Detection of nucleic acid sequences

Abstract

The present disclosure provides an enzyme-free, amplification-free optical method to detect ultra-low concentrations of microbial nucleic acids. The technique relies on detecting high signal-to-noise signals (e.g., fluorescent readouts of hybridization) between target nucleic acid sequences and complementary probes immobilized on the surface of a detection cell. The technique simplifies and accelerates diagnosis workflow, significantly reduces costs, and outperforms existing methods that rely heavily on the use of enzymes, are often time-consuming, and require specialized equipment and trained personnel.

Description

DETECTION OF NUCLEIC ACID SEQUENCESCROSS REFERENCE TO RELATED APPLCATION

[0001] The instant application claims priority to U.S. Provisional Patent Application No. 63 / 678,502, filed on August 1, 2024, the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND OF THE DISCLOSURE

[0002] Pathogenic infections can severely impact human health leading to lifethreatening conditions and disease outbreaks. Consequently, the ability to detect pathogens such as bacteria, viruses, fungi, and parasites from infected individuals or food or environmental samples is crucial for public health, understanding disease etiology, tracking antimicrobial resistance, and developing targeted therapeutics. This necessitates concerted efforts to efficiently diagnose different pathogens to control their spread and facilitate treatment. Currently, diagnostic tests are based on nucleic acid or antigen detection. Nucleic acid-based detection methods have revolutionized the field of diagnostics, offering sensitive and specific identification of infectious agents. These techniques leverage the genetic material of pathogens, such as DNA or RNA, relying on the principles of nucleic acid hybridization wherein complementary nucleic acid probes bind to target sequences, followed by the detection of the hybridized complexes. With the limitations of detection sensitivity, many of these methods, if not all, use polymerase chain reaction (PCR), reverse transcription PCR or loop-mediated isothermal amplification (LAMP)— based amplification methods to increase the sample concentration. Amplification of the target nucleic acids to boost sensitivity involves lengthy procedures that are expensive and complex and require specialized equipment and trained personnel and are therefore not conducive for swift point- of-care deployment. Additionally, preamplification increases the risk of contamination during multiple steps. Moreover, in the event of a widespread disease outbreak or pandemic, the specific enzymes and reagents are in high demand and navigating complex supply chains becomes highly limiting and challenging. Nonetheless, hybridization-based methods continue to evolve with advancements in technology, including the development of novel probes, detection platforms, and multiplexing strategies. There is a need for developing amplification-free simple diagnostic techniques with high sensitivity that could accelerate diagnosis workflow, and significantly reduce costs with multiplexing capabilities and point- of-care applications.BRIEF SUMMARY OF THE DISCLOSURE

[0003] The present disclosure provides an enzyme-free, amplification-free optical method to detect ultra-low concentrations of microbial nucleic acids. The technique relies on detecting high signal-to-noise signals (e.g., fluorescent readouts of hybridization) between target nucleic acid sequences and complementary probes immobilized on the surface of a detection cell. The technique simplifies and accelerates diagnosis workflow, significantly reduces costs, and outperforms existing methods that rely heavily on the use of enzymes, are often time-consuming, and require specialized equipment and trained personnel.

[0004] The present disclosure provides single-molecule detection of nucleic acid sequences. After extraction of target nucleic acids, a sample comprising the nucleic acids is incubated in an engineered detection chamber and then washed with a solution containing a DNA-binding dyes that allow the detection of the remaining substrate-bound sequences. Those bound sequences hybridize with a specific short nucleic acid (e.g., DNA, RNA, or PNA) probe immobilized on the sample chamber substrate. The sample chamber is passivated (e.g., coated with PEG or other reagents) such that it inhibits non-specific binding of nucleic acids. The probes are immobilized to the substrate (e.g., sample chamber) either through an avidin-biotin bridge, covalently, or through other means. In various embodiments, fluorescence from the hybridized surface-bound sequences is collected using a microscope objective and detected on a camera. To establish detection, this fluorescence is compared to that in a negative control where the sample is not introduced, or the probes are not specific, or both. A positive control using probes targeting a host sample could also be used. The attached figures illustrate the principle of the detection as well as experimental demonstration of detection as low as about 1 aM concentrations (-600 target molecules in 1 ml of sample) or lower.

[0005] In an aspect, the present disclosure provides a method for detecting one or more nucleic acid sequences in a sample. The sample may be various media comprising the one or more nucleic acid sequences.BRIEF DESCRIPTION OF THE FIGURES

[0006] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0007] Figure 1. (a) Schematic diagram emphasizing the concept of detection in the enlarged inset. The target microbial sequence hybridizes with the substrate-bound specificprobes and recruits a DNA-binding dye making the hybridized sequence brighter. Light emitted from individual molecules is detected on a camera or photon detector, (b) Schematic showing customized sample chamber substrate in the enlarged inset. The substrate is passivated using PEG (e.g., covalently bound) or other passivating agents and / or other surfactants that inhibit non-specific binding of other molecules. A small fraction of the PEG is conjugated to biotin which recruits the biotin-labelled probes through a streptavidin bridge, (c) Images of a substrate to which the single-stranded probe is attached in the presence of the DNA-binding dye only (left) and the target sequence and the dye (right). Both images have the same intensity scale, and the substrate is incubated with a 1 fM concentration of the nucleic acid solution. Scale bars are 5 pm. (d) Intensities of individual spots recorded in the presence of the target and which have intensities larger than the largest signal recorded in the presence of the probe only are plotted in the histogram at the bottom.

[0008] Figure 2. Histograms of intensities of individual spots from samples where the nucleic acids are incubated with the substrate at concentrations of (a) 100 aM (b)10 aM, and (c)l aM (top to bottom). At 1 aM, there are as few as -600 molecules in a volume of 1ml. (d) Images of a substrate to which the single-stranded probe is attached in the presence of the DNA-binding dye only (left) and the SARS-COV-2 RNA and the dye (right). Both images have the same intensity scale and the substrate is incubated with a IpM concentration of the nucleic acid solution. Scale bars are 5pm. (e) Intensities of individual spots recorded in the presence of the target and which have intensities larger than the largest signal recorded in the presence of the probe only are plotted in the histogram at the bottom.

[0009] Figure 3. (a) Bacteriophage lambda DNA was deposited on the surface of a non-passivated chamber (a) and a passivated chamber (b). In both cased the chambers were washed several times after depositing the DNA, and then supplemented with the intercalating dye for visualization. Scale bars are 5pm.

[0010] Figure 4. The DNA-binding dye (SYBRSafe) shows enhanced fluorescence when bound to 10 nM double-stranded DNA (dsDNA) when compared to 10 nM singlestranded DNA (ssDNA).

[0011] Figure 5. Histograms of intensities of individual fluorescent spots from samples where the probe is attached to the substrate in the presence of the DNA-binding dye only (striped, tan) or the dye and a mismatched target (dotted, green), both at IfM concentration. The distributions are indistinguishable emphasizing the specificity of the detection.

[0012] Figure 6. Schematic illustrating ways in which the signal from individual target nucleic acid sequences is enhanced through binding of the dye to other regions of the sequence.

[0013] Figure 7. Schematic diagram emphasizing the concept of detection using flowing microbeads to which the probe is attached. The target microbial sequence hybridizes with the substrate-bound specific probes and recruits a DNA-binding dye making the hybridized sequence brighter. As the microbeads flow across the region illuminated by the excitation light, fluorescence emitted from individual beads is collected and detected on a photon detector. Beads with hybridized sequences will be brighter. The enlarged inset shows the microbead passivated using PEG (e.g., covalently bound) or other passivating agents and / or other surfactants that inhibit non-specific binding of other molecules. A small fraction of the PEG is conjugated to biotin which recruits the biotin-labelled probes through a streptavidin bridge.DETAILED DESCRIPTION OF THE DISCLOSURE

[0014] Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, process step, and electronic changes may be made without departing from the scope of the disclosure.

[0015] As used herein, unless otherwise indicated, “about”, “substantially”, or “the like”, when used in connection with a measurable variable (such as, for example, a parameter, an amount, a temporal duration, or the like) or a list of alternatives, is meant to encompass variations of and from the specified value including, but not limited to, those within experimental error (which can be determined by, e.g., a given data set, an art accepted standard, etc. and / or with, e.g., a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as, for example, variations of + / - 10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value), insofar such variations in a variable and / or variations in the alternatives are appropriate to perform in the instant disclosure. As used herein, the term “about” may mean that the amount or value in question is the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, compositions, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors,rounding off, measurement error, or the like, or other factors known to those of skill in the art such that equivalent results or effects are obtained. In general, an amount, size, composition, parameter, or other quantity or characteristic, or alternative is “about” or “the like,” whether or not expressly stated to be such. It is understood that where “about,” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0016] Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value) of a range. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “0.1% to 5%” should be interpreted to include not only the explicitly recited values of 0.1% to 5%, but also, unless otherwise stated, include individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5% to 1.1%; 0.5% to 2.4%; 0.5% to 3.2%, and 0.5% to 4.4%, and other possible sub-ranges) within the indicated range. It is also understood (as presented above) that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about, it will be understood that the particular value forms a further disclosure. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0017] The articles “a” and “an” are used in this disclosure to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0018] As used herein, unless otherwise stated or indicated, “s” refers to second(s), “min” refers to minute(s), and “h” refers to hour(s).

[0019] As used herein, unless otherwise stated, the term “group” refers to a chemical entity that is monovalent (i.e., has one terminus that can be covalently bonded to other chemical species), divalent, or polyvalent (i.e., has two or more termini that can be covalentlybonded to other chemical species). The term “group” also includes radicals (e.g., monovalent and multivalent, such as, for example, divalent radicals, trivalent radicals, and the like).Illustrative examples of groups include:

[0020] The present disclosure provides an enzyme-free, amplification-free optical method to detect ultra-low concentrations of microbial nucleic acids. The technique relies on detecting high signal-to-noise signals (e.g., fluorescent readouts of hybridization) between target nucleic acid sequences and complementary probes immobilized on the surface of a detection cell. The technique simplifies and accelerates diagnosis workflow, significantly reduces costs, and outperforms existing methods that rely heavily on the use of enzymes, are often time-consuming, and require specialized equipment and trained personnel.

[0021] The present disclosure provides single-molecule detection of nucleic acid sequences. After extraction of target nucleic acids, a sample comprising the nucleic acids is incubated in an engineered detection chamber and then washed with a solution containing a DNA-binding dyes that allow the detection of the remaining substrate-bound sequences. Those bound sequences hybridize with a specific short nucleic acid (e.g., DNA, RNA, or PNA) probe immobilized on the sample chamber substrate. The sample chamber is passivated (e.g., coated with PEG or other reagents) such that it inhibits non-specific binding of nucleic acids. The probes are immobilized to the substrate (e.g., sample chamber) either through an avidin-biotin bridge, covalently, or through other means. In various embodiments, fluorescence from the hybridized surface-bound sequences is collected using a microscope objective and detected on a camera. To establish detection, this fluorescence is compared to that in a negative control where the sample is not introduced, or the probes are not specific, or both. A positive control using probes targeting a host sample could also be used. The attached figures illustrate the principle of the detection as well as experimental demonstration of detection as low as about 1 aM concentrations (-600 target molecules in 1 ml of sample) or lower.

[0022] In an aspect, the present disclosure provides a method for detecting one or more nucleic acid sequences in a sample. The sample may be various media comprising the one or more nucleic acid sequences.

[0023] The method of the present disclosure allows for a non-specific reporter (e.g., dye) to be rendered specific by: (i) specific binding of a target to a substrate-immobilizedprobe; (ii) blocking / passivation of the substrate so that only specific binding occurs; and (iii) single molecule / single particle detection such that the small signal from the target probe hybrid can be discerned from background (to which the probe-only signal contributes appreciably).

[0024] In various examples, a method of the present disclosure comprises contacting a sample comprising or suspected of comprising one or more nucleic acid sequences with one or more probes. Upon contacting the one or more nucleic acid sequences with the probes, the probes will form a hybrid. In various examples, the one or more probes are disposed on a substrate, where the substrate has one or more passivated surfaces. In various other examples, the one or more probes are a component of a mixture. In such an example, the one or more probes and sample are mixed; then, after mixing, the mixture is contacted with a substrate having one or more passivated surfaces, such that the one or more probes and one or more probes hybridized with the one or more nucleic acid sequences bind to the surface. Alternatively, the one or more probes may be added directly into the sample. In various examples, when the one or more probes are added directly to the sample, the sample may be in a vessel comprising a substrate with one or more passivated surfaces and the one or more probes (and eventual probe hybrids) may bind to the substrate A baseline signal or control signal is measured. The baseline signal or control signal may be a signal (e.g., fluorescence signal) where dye is not bound to the hybrids and / or of dye alone and / or of the dye in the presence of the probes without the presence of the sample (e.g., in the presence of an unhybridized probe). The hybrids are contacted with one or more dyes, and then an interaction signal is measured following the interaction of the one or more dyes with the hybrid. The interaction signal corresponds to a signal (e.g., fluorescence signal) measured of the hybrids interacting with the one or more dyes. The interaction signal and the baseline signal can be compared and a change in signal can indicate in the presence or absence of the one or more nucleic acid. For example, if fluorescence signals are being measured, an increase in fluorescence following the binding of the one or more dyes (relative to the signal of dye alone and / or hybrids in the absence of dye and / or signal of dye in the presence of nonhybridized probes ) can correspond to the presence of the one or more nucleic acid sequences.

[0025] Various probes may be used. For example, the probes may comprise a singlestranded nucleic acid strand complementary to at least a portion of the one or more nucleic acid sequences. Various single-stranded nucleic acid strands may be used. For example, the single-stranded nucleic acid strand may be a single-stranded DNA, an RNA, or a PNA.

[0026] For example, method may be implemented in various ways. For example, the method may comprise single molecule imaging via microscopy. In other examples, the method may comprise utilizing flow. The substrate (e.g., bead) having the dyes and hybrids may be detected in a flow system. See, e.g., Figure 7.

[0027] In various examples, a method of the present disclosure comprises contacting a sample comprising or suspected of comprising one or more nucleic acids sequences with one or more probes. The contacting may occur in a sample chamber having one or more passivated surfaces and containing one or more dyes. Upon the one or more nucleic acids sequences contacting the one or more probes, the one or more nucleic acids and the one or more probes form a hybrid to which the one or more dyes can interact. Following formation of the hybrid, an interaction signal is measured following the interaction with the one or more dyes and the hybrid. The measured interaction signal is compared to a baseline signal where there is no interaction of the dye with the hybrids (e.g., a control sample comprising the probes and sample in the absence of a dye, a control comprising the probes and the one or more dyes in the absence of the sample, and / or a control comprising the sample and the one or more dyes in the absence of the probes). In various examples, the method does not comprise utilization of any enzymes.

[0028] Various samples may be analyzed using a method of the present disclosure. For example, the sample may be an environmental sample. Limiting examples of environmental samples include, but are not limited to, water from an environmental source (e.g., wastewater, water from a body of water, etc.), soil, or the like. In other examples, the sample is a biological sample. Examples of biological samples include, but are not limited to, material from a nasopharyngeal swab, blood, saliva, tissue, urine, material from a mucosal biopsy, intestinal fluids, cells, or the like. In other examples, the sample may be food or something otherwise consumable by a human or an animal.

[0029] The sample may comprise or be suspected of comprising one or more nucleic acid sequences. The one or more nucleic acid sequences may be referred to as target nucleic acid sequences. The target nucleic acid sequence may be DNA, RNA, or PNA. The one or more nucleic acids may be associated with a virus, a bacterium, a fungus, or protist (e.g., DNA or RNA may be viral or bacterial). In various examples, the nucleic acids may be associated with SARS-CoV-2, Influenza virus, Enterovirus, Paramyxovirus, Salmonella, Campylobacter, Escherichia coli, Helicobacter pylori, Neisseria gonorrhoeae, Neisseria meningitides, Staphylococcus aureus, or the like.

[0030] In various examples, if the sample comprises one or more cells containing the target nucleic acid sequence, the cells may be lysed to such that the target nucleic acid sequence is released from the cell such that it can be detected by a method of the present disclosure.

[0031] The method may comprise utilizing a substrate (e.g., sample chamber or bead (e.g., microparticle, bead, resin, or nanoparticle, or the like)). If the substrate is a sample chamber, the sample chamber may have one or more substrates. Each of the one or more surfaces or substrates (e.g., microparticle, bead, resin, or nanoparticle) may be passivated. Passivation of the one or more surfaces or substrates inhibits or prevents or substantially inhibits or substantially prevents or at least partially inhibits or at least partially prevents nonspecific interactions. For example, the surfaces may first be plasma etched and hydroxylated using a hydroxylation agent, such as, for example, KOH. Following hydroxylation, the hydroxyl groups may be reacted with one or more molecules comprising an amine, resulting in the amination of the one or more surfaces or substrates (i.e., one or more amine groups suitable for nucleophilic reaction). The amine groups may then be reacted with one or more passivator groups comprising an electrophilic group. For example, the passivator group may be a polyethyleneglycol (PEG) group functionalized with an electrophilic group suitable for reaction with an amine. The PEG may have a molecular weight of 100,000 Da or less, 50,000 or less, 30,000 Da or less, 25,000 Da or less, 20,000 Da or less, 19,000 Da or less, 18,000 Da or less, 17,000 Da or less, 16,000 Da or less, 15,000 Da or less, 14,000 Da or less, 13,000 Da or less, 12,000 Da or less, 11,000 Da or less, 10,000 Da or less, 9,000 Da or less, 8,000 Da or less, 7,000 Da or less, 6,000 Da or less, 5,000 Da or less, 4000 Da or less, 3000 Da or less, 2000 Da or less, or 1000 Da or less. For example, the PEG group may be functionalized with an alkyl succinimidyl group (e.g., a succinimidyl valerate PEG). The passivators may be further functionalized with one or more anchoring group, which can be used as a group upon which the one or more probes may connected through. Passivation may be repeated one or more times. If passivation is repeated, different lengths of PEG may be used (e.g., MS4-PEG or mPEG SVA (e.g., having a molecular weight of 333 Da or 2000 Da). Additionally, the surfaces and / or substrates may be treated with Tween 20, Pluronic F127, Pluronic F68, or similar surfactant. Additionally, PEGylation and / or treatment with Tween 20, Pluronic F127, Pluronic F68, or similar surfactant may enhance the quality of PEGylation.

[0032] The chemical composition of the substrate may determine the method of passivation used. For example, glass microbeads can be similarly passivated andfunctionalized akin to a glass slide, whereas polystyrene microbeads can be passivated and functionalized using carboxyl groups on their surface.

[0033] Various other methods of passivation may be utilized or combined with PEGylation. For example, passivation may comprise single-step PEG-silane passivation; passivation with bovine serum albumin; passivation with polylysine and PEG; passivation with a lipid bilayer; passivation with dextran. For example, single-step PEG-silane passivation comprises grafting PEG-silane (PEG-Si(OEt)3) on a surface (e.g., a glass surface) via alcohol condensation between unhydrolyzed PEG-silane and silanol groups to form a siloxane group and a portion of the PEG groups may be further substituted with an anchoring group. For example, passivation with bovine serum albumin (BSA) comprises contacting the surface with BSA and BSA functionalized with an anchoring group (e.g., Biotin- functionalized BSA). Use of BSA passivation may be further enhanced by contacting the surface with a non-ionic surfactant. For example, passivation with polylysine and PEG may comprise contacting the surface with polylysine-PEG and polylysine functionalized with an anchoring group (e.g., polylysine functionalized with a biotin group). Use of polylysine passivation may be further enhanced by contacting the surface with a non-ionic surfactant. For example, passivation with lipid bilayers may comprise contacting the surface with lipids suitable to form a lipid bilayer and lipids functionalized with an anchoring group (e.g., a lipid functionalized with a biotin group). For example, passivation with dextran may comprise contacting the surface with dextran and dextran functionalized with an anchoring group (e.g., dextran functionalized with a biotin group).

[0034] Various substrates may be used. For example, the substrate is a glass slide, a glass coverslip, plastic slide, plastic coverslip, a microbead, a microparticle, a nanoparticle, or the like. For example, a substrate may be a sample chamber, which may comprise one or more glass surfaces or substrates. Particle or bead-type substrates may be suitable for use in measuring the signal via a flow method (see, e.g., Figure 7). Coverslips, slides, and sample chambers may be suitable for use with other detection chambers (see, e.g., Figure 1.). Various examples of plastic may be used. For example, the plastic may an ultraviolet transmittable plastic, such as, for example, polystyrene, acrylic, or vinyl.

[0035] Various anchoring groups may be used. The anchoring groups allow for covalent or non-covalent interaction to the one or more probes. The one or more probes may be connected to the anchoring group of the passivator group through one or more connecting groups and / or anchoring groups attached to the one or more probes. For example, the passivator may comprise an anchoring group and the probe may comprise an anchoringgroup, and the two anchoring groups are attached via a connecting group. Non-limiting examples of anchoring groups include biotin groups, alkynes, azides, thiols, amines, acrydite groups, and the like. When the anchoring group is a biotin group, the passivator group may comprise a biotin group and the probe may comprise a biotin group, and the passivator group and the probe are connected via a connecting group and the connecting group is a streptavidin molecule. In various other examples, the probe and passivator groups are connected through a triazole formed from a click reaction (e.g., a reaction between an azide and an alkyne). In such an example, the probe may comprise the alkyne and the passivator group may comprise the azide, or, alternatively, the probe may comprise the azide and the passivator group may comprise the alkyne. In various other examples, the one or more probes may be covalently attached to the passivator group. For example, the one or more probe and passivator may be attached through a substitution reaction, through acylation chemistry, or additional reaction (e.g., Michael addition or Michael-like addition). For example, the one or more probes may be functionalized with a nucleophilic group, such as an amine or thiol, and reaction with an electrophile (e.g., a carboxylic acid (which may be activated) or mal eimide) on the passivator group. Alternatively, the one or more probes may have an electrophilic group, such as, for example, an acrydite group and the passivator may be functionalized with a nucleophilic group, such as, for example, a thiol.

[0036] The probe may comprise an anchoring group to which the probe is connected to the passivator group, either through an anchoring group of the passivator group or through a connecting group attached to the anchoring group of the passivator group. The probe may comprise 10 to 100 nucleobases, including all values and ranges therebetween (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100). At least a portion of the probe is complementary to at least a portion of the target nucleic acid of the sample.

[0037] Upon contacting the nucleic acids of the sample with the one or more probes, a nucleic acid will hybridize with a probe resulting in a hybrid. The contacting the sample with the one or more probes may be done for any length of time that is sufficient to form a hybrid (e.g., about one second to several days (e.g., 30 minutes to about 3 hours), including all second values and ranges therebetween). The hybrid may then be contacted with one or more dyes to detect the formation of the hybrid formation. Hybrid formation is indication thatthe target nucleic acid is present in the sample. In various examples, only a portion of the target nucleic acid hybridizes with the probe or a portion of the probe.

[0038] Various dyes may be used. The dyes may be non-specific to either the one or more probes and / or to the one or more nucleic acids of the sample. The dyes may be fluorescent dyes. For example, the dye may be intercalating dyes. Examples of intercalating dyes include, but are not limited to SYBR dyes (e.g., SYBRSafe, SYBR Green 1, SYBR Gold), Picogreen, Evagreen, Malachite Green, Brilliant Green, SYTOX Green, SYTOX Orange, GelRed, GelGreen, Quantifluor, LC Green, Acridine Orange, Ethidium Bromide, Propidium Iodide, Hoechst 33342, Hoechst 34580, Hoechst 33258, DAPI, 7- aminoactinomycin D (7-AAD), Propidium Monozaide (PMA), PMAxx, 3,3’- Diethylthiadicarbocyanine, iodide (DiSC2 (5)), SYTO-9, SYTO-13, SYTO-16, SYTO-24, SYTO-60, SYTO-62, SYTO-64, SYTO-82, YOYO-1, YO-Pro-1, YO-Pro-2, YO-Pro-3, Thiazole Orange (TO), TOTO-1, TOTO-3, TO-Pro-3, POPO-3, PO-Pro-3, BEBO, BOBO-3, DiYO-1, DiTO-1, and the like, and any combination thereof.

[0039] The signal produced by the contacting of the dye with the hybrid (i.e., interaction signal may be detected by various means. For example, the interaction signal by detected by camera, photon detector, a fluorimeter, or other similar detection system.

[0040] In various examples, a method of the present disclosure does not include an amplification technique. Examples of amplification techniques include, but are not limited to, PCR, loop-mediated isothermal amplification (LAMP), Recombinase Polymerase Amplification (RPA), Helicase-Dependent Amplification (HD A), Strand Displacement Amplification (SDA), nucleic acid sequence-based amplification (NASBA), self-sustained sequence replication (3 SR), rolling circle amplification (RCA), ligase chain reaction (LCR), Nick Displacement Amplification (ND A), Multiple Displacement Amplification (MDA), Multiplex Ligation-dependent Probe Amplification (MLP A), Single Primer Isothermal Amplification (SPIA), Transcription-Mediated Amplification (TMA), and the like. That is, the target nucleic acid can be detected without further amplification of the target nucleic acid, but by detection of the hybridized nucleic acid to the probe by utilizing a dye. In various other examples, the method does not utilize enzymes (e.g., the method is enzyme-free).

[0041] The target nucleic acid can be detected at low concentrations. For examples, the one or more nucleic acids of the sample have a concentration of less than 100 nM, less than 10 nM, less than 1 nM, less than 100 fM, less than 10 fM, less than 1 fM, less than 100 aM, less than 10 aM, less than 9 aM, less than 8 aM, less than 7 aM, less than 6 aM, less than 5 aM, less than 4 aM, less than 3 aM, less than 2 aM, about 1 aM, or less than 1 aM. Invarious examples, the probe can be at low concentrations. For examples, the one or more probes may be present at a concentration of less than 100 nM, less than 10 nM, less than 1 nM, less than 100 fM, less than 10 fM, less than 1 fM, less than 100 aM, less than 10 aM, less than 9 aM, less than 8 aM, less than 7 aM, less than 6 aM, less than 5 aM, less than 4 aM, less than 3 aM, less than 2 aM, about 1 aM, or lower than 1 aM.

[0042] In an aspect, the present disclosure provides a kit. The kit may comprise the materials necessary to create a substrate of the present disclosure or to perform a method of the present disclosure. The kit may also include instructions for use.

[0043] For example, a kit may comprise the components necessary to functionalize a substrate, such as, for example, materials to passivate the surface, materials to attach a probe, and one or more dyes. The kit may also comprise a substrate and / or all other reagents and / or the materials to functionalize a desired substrate. In various examples, the substrate and / or surfaces of the substrate may come in the kit already passivated. Further, the kit may comprise a passivated substrate where the probes are already attached to the substrates.

[0044] The steps of the method described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of the present invention. Thus, in an embodiment, the method consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of such steps.

[0045] The following Statements are provided as non-limiting examples of the present disclosure.Statement 1. A method for detecting nucleic acid sequences in a sample, comprising measuring a baseline signal; contacting a sample comprising or suspected of comprising one or more nucleic acids sequences with one or more probes disposed on a substrate having one or more passivated surfaces, wherein upon the one or more nucleic acids sequences contacting the one or more probes the one or more nucleic acid sequences and the one or more probes form one or more hybrids; contacting the hybrids with one or more dyes; and measuring an interaction signal following an interaction with the one or more dyes and the one or more hybrids, wherein each probe comprises a single-stranded nucleic acid sequence complementary to at least a portion of the one or more nucleic acid sequences. In various examples, the method does not comprise utilization of any enzymes.Statement 2. A method according to Statement 1, wherein the substrate is a glass slide, a glass coverslip, a plastic slide, a plastic coverslip, a microbead, a microparticle, a nanoparticle, or the like.Statement 3. A method according to Statement 1 or Statement 2, wherein the single-stranded nucleic acid sequence is single-stranded DNA, RNA, or PNA.Statement 4. A method according to any one of the preceding Statements, wherein the sample is a material from a nasopharyngeal swab, blood, saliva, tissue, urine, material from a mucosal biopsy, intestinal fluids, cells, food, water, or an environmental sample.Statement 5. A method according to any one of the preceding Statements, wherein the one or more passivated surfaces are passivated with polyethylene glycol (PEG) groups.Statement 6. A method according to Statement 5, wherein the PEG group has a molecule weight of 100,000 Da or less (e.g., 100,000 Da or less, 50,000 or less, 30,000 Da or less, 25,000 Da or less, 20,000 Da or less, 19,000 Da or less, 18,000 Da or less, 17,000 Da or less, 16,000 Da or less, 15,000 Da or less, 14,000 Da or less, 13,000 Da or less, 12,000 Da or less, 11,000 Da or less, 10,000 Da or less, 9,000 Da or less, 8,000 Da or less, 7,000 Da or less, 6,000 Da or less, 5,000 Da or less, 4000 Da or less, 3000 Da or less, 2000 Da or less, or 1000 Da or less).Statement 7. A method according to any one of the preceding Statements, wherein the PEG groups comprise a biotin group or streptavidin group.Statement 8. A method according to any one of Statements 5 to 7, wherein the PEG group comprises an electrophile or nucleophile or other functional group suitable for a substitution or addition reaction.Statement 9. A method according to any one of the preceding Statements, wherein the probe further comprises a biotin group or a streptavidin group.Statement 10. A method according to Statement 8, wherein the probe further comprises a group suitable for a substitution or addition reaction with the electrophile or nucleophile of the PEG group.Statement 11. A method according to Statement 9 or Statement 10, wherein the one or more probes are non-covalently connected to a streptavidin molecule, wherein the streptavidin molecule is further non-covalently connected to the biotin group of the PEG groups.Statement 12. A method according to any one of Statements 1 to 4, wherein the one or more passivated surfaces are passivated with BSA, dextran, polylysine, or the like, wherein the BSA, dextran, polylysine, or the like comprises i) an electrophile or nucleophile or otherfunctional group suitable for a substitution or addition reaction, or ii) a biotin group or a streptavidin group.Statement 13. A method according to any one of the preceding Statements, wherein the one or more dyes are fluorescent dyes.Statement 14. A method according to any one of the preceding Statements, wherein the one or more dyes are intercalating dyes.Statement 15. A method according to Statement 14, wherein the one or more dyes are chosen from SYBRSafe, SYBR Green 1, SYBR Gold, Picogreen, Evagreen, Malachite Green, Brilliant Green, SYTOX Green, SYTOX Orange, GelRed, GelGreen, Quantifluor, LC Green, Acridine Orange, Ethidium Bromide, Propidium Iodide, Hoechst 33342, Hoechst 34580, Hoechst 33258, DAPI, 7-aminoactinomycin D (7-AAD), Propidium Monozaide (PMA), PMAxx, 3,3 ’-Diethylthiadicarbocyanine, iodide (DiSC2 (5)), SYTO-9, SYTO-13, SYTO-16, SYTO-24, SYTO-60, SYTO-62, SYTO-64, SYTO-82, YOYO-1, YO-Pro-1, YO-Pro-2, YO- Pro-3, Thiazole Orange (TO), TOTO-1, TOTO-3, TO-Pro-3, POPO-3, PO-Pro-3, BEBO, BOBO-3, DiYO-1, DiTO-1, and the like, and any combination thereof.Statement 16. A method according to any one of the preceding Statements, further comprising comparing the interaction signal and baseline signal to determine if one or more hybrids have formed.Statement 17. A method according to any one of the preceding Statements, wherein the method does not comprise utilizing an amplification technique.Statement 18. A method according to any one of the preceding Statements, wherein the one or more nucleic acid sequences are DNA.Statement 19. A method according to any one of Statements 1-17, wherein the one or more nucleic acid sequences are RNA.Statement 20. A method according to any one of Statements 1-17, wherein the one or more nucleic acid sequences are PNA.Statement 21. A method according to any one of the preceding Statements, wherein the one or more nucleic acids are associated with a virus, a fungus, protist, or a bacterium.Statement 22. A method according to any one of the preceding Statements, wherein the one or more nucleic acids are associated with SARS-CoV-2, Influenza virus, Enterovirus,Paramyxovirus, Salmonella, Campylobacter, Escherichia Coli, Helicobacter pylori, Neisseria gonorrhoeae, Neisseria meningitides, Staphylococcus aureus, or the like.Statement 23. A method according to any one of the preceding Statements, wherein the one or more nucleic acids of the sample have a concentration of less than 100 nM, less than 10 nM, less than 1 nM, less than 100 fM, less than 10 fM, less than 1 fM, less than 100 aM, less than 10 aM, less than 9 aM, less than 8 aM, less than 7 aM, less than 6 aM, less than 5 aM, less than 4 aM, less than 3 aM, less than 2 aM, about 1 aM, or less than 1 aM.Statement 24. A method according to any one of the preceding Statements, wherein the contacting the sample with the one or more probes is done for at least about one second to several days (e.g., 30 minutes to about 3 hours).Statement 25. A method according to any one of the preceding Statements, wherein the probe comprises about 10 to about 100 nucleobases, including all integer values and ranges therebetween.Statement 26. A method according to any one of the preceding Statements, wherein the one or more probes have a concentration of less than 100 nM, less than 10 nM, less than 1 nM, less than 100 fM, less than 10 fM, less than 1 fM, less than 100 aM, less than 10 aM, less than 9 aM, less than 8 aM, less than 7 aM, less than 6 aM, less than 5 aM, less than 4 aM, less than 3 aM, less than 2 aM, about 1 aM, or less than 1 aM.Statement 27. A method according to any one of the preceding Statements, wherein the method comprises imaging a single hybrid.Statement 28. A method according to any one of the preceding Statements, wherein the method comprises imaging a single particle.Statement 29. A method according to any one of the preceding Statements, wherein the one or more dyes are contacted to the one or more probes prior to hybrid formation.Statement 30. A method according to any one of the preceding Statements, wherein the method further comprises imaging one or more hybrids contacted with the one or more dyes.Statement 31. A method for detecting nucleic acid sequences in a sample, comprising measuring a baseline signal; contacting a sample comprising or suspected of comprising one or more nucleic acids sequences with one or more probes, wherein the one or more probes are either i) disposed on a substrate having one or more passivated surfaces prior to contacting the sample or ii) the one or more probes are covalently or non-covalently attached to asubstrate having one or more passivated surfaces after contacting the sample, wherein upon the one or more nucleic acids sequences contacting the one or more probes, the one or more nucleic acid sequences and the one or more probes form one or more hybrids; contacting the hybrids with one or more dyes; and measuring an interaction signal following an interaction with the one or more dyes and the one or more hybrids, wherein each probe comprises a single-stranded nucleic acid sequence complementary to at least a portion of the one or more nucleic acid sequences. In various examples, the method does not comprise utilization of any enzymes.Statement 32. A method according to Statement 31, wherein the one or more probes are disposed on a substrate having one or more passivated surfaces prior to contacting the sample.Statement 33. A method according to Statement 31, wherein the one or more probes are covalently or non-covalently attached to a substrate having one or more passivated surfaces after contacting the sample.Statement 34. A method according to any one of Statements 31-33, wherein the substrate is a glass slide, a glass coverslip, a plastic slide, a plastic coverslip, a microbead, a microparticle, a nanoparticle, or the like.Statement 35. A method according to any one of Statements 31-34, wherein the singlestranded nucleic acid sequence is single-stranded DNA, RNA, or PNA.Statement 36. A method according to any one of Statements 31-35, wherein the sample is a material from a nasopharyngeal swab, blood, saliva, tissue, urine, material from a mucosal biopsy, intestinal fluids, cells, food, water, or an environmental sample.Statement 37. A method according to any one of Statements 31-36, wherein the one or more passivated surfaces are passivated with polyethylene glycol (PEG) groups.Statement 38. A method according to Statement 37, wherein the PEG group has a molecule weight of 100,000 Da or less (e.g., 100,000 Da or less, 50,000 or less, 30,000 Da or less, 25,000 Da or less, 20,000 Da or less, 19,000 Da or less, 18,000 Da or less, 17,000 Da or less, 16,000 Da or less, 15,000 Da or less, 14,000 Da or less, 13,000 Da or less, 12,000 Da or less, 11,000 Da or less, 10,000 Da or less, 9,000 Da or less, 8,000 Da or less, 7,000 Da or less, 6,000 Da or less, 5,000 Da or less, 4000 Da or less, 3000 Da or less, 2000 Da or less, or 1000 Da or less).Statement 39. A method according to Statement 37 or Statement 38, wherein the PEG groups comprise a biotin group or streptavidin group.Statement 40. A method according to any one of Statements 37-39, wherein the PEG group comprises an electrophile or nucleophile or other functional group suitable for a substitution or addition reaction.Statement 41. A method according to any one of Statements 31-40, wherein the probe further comprises a biotin group or a streptavidin group.Statement 42. A method according to Statement 41, wherein the probe further comprises a group suitable for a substitution or addition reaction with the electrophile or nucleophile of the PEG group.Statement 43. A method according to Statement 41 or Statement 42, wherein the one or more probes are non-covalently connected to a streptavidin molecule, wherein the streptavidin molecule is further non-covalently connected to the biotin group of the PEG groups.Statement 44. A method according to any one of Statements 31-37, wherein the one or more passivated surfaces are passivated with BSA, dextran, polylysine, or the like, wherein the BSA, dextran, polylysine, or the like comprises i) an electrophile or nucleophile or other functional group suitable for a substitution or addition reaction, or ii) a biotin group or a streptavidin group.Statement 45. A method according to any one of Statements 31-38, wherein the one or more dyes are fluorescent dyes.Statement 46. A method according to any one of Statements 31-45, wherein the one or more dyes are intercalating dyes.Statement 47. A method according to Statement 46, wherein the one or more dyes are chosen from SYBRSafe, SYBR Green 1, SYBR Gold, Picogreen, Evagreen, Malachite Green, Brilliant Green, SYTOX Green, SYTOX Orange, GelRed, GelGreen, Quantifluor, LC Green, Acridine Orange, Ethidium Bromide, Propidium Iodide, Hoechst 33342, Hoechst 34580, Hoechst 33258, DAPI, 7-aminoactinomycin D (7-AAD), Propidium Monozaide (PMA), PMAxx, 3,3 ’-Diethylthiadicarbocyanine, iodide (DiSC2 (5)), SYTO-9, SYTO-13, SYTO-16, SYTO-24, SYTO-60, SYTO-62, SYTO-64, SYTO-82, YOYO-1, YO-Pro-1, YO-Pro-2, YO- Pro-3, Thiazole Orange (TO), TOTO-1, TOTO-3, TO-Pro-3, POPO-3, PO-Pro-3, BEBO, BOBO-3, DiYO-1, DiTO-1, and the like, and any combination thereof.Statement 48. A method according to any one of Statements 31-46, further comprising comparing the interaction signal and baseline signal to determine if one or more hybrids have formed.Statement 49. A method according to any one of Statements 31-48, wherein the method does not comprise utilizing an amplification technique.Statement 50. A method according to any one of Statements 31-49, wherein the one or more nucleic acid sequences are DNA.Statement 51. A method according to any one of Statements 31-49, wherein the one or more nucleic acid sequences are RNA.Statement 52. A method according to any one of Statements 31-49, wherein the one or more nucleic acid sequences are PNA.Statement 53. A method according to any one of Statements 31-51, wherein the one or more nucleic acids are associated with a virus, a fungus, protist, or a bacterium.Statement 54. A method according to any one of Statements 31-51, wherein the one or more nucleic acids are associated with SARS-CoV-2, Influenza virus, Enterovirus, Paramyxovirus, Salmonella, Campylobacter, Escherichia Coli, Helicobacter pylori, Neisseria gonorrhoeae, Neisseria meningitides, Staphylococcus aureus, or the like.Statement 55. A method according to any one of Statements 31-53, wherein the one or more nucleic acids of the sample have a concentration of less than 100 nM, less than 10 nM, less than 1 nM, less than 100 fM, less than 10 fM, less than 1 fM, less than 100 aM, less than 10 aM, less than 9 aM, less than 8 aM, less than 7 aM, less than 6 aM, less than 5 aM, less than 4 aM, less than 3 aM, less than 2 aM, about 1 aM, or less than 1 aM.Statement 56. A method according to any one of Statements 31-55, wherein the contacting the sample with the one or more probes is done for at least about one second to several days (e.g., 30 minutes to about 3 hours).Statement 57. A method according to any one of Statements 31-56, wherein the probe comprises about 10 to about 100 nucleobases, including all integer values and ranges therebetween.Statement 58. A method according to any one of Statements 31-57, wherein the one or more probes have a concentration of less than 100 nM, less than 10 nM, less than 1 nM, less than 100 fM, less than 10 fM, less than 1 fM, less than 100 aM, less than 10 aM, less than 9 aM,less than 8 aM, less than 7 aM, less than 6 aM, less than 5 aM, less than 4 aM, less than 3 aM, less than 2 aM, about 1 aM, or less than 1 aM.Statement 59. A method according to any one of Statements 31-58, wherein the method comprises imaging a single hybrid.Statement 60. A method according to any one of Statements 31-59, wherein the method comprises imaging a single particle.Statement 61. A method according to any one of Statements 31-60, wherein the one or more dyes are contacted to the one or more probes prior to hybrid formation.Statement 62. A method according to any one of Statements 31-61, wherein the method further comprises imaging one or more hybrids contacted with the one or more dyes.

[0046] The following example is presented to illustrate the present disclosure. It is not intended to be limiting in any way.EXAMPLE

[0047] This example provides a description of a method of the present disclosure.

[0048] Described herein is a proof-of-concept amplification-free, single-molecule technique to detect specific nucleic acid sequences with extremely high sensitivity.Moreover, this simple and versatile imaging platform can directly detect a few hundred molecules of target nucleic acids with high specificity and fidelity. The instant method relies on direct hybridization between specific segments of target nucleic acid and complementary probes immobilized on the surface of a customized sample chamber. The schematic in Figure la depicts the working principle of the instant technique, whereby successful hybridization events lead to the trapping of target microbial genetic material on the glass chamber surface. Subsequent exposure to cyanine-based intercalator dyes (e.g. SYBRSafe) followed by objective-based total internal reflection fluorescence (TIRF) imaging of the sample chamber surface results in a high signal -to-noise fluorescent signal, thereby confirming the presence of the specific nucleic acid target. The immobilization of short DNA probes on the glass surface of sample chambers may introduce unwanted surface artefacts or nonspecific binding of fluorescent species or non-specific DNA contamination all of which could contribute to falsepositive detection. It is therefore crucial to block such non-specific biological molecules, such as proteins, cells, and DNA by surface passivation. Polyethylene glycol (PEG) coating was used for passivating the sample chambers because the PEG chains shield the surface andminimize nonspecific interactions (Figure 3). Briefly, PEG attachment involved hydroxylation by KOH etching and plasma cleaning. The free hydroxyl groups are required for the amino-silanization reaction. Subsequently, surface functionalization with amine groups was done using 3-aminopropyl-triethoxysilane (APTES) via silane chemistry to facilitate the covalent interaction of succinimidyl valerate PEG (mPEG SV A) via N- hydroxysuccinimide ester-amino reaction. Before treatment with mPEG spiked with biotin- PEG, a multistep cleaning of the glass coverslips was done to remove fluorescent organic molecules on the surface and other dirt which could interfere with the single-molecule fluorescent measurements. PEG is biologically inert and its properties can be tuned by varying the length and density of PEG chains to modulate surface properties, such as hydration, stiffness, and protein repellency. The walls of the sample chamber were also passivated with surfactants such that there was no loss of microbial nucleic acid to the walls of the chamber when the sample was loaded. The biotinylated single-stranded DNA (ssDNA) probes were directly attached to the biotin PEG through a streptavidin bridge as shown in schematic in Figure lb. The interaction between streptavidin and biotin is one of the strongest non-covalent interactions known in nature and involves hydrogen bonds, hydrophobic interactions, and van der Waals forces.

[0049] Upon surface passivation, the sample chamber was assembled as discussed in the methods section. To demonstrate proof-of-concept of the technique, varying concentrations of the biotinylated 50-base ssDNA probe (nanomolar to attomolar) were mixed with its equivalent concentration of complementary 50-base DNA sequence in a hybridization buffer, heated and cooled slowly. Separately, bulk fluorescence measurement with commercially available SYBRsafe showed an enhanced fluorescence signal (> 3 times) of 10 nM DNA:DNA hybrid compared to 10 nM single-stranded (ssDNA) probes (Figure 4) showcasing the efficacy of SYBRsafe intercalator as a strong reporter of successful hybridization. The original mixed samples were then incubated in the sample chamber. The control chamber was incubated with the ssDNA probe alone. Upon formation of DNA:DNA hybrid and its successful anchoring to the surface of the sample chamber through the biotin- streptavidin-biotin bridge, the samples were washed thoroughly to remove non-specific binding, if any, and then incubated with SYBRsafe dyes. The light emitted from individual molecules was detected on an sCMOS camera and analyzed for the intensity of individual bright spots. A typical probe of about 50 bases could accommodate around 25 fluorescent intercalators and significantly enhance the fluorescent signal. Because intercalators are not sequence-selective, extra caution was taken to eliminate the nonspecific binding of unwantednucleic acids to the surface by washing steps and optimized PEG passivated surfaces. The intercalator dye also interacted with ssDNA (Figure 4) but emitted low fluorescence served to our great advantage. The single-stranded segments that span the hybridized duplex of the microbial nucleic acid may form secondary structures or may have single-stranded stretches which will then interact with the dye and enormously enhance the emitted fluorescent signal displaying significantly higher signal-to-noise ratio. When extracting nucleic acids from microbes, the design of the probes should be such that apart from the interaction energies, accessibility of the target regions, oligo length, protein occupancy and chemical modifications of the nucleic acids should be considered.

[0050] To demonstrate the proof-of-concept and determine the sensitivity of the enzyme-free, amplification-free method, varying concentrations of the target nucleic acid (50 bases) were incubated with the corresponding ssDNA probe (50 bases) from nanomolar (data not shown) to 1 femtomolar (Figure 1c, Id) to 100, 10 and 1 attomolar (Figure 2a-c). The optimal duration of incubation of the sample in the chamber depends on the concentrations of the nucleic acids. Ultralow concentration (attomolar) required 1 to 3 hrs of incubation while for higher concentrations of nucleic acids (nanomolar), 30-60 mins was sufficient. Several images were obtained for each concentration, taking advantage of the tiling feature in a motorized microscope to significantly decrease image acquisition time. The intensity of the individual spots was analyzed in Fiji after background subtraction. The intensities of individual spots recorded in the presence of the target nucleic acid, and which have intensities larger than the largest signal recorded in the presence of the probe-only sample were plotted as a histogram and represented in Figures Id, 2a-c. The imaging data showed a similar increase in fluorescent intensity (>3 times) as observed in bulk measurements. This technique is ultrasensitive such that the current design allows us to easily detect 1 attomolar concentration (600 molecules in 1 mL) of target nucleic acid in a highly specific manner (Figure 2c). Additionally, several different ssDNA probes could be implanted in the sample chamber for a given microbial genome and fragment the microbial nucleic acid. In that case, zepto-molar sensitivity could be easily achieved eliminating the need for PCR-or-LAMP- based amplification methods for ultra-low titer samples.

[0051] To determine the specificity of the optical method for detecting target nucleic acids, 50-base DNA were incubated with a mismatched ssDNA probe (50 bases) and found that the distributions were indistinguishable between the ssDNA probe alone with the fluorescent intercalator and sample containing the probe and mismatched target with the intercalator (Figure 5). This demonstrates that the detection capabilities of our method arehighly specific and do not show spurious signals or false positives even at ultra-low concentrations. To further demonstrate the robustness and utility of the diagnostic method, synthetic SARS-CoV-2 RNA was incubated with complementary ssDNA probes (25 bases) in hybridization buffer as discussed in the methods section. A significant enhancement in fluorescent signal intensity of the spots (> 8 times) was found when compared to ssDNA probe-only samples (Figure 2d, 2e), thereby demonstrating the efficacy and robustness of the instant method. The enhanced signal was due to intercalation at the DNA:RNA hybrid stretch, along the single-stranded stretches of RNA, or local secondary structure formation in the RNA overhangs.

[0052] In summary, the instant amplification-free technique fills a significant market gap while addressing the shortcomings of the current commercial methods. Amplification processes, such as polymerase chain reaction (PCR), can be time-consuming and expensive. The innovation eliminates the need for these additional steps, resulting in quicker and more cost-effective assays. These results show that the enzyme-free, amplification-free method can optically detect specific and ultra-low concentrations (1 attomolar or lower) of target nucleic acids robustly and efficiently. The simple, sensitive, and cost-effective method will allow for widespread adoption in diverse settings such as the point-of-care application in the clinic or during a global health crisis such as a pandemic or an endemic in resource-limited environments. Taken together, the instant optical method is well poised to revolutionize the area of nucleic-acid-based disease diagnostics, point-of-care applications, and environmental samples and will be a major game changer.

[0053] Methods

[0054] Chemicals: Alconox, Distilled or MilliQ water, ethanol, methanol, potassium hydroxide, acetic acid, 3 -aminopropyltri ethoxy silane / 3 -aminopropyltrimethoxy silane, sodium bicarbonate, mPEG-SVA (MW 5000), biotin-PEG-SVA (MW 5000), streptavidin, biotinylated DNA probes, synthetic SARS-CoV-2 control RNA.

[0055] Surface Passivation and assembly of the detection chamber, (a) Cleaning of slides and Coverslips: The microscope slides were scrubbed with Alconox detergent and rinsed thoroughly with distilled water. The coverslips and slides were then placed in Coplin glass staining jars and sonicated with ethanol for 10 mins, followed by rinsing with fresh ethanol solution. Subsequently, the slides and coverslips were rinsed with distilled water to remove any ethanol residue. The water in the jars was replaced with freshly prepared IM KOH solution for etching. The slides and coverslips were sonicated for 20 to 30 minutes. Thereafter, to remove the KOH traces, the slides were rinsed at least 3 times with distilledwater. Clean Nitrogen gas was used to dry the slides and coverslips. During drying, the coverslips were held with Teflon-coated flat-edged tweezers against clean N2 gas. This was followed by plasma cleaning of the coverslips for 15 mins at 30W set at 200 mTorr pressure. Both the slides and coverslips were rinsed with Milli Q water and then dried, (b) Aminosilanization of slides and cover slips: The surface of coverslips was functionalized with the amine group via the aminosilanization chemistry. The amino-silanization solution is prepared by adding 3 -aminopropyl tri ethoxy silane (APTES) to a mixture of methanol and acetic acid (20: 1). The cleaned coverslips and slides were rinsed with methanol. Subsequently, the methanol solution in the staining jars was replaced with freshly prepared amino-silanization solution and incubated for 30 minutes with brief sonication for 1 minute. The aminosilanization reagent was subsequently replaced with methanol and washed 3 times with methanol, (c) Surface PEGylation: The freshly prepared aminosilanized glass surface was passivated by conjugating polyethylene glycol (PEG) succinimidyl valerate at pH 8.5 overnight. Briefly, the amino-silanized slides and coverslips were dried using N2 gas and placed in a humidified chamber with the to-be PEGylated surface facing upwards. The moist environment in the humidified chamber would prevent the drying up of the PEGylation solution. The PEGylation solution was prepared by dissolving methoxy PEG succinimidyl valerate (mPEG- SVA) (250 mg / ml) and biotin-PEG succinimidyl valerate (biotin-PEG SVA) in the ratio 10:1 in freshly-prepared 0.1 M sodium bicarbonate buffer (pH 8.5). The PEGylation solution (~ 100 pl) was placed in between the slide and coverslip and left overnight. The slides and coverslips were carefully disassembled and the PEGylated surface was appropriately labelled, rinsed with distilled water and dried using N2 gas. (d) Assembly of the detection chamber: Circular plexiglass cylinders (1 cm internal diameter, 2 cm height), were cleaned thoroughly and surface-coated with Pluronic F68 polyol. The cylinders were glued to the PEG-passivated glass coverslip surface using Epoxy glue. Each detection unit is comprised of two chambers - sample and control. Before sample incubation, the detection chambers were incubated with a streptavidin solution for 30 minutes and washed thrice with buffer, (e) Hybridization experiment: The hybridization buffer for DNA: DNA hybridization comprised 10 mM Tris buffer and 100 mM NaCl. In the case of DNA: RNA hybridization, the hybridization buffer consisted of 50% Formamide, 40 mM Piperazine- 1, 4- bis (2-ethanesulphonic acid) [PIPES], 400 mM NaCl and 1 mM ethylenediaminetatracetic acid [EDTA] in RNAase / DNAase-free distilled water. A stock solution of 100 pM of the biotinylated DNA probe was diluted to several different concentrations (from nM to aM) and incubated with its complementary DNA strand diluted to the same concentration. The testsample and control were heated at 95°C for 5 mins if the test sample is DNA and to 65 °C if the test sample is RNA, followed by slow cooling and then incubated in the detection chamber. The probe-only sample was used to normalize the data and report successful hybridization if the intensities in the sample chamber observed were significantly greater than the control chamber, (f) Image acquisition: Samples were imaged in total internal reflection fluorescence (TIRF) mode using an inverted motorized Nikon TIRF microscope consisting of a Ti stand with perfect focus, 100 x 1.49 NA TIRF objective lens, 488nm laser line from the Omicron BrixXHUB ultra-high integrated multimode light engine and Hamamatsu ORCA- Flash4.0 V3 Digital sCMOS camera. Samples were imaged in a hybridization buffer containing intercalating dye (SYBRSafe). The microscope control and image acquisition were performed by the NIS Elements software (Nikon Instruments). To ensure homogenous illumination, only the central quarter of the chip of field size 512 x 512 pixels (35 pm X 35 pm) was used for imaging. Tiling (10 X 10) of fields was performed to decrease the overall image acquisition time. The intensity of the fluorescent spots after background subtraction was measured using Fiji (https: / / imagej.net / software / fiji / ).

[0056] Although the present disclosure has been described with respect to one or more particular embodiments and / or examples, it will be understood that other embodiments and / or examples of the present disclosure may be made without departing from the scope of the present disclosure.

Claims

CLAIMS:

1. A method for detecting nucleic acid sequences in a sample, comprising measuring a baseline signal; contacting a sample comprising or suspected of comprising one or more nucleic acids sequences with one or more probes, wherein upon the one or more nucleic acids sequences contacting the one or more probes the one or more nucleic acid sequences and the one or more probes form one or more hybrids, wherein the one or more probes are either i) disposed on a substrate having one or more passivated surfaces prior to contacting the sample or ii) the one or more probes are covalently or non-covalently attached to a substrate having one or more passivated surfaces after contacting the sample; contacting the hybrids with one or more dyes; and measuring an interaction signal following an interaction with the one or more dyes and the one or more hybrids, wherein each probe comprises a single-stranded nucleic acid sequence complementary to at least a portion of the one or more nucleic acid sequences.

2. The method according to claim 1, wherein the one or more probes are disposed on a substrate having one or more passivated surfaces prior to contacting the sample.

3. The method according to claim 1, wherein the one or more probes are covalently or non- covalently attached to a substrate having one or more passivated surfaces after contacting the sample.

4. The method according to claim 1, wherein the substrate is a glass slide, a glass coverslip, a plastic coverslip, a plastic slide, a microbead, a microparticle, a nanoparticle, or the like.

5. The method according to claim 1, wherein the single-stranded nucleic acid sequence is single-stranded DNA, RNA, or PNA.

6. The method according to claim 1, wherein the sample is a material from a nasopharyngeal swab, blood, saliva, tissue, urine, material from a mucosal biopsy, intestinal fluids, cells, food, water, or an environmental sample.

7. The method according to claim 1, wherein the one or more passivated surfaces are passivated with polyethylene glycol (PEG) groups.

8. The method according to claim 7, wherein the PEG group has a molecule weight of 100,000 Da or less.

9. The method according to claim 7, wherein the PEG groups comprise a biotin group or streptavidin group.

10. The method according to claim 1, wherein the PEG group comprises an electrophile or nucleophile or other functional group suitable for a substitution or addition reaction.

11. The method according to claim 1, wherein the probe further comprises a biotin group or a streptavidin group.

12. The method according to claim 11, wherein the probe further comprises a group suitable for a substitution or addition reaction with the electrophile or nucleophile of the PEG group.

13. The method according to claim 11, wherein the one or more probes are non-covalently connected to a streptavidin molecule, wherein the streptavidin molecule is further non- covalently connected to the biotin group of the PEG groups.

14. The method according to claim 1, wherein the one or more passivated surfaces are passivated with BSA, dextran, polylysine, or the like, wherein the BSA, dextran, or polylysine comprises i) an electrophile or nucleophile or other functional group suitable for a substitution or addition reaction, or ii) a biotin group or a streptavidin group.

15. The method according to claim 1, wherein the one or more dyes are fluorescent dyes.

16. The method according to claim 1, wherein the one or more dyes are intercalating dyes.

17. The method according to claim 16, wherein the one or more dyes are chosen from SYBRSafe, SYBR Green 1, SYBR Gold, Picogreen, Evagreen, Malachite Green, Brilliant Green, SYTOX Green, SYTOX Orange, GelRed, GelGreen, Quantifluor, LC Green, AcridineOrange, Ethidium Bromide, Propidium Iodide, Hoechst 33342, Hoechst 34580, Hoechst 33258, DAPI, 7-aminoactinomycin D (7-AAD), Propidium Monozaide (PMA), PMAxx, 3,3 ’-Diethylthiadicarbocyanine, iodide (DiSC2 (5)), SYTO-9, SYTO-13, SYTO-16, SYTO- 24, SYTO-60, SYTO-62, SYTO-64, SYTO-82, YOYO-1, YO-Pro-1, YO-Pro-2, YO-Pro-3, Thiazole Orange (TO), TOTO-1, TOTO-3, TO-Pro-3, POPO-3, PO-Pro-3, BEBO, BOBO-3, DiYO-1, DiTO-1, and any combination thereof.

18. The method according to claim 1, further comprising comparing the interaction signal and baseline signal to determine if one or more hybrids have formed.

19. The method according to claim 1, wherein the method does not comprise utilizing an amplification technique.

20. The method according to claim 1, wherein the one or more nucleic acid sequences are DNA.

21. The method according to claim 1, wherein the one or more nucleic acid sequences are RNA.

22. The method according to claim 1, wherein the one or more nucleic acid sequences are PNA.

23. The method according to claim 1, wherein the one or more nucleic acids are associated with a virus, a fungus, protist, or a bacterium.

24. The method according to claim 1, wherein the one or more nucleic acids are associated with SARS-CoV-2, Influenza virus, Enterovirus, Paramyxovirus, Salmonella, Campylobacter, Escherichia Coli, Helicobacter pylori, Neisseria gonorrhoeae, Neisseria meningitides, or Staphylococcus aureus.

25. The method according to claim 1, wherein the one or more nucleic acids of the sample have a concentration of less than 100 nM.

26. The method according to claim 1, wherein the contacting the sample with the one or more probes is done for at least about one second to several days.

27. The method according to claim 1, wherein the probe comprises about 10 to about 100 nucleobases.

28. The method according to claim 1, wherein the one or more probes have a concentration of less than 100 nM.

29. The method according to claim 1, wherein the method comprises imaging a single hybrid.

30. The method according to claim 1, wherein the method comprises imaging a single particle.

31. The method according to claim 1, wherein the one or more dyes are contacted to the one or more probes prior to hybrid formation.

32. The method according to claim 1, wherein the method further comprises imaging one or more hybrids contacted with the one or more dyes.

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