a second electrode, wherein the housing is configured for retaining an electrolyte solution such that the first and second electrodes are at least partially immersed in the electrolyte solution and in electric communication during use.

[0011] In some aspects, the first and second electrodes are connected via a potentiostat or a galvanostat.

[0012] In some aspects, the housing further contains a reference electrode connected to the first and second electrodes disposed such that the reference electrode is at least partially immersed in the electrolyte solution and in electric communication with the first and second electrodes during use.

[0013] In some aspects, the second electrode comprises platinum. In some aspects, the reference electrode comprises silver. In some aspects, the probe nucleotide sequence is from about 10 to about 25 nucleotides long.

[0014] In some aspects, the nanotube is multi-wall functionalized. In some aspects, the nanotube has an outer diameter of about 7 nm to about 15 nm. In some aspects, the nanotube has an inner diameter of about 3 nm to about 6 nm. In some aspects, the nanotube is from about 0.5 nm to about 100 nm in length.

[0015] Another embodiment of the present disclosure provides a method for detecting a target nucleotide sequence in a liquid sample, comprising: incubating a first electrode in the liquid sample, wherein the first electrode comprises a probe for the target nucleotide sequence, each probe comprising a carbon nanotube labeled with a probe nucleotide sequence complementary to the target sequence, wherein said probe nucleotide sequence comprises a plurality of guanine nucleotide bases;

connecting the first electrode to a second electrode in a device comprising a housing and an electrolyte solution, such that the first and second electrodes are at least partially immersed in the electrolyte solution and in electric communication with each other; and determining the conductivity of the first electrode, wherein a decrease in conductivity of the first electrode relative to a negative control indicates the presence of the target nucleotide sequence in the liquid sample.

[0016] In some aspects, the liquid sample comprises a bodily fluid, such as plasma, from a subject. In some aspects, the subject is suspected of having been exposed to a virus or bacterium, or suspected of having cancer. In some aspects, the subject is suspected of having been exposed to HIV, a hepatitis virus, an influenza virus, M. tuberculosis, E. coli, or Chlamydia, or is suspected of having cancer. In some aspects, the subject is suspected of having been exposed to HIV. In some aspects, the subject is undergoing treatment for HIV infection. In some aspects, the liquid sample comprises fewer than 50,000 copies/ml of the target nucleotide sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1A-E illustrate methods and devices for detecting target

polynucleotides as described herein.

[0018] FIG. 2 presents data showing that binding of polynucleotides (e.g., viral RNA) to the probes described herein changes the potentials between electrodes.

[0019] FIG. 3 presents data showing the correlation between polynucleotide concentration and the change of potential.

[0020] FIG. 4A-B show the signals detected for a positive control and a testing virus with a carbon foam on the electrode. DETAILED DESCRIPTION

[0021] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented here.

[0022] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0023] As used herein, "about" will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, "about" will mean up to plus or minus 10% of the particular term.

[0024] Described herein are devices and methods for detecting target

polynucleotides in a sample, such as a physiolaogical sample from a subject, such as a blood sample. The target polynucleotides can be any polynucleotides of interest, such as any polynucleotides of diagnositc interest, or any polynucleotide associated with a disese or condition, with risk of developing a disese or condition, or with progression or remission of a disese or condition. In some embodiments, the target polynucleotide is associated with a pathogenic organism, such as a disease-causing or contamination organism, such as a virus or bacterium or fungus (e.g. , HIV, hepatitis, influenza, tuburculosis, E. coli, Chlamydia , etc.), or with a tumor (such as DNA or RNA specific to a tumor cell, such as having a mutation specific to the tumor cell or being overexpressed by the tumor cell). When the target polynucleotide is associated with a pathogenic organism associated with a disease state, the devices and methods described herein can be used to quickly diagnosis disease and/or provide information as to disease state. When the target polynucleotide is associated with a pathogenic organism associated with a contaminating organism, the devices and methods described herein can be used to quickly identify whether a sample is contaminated and/or provide information as to the level of contamination. When the the target polunucleotide is associated with a tumor, the devices and methods described herein can be used to quickly diagnosis the cancer and/or provide information as to progression or regression thereof.

[0025] The devices described herein include a probe for a target nucleotide sequence (a target polynucleotide) comprising a carbon nanotube labeled with a probe nucleotide sequence that is complementary to the target nucleotide sequence. For example, the probe nucleotide sequence may be bound via a biotin-avidin or biotin- streptavidin linkage to a surface of the carbon nanotube. A plurality of such probes may be provided on an electrode, such as a graphic electrode. Such an electrode may be used in a device, such as a device as generally depicted in FIG. 1A-E. For example, a device may comprise a housing that contains such an electrode, connected to a second electrode, such that the electrodes can be at least partially immersed in an electrolyte solution and an in electric communication during use.

[0026] The methods described herein involve incubating an electrode or probe as described herein in a liquid sample, such as a liquid sample suspected of containing the target nucleotide sequence (the target polynucleotide), such as a physiological sample from a subject or a liquid sample obtained or prepared from a material suspected of being contaminated. Binding of target polynucleotide from the sample to the probe alters the conductivity of the electrode. For example, binding of target polynucleotide from the sample to the probe may decrease its conductivity, thus permitting detection and/or quantification of target polynucleotide present in the sample. This method can be very sensitive, as the binding of a small number of target nucleotide sequences can lead to a detectable change in conductivity.

[0027] Exemplary embodiments of the devices and methods are described herein with reference to the figures.

[0028] FIG. 1 illustrates one embodiment of the devices and methods. FIG. 1A shows a nanotube (101) labeled with (i.e., linked to) a probe nucleotide sequence (104). The probe nucleotide sequence can be linked to the nanotube directly or through a linker. In FIG. 1A, the probe nucleotide sequence 104 is attached (e.g., conjugated) to a biotin moiety (103), which can bind to a streptavidin moiety (102) that is covalently attached to the nanotube 101. Using a linkage that can be separated or cleaved to label the nanotube with the probe nucleotide sequence, such as biotin/ streptavidin linkage or any other binding pairs or cleavable linkage, allows removal and/or replacement of the probe nucleotide sequence, so that the nanotube itself can be reused. Thus, while FIG. 1A illustrates an embodiment with a biotin/ streptavidin linkage, also included within the scope of the devices and methods described herein are embodiments where the nanotube is labeled with the probe nucleotide sequence via any binding pair or linkage, including binding pairs and linkages that can be separated or cleaved to release the probe nucleotide sequence from the nanotube, and, optionally, can to permit subsequent labeling of the nanotube with a different probe nucleotide sequence. Other examples of such binding pairs or linkages include, without limitation, antibody and antigen.

[0029] As illustrated in FIG. IB, a plurality of nanotubes can be attached to an electrode, to provide an electrode probe (or "test probe"), as illustrated as 105 in FIG. IB. In some embodiments, the surface area of the electrode is enhanced or increased by employing, for instance, metal foam, steel wool, or nets (see FIG. 1C), in order to permit attachment of a larger number of nanotubes.

[0030] As illustrated in FIG. ID, a test electrode probe 110 as described herein can be immersed in a liquid sample (111) known or suspected to include a target nucleotide sequence ("target polynucleotide") complementary to the probe nucleotide sequence, such that nucleotide sequences present in the sample will bind to probe nucleotide sequence on the electrode, resulting in an electrode having target polynucleotides bound thereto. Electrodes having target polynucleotides bound thereto have a different conductivity than electrodes not having target polynucleotides bound thereto.

[0031] As illustrated in FIG. IB and FIG. IE, a test electrode can be used in a device to detect and/or quantitate target polynucleotide present in the sample, based on an assessment of the conductivity of the test electrode. A device may include a housing (shown as 114 in FIG. IE) capable of containing an electrolyte solution (shaded areas in FIG. IB and IE), such as a buffer. The device and components are configured such that when the test electrode is inserted into the device, at least part of it can be at least partially immersed in electrolyte solution. The device also includes a counter electrode (107) that also can be at least partially immersed in the electrolyte solution, and that can be electrically connected to the test electrode, optionally through a meter (109).

[0032] Once the elements of the electric circuit of the device are connected, a potential is generated due to oxidation of nucleotide bases of the probe and/or target nucleotide sequences, such as oxidation of guanine bases present in the probe nucleotide sequence and/or the target nucleotide sequence. A meter can be used to detect and measure the potential.

[0033] The potential generated by such oxidation depends, for example, on the number of guanine bases present, which in turn depends on the specific

polynucleotides present, and also depends on whether the probe nucleotide sequence is bound to a target nucleotide sequence. Therefore, a test electrode that does not have target polynucleotide bound to its probe polynucleotides will generate a different potential from a test electrode probe that does have target polynucleotides bound to its probe polynucleotides. Moreover, the potential also depends on how many target nucleotide sequences are bound to probe nucleotide sequences, and so provides information on the quantity of target polynucleotides in the sample. Thus, the measured potential can provide a qualitative and quantitative assessment of the presence and amount of target polynucleotide present in the sample. Therefore, the present devices and methods are capable of detecting and quantitating target nucleotide sequence in the sample, to give an indication of viral load, disease state or progression, contamination, etc.

[0034] Optionally, a device as described herein can further include a reference electrode (shown as 108 in FIG. IB). As readily appreciated by the skilled artisan, the reference electrode can improve the sensitivity and/or reliability of the device with regard to detection and measurement of the potential changes.

[0035] Further details of specific aspects of the device and methods described herein are provided below.

Nanotubes

[0036] Nanotubes are typically, but not necessarily or exclusively, carbon molecules and have properties that make them potentially useful in a wide variety of applications in electronics, optics, optoelectronics, biological sensing and drug delivery. They exhibit extraordinary strength and excellent electrical properties. The name is derived from their size, since the diameter of a nanotube can be on the order of a few nanometers (approximately 50,000 times smaller than the width of a human hair), while they can be up to several centimeters in length. There are two main types of nanotubes: single- walled carbon nanotubes (SWCNTs) and multi -walled carbon nanotubes (MWCNTs). Bulk synthesized nanotubes naturally group into "ropes" due to strong Van der Waals forces.

[0037] As used herein the term "nanotube" refers to a cylindrical tubular structure of which the most inner diameter size lies between 0.5 nm and 1000 nm. Various types of nanotubes can be used in the devices disclosed herein. In some aspect, the nanotubes are semiconducting nanotubes, such as carbon nanotubes.

[0038] In some aspects, the outer diameter of a nanotube is not greater than about 1000 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 19 nm, 18 nm, 17 nm, 16 nm, 15 nm, 14 nm, 13 nm, 12 nm, 1 1 nm, or 10 nm. In some aspects, the outer diameter of the nanotube is greater than about 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 1 1 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, or 100 nm. In some aspects, the outer diameter of the nanotube is from about 3 nm to about 100 nm, or from about 4 nm to about 50 nm, or from about 5 nm to about 30 nm, or from about 5 nm to about 25 nm, or from about 6 nm to about 20 nm, or from about 7 nm to about 15 nm.

[0039] In some aspects, the inner diameter of a nanotube is not greater than about 200 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 19 nm, 18 nm, 17 nm, 16 nm, 15 nm, 14 nm, 13 nm, 12 nm, 1 1 nm, or 10 nm. In some aspects, the inner diameter of the nanotube is greater than about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 1 1 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, or 100 nm. In some aspects, the inner diameter of the nanotube is from about 0.5 nm to about 50 nm, or from about 1 nm to about 30 nm, or from about 2 nm to about 10 nm, or from about 3 nm to about 6 nm.

[0040] In some aspects, the length of the nanotube is not greater than about 1000 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 19 nm, 18 nm, 17 nm, 16 nm, 15 nm, 14 nm, 13 nm, 12 nm, 11 nm, or 10 nm. In some aspects, the outer diameter of the nanotube is greater than about 0.5 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 40 nm, 50 nm, or 100 nm. In some aspects, the length of the nanotube is from about 0.5 nm to about 500 nm, or from about 0.5 nm to about 200 nm, or from about 1 nm to about 100 nm.

[0041] In one embodiment, the nanotubes are carbon nanotubes. A "carbon nanotube" is a member of the fullerene structural family, which also includes buckyballs. Whereas buckyballs are spherical in shape, a carbon nanotube is cylindrical, with at least one end typically capped with a hemisphere of the buckyball structure. Carbon nanotubes are composed primarily or entirely of sp2 bonds, similar to those of graphite. This bonding structure, stronger than the sp3 bonds found in diamond, provides the molecules with their unique strength. Single-walled CNTs (SCNT) or multi-walled CNTs (MCNTs) are examples of well-studied CNT structures.

[0042] Carbon nanotubes are generally produced by three main techniques, arc discharge (Iijima et al. Nature, 1991, 354, 56-58 and Ebbesen et al. Nature 1992, 358, 220-222), laser ablation (Anazawa et al. U.S. Pat. No. 7,132,039, Guo et al. Chemical Physics Letters, 1995, 243, 49-54, A. Thess et al. Science 1996, 273, 483-487) and chemical vapor deposition (Lee, et al. U.S. Pat. No. 6,350,488; Hirakata et al. U.S. Pat. No. 6,936,228, Resasco, et al. U.S. Pat. No. 6,333,016, Awasthi et al. J. Nanosci. Nanotechnol. 2005, 5(10), 1616-36). The arc discharge method involves a carbon vapor as a precursor to CNTs that is created by an arc discharge between two carbon electrodes. The carbon nanotubes can be synthesized from the resulting carbon vapor. Laser ablation technique involves a high-power laser beam coming in contact with a volume of carbon-rich gas (methane or carbon monoxide, for example). Laser ablation produces a small amount of clean nanotubes, whereas arc discharge generally produces large quantities of impure CNTs. Chemical vapor deposition (CVD) often utilizes a catalyst nanoparticle and two gases which are bled into the reactor: a process gas (such as ammonia, nitrogen, hydrogen, etc.) and a carbon-containing gas (such as acetylene, ethylene, ethanol, methane, etc.). Nanotubes grow at the sites of catalyst nanoparticle as the carbon-containing gas is broken apart. It is generally believed that oversaturated carbon is diffused to the edges of the catalyst particle wherein this leads to the growth of CNTs. The catalyst particles either stay at the tips of the growing nanotube during the growth process, or remain at the nanotube base, depending on the adhesion between the catalyst particle and the substrate on which the catalyst particle is disposed. Therefore, in order to control the diameter and location of the CNTs in general, one must use uniformly sized catalyst particles at predefined locations.

[0043] Single-walled nanotubes exhibit important electric properties that are not shared by the multi -walled carbon nanotube (MWNT) variant. Semiconducting single-walled carbon nanotubes, exhibiting ballistic electron transport properties and exceptionally high current carrying capability, are direct bandgap materials (Javey, et al. Nature, 2003, 424, 654-657, Moon, et al. Nanotechnology, 2007, 18, 235201, Dresselhaus, et al. "Carbon Nanotubes", Springer, Berlin, 2001, Itkis, et al. Science, 2006, 312(5772), 413-416, Connell, et al. Science, 2002, 297, 593-596). Additionally, the spectral absorption range spans from the visible to the infrared (Javey, et al, supra, Dresselhaus, et al, supra, Hagen, et al. Nano Letters, 2003, 3, 383-388, Misewich, et al. Science, 2003, 300, 783-786). A recent study has indicated that the absorption coefficient of CNTs is extremely high, at least one order of magnitude greater than that of mercury cadmium telluride, the most popular photoconductor for 2D arrays of IR photodetectors (Saito, et al. "Physical Properties of Carbon

Nanotubes", Imperial College, London, 1998). This has led to widespread

investigation of the use of CNTs for optoelectronic applications (Saito, et al, supra, Freitag, et al. Nano Letters, 2003, 3, 1067-1071, Lee J. U. Applied Physics Letters, 2005, 87, 073101, Wei, et al. Nano Letters, 2007, 7, 2317-2321, Chen, et al. Science, 2005, 310(5751), 1171-1174).

[0044] In some embodiments, the carbon nanotubes are functionalized. Methods of functionalizing nanotubes are well known in the art. See e.g. , Bhattacharyya et al, Nanotechnology, 23 (2012); 385304 (8 pp).

Electrodes and Meters

[0045] Electrodes useful in accordance with the devices and methods described herein, including the test electrode, counter electrode and reference electrode, can be obtained commercially or prepared using methods and materials known in the art. For instance, the electrode probe can be made of graphite or reticulated vitreous carbon form (2-10% density), 50-200 PPI (parts per inch) or larger, without limitation. In some embodiments, the counter electrode is, for instance, prepared with platinum. In some embodiments, the reference electrode is, for instance, prepared with silver chloride.

[0046] Any meter than can be used to measure current change, potential change, or conductivity change can be used in accordance with the devices and methods described herein. In one aspect, the meter is a potentiostat. In another aspect, the meter is a galvanostat. In another aspect, the meter measures a current with optionally an external voltage source.

Nucleotide Sequences

[0047] A probe nucleotide sequence is typically a single-stranded polynucleotide sequence, and may be a RNA sequence or a DNA sequence, or any other type of nucleotide-based sequence, including a PNA sequence (a peptide nucleic acid sequence). In some aspects, the probe consists of from about 5 nucleotides to about 100 nucleotides, or from about 7 nucleotides to about 50 nucleotides, or from about 10 nucleotides to about 30 nucleotides or from about 15 nucleotides to about 25 nucleotides.

[0048] A probe nucleotide sequence is designed and/or selected to be

complementary to the target nucleotide sequence, e.g. , to have sufficient

complementarity to the target nucleotide sequence such that target nucleotide sequence will bind to probe nucleotide sequence on the electrodes, and will remain bound thereto during use in the methods described herein. While in some embodiments the probe nucleotide sequence may be 100% complementary to the target nucleotide sequence, such a high degree of complementarity is not necessary. Those skilled in the art can design suitable probe nucleotide sequences based on the target nucleotide sequence to, e.g. , cover a range of sequence diversity and the conditions being used.

[0049] As noted above, the target polynucleotides can be any polynucleotides of interest, such as any polynucleotides of diagnositc interest, or any polynucleotide associated with a disese or condition, with risk of developing a disese or condition, or with progression or remission of a disese or condition. In some embodiments, the target polynucleotide is associated with a pathogenic organism, such as a disease- causing or contamination organism (e.g. , HIV, hepatitis, influenza, tuburculosis, E. coli, etc.), or with a tumor. When the target polynucleotide is associated with a pathogenic organism associated with a disease state, the devices and methods described herein can be used to quickly diagnosis disease and/or provide information as to disease state. When the target polynucleotide is associated with a pathogenic organism associated with a contaminating organism, the devices and methods described herein can be used to quickly identify whether a sample is contaminated and/or provide information as to the level of contamination. When the the target polunucletodie is associated with a tumor, the devices and methods described herein can be used to quickly diagnosis the cancer and/or provide information as to progression or regression thereof.

[0050] In some aspects, the probe nucleotide sequence has a guanine content of at least about 1/12 based on the total number of nucleotide bases in the sequence, such as at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% , or at least 50% guanine bases. In some aspects, the probe has one or more guanine-rich regions. As used herein, a "guanine-rich region" refers to a short span of the nucleotide sequence (e.g. , 3-6 nucleotides) at least 50% of which is comprised of guanine bases (e.g., 2 or 3 out of 3; 2, 3 or 4 out of 4; 3, 4, or 5 out of 5, or 3, 4, 5, or 6 out of 6).

[0051] Some examples of suitable probe nucleotide sequences for detecting a target HIV nucleotide sequence are set forth in Table 1 below.

Samples

[0052] In some aspects, the sample comprises a physiological sample from a subject, such as a human or animal subject, such as blood, plasma, saliva, cerebrospinal fluid, or tissue. In some aspects, the sample comprises a sample of a material suspected of being contaminated, such as a material intended for human or animal consumption or a material intended to be administered to a human or animal for diagnostic, therapeutic, prophylactic, or nutritive purposes, such as a food or medical product. In some aspects, the sample comprises a long-term cultured cell line. [0053] In some aspects, the subject is a human or animal subject known or suspected to have been exposed to the pathogenic microorganism associated with the target polynucleotide (e.g., HIV, hepatitis, influenza, tuburculosis, E. coli, etc.). In some aspects, the subject is undergoing treatment for infection by the pathogenic microorganism associated with the target polynucleotide. In some aspects, the subject is a human or animal subject known or suspected to have a tumor associated with the target polynucleotide, and/or is undergoing treatment for cancer associated with a tumor associated with the target polynucleotide.

Qualitative and Quantitative Methods

[0054] As set forth above, the devices and methods described herein are useful for detecting and quantitating a target nucleotide sequence present in a sample, and can do so at high sensitivity, to detect and/or quantitate target polynucleotide present at relatively low concentration or abundance.

[0055] The present technology is highly sensitive in detecting target nucleotide sequences. In one aspect, a target nucleotide sequence can be detected and/or quantitated at a concentration of the target nucleotide sequence in a sample lower than about 100,000 copies/mL, or lower than about 50,000 copies/mL, or lower than about 40,000 copies/mL, or lower than about 30,000 copies/mL, or lower than about 20,000 copies/mL, or lower than about 10,000 copies/mL, or lower than about 5,000 copies/mL, or lower.

EXAMPLES

[0056] The devices and methods described herein are further illustrated by the following examples, which should not be construed as limiting in any way.

Example 1

Electrode Preparation

[0057] One milligram carbon nanotubes (CNTs) are weighed out and dissolved in 1 mL of PBS in a 1.7 mL Eppy tube. 1,2-dichloroethane (EDC) (-20° Molecular Bio lab) is dissolved in PBS to obtain 0.4 mg of EDC solution to be added to the CNT solution. Likewise, 0.6 mg of N-Hydroxysuccinimide (NHS) is added.

[0058] This mixture is sonicated for 45 min by floating the Eppy tube on a foam pad in water to activate carboxylic groups/NHS ester. An 110 μΐ aliquot of the resulting activated CNT solution is added into each of a plurality of reaction tubes and an untreated pencil lead is immersed in each for 1 hr.

[0059] A solution of 20 μg/ml Streptavidin (LL-Strep) is prepared in PBS. The CNT-PGE(pyrolytic graphite electrode) prepared as described above is rinsed with PBS for 10 seconds by placing the PGE in 110 μΐ of PBS, and then inverted to dry for 5 minutes. The CNT-PGE is immersed in 110 μΐ of the streptavidin (STR) solution for 3 hours. Unbound streptavidin is removed by rinsing the electrode with 110 μΐ PBS for 10 seconds, and then the electrode is inverted to dry for 5 minutes.

[0060] Biotinylated probes (such as BS01-BS04 in Table 1) are prepared, and a solution of 150 μg/mL biotinylated probe inABS buffer (0.5M acetate, 20mM NaCl, pH 4.8) is prepared. Electrode prepared as described above (CNT-PGE-STR electrode) is immersed in 110 μΐ of the 150 μg/mL biotinylated probe/ ABS solution for 30 minutes, and the electrode is rinsed for 10 seconds with 110 μΐ ABS and inverted to dry for 5 minutes. Due to the binding between the biotin moieties on the probes and streptavidin moieties on the CNTs of the electrodes, the probe sequences readily attached to the electrodes. This results in electrodes comprising CNTs labeled with probe nucleotide sequences.

Hybridization

[0061] In general, the prepared electrode is immersed in a test solution in a reaction tube for a period of time, such as from 5 minutes to 1 hour, such as 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, or one hour. For experimental purposes, the test solution may contain 50,000 virus particles in a Tris-HCl buffer solution (20mM Tris-HCl, 20mM NaCl, pH 7.0) (TBS) with 0.1% Tween 20, and the prepared electrode may be immersed in 110 μΐ virus/TBS solution for 1 hr.

Additionally or alternatively, test solutions are prepared containing target HIV RNA (sonicated NL4-3), positive control polynucleotides, or negative control

polynucleotides (as shown in Table 1) at concentrations equivalent to a viral load of -100,000 HIV RNA copies/ml, in ABS, and provided in a reaction tube into which a prepared electrode is immersed for 5-10 minutes. The hybridized electrode is then thoroughly rinsed in TBS buffer. Assay

[0062] An electric circuit is created by immersing a test electrode (prepared as described above), a counter platinum (Pt) electrode and an Ag/CL reference electrode in a buffer solution, and connecting the electrodes electrically to each other and to a potentiostat (FIG. IB). The potentiostat is set up such that a working electrode clamp is attached to the test electrode, the reference electrode clamp is attached to the Ag/Cl electrode, and the counter electrode clamp is attached to the Pt electrode. Also, it is ensured that the Pt wire and PGE are both submersed sufficiently in TBS solution and not touching. The reference electrode is kept as close as possible to the reaction tube.

[0063] DPV (differential pulse voltammetry) measurements are performed in ABS by scanning in a range of +0.50V to + 1.40V w/ 50mV modulation amp and 8mV step potential.

[0064] The strength of the signals on the potentiostat are caused at least in part by oxidation of guanine bases present in the nucleotide sequences.

Probes

[0065] The probes (BS01-BS04 in Table 1) were designed to contain guanine-rich regions and target HIV RNA. ("bio" designates biotinylated probes)

Table 1. Oligonucleotide probe sequences

[0066] Results using electrodes labeled with the probes BS01-BS04 set forth in Table 1 are shown in FIG 2. As shown, electrodes bound to target HIV RNA (Virus-1) or positive control nucleotide sequence (POS-1) exhibited quantitatable potential shifts as compared to electrodes incubated with negative control polynucleotides (NEG-1), with conductivity (measured in Amp) lower in the viral samples as compared to the negative control for each of the probe nucleotides tested.

[0067] As shown in FIG. 3, higher levels of target polynucleotide (e.g. , viral RNA) resulted in a stronger decrease in conductivity, but the method was able to detect target polynucleotide (e.g., viral RNA) at a low concentration.

Example 2

[0068] This example tests the use of carbon foam on the surface of an electrode which is useful in attaching carbon nanotubes. The experimental procedure was similar to that used in Example 1. The carbon foam used was made by ERG

Aerospace Corporation (Emeryville, CA) and the nanotubes were obtained from Sigma-Aldrich.

[0069] FIG. 4A-B show the results of the testing. The positive control showed a significant signal peak around 0.9 volts, and the detection of a testing virus was also apparent even though the magnitude of signal was slightly lower compared to the positive control.

* * *

[0070] The present invention is not to be limited in terms of the particular embodiments described in this application, which are intended as illustrations of specific aspects of the invention. Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses to those enumerated herein will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the invention. Thus, it is to be understood that this invention is not limited to particular methods, reagents, compounds compositions or biological systems described herein, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.