Methods, systems and compositions for nucleic acid detection

Abstract

Provided herein are methods, compositions, and systems for detecting nucleic acids. The methods, compositions, and systems may comprise nanoparticles comprising oligonucleotides. The oligonucleotide may anneal with a target nucleic acid. The nanoparticles may comprise an optical parameter that can change upon reaction with a target nucleic acid.

Description

[0001] Cross-reference

[0002] This application claims priority to U.S. Provisional Application No. 63 / 370,218, filed Aug. 2, 2022, which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application is being filed with an electronic sequence listing. The sequence listing is provided as a file entitled 64978-701.601.xml, created on Jul. 31, 2023, and having a size of 21,233 bytes. The information on the electronic sequence listing is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0005] Nucleic acids are present in many organisms and allow organisms to replicate or encode specific proteins. For example, viruses can use nucleic acids to replicate, and the presence of viral nucleic acids in an object can indicate the presence of a virus in the object. SUMMARY OF THE INVENTION

[0006] In one aspect, the present disclosure provides a method for processing or analyzing a sample, the method comprising: (a) contacting the sample with a composition comprising one or more nanoparticles to provide a test composition, wherein the one or more nanoparticles are assembled with one or more oligonucleotides, and wherein the one or more oligonucleotides hybridize to one or more target nucleic acids that may be present in the body sample; forming a nanoparticle matrix from the one or more nanoparticles hybridized to the one or more target nucleic acids in the presence of the one or more target nucleic acids; (c) determining an optical parameter of the test composition, the optical parameter indicating the presence or absence of one or more nucleic acids in the sample. In some embodiments, the optical parameter is determined by color spacing analysis. In some embodiments, the optical parameter comprises absorption, transmission, scattering, or reflection of light at a wavelength or wavelength range. In some embodiments, the optical parameter comprises a photometric parameter (e.g., brightness of a color), a saturation parameter (e.g., intensity of a color), or a hue parameter (e.g., chromaticity of a color). In some embodiments, (c) further comprises comparing the optical parameter of the test composition with a corresponding optical parameter determined from a corresponding reference composition. The method determines the presence or absence of one or more target nucleic acids in the sample with a sensitivity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the method determines the presence or absence of one or more target nucleic acids in the sample with a specificity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the method determines the presence or absence of one or more target nucleic acids in the sample with an accuracy of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the sample is selected from: a blood sample, a serum sample, a plasma sample, a saliva sample, a fecal sample, a sputum sample, a urine sample, a semen sample, a vaginal fluid sample, a cerebrospinal fluid sample, a sweat sample, a cell sample, and a tissue sample. In some embodiments, the sample is from a mammal (e.g., a human). In some embodiments, the sample is from an animal. In some embodiments, the sample is from a plant. In some embodiments, the sample comprises a lysis solution. In some embodiments, (b) comprises contacting the test composition with a nanoparticle condensing agent and / or a salt. In some embodiments, the condensing agent comprises magnesium chloride. In some embodiments, at least about 40% of the nucleotides of the one or more oligonucleotides are guanine or cytosine. In some embodiments, about 40% to about 60% of the nucleotides of the one or more oligonucleotides are guanine or cytosine. In some embodiments, the one or more oligonucleotides are characterized by a melting temperature (Tm) of at least about 65 degrees Celsius (°C). In some embodiments, the one or more oligonucleotides are characterized by a Tm of about 65°C to about 75°C.In some embodiments, one or more oligonucleotides comprise a conjugate moiety at the 5'-end. In some embodiments, the conjugate moiety is a 5'-thiol. In some embodiments, the 5'-thiol comprises a thioalkyl group, such as a thiohexyl group. In some embodiments, one, two, three, four, five, or six oligonucleotides are assembled to a nanoparticle in one or more nanoparticles. In some embodiments, each (e.g., independently) of the one or more nanoparticles is assembled with one, two, three, four, five, or six oligonucleotides. In some embodiments, one or more nanoparticles comprise gold. In some embodiments, one or more nanoparticles are characterized by an average size of from about 10 nanometers (nm) to about 200 nm. In some embodiments, one or more oligonucleotides are 16 to 24 nucleotides in length. In some embodiments, one or more oligonucleotides associate with one or more target nucleic acids such that the distance between two adjacent nanoparticles of the one or more nanoparticles corresponds to about 50 to about 70 nucleotides. In some embodiments, one or more oligonucleotides comprise two oligonucleotides, wherein the first oligonucleotide hybridizes to a first region of the target nucleic acid and the second oligonucleotide hybridizes to a second region of the target nucleic acid. In some embodiments, the distance between the first region and the second region of the target nucleic acid is about 50 to 70 nucleotides. In some embodiments, one or more oligonucleotides hybridize to 10 to 30 nucleotides of one or more target nucleic acids. In some embodiments, one or more nanoparticles form aggregates in the absence of one or more target nucleic acids. In some embodiments, one or more target nucleic acids are from one or more viruses or one or more bacteria. In some embodiments, one or more target nucleic acids are not from a coronavirus. In some embodiments, one or more target nucleic acids are not from SARS-CoV-2 or one or more of its variants. In some embodiments, one or more viruses include influenza virus or human papillomavirus. In some embodiments, one or more bacteria include Salmonella. In some embodiments, Salmonella includes Salmonella enterica. In some embodiments, Salmonella includes one or more Salmonella strains or serotypes. In some embodiments, one or more Salmonella strains or serotypes comprise one or more members selected from Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella pullorum. In some embodiments, one or more target nucleic acids are associated with one or more diseases or conditions. In some embodiments, one or more diseases or conditions include infectious diseases, cancer, or degenerative diseases. In some embodiments, one or more target nucleic acids encode a polypeptide or protein. In some embodiments, one or more target nucleic acids comprise DNA or RNA. In some embodiments, the DNA is genomic DNA. In some embodiments, the RNA is genomic RNA. In some embodiments, the RNA is double-stranded RNA or single-stranded RNA.In some embodiments, the RNA is double-stranded DNA or single-stranded DNA. In some embodiments, one or more target nucleic acids are from human papillomavirus (HPV) or one or more variants thereof. In some embodiments, one or more target nucleic acids comprise one or more members selected from the following: the L1 capsid protein of HPV, the L2 capsid protein of HPV, the E6 protein of HPV, the E7 protein of HPV, and fragments of any of the foregoing. In some embodiments, one or more target nucleic acids are from Salmonella. In some embodiments, Salmonella includes Salmonella enterica. In some embodiments, Salmonella includes one or more Salmonella strains or serotypes. In some embodiments, one or more Salmonella strains or serotypes comprise one or more members selected from Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella pullorum. In some embodiments, one or more oligonucleotides comprise sequences selected from SEQ ID NO: 1-18.

[0007] In one aspect, the present disclosure provides a composition for detecting one or more target nucleic acids, the composition comprising: one or more nanoparticles assembled with one or more oligonucleotides, wherein the one or more oligonucleotides are complementary to the one or more target nucleic acids, wherein, in the presence of the one or more target nucleic acids, the one or more nanoparticles form a nanoparticle matrix, and wherein the nanoparticle matrix has different optical parameters compared to a solution comprising the corresponding nanoparticles not in the nanoparticle matrix.

[0008] In another aspect, the present disclosure provides a composition for detecting a target nucleic acid, the composition comprising: one or more nanoparticles assembled with one or more oligonucleotides, wherein a first oligonucleotide among the one or more oligonucleotides is complementary to the target nucleic acid, and wherein a second oligonucleotide among the one or more oligonucleotides is complementary to the target nucleic acid at a second sequence, wherein the one or more nanoparticles comprise gold, and wherein, in the presence of one or more target nucleic acids, the one or more nanoparticles form a nanoparticle matrix. In some embodiments, at least about 40% (e.g., from about 40% to about 60%) of the nucleotides of the one or more oligonucleotides are guanine or cytosine. In some embodiments, the one or more oligonucleotides are characterized by a melting temperature (Tm) of at least about 65 degrees Celsius (°C) (e.g., from about 65°C to about 75°C). In some embodiments, the one or more oligonucleotides comprise a conjugated moiety at the 5' end. In some embodiments, the conjugated moiety is a 5'-thiol. In some embodiments, the 5'-thiol is a thioalkyl group. In some embodiments, the thioalkyl is a thiohexyl group. In some embodiments, the nanoparticles among the one or more nanoparticles are assembled with one, two, three, four, five, or six oligonucleotides. In some embodiments, each of the one or more nanoparticles (e.g., independently) is assembled with one, two, three, four, five, or six oligonucleotides. In some embodiments, the one or more nanoparticles comprise gold. In some embodiments, the one or more nanoparticles are characterized by an average size of from about 10 nanometers (nm) to about 200 nm. In some embodiments, the one or more oligonucleotides are 16 to 24 nucleotides in length. In some embodiments, the one or more oligonucleotides associate with the one or more target nucleic acids such that the distance between two adjacent nanoparticles of the one or more nanoparticles corresponds to from about 50 to about 70 nucleotides. In some embodiments, the one or more oligonucleotides comprise two oligonucleotides, wherein the first oligonucleotide hybridizes to a first region of the target nucleic acid, and the second oligonucleotide hybridizes to a second region of the target nucleic acid. In some embodiments, the distance between the first region and the second region of the target nucleic acid is from about 50 to 70 nucleotides. In some embodiments, the one or more target nucleic acids are from one or more viruses or one or more bacteria. In some embodiments, the one or more target nucleic acids are not from a coronavirus. In some embodiments, the one or more target nucleic acids are not from SARS-CoV-2 or one or more of its variants. In some embodiments, the one or more viruses include influenza virus or human papillomavirus. In some embodiments, the one or more bacteria include Salmonella. In some embodiments, Salmonella includes Salmonella enterica. In some embodiments, Salmonella includes one or more Salmonella strains or serotypes.In some embodiments, one or more Salmonella strains or serotypes comprise one or more members selected from Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella pullorum. In some embodiments, one or more target nucleic acids are associated with one or more diseases or conditions. In some embodiments, one or more diseases or conditions include infectious diseases, cancers, or degenerative diseases. In some embodiments, one or more target nucleic acids encode polypeptides or proteins. In some embodiments, one or more target nucleic acids include DNA or RNA. In some embodiments, the DNA is genomic DNA. In some embodiments, the RNA is genomic RNA. In some embodiments, the RNA is double-stranded RNA or single-stranded RNA. In some embodiments, the RNA is double-stranded DNA or single-stranded DNA. In some embodiments, one or more target nucleic acids are from human papillomavirus (HPV) or one or more variants thereof. In some embodiments, one or more target nucleic acids comprise one or more members selected from: the L1 capsid protein of HPV, the L2 capsid protein of HPV, the E6 protein of HPV, the E7 protein of HPV, and fragments of any of the foregoing. In some embodiments, one or more target nucleic acids are from Salmonella. In some embodiments, Salmonella includes Salmonella enterica. In some embodiments, Salmonella includes one or more Salmonella strains or serotypes. In some embodiments, one or more Salmonella strains or serotypes comprise one or more members selected from Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella pullorum. In some embodiments, one or more oligonucleotides comprise sequences selected from SEQ ID NO:1-18.

[0009] In one aspect, the present disclosure provides a kit for identifying the presence of a target nucleic acid, the kit comprising: (i) one or more gold nanoparticles assembled with one or more oligonucleotides, (ii) a condensation solution, (iii) instructions for using the one or more gold nanoparticles assembled with one or more oligonucleotides.

[0010] In one aspect, the present disclosure provides a kit for identifying the presence of a target nucleic acid, the kit comprising: (i) the composition described elsewhere herein, (ii) a condensation solution, (iii) instructions for using the one or more gold nanoparticles assembled with one or more oligonucleotides.

[0011] Those skilled in the art will readily appreciate the additional aspects and advantages of the present disclosure from the following detailed embodiments, in which only illustrative embodiments of the present disclosure are shown and described. As will be recognized, the present disclosure is capable of other different embodiments, and several details thereof can be modified in various obvious aspects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative rather than restrictive.

[0012] Incorporation by reference

[0013] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. If the cited publications, patents, or patent applications conflict with the disclosure in the specification, the specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The patent or application document contains at least one color drawing. After request and payment of the necessary fees, the patent office will provide a copy of the color drawing of this patent or patent application publication. The novel features of the present invention are set forth in detail in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed embodiments that set forth illustrative embodiments utilizing the principles of the present invention and the drawings (also referred to herein as "figures"), in which:

[0015] Figures 1A - 1B An exemplary schematic diagram of the method disclosed herein is shown.

[0016] Figure 2A A representation of a negative sample is shown. Figure 2B A representation of a positive sample is shown. Figure 2C A series of cuvettes with increasing amounts of positive signal are shown.

[0017] Figure 3 An exemplary nanoparticle configuration is shown.

[0018] Figure 4 The ultraviolet-visible spectrum of gold nanoparticles conjugated with oligonucleotides is shown.

[0019] Figure 5 An exemplary apparatus for performing the method of the present disclosure is shown.

[0020] Figure 6 The assay results for detecting HPV nucleic acid sequences are shown.

[0021] Figures 7A - 7C The assay data for detecting HPV using optical density are shown. Figure 7A The average measured values of the samples are shown.Figure 7B Displays the curves of each sample in the CasKi assay, Figure 7C Displays the curves of each sample in the HeLa assay.

[0022] Figures 8A - 8B Displays the assay data for detecting HPV using optical density. Figure 8A Displays the ROC curve of the assay using CasKi cells, Figure 8B Displays the ROC curve of the assay using HeLa cells.

[0023] Figure 9 Displays the assay data for detecting Salmonella using optical density.

[0024] Figure 10 Displays the assay data for detecting Salmonella using optical density.

[0025] Figure 11 Displays the ROC curve of the assay for detecting Salmonella using optical density.

[0026] Figure 12 Displays a computer control system programmed or otherwise configured to implement the methods provided herein. Detailed Description

[0027] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will be apparent to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0028] Nucleic acid detection has wide applications for human health. Specifically, nucleic acids are present in an object and can be associated with a disease condition or pathogen infection. Nucleic acids are present in organisms or microorganisms for replication and protein expression. The detection of specific nucleic acids can indicate the presence of a particular microorganism in a sample / object and can be used to diagnose an object suffering from a condition. Human nucleic acid detection can also be used to identify mutations in an object's genome and can indicate a disease, disorder, or other condition, such as cancer. The detection of specific nucleic acids can involve complex reactions that may require specific conditions and may still provide inaccurate results.

[0029] The present disclosure provides methods, systems, and compositions for detecting specific nucleic acids that are rapid, easy to use, and provide accurate results. The methods can provide an alternative to other nucleic acid detection techniques and can be performed without a thermal cycler or other instrument for heating and / or cooling nucleic acids. The methods can also be performed without using enzymes that allow kits or compositions to be more easily stored and have a longer shelf life. The methods can be performed without nucleic acid extension or amplification. Thus, compared to methods using nucleic acid amplification, the disclosed methods can involve simpler and more robust sample preparation and can be less susceptible to reagent degradation. These methods and compositions can be used without the need for complex or expensive optical instruments that would otherwise generally not be accessible to the public or would be prohibitively expensive for a member of the general public to obtain.

[0030] Compared to other detection methods, these methods and compositions can allow for a rapid test for the presence of nucleic acids and can, for example, generate results within 5 minutes or less. Additionally, the production cost of the compositions can be lower than other tests of similar accuracy and can be less expensive for the test to be performed. The methods can also have other advantages over methods of similar accuracy. For example, the methods can eliminate the need to purify nucleic acids prior to the assay, thereby reducing the time required to generate results. Thus, the methods can be performed without microbial culturing or storing RNA samples.

[0031] Provided herein are methods, systems, and compositions for nucleic acid analyte detection. In one aspect, the present disclosure provides a composition for detecting one or more target nucleic acids (DNA or RNA), the composition comprising: one or more nanoparticles assembled with one or more oligonucleotides, wherein the one or more oligonucleotides are configured to bind to the one or more target nucleic acids, and wherein in the presence of the one or more target nucleic acids, the one or more nanoparticles form a nanoparticle matrix. The nanoparticle matrix can be detected via an optical property or parameter of the nanoparticle matrix. The one or more nanoparticles in solution can have an optical property different from that of the nanoparticle matrix such that the formation of the nanoparticle matrix from the one or more nanoparticles can be identified via the optical property or parameter. Each of the one or more oligonucleotides can comprise from about 16 to about 24 nucleotides. The one or more oligonucleotides can associate with the one or more target nucleic acids such that the (average) distance between two adjacent nanoparticles in the one or more nanoparticles corresponds to from about 50 to about 70 nucleotides. In some cases, the one or more oligonucleotides comprise two oligonucleotides, wherein the first oligonucleotide hybridizes to a first region of the target nucleic acid and the second oligonucleotide hybridizes to a second region of the target nucleic acid. In some embodiments, the distance between the first region and the second region of the target nucleic acid is from about 50 to 70 nucleotides.

[0032] The present disclosure provides a method for processing or analyzing a sample of an object, the method comprising: (a) contacting a bodily sample with a composition comprising one or more nanoparticles assembled with one or more oligonucleotides to provide a test composition, wherein the one or more oligonucleotides; and (2) are configured to bind to one or more target nucleic acids, if present, in the sample; (b) subjecting the test composition of (a) to conditions sufficient to induce aggregation of the one or more nanoparticles in the absence of the one or more target nucleic acids, wherein in the presence of the one or more target nucleic acids, the one or more nanoparticles form a nanoparticle matrix; (c) determining an optical parameter of the test composition that indicates the presence or absence of the one or more target nucleic acids in the sample. Each of the one or more oligonucleotides may comprise from about 16 to about 24 nucleotides. The one or more oligonucleotides may associate with the one or more target nucleic acids such that the (average) distance between two adjacent nanoparticles in the one or more nanoparticles corresponds to from about 50 to about 70 nucleotides.

[0033] In various aspects disclosed herein, the method can be used to determine the presence of a nucleic acid sequence or an infectious pathogen in an object or sample. The method can be used to determine the presence of an infectious pathogen in an object or to diagnose the presence of a disorder or disease.

[0034] In various aspects, one or more oligonucleotides are assembled with the nanoparticles. The one or more oligonucleotides may be complementary to a specific sequence. The one or more oligonucleotides may anneal to a target sequence. In some embodiments, annealing includes generating a duplex between two nucleic acid strands that form hydrogen bonds between complementary bases. After annealing of the one or more oligonucleotides to the target, the resulting product can be detected to determine the presence of the target sequence. The oligonucleotides can be designed to bind, anneal, or hybridize to a specific sequence. For example, the one or more oligonucleotides may anneal to a viral sequence. The one or more oligonucleotides may anneal to a bacterial sequence. The one or more oligonucleotides may anneal to a fungal sequence. The one or more oligonucleotides may anneal to a human sequence. These oligonucleotide sequences can be generated by analyzing the target sequence and generating a sequence that is partially or fully complementary to the target nucleic acid. The one or more oligonucleotides assembled with a given nanoparticle may comprise the same sequence or different sequences. The nanoparticle solution may also comprise nanoparticles having the same oligonucleotides or different oligonucleotides. Figure 3 Illustrated are example nanoparticles that can be used in the methods, compositions, and systems of the present disclosure. The single-nanomulticomposite shows a nanoparticle with different oligonucleotides attached to one nanoparticle. The multi-nanomulticomposite shows multiple oligonucleotide molecules attached to multiple nanoparticles.

[0035] One or more oligonucleotides can include specific or particular characteristics. One or more oligonucleotides can include a melting temperature. The melting temperature can be the temperature at which one or more oligonucleotides remain annealed to a target nucleic acid at a specific assay temperature or reaction temperature. The melting temperature can be the temperature at which one or more oligonucleotides do not remain annealed to a target nucleic acid at a specific assay temperature or reaction temperature. One or more oligonucleotides can have a melting temperature (Tm) of at least about 65 degrees Celsius (°C). One or more oligonucleotides can have a Tm of about 65°C to about 75°C. One or more oligonucleotides can have a Tm that is no more than 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, or 80°C or less. One or more oligonucleotides can have a Tm that is greater than 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, or 80°C or more. The Tm of one or more oligonucleotides can be related to the guanine or cytosine (GC) content of the oligonucleotide. One or more oligonucleotides can contain a specific percentage of a given nucleotide. For example, an oligonucleotide among one or more oligonucleotides can contain at least 20% guanine. An oligonucleotide among one or more oligonucleotides can contain at least 30% guanine. At least 40% of the nucleotides in the oligonucleotide can be guanine or cytosine. At least 45% of the nucleotides in the oligonucleotide can be guanine or cytosine. At least 50% of the nucleotides in the oligonucleotide can be guanine or cytosine. At least 55% of the nucleotides in the oligonucleotide can be guanine or cytosine. At least 60% of the nucleotides in the oligonucleotide can be guanine or cytosine. About 40% to about 60% of the nucleotides in one or more oligonucleotides can be guanine or cytosine. One or more oligonucleotides can include minimal secondary structure. For example, one or more oligonucleotides can not have a hairpin or self-anneal. One or more oligonucleotides can include minimal interaction with each other and can be designed such that the oligonucleotides among one or more oligonucleotides do not anneal to each other.

[0036] One or more oligonucleotides can contain one or more reactive groups or conjugation moieties. The reactive group or conjugation moiety can allow the oligonucleotide to conjugate, attach, or otherwise assemble to another molecule. The oligonucleotide can contain a conjugation moiety that allows it to conjugate to a nanoparticle. One or more oligonucleotides can contain a conjugation moiety at the 5' end. One or more oligonucleotides can contain a conjugation moiety at the 3' end. The conjugation moiety can be a thiol, such as a 3'-thiol or 5'-thiol, such as a thiol group alkyl (e.g., thiol group hexyl).

[0037] One or more oligonucleotides can comprise a specific or particular sequence. One or more oligonucleotides can comprise the sequences of Table 1. The oligonucleotide can comprise a sequence that is the same as or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide can comprise a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% the same as or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide can comprise a sequence that is at least 90% the same as or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide can comprise a sequence that is at least 91% the same as or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide can comprise a sequence that is at least 92% the same as or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide can comprise a sequence that is at least 93% the same as or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide can comprise a sequence that is at least 94% the same as or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide can comprise a sequence that is at least 95% the same as or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide can comprise a sequence that is at least 96% the same as or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide can comprise a sequence that is at least 97% the same as or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide can comprise a sequence that is at least 98% the same as or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide can comprise a sequence that is at least 99% the same as or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide can comprise no more than three, no more than two, or no more than one alteration relative to a sequence selected from SEQ ID NOs: 1-18 or its complementary sequence. The oligonucleotide can comprise no more than three alterations relative to a sequence selected from SEQ ID NOs: 1-18 or its complementary sequence. The oligonucleotide can comprise no more than two alterations relative to a sequence selected from SEQ ID NOs: 1-18 or its complementary sequence. The oligonucleotide can comprise no more than one alteration relative to a sequence selected from SEQ ID NOs: 1-18 or its complementary sequence. No more than three, no more than two, or no more than one alteration can include substitution, addition, deletion, or a combination thereof. No more than three, no more than two, or no more than one alteration can be a substitution. One or more oligonucleotides can comprise degenerate bases or modified bases. The degenerate bases can be a first base on a first oligonucleotide and a second base on a second oligonucleotide. For example, the sequence can comprise a degenerate base at a position represented by the letter "K". In the first oligonucleotide, guanine can be present at the position represented by the letter "K", while in the second oligonucleotide, threonine can be present at the position represented by the letter "K".Thus, the sequence can represent a mixture of a first oligonucleotide and a second oligonucleotide. The use of degenerate bases in an oligonucleotide sequence can allow one or more nucleotides to bind to a greater variety of sequences, such as variant sequences.

[0038] Table 1

[0039]

[0040]

[0041] In various embodiments, one or more oligonucleotides are configured to bind to one or more target nucleic acids. Binding to one or more target nucleic acids can include annealing (or hybridizing) to the target nucleic acid under conditions that produce a double-stranded nucleic acid complex. For example, one or more oligonucleotides can be configured to bind to one or more targets based at least in part on the complementarity of the oligonucleotide to the target nucleic acid. In some embodiments, one or more oligonucleotides hybridize to a target nucleic acid if the oligonucleotide comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to the target nucleic acid.

[0042] One or more oligonucleotides may be capable of annealing to a nucleic acid associated with a disease such as cancer. The nucleic acid target may be associated with a degenerative disease. The nucleic acid target may be a nucleic acid from or derived from an infectious agent. For example, one or more oligonucleotides may be capable of annealing to a nucleic acid comprising a sequence from or derived from a human papillomavirus (HPV) gene. One or more oligonucleotides may be capable of annealing to a nucleic acid comprising a sequence indicative of the presence of HPV in a subject. One or more oligonucleotides may be capable of annealing to a nucleic acid comprising a sequence from the L1 capsid gene of HPV, the L2 capsid gene of HPV, the E6 gene of HPV, the E7 gene of HPV, and fragments thereof. One or more oligonucleotides may be capable of annealing to a nucleic acid encoding a protein or polypeptide. For example, the nucleic acid encoding a protein or polypeptide may encode the L1 capsid protein of HPV, the L2 capsid protein of HPV, the E6 protein of HPV, the E7 protein of HPV, or fragments thereof. One or more oligonucleotides may be capable of annealing to a nucleic acid comprising a sequence indicative of the presence of Salmonella in a subject. One or more oligonucleotides may be capable of annealing to a nucleic acid comprising a sequence from or derived from a Salmonella species. One or more oligonucleotides may be capable of annealing to a nucleic acid comprising a sequence from or derived from Salmonella enterica. One or more oligonucleotides may be capable of annealing to a nucleic acid comprising a sequence from or derived from a different species or serovar of Salmonella. For example, one or more oligonucleotides may be capable of annealing to a nucleic acid comprising a sequence from or derived from Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, or Salmonella pullorum. One or more oligonucleotides may be capable of annealing to a nucleic acid comprising a sequence common between two or more different Salmonella species or serovars. For example, one or more oligonucleotides may be capable of annealing to a nucleic acid comprising a sequence common between Salmonella gallinarum and Salmonella pullorum. In another example, the nucleic acid may comprise a sequence common among multiple members of the genus Salmonella or multiple strains of Salmonella enterica. For example, one or more oligonucleotides may bind to a sequence present in multiple strains of Salmonella enterica and thus indicate the presence of at least one Salmonella strain.

[0043] Combinations of one or more oligonucleotides can be used to detect specific targets. For example, in a given mixture, there can be multiple oligonucleotides with different sequences. In some cases, one or more oligonucleotides include a first oligonucleotide and a second oligonucleotide, where the first oligonucleotide contains a sequence that is the same as or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or complementary to the sequence of SEQ ID NO:9, and the second oligonucleotide contains a sequence that is the same as or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or complementary to the sequence of SEQ ID NO:10. In some cases, one or more oligonucleotides include a first oligonucleotide and a second oligonucleotide, where the first oligonucleotide contains the same sequence as SEQ ID NO:9, and the second oligonucleotide contains the same sequence as SEQ ID NO:10. In some cases, one or more oligonucleotides include a first oligonucleotide and a second oligonucleotide, where the first oligonucleotide contains a sequence that is the same as or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or complementary to the sequence of SEQ ID NO:11, and the second oligonucleotide contains a sequence that is the same as or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or complementary to the sequence of SEQ ID NO:12. In some cases, one or more oligonucleotides include a first oligonucleotide and a second oligonucleotide, where the first oligonucleotide contains the same sequence as SEQ ID NO:11, and the second oligonucleotide contains the same sequence as SEQ ID NO:12. In some cases, one or more oligonucleotides include a first oligonucleotide and a second oligonucleotide, where the first oligonucleotide contains a sequence that is the same as or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or complementary to the sequence of SEQ ID NO:13, and the second oligonucleotide contains a sequence that is the same as or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or complementary to the sequence of SEQ ID NO:14. In some cases, one or more oligonucleotides include a first oligonucleotide and a second oligonucleotide, where the first oligonucleotide contains the same sequence as SEQ ID NO:13, and the second oligonucleotide contains the same sequence as SEQ ID NO:14.In some cases, one or more oligonucleotides include a first oligonucleotide and a second oligonucleotide, the first oligonucleotide comprising a sequence that is the same as or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or complementary to the sequence of SEQ ID NO:15, and the second oligonucleotide comprising a sequence that is the same as or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or complementary to the sequence of SEQ ID NO:16. In some cases, one or more oligonucleotides include a first oligonucleotide and a second oligonucleotide, the first oligonucleotide comprising the same sequence as SEQ ID NO:15, and the second oligonucleotide comprising the same sequence as SEQ ID NO:16. In some cases, one or more oligonucleotides include a first oligonucleotide and a second oligonucleotide, the first oligonucleotide comprising a sequence that is the same as or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or complementary to the sequence of SEQ ID NO:17, and the second oligonucleotide comprising a sequence that is the same as or at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or complementary to the sequence of SEQ ID NO:18. In some cases, one or more oligonucleotides include a first oligonucleotide and a second oligonucleotide, the first oligonucleotide comprising the same sequence as SEQ ID NO:17, and the second oligonucleotide comprising the same sequence as SEQ ID NO:18. Combinations of two or more oligonucleotides can allow multiple oligonucleotides to bind to a target nucleic acid. By binding multiple oligonucleotides to the same nucleic acid target, a structure comprising multiple nanoparticles can be formed and a matrix or colloidal solution can be allowed to be generated. Since one or more oligonucleotides can bind to nanoparticles, multiple nanoparticles can bind to a single target, and similarly, multiple targets can bind to a single nanoparticle having multiple conjugated oligonucleotides. The binding event can generate a matrix formed by the nanoparticles, the target nucleic acid, and one or more oligonucleotides. The matrix can have optical properties different from those of the solution without the matrix, and thus the formation of the matrix can be detectable by observing the optical properties of the solution.

[0044] The optical properties of the matrix can be adjusted or can depend on the distance between adjacent nanoparticles. The distance between adjacent nanoparticles can be adjusted via the length of the oligonucleotide. This distance can be adjusted by the number of nucleotides separating the target sequences on a given nucleic acid target. For example, as described herein, a variety of oligonucleotides can bind to a nucleic acid target. A first oligonucleotide can bind to a first sequence that is a plurality of nucleotides from the binding position of a second oligonucleotide. The first oligonucleotide can bind to a sequence that is at least 30 nucleotides from the sequence to which the second oligonucleotide binds. The first oligonucleotide can bind to a sequence that is no more than 30 nucleotides from the sequence to which the second oligonucleotide binds. The first oligonucleotide can bind to a sequence that is at least 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 or more nucleotides from the sequence to which the second oligonucleotide binds. The first oligonucleotide can bind to a sequence that is no more than 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 or more nucleotides from the sequence to which the second oligonucleotide binds. The first oligonucleotide can bind to a sequence that is 50 to 70 nucleotides from the sequence to which the second oligonucleotide binds. The length of the oligonucleotide can alter the ability to form a matrix based on the physical properties of the nanoparticle and the oligonucleotide. For example, an oligonucleotide that is too short and has an insufficient spacing between adjacent binding positions can cause adjacent nanoparticles to come into direct contact or can sterically inhibit matrix formation. Similarly, the size of the nanoparticle can be related to the distance separating the nanoparticles. For example, smaller nanoparticles can allow for a smaller distance between adjacent nanoparticles while still generating a matrix when binding to the target nucleic acid. Thus, the generation of a matrix when binding to the target nucleic acid can be sufficient to detect the presence of the target nucleic acid, regardless of the specific oligonucleotide length and nucleotide separation distance.

[0045] One or more oligonucleotides can comprise a certain length or number of nucleotides. For example, one or more oligonucleotides can be at least 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 or more nucleotides in length. For example, one or more oligonucleotides can be no more than 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 or fewer nucleotides in length. The oligonucleotide can be about 16 to about 24 nucleotides in length. The oligonucleotide can be about 10 to about 20 nucleotides in length. The oligonucleotide can be about 20 to about 30 nucleotides in length.

[0046] The systems, methods, and compositions of the present disclosure can comprise nanoparticles. The nanoparticles can be used to detect the presence of a target nucleic acid. The nanoparticles can comprise one or more oligonucleotides or otherwise be assembled with one or more oligonucleotides. Assembly can mean that the nanoparticles and the oligonucleotides are conjugated to each other. Assembly can mean that the nanoparticles and the oligonucleotides are linked to each other. In some embodiments, the nanoparticles in one or more nanoparticles are assembled with one, two, three, four, five, or six oligonucleotides. In some embodiments, the nanoparticles in one or more nanoparticles are assembled with at least one, two, three, four, five, or six or more oligonucleotides. Each of one or more nanoparticles can be (e.g., independently) assembled with one, two, three, four, five, or six oligonucleotides. For example, a first nanoparticle can comprise a first oligonucleotide and a second oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide comprise different sequences. For example, a first nanoparticle can comprise a first oligonucleotide, and a second nanoparticle can comprise a second oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide comprise different sequences. In another example, a first nanoparticle can comprise a first oligonucleotide, and a second nanoparticle can comprise a second oligonucleotide, wherein the first oligonucleotide and the second oligonucleotide comprise the same sequence.

[0047] One or more nanoparticles can comprise various materials. The nanoparticles can comprise gold. The nanoparticles can comprise metals. The metals can have optical properties based on coordination chemistry. The metals can have optical properties based on reflectivity, absorption or transmission at specific wavelengths. The nanoparticles can comprise materials including optical properties (e.g., reflectivity, transmittance). The optical properties of the nanoparticles can allow the particles to be detected in solution. The optical properties can be changed in the presence of a target nucleic acid. For example, in the presence of a target nucleic acid, the nanoparticles can produce a nanoparticle matrix having optical properties different from those of a single nanoparticle.

[0048] Various compositions may include the use of gold nanoparticles. Gold is a practical element for nanoscience given its unique stability as a pure metal. Gold nanoparticles are appreciated for their optical and electronic properties such as those described in https: / / www.sigmaaldrich.com / BR / pt / technical-documents / technical-article / materials-science-and-engineering / biosensors-and-imaging / gold-nanoparticles, gold having outstanding optical properties such as high extinction coefficient, chemical stability, water solubility, localized surface plasmon resonance, and intrinsic photostability. The interaction or breaking of the chemical bond between the nanoscale particles and the oligonucleotide allows the revelation of positive or negative results. An observation evidenced by a departure from its original color implies a negative result. The strength of the binding between the conjugate and the nucleic acid being studied can determine the sensitivity and specificity of the method.

[0049] The reddish appearance that is characteristic of gold nanoparticles is related to their reduced volume and the large number of electrons on their surface. According to theory, the luminescent display is produced by the incidence of light on the surface of the conduction band nanoparticles (plasmon resonance), which propagates with waves associated with characteristic reception values. Surface resonance bands in the visible region can be observed, which are produced by the lattice oscillations of the electrons in this region relative to the metal ions. Therefore, in the presence of particle agglomerates or when the diameter increases, a solution color change from red to blue can be observed.

[0050] The optical and electronic properties of gold nanoparticles can be tuned by changing their size, shape, surface chemistry, or state of aggregation. Optical interactions and developments can be determined by their size and dimensions. A light beam propagating near the colloidal nanoparticles can interact with free electrons and induce oscillations of the electron charge signal. This phenomenon is directly related to the frequency of visible light. Short gold nanoparticles (about 30 nm) have an absorption wavelength in the blue-green part (450 nm spectrum), while red light in the spectrum is reflected at 700 nm. When red light is absorbed and blue light is reflected, the solution appears blue or purple in color. As the particle size increases towards the mass limit, the surface plasmon resonance wavelength shifts, and more visible light is reflected, making the nanoparticles appear light-colored or translucent. Surface plasmon resonance is flexible and changes depending on its application, size, or shape. The chemical interaction of gold nanoparticles when in contact with saline solution or excess salt brings neutrality to the reaction, causing nanoparticle aggregation. This results in a change from the original red color to blue. This problem can be corrected by desalination or protected by coating with polymers, small molecules, and specific biorecognition molecules.

[0051] Gold nanoparticles (AuNP) can contain surface citrate molecules in solution. These surface citrate molecules can be replaced with differentially thiol-functionalized oligonucleotides such as antisense oligonucleotides (ASO). The combination of the intrinsic optical properties of AuNP with the targeting ability of oligonucleotides can be used to develop a selective detection platform. The reactivity of the oligonucleotide with the target can be correlated with the surface resonance in the visible region without the need for any expensive instrumentation techniques. Optical changes can be observed either by the naked eye or using a camera or other image analysis techniques without the need to observe specific wavelengths with more expensive instrumentation. Additionally, the ratio of the oligonucleotide (e.g., the ratio of ASO to AuNP (ASO / AuNP)) can be adjusted to tune the sensitivity of the biosensor to the target. The increase in AuNP-ASO sensitivity can be monitored by changing the incubation time using a defined concentration of the target analyte (DNA / RNA), knowing that the optimal sensitivity is achieved at 37 °C. The relative sensitivity of the gold nanoparticles coated with ASO is observed by monitoring the relatively increased absorbance at 660 nm. This enables the evaluation of different colloidal distribution profiles within the analyzed range.

[0052] One or more nanoparticles can be characterized by size. One or more nanoparticles can be characterized by an average size of about 10 nanometers (nm). One or more nanoparticles can be characterized by an average size of about 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm. One or more nanoparticles can be characterized by an average size of at least 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm or greater. One or more nanoparticles can be characterized by an average size of no more than 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm or smaller. One or more nanoparticles can be characterized by a size of about 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm. One or more nanoparticles can be characterized by a size of at least about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm or greater. One or more nanoparticles can be characterized by a size of no more than about 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm or smaller.

[0053] The sample can be a biological sample. The sample can be derived from a biological sample. The biological sample can be, for example, a blood sample, a serum sample, a plasma sample, a saliva sample, a fecal sample, a sputum sample, a urine sample, a semen sample, a vaginal fluid sample, a cerebrospinal fluid sample, a sweat sample, a cell sample, and a tissue sample. The biological sample can be a fluid sample. The fluid sample can be blood or plasma. The sample can be from or derived from an animal. The sample can be from or derived from a mammal. The sample can be from or derived from a plant. The sample can be from or derived from a human. The sample can contain nucleic acids.

[0054] Nucleic acid target

[0055] The nucleic acid targets of the present disclosure can be from a sample. The biological sample can be a sample from or derived from a subject. The sample can contain any number of macromolecules, such as cellular macromolecules. The sample can contain multiple cells. The sample can be a tissue sample, such as a biopsy sample, a core biopsy sample, a needle aspiration sample, or a fine needle aspiration sample. The sample can be a tumor sample. The sample can be a fluid sample, such as a blood sample, a plasma sample, a urine sample, or a saliva sample. The sample can be a skin sample. The biological sample can be a cheek swab. The sample can be a plasma or serum sample. The sample can contain one or more cells. The one or more cells can be from or derived from a tumor. The biological sample can be, for example, blood, plasma, serum, urine, saliva, mucosal excretions, sputum, feces, or tears.

[0056] The nucleic acid target can be from or derived from one or more cells. The nucleic acid target can include deoxyribonucleic acid (DNA). The DNA can be any kind of DNA, including genomic DNA. The nucleic acid target can be viral DNA. The nucleic acid target can include ribonucleic acid (RNA). The RNA can be any kind of RNA, including messenger RNA, transfer RNA, ribosomal RNA, and microRNA. The RNA can be viral RNA. The nucleic acid can include human genomic sequences. The nucleic acid can include animal genomic sequences. The nucleic acid can include plant genomic sequences. The nucleic acid can include fungal genomic sequences. The nucleic acid can include archaeal genomic sequences. The nucleic acid can include pathogen-related sequences. The nucleic acid can include wild-type sequences. The nucleic acid can include variant sequences.

[0057] One or more target nucleic acids can be of any length. The target nucleic acid can be, for example, up to 1, 2, 3, 4, 5, 10, 20, 50, 100, 500, 1000, 5000, 10000, 50000, or 100000 nucleotides or more.

[0058] In some cases, the target nucleic acid can include a gene or a portion thereof. The nucleic acid target can include a gene whose detection can be used to diagnose one or more diseases. The gene can be a viral gene or a bacterial gene, and its detection can be used to identify the presence or absence of a pathogen in a subject. In some cases, the methods of the present disclosure can be used to detect the presence or absence of one or more infectious agents (e.g., viruses, bacteria, fungi) in a subject. The nucleic acid target can be a human gene.

[0059] The nucleic acid target can be associated with a disease, such as cancer. The nucleic acid target can be associated with a degenerative disease. The nucleic acid target can be a nucleic acid from or derived from an infectious agent. For example, the nucleic acid target can include a sequence from or derived from a human papillomavirus (HPV) gene. The nucleic acid target can include a sequence that is present in a subject with HPV. The nucleic acid target can include sequences from the L1 capsid gene of HPV, the L2 capsid gene of HPV, the E6 gene of HPV, the E7 gene of HPV, and fragments thereof. The nucleic acid target can encode a protein or polypeptide. For example, the nucleic acid target can encode the L1 capsid protein of HPV, the L2 capsid protein of HPV, the E6 protein of HPV, the E7 protein of HPV, or fragments thereof. The nucleic acid target can include a sequence from or derived from a Salmonella species. The nucleic acid target can include a sequence from or derived from Salmonella enterica. The nucleic acid target can include sequences from or derived from different species or serotypes of Salmonella. For example, the nucleic acid target can include a sequence from or derived from Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum; or Salmonella pullorum. The nucleic acid target can include a sequence that is shared between two or more different species or serotypes of Salmonella. For example, the nucleic acid target can include a sequence shared by Salmonella gallinarum and Salmonella pullorum. In another example, the nucleic acid can include a sequence that is shared among multiple members of the genus Salmonella or multiple strains of Salmonella enterica. For example, one or more oligonucleotides can bind to a sequence present in multiple strains of Salmonella enterica and thus indicate the presence of at least one Salmonella strain.

[0060] In some cases, the method can be carried out by using a composition as disclosed elsewhere herein. The method can be used to carry out a reaction. The reaction can include a hybridization reaction. For example, the composition can contain nucleic acid and hybridize with another nucleic acid. The method can include inducing or causing the aggregation of one or more nanoparticles. The method can include inducing or causing the formation of a nanoparticle matrix of one or more nanoparticles. The method can include adding a solution. The method can include adding a coagulant. The solution (e.g., a coagulation solution) and / or the coagulant can cause nanoparticle aggregation. For example, the presence of salt can induce the aggregation of nanoparticles. The solution can cause the formation of a nanoparticle matrix. The aggregation or formation of the nanoparticle matrix can depend on the structure or molecule associated with the nanoparticle. The nanoparticle can contain one or more oligonucleotides, which can interact to form aggregates or a matrix. In the presence of a molecule (such as a complementary sequence) that binds to one or more oligonucleotides, aggregation or matrix formation can be prevented or inhibited. The coagulant can include magnesium chloride.

[0061] Compared with the formation of a nanoparticle matrix, the formation of nanoparticle aggregates can be detected and used to detect one or more target nucleic acids. A solution can be added to the mixture, which causes nanoparticle aggregation or alternatively forms a nanoparticle matrix. The formation of aggregates and the formation of a nanoparticle matrix can depend on the presence of the target nucleic acid. In the absence of the target nucleic acid, the nanoparticles can form aggregates, while in the presence of the target nucleic acid, the nanoparticles can form a matrix. The aggregates and the matrix can have different optical properties and can therefore be detectable and distinguishable based on these optical properties. Since aggregation or matrix formation depends on the presence of the target nucleic acid, detecting the aggregates or the matrix can indicate the presence of the target nucleic acid.

[0062] The method can include detecting or determining an optical parameter of a solution or composition. Detecting or determining can include using a sensor to detect a wavelength. Detecting or determining can include using a camera. Detecting or determining can include image analysis techniques. The optical parameter can include absorption, transmission, scattering, or reflection of light or other waves at a wavelength or wavelength range. The optical parameter can include a photometric parameter (e.g., the brightness of a color), a saturation parameter (e.g., the intensity of a color), or a hue parameter (e.g., the shade of a color). Determining can include using color spacing analysis. The optical parameter can be binned based on a wavelength or wavelength range. For example, the amount of red or green in a color can be parameterized. Based on color theory, a color cannot be both red and green simultaneously, allowing for the generation of a single parameter based on a red - green scale. Parameterization can assign values to the redness or greenness of a value, where the amount of red is parameterized as a positive number and the amount of green. In the same way, the amount of yellow or blue in a color can be parameterized. Analysis of red - green and yellow - blue can indicate a parameterized color, which can be used for other analyses. The photometric or brightness of the solution can also be parameterized. The optical parameter can be an optical density, e.g., the optical density at 520 nm or 560 nm. By observing the optical density of a sample, the sample can be termed positive or negative. A negative control sample, a biological control sample, or a positive control can also be used to calibrate the method or allow for normalization or reference of the sample. For example, it can be observed that the optical parameter is significantly (e.g., statistically significantly) lower or higher than the negative control. This significant difference can be used to determine whether the sample is a negative or positive sample. The methods of the present disclosure can be used to detect various wavelengths.For example, the detection can be carried out using wavelengths at approximately 300 nm, 305 nm, 310 nm, 315 nm, 320 nm, 325 nm, 330 nm, 335 nm, 340 nm, 345 nm, 350 nm, 355 nm, 360 nm, 365 nm, 370 nm, 375 nm, 380 nm, 385 nm, 390 nm, 395 nm, 400 nm, 405 nm, 410 nm, 415 nm, 420 nm, 425 nm, 430 nm, 435 nm, 440 nm, 445 nm, 450 nm, 455 nm, 460 nm, 465 nm, 470 nm, 475 nm, 480 nm, 485 nm, 490 nm, 495 nm, 500 nm, 505 nm, 510 nm, 515 nm, 520 nm, 525 nm, 530 nm, 535 nm, 540 nm, 545 nm, 550 nm, 555 nm, 560 nm, 565 nm, 570 nm, 575 nm, 580 nm, 585 nm, 590 nm, 595 nm, 600 nm, 605 nm, 610 nm, 615 nm, 620 nm, 625 nm, 630 nm, 635 nm, 640 nm, 645 nm, 650 nm, 655 nm, 660 nm, 665 nm, 670 nm, 675 nm, 680 nm, 685 nm, 690 nm, 695 nm, 700 nm, 705 nm, 710 nm, 715 nm, 720 nm, 725 nm, 730 nm, 735 nm, 740 nm, 745 nm, 750 nm, 755 nm, 760 nm, 765 nm, 770 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm, 805 nm, 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 845 nm, 850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm, 895 nm or 900 nm.For example, the detection can be carried out using the following wavelengths: at least 300 nm, 305 nm, 310 nm, 315 nm, 320 nm, 325 nm, 330 nm, 335 nm, 340 nm, 345 nm, 350 nm, 355 nm, 360 nm, 365 nm, 370 nm, 375 nm, 380 nm, 385 nm, 390 nm, 395 nm, 400 nm, 405 nm, 410 nm, 415 nm, 420 nm, 425 nm, 430 nm, 435 nm, 440 nm, 445 nm, 450 nm, 455 nm, 460 nm, 465 nm, 470 nm, 475 nm, 480 nm, 485 nm, 490 nm, 495 nm, 500 nm, 505 nm, 510 nm, 515 nm, 520 nm, 525 nm, 530 nm, 535 nm, 540 nm, 545 nm, 550 nm, 555 nm, 560 nm, 565 nm, 570 nm, 575 nm, 580 nm, 585 nm, 590 nm, 595 nm, 600 nm, 605 nm, 610 nm, 615 nm, 620 nm, 625 nm, 630 nm, 635 nm, 640 nm, 645 nm, 650 nm, 655 nm, 660 nm, 665 nm, 670 nm, 675 nm, 680 nm, 685 nm, 690 nm, 695 nm, 700 nm, 705 nm, 710 nm, 715 nm, 720 nm, 725 nm, 730 nm, 735 nm, 740 nm, 745 nm, 750 nm, 755 nm, 760 nm, 765 nm, 770 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm, 805 nm, 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 845 nm, 850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm, 895 nm or 900 nm, or greater.For example, the detection can be performed using the following wavelengths: not more than 300 nm, 305 nm, 310 nm, 315 nm, 320 nm, 325 nm, 330 nm, 335 nm, 340 nm, 345 nm, 350 nm, 355 nm, 360 nm, 365 nm, 370 nm, 375 nm, 380 nm, 385 nm, 390 nm, 395 nm, 400 nm, 405 nm, 410 nm, 415 nm, 420 nm, 425 nm, 430 nm, 435 nm, 440 nm, 445 nm, 450 nm, 455 nm, 460 nm, 465 nm, 470 nm, 475 nm, 480 nm, 485 nm, 490 nm, 495 nm, 500 nm, 505 nm, 510 nm, 515 nm, 520 nm, 525 nm, 530 nm, 535 nm, 540 nm, 545 nm, 550 nm, 555 nm, 560 nm, 565 nm, 570 nm, 575 nm, 580 nm, 585 nm, 590 nm, 595 nm, 600 nm, 605 nm, 610 nm, 615 nm, 620 nm, 625 nm, 630 nm, 635 nm, 640 nm, 645 nm, 650 nm, 655 nm, 660 nm, 665 nm, 670 nm, 675 nm, 680 nm, 685 nm, 690 nm, 695 nm, 700 nm, 705 nm, 710 nm, 715 nm, 720 nm, 725 nm, 730 nm, 735 nm, 740 nm, 745 nm, 750 nm, 755 nm, 760 nm, 765 nm, 770 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm, 805 nm, 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 845 nm, 850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm, 895 nm or 900 nm, or less. The detection can be performed by obtaining a spectrum of any subset of the wavelength range from 300 nm to 900 nm. For example, the detection can be performed from 450 nm to 700 nm.

[0063] The detection can be performed using a plate reader, a spectrophotometer or other instrument capable of detecting light or UV waves. For example, the instrument can include a detector that can quantify the amount of light received. The instrument can use a monochromator to direct a specific wavelength to the sample or to observe a given wavelength from the sample.

[0064] The method can include comparing the optical parameters of a test solution with a reference or control solution. The optical parameters of the test solution and the reference solution can be determined and compared. Based on the comparison of the test solution and the reference solution, the detection of the target nucleic acid can be determined. For example, the reference solution can be a positive control solution and contain the target nucleic acid. This reference solution can include optical parameters. If the solution contains the target nucleic acid, the test solution can also be analyzed, and the optical parameters of the test solution and the reference solution can be the same (or substantially similar). The reference solution can also be a negative control type solution in which the target nucleic acid is absent or there is another known sequence that is not the target sequence. The optical parameters of the test solution and the reference solution can be compared, and a determination result can be generated based on the similarity of the optical parameters.

[0065] The aggregated nanoparticles or nanoparticle matrix can include optical parameters that are different from one or more of the nanoparticles in the solution. Because the optical parameters can be different when the aggregated nanoparticles (or nanoparticle matrix) are compared with the nanoparticles in the solution, it can be possible to detect the presence of the aggregated nanoparticles or nanoparticle matrix and distinguish a solution having aggregated particles (or nanoparticle matrix) from a solution having non-aggregated nanoparticles or individual nanoparticles in the solution. The detection of the target nucleic acid can use this difference to determine whether the target nucleic acid is present. The target nucleic acid can inhibit the formation of the aggregate or matrix such that the lack of the aggregate or matrix can indicate the presence of the target nucleic acid.

[0066] For example, biosensor homogeneous dispersion (AuNP-ASO; no target nucleic acid; no aggregating agent): can be characterized by the maximum distance from the AuNP-ASO where minimal aggregation of the particles is observed. Reactivity can be visualized by a change in the reddish portion. Positive heterogeneous dispersion (AuNP-ASO + target RNA / DNA + MgCl2): can be characterized by the controlled distance of the AuNP-ASO that forms a bridge by recognizing the genetic material (RNA / DNA). The annealing distance can include a minimum number of bases (60 ± 10 nucleotides). In the presence of MgCl2, partial aggregation of the particles can be observed and is associated with an opaque gray / purple visualization. Negative heterogeneous dispersion (AuNP-ASO + MgCl2): can be characterized by the high aggregation of the AuNP-ASO in the presence of MgCl2 and is associated with a visualization that changes from reddish to gray / translucent blue. Based on the color of the solution (at least based on aggregation), different dispersion patterns can be distinguished and the presence of the target nucleic acid can be determined.

[0067] The method can determine the presence or absence of one or more target nucleic acids in a bodily sample with a sensitivity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The method can determine the presence or absence of one or more target nucleic acids in a bodily sample with a sensitivity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The method can determine the presence or absence of one or more target nucleic acids in a bodily sample with a sensitivity of at least 95%, 96%, 97%, 98% or 99%. The method determines the presence or absence of one or more target nucleic acids in a bodily sample with an accuracy of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0068] The method can be performed using a device that permits the performance of the method. Figure 5 An example device is shown. The inlet of the device can be used to add the sample and the lysis solution. Then, this solution is allowed to flow into a mixing module, a heating chamber, and a filter such that the sample solution contains the extracted nucleic acid. The sample is allowed to flow into a pool where a biosensor (e.g., an AuNP containing an oligonucleotide) is added. This permits mixing in the mixing module and then flowing into a new pool for the addition of a display solution (e.g., a flocculating solution). Then, this solution is mixed in the mixing module and then flows into a new pool for optical reading, such as RGB reading or determining another optical parameter, such as one described elsewhere herein.

[0069] The method can be performed in a plate containing one or more wells. For example, the method can use a 96-well plate. For example, when a given sample is present in each well, multiple assays can be performed simultaneously. Then, the wells can be analyzed using a plate reader, and an output can be provided for each sample. Thus, the assay permits rapid and efficient multiplexing, which can reduce wait times and increase throughput. The method can also be performed using a cuvette or a tube or other container. For example, the method can be performed by mixing the sample in a tube (e.g., a centrifuge tube or a microcentrifuge tube). Then, the sample can be added to a cuvette for reading via a spectrophotometer or other instrument capable of detecting optical properties.

[0070] The ability of a method and composition to identify the presence of a target nucleic acid can be measured according to the accuracy of the assay, the sensitivity of the assay, the specificity of the assay, the positive predictive value (PPV) of the assay, the negative predictive value (NPV) of the assay, or the "area under the curve" (AUC) (e.g., the area under the receiver operating characteristic (ROC) curve). As used herein, accuracy is a measure of the fraction of misclassified samples. Accuracy can be calculated as the total number of correctly classified samples divided by, for example, the total number of samples in the test population. Sensitivity is a measure of the "true positives" predicted to be positive by the test, and can be calculated as the number of correctly identified cancer samples divided by the total number of cancer samples. Specificity is a measure of the "true negatives" predicted to be negative by the test, and can be calculated as the number of correctly identified normal samples divided by the total number of normal samples. The AUC is a measure of the area under the receiver operating characteristic curve, which is a plot of sensitivity versus false positive rate (1 - specificity). The larger the AUC, the more powerful the predictive value of the test. In some embodiments, the method can identify the presence of a target nucleic acid with an area under the curve (AUC) of at least about 0.9. In some embodiments, the method can identify the presence of a target nucleic acid with an area under the curve (AUC) of at least about 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or higher. In some embodiments, the method can identify the presence of a target nucleic acid with an area under the curve (AUC) of at least about 0.50, at least about 0.55, at least about 0.60, at least about 0.65, at least about 0.70, at least about 0.75, at least about 0.80, at least about 0.85, at least about 0.90, at least about 0.95, or greater. Other useful measures of the utility of the test include the "positive predictive value" and the "negative predictive value", where the "positive predictive value" is the percentage of actual positives that test positive, and the "negative predictive value" is the percentage of actual negatives that test negative. In some embodiments, the method can identify the presence of a target nucleic acid with a positive predictive value of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or greater. In some embodiments, the method can identify the presence of a target nucleic acid with a negative predictive value of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or greater. In some embodiments, the methods described herein exhibit an accuracy of at least about 75%, such as at least about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, or about 100% accuracy.For example, the method can identify the presence of a target nucleic acid in at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or a greater area. In other embodiments, the method can identify the presence of a target nucleic acid with at least about 75% specificity, such as at least about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99% or about 100% specificity. For example, the method can identify the presence of a target nucleic acid with at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or greater specificity. In some embodiments, the method can identify the presence of at least about 75% of the target nucleic acid, such as at least about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99% or about 100% sensitivity. For example, the method can identify the presence of a target nucleic acid with at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or greater sensitivity. In other embodiments, the method can identify the presence of a target nucleic acid with at least about 75% specificity and sensitivity each, such as at least about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99% or about 100% specificity and sensitivity (e.g., at least about 80% specificity and at least about 80% sensitivity, or for example, at least about 80% specificity and at least about 95% sensitivity).

[0071] The methods of the present disclosure can be performed in a short period of time and can be faster than other methods with similar metrics of accuracy. For example, the methods of the present disclosure can be performed in no more than 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes or less. Starting from pre-assembled nanoparticles (e.g., one or more nanoparticles assembled to one or more oligonucleotides), the method can be performed in less than 60 minutes. Starting from pre-assembled nanoparticles (e.g., one or more nanoparticles assembled to one or more oligonucleotides), the method can be performed in no more than 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes or less. Starting from one or more nanoparticles not assembled to one or more oligonucleotides, the method can be performed in no more than 60 minutes, 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes or less.

[0072] Kit

[0073] The present disclosure provides kits for performing the methods of the present disclosure. The present disclosure also provides kits comprising the compositions described in the present disclosure. The kits can comprise nanoparticles and oligonucleotides as described elsewhere herein. For example, the kits can comprise nanoparticles assembled to one or more oligonucleotides. The kits can comprise a condensation solution. The kits can comprise a tube that contains the nanoparticles.

[0074] The kits can comprise instructions for using any of the foregoing methods described herein. The kits can comprise solutions or other components that can be used as standards, negative or positive controls. For example, the kits can comprise a standard that can be used as a baseline for color analysis or determination of optical parameters. The kits can comprise swabs or other implements for collecting samples from a subject. The kits can comprise means for adding solutions to run the methods of the present disclosure or otherwise run the methods of the present disclosure.

[0075] Computer control system

[0076] The present disclosure provides a computer control system programmed to implement the methods of the present disclosure. Figure 12 Shown is a computer system 1201, which is programmed or otherwise configured to perform portions of the methods disclosed elsewhere herein. The computer system 1201 can regulate various aspects of the present disclosure, such as, for example, processing images of samples, determining and processing optical parameters, receiving images from a user, processing the images such that colors and color differences between the sample and a reference color can be identified, processing the images using an edge detector algorithm, outputting results regarding the presence of nucleic acids for the user. The computer system 1201 can be an electronic device of the user or a computer system remotely located relative to the electronic device. The electronic device can be a mobile electronic device.

[0077] The computer system 1201 includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") 1205, which can be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system 1201 also includes a memory or storage location 1210 (e.g., random access memory, read-only memory, flash memory), an electronic storage unit 1215 (e.g., hard disk), a communication interface 1220 for communicating with one or more other systems (e.g., network adapter), and peripheral devices 1225 (such as caches, other memories, data storage, and / or electronic display adapters). The memory 1210, storage unit 1215, interface 1220, and peripheral devices 1225 communicate with the CPU 1205 via a communication bus (solid lines), such as a motherboard. The storage unit 1215 can be a data storage unit (or data repository) for storing data. With the aid of the communication interface 1220, the computer system 1201 can be operably coupled to a computer network ("network") 1230. The network 1230 can be the Internet, an intranet, and / or an extranet, or an intranet and / or extranet that communicates with the Internet. In some cases, the network 1230 is a telecommunications and / or data network. The network 1230 can include one or more computer servers that can enable distributed computing, such as cloud computing. In some cases, with the aid of the computer system 1201, the network 1230 can implement a peer-to-peer network, which can enable devices coupled to the computer system 1201 to act as clients or servers.

[0078] The CPU 1205 can execute a series of machine-readable instructions, which can be embodied in a program or software. The instructions can be stored in a storage location, such as the memory 1210. The instructions can be directed to the CPU 1205, which can then program or otherwise configure the CPU 1205 to implement the methods of the present disclosure. Examples of operations performed by the CPU 1205 can include fetching, decoding, executing, and writing back.

[0079] The CPU 1205 can be part of a circuit (such as an integrated circuit). One or more other components of the system 1201 can be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC).

[0080] The storage unit 1215 can store files, such as drivers, libraries, and saved programs. The storage unit 1215 can store user data, such as user preferences and user programs. In some cases, the computer system 1201 can include one or more additional data storage units external to the computer system 1201, such as on a remote server that communicates with the computer system 1201 via an intranet or the Internet.

[0081] The computer system 1201 can communicate with one or more remote computer systems via the network 1230. For example, the computer system 1201 can communicate with the remote computer systems of users (such as patients, healthcare providers, test administrators). Examples of remote computer systems include personal computers (such as portable PCs), tablets or tablet PCs (such as, iPad, Galaxy Tab), telephones, smartphones (such as, iPhone, Android - enabled devices, ) or personal digital assistants. Users can access the computer system 1201 via the network 1230.

[0082] The methods described herein can be implemented by machine (e.g., computer processor) - executable code stored at an electronic storage location of the computer system 1201 (such as on the memory 1210 or the electronic storage unit 1215). The machine - executable or machine - readable code can be provided in the form of software. During use, the code can be executed by the processor 1205. In some cases, the code can be retrieved from the storage unit 1215 and stored on the memory 1210 for ready access by the processor 1205. In some cases, the electronic storage unit 1215 can be excluded, and the machine - executable instructions are stored on the memory 1210.

[0083] The code can be pre - compiled and configured for use with a machine having a processor suitable for executing the code, or can be compiled during runtime. The code can be provided in a programming language, and the programming language can be chosen such that the code can be executed in a pre - compiled or just - in - time compiled manner.

[0084] Aspects of the systems and methods provided herein, such as computer system 1201, may be embodied in programming. Various aspects of technology may be considered "products" or "articles of manufacture" that are typically carried or embodied in a type of machine-readable medium in the form of machine (or processor) executable code and / or associated data. Machine executable code may be stored on an electronic storage unit, such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. A "storage" type medium may include any or all tangible memories of a computer, processor, etc., or associated modules, such as various semiconductor memories, tape drives, disk drives, etc., which can provide non-transitory storage for software programming at any time. All or part of the software can sometimes be communicated via the Internet or various other telecommunications networks. For example, such communication can enable the software to be loaded from one computer or processor to another, e.g., from an administrative server or host to a computer platform of an application server. Thus, another type of medium that can carry software elements includes optical, electrical, and electromagnetic waves, such as those used over wired and optical landline networks and over various air links on a physical interface between local devices. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., can also be considered media that carry software. As used herein, unless restricted to non-transitory, tangible "storage" media, the term such as computer or machine "readable medium" refers to any medium that participates in providing instructions to a processor for execution.

[0085] Thus, machine-readable media, such as computer-executable code, can take many forms, including but not limited to tangible storage media, carrier media, or physical transmission media. Non-volatile storage media includes, for example, optical or magnetic disks (such as any storage device in any computer, etc.), which can be used to implement databases, etc., as shown in the accompanying drawings. Volatile storage media includes dynamic memory, such as the main memory of such a computer platform. Tangible transmission media includes coaxial cables; copper wire and optical fiber, including the wires that make up the internal bus of a computer system. Carrier transmission media can take the form of electrical or electromagnetic signals, or acoustic or optical waves, such as acoustic or optical waves generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer-readable media include, for example: floppy disks, flexible disks, hard disks, magnetic tapes, any other magnetic media, CD-ROMs, DVDs or DVD-ROMs, any other optical media, punched cards, paper tapes, any other physical storage media with a pattern of holes, RAM, ROM, PROM, and EPROM, FLASH-EPROM, any other storage chip or cartridge, carriers that transmit data or instructions, cables or links that transmit such carriers, or any other medium from which a computer can read programming code and / or data. Many of these forms of computer-readable media can participate in transferring one or more sequences of one or more instructions to a processor for execution.

[0086] The computer system 1201 can include or communicate with an electronic display 1235, which includes a user interface (UI) 1240 for providing, for example, the results of a method, optical parameters of a solution, or an image. Examples of UIs include but are not limited to graphical user interfaces (GUIs) and web-based user interfaces.

[0087] The methods and systems of the present disclosure can be implemented by one or more algorithms. The algorithms can be implemented in software when executed by a central processing unit 1205. The algorithms can, for example, determine the optical parameters of a solution via image analysis, perform a comparison of optical parameters, or normalize optical parameters against a baseline or control.

[0088] List of Embodiments Embodiment 1. A method for processing or analyzing a sample, the method comprising:

[0089] (a) contacting the sample with a composition comprising one or more nanoparticles, the one or more nanoparticles being assembled with one or more oligonucleotides, wherein the one or more oligonucleotides hybridize to one or more target nucleic acids that may be present in the sample;

[0090] (b) In the presence of one or more target nucleic acids, a nanoparticle matrix is formed from the one or more nanoparticles hybridized to the one or more target nucleic acids;

[0091] (c) Determine an optical parameter of the test composition, the optical parameter indicating the presence or absence of the one or more nucleic acids in the sample.

[0092] Embodiment 2. The method according to Embodiment 1, wherein the optical parameter is determined by color space analysis.

[0093] Embodiment 3. The method according to Embodiment 1 or Embodiment 2, wherein the optical parameter includes absorption, transmission, scattering, or reflection of light at a wavelength or wavelength range.

[0094] Embodiment 4. The method according to any one of Embodiments 1-3, wherein the optical parameter includes a photometric parameter (e.g., brightness of a color), a saturation parameter (e.g., intensity of a color), or a hue parameter (e.g., chromaticity of a color).

[0095] Embodiment 5. The method according to any one of Embodiments 1-4, wherein (c) further comprises comparing the optical parameter of the test composition with the corresponding optical parameter determined from a corresponding reference composition.

[0096] Embodiment 6. The method according to any one of Embodiments 1-5, wherein the method determines the presence or absence of the one or more target nucleic acids in the sample with a sensitivity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0097] Embodiment 7. The method according to any one of Embodiments 1-6, wherein the method determines the presence or absence of the one or more target nucleic acids in the sample with a specificity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0098] Embodiment 8. The method according to any one of Embodiments 1-7, wherein the method determines the presence or absence of the one or more target nucleic acids in the sample with an accuracy of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0099] Embodiment 9. The method according to any one of Embodiments 1-8, wherein the sample is selected from: blood sample, serum sample, plasma sample, saliva sample, fecal sample, sputum sample, urine sample, semen sample, vaginal fluid sample, cerebrospinal fluid sample, sweat sample, cell sample, and tissue sample.

[0100] Embodiment 10. The method according to any one of Embodiments 1-9, wherein the sample is from a mammal (e.g., human).

[0101] Embodiment 11. The method according to any one of Embodiments 1-10, wherein the sample is from an animal.

[0102] Embodiment 12. The method according to any one of Embodiments 1-11, wherein the sample is from a plant.

[0103] Embodiment 13. The method according to any one of Embodiments 1-12, wherein the sample comprises a lysis solution.

[0104] Embodiment 14. The method according to any one of Embodiments 1-13, wherein (b) comprises contacting the test composition with a nanoparticle condensing agent and / or a salt.

[0105] Embodiment 15. The method according to Embodiment 14, wherein the condensing agent comprises magnesium chloride.

[0106] Embodiment 16. The method according to any one of Embodiments 1-15, wherein at least about 40% of the nucleotides of the one or more oligonucleotides are guanine or cytosine.

[0107] Embodiment 17. The method according to any one of Embodiments 1-15, wherein about 40% to about 60% of the nucleotides of the one or more oligonucleotides are guanine or cytosine.

[0108] Embodiment 18. The method according to any one of Embodiments 1-17, wherein the one or more oligonucleotides are characterized by a melting temperature (Tm) of at least about 65 degrees Celsius (°C). Embodiment 19. The method according to any one of Embodiments 1-17, wherein the one or more oligonucleotides are characterized by a Tm of about 65°C to about 75°C.

[0109] Embodiment 20. The method according to any one of Embodiments 1-19, wherein the one or more oligonucleotides comprise a conjugated moiety at the 5'-end.

[0110] Embodiment 21. The method according to Embodiment 20, wherein the conjugated moiety is 5'-thiol.

[0111] Embodiment 22. The method according to Embodiment 21, wherein the 5'-thiol comprises a thioalkyl group, such as thiohexyl.

[0112] Embodiment 23. The method according to any one of embodiments 1-22, wherein the nanoparticles in the one or more nanoparticles are assembled with one, two, three, four, five, or six oligonucleotides.

[0113] Embodiment 24. The method according to any one of embodiments 1-23, wherein each of the one or more nanoparticles is (e.g., independently) assembled with one, two, three, four, five, or six oligonucleotides.

[0114] Embodiment 25. The method according to any one of embodiments 1-24, wherein the one or more nanoparticles comprise gold.

[0115] Embodiment 26. The method according to any one of embodiments 1-25, wherein the one or more nanoparticles are characterized by an average size of about 10 nanometers (nm) to about 200 nm.

[0116] Embodiment 27. The method according to any one of embodiments 1-26, wherein the one or more oligonucleotides are 16 to 24 nucleotides in length.

[0117] Embodiment 28. The method according to any one of embodiments 1-27, wherein the one or more oligonucleotides associate with the one or more target nucleic acids such that the distance between two adjacent nanoparticles of the one or more nanoparticles corresponds to about 50 to about 70 nucleotides.

[0118] Embodiment 29. The method according to any one of embodiments 1-28, wherein the one or more oligonucleotides comprise two oligonucleotides, wherein the first oligonucleotide hybridizes to a first region of the target nucleic acid, and the second oligonucleotide hybridizes to a second region of the target nucleic acid. Embodiment 30. The method according to embodiment 29, wherein the distance between the first region and the second region of the target nucleic acid is about 50 to 70 nucleotides.

[0119] Embodiment 31. The method according to any one of embodiments 1-30, wherein the one or more oligonucleotides hybridize to 10 to 30 nucleotides of the one or more target nucleic acids.

[0120] Embodiment 32. The method according to any one of embodiments 1-31, wherein, in the absence of the one or more target nucleic acids, the one or more nanoparticles form aggregates.

[0121] Embodiment 33. The method according to any one of embodiments 1-32, wherein the one or more target nucleic acids are from one or more viruses or one or more bacteria.

[0122] Embodiment 34. The method according to embodiment 33, wherein the one or more target nucleic acids are not from a coronavirus.

[0123] Embodiment 35. The method according to embodiment 33, wherein the one or more target nucleic acids are not from SARS-CoV-2 or one or more of its variants.

[0124] Embodiment 36. The method according to any one of embodiments 33-35, wherein the one or more viruses include an influenza virus or a human papillomavirus.

[0125] Embodiment 37. The method according to any one of embodiments 33-36, wherein the one or more bacteria include Salmonella.

[0126] Embodiment 38. The method according to embodiment 37, wherein the Salmonella includes Salmonella enterica.

[0127] Embodiment 39. The method according to embodiment 37, wherein the Salmonella includes one or more Salmonella strains or serotypes.

[0128] Embodiment 40. The method according to embodiment 39, wherein the one or more Salmonella strains or serotypes include one or more members selected from Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella pullorum.

[0129] Embodiment 41. The method according to any one of embodiments 1-40, wherein the one or more target nucleic acids are associated with one or more diseases or conditions.

[0130] Embodiment 42. The method according to embodiment 41, wherein the one or more diseases or conditions include infectious diseases, cancer, or degenerative diseases.

[0131] Embodiment 43. The method according to any one of embodiments 1-42, wherein the one or more target nucleic acids encode a polypeptide or a protein.

[0132] Embodiment 44. The method according to any one of claims 1-43, wherein the one or more target nucleic acids include DNA or RNA.

[0133] Embodiment 45. The method according to embodiment 44, wherein the DNA is genomic DNA.

[0134] Embodiment 46. The method according to embodiment 44, wherein the RNA is genomic RNA.

[0135] Embodiment 47. The method according to embodiment 44, wherein the RNA is double-stranded RNA or single-stranded RNA.

[0136] Embodiment 48. The method according to embodiment 44, wherein the RNA is double-stranded DNA or single-stranded DNA.

[0137] Embodiment 49. The method according to any one of embodiments 1-48, wherein the one or more target nucleic acids are from human papillomavirus (HPV) or one or more variants thereof.

[0138] Embodiment 50. The method according to any one of claims 1-49, wherein the one or more target nucleic acids comprise one or more members selected from the following: the L1 capsid protein of HPV, the L2 capsid protein of HPV, the E6 protein of HPV, the E7 protein of HPV, and fragments of any of them.

[0139] Embodiment 51. The method according to any one of embodiments 1-50, wherein the one or more target nucleic acids are from Salmonella.

[0140] Embodiment 52. The method according to embodiment 51, wherein the Salmonella includes Salmonella enterica.

[0141] Embodiment 53. The method according to embodiment 51, wherein the Salmonella includes one or more Salmonella strains or serotypes.

[0142] Embodiment 54. The method according to embodiment 53, wherein the one or more Salmonella strains or serotypes comprise one or more members selected from Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella pullorum.

[0143] Embodiment 55. The method according to any one of embodiments 1-54, wherein the one or more oligonucleotides comprise sequences selected from SEQ ID NO: 1-18.

[0144] Embodiment 56. A composition for detecting one or more target nucleic acids, the composition comprising:

[0145] One or more nanoparticles assembled with one or more oligonucleotides, wherein the one or more oligonucleotides are complementary to the one or more target nucleic acids,

[0146] wherein, in the presence of the one or more target nucleic acids, the one or more nanoparticles form a nanoparticle matrix, and the nanoparticle matrix has different optical parameters compared to a solution containing the corresponding nanoparticles not in the nanoparticle matrix.

[0147] Embodiment 57. A composition for detecting a target nucleic acid, the composition comprising:

[0148] One or more nanoparticles assembled with one or more oligonucleotides, wherein a first oligonucleotide among the one or more oligonucleotides is complementary to the target nucleic acid, and wherein a second oligonucleotide among the one or more oligonucleotides is complementary to the target nucleic acid at a second sequence, and wherein the one or more nanoparticles comprise gold,

[0149] wherein, in the presence of the one or more target nucleic acids, the one or more nanoparticles form a nanoparticle matrix.

[0150] Embodiment 58. The composition according to any one of Embodiments 56 or 57, wherein at least about 40% (e.g., from about 40% to about 60%) of the nucleotides of the one or more oligonucleotides are guanine or cytosine.

[0151] Embodiment 59. The composition according to any one of Embodiments 56-58, wherein the one or more oligonucleotides are characterized by a melting temperature (Tm) of at least about 65 degrees Celsius (°C) (e.g., from about 65°C to about 75°C).

[0152] Embodiment 60. The composition according to any one of Embodiments 56-59, wherein the one or more oligonucleotides comprise a conjugated moiety at the 5'-end.

[0153] Embodiment 61. The composition according to Embodiment 60, wherein the conjugated moiety is a 5'-thiol.

[0154] Embodiment 62. The composition according to Embodiment 61, wherein the 5'-thiol is a thioalkyl group.

[0155] Embodiment 63. The composition according to Embodiment 62, wherein the thioalkyl is a hexylthio group.

[0156] Embodiment 64. The composition according to any one of Embodiments 56-63, wherein the nanoparticles among the one or more nanoparticles are assembled with one, two, three, four, five, or six oligonucleotides.

[0157] Embodiment 65. The composition according to any one of Embodiments 56-64, wherein each (e.g., independently) of the one or more nanoparticles is assembled with one, two, three, four, five, or six oligonucleotides.

[0158] Embodiment 66. The composition according to any one of Embodiments 56-65, wherein the one or more nanoparticles comprise gold.

[0159] Embodiment 67. The composition according to any one of embodiments 56-66, wherein the one or more nanoparticles are characterized by an average size of about 10 nanometers (nm) to about 200 nm.

[0160] Embodiment 68. The composition according to any one of embodiments 56-67, wherein the one or more oligonucleotides are 16 to 24 nucleotides in length.

[0161] Embodiment 69. The composition according to any one of embodiments 56-68, wherein the one or more oligonucleotides associate with the one or more target nucleic acids such that the distance between two adjacent nanoparticles of the one or more nanoparticles corresponds to about 50 to about 70 nucleotides.

[0162] Embodiment 70. The composition according to any one of embodiments 56-69, wherein the one or more oligonucleotides comprise two oligonucleotides, wherein the first oligonucleotide hybridizes to a first region of the target nucleic acid and the second oligonucleotide hybridizes to a second region of the target nucleic acid.

[0163] Embodiment 71. The composition according to embodiment 70, wherein the distance between the first region and the second region of the target nucleic acid is about 50 to 70 nucleotides.

[0164] Embodiment 72. The composition according to any one of embodiments 56-69, wherein the one or more target nucleic acids are from one or more viruses or one or more bacteria.

[0165] Embodiment 73. The composition according to embodiment 72, wherein the one or more target nucleic acids are not from coronaviruses.

[0166] Embodiment 74. The composition according to embodiment 73, wherein the one or more target nucleic acids are not from SARS-CoV-2 or one or more of its variants.

[0167] Embodiment 75. The composition according to any one of embodiments 68-74, wherein the one or more viruses include influenza virus or human papillomavirus.

[0168] Embodiment 76. The composition according to embodiment 73, wherein the one or more bacteria include Salmonella.

[0169] Embodiment 77. The composition according to embodiment 76, wherein the Salmonella includes Salmonella enterica.

[0170] Embodiment 78. The composition according to embodiment 77, wherein the Salmonella genus comprises one or more Salmonella strains or serotypes.

[0171] Embodiment 79. The composition according to embodiment 78, wherein the one or more Salmonella strains or serotypes comprise one or more members selected from Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella pullorum.

[0172] Embodiment 80. The composition according to any one of embodiments 56-79, wherein the one or more target nucleic acids are associated with one or more diseases or conditions.

[0173] Embodiment 81. The composition according to embodiment 80, wherein the one or more diseases or conditions include infectious diseases, cancer, or degenerative diseases.

[0174] Embodiment 82. The composition according to any one of embodiments 56-81, wherein the one or more target nucleic acids encode a polypeptide or protein.

[0175] Embodiment 83. The composition according to any one of claims 56-82, wherein the one or more target nucleic acids comprise DNA or RNA.

[0176] Embodiment 84. The composition according to embodiment 83, wherein the DNA is genomic DNA.

[0177] Embodiment 85. The composition according to embodiment 83, wherein the RNA is genomic RNA.

[0178] Embodiment 86. The composition according to embodiment 83, wherein the RNA is double-stranded RNA or single-stranded RNA.

[0179] Embodiment 87. The composition according to embodiment 83, wherein the RNA is double-stranded DNA or single-stranded DNA.

[0180] Embodiment 88. The composition according to any one of embodiments 56-87, wherein the one or more target nucleic acids are from human papillomavirus (HPV) or one or more variants thereof. Embodiment 89. The composition according to any one of claims 56-88, wherein the one or more target nucleic acids comprise one or more members selected from the following: the L1 capsid protein of HPV, the L2 capsid protein of HPV, the E6 protein of HPV, the E7 protein of HPV, and fragments of any of them.

[0181] Embodiment 90. The composition according to any one of embodiments 56 - 89, wherein the one or more target nucleic acids are from Salmonella.

[0182] Embodiment 91. The composition according to embodiment 90, wherein the Salmonella includes Salmonella enterica.

[0183] Embodiment 92. The composition according to embodiment 90, wherein the Salmonella includes one or more Salmonella strains or serotypes.

[0184] Embodiment 93. The composition according to embodiment 92, wherein the one or more Salmonella strains or serotypes include one or more members selected from Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella pullorum.

[0185] Embodiment 94. The composition according to any one of embodiments 56 - 93, wherein the one or more oligonucleotides include sequences selected from SEQ ID NO: 1 - 18.

[0186] Embodiment 95. A kit for identifying the presence of a target nucleic acid, the kit comprising: (i) one or more gold nanoparticles assembled with one or more oligonucleotides, (ii) a condensation solution, (iii) instructions for using the one or more gold nanoparticles assembled with one or more oligonucleotides.

[0187] Embodiment 96. A kit for identifying the presence of a target nucleic acid, the kit comprising: (i) the composition according to any one of embodiments 56 to 94, (ii) a condensation solution, (iii) instructions for using the one or more gold nanoparticles assembled with one or more oligonucleotides.

[0188] Examples

[0189] Example 1: Generation of nanoparticles containing oligonucleotides.

[0190] Gold nanoparticles containing antisense oligonucleotides (ASOs) are generated by conjugating nucleic acids to the surface of the nanoparticles. The binding of the nucleic acid sequence to the surface of the nanoparticle occurs through a covalent bond that replaces the stabilizing citrate group with the ASO. The conjugation step is carried out by linking the oligonucleotide to the gold particle. Each ASO molecule is capable of forming a covalent bond through the thiol group in only a single gold particle.

[0191] First, the gold nanoparticles are combined with the ASO by adding citric acid and hydrochloric acid to the nanoparticles and the ASO. The solution is homogenized by stirring to allow conjugation to occur. Then, the solution is centrifuged to remove free ASO molecules (e.g., ASO not bound to the gold particles). After centrifugation, the supernatant is discarded and the pellet is resuspended in HEPES buffer. To improve signal sensitivity, the final product contains no free ASO molecules and all of the gold should be bound to the ASO.

[0192] The conjugation process is carried out using a 2 mL suspension of gold nanoparticles (AuNP) with a concentration of 1 mM of gold atoms and an average particle size of 20 nm. 36.6 μL of ASO with a concentration of 50 μM is added to obtain a ratio of 1 particle to 900 nucleic acid strands. After stirring at 350 rpm for 10 minutes at room temperature, 10 μL of sodium citrate solution (500 mM, pH 3.0) and 5 μL of hydrochloric acid solution (1 M) are added to adjust the pH to 3.0. After this step, the solution is stirred at 350 rpm for 20 minutes and then centrifuged at 14,000 rpm for 15 minutes. The supernatant is discarded and the pellet is resuspended in 2 mL of HEPES buffer (10 mM).

[0193] Figure 4 Multiple UV-visible curves of the nanoparticles conjugated to the ASO are shown. The nanoparticles conjugated to the ASO show a large peak in the UV-visible spectrum, while the individual nanoparticles without ASO do not show a UV-visible spectrum. Notably, this can also be observed by the naked eye, where the AuNP-ASO solution is light red, while the solution of the unconjugated nanoparticles is typically colorless and transparent.

[0194] Figure 3 An example schematic diagram of the nanoparticle-ASO that can be prepared using this method is shown. As shown, a single nanoparticle can be attached with multiple different oligonucleotides ("single nanoparticle multiplex"). Additionally, multiple oligonucleotides can be attached to different nanoparticles ("multiple nanoparticle multiplex"), where multiple nanoparticles can be attached to oligonucleotides with the same sequence and can also be attached to oligonucleotides with different sequences.

[0195] Example 2: Detection of nucleic acids.

[0196] Two types of samples are analyzed via RT-PCR and AuNP is used as the detection agent. The AuNP containing the oligonucleotide is incubated with two different solutions, one solution containing the target analyte (and detectable via RT-PCR) and the other solution without the target nucleic acid. After incubating the AuNP with the sample solution, a revealing solution containing magnesium chloride is added. Additionally, a standard solution serving as a baseline for color measurement is generated. Figures 1A - 1BAn exemplary schematic diagram of the process is shown. In Figure 1A , the tube schematic 1 shows a sample tube containing a solution of AuNP (circles) and oligonucleotides (helices). The tube schematic 2 shows the addition of DNA / RNA from the sample to tube schematic 1, where the DNA / RNA is obtained by digesting or extracting nucleic acids from a virus or other biological object containing DNA / RNA. The DNA / RNA is allowed to interact with the oligonucleotides and possibly anneal. In the tube schematic Figure 3 , depending on the presence of the target nucleic acid, a revealing solution is added to the tube in tube schematic 2 to induce observable changes in the tube, such as promoting the condensation of the nucleic acid structure. Figure 1B The tube schematic in Figure 4 shows the result from tube 3, where there is no target nucleic acid. The tube becomes clear / transparent, indicating a negative result. Alternatively, the tube schematic Figure 5 shows the result from tube 3, where there is target nucleic acid. The test tube becomes opaque, indicating a positive result. Figures 2A - 2B shows an exemplary structural schematic diagram of a positive sample and a negative sample. In the negative sample, the revealing solution causes the particles to aggregate and makes the solution turn translucent light gray ( Figure 2A ). Alternatively, when the oligonucleotide is able to bind to the target nucleic acid, the revealing solution induces the nucleic acid and the nanoparticles to form a particle matrix, which is turbid light red. Figure 2C shows a series of test tubes with solutions, where the test tubes on the left show particle aggregation and a negative signal, and the test tubes on the right show a positive signal indicated by the light red color of the solution.

[0197] Initially, the standard was defined by separately identifying the primary colors of red, green, and blue. From this, the CIELAB color space was determined, which is a system for evaluating elements of lightness or clarity, hue or chromaticity, and saturation or colorfulness. Lightness (L*) is the brightness value (Y-axis) that varies approximately from white to black, assuming the value of absolute black is 0 (zero) and the value of full white is 100. Hue is represented by the primary colors and is identified by the value of a*, ranging from green to red, and the value of b* can vary from blue to yellow, representing in a way similar to color perception. The L*a*b* color space was created using the opposite color theory, where two colors cannot be both green and red, nor both yellow and blue at the same time. L* indicates lightness, and a* and b* are chromaticity coordinates. L* = lightness; a* = red / green coordinate (+a indicates red and -a indicates green); b* = yellow / blue coordinate (+b indicates yellow and -b indicates blue); color difference is defined by a numerical comparison between a sample and a standard. The absolute difference in color coordinates between a sample and a target is called Delta (Δ). The Delta of L* (ΔL), a* (Δa), and b* (Δb) can be negative (-) or positive (+). However, the total difference Delta E (ΔE) is always positive. They are expressed as: ΔL* = difference between brighter and darker (+ = brighter, - = darker); Δa* = difference between red and green (+ = more red, - = more green); Δb* = difference between yellow and blue (+ = more yellow, - = more blue); ΔE* = total color difference;

[0198] To determine the total color difference between the three coordinates, the following formula is used: ΔE* = [(ΔL*)2 + (Δa*)2 + (Δb*)2]1 / 2. Comparing RT-PCR positive samples and RT-PCR negative samples, each Δ coordinate has a significant difference, and it is proven that color analysis can distinguish between positive and negative samples.

[0199] Example 3: Detection of nucleic acids in patient samples.

[0200] Collect samples from patients using a sterile nasopharyngeal swab and immediately place them into tubes containing PBS medium. Homogenize the samples to depolymerize the cells from the swabs. Transfer 500 μL from the collection tube to a tube containing the same volume (v / v) of lysis solution; homogenize the samples and then let stand for 5 minutes for the extraction reaction to occur. Then transfer the entire volume from the microtube into a syringe (with a 0.22 μm filter into the syringe) and apply it directly to the biosensor by dripping; then add 40 μL of the revelation solution. After waiting for 1 minute, read the test result. There are three possible results. If the target nucleic acid is present, the color developed will be dark pink / red. If the target reagent is absent, the color developed will be blue / light gray. When neither of the above colors is developed, or if the color does not match the colors on the printed control color chart, the test is considered invalid. This result may be due to insufficient sample volume or incorrect procedure execution, and the test can be repeated to obtain a valid result. Negative control swabs and positive control swabs can also be obtained, and similar steps can be performed for quality control. Figure 5 An example detection system used in this embodiment is shown. As shown, the sample is added to the inlet, mixed and homogenized for 5 minutes, then moved to the heating chamber and passed through the filter. Then a biosensor solution containing nanoparticles assembled to oligonucleotides is added via the port and mixed with the sample. After mixing with the biosensor, then the revelation solution is added to the port and combined with the sample (and the biosensor). Then the resulting solution is pushed into a new reservoir where RGB reading (or reading another optical parameter) can be performed. The system can use a pump (such as a syringe pump) to move the solution through the system.

[0201] Example 4: Generation of nanoparticles and detection of HPV

[0202] Prepare nanoparticles based on the protocol described in Example 1. A variety of types of nanoparticles were produced, including nanoparticles with different ASOs. First, perform the protocol using ASO No. 1 (SEQ ID NO:1) to generate nanoparticles with ASO No. 1. Then repeat the process for ASO Nos. 3, 4, and 6 in 4 vials with 2 mL of suspension each (SEQ ID NOs: 3, 4, and 6 respectively), with each vial having one ASO. Mix the four suspensions in equal amounts, 2 mL each, for a total of 8 mL, containing a mixture of particles conjugated with 4 ASOs, to generate the biosensor solution. Store the biosensor in a refrigerator at 4 - 8 °C until use with clinical samples.

[0203] The biosensor solution has a clear solution appearance, is light red in color, and has a maximum UV-vis absorption peak at 530 nm. Additionally, a control sequence corresponding to the HPV virus sequence ("PROBE") was used to verify the hybridization of the ASO with the complementary sequence. After homogenization, the solution was applied to agarose gel electrophoresis and tested with free samples, ASO, and PROBE, without AuNP. PCR for ASO signal amplification and differentiation was performed at two concentrations to confirm binding. The ASO tested had 20 bases and the PROBE had 100 bases.

[0204] Figure 6 Data obtained by detecting the presence of HPV RNA using the biosensor solution are shown. HPV RNA is expressed in the genome of CasKi (squamous cell carcinoma) cells, and the biosensor solution (i.e., the solution contained in AuNP ASO) was applied to the cells. L929 cells (connective tissue mouse tissue) that do not have HPV-RNA were also cultured. RNA was extracted from CasKi and L929 cells and measured using the biosensor. A PBS sample was also measured as a negative control / background signal. The spectral data of the samples were read, parameterized, and normalized using an algorithm to generate a spectral score. As Figure 6 shown, samples containing extracted CasKi RNA ("CasKi-20ng", ""CasKi-10ng", "CasKi-5ng") showed significantly higher spectral scores compared to samples containing extracted L929 RNA ("929-20ng", "929-10ng", "929-5ng"), and samples containing extracted L929 RNA ("929-20ng", "929-10ng", "929-5ng") showed spectra similar to those of samples with only PBS ("negative control"). Thus, HPV-positive samples are distinguishable from HPV-RNA negative samples. Additionally, measurements performed on nucleic acids different from the target nucleic acid yielded results consistent with those of negative samples (e.g., PBS only), demonstrating the specificity of the recognition of the target sequence.

[0205] Example 5: Detection of nucleic acids using optical density 。

[0206] First, a sample of bacteria is generated by culturing in a BOD (Biochemical Oxygen Demand) chamber at 37 °C for approximately 16 hours (e.g., overnight) until the optical density reaches 0.5. The sample is initially diluted between 100x and 100,000x with 0.1% peptone water and then a serial dilution curve is made. Then the diluted sample is incubated in a dry heat bath at 95 °C for 5 minutes. After incubation, the sample is placed on ice to stop boiling. Then the sample is vortexed for 10 seconds and then placed on ice. Then the sample is added to a 96-well plate by pipetting 100 μl of the sample. Based on the following table, in addition to the test samples, a negative control and a biosensor-only sample are generated:

[0207] Table 2: Sample Preparation Guide

[0208]

[0209] Various samples are added to the plate and incubated at room temperature for 5 minutes. A revealing solution is generated by mixing 0.79 g of MgCl2 in water to a total volume of 50 mL. 10 μl of the revealing solution is added to the wells and incubated at room temperature for 2 - 4 minutes, and then readings are taken using absorbance readings at 520 nm. Then the optical density at 520 nm is obtained and can be analyzed using the following formula

[0210]

[0211] After calculation, the cut-off value (2 standard deviations lower than the negative control) is determined. Readings above the cut-off value are considered negative and readings below the cut-off value are considered positive.

[0212] Example 6: Detection of HPV using optical density measurement

[0213] HPV samples are generated in a similar manner as described in Example 6. Using three biological replicates, samples of the Caski cell line + HPV-16 and the HeLa cell line with HPV-18 are diluted to form a serial dilution curve. Then the AuNP-ASO biosensor is added to the solution and used to detect the presence of HPV, the level of which is statistically significant compared to the negative control of the biosensor alone (i.e., absence of target nucleic acid). Reference samples are also used to evaluate the colloidal dispersion and determine the range of indeterminates. Figure 7A Shows the results of an assay for detecting HPV using the optical density (OD 560 ) of the detection signal at 560 nm. Figure 7B Shows the OD 560 values of biological replicates of CasKi cells, reference sensors, and biosensor-only samples, and Figure 7C shows the OD 560The respective values. Table 3 shows the analytical performance of this assay.

[0214] Table 3: Analytical Performance of the HPV Assay

[0215]

[0216] Figure 8A and Figure 8B shows the ROC (Receiver Operating Characteristic) curves of dilutions of the Caski cell line with HPV and the HeLa cell line with HPV. The AUC (Area Under the Curve) for the assay performed with the Caski cell line at (about 400 copies of HPV / cell) is 1, and the AUC for the HeLa cell line (about 40 copies of HPV / cell) is 0.9750.

[0217] Example 7. Detection of Salmonella

[0218] Nanoparticles were generated based on the protocol described in Example 1. Additionally, control samples were generated. Dilutions were mixed with AuNP - ASO and the optical density of the samples was read. Figure 9 shows the optical density normalized to a reference relative to various dilutions of Salmonella and Escherichia coli (E. coli). Notably, the E. coli samples (squares) show a normalized optical density greater than 1, while the Salmonella samples (circles) show a normalized optical density less than 0.9905. 10 6 Salmonella samples of 10 or greater experience the prozone phenomenon and thus generate different signals, as well as multiple signals. Figure 10 shows a plot of colony - forming units per milliliter (“CFU / ml”) versus the processed optical density parameter. As shown, Salmonella samples diluted below 10 6 cfu / ml are below the cut - off value (cut - off value = 2 times the standard deviation of the negative control), while E. coli samples are above the cut - off value. Figure 11 shows the ROC curve analysis, where the AUC is 1. Table 4 shows the analytical performance of the Salmonella assay.

[0219] Table 4: Analytical Performance of the Salmonella Assay

[0220]

[0221]

[0222] This method can be performed on food samples to detect the presence of Salmonella in the food samples. This method can also determine the quantity of Salmonella in the food samples.

[0223] While the preferred embodiments of the present invention have been shown and described herein, those skilled in the art will understand that these embodiments are provided by way of example only. The present invention is not limited to the specific examples provided in the specification. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the present invention. In addition, it should be understood that all aspects of the present invention are not limited to the specific descriptions, configurations, or relative proportions described herein, which depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention. Accordingly, the present invention should also cover any such alternatives, modifications, variations, or equivalents. The following claims are intended to define the scope of the present invention and cover the methods and structures within the scope of these claims and their equivalents.

Claims

1. A method for processing or analyzing a sample, the method comprising: a. contacting the sample with a composition comprising one or more nanoparticles to provide a test composition, wherein the one or more nanoparticles are assembled with one or more oligonucleotides, and wherein the one or more oligonucleotides hybridize to one or more target nucleic acids that may be present in the sample; b. forming a nanoparticle matrix if the one or more target nucleic acids are present, the nanoparticle matrix comprising the one or more nanoparticles that are hybridized to the one or more target nucleic acids; c. determining an optical parameter of the test composition, the optical parameter indicating the presence or absence of the one or more target nucleic acids in the sample.

2. The method according to claim 1, wherein the optical parameter is determined by color space analysis.

3. The method according to claim 1 or claim 2, wherein the optical parameter comprises absorption, transmission, scattering or reflection of light at a wavelength or wavelength range.

4. The method according to any one of claims 1-3, wherein the optical parameter comprises a photometric parameter (e.g., brightness of a color), a saturation parameter (e.g., intensity of a color) or a hue parameter (e.g., chromaticity of a color).

5. The method according to any one of claims 1-4, wherein (c) further comprises comparing the optical parameter of the test composition with a corresponding optical parameter determined from a corresponding reference composition.

6. The method according to any one of claims 1-5, wherein the sample is selected from: a blood sample, a serum sample, a plasma sample, a saliva sample, a fecal sample, a sputum sample, a urine sample, a semen sample, a vaginal fluid sample, a cerebrospinal fluid sample, a sweat sample, a cell sample, a tissue sample or a food sample.

7. The method according to any one of claims 1-6, wherein (b) comprises contacting the test composition with a nanoparticle condensing agent and / or a salt.

8. The method according to claim 7, wherein the condensing agent comprises magnesium chloride.

9. The method according to any one of claims 1-8, wherein the melting temperature (Tm) of the one or more oligonucleotides is at least about 65 degrees Celsius (°C).

10. The method according to any one of claims 1-9, wherein the Tm of the one or more oligonucleotides is from about 65 °C to about 75 °C.

11. The method according to any one of claims 1-10, wherein the nanoparticles in the one or more nanoparticles are assembled with one, two, three, four, five or six oligonucleotides, optionally wherein each of the oligonucleotides is the same or different.

12. The method according to any one of claims 1-11, wherein the one or more nanoparticles comprise gold.

13. The method according to any one of claims 1-12, wherein the average size of the one or more nanoparticles is from about 10 nanometers (nm) to about 200 nm.

14. The method according to any one of claims 1-13, wherein each of the one or more oligonucleotides is from about 16 to about 24 nucleotides in length.

15. The method according to any one of claims 1-14, wherein the one or more oligonucleotides comprise two oligonucleotides, wherein the first oligonucleotide hybridizes to a first region of the target nucleic acid, and the second oligonucleotide hybridizes to a second region of the target nucleic acid.

16. The method according to claim 15, wherein the distance between the first region and the second region of the target nucleic acid is about 50 to about 70 nucleotides.

17. The method according to any one of claims 1-16, wherein each of the one or more oligonucleotides hybridizes to about 10 to about 30 nucleotides of the one or more target nucleic acids.

18. The method according to any one of claims 1-17, wherein In the absence of the one or more target nucleic acids, the one or more nanoparticles form aggregates.

19. The method according to any one of claims 1-18, wherein the one or more target nucleic acids comprise viral nucleic acid and / or bacterial nucleic acid.

20. The method according to claim 19, wherein the one or more target nucleic acids are not coronavirus nucleic acid.

21. The method according to any one of claims 19-20, wherein the one or more target nucleic acids comprise viral nucleic acid, and wherein the viral nucleic acid comprises influenza and / or human papillomavirus nucleic acid.

22. The method according to any one of claims 19-21, wherein the one or more target nucleic acids comprise bacterial nucleic acid, wherein the bacterial nucleic acid comprises Salmonella nucleic acid.

23. The method according to claim 22, wherein the Salmonella comprises one or more Salmonella strains or serotypes.

24. The method according to claim 23, wherein the one or more Salmonella strains or serotypes comprise Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum or Salmonella pullorum, or a combination of two or more thereof.

25. The method according to any one of claims 1-24, wherein the presence of the one or more target nucleic acids is associated with one or more diseases or conditions of an object comprising the one or more target nucleic acids.

26. The method according to any one of claims 1-25, wherein the one or more target nucleic acids are human papillomavirus (HPV) nucleic acid or nucleic acid of one or more variants of HPV.

27. The method according to any one of claims 1-26, wherein the one or more target nucleic acids comprise the L1 capsid protein of HPV, the L2 capsid protein of HPV, the E6 protein of HPV or the E7 protein of HPV, or a fragment and / or a combination of two or more thereof.

28. The method according to any one of claims 1-27, wherein the one or more oligonucleotides comprise a sequence selected from SEQ ID NO: 1-18, or a sequence at least 90% identical to a sequence selected from SEQ ID NO: 1-18.

29. A composition for detecting one or more target nucleic acids, the composition comprising: a. One or more nanoparticles assembled with one or more oligonucleotides, wherein the one or more oligonucleotides are complementary to the one or more target nucleic acids, b. Wherein, in the presence of the one or more target nucleic acids, the one or more nanoparticles form a nanoparticle matrix comprising the one or more nanoparticles and the one or more target nucleic acids, and wherein the nanoparticle matrix has different optical parameters compared to a solution comprising the corresponding nanoparticles not bound to the one or more target nucleic acids.

30. A composition for detecting a target nucleic acid, the composition comprising: a. One or more nanoparticles assembled with one or more oligonucleotides, wherein a first oligonucleotide among the one or more oligonucleotides is complementary to the target nucleic acid at a first sequence of the target nucleic acid, and wherein a second oligonucleotide among the one or more oligonucleotides is complementary to the target nucleic acid at a second sequence of the target nucleic acid, and wherein the one or more nanoparticles comprise gold. b. Wherein, in the presence of the one or more target nucleic acids, the one or more nanoparticles form a nanoparticle matrix.

31. The composition according to any one of claims 29 - 30, wherein the nanoparticles among the one or more nanoparticles are assembled with one, two, three, four, five, or six oligonucleotides.

32. The composition according to any one of claims 29 - 31, wherein each of the one or more nanoparticles (e.g., independently) is assembled with one, two, three, four, five, or six oligonucleotides.

33. The composition according to any one of claims 29 - 32, wherein the one or more nanoparticles comprise gold.

34. The composition according to any one of claims 29 - 33, wherein the average size of the one or more nanoparticles is from about 10 nanometers (nm) to about 200 nm.

35. The composition according to any one of claims 29 - 34, wherein the one or more oligonucleotides are from about 16 to about 24 nucleotides in length.

36. The composition according to any one of claims 29 - 35, wherein the one or more target nucleic acids include viral nucleic acid and / or bacterial nucleic acid.

37. The composition according to claim 36, wherein the one or more target nucleic acids are not coronavirus nucleic acid.

38. The composition according to any one of claims 36 - 37, wherein the one or more target nucleic acids include viral nucleic acid, and wherein the viral nucleic acid includes influenza virus nucleic acid and / or human papillomavirus nucleic acid.

39. The composition according to claim 36, wherein the one or more target nucleic acids include bacterial nucleic acid, and wherein the bacterial nucleic acid includes Salmonella nucleic acid.

40. The composition according to any one of claims 29 - 39, wherein the one or more oligonucleotides comprise a sequence selected from SEQ ID NO: 1 - 18, or a sequence at least 90% identical to a sequence selected from SEQ ID NO: 1 - 18.

41. A kit for identifying the presence of a target nucleic acid, the kit comprising: (i) one or more gold nanoparticles assembled with one or more oligonucleotides, (ii) a condensation solution, (iii) instructions for using the one or more gold nanoparticles assembled with one or more oligonucleotides.

42. A kit for identifying the presence of a target nucleic acid, the kit comprising: (i) a composition according to any one of claims 29-40, (ii) a condensation solution, (iii) instructions for using the one or more gold nanoparticles assembled with one or more oligonucleotides.

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