WO2025054393A1 - Method and device for detecting diseases, pathogens and parasites - Google Patents

Abstract

The disclosure describes a method and device for detecting diseases, pathogens and parasites. The method and device utilize bio-nanosensors comprising carbon nanotubes, where hybridization of polynucleotide probes (e.g., cDNAs) on the carbon nanotubes to a target polynucleotide (e.g, an mRNA or a non-coding RNA such as a miRNA) or nucleic acid sequence associated with a disease, a pathogen or a parasite generates electrical signals (e.g., voltage) that are processed and analyzed to detect the absence or presence of the disease, the pathogen or the parasite. The method and device can also quantify the level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in the sample. The method and device can provide diagnosis and prognosis of diseases and infections.

Description

Method and Device for Detecting Diseases, Pathogens and Parasites

Cross-Reference to Related Applications

This application claims priority to U.S. Provisional Application Ser. No. 63/581,333 filed September 8, 2023, which is incorporated by reference herein in its entirety.

Statement of Government Support

[0001] This invention was made with government support under Grant No. NIH-RADx- 12543 awarded by the National Institutes of Health. The government has certain rights in the invention.

Background of the Disclosure

[0002] Early diagnosis of diseases, pathogens and parasites can play an important role in treatment planning, medical care and outcome. Detection of polynucleotides such as messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs, such as microRNAs [miRNAs] and long non-coding RNAs [IncRNAs]) associated with diseases, pathogens and parasites, and quantification of the levels of such polynucleotides, pathogens and parasites, would be very useful in the diagnosis and prognosis of diseases. Non-coding RNAs were long regarded as “junk” transcripts until the discovery that many of them regulate the expression of many genes or proteins, regulate many cellular processes and functions, and play an important role in many diseases.

[0003] As an example, miRNAs play an important role in the development and regulation of cancers. For instance, in the carcinogenic process, tumor suppressor genes are downregulated by oncogenic miRNAs, whereas the expression of oncogenes is increased due to reduced levels of tumor suppressor miRNAs. Early diagnosis of cancers and timely and informed treatment of cancers have a great impact on the health and survival of cancer patients. Most cancer diagnoses are currently based on tissue biopsies, liquid biopsies including blood tests for tumor markers such as CEA and CA19-9, and imaging techniques such as computerized tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET) and ultrasound imaging. The current techniques have limitations, including the invasiveness of obtaining tissue biopsies, the low sensitivity and specificity of liquid biopsies, the difficulty of making early diagnosis with imaging techniques, and the high cost of imaging machines. Regarding the current methods for detecting miRNAs, quantitative PCR (qPCR) requires complicated steps for sample preparation, and microarray is expensive, has low sensitivity and specificity, and involves long processing time to obtain test results.

Summary of the Disclosure

[0004] The disclosure describes a method and device that can quickly detect diseases, pathogens and parasites with high sensitivity and specificity in a non-invasive, easy-to-use and low-cost manner. The method and device utilize bio-nanosensors comprising carbon nanotubes, where hybridization of polynucleotide probes (e.g., complementary DNAs [cDNAs]) on the carbon nanotubes to a target polynucleotide (e.g, mRNA, miRNA or DNA) or nucleic acid sequence associated with a disease, a pathogen or a parasite generates electrical signals (e.g., voltage) that are processed and analyzed to detect the absence or presence of the disease, the pathogen or the parasite. The method and device do not require amplification (e.g., by PCR) of the target polynucleotide or nucleic acid sequence, nor extraction of the target polynucleotide or nucleic acid sequence from cells or exosomes, although extracted samples can also be used. A sample, such as a biological sample (e.g., saliva or blood) or an environmental sample (e.g., a water sample), which may optionally be diluted in an aqueous solvent such as water (e.g., purified water), is simply added to the bio- nanosensors. The method and device can also quantify the level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in the sample. In addition to providing early diagnosis of the disease, the method and device can determine the status (e.g., the severity and stage) of the disease and the efficacy of the current treatment, and can monitor the progress of the disease and any resistance to the treatment, and thereby can provide prognosis of the disease and inform treatment of it. Additional testing with one or more other methods can be performed to confirm the diagnosis or prognosis provided by the method and device of the disclosure. The portable device can be used at a point of care (e.g., at a medical or veterinary facility) or in the field (e.g., at a mobile medical clinic or a farm).

Brief Description of the Drawings

[0005] A better understanding of features and advantages of the present disclosure will be obtained by reference to the following detailed description, which sets forth illustrative embodiments of the disclosure, and the accompanying drawings.

[0006] Fig. 1 shows an embodiment of the biodetection device 100 for detecting absence or presence of a disease, a pathogen or a parasite, wherein the biodetection device is connected to a laptop computer 190. [0007] Figs. 2A and 2B show that the addition of ultrapure water (UPW, negative control) and 1 :20 diluted pooled saliva to bio-nanosensors primed with a cDNA probe for synthetic miR-21 yielded little change in voltage, whereas the addition of synthetic miR-21 at 10⁷ copies/pL in UPW or 1:20 diluted pooled saliva to the cDNA-primed bio-nanosensors yielded significant changes in voltage.

[0008] Figs. 3A and 3B show that the addition of 1 :20 diluted individual saliva to bio- nanosensors primed with a cDNA probe for synthetic miR-21 yielded little change in voltage, whereas the addition of synthetic miR-21 at I O³ copies/pL or 10⁴ copies/pL in 1 :20 diluted individual saliva to the cDNA-primed bio-nanosensors yielded significant changes in voltage.

[0009] Figs. 4A and 4B show that the addition of synthetic miR-21 at 10⁴ copies/ L, 10⁷ copies/pL or 10¹⁰ copies/pL in 1 :20 diluted pooled saliva to cDNA-primed bio- nanosensors resulted in an increasingly greater average AV. Fig. 4C shows that the log values of the three different concentrations of synthetic miR-21 had an essentially linear correlation with the average AV.

[0010] Figs. 5A and 5B show that the addition of synthetic miR-21 at 10² copies/pL, 10³ copies/pL or 10⁴ copies/pL in 1:20 diluted individual saliva to cDNA-primed bio- nanosensors resulted in an increasingly greater average AV. The vertical rectangle in Fig. 5A indicates the portion of the plot where the AV was determined for each copy number of synthetic miR-21. Fig. 5C shows that the log values of the three different concentrations of synthetic miR-21 had a substantially linear correlation with the average AV.

[0011] Figs. 6A and 6B show that the addition of ultrapure water (UPW, negative control) to a bio-nanosensor primed with a cDNA targeting Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) RNA yielded little change in voltage, whereas the addition of a sample prepared by extraction of viral RNA from the serum of PRRSV-infected pigs to cDNA-primed bio-nanosensors resulted in a significant AV.

[0012] Fig. 7A shows that the addition of 1:20 diluted saliva from an uninfected subject to bio-nanosensors primed with a cDNA specific for SARS-CoV-2 RNA at around 350 seconds resulted in a reduction in voltage (or no change in voltage), which was deemed Negative, whereas Fig. 7B shows that the addition of 1 :20 diluted saliva from a subject infected with SARS-CoV-2 to cDNA-primed bio-nanosensors at around 350 seconds resulted in a significant increase in voltage, which was deemed Positive.

[0013] The green curve in Fig. 8 shows that the addition of 1:10 diluted saliva not spiked with SARS-CoV-2 RNA to bio-nanosensors primed with a cDNA specific for SARS-CoV-2 RNA resulted in a reduction in voltage or no change in voltage, which was deemed Negative, whereas the red curve in Fig. 8 shows that the addition of 1 :10 diluted saliva spiked with SARS-CoV-2 RNA to cDNA-primed bio-nanosensors resulted in a significant increase in voltage, which was deemed Positive.

[0014] Fig. 9 shows a 64 sensor array for use in the device of the present invention.

[0015] Fig. 10 shows a high throughput version of the device of the present invention.

[0016] Fig. 11A and Fig. 11B show detection of Potato Virus Y in a sample (Fig. 1 IB) compared to a control sample (no Potato Virus Y, Fig. 11 A).

Detailed Description of the Disclosure

[0017] While various embodiments of the present disclosure are described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications and changes to, and variations and substitutions of, the embodiments described herein will be apparent to those skilled in the art without departing from the disclosure. It is understood that various alternatives to the embodiments described herein, including materials and methods similar or equivalent to those described herein, may be employed in practicing the disclosure. It is also understood that every embodiment of the disclosure may optionally be combined with any one or more of the other embodiments described herein which are consistent with that embodiment.

[0018] Where a combination is disclosed, it is understood that each possible subcombination of the elements of that combination is also disclosed. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed.

[0019] Where elements are presented in list format or as alternative members of a group (e.g., a Markush group), it is understood that each possible subgroup of the elements is also disclosed, and any one or more elements can be removed from that list or group.

[0020] Where a range of values is recited, it is understood that each intervening integer value and each fraction thereof, as well as each subrange, between the recited upper and lower limits of that range are specifically disclosed. Where a value has an inherent limit, that inherent limit is specifically disclosed. Where a value is explicitly recited, it is understood that values which are about the same as the recited value are specifically disclosed. [0021] It is also understood that, unless clearly indicated to the contrary, in any method described or claimed herein that includes more than one act or step, the order of the acts or steps of the method is not necessarily limited to the order in which the acts or steps of the method are recited, but the disclosure encompasses embodiments in which the order is so limited.

[0022] It is further understood that, in general, where an embodiment in the description or the claims is referred to as comprising one or more features, the disclosure also encompasses embodiments that consist of, or consist essentially of, such feature(s).

[0023] It is also understood that any embodiment of the disclosure, e.g., any embodiment found within the prior art, can be explicitly excluded from the claims, regardless of whether or not the specific exclusion is recited in the specification.

[0024] Headings are included herein for reference and to aid in locating certain sections. Headings are not intended to limit the scope of the embodiments and concepts described in the sections under those headings, and those embodiments and concepts may have applicability in other sections throughout the entire disclosure.

[0025] All patent literature and all non-patent literature cited herein are incorporated herein by reference in their entirety to the same extent as if each patent literature or non-patent literature were specifically and individually indicated to be incorporated herein by reference in its entirety.

Definitions

[0026] Unless defined otherwise or clearly indicated otherwise by their use herein, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd Ed., John Wiley and Sons, New York (1994), and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, New York (1991), provide dictionary definitions of many terms used in the biotechnology art.

[0027] As used in the specification and the appended claims, the indefinite articles “a” and “an” and the definite article “the” can include plural referents as well as singular referents unless specifically stated otherwise or the context clearly indicates otherwise.

[0028] The terms “or/and” and “and/or” mean “either ... or . . ., or both . . . and . . .” when referring to two elements, and mean “either . . ., ... or . . ., or any combination or all thereof’ when referring to three or more elements. As an example, the phrase “A or/and B” means “either A or B, or both A and B”, and the phrase “A, B or/and C” means “either A, B or C, or any combination or all thereof’.

[0029] As used in the specification and the claims, all transitional terms such as “comprising”, “containing”, “having”, “including”, “composed of”, and the like are open- ended and inclusive, that is, mean including but not limited to and do not exclude additional, unrecited element(s) or method step(s). Only the transitional term “consisting of’ is closed, that is, excludes any additional, unrecited element or method step, and the transitional term “consisting essentially of” is semi-closed, that is, only allows inclusion of additional, unrecited element(s) or method step(s) that do not materially affect the basic and novel characteristic(s) of that particular embodiment.

[0030] The term “exemplary” as used herein means “serving as an example, instance or illustration”. Any embodiment or feature characterized herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments or features.

[0031] The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within one standard deviation. In some embodiments, when no particular margin of error (e.g., a standard deviation to a mean value given in a chart or table of data) is recited, the term “about” or “approximately” means that range which would encompass the recited value and the range which would be included by rounding up or down to the recited value as well, taking into account significant figures. In certain embodiments, the term “about” or “approximately” means within 10% or 5% of the specified value.

Whenever the term “about” or “approximately” precedes the first numerical value in a series of two or more numerical values or in a series of two or more ranges of numerical values, the term “about” or “approximately” applies to each one of the numerical values in that series of numerical values or in that series of ranges of numerical values.

[0032] Whenever the term “at least” or “greater than” precedes the first numerical value in a series of two or more numerical values, the term “at least” or “greater than” applies to each one of the numerical values in that series of numerical values.

[0033] Whenever the term “no more than” or “less than” precedes the first numerical value in a series of two or more numerical values, the term “no more than” or “less than” applies to each one of the numerical values in that series of numerical values. [0034] The term “medical conditions” (or “conditions” for brevity) includes diseases and disorders. The terms “diseases” and “disorders” are used interchangeably herein.

[0035] The term “subject” refers to an animal, including a mammal, such as a primate (e.g., a human, a chimpanzee or a monkey), a rodent (e.g., a rat, a mouse, a gerbil or a hamster), a lagomorph (e.g., a rabbit), a swine (e.g., a pig), an equine (e.g., a horse), a canine (e.g., a dog) or a feline (e.g., a cat). The terms “subject” and “patient” may be used interchangeably herein in reference to a subject/patient (e.g., a mammalian subject/patient such as a human subject/ patient) having a medical condition.

[0036] The term “polynucleotide” refers to a polymer composed of nucleotide units. Polynucleotides can contain naturally occurring nucleic acids (e.g., deoxyribonucleic acid [“DNA”] and ribonucleic acid [“RNA”]), or/and nucleic acid analogs. Polynucleotides containing one or more nucleic acid or nucleotide analogs are sometimes called “aptamers”. Nucleic acid or nucleotide analogs include without limitation those which have a non- naturally occurring base/nucleobase, have a sugar or non-sugar moiety other than 2’- deoxyribose or ribose, or engage in linkages with other nucleotides other than the naturally occurring phosphodiester bond, or a combination or all thereof. Non-limiting examples of nucleic acid or nucleotide analogs include xeno(biotic) nucleic acids (XNAs) having a backbone other than the naturally occurring sugar-phosphate backbone present in DNA or RNA (e.g., 2’-O-substituted ribonucleotides [e.g., 2’-O-methyl ribonucleotides and 2’-O-(2- methoxyethyl) ribonucleotides], cyclohexene nucleic acids [CeNAs], 2’-deoxy-2’- fluoroarabino nucleic acids [FANAs], glycol nucleic acids [GNAs], 1,5-anhydrohexitol nucleic acids [HNAs], locked nucleic acids [LNAs] (also called bridged nucleic acids [BNAs]), morpholino nucleic acids [MNAs], peptide nucleic acids [PNAs], and threose nucleic acids [TNAs]), 5-methylcytosine, 5-methyluracil, phosphorothioates/thiophosphates, phosphorodithioates, phosphoramidates, phosphorodiamidates, boranophosphates, phosphorotriesters, methylphosphonates, chiral-methyl phosphonates, and the like. DNA and RNA polynucleotides can be synthesized using a DNA or RNA polymerase or an automated DNA or RNA synthesizer. Polynucleotides containing nucleic acid analogs can be synthesized using, e.g., an engineered DNA or RNA polymerase that recognizes the nucleic acid analogs, a phosphoramidite strategy, or an automated peptide synthesizer in the case of PNAs. The term “nucleic acid molecule” typically refers to a larger polynucleotide. The term “oligonucleotide” typically refers to a shorter polynucleotide. In certain embodiments, an oligonucleotide contains no more than about 100 nucleotides. In some embodiments, when a polynucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes the corresponding, or the complementary, RNA sequence (i.e., A, U, G, C) in which “U” replaces “T”.

[0037] The term “cDNA” refers to a DNA sequence that is complementary to a target polynucleotide (e.g., an mRNA or a non-coding RNA such as a miRNA), in either singlestranded or double- stranded form. In some embodiments, a cDNA is single-stranded DNA (ssDNA).

[0038] The term “complementary” refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and furthermore the contact surface characteristics are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide. Complementarity of polynucleotides typically refers to C/G and A/T (or U in the case of RNA) base pairings between antiparallel DNA/DNA, DNA/RNA or RNA/RNA sequences. Thus, the polynucleotide whose sequence is 5’-TATAC-3’ is complementary to a polynucleotide whose sequence is 5’-GTATA-3’. A nucleotide sequence is “substantially complementary” to a reference nucleotide sequence if the sequence complementary to the subject nucleotide sequence is substantially identical to the reference nucleotide sequence.

[0039] The term “hybridizing specifically to”, “specific hybridization” or “selectively hybridize to” refers to the binding, duplexing or hybridizing of a polynucleotide (e.g., a nucleic acid molecule) preferentially to a particular nucleotide sequence under stringent conditions (e.g., highly stringent conditions) when that sequence is present in a mixture of (e.g., total cellular) DNA or/and RNA.

[0040] The term “stringent conditions” refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes, Part I, Chapter 2 in “Overview of principles of hybridization and the strategy of nucleic acid probe assays”, Elsevier (New York, 1993). In certain embodiments, highly stringent hybridization conditions are about 5 °C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. In certain embodiments, very stringent conditions are equal to the Tm for a particular probe.

Method for detecting diseases, pathogens and parasites

[0041] The disclosure describes a method and device that can quickly detect diseases, pathogens and parasites with high sensitivity and specificity in a non-invasive, easy-to-use and low-cost manner. The method and device utilize bio-nanosensors comprising carbon nanotubes, where hybridization of polynucleotide probes (e.g., complementary DNAs [cDNAs]) on the carbon nanotubes to a target polynucleotide (e.g, mRNA, miRNA or DNA) or nucleic acid sequence associated with a disease, a pathogen or a parasite generates electrical signals (e.g., voltage) that are processed and analyzed to detect the absence or presence of the disease, the pathogen or the parasite. The method and device do not require amplification (e.g., by PCR) of the target polynucleotide or nucleic acid sequence, nor extraction of the target polynucleotide or nucleic acid sequence from cells or exosomes, although extracted samples can also be used. A sample, such as a biological sample (e.g., saliva or blood) or an environmental sample (e.g., a water sample), or an agricultural or plant sample, which may optionally be diluted in an aqueous solvent such as water (e.g., purified water), is simply added to the bio-nanosensors. The method and device can also quantify the level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in the sample. In addition to providing early diagnosis of the disease, the method and device can determine the status (e.g., the severity and stage) of the disease and the efficacy of the current treatment, and can monitor the progress of the disease and any resistance to the treatment, and thereby can provide prognosis of the disease and inform treatment of it. Additional testing with one or more other methods can be performed to confirm the diagnosis or prognosis provided by the method and device of the disclosure. The portable device can be used at a point of care (e.g., at a medical or veterinary facility) or in the field (e.g., at a mobile medical clinic or a farm).

[0042] Some embodiments of the method relate to a method for detecting absence or presence of a disease, a pathogen or a parasite, comprising: contacting a bio-nanosensor with a sample (test sample), wherein the bio-nanosensor comprises carbon nanotubes, polynucleotide probes are associated with or bound to an outer surface of the carbon nanotubes, the polynucleotide probes are complementary to a target polynucleotide or nucleic acid sequence, and the target polynucleotide or nucleic acid sequence is associated with a disease, a pathogen or a parasite; measuring a change in an electrical property (e.g., voltage, electrical conductivity/ conductance, electric current flow, or electrical resistivity/resistance) resulting from contacting the bio-nanosensor with the sample; comparing the measured (test) change in the electrical property to a reference change in the electrical property resulting from contacting a bio-nanosensor with a sample (reference sample) from a subject known not to have the disease, the pathogen or the parasite, or a sample (reference sample) known not to have the pathogen or the parasite; and determining absence or presence of the disease, the pathogen or the parasite based on comparing the test and reference changes in the electrical property.

[0043] In some embodiments, a change in voltage (AV) resulting from contacting the bionanosensor with the test sample (test AV) is measured, the test AV is compared to a reference AV, and the absence or presence of the disease, the pathogen or the parasite is determined based on comparing the test AV to the reference AV.

[0044] In some embodiments, the reference AV is determined from contacting a plurality of bio-nanosensors with a plurality of samples from a plurality of subjects known not to have the disease, the pathogen or the parasite, or a plurality of samples known not to have the pathogen or the parasite.

[0045] In some embodiments, the target polynucleotide or nucleic acid sequence is associated with promotion of the disease and the test AV resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is greater than the reference AV, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the disease. In certain embodiments, the disease is a tumor or cancer and the target polynucleotide or nucleic acid sequence is an oncogenic nucleic acid sequence or polynucleotide (e.g., messenger RNA [mRNA] or non-coding RNA [ncRNA] such as microRNA [miRNA] or long non-coding RNA [IncRNA]).

[0046] In other embodiments, the target polynucleotide or nucleic acid sequence is associated with protection against or inhibition of the disease and the test AV resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is less than the reference AV, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the disease. In certain embodiments, the disease is a tumor or cancer and the target polynucleotide or nucleic acid sequence is a tumor suppressor nucleic acid sequence or polynucleotide (e.g., mRNA or ncRNA such as miRNA or IncRNA).

[0047] In further embodiments, the target polynucleotide or nucleic acid sequence is derived from the pathogen or is a host-derived polynucleotide whose level increases in response to infection with the pathogen, and the test AV resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is greater than the reference AV, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the pathogen. In other embodiments, the target polynucleotide or nucleic acid sequence is a host-derived polynucleotide whose level reduces in response to infection with the pathogen and the test AV resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is less than the reference AV, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the pathogen.

[0048] In additional embodiments, the target polynucleotide or nucleic acid sequence is derived from the parasite or is a host-derived polynucleotide whose level increases in response to infection with the parasite, and the test AV resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is greater than the reference AV, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the parasite. In other embodiments, the target polynucleotide or nucleic acid sequence is a host-derived polynucleotide whose level reduces in response to infection with the parasite and the test AV resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is less than the reference AV, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the parasite.

[0049] In other embodiments, the test AV is substantially similar to (e.g., within about 10% or 20% of) the reference AV, indicating absence of the disease, the pathogen or the parasite. [0050] In some embodiments, the method comprises: contacting a plurality of (e.g., about 4, 8, 16, 32, 64 or more) bio-nanosensors with the sample; comparing the test AV generated by each of the bio-nanosensors to the reference AV ; and determining presence of the disease, the pathogen or the parasite if comparing the test AV to the reference AV for a majority of the bio-nanosensors indicates presence of the disease, the pathogen or the parasite, or determining absence of the disease, the pathogen or the parasite if comparing the test AV to the reference AV for a majority or half of the bio- nanosensors indicates absence of the disease, the pathogen or the parasite.

[0051] In addition to measuring the change in voltage (AV), the method for detecting the absence or presence of a disease, a pathogen or a parasite can measure the rate of change in voltage, including the slope of the voltage vs. time curve and the steepness of such a slope.

[0052] In some embodiments, the polynucleotide probes are a plurality of a particular polynucleotide probe.

[0053] In other embodiments, the polynucleotide probes are a plurality of two or more different polynucleotide probes complementary to a particular target polynucleotide or nucleic acid sequence. Using multiple probes for the same target may increase the accuracy of the method.

[0054] In further embodiments, the polynucleotide probes are a plurality of two or more different polynucleotide probes complementary to two or more different target polynucleotides or/and nucleic acid sequences which are associated with a particular disease, pathogen or parasite. In certain embodiments, the two or more different polynucleotide probes are complementary to two or more different conserved nucleic acid sequences of the genome of a pathogen or a parasite, such as a virus (e.g., SARS-CoV-2 or HIV-1).

[0055] In additional embodiments, the polynucleotide probes are a plurality of two or more different polynucleotide probes complementary to two or more different target polynucleotides or/and nucleic acid sequences which are associated with two or more different diseases, pathogens or parasites.

[0056] In some embodiments, the method utilizes polynucleotide probes complementary to a particular target polynucleotide or nucleic acid sequence to determine absence or presence of a particular disease, pathogen or parasite. This may occur if, e.g., a particular target polynucleotide or nucleic acid sequence is associated with a specific disease, pathogen or parasite, or another method is performed in conjunction with the method described herein to determine absence or presence of the disease, pathogen or parasite.

[0057] In other embodiments, the method utilizes a plurality of different polynucleotide probes complementary to a plurality or panel of (e.g., about 2-10, or about 3, 4, 5, 6 or 7) different target polynucleotides or/and nucleic acid sequences to determine absence or presence of a particular disease, pathogen or parasite. Hybridization between the different polynucleotide probes and the plurality or panel of different target polynucleotides or/and nucleic acid sequences can be evaluated in the same test or separate tests.

[0058] The polynucleotide probes can be any suitable polynucleotide probes for hybridizing to the target polynucleotide or nucleic acid sequence. In some embodiments, the polynucleotide probes are selected from single-stranded polynucleotides comprising DNA residues (e.g., complementary DNAs [cDNAs]) or RNA residues, and optionally unnatural bonds (e.g., phosphorothioate/thiophosphate or phosphorodiamidate bonds) linking the nucleotides; single-stranded polynucleotides comprising nucleotide analogs (e.g., xeno nucleic acids [XNAs] such as locked nucleic acids [LNAs]) and optionally unnatural bonds (e.g., phosphorothioate/ thiophosphate or phosphorodiamidate bonds) linking the nucleotides; and single-stranded polynucleotides comprising DNA residues or RNA residues, and nucleotide analogs (e.g., XNAs such as LNAs), and optionally unnatural bonds (e.g., phosphorothioate/thiophosphate or phosphorodiamidate bonds) linking the nucleotides. In certain embodiments, the polynucleotide probes are cDNAs. In some embodiments, the polynucleotide probes comprise about 10-35, 15-30 or 20-25 nucleotides.

[0059] The polynucleotide probes can be associated with or bound to the outer surface of the carbon nanotubes by any suitable means. In some embodiments, the polynucleotide probes are associated with or bound to the outer surface of the carbon nanotubes by van der Waals force. In some embodiments, the polynucleotide probes have a number of (e.g., about 5-10) additional nucleotide residues at the 5’ or 3’ end of the probes which are designed to increase van der Waals interaction with the carbon nanotubes and not to hybridize to the target polynucleotide or nucleic acid sequence.

[0060] In other embodiments, the carbon nanotubes are impregnated with gold nanoparticles and the polynucleotide probes have a thiol group (e.g., a thiol group such as a cysteamine group which is part of a phosphorodiamidate group at the 3’ or 5’ end of the probes) which adsorbs onto the gold nanoparticles. [0061] In some embodiments, the bio-nanosensor(s) is/are prepared with the polynucleotide probes primed on the outer surface of the carbon nanotubes.

[0062] In other embodiments, the bio-nanosensor(s) is/are contacted with the polynucleotide probes shortly (e.g., within about 3, 5 or 10 minutes) prior to contacting the bio-nanosensor(s) with the sample. In some embodiments, the method further comprises subtracting the AV resulting from contacting the bio-nanosensor(s) with the polynucleotide probes from the test AV resulting from contacting the bio-nanosensor(s) with the sample.

[0063] The target polynucleotide or nucleic acid sequence can be any suitable target polynucleotide or nucleic acid sequence for detecting the disease, the pathogen or the parasite. For example, the target polynucleotide or nucleic acid sequence can be any type of DNA (e.g., ssDNA, dsDNA, or cell-free or circulating DNA) or any type of RNA (e.g., ssRNA, dsRNA, mRNA, sRNA, rRNA, snRNA, snoRNA, piRNA, miRNA, shRNA, siRNA, or IncRNA). In some embodiments, the target polynucleotide is mRNA, ncRNA (e.g., miRNA or IncRNA), other single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA). In certain embodiments, the target polynucleotide is miRNA. In other embodiments, the target polynucleotide is cell-free DNA (cfDNA), such as cfDNA released by tumor or cancer cells. In further embodiments, the target nucleic acid sequence is a DNA or RNA sequence of the genome of a pathogen (e.g., a virus or a bacterium) or a parasite (e.g., a protozoan).

[0064] Table 1 lists the level of microRNAs (miRNAs) in a biological sample (e.g., blood, plasma, serum or saliva) from human subjects having particular diseases or infections relative to that from healthy human subjects.

Table 1. Levels of microRNAs in diseases

CNS - central nervous system

COPD = chronic obstructive pulmonary disease

EBV - Epstein-Barr virus [0065] Panels of different target polynucleotides or nucleic acid sequences can be created for different diseases, pathogens and parasites.

[0066] In some embodiments, the carbon nanotubes are or comprise single-wall carbon nanotubes (SWCNTs). The SWCNTs can have any suitable dimensions. In certain embodiments, the SWCNTs have a diameter of about 0.5-5 nm and a length of about 2-30 microns. In certain embodiments, the SWCNTs have a purity of at least about 95% or 98%.

[0067] In other embodiments, the carbon nanotubes are or comprise multi-wall carbon nanotubes (MWCNTs). The MWCNTs can have any suitable number of walls. In certain embodiments, the MWCNTs have about 2-6 or 6-12 substantially concentric layers of graphene. The MWCNTs can have any suitable dimensions. In certain embodiments, the MWCNTs have a diameter of about 5-50 nm or 50-100 nm and a length of about 2-30 microns. In certain embodiments, the MWCNTs have a purity of at least about 95% or 98%.

[0068] The sample can be any suitable sample for detecting the disease, the pathogen or the parasite. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is or comprises saliva, blood, plasma, serum, cerebrospinal fluid, urine, stool, buccal scrape or nasal scrape. In certain embodiments, the biological sample is or comprises saliva, blood, plasma or serum. The biological sample can be diluted with an aqueous solvent such as water (e.g., purified water) prior to being added to or contacting the bio-nanosensor(s).

[0069] In other embodiments, the sample is an environmental sample or an agricultural or plant sample. In some embodiments, the environmental sample is a water sample from a water-treatment facility (e.g., a sample of wastewater or treated water), an industrial facility (e.g., a sample of wastewater), a business, a domestic residence, a body of water (e.g., a lake, a river or a stream), or a pool of water (e.g., a puddle).

[0070] The method described herein can detect a wide range of diseases, pathogens and parasites. In some embodiments, the method can detect the absence or presence of a disease, a pathogen or a parasite with an accuracy of at least about 90%, 95% or 98%. Non-limiting examples of diseases include tumors, cancers, bone disorders, cardiovascular disorders, cerebrovascular disorders, fibrotic disorders, immune-related disorders (e.g., inflammatory disorders, autoimmune disorders, allergies and immunodeficiency), liver and hepatobiliary disorders, gastrointestinal disorders, metabolic disorders, neurological disorders (including neurodegenerative disorders and neurodevelopmental disorders), eye disorders, genetic disorders, and disorders in response to infections (e.g., sepsis). In certain embodiments, the disease is a tumor or cancer. Examples of pathogens include without limitation viruses, viroids, bacteria, protozoa, fungi and algae. Examples of parasites include without limitation protozoa, helminths and insects.

[0071 ] In addition to detecting the absence or presence of a disease, a pathogen or a parasite, the method described herein can quantify the level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in the sample based on the test AV. In some embodiments, the level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in the sample is calculated using an equation (e.g., a substantially linear equation) formulated from changes in voltage resulting from contacting a plurality of bio-nanosensors with a plurality of samples containing known levels of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite.

[0072] In addition to providing diagnosis of a disease or infection, the method described herein can provide prognosis of the disease or infection and inform treatment of it. The method can determine the status (e.g., the severity or/and the stage) of the disease or infection, and the efficacy of any ongoing treatment, based on the level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in the sample. For example, the method can quantify the level of certain miRNAs whose level varies with the stage (e.g., localized, invasive or metastasized) of certain cancers, and which are associated with resistance to certain chemotherapeutic drugs. In some embodiments, the status of the disease or infection and the efficacy of any ongoing treatment are determined based on comparison of the present level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in the present sample to a, or the, previous level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in a, or the, previous sample. For instance, an improvement in the level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite compared to a, or the, previously measured level of the same may indicate improvement in the disease or infection and that the treatment should be continued, while a worsening of the level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite compared to a, or the, previously measured level of the same may indicate worsening of the disease or infection and that the dosage or/and the dosing frequency of the drug(s) being used should be increased, or/and other drug(s) should be used to treat the disease or infection. Device for detecting diseases, pathogens and parasites

[0073] The disclosure further describes a device that performs the method for detecting diseases, pathogens and parasites described herein. The entire disclosure relating to the method also applies to the device.

[0074] Some embodiments of the disclosure relate to a biodetection device for detecting absence or presence of a disease, a pathogen or a parasite, comprising: a biosensor module comprising bio-nanosensors, wherein: each bio-nanosensor comprises carbon nanotubes and graphene; polynucleotide probes complementary to a target polynucleotide or nucleic acid sequence are associated with an outer surface of the carbon nanotubes; the target polynucleotide or nucleic acid sequence is associated with a disease, a pathogen or a parasite; the bio-nanosensors are configured to generate an analog electrical signal when the polynucleotide probes on the carbon nanotubes hybridize to the target polynucleotide or nucleic acid sequence; the graphene facilitates thermal and electrical conduction; and the biosensor module is configured to receive or contact a sample; a heater configured to heat the biosensor module to an optimum temperature for hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence and to maintain the biosensor module at the optimum temperature during testing; a relay configured to control the temperature of the heater and the biosensor module; a thermistor configured to measure the temperature of the heater and the biosensor module; an AD (analog-to-digital) converter configured to convert analog electrical signals from the biosensor module into digital electrical signals; and a microcontroller configured to receive AD-converted electrical signals and data from the biosensor module via the AD converter, to transfer data, to obtain temperature readings from the thermistor, and to control the heater via control of the relay; wherein the biodetection device is configured to connect to a computer device and to perform the method for detecting absence or presence of a disease, a pathogen or a parasite described herein.

[0075] Fig. 1 shows an embodiment of the biodetection device 100 connected to a laptop computer 190 as the computer device. [0076] The analog electrical signal generated by the bio-nanosensors when the polynucleotide probes on the carbon nanotubes hybridize to the target polynucleotide or nucleic acid sequence relates to an electrical property such as voltage, electrical conductivity /conductance, electric current flow, or electrical resistivity /resistance. In some embodiments, the electrical signal is signal voltage, and the microcontroller receives voltage vs time data from the biosensor module via the AD converter.

[0077] In some embodiments, the biodetection device further comprises a connector board electrically connected to the biosensor module and the AD converter and configured to receive analog electrical signals from the biosensor module and to transfer the signals to the AD converter.

[0078] In some embodiments, the biodetection device further comprises a samplecollection module configured to collect a sample and to bring the sample into contact with the bio-nanosensors. The sample can be diluted with an aqueous solvent such as water (e.g., purified water).

[0079] In some embodiments, the computer device comprises: software installed in the computer device or obtained from a remote server or the digital cloud, and configured to provide instructions for operating the biodetection device and performing the method for detecting a disease, a pathogen or a parasite, obtain data (e.g., voltage vs time) from the microcontroller of the biodetection device, process data (e.g., calculate the change in voltage [AV] between two timepoints), analyze data (e.g., analyze AV data for trends or patterns), and formulate and provide results; a memory coupled to the software and configured to store information, data and instructions; and a processor coupled to the software and the memory and configured to execute instructions and operations, to process and analyze data, and to perform the method for detecting a disease, a pathogen or a parasite.

[0080] In some embodiments, data processing or/and data analysis is/are performed by the software in the digital cloud. In further embodiments, instructions and operations provided by the software are executed by a processor in the biodetection device.

[0081] The computer device can be any suitable computer device for communicating with and operating the biodetection device. In some embodiments, the computer device is a laptop computer, a desktop computer, a tablet or a smartphone. [0082] The biodetection device can be connected to the computer device via a cable, such as a USB Type-A or Type-C cable. Alternatively, the biodetection device can be connected to the computer device via a wireless connection, such as Wi-Fi or Bluetooth.

[0083] In some embodiments, the optimum temperature for testing is about 45-70 °C, 50-70 °C or 55-65 °C. In further embodiments, the optimum temperature for testing is about 50-55 °C, 55-60 °C or 60-65 °C. In certain embodiments, the optimum temperature for testing is about 55 °C, 60 °C or 65 °C.

[0084] In some embodiments, the carbon nanotubes are or comprise single-wall carbon nanotubes (SWCNTs). The SWCNTs can have any suitable dimensions. In certain embodiments, the SWCNTs have a diameter of about 0.5-5 nm and a length of about 2-30 microns. In certain embodiments, the SWCNTs have a purity of at least about 95% or 98%.

[0085] In other embodiments, the carbon nanotubes are or comprise multi-wall carbon nanotubes (MWCNTs). The MWCNTs can have any suitable number of walls. In certain embodiments, the MWCNTs have about 2-6 or 6-12 substantially concentric layers of graphene. The MWCNTs can have any suitable dimensions. In certain embodiments, the MWCNTs have a diameter of about 5-50 nm or 50-100 nm and a length of about 2-30 microns. In certain embodiments, the MWCNTs have a purity of at least about 95% or 98%.

[0086] In some embodiments, the graphene of each bio-nanosensor is a multi-layer graphene or nano-graphite platelet comprising about 5-30 layers of graphene, or about 5-10, 10-20 or 20-30 layers of graphene.

[0087] The biosensor module can contain any suitable number of bio-nanosensors for detecting one or more compounds, diseases, pathogens or parasites. In some embodiments, the biosensor module comprises about 4, 8, 16, 32, 64, 128, 256, 512, or more bio- nanosensors. In some embodiments, a disposable cartridge may be used such as shown in Figure 9 with 64 sensors. Additional sensor allow the user to test significantly more and different analytes simultaneously from a single sample in a single test run.

In another alternative embodiment, the sample can be added from a single syringe and then the liquid will be flowing onto the sensor cartridge. The syringe may have a filter for filtering out solid particles in the sample and a water-ball for diluting the sample. The syringe may be disposable. The syringe may also have a heating step where the sample can be pre-heated if the user required by a heater coil in the reader unit (e.g, for denaturing the double stranded DNA as well as for lysing cells in the sample before it comes in contact with the sensor). The syringe may be operated mechanically by a stepper motor system. [0088] To avoid sample contamination, the biosensor module and the optional samplecollection module can be single-use/disposable.

[0089] In certain embodiments, the biosensor module is manufactured with the polynucleotide probes primed on the outer surface of the carbon nanotubes. In other embodiments, the polynucleotide probes are added to the bio-nanosensors shortly (e.g., within about 3, 5 or 10 minutes) prior to addition of the sample to the bio-nanosensors.

[0090] In some embodiments, the software sets a threshold value of AV (e.g., about 0.015 or 0.020 mV, or about 0.02 V) between two (e.g., pre-determined) timepoints equal to or above which the AV calculated for a particular bio-nanosensor is deemed a positive reading and below which the AV is deemed a negative reading, and the software determines a positive result (presence of a disease, a pathogen or a parasite) if the majority of the bio-nanosensors provide positive readings or a negative result (absence of a disease, a pathogen or a parasite) if the majority or half of the bio-nanosensors provide negative readings.

[0091] In some embodiments, the biodetection device further comprises a light-emitting diode (LED) which indicates a qualitative result of the test by a different color of light, such as a red light for a positive result, a green light for a negative result, or a yellow or orange light for an inconclusive result. In certain embodiments, the microcontroller controls the LED.

In alternative embodiments, the biodetection device can comprise a temperature control circuit, such as a MOSFET (Metal Oxide Semiconductor Field Effect Transistor) switching device for turning a heating circuit or a cooling fan on and off in order to control the temperature of the device while in use. In a related alternative embodiment, the sensor material itself can be used as the heating device.

[0092] In additional embodiments, the computer device provides a qualitative result of the test, and optionally a quantitative result of the test (e.g., the level of the target polynucleotide or nucleic acid sequence, a pathogen or a parasite in the sample), on the computer device, such as on the screen or in a Results page or file of the computer device.

[0093] In further embodiments, the computer device provides a qualitative result of the test, and optionally a quantitative result of the test, to the subject providing the sample if a biological sample and the person (e.g., a medical or veterinary practitioner) overseeing the test, and optionally a government or health authority, agency or department if reporting of such result(s) thereto is required. [0094] In some embodiments, the biodetection device is capable of providing a qualitative result or/and a quantitative result of the test within about 15 or 20 minutes after addition of the sample to the biosensor module.

[0095] The biodetection device can be plugged into an electrical socket. Alternatively, the biodetection device can be powered by a battery, which enables use of the portable biodetection device in the field.

[0096] The biodetection device can be used at a point of care, such as at a medical office, a medical clinic, an out-patient clinic, a hospital, a pharmacy, a laboratory, a nursing home, a veterinary office, a veterinary clinic or a veterinary hospital. The portable biodetection device can also be used in the field, such as at a mobile medical clinic, a humanitarian medical clinic, a mobile veterinary practice or clinic, or a farm.

The embodiments above, as well as others, may be combined such that the device and system may be designed as a high throughput system that can analyze a large number of samples for the same analyte in 5-30 minutes as shown in Fig. 10. In a high throughput configuration the number of sensors is generally from 8 to 96 or more. A printed circuit board on which the sensors are attached can contain the disposable heating elements and the MOSFET system described above can control the temperatures of each sensor. In another embodiment some of the sensors can be primed and heated to a different temperature so that testing can be performed for multiple samples for 2 or more analytes simultaneously. All samples will be separately pipetted on to the sensors mechanically. Samples can be collected and added to disposable vials with a QR code printed. The high throughput device can have a preheating step where the sample vials will be pre-heated after the QR-code has been read by the software. The adding of sample to the sensor can be performed 8 at a time or any multiples of 8 . The software will start gathering data as soon it has detected that there is sample on the senor surface.

Representative embodiments

[0097] The following embodiments of the present disclosure are provided by way of illustration and example:

1. A method for detecting absence or presence of a disease, a pathogen or a parasite, comprising: contacting a bio-nanosensor with a sample, wherein the bio-nanosensor comprises carbon nanotubes, polynucleotide probes are associated with or bound to an outer surface of the carbon nano tubes, the polynucleotide probes are complementary to a target polynucleotide or nucleic acid sequence, and the target polynucleotide or nucleic acid sequence is associated with a disease, a pathogen or a parasite; measuring a change in voltage (AV) resulting from contacting the bio-nanosensor with the sample (test AV); comparing the test AV to a reference AV resulting from contacting a bio-nanosensor with a sample from a subject known not to have the disease, the pathogen or the parasite, or a sample known not to have the pathogen or the parasite; and determining absence or presence of the disease, the pathogen or the parasite based on comparing the test AV to the reference AV.

2. The method of embodiment 1 , wherein the reference AV is determined from contacting a plurality of bio-nanosensors with a plurality of samples from a plurality of subjects known not to have the disease, the pathogen or the parasite, or a plurality of samples known not to have the pathogen or the parasite.

3. The method of embodiment 1 or 2, wherein the target polynucleotide or nucleic acid sequence is associated with promotion of the disease and the test AV resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is greater than the reference AV, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the disease.

4. The method of embodiment 3, wherein the disease is a tumor or cancer and the target polynucleotide or nucleic acid sequence is an oncogenic nucleic acid sequence or polynucleotide (e.g., messenger RNA [mRNA] or non-coding RNA [ncRNA] such as microRNA [miRNA] or long non-coding RNA [IncRNA]).

5. The method of embodiment 1 or 2, wherein the target polynucleotide or nucleic acid sequence is associated with protection against or inhibition of the disease and the test AV resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is less than the reference AV, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the disease. 6. The method of embodiment 5, wherein the disease is a tumor or cancer and the target polynucleotide or nucleic acid sequence is a tumor suppressor nucleic acid sequence or polynucleotide (e.g., mRNA or ncRNA such as miRNA or IncRNA).

7. The method of embodiment 1 or 2, wherein the target polynucleotide or nucleic acid sequence is derived from the pathogen or is a host-derived polynucleotide whose level increases in response to infection with the pathogen, and the test AV or AV/R where R is the starting resistance of the biosensor without any sample, resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is greater than the reference AV or AV/R, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the pathogen.

8. The method of embodiment 1 or 2, wherein the target polynucleotide or nucleic acid sequence is a host-derived polynucleotide whose level reduces in response to infection with the pathogen and the test AV or AV/R resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is less than the reference AV or AV/R, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the pathogen.

9. The method of embodiment 1 or 2, wherein the target polynucleotide or nucleic acid sequence is derived from the parasite or is a host-derived polynucleotide whose level increases in response to infection with the parasite, and the test AV or AV/R resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is greater than the reference AV or AV/R, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the parasite.

10. The method of embodiment 1 or 2, wherein the target polynucleotide or nucleic acid sequence is a host-derived polynucleotide whose level reduces in response to infection with the parasite and the test AV or AV/R resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is less than the reference AV or AV/R, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the parasite. 11. The method of embodiment 1 or 2, wherein the test AV is substantially similar to (e.g., within about 10% or 20% of) the reference AV or AV/R, indicating absence of the disease, the pathogen or the parasite.

12. The method of any one of the preceding embodiments, which comprises: contacting a plurality of (e.g., about 4, 8, 16, 32, 64 or more) bio-nanosensors with the sample; comparing the test AV generated by each of the bio-nanosensors to the reference AV ; and determining presence of the disease, the pathogen or the parasite if comparing the test AV to the reference AV for a majority of the bio-nanosensors indicates presence of the disease, the pathogen or the parasite, or determining absence of the disease, the pathogen or the parasite if comparing the test AV to the reference AV for a majority or half of the bio-nanosensors indicates absence of the disease, the pathogen or the parasite.

13. The method of any one of the preceding embodiments, wherein the polynucleotide probes are a plurality of a particular polynucleotide probe.

14. The method of any one of embodiments 1 to 12, wherein the polynucleotide probes are a plurality of two or more different polynucleotide probes complementary to a particular target polynucleotide or nucleic acid sequence.

15. The method of any one of embodiments 1 to 12, wherein the polynucleotide probes are a plurality of two or more different polynucleotide probes complementary to two or more different target polynucleotides or/and nucleic acid sequences which are associated with a particular disease, pathogen or parasite.

16. The method of embodiment 15, wherein the two or more different polynucleotide probes are complementary to two or more different conserved nucleic acid sequences of the genome of a pathogen or a parasite, such as a virus (e.g., SARS-CoV-2 or HIV-1).

17. The method of any one of embodiments 1 to 12, wherein the polynucleotide probes are a plurality of two or more different polynucleotide probes complementary to two or more different target polynucleotides or/and nucleic acid sequences which are associated with two or more different diseases, pathogens or parasites.

18. The method of any one of the preceding embodiments, which uses polynucleotide probes complementary to a particular target polynucleotide or nucleic acid sequence to determine absence or presence of a particular disease, pathogen or parasite. 19. The method of any one of embodiments 1 to 17, which uses a plurality of different polynucleotide probes complementary to a plurality or panel of (e.g., about 2-10, or about 3, 4, 5, 6 or 7) different target polynucleotides or/and nucleic acid sequences to determine absence or presence of a particular disease, pathogen or parasite, whether in the same test or separate tests.

20. The method of any one of the preceding embodiments, wherein the polynucleotide probes are selected from single-stranded polynucleotides comprising DNA residues (e.g., complementary DNAs [cDNAs]) or RNA residues, and optionally unnatural bonds (e.g., phosphorothioate/thiophosphate or phosphorodiamidate bonds) linking the nucleotides; singlestranded polynucleotides comprising nucleotide analogs (e.g., xeno nucleic acids [XNAs] such as locked nucleic acids [LNAs]) and optionally unnatural bonds (e.g., phosphorothioate/ thiophosphate or phosphorodiamidate bonds) linking the nucleotides; and single-stranded polynucleotides comprising DNA residues or RNA residues, and nucleotide analogs (e.g., XNAs such as LNAs), and optionally unnatural bonds (e.g., phosphorothioate/thiophosphate or phosphorodiamidate bonds) linking the nucleotides.

21. The method of embodiment 20, wherein the polynucleotide probes are cDNAs.

22. The method of embodiment 20 or 21 , wherein the polynucleotide probes comprise about 10-35, 15-30 or 20-25 nucleotides.

23. The method of any one of the preceding embodiments, wherein the polynucleotide probes are associated with or bound to the outer surface of the carbon nanotubes by van der Waals force.

24. The method of embodiment 23, wherein the polynucleotide probes have a number of (e.g., about 5-10) additional nucleotide residues at the 5’ or 3’ end of the probes which are designed to increase van der Waals interaction with the carbon nanotubes and not to hybridize to the target polynucleotide or nucleic acid sequence.

25. The method of any one of embodiments 1 to 22, wherein the carbon nanotubes are impregnated with gold nanoparticles and the polynucleotide probes have a thiol group (e.g., a thiol group such as a cysteamine group which is part of a phosphorodiamidate group at the 3’ or 5 ’ end of the probes) which adsorbs onto the gold nanoparticles.

26. The method of any one of the preceding embodiments, wherein: the target polynucleotide is mRNA, ncRNA (e.g., sRNA, miRNA or IncRNA), other single-stranded RNA (ssRNA), single- stranded DNA (ssDNA), or double- stranded DNA (dsDNA); or the target nucleic acid sequence is a DNA or RNA sequence of the genome of a pathogen (e.g., a virus or a bacterium) or a parasite (e.g., a protozoan).

27. The method of embodiment 26, wherein the target polynucleotide is miRNA.

28. The method of any one of the preceding embodiments, wherein the carbon nanotubes are or comprise single-wall carbon nanotubes (SWCNTs).

29. The method of embodiment 28, wherein the SWCNTs have a diameter of about 0.5-5 nm and a length of about 2-30 microns.

30. The method of any one of embodiments 1 to 27, wherein the carbon nanotubes are or comprise multi-wall carbon nanotubes (MWCNTs).

31. The method of embodiment 30, wherein the MWCNTs have: about 2-6 or 6-12 substantially concentric layers of graphene; a diameter of about 5-50 nm or 50-100 nm; and a length of about 2-30 microns.

32. The method of any one of the preceding embodiments, wherein the sample is a biological sample.

33. The method of embodiment 32, wherein the biological sample is or comprises saliva, blood, plasma, serum, cerebrospinal fluid, urine, stool, buccal scrape or nasal scrape, optionally soaked in water.

34. The method of any one of embodiments 1 to 31, wherein the sample is an environmental sample, optionally soaked in water.

35. The method of embodiment 34, wherein the environmental sample is a water sample from a water-treatment facility (e.g., a sample of wastewater or treated water), an industrial facility (e.g., a sample of wastewater), a business, a domestic residence, a body of water (e.g., a lake, a river or a stream), or a pool of water (e.g., a puddle).

36. The method of any one of the preceding embodiments, wherein the disease is selected from tumors, cancers, bone disorders, cardiovascular disorders, cerebrovascular disorders, fibrotic disorders, immune-related disorders (e.g., inflammatory disorders, autoimmune disorders and allergies), liver and hepatobiliary disorders, gastrointestinal disorders, metabolic disorders, neurological disorders (including neurodegenerative disorders), eye disorders, genetic disorders, and disorders in response to infections (e.g., sepsis).

37. The method of embodiment 36, wherein the disease is a tumor or cancer. 38. The method of any one of embodiments 1 to 35, wherein the pathogen is selected from viruses, viroids, bacteria, protozoa, fungi and algae.

39. The method of any one of embodiments 1 to 35, wherein the parasite is selected from protozoa, helminths and insects.

40. The method of any one of the preceding embodiments, which can detect the absence or presence of the disease, the pathogen or the parasite with an accuracy of at least about 90%, 95% or 98%.

41. The method of any one of the preceding embodiments, wherein the bio-nanosensor(s) is/are prepared with the polynucleotide probes primed on the outer surface of the carbon nano tubes.

42. The method of any one of embodiments 1 to 40, further comprising contacting the bio- nanosensor(s) with the polynucleotide probes shortly (e.g., within about 3, 5 or 10 minutes) prior to contacting the bio-nanosensor(s) with the sample.

43. The method of embodiment 42, further comprising subtracting the AV or AV/R resulting from contacting the bio-nanosensor(s) with the polynucleotide probes from the test AV or AV/R resulting from contacting the bio-nanosensor(s) with the sample.

44. The method of any one of the preceding embodiments, further comprising quantifying the level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in the sample based on the test AV pr AV/R.

45. The method of embodiment 44, wherein the level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in the sample is calculated using an equation (e.g., a substantially linear equation) formulated from changes in voltage resulting from contacting a plurality of bio-nanosensors with a plurality of samples containing known levels of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite.

46. The method of embodiment 44 or 45, further comprising determining the status (e.g., the severity or/and the stage) of the disease, and the efficacy of any ongoing treatment, based on the level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in the sample.

47. The method of embodiment 46, wherein the status of the disease and the efficacy of any ongoing treatment are determined based on comparison of the present level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in the present sample to a, or the, previous level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in a, or the, previous sample.

48. A biodetection device for detecting absence or presence of a disease, a pathogen or a parasite, comprising: a biosensor module comprising bio-nanosensors, wherein: each bio-nanosensor comprises carbon nanotubes and graphene; polynucleotide probes complementary to a target polynucleotide or nucleic acid sequence are associated with an outer surface of the carbon nanotubes; the target polynucleotide or nucleic acid sequence is associated with a disease, a pathogen or a parasite; the bio-nanosensors are configured to generate an analog electrical signal when the polynucleotide probes on the carbon nanotubes hybridize to the target polynucleotide or nucleic acid sequence; the graphene facilitates thermal and electrical conduction; and the biosensor module is configured to receive or contact a sample; a heater configured to heat the biosensor module to an optimum temperature for hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence and to maintain the biosensor module at the optimum temperature during testing; a relay configured to control the temperature of the heater and the biosensor module; a thermistor configured to measure the temperature of the heater and the biosensor module; an AD (analog-to-digital) converter configured to convert analog electrical signals from the biosensor module into digital electrical signals; and a microcontroller configured to receive AD-converted electrical signals and data from the biosensor module via the AD converter, to transfer data, to obtain temperature readings from the thermistor, and to control the heater via control of the relay; wherein the biodetection device is configured to connect to a computer device and to perform the method of any one of embodiments 1 to 47.

49. The biodetection device of embodiment 48, further comprising a connector board electrically connected to the biosensor module and the AD converter and configured to receive analog electrical signals from the biosensor module and to transfer the signals to the AD converter. 50. The biodetection device of embodiment 48 or 49, further comprising a sample-collection module configured to collect a sample and to bring the sample into contact with the bionanosensors.

51. The biodetection device of any one of embodiments 48 to 50, wherein the computer device comprises: software installed in the computer device or obtained from a remote server or the digital cloud, and configured to provide instructions for operating the biodetection device and performing the method of any one of embodiments 1 to 47, obtain data (e.g., voltage vs time) from the microcontroller of the biodetection device, process data (e.g., calculate the change in voltage [AV] between two timepoints), analyze data (e.g., analyze AV data for trends or patterns), and formulate and provide results; a memory coupled to the software and configured to store information, data and instructions; and a processor coupled to the software and the memory and configured to execute instructions and operations, to process and analyze data, and to perform the method of any one of embodiments 1 to 47.

52. The biodetection device of any one of embodiments 48 to 51 , wherein the biodetection device is configured to connect to the computer device via a cable, such as a USB Type-A or Type-C cable.

53. The biodetection device of any one of embodiments 48 to 51, wherein the biodetection device is configured to connect to the computer device via a wireless connection, such as Wi-Fi or Bluetooth.

54. The biodetection device of any one of embodiments 48 to 53, wherein the computer device is a laptop computer, a desktop computer, a tablet or a smartphone.

55. The biodetection device of any one of embodiments 48 to 54, wherein the bio- nanosensors generate an analog signal voltage when the polynucleotide probes on the carbon nanotubes hybridize to the target polynucleotide or nucleic acid sequence, and the microcontroller receives voltage vs time data from the biosensor module via the AD converter.

56. The biodetection device of any one of embodiments 48 to 55, wherein the optimum temperature for testing is about 45-70 °C, 50-70 °C or 55-65 °C.

57. The biodetection device of any one of embodiments 48 to 56, wherein the carbon nanotubes are or comprise single-wall carbon nanotubes (SWCNTs). 58. The biodetection device of embodiment 57, wherein the SWCNTs have a diameter of about 0.5-5 nm and a length of about 2-30 microns.

59. The biodetection device of any one of embodiments 48 to 56, wherein the carbon nanotubes are or comprise multi-wall carbon nanotubes (MWCNTs) comprising about 2-6 or 6- 12 substantially concentric layers of graphene.

60. The biodetection device of embodiment 59, wherein the MWCNTs have a diameter of about 5-50 nm or 50-100 nm and a length of about 2-30 microns.

61. The biodetection device of any one of embodiments 48 to 60, wherein the graphene of each bio-nanosensor is a multi-layer graphene or nano-graphite platelet comprising about 5-30 layers of graphene, or about 5-10, 10-20 or 20-30 layers of graphene.

62. The biodetection device of any one of embodiments 48 to 61 , wherein the biosensor module comprises about 4, 8, 16, 32, 64, 128 or more bio-nanosensors.

63. The biodetection device of any one of embodiments 48 to 62, wherein the biosensor module and the optional sample-collection module are disposable.

64. The biodetection device of any one of embodiments 48 to 63, wherein the biosensor module is manufactured with the polynucleotide probes primed on the outer surface of the carbon nano tubes.

65. The biodetection device of any one of embodiments 48 to 63, wherein the polynucleotide probes are added to the bio-nanosensors shortly (e.g., within about 3, 5 or 10 minutes) prior to addition of the sample to the bio-nanosensors.

66. The biodetection device of any one of embodiments 51 to 65, wherein the software sets a threshold value of AV (e.g., about 0.015 or 0.020 mV, or about 0.02 V) between two (e.g., predetermined) timepoints equal to or above which the AV calculated for a particular bio-nanosensor is deemed a positive reading and below which the AV is deemed a negative reading, and the software determines a positive result (presence of a disease, a pathogen or a parasite) if the majority of the bio-nanosensors provide positive readings or a negative result (absence of a disease, a pathogen or a parasite) if the majority or half of the bio-nanosensors provide negative readings.

67. The biodetection device of any one of embodiments 48 to 66, which further comprises a light-emitting diode (LED) which indicates a qualitative result of the test by a different color of light, such as a red light for a positive result, a green light for a negative result, or a yellow or orange light for an inconclusive result. 68. The biodetection device of any one of embodiments 48 to 67, wherein the computer device provides a qualitative result of the test, and optionally a quantitative result of the test (e.g., the level of the target polynucleotide or nucleic acid sequence, a pathogen or a parasite in the sample), on the computer device, such as on the screen or in a Results page or file of the computer device.

69. The biodetection device of any one of embodiments 48 to 68, wherein the computer device provides a qualitative result of the test, and optionally a quantitative result of the test, to the subject providing the sample if a biological sample and the person (e.g., a medical or veterinary practitioner) overseeing the test, and optionally a government or health authority, agency or department if reporting of such result(s) thereto is required.

70. The biodetection device of any one of embodiments 48 to 69, which is capable of providing a qualitative result or/and a quantitative result of the test within about 15 or 20 minutes after addition of the sample to the biosensor module.

71. The biodetection device of any one of embodiments 48 to 70, which can be plugged into an electrical socket or powered by a battery.

72. The biodetection device of any one of embodiments 48 to 71 , which is portable.

73. The biodetection device of any one of embodiments 48 to 72, which is operable at a point of care, such as at a medical office, a medical clinic, an out-patient clinic, a hospital, a pharmacy, a nursing home, a veterinary office, a veterinary clinic or a veterinary hospital.

74. The biodetection device of any one of embodiments 48 to 72, which is operable in the field, such as at a mobile medical clinic, a humanitarian medical clinic, a mobile veterinary practice or clinic, or a farm.

Examples

[0098] The following examples are intended only to illustrate the disclosure. Other procedures, methodologies, techniques, conditions and reagents may alternatively be used as appropriate.

Example 1. Detection of miR-21 in saliva using biodetection device

Materials and method

[0099] Saliva specimens were collected from volunteers. Tests were conducted on both pooled saliva and individual saliva. Briefly, pooled and individual saliva samples were mixed well by vortexing to obtain homogenous solutions. 100 pL of pooled and individual saliva was added to 2 mL of ultrapure water (UPW), which is RNase- and DNase-free, to a 1 :20 dilution. Synthetic microRNA 21 (miR-21), which functions as an oncogenic miRNA in many cancers, and its complementary DNA (cDNA) were obtained from Integrated DNA Technologies (Coralville, Iowa). Synthetic MiR-21: 5’- UAGCUUAUC AG ACUG AUGUUGA-3 ’ .

The cDNA of the synthetic miR-21: 5’-TCAACATCAGTCTGATAAGCTA-3’.

Desired concentrations of miR-21 samples such as IO¹⁰ copies/pL, 10⁷ copies/pL, 10⁴ copies/pL and 10² copies/pL were prepared by adding synthetic miR-21 to 1 :20 diluted pooled saliva. The cDNA of synthetic miR-21 was prepared at 10ⁿ copies/pL and used as a probe to hybridize to the synthetic miR-21 in all the tests.

[00100] The biosensor module of the biodetection device contained eight bio-nanosensors attached to a breadboard that was connected to a balanced and feedback-controlled electronic circuit. Each bio-nanosensor contained multi-wall carbon nanotubes and graphene. The temperature for hybridization between the synthetic miR-21 and its cDNA was set at around 47-50 °C. Once the biosensor module reached the temperature for testing, 5 pL of the miR- 21 cDNA probe (10¹¹ copies/pL) was pipetted onto each bio-nanosensor starting at 10 seconds of the experimental time, with a 10 second interval between drops (at 10 to 80 seconds). Depending on the experimental design, generally starting at 310 seconds, 5 pL of 1 :20 diluted saliva sample and spiked samples of synthetic miR-21 of varying concentrations were pipetted onto the bio-nanosensors at 10 second intervals. The tests were completed in about 700 seconds. Output voltage for the eight bio-nanosensors was collected and analyzed. The result of the hybridization between the synthetic miR-21 and its cDNA was analyzed based on the change in voltage (AV) resulting therefrom.

Results

[00101 ] In a first experiment, the miR-21 cDNA probe was added to the eight bio- nanosensors starting at 10 seconds of the experimental time as described above. At 310 seconds UPW was pipetted onto the first bio-nanosensor, at 320 seconds synthetic miR-21 at 10⁷ copies/pL in UPW was pipetted onto the second bio-nanosensor, at 330 seconds 1:20 diluted pooled saliva was pipetted onto the third bio-nanosensor, and synthetic miR-21 at 10⁷ copies/pL in 1:20 diluted pooled saliva was pipetted onto the remaining five bio-nanosensors at 10 second intervals. The average of the five AV values for synthetic miR-21 in pooled saliva was calculated. Figs. 2A and 2B show that the addition of UPW (negative control) and 1 :20 diluted pooled saliva to the cDNA-primed bio-nanosensors yielded little change in voltage. By contrast, Figs. 2A and 2B show that the addition of synthetic miR-21 at 10⁷ copies/pL in UPW or 1:20 diluted pooled saliva to the cDNA-primed bio-nanosensors yielded significant changes in voltage.

[00102] In a second experiment, the miR-21 cDNA probe was added to the eight bio- nanosensors starting at 10 seconds of the experimental time as described above. Starting at 310 seconds and then at 10 second intervals, 1:20 diluted individual saliva was pipetted onto the first two bio-nanosensors, and synthetic miR-21 at 10³ copies/p L or 10⁴ copies/p L in 1 :20 diluted individual saliva was pipetted onto three consecutive bio-nanosensors for each spiked sample. The average of the three AV values for synthetic miR-21 at 10³ copies/pL or 10⁴ copies/pL was calculated. Figs. 3A and 3B show that the addition of 1 :20 diluted individual saliva to the cDNA-primed bio-nanosensors yielded little change in voltage. By contrast, Figs. 3A and 3B show that the addition of synthetic miR-21 at 10³ copies/pL or 10⁴ copies/pL in 1 :20 diluted individual saliva to the cDNA-primed bio-nanosensors yielded significant changes in voltage.

[00103] In a third experiment, the miR-21 cDNA probe was added to the eight bio- nanosensors starting at 10 seconds of the experimental time as described above. Starting at 310 seconds and then at 10 second intervals, 1:20 diluted pooled saliva was pipetted onto the first two bio-nanosensors, and synthetic miR-21 at 10¹⁰ copies/pL, 10⁷ copies/pL or 10⁴ copies/pL in 1 :20 diluted pooled saliva was pipetted onto two consecutive bio-nanosensors for each spiked sample. The average AV for the 1 :20 diluted pooled saliva control/baseline was subtracted from the average AV for each copy number of synthetic miR-21, and the average AV for each copy number of synthetic miR-21 after baseline subtraction was plotted versus the time of experiment. For each copy number of synthetic miR-21 , AV was determined based on the peak at around 400 seconds. Figs. 4A and 4B show that the addition of synthetic miR-21 at 10⁴ copies/pL, 10⁷ copies/pL or 10¹⁰ copies/pL in 1:20 diluted pooled saliva to the cDNA-primed bio-nanosensors resulted in an increasingly greater average AV. Fig. 4C shows that the log values of the three different concentrations of synthetic miR-21 had an essentially linear correlation with the average AV.

[00104] In a fourth experiment, the miR-21 cDNA probe was added to the eight bio- nanosensors starting at 10 seconds of the experimental time as described above. Starting at 310 seconds and then at 10 second intervals, 1 :20 diluted individual saliva was pipetted onto the first two bio-nanosensors, and synthetic miR-21 at 10⁴ copies/pL, 10³ copies/pL or 10² copies/pL in 1:20 diluted individual saliva was pipetted onto two consecutive bio- nanosensors for each spiked sample. The average AV for the 1 :20 diluted individual saliva control/baseline was subtracted from the average AV for each copy number of synthetic miR- 21, and the average AV for each copy number of synthetic miR-21 after baseline subtraction was plotted versus the time of experiment. The vertical rectangle in Fig. 5A indicates the portion of the plot where the AV was determined for each copy number of synthetic miR-21. Figs. 5A and 5B show that the addition of synthetic miR-21 at 10² copies/pL, 10³ copies/pL or 10⁴ copies/pL in 1 :20 diluted individual saliva to the cDNA-primed bio-nanosensors resulted in an increasingly greater average AV. Fig. 5C shows that the log values of the three different concentrations of synthetic miR-21 had a substantially linear correlation with the average AV.

Example 2. Detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) using biodetection device

[00105] Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) can spread infections among pigs via direct contact or respiratory aerosols. Viral RNA was extracted from the serum of PRRS V-infected animals with TRIzol reagent. A cDNA targeting PRRSV RNA was added to four bio-nanosensors starting from 10 seconds to 40 seconds of the experimental time. Ultrapure water was added to the first bio-nanosensor at 210 seconds, and the extracted viral RNA sample was added to the three remaining bio-nanosensors at 220, 230 and 240 seconds. The average of the three AV values for PRRSV RNA was calculated. Figs. 6A and 6B show that the addition of ultrapure water (UPW, negative control) to a cDNA-primed bio-nanosensor yielded little change in voltage, whereas the addition of the extracted viral RNA sample to the cDNA-primed bio-nanosensors yielded a significant AV.

Example 3. Detection of SARS-CoV-2 in saliva of infected subject using biodetection device

[00106] A cDNA specific for the RNA of SARS-CoV-2, the causative agent of COVID-19, was added to bio-nanosensors. About 300 seconds later at around 350 seconds, 3 mL of 1:20 diluted saliva from an uninfected subject was added to four cDNA-primed bio-nanosensors, and 3 mL of 1 :20 diluted saliva from a subject infected with SARS-CoV-2 was added to four cDNA-primed bio-nanosensors. Six of the eight bio-nanosensors were primed with 100 nM/5 L cDNA specific for SARS-CoV-2 RNA, and the other two bio-nanosensors served as positive control by being primed with 100 nM/5 pL cDNA specific for RNaseP RNA in saliva. Fig. 7A shows that the addition of 1:20 diluted saliva from an uninfected subject to cDNA-primed bio-nanosensors at around 350 seconds resulted in a reduction in voltage (or no change in voltage), which was deemed Negative, whereas Fig. 7B shows that the addition of 1:20 diluted saliva from a subject infected with SARS-CoV-2 to cDNA-primed bio- nanosensors at around 350 seconds resulted in a significant increase in voltage, which was deemed Positive.

Example 4. Detection of SARS-CoV-2 spiked into saliva using biodetection device

[00107] 6.6 mL of 1 : 10 diluted saliva not spiked with SARS-CoV-2 RNA was added to four cDNA-primed bio-nanosensors, and test data was collected about 6-7 min later. Likewise, 6.6 mL of 1 : 10 diluted saliva spiked with SARS-CoV-2 RNA was added to four cDNA- primed bio-nanosensors, and test data was collected about 6-7 min later. Six of the eight bio- nanosensors were primed with 100 nM/5 pL cDNA specific for SARS-CoV-2 RNA, and the other two bio-nanosensors served as positive control by being primed with 100 nM/5 L cDNA specific for RNaseP RNA in saliva. The green curve in Fig- 8 shows that the addition of 1:10 diluted saliva not spiked with SARS-CoV-2 RNA to cDNA-primed bio-nanosensors resulted in a reduction in voltage or no change in voltage, which was deemed Negative, whereas the red curve in Fig. 8 shows that the addition of 1 :10 diluted saliva spiked with SARS-CoV-2 RNA to cDNA-primed bio-nanosensors resulted in a significant increase in voltage, which was deemed Positive.

Example 5: Detection of pathogenic biomarker Potato Virus Y (PVY) from plant tissues using Tearing and Steeping Methods (TSM)

A sample of plant leaf, plant seed, or tuber is prepared by tearing a leaf or skin of a potato and soaking it in water for 2-60 minutes. Alternatively, the leaf, seed, or tuber is smashed and a sample is extracted with buffer solution. The prepared sample is added to the sample jar or syringe and applied to the above testing device. The PVY c-DNA sequence which we prime our sensor is 5'-TTC ATC TCC ATC CAT CAT AAC CC-3'. In approximately 10 minutes the test is complete, the data analyzed by the device.

The device calculates the change of voltage/resistance over time. As shown in Figs. 11 A and 1 IB, the difference between positive data (e.g., sample is PVY positive, Fig. 1 IB) and negative data (e.g., sample is PVY negative, Fig. HA) indicates the presence of the potato virus. [00108] It is understood that, while particular embodiments have been illustrated and described, various modifications may be made thereto and are contemplated herein. It is also understood that the disclosure is not limited by the specific examples provided herein. The description and illustration of embodiments and examples of the disclosure herein are not intended to be construed in a limiting sense. It is further understood that all aspects of the disclosure are not limited to the specific depictions, configurations or relative proportions set forth herein, which may depend upon a variety of conditions and variables. Various modifications and variations in form and detail of the embodiments and examples of the disclosure will be apparent to a person skilled in the art. It is therefore contemplated that the disclosure also covers any and all such modifications, variations and equivalents.

Claims

What Is Claimed Is:

1. A method for detecting absence or presence of a disease, a pathogen or a parasite, comprising: contacting a bio-nanosensor with a sample, wherein the bio-nanosensor comprises carbon nanotubes, polynucleotide probes are associated with or bound to an outer surface of the carbon nanotubes, the polynucleotide probes are complementary to a target polynucleotide or nucleic acid sequence, and the target polynucleotide or nucleic acid sequence is associated with a disease, a pathogen or a parasite; measuring a change in voltage (AV) resulting from contacting the bio-nanosensor with the sample (test AV); comparing the test AV to a reference AV resulting from contacting a bio-nanosensor with a sample from a subject known not to have the disease, the pathogen or the parasite, or a sample known not to have the pathogen or the parasite; and determining absence or presence of the disease, the pathogen or the parasite based on comparing the test AV to the reference AV.

2. The method of claim 1, wherein the reference AV is determined from contacting a plurality of bio-nanosensors with a plurality of samples from a plurality of subjects known not to have the disease, the pathogen or the parasite, or a plurality of samples known not to have the pathogen or the parasite.

3. The method of claim 1 or 2, wherein the target polynucleotide or nucleic acid sequence is associated with promotion of the disease and the test AV resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is greater than the reference AV, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the disease.

4. The method of claim 3, wherein the disease is a tumor or cancer and the target polynucleotide or nucleic acid sequence is an oncogenic nucleic acid sequence or polynucleotide (e.g., messenger RNA [mRNA] or non-coding RNA [ncRNA] such as microRNA [miRNA] or long non-coding RNA [IncRNA]).

5. The method of claim 1 or 2, wherein the target polynucleotide or nucleic acid sequence is associated with protection against or inhibition of the disease and the test AV resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is less than the reference AV, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the disease.

6. The method of claim 5, wherein the disease is a tumor or cancer and the target polynucleotide or nucleic acid sequence is a tumor suppressor nucleic acid sequence or polynucleotide (e.g., mRNA or ncRNA such as miRNA or IncRNA).

7. The method of claim 1 or 2, wherein the target polynucleotide or nucleic acid sequence is derived from the pathogen or is a host-derived polynucleotide whose level increases in response to infection with the pathogen, and the test AV resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is greater than the reference AV, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the pathogen.

8. The method of claim 1 or 2, wherein the target polynucleotide or nucleic acid sequence is a host-derived polynucleotide whose level reduces in response to infection with the pathogen and the test AV resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is less than the reference AV, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the pathogen.

9. The method of claim 1 or 2, wherein the target polynucleotide or nucleic acid sequence is derived from the parasite or is a host-derived polynucleotide whose level increases in response to infection with the parasite, and the test AV resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is greater than the reference AV, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the parasite.

10. The method of claim 1 or 2, wherein the target polynucleotide or nucleic acid sequence is a host-derived polynucleotide whose level reduces in response to infection with the parasite and the test AV resulting from hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence is less than the reference AV, optionally by a certain absolute amount (e.g., by at least about 0.015 or 0.020 mV, or by at least about 0.02 V) or by a certain relative amount (e.g., by at least about 20%, 30%, 50% or 100%), indicating presence of the parasite.

11. The method of claim 1 or 2, wherein the test AV is substantially similar to (e.g., within about 10% or 20% of) the reference AV, indicating absence of the disease, the pathogen or the parasite.

12. The method of any one of the preceding claims, which comprises: contacting a plurality of (e.g., about 4, 8, 16, 32, 64 or more) bio-nanosensors with the sample; comparing the test AV generated by each of the bio-nanosensors to the reference AV; and determining presence of the disease, the pathogen or the parasite if comparing the test AV to the reference AV for a majority of the bio-nanosensors indicates presence of the disease, the pathogen or the parasite, or determining absence of the disease, the pathogen or the parasite if comparing the test AV to the reference AV for a majority or half of the bio-nanosensors indicates absence of the disease, the pathogen or the parasite.

13. The method of any one of the preceding claims, wherein the polynucleotide probes are a plurality of a particular polynucleotide probe.

14. The method of any one of claims 1 to 12, wherein the polynucleotide probes are a plurality of two or more different polynucleotide probes complementary to a particular target polynucleotide or nucleic acid sequence.

15. The method of any one of claims 1 to 12, wherein the polynucleotide probes are a plurality of two or more different polynucleotide probes complementary to two or more different target polynucleotides or/and nucleic acid sequences which are associated with a particular disease, pathogen or parasite.

16. The method of claim 15, wherein the two or more different polynucleotide probes are complementary to two or more different conserved nucleic acid sequences of the genome of a pathogen or a parasite, such as a virus (e.g., SARS-CoV-2 or HIV-1).

17. The method of any one of claims 1 to 12, wherein the polynucleotide probes are a plurality of two or more different polynucleotide probes complementary to two or more different target polynucleotides or/and nucleic acid sequences which are associated with two or more different diseases, pathogens or parasites.

18. The method of any one of the preceding claims, which uses polynucleotide probes complementary to a particular target polynucleotide or nucleic acid sequence to determine absence or presence of a particular disease, pathogen or parasite.

19. The method of any one of claims 1 to 17, which uses a plurality of different polynucleotide probes complementary to a plurality or panel of (e.g., about 2-10, or about 3, 4, 5, 6 or 7) different target polynucleotides or/and nucleic acid sequences to determine absence or presence of a particular disease, pathogen or parasite, whether in the same test or separate tests.

20. The method of any one of the preceding claims, wherein the polynucleotide probes are selected from single-stranded polynucleotides comprising DNA residues (e.g., complementary DNAs [cDNAs]) or RNA residues, and optionally unnatural bonds (e.g., phosphorothioate/thiophosphate or phosphorodiamidate bonds) linking the nucleotides; singlestranded polynucleotides comprising nucleotide analogs (e.g., xeno nucleic acids [XNAs] such as locked nucleic acids [LNAs]) and optionally unnatural bonds (e.g., phosphorothioate/ thiophosphate or phosphorodiamidate bonds) linking the nucleotides; and single-stranded polynucleotides comprising DNA residues or RNA residues, and nucleotide analogs (e.g., XNAs such as LNAs), and optionally unnatural bonds (e.g., phosphorothioate/thiophosphate or phosphorodiamidate bonds) linking the nucleotides.

21. The method of claim 20, wherein the polynucleotide probes are cDNAs.

22. The method of claim 20 or 21 , wherein the polynucleotide probes comprise about 10-35, 15-30 or 20-25 nucleotides.

23. The method of any one of the preceding claims, wherein the polynucleotide probes are associated with or bound to the outer surface of the carbon nanotubes by van der Waals force.

24. The method of claim 23, wherein the polynucleotide probes have a number of (e.g., about 5-10) additional nucleotide residues at the 5’ or 3’ end of the probes which are designed to increase van der Waals interaction with the carbon nanotubes and not to hybridize to the target polynucleotide or nucleic acid sequence.

25. The method of any one of claims 1 to 22, wherein the carbon nanotubes are impregnated with gold nanoparticles and the polynucleotide probes have a thiol group (e.g., a thiol group such as a cysteamine group which is part of a phosphorodiamidate group at the 3’ or 5’ end of the probes) which adsorbs onto the gold nanoparticles.

26. The method of any one of the preceding claims, wherein: the target polynucleotide is mRNA, ncRNA (e.g., sRNA, miRNA or IncRNA), other single-stranded RNA (ssRNA), single- stranded DNA (ssDNA), or double- stranded DNA (dsDNA); or the target nucleic acid sequence is a DNA or RNA sequence of the genome of a pathogen (e.g., a virus or a bacterium) or a parasite (e.g., a protozoan).

27. The method of claim 26, wherein the target polynucleotide is miRNA.

28. The method of any one of the preceding claims, wherein the carbon nanotubes are or comprise single-wall carbon nanotubes (SWCNTs).

29. The method of claim 28, wherein the SWCNTs have a diameter of about 0.5-5 nm and a length of about 2-30 microns.

30. The method of any one of claims 1 to 27, wherein the carbon nanotubes are or comprise multi- wall carbon nanotubes (MWCNTs).

31. The method of claim 30, wherein the MWCNTs have: about 2-6 or 6-12 substantially concentric layers of graphene; a diameter of about 5-50 nm or 50-100 nm; and a length of about 2-30 microns.

32. The method of any one of the preceding claims, wherein the sample is a biological sample.

33. The method of claim 32, wherein the biological sample is or comprises saliva, blood, plasma, serum, cerebrospinal fluid, urine, stool, buccal scrape or nasal scrape.

34. The method of any one of claims 1 to 31, wherein the sample is an environmental sample.

35. The method of claim 34, wherein the environmental sample is a water sample from a water-treatment facility (e.g., a sample of wastewater or treated water), an industrial facility (e.g., a sample of wastewater), a business, a domestic residence, a body of water (e.g., a lake, a river or a stream), or a pool of water (e.g., a puddle).

36. The method of any one of the preceding claims, wherein the disease is selected from tumors, cancers, bone disorders, cardiovascular disorders, cerebrovascular disorders, fibrotic disorders, immune-related disorders (e.g., inflammatory disorders, autoimmune disorders and allergies), liver and hepatobiliary disorders, gastrointestinal disorders, metabolic disorders, neurological disorders (including neurodegenerative disorders), eye disorders, genetic disorders, and disorders in response to infections (e.g., sepsis).

37. The method of claim 36, wherein the disease is a tumor or cancer.

38. The method of any one of claims 1 to 35, wherein the pathogen is selected from viruses, viroids, bacteria, protozoa, fungi and algae.

39. The method of any one of claims 1 to 35, wherein the parasite is selected from protozoa, helminths and insects.

40. The method of any one of the preceding claims, which can detect the absence or presence of the disease, the pathogen or the parasite with an accuracy of at least about 90%, 95% or 98%.

41. The method of any one of the preceding claims, wherein the bio-nanosensor(s) is/are prepared with the polynucleotide probes primed on the outer surface of the carbon nanotubes.

42. The method of any one of claims 1 to 40, further comprising contacting the bio- nanosensor(s) with the polynucleotide probes shortly (e.g., within about 3, 5 or 10 minutes) prior to contacting the bio-nanosensor(s) with the sample.

43. The method of claim 42, further comprising subtracting the AV resulting from contacting the bio-nanosensor(s) with the polynucleotide probes from the test AV resulting from contacting the bio-nanosensor(s) with the sample.

44. The method of any one of the preceding claims, further comprising quantifying the level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in the sample based on the test AV.

45. The method of claim 44, wherein the level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in the sample is calculated using an equation (e.g., a substantially linear equation) formulated from changes in voltage resulting from contacting a plurality of bio-nanosensors with a plurality of samples containing known levels of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite.

46. The method of claim 44 or 45, further comprising determining the status (e.g., the severity or/and the stage) of the disease, and the efficacy of any ongoing treatment, based on the level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in the sample.

47. The method of claim 46, wherein the status of the disease and the efficacy of any ongoing treatment are determined based on comparison of the present level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in the present sample to a, or the, previous level of the target polynucleotide or nucleic acid sequence, the pathogen or the parasite in a, or the, previous sample.

48. A biodetection device for detecting absence or presence of a disease, a pathogen or a parasite, comprising: a biosensor module comprising bio-nanosensors, wherein: each bio-nanosensor comprises carbon nanotubes and graphene; polynucleotide probes complementary to a target polynucleotide or nucleic acid sequence are associated with an outer surface of the carbon nanotubes; the target polynucleotide or nucleic acid sequence is associated with a disease, a pathogen or a parasite; the bio-nanosensors are configured to generate an analog electrical signal when the polynucleotide probes on the carbon nanotubes hybridize to the target polynucleotide or nucleic acid sequence; the graphene facilitates thermal and electrical conduction; and the biosensor module is configured to receive or contact a sample; a heater configured to heat the biosensor module to an optimum temperature for hybridization between the polynucleotide probes and the target polynucleotide or nucleic acid sequence and to maintain the biosensor module at the optimum temperature during testing; a relay configured to control the temperature of the heater and the biosensor module; a thermistor configured to measure the temperature of the heater and the biosensor module; an AD (analog-to-digital) converter configured to convert analog electrical signals from the biosensor module into digital electrical signals; and a microcontroller configured to receive AD-converted electrical signals and data from the biosensor module via the AD converter, to transfer data, to obtain temperature readings from the thermistor, and to control the heater via control of the relay; wherein the biodetection device is configured to connect to a computer device and to perform the method of any one of claims 1 to 47.

49. The biodetection device of claim 48, further comprising a connector board electrically connected to the biosensor module and the AD converter and configured to receive analog electrical signals from the biosensor module and to transfer the signals to the AD converter.

50. The biodetection device of claim 48 or 49, further comprising a sample-collection module configured to collect a sample and to bring the sample into contact with the bio-nanosensors.

51. The biodetection device of any one of claims 48 to 50, wherein the computer device comprises: software installed in the computer device or obtained from a remote server or the digital cloud, and configured to provide instructions for operating the biodetection device and performing the method of any one of claims 1 to 47, obtain data (e.g., voltage vs time) from the microcontroller of the biodetection device, process data (e.g., calculate the change in voltage [AV] between two timepoints), analyze data (e.g., analyze AV data for trends or patterns), and formulate and provide results; a memory coupled to the software and configured to store information, data and instructions; and a processor coupled to the software and the memory and configured to execute instructions and operations, to process and analyze data, and to perform the method of any one of claims 1 to 47.

52. The biodetection device of any one of claims 48 to 51 , wherein the biodetection device is configured to connect to the computer device via a cable, such as a USB Type-A or Type-C cable.

53. The biodetection device of any one of claims 48 to 51, wherein the biodetection device is configured to connect to the computer device via a wireless connection, such as Wi-Fi or Bluetooth.

54. The biodetection device of any one of claims 48 to 53, wherein the computer device is a laptop computer, a desktop computer, a tablet or a smartphone.

55. The biodetection device of any one of claims 48 to 54, wherein the bio-nanosensors generate an analog signal voltage when the polynucleotide probes on the carbon nanotubes hybridize to the target polynucleotide or nucleic acid sequence, and the microcontroller receives voltage vs time data from the biosensor module via the AD converter.

56. The biodetection device of any one of claims 48 to 55, wherein the optimum temperature for testing is about 45-70 °C, 50-70 °C or 55-65 °C.

57. The biodetection device of any one of claims 48 to 56, wherein the carbon nanotubes are or comprise single-wall carbon nanotubes (SWCNTs).

58. The biodetection device of claim 57, wherein the SWCNTs have a diameter of about 0.5- 5 nm and a length of about 2-30 microns.

59. The biodetection device of any one of claims 48 to 56, wherein the carbon nanotubes are or comprise multi-wall carbon nanotubes (MWCNTs) comprising about 2-6 or 6-12 substantially concentric layers of graphene.

60. The biodetection device of claim 59, wherein the MWCNTs have a diameter of about 5- 50 nm or 50-100 nm and a length of about 2-30 microns.

61. The biodetection device of any one of claims 48 to 60, wherein the graphene of each bionanosensor is a multi-layer graphene or nano-graphite platelet comprising about 5-30 layers of graphene, or about 5-10, 10-20 or 20-30 layers of graphene.

62. The biodetection device of any one of claims 48 to 61 , wherein the biosensor module comprises about 4, 8, 16, 32, 64, 128 or more bio-nanosensors.

63. The biodetection device of any one of claims 48 to 62, wherein the biosensor module and the optional sample-collection module are disposable.

64. The biodetection device of any one of claims 48 to 63, wherein the biosensor module is manufactured with the polynucleotide probes primed on the outer surface of the carbon nano tubes.

65. The biodetection device of any one of claims 48 to 63, wherein the polynucleotide probes are added to the bio-nanosensors shortly (e.g., within about 3, 5 or 10 minutes) prior to addition of the sample to the bio-nanosensors.

66. The biodetection device of any one of claims 51 to 65, wherein the software sets a threshold value of AV (e.g., about 0.015 or 0.020 mV, or about 0.02 V) between two (e.g., predetermined) timepoints equal to or above which the AV calculated for a particular bio-nanosensor is deemed a positive reading and below which the AV is deemed a negative reading, and the software determines a positive result (presence of a disease, a pathogen or a parasite) if the majority of the bio-nanosensors provide positive readings or a negative result (absence of a disease, a pathogen or a parasite) if the majority or half of the bio-nanosensors provide negative readings.

67. The biodetection device of any one of claims 48 to 66, which further comprises a lightemitting diode (LED) which indicates a qualitative result of the test by a different color of light, such as a red light for a positive result, a green light for a negative result, or a yellow or orange light for an inconclusive result.

68. The biodetection device of any one of claims 48 to 67, wherein the computer device provides a qualitative result of the test, and optionally a quantitative result of the test (e.g., the level of the target polynucleotide or nucleic acid sequence, a pathogen or a parasite in the sample), on the computer device, such as on the screen or in a Results page or file of the computer device.

69. The biodetection device of any one of claims 48 to 68, wherein the computer device provides a qualitative result of the test, and optionally a quantitative result of the test, to the subject providing the sample if a biological sample and the person (e.g., a medical or veterinary practitioner) overseeing the test, and optionally a government or health authority, agency or department if reporting of such result(s) thereto is required.

70. The biodetection device of any one of claims 48 to 69, which is capable of providing a qualitative result or/and a quantitative result of the test within about 15 or 20 minutes after addition of the sample to the biosensor module.

71. The biodetection device of any one of claims 48 to 70, which can be plugged into an electrical socket or powered by a battery.

72. The biodetection device of any one of claims 48 to 71, which is portable.

73. The biodetection device of any one of claims 48 to 72, which is operable at a point of care, such as at a medical office, a medical clinic, an out-patient clinic, a hospital, a pharmacy, a nursing home, a veterinary office, a veterinary clinic or a veterinary hospital.

74. The biodetection device of any one of claims 48 to 72, which is operable in the field, such as at a mobile medical clinic, a humanitarian medical clinic, a mobile veterinary practice or clinic, or a farm.