Multiplex target detection using graphene-based FET sensory array

Abstract

This disclosure generally relates to graphene-based diagnostic sensor devices. More particularly, the disclosure relates to graphene-based diagnostic sensor devices for the multiplex detection of target nucleic acids in fluid samples, including biological fluid samples. In certain aspects, the graphene-based multiplex diagnostic devices disclosed herein comprise a graphene-based FET sensor array chip, wherein each sensor in the array comprises an independent sensing regions, each sensing region having a nucleic acid probe attached to a graphene monolayer, for hybridizing and detecting a target nucleic acid.

Description

MULTIPLEX TARGET DETECTION USING GRAPHENE-BASED FET SENSOR ARRAYCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 671 ,309, filed July 15, 2024, the content of which is hereby incorporated by reference in its entirety into this disclosure.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] This application contains a Sequence listing that has been submitted in a computer readable format and is hereby incorporated by reference in its entirety. The XML file, created on July 14, 2025, is named 67664WO01_SequenceListing.xml, and is 41 ,827 bytes in size.FIELD OF THE INVENTION

[0003] The present technology relates in general to graphene-based FET sensor array devices for the point-of-care multiplex detection of multiple infectious agents and antimicrobial resistance. More particularly, the present technology relates to graphenebased FET sensor array devices for the multiplex detection of target nucleic acids in fluid samples, including biological fluid samples derived from infectious agents.BACKGROUND

[0004] Many of the deadliest infectious diseases in the world can be prevented through timely diagnosis and treatment. However, current diagnostic tools have several limitations, including an inability to rapidly and accurately detect the presence or absenceof multiple infectious agents (and optionally antimicrobial resistance) simultaneously, efficiently, and at the point-of-care.

[0005] Therefore, alternative diagnostic tools are urgently needed, and the present disclosure provides graphene-based diagnostic sensor devices for the multiplex point-of- care detection of multiple target nucleic acids in biological samples.

[0006] Limitations and disadvantages of traditional sensors will become apparent to one of skill in the art, through comparison of such devices with certain aspects of the devices set forth in the remainder of this disclosure, including with reference to the claims.BRIEF SUMMARY

[0007] The present disclosure generally relates to graphene-based FET sensor arrays for the simultaneous multiplex detection of multiple target nucleic acids, including nucleic acids derived from infectious agents (and optionally genes related to the development of antimicrobial resistance). In this aspect, each sensor in the graphene-based FET sensor array comprises an independent sensing region having a graphene monolayer, and a unique nucleic acid probe bound to the graphene monolayer, wherein the nucleic acid probe is complementary to a specified target nucleic acid.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1 illustrates a top view of an example graphene-based FET sensor array, in accordance with various example implementations of this disclosure.DETAILED DESCRIPTION

[0009] While the present technology will be described in connection with one or more preferred embodiments, it will be understood by those skilled in the art that the technology is not limited to only those particular embodiments. To the contrary, the presently described technology includes all alternatives, modifications, and equivalents as may be included within the spirit and scope of the appended claims.

[0010] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.

[0011] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0012] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0013] Generally, the present disclosure is directed to devices incorporating a graphene-based FET sensor array for the multiplex detection of multiple target nucleic acids in fluid samples. In one aspect, the graphene-based FET sensor array (or sensor array chip) is coupled to a sample collector. Each independent sensor of the graphene-based FET sensor array comprises a sensing region having a graphene monolayer, and a unique nucleic acid probe bound to the graphene monolayer.

[0014] The nucleic acid probes used herein are generally single stranded synthetic deoxyribonucleic acid, and are complementary to target nucleic acids of interest. As used herein, the term “complementary” does not require that a sequence (e.g., a nucleic acid probe) is complementary over the full-length of a complementary strand (e.g., the target nucleic acid of interest), and can encompasses a sequence that is complementary to a portion of another sequence.

[0015] For example, a nucleic acid probe sequences can be complementary to a target nucleic acid sequence over a length ranging from about 2 to about 100 consecutive (contiguous) nucleotides, or any integer between 2 and 100. In some embodiments, the nucleic acid probe can be complementary to the target nucleic acid other over a length ranging from about 15 to about 30 consecutive (contiguous) nucleotides, or any integer between 15 and 30.

[0016] The nucleic acid probes used herein are complementary to target nucleic acids of interest such that the nucleic acids probes are capable of hybridizing (i.e. , capable of forming a stable double stranded duplex via Watson-Crick base-pairing) with target nucleic acids. Despite some amount of mismatches, nucleic acid probe sequences generally have the ability to selectively hybridize to the designated target nucleic acid under appropriate conditions such as, for example, stringent and highly stringent conditions, such as those generally known by those of ordinary skill in the art.

[0017] The diagnostic sensor devices disclosed herein are for the multiplex detection of target nucleic acids in biological fluid, biological solid, non-biological fluid, or non-biological solid samples. The term target nucleic acid generally refers to a nucleic acid of interest, and can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and can be double stranded or single stranded.

[0018] Target nucleic acids of interest include nucleic acids derived from infectious agents, including, for example, viral, bacterial, protozoa, and / or fungal infectious agents. Target nucleic acids can also be derived from genes conferring antimicrobial drug resistance. In some embodiments, the target nucleic acid may comprise genomic DNA, genomic RNA, mRNA, or cDNA wherein target DNA is created using isolated transcripts from a biological sample (i.e., wherein mRNA is reverse transcribed into using conventional techniques).

[0019] Target nucleic acids derived from bacterial sources include nucleic acids derived from, for example, Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Haemophilus influenzae, Haemophilus parainfluenzae, Stenotrophomonas maltophilia, Chlamydophila pneumoniae, Moraxella catarrhalis, Achromobacter, Serratia marcescens, Escherichia coli, Acinetobacter, Enterobacter cloacae, Proteus mirabilis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium abscessus, Mycobacterium kansasii, and Mycobacterium gordonae.

[0020] Target nucleic acids derived from viral sources include, for example, nucleic acids derived from either DNA viruses or RNA viruses. DNA viruses include, for example, Adenoviruses (e.g., Human adenoviruses (types 3, 4, and 7)), Herpesviruses (e.g., Herpes simplex, varicella zoster, Epstein-Barr virus, cytomegalovirus, and Kaposi’s sarcoma), Poxviruses (e.g., Vaccinia virus), Parvoviruses (e.g., Human parvovirus),Papovaviruses (e.g., Papilloma virus), and Hepadnaviruses (e.g., Hepatitis B virus). RNA viruses include, for example, Orthomyxoviruses (e.g., Influenza virus), Paramyxoviruses (e.g., Mumps, measles, respiratory syncytial virus), Coronaviruses (e.g., common cold viruses), Picornaviruses (e.g., Polio, coxsackie, hepatitis A, and rhinovirus), Reoviruses (e.g., Rotavirus, and reovirus), Togaviruses (e.g., Rubella, and arthropod-borne encephalitis), Flaviviruses (e.g., arthropod-borne viruses (yellow fever, dengue fever), Arenaviruses (e.g., Lymphocytic choriomeningitis, and Lassa fever), Rhabdoviruses (e.g., Rabies), and Retroviruses (e.g., Human T-cell leukemia virus, and HIV).

[0021] Target nucleic acids derived from fungal sources include, for example, nucleic acid derived from Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Paracoccidioides, Aspergillus, Mucormycetes, Pneumocyctis, Taloromyces, and Sporothrix.

[0022] Target nucleic acids derived from protozoal sources include, for example, nucleic acid derived from Sarcodina (e.g., Entamoeba), Mastigophora (e.g., Giardia, and Leishmania), Ciliophora (e.g., Balantidium), and Sporozoa (e.g., Plasmodium, and Cryptosporidium).

[0023] Target nucleic acids can also be derived from genes conferring antimicrobial drug resistance, including, for example, nucleic acid derived from extended-spectrum p- lactamases, CTX-M beta-lactamases, Carbapenemases, Klebsiella pneumoniae carbapenemase, New Delhi metallo beta lactamase, OXA-48-like carbapenemases, Verona integron-encoded metallo-p-lactamase, IMP-type metallo-p-lactamase, and methicillin resistance.

[0024] In one aspect of the disclosure, devices are provided having a graphene-based FET sensor array chip coupled to a sample collector, wherein each sensor in the sensor array has a separate and independent sensing region. Each separate and independent sensing region further comprises a graphene monolayer, and a unique nucleic acid probe bound to the graphene monolayer. Figure 1 illustrates a top view of an example graphenebased FET sensor array, in accordance with various example implementations of this disclosure. In this example, the terminals of the graphene-based FET sensor array comprise a common source terminal 101 , a common gate terminal 107 and 98 drain terminals 103. Each of the 98 drain terminals 103 is connected to a graphene sensor 105.

[0025] In the embodiment illustrated in Figure 1 , the sensors 105 are in 20 groups of 4 sensors, 8 groups of 2 sensors, and 2 individual sensors. This particular grouping of 98 sensors 105 is an example design, and a smaller or larger number of sensors is also within the scope of this disclosure. For example, the sensor array can comprise from 60 to 120 sensors, from 70 to 120 sensors, from 80 to 120, from 90 to 120 sensors, from 60 to 100 sensors, from 70 to 100 sensors, from 80 to 100, or from 90 to 100 sensors. Further, the response of each sensor 105 may be measured by the corresponding drain 103, and a reader may read all sensors 105 simultaneously, or in groups.

[0026] Each sensor in the graphene-based FET sensor array comprises an independent sensing region having a nucleic acid probe capable of hybridizing with one or more target nucleic acids. For example, the following Table provides a representative list of nucleic acid sequences that can be used as either probes (wherein the complementof the listed sequence is the target) or targets (wherein the complement of the listed sequence can be used as a probe):

[0027] Additional nucleic acid sequences that can be used as either probes (wherein the complement of the listed sequence is the target) or targets (wherein the complement of the listed sequence can be used as a probe) for the detection of pathogens can be found in the following reference: Ma et al. BMC Microbiology (2020) 20:177 (https: / / doi.org / 10.1186 / s12866-020-01842-3). The entirety of this reference is incorporated by reference herein.

[0028] Additional nucleic acid sequences that can be used as either probes (wherein the complement of the listed sequence is the target) or targets (wherein the complement of the listed sequence can be used as a probe) for the detection of Antimicrobial Resistance Genes can be found in the following reference: European Reference Laboratory for Antimicrobial Resistance (EURL-AR, Technical University of Denmark, National Food Institute). List of Primers for Detection of Antimicrobial Resistance Genes. (Available online: https: / / www.eurl-ar.eu / CustomerData / Files / Folders / 25- resourcer / 459_primerliste-til-web-07-11 -2018.pdf).) The entirety of this reference is incorporated by reference herein.

[0029] The nucleic acid sequences listed and disclosed herein can be used as probes in the devices described herein, wherein the complement of the listed sequence is the target. Likewise, the nucleic acid sequences listed and disclosed herein can be targets of interest, wherein the complement of the listed sequence can be used as a probe in the devices described herein.

[0030] In certain aspects, the nucleic acid sequences listed and disclosed herein, when used as a probe in the devices disclose herein, can be 5’ or 3’ terminally modified. For example, the nucleic acid sequences listed and disclosed herein, when used as a probe in the devices disclose herein, can be can be 5’ or 3’ terminally modified with: NH2- poly C; NH2-poly A; NH2-poly T; or NH2-poly G (wherein the poly C, A, T, or G comprises from 4 to 6 bases, 4 to 8 bases, 4 to 10 bases, 4 to 12 bases, 4 to 14 bases, 4 to 16 bases, 4 bases, 5 bases, 6 bases, 8 bases, 10 bases, 11 bases, or 12 bases).

[0031] In certain aspects, each sensor in the graphene-based FET sensor array comprises an independent sensing region having a nucleic acid probe, wherein the nucleic acid probe is an isolated / synthetic nucleic acid probe having a sequence that is 100% complementary, or from 90% to 99% complementary, to a sequence found within a specified target nucleic acid. In another aspect, the nucleic acid probe is an isolated / synthetic nucleic acid probe having a sequence that is 90% complementary, 91% complementary, 92% complementary, 93% complementary, 94% complementary, 95% complementary, 96% complementary, 97% complementary, 98% complementary, 99% complementary to a sequence found within a specified target nucleic acid.

[0032] In another aspect, one or more sensors in the graphene-based FET sensor array comprise multiple different isolated / synthetic nucleic acid probes for identifying thepresence of multiple infectious agent species within the same genus. For example, the sensor region of one or more sensors, within the graphene-based FET sensor array, can comprise 2 different isolated / synthetic nucleic acid probes for identifying the presence of2 different infectious agent species within the same genus. In a further aspect, the sensor region of one or more sensors, within the graphene-based FET sensor array, can comprise 3 different isolated / synthetic nucleic acid probes for identifying the presence of3 different infectious agent species within the same genus. In a still further aspect, the sensor region of one or more sensors, within the graphene-based FET sensor array, can comprise 4 or more different isolated / synthetic nucleic acid probes for identifying the presence of 4 or more different infectious agent species within the same genus.

[0033] In one aspect, the graphene-based FET sensor array chip comprises multiple independent sensing regions for the simultaneous detection of target nucleic acids derived from Streptococcus pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Haemophilus influenzae, Haemophilus parainfluenzae, Stenotrophomonas maltophilia, Chlamydophila pneumoniae, Moraxella catarrhalis, Achromobacter, Serratia marcescens, Escherichia coli, Acinetobacter, Enterobacter cloacae, Proteus mirabilis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium gordonae, or combinations thereof.

[0034] In yet another aspect, the graphene-based FET sensor array chip comprises multiple independent sensing regions for the simultaneous detection of target nucleic acids derived from genes conferring antimicrobial drug resistance, including, for example, extended-spectrum p-lactamases, CTX-M beta-lactamases, Carbapenemases,Klebsiella pneumoniae carbapenemase, New Delhi metallo beta lactamase, OXA-48-like carbapenemases, Verona integron-encoded metallo-p-lactamase, IMP-type metallo-p- lactamase, and methicillin resistance.

[0035] The present invention should not be considered limited to the particular examples described above. Various modifications, equivalent processes, as well as numerous structures to which the present disclosure may be applicable, and which fall within the general scope of the disclosure, will be readily apparent to those of skill in the art to which the present disclosure is directed upon review of the instant specification.

[0036] What is more, as will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several aspects, without departing from the scope or spirit of the present disclosure.

Claims

CLAIMSWhat is claimed is:1 . A diagnostic device for the simultaneous detection of multiple target nucleic acids, the device comprising: a graphene-based FET sensor array, wherein each sensor in the sensor array has an independent sensing region comprising: a graphene monolayer; and a unique nucleic acid probe bound to the graphene monolayer; wherein the nucleic acid probe is a synthetic deoxyribonucleic acid hybridizing to a target nucleic acid derived from an infectious agent, a gene related to the development of antimicrobial resistance, or a combination thereof.

2. The diagnostic device of claim 1 , wherein the infectious agent is viral, bacterial, protozoan, or fungal.

3. The diagnostic device of claim 2, wherein the viral infectious agent is a DNA virus or RNA virus.

4. The diagnostic device of claim 3, wherein the DNA virus is an Adenovirus, a Herpesvirus, a Poxvirus, a Parvovirus, a Hepadnavirus, or a combination thereof.

5. The diagnostic device of claim 3, wherein the RNA virus is an Orthomyxovirus, a Paramyxovirus, a Picornavirus, a Reovirus, a Togavirus, a Flavivirus, an Arenavirus, or a Retrovirus.

6. The diagnostic device of claim 2, wherein the bacterial infectious agent is Streptococcus, Staphylococcus, Pseudomonas, Klebsiella, Haemophilus, Stenotrophomonas, Chlamydophila, Moraxella, Achromobacter, Serratia, Escherichia, Acinetobacter, Enterobacter, Proteus, Mycobacterium, Mycobacterium, Mycobacterium, Mycobacterium, Mycobacterium, or a combination thereof.

7. The diagnostic device of claim 2, wherein the fungal infectious agent is Candida, Blastomyces, Coccidioides, Cryptococcus, Histoplasma, Paracoccidioides, Aspergillus, Mucormycetes, Pneumocyctis, Taloromyces, Sporothrix, or a combination thereof.

8. The diagnostic device of claim 2, wherein the protozoan infectious agent is Sarcodina, Mastigophora, Ciliophora, Sporozoa, or a combination thereof.

9. The diagnostic device of claim 1 , wherein the gene related to the development of antimicrobial resistance is an extended-spectrum p-lactamase, a CTX-M beta-lactamase, a Carbapenemase, a Klebsiella pneumoniae carbapenemase, a New Delhi metallo beta lactamase, a OXA-48-like carbapenemase, a Verona integron- encoded metallo-p-lactamase, an IMP-type metallo-p-lactamase, or a combination thereof.

10. The diagnostic device of claim 1 , wherein the diagnostic device simultaneously detects the presence or absence of target nucleic acid derived from S. pneumoniae, S. aureus, P. aeruginosa, K. pneumoniae, H. influenzae, Haemophilus parainfluenzae, Stenotrophomonas maltophilia, Chlamydophila pneumoniae, Moraxella catarrhalis, Achromobacter, Serratia marcescens, Escherichia coli, Acinetobacter, Enterobacter cloacae, Proteus mirabilis, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium abscessus, Mycobacterium kansasii, and Mycobacterium gordonae.11 . The diagnostic device of claim 1 , wherein the diagnostic device detects the presence or absence of target nucleic acids derived from one or more genes related to the development of antimicrobial resistance.

12. The diagnostic device of claim 1 , wherein the graphene-based FET sensor array comprises from 80 to 100 individual sensors.

13. The diagnostic device of claim 1 , wherein the graphene-based FET sensor array comprises from 90 to 100 individual sensors.

14. The diagnostic device of claim 1 , wherein the graphene-based FET sensor array comprises separate groups of sensors sharing a common source terminal.

15. The diagnostic device of claim 14, wherein the graphene-based FET sensor array comprises a group of 4 sensors, a group of 3 sensors, a group of 2 sensors, or combinations thereof.

16. The diagnostic device of any of claims 1 -10, wherein the nucleic acid probe bound to the graphene monolayer is 100% complementary to the target nucleic acid.

17. The diagnostic device of any of claims 1 -10, wherein the nucleic acid probe bound to the graphene monolayer is from 90% to 99% complementary to the target nucleic acid.

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