Methods, systems, and compositions for the detection of nucleic acids

Abstract

Provided herein are methods, compositions, and systems for detecting nucleic acids. The methods, compositions, and systems may include nanoparticles containing oligonucleotides. The oligonucleotides may anneal to target nucleic acids. The nanoparticles may include optical parameters that can change upon reaction with the target nucleic acids.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 370,218, filed August 2, 2022, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application is submitted with a Sequence Listing in electronic format. The Sequence Listing was submitted as a file entitled 64978-701.601.xml, created on July 31, 2023, and is 21,233 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. [Background technology]

[0003] Nucleic acids are found in many organisms and may enable the organism to replicate or encode specific proteins. For example, viruses may use nucleic acids to replicate, and the presence of viral nucleic acids in a subject may indicate the presence of the virus in the subject. Summary of the Invention

[0004] 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 incorporating one or more oligonucleotides to provide a test composition, wherein the one or more oligonucleotides hybridize to one or more target nucleic acids, if present, in the biological sample; and (c) 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; and (c) determining an optical parameter of the test composition indicative of the presence or absence of the one or more nucleic acids in the sample. In some embodiments, the optical parameter is determined by color space analysis. In some embodiments, the optical parameter comprises absorption, transmission, scattering, or reflection of light of a wavelength or a range of wavelengths. In some embodiments, the optical parameter comprises a luminosity parameter (e.g., color brightness), a saturation parameter (e.g., color intensity), or a hue parameter (e.g., hue). 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. In some embodiments, the method determines the presence or absence of one or more target nucleic acids in a 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 a 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 a 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 derived from a mammal (e.g., a human).In some embodiments, the sample is derived from an animal. In some embodiments, the sample is derived 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 in the one or more oligonucleotides are guanine or cytosine. In some embodiments, about 40% to about 60% of the nucleotides in 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, the one or more oligonucleotides comprise a conjugate moiety at the 5'-terminus. In some embodiments, the conjugate moiety is a 5'-thiol. In some embodiments, the 5'-thiol comprises a thioalkyl, such as thiohexyl. In some embodiments, one nanoparticle of the one or more nanoparticles incorporates 1, 2, 3, 4, 5, or 6 oligonucleotides. In some embodiments, the one or more nanoparticles each (e.g., independently) incorporate 1, 2, 3, 4, 5, or 6 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 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 are associated 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, the one or more oligonucleotides comprise two oligonucleotides, where a first oligonucleotide hybridizes to a first region of the target nucleic acid and a second oligonucleotide hybridizes to a second region of the target nucleic acid. In some embodiments, the distance between the first and second regions of the target nucleic acid is approximately 50-70 nucleotides. In some embodiments, the one or more oligonucleotides hybridize to 10-30 nucleotides of the one or more target nucleic acids. In some embodiments, in the absence of the one or more target nucleic acids, the one or more nanoparticles form aggregates. In some embodiments, the one or more target nucleic acids are derived from one or more viruses or one or more bacteria. In some embodiments, the one or more target nucleic acids are not derived from a coronavirus. In some embodiments, the one or more target nucleic acids are not derived from SARS-CoV-2 or one or more variants thereof. In some embodiments, the one or more viruses comprise influenza virus or human papillomavirus. In some embodiments, the one or more bacteria comprise Salmonella. In some embodiments, the Salmonella comprises Salmonella enterica. In some embodiments, the Salmonella comprises one or more strains or serotypes of Salmonella. In some embodiments, the one or more strains or serotypes of Salmonella comprise one or more members selected from the group consisting of Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella pullorum. In some embodiments, the one or more target nucleic acids are associated with one or more diseases or conditions. In some embodiments, the one or more diseases or conditions comprise an infectious disease, cancer, or a degenerative disease. In some embodiments, the one or more target nucleic acids encode a polypeptide or protein.In some embodiments, the 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, the one or more target nucleic acids are derived from a human papillomavirus (HPV) or one or more variants thereof. In some embodiments, the one or more target nucleic acids comprise one or more members selected from an HPV L1 capsid protein, an HPV L2 capsid protein, an HPV E6 protein, an HPV E7 protein, and fragments of any thereof. In some embodiments, the one or more target nucleic acids are derived from Salmonella. In some embodiments, the Salmonella comprises Salmonella enterica. In some embodiments, the Salmonella comprises one or more strains or serotypes of Salmonella. In some embodiments, the one or more Salmonella strains or serotypes comprise one or more members selected from the group consisting of Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella prorum. In some embodiments, the one or more oligonucleotides comprise a sequence selected from SEQ ID NOs: 1-18.

[0005] In one aspect, the present disclosure provides a composition for detecting one or more target nucleic acids, the composition comprising one or more nanoparticles having incorporated therein one or more oligonucleotides, wherein the one or more oligonucleotides are complementary 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 comprising different optical parameters compared to a solution comprising corresponding nanoparticles that are not in the nanoparticle matrix.

[0006] In another aspect, the present disclosure provides a composition for detecting a target nucleic acid, the composition comprising: one or more nanoparticles incorporating one or more oligonucleotides, wherein a first oligonucleotide of the one or more oligonucleotides is complementary to the target nucleic acid and a second oligonucleotide of the one or more oligonucleotides is complementary in a second sequence to the target nucleic acid; and the one or more nanoparticles comprise gold; and forming a nanoparticle matrix with the one or more nanoparticles in the presence of the one or more target nucleic acids. In some embodiments, at least about 40% (e.g., 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., about 65°C to about 75°C). In some embodiments, the one or more oligonucleotides comprise a conjugate moiety at the 5'-terminus. In some embodiments, the conjugate 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, one nanoparticle of the one or more nanoparticles incorporates 1, 2, 3, 4, 5, or 6 oligonucleotides. In some embodiments, the one or more nanoparticles each (e.g., independently) incorporate 1, 2, 3, 4, 5, or 6 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 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 are associated 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, the one or more oligonucleotides comprise two oligonucleotides, where a first oligonucleotide hybridizes to a first region of the target nucleic acid and a second oligonucleotide hybridizes to a second region of the target nucleic acid. In some embodiments, the distance between the first and second regions of the target nucleic acid is approximately 50-70 nucleotides. In some embodiments, the one or more target nucleic acids are derived from one or more viruses or one or more bacteria. In some embodiments, the one or more target nucleic acids are not derived from a coronavirus. In some embodiments, the one or more target nucleic acids are not derived from SARS-CoV-2 or one or more variants thereof. In some embodiments, the one or more viruses comprise influenza virus or human papillomavirus. In some embodiments, the one or more bacteria comprise Salmonella. In some embodiments, the Salmonella comprises Salmonella enterica. In some embodiments, the Salmonella comprises one or more strains or serotypes of Salmonella. In some embodiments, the one or more Salmonella strains or serotypes comprise one or more members selected from the group consisting of Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella proliferum. In some embodiments, the one or more target nucleic acids are associated with one or more diseases or conditions. In some embodiments, the one or more diseases or conditions comprise an infectious disease, cancer, or a degenerative disease. In some embodiments, the one or more target nucleic acids encode a polypeptide or protein. In some embodiments, the 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, the one or more target nucleic acids are derived from a human papillomavirus (HPV) or one or more variants thereof.In some embodiments, the one or more target nucleic acids comprise one or more members selected from HPV L1 capsid protein, HPV L2 capsid protein, HPV E6 protein, HPV E7 protein, and fragments of any thereof. In some embodiments, the one or more target nucleic acids are derived from Salmonella. In some embodiments, the Salmonella comprises Salmonella enterica. In some embodiments, the Salmonella comprises one or more strains or serotypes of Salmonella. In some embodiments, the one or more strains or serotypes of Salmonella comprise one or more members selected from the group consisting of Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella prorum. In some embodiments, the one or more oligonucleotides comprise a sequence selected from SEQ ID NOs: 1-18.

[0007] In another aspect, the present disclosure provides a kit for identifying the presence of a target nucleic acid, the kit including: (i) one or more gold nanoparticles having one or more oligonucleotides incorporated therein; (ii) a condensation solution; and (iii) instructions for using the one or more gold nanoparticles having one or more oligonucleotides incorporated therein.

[0008] In another aspect, the present disclosure provides a kit for identifying the presence of a target nucleic acid, the kit comprising: (i) a composition described elsewhere herein; (ii) a condensation solution; and (iii) instructions for using the one or more gold nanoparticles having one or more oligonucleotides incorporated therein.

[0009] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its various details can be modified in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0010] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over any such conflicting material. [Brief explanation of the drawings]

[0011] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "Figures" and "FIGs.")

[0012] [Figure 1A] Figure 1A shows a schematic diagram of one example of the method disclosed herein. [Figure 1B] FIG. 1B shows a schematic diagram of one example of the method disclosed herein. [Figure 2A] Figure 2A shows a depiction of a negative sample. [Figure 2B]Figure 2B shows a depiction of a positive sample. [Figure 2C] FIG. 2C shows a series of cuvettes with increasing amounts of positive signal. [Figure 3] 1 shows an example of the nanoparticle configuration. [Figure 4] 1 shows the UV-visible spectrum of gold nanoparticles conjugated to oligonucleotides. [Figure 5] 1 illustrates an example of an apparatus for carrying out the methods of the present disclosure. [Figure 6] 1 shows the results of an assay that detects HPV nucleic acid sequences. [Figure 7A] Figures 7A-7C show data from an assay for the detection of HPV using optical density, with Figure 7A showing the average measured values for the samples. [Figure 7B] Figures 7A-7C show data from an assay for HPV detection using optical density, and Figure 7B shows the curves for each sample in the CasKi assay. [Figure 7C] Figures 7A-7C show data from an assay for detecting HPV using optical density, with Figure 7C showing the curves for each sample in the HeLa assay. [Figure 8A] Figures 8A-8B show data from an assay for the detection of HPV using optical density. Figure 8A shows the ROC curve for the assay using CasKi cells. [Figure 8B] Figures 8A-8B show data from an assay for the detection of HPV using optical density, and Figure 8B shows the ROC curve for the assay using HeLa cells. [Figure 9] 1 shows data from an assay for the detection of Salmonella using optical density. [Figure 10] 1 shows data from an assay for the detection of Salmonella using optical density. [Figure 11] 1 shows the ROC curve of an assay for the detection of Salmonella using optical density. [Figure 12] 1 illustrates a computer control system that is programmed or otherwise configured to implement the methods provided herein. DETAILED DESCRIPTION OF THE INVENTION

[0013] While 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. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It will be understood that various alternatives to the embodiments of the invention described herein may be utilized.

[0014] The detection of nucleic acids has a wide range of applications for human health. Specifically, nucleic acids may be present in a subject and associated with a disease state or pathogenic infection. Nucleic acids exist in organisms or microorganisms for replication and protein expression. The detection of specific nucleic acids may indicate the presence of specific microorganisms in a sample / subject and may be used to diagnose a subject with a disease. The detection of human nucleic acids may also be used to identify mutations in a subject's genome, which may be indicative of a disease, disorder, or other condition, such as cancer. The detection of specific nucleic acids may require complex reactions that may require specific conditions and still provide inaccurate results.

[0015] The present disclosure provides methods, systems, and compositions that enable the detection of specific nucleic acids that are rapid, easy to use, and provide accurate results. The methods may provide an alternative to other nucleic acid detection technologies and may be performed without a thermocycler or other equipment that heats and / or cools nucleic acids. The methods may also be performed without the use of enzymes, allowing the kit or composition to be stored more easily and maintain a longer shelf life. The methods may be performed without nucleic acid extension or amplification. Thus, the disclosed methods may involve simpler and more robust sample preparation and may be less susceptible to reagent degradation compared to methods that use nucleic acid amplification. The methods and compositions may be used without the need for complex, expensive, or cost-prohibitive optical equipment that is otherwise not generally available to the public or difficult for the general public to obtain.

[0016] The present method and composition can enable rapid testing for the presence of nucleic acid compared to other detection methods, for example, can produce results within 5 minutes or less.In addition, the manufacturing cost of the composition can be lower than other tests of similar accuracy, making it cheaper to perform the test.The present method can also have other advantages over methods of similar accuracy.For example, the present method can eliminate the need for nucleic acid purification before assay, reducing the time required to generate results.In addition, the present method can be performed without microbial culture or preservation of RNA samples.

[0017] Methods, systems, and compositions for detecting nucleic acid analytes are provided herein. In some embodiments, the present disclosure provides a composition for detecting one or more target nucleic acids (DNA or RNA), the composition comprising one or more nanoparticles incorporating one or more oligonucleotides, the one or more oligonucleotides configured to bind one or more target nucleic acids, and the one or more nanoparticles forming a nanoparticle matrix in the presence of the one or more target nucleic acids. The nanoparticle matrix can be detected via optical properties or parameters of the nanoparticle matrix. One or more nanoparticles in solution can have optical properties different from the nanoparticle matrix, such that the formation of a nanoparticle matrix from the one or more nanoparticles can be identified via optical properties or parameters. The one or more oligonucleotides can each comprise about 16 to about 24 nucleotides. The one or more oligonucleotides are associated with the one or more target nucleic acids such that the (e.g., average) distance between two adjacent nanoparticles of the one or more nanoparticles corresponds to about 50 to about 70 nucleotides. In some cases, the one or more oligonucleotides comprise two oligonucleotides, a first oligonucleotide hybridizing to a first region of the target nucleic acid and a second oligonucleotide hybridizing to a second region of the target nucleic acid. In some embodiments, the distance between the first and second regions of the target nucleic acid is about 50 to 70 nucleotides.

[0018] Provided herein are methods for processing or analyzing a subject sample, the methods comprising: (a) contacting a biological sample with a composition comprising one or more nanoparticles incorporating 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 when 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 the one or more nanoparticles form a nanoparticle matrix in the presence of the one or more target nucleic acids; and (c) determining an optical parameter of the test composition indicative of the presence or absence of the one or more target nucleic acids in the sample. The one or more oligonucleotides may each comprise about 16 to about 24 nucleotides. The one or more oligonucleotides are associated with the one or more target nucleic acids such that the (e.g., average) distance between two adjacent nanoparticles of the one or more nanoparticles corresponds to about 50 to about 70 nucleotides.

[0019] In various embodiments disclosed herein, the methods may be used to determine the presence of a nucleic acid sequence or a transmissible pathogen in a subject or sample. The methods may be used to determine the presence of a transmissible pathogen in a subject or to diagnose the presence of a disorder or disease.

[0020] In various embodiments, one or more oligonucleotides are incorporated into the nanoparticles. The one or more oligonucleotides may be complementary to a specific sequence. One or more nucleotides may anneal to a target sequence. In some embodiments, annealing involves creating a duplex between two strands of nucleic acid, forming hydrogen bonds between complementary bases. After one or more oligonucleotides anneal 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, one or more oligonucleotides may anneal to a viral sequence, one or more oligonucleotides may anneal to a bacterial sequence, one or more oligonucleotides may anneal to a fungal sequence, or one or more oligonucleotides may anneal to a human sequence. These oligonucleotide sequences can be generated by analyzing the target sequence and generating sequences that are partially or fully complementary to the target nucleic acid. The one or more oligonucleotides assembled into a given nanoparticle can contain the same or different sequences. A solution of nanoparticles can also contain nanoparticles with the same or different oligonucleotides. Figure 3 shows exemplary nanoparticles that can be used in the methods, compositions, and systems of the present disclosure. A single nanomultiplex refers to a nanoparticle with different oligonucleotides attached to a single nanoparticle. A multi-nanopluplex refers to multiple oligonucleotide molecules attached to multiple nanoparticles.

[0021] The one or more oligonucleotides may comprise specific or particular characteristics. The one or more oligonucleotides may have a melting temperature. The melting temperature may be a temperature at which the one or more oligonucleotides remain annealed to the target nucleic acid at a particular assay or reaction temperature. The melting temperature may be a temperature at which the one or more oligonucleotides do not remain annealed to the target nucleic acid at a particular assay or reaction temperature. The one or more oligonucleotides may have a melting temperature (Tm) of at least about 65 degrees Celsius (°C). The one or more oligonucleotides may have a Tm of about 65°C to about 75°C. The one or more oligonucleotides may have a Tm of 65°C or less, 66°C or less, 67°C or less, 68°C or less, 69°C or less, 70°C or less, 71°C or less, 72°C or less, 73°C or less, 74°C or less, 75°C or less, 76°C or less, 77°C or less, 78°C or less, 79°C or less, or 80°C or less. One or more oligonucleotides may have a Tm of greater than 65°C, greater than 66°C, greater than 67°C, greater than 68°C, greater than 69°C, greater than 70°C, greater than 71°C, greater than 72°C, greater than 73°C, greater than 74°C, greater than 75°C, greater than 76°C, greater than 77°C, greater than 78°C, greater than 79°C, or greater than 80°C, or greater. The Tm of one or more nucleotides may be related to the guanine or cytosine (GC) content of the oligonucleotide. One or more oligonucleotides may contain a specific percentage of a given nucleotide. For example, one or more oligonucleotides may contain at least 20% guanine. One or more oligonucleotides may contain at least 30% guanine. At least 40% of the nucleotides of the oligonucleotide may be guanine or cytosine. At least 45% of the nucleotides of the oligonucleotide may be guanine or cytosine. At least 50% of the nucleotides of the oligonucleotide may be guanine or cytosine. At least 55% of the nucleotides of the oligonucleotide can be guanine or cytosine.At least 60% of the nucleotides of the oligonucleotide can be guanine or cytosine.About 40% to about 60% of the nucleotides of the one or more oligonucleotides may be guanine or cytosine. The one or more oligonucleotides may contain minimal secondary structure. For example, the one or more oligonucleotides may not have a hairpin or self-annealing. The one or more oligonucleotides may contain minimal interactions with each other, and one of the one or more oligonucleotides may be designed not to anneal with each other.

[0022] One or more oligonucleotides may contain one or more reactive groups or conjugate moieties. The reactive groups or conjugate moieties may allow the oligonucleotides to be conjugated, attached, or otherwise incorporated into another molecule. The oligonucleotides may contain conjugation moieties that allow for conjugation to nanoparticles. One or more oligonucleotides may contain a conjugate moiety at the 5' end. One or more oligonucleotides may contain a conjugate moiety at the 3' end. The conjugate moiety may be a thiol, e.g., a 3' thiol, or a 5'-thiol, such as a thioalkyl (e.g., thiohexyl).

[0023] The one or more oligonucleotides may comprise a particular or specific sequence. The one or more oligonucleotides may comprise a sequence in Table 1. The oligonucleotides may comprise a sequence identical to or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotides may comprise a sequence 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 to or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotides may comprise a sequence at least 90% identical to or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotides may comprise a sequence at least 91% identical to or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotides may comprise a sequence at least 92% identical to or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotides may comprise a sequence at least 93% identical to or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide may comprise a sequence at least 94% identical to or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide may comprise a sequence at least 95% identical to or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide may comprise a sequence at least 96% identical to or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide may comprise a sequence at least 97% identical to or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide may comprise a sequence at least 98% identical to or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide may comprise a sequence at least 99% identical to or complementary to a sequence selected from SEQ ID NOs: 1-18. The oligonucleotide may comprise no more than three, no more than two, or no more than one modification to a sequence selected from SEQ ID NOs: 1-18 or its complementary sequence.An oligonucleotide may contain no more than three modifications relative to a sequence selected from SEQ ID NOs: 1-18 or its complementary sequence. An oligonucleotide may contain no more than two modifications relative to a sequence selected from SEQ ID NOs: 1-18 or its complementary sequence. An oligonucleotide may contain no more than one modification relative to a sequence selected from SEQ ID NOs: 1-18 or its complementary sequence. The no more than three, no more than two, or no more than one modification may include substitutions, additions, deletions, or combinations thereof. The no more than three, no more than two, or no more than one modification may be substitutions. One or more oligonucleotides may contain degenerate bases or modified bases. A degenerate base may be a first base on a first oligonucleotide and a second base on a second oligonucleotide. For example, a sequence may contain a degenerate base at the position indicated by the letter "K." In a first oligonucleotide, a guanine may be present at the position indicated by the letter "K," and in a second oligonucleotide, a threonine may be present at the position indicated by the letter "K." Thus, a sequence may represent a mixture of a first and a second oligonucleotide. The use of degenerate bases in the sequence of an oligonucleotide may allow one or more nucleotides to be attached to a wider variety of sequences, including variant sequences.

[0024] [Table 1]

[0025] 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 on the complementarity of the oligonucleotide to the target nucleic acid. In some embodiments, one or more oligonucleotides hybridize to a target nucleic acid when the oligonucleotide has 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.

[0026] The 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 an infectious pathogen or a nucleic acid derived from an infectious pathogen. For example, the one or more oligonucleotides may be capable of annealing to a nucleic acid comprising a sequence derived from a human papillomavirus (HPV) gene or a sequence derived from HPV. The one or more oligonucleotides may be capable of annealing to a nucleic acid comprising a sequence indicating the presence of HPV in a subject. The one or more oligonucleotides may be capable of annealing to a nucleic acid comprising a sequence derived from the HPV L1 capsid gene, the HPV L2 capsid gene, the HPV E6 gene, the HPV E7 gene, and fragments thereof. The 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 HPV L1 capsid protein, the HPV L2 capsid protein, the HPV E6 protein, the HPV E7 protein, or fragments thereof. The one or more oligonucleotides may be capable of annealing to a nucleic acid containing a sequence indicative of the presence of Salmonella in a subject. The one or more oligonucleotides may be capable of annealing to a nucleic acid containing a sequence from or derived from a Salmonella species. The one or more oligonucleotides may be capable of annealing to a nucleic acid containing a sequence from or derived from Salmonella enterica. The one or more oligonucleotides may be capable of annealing to a nucleic acid containing a sequence from or derived from a different Salmonella species or serovar. For example, the one or more oligonucleotides may be capable of annealing to a nucleic acid containing a sequence from or derived from Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, or Salmonella prorum.The one or more oligonucleotides may be capable of annealing to a nucleic acid containing a sequence shared between two or more different species or serovars of the genus Salmonella. For example, the one or more oligonucleotides may be capable of annealing to a nucleic acid containing a sequence shared by Salmonella gallinarum and Salmonella prorum. In another example, the nucleic acid may contain a sequence shared between multiple members of the genus Salmonella or multiple strains of Salmonella enterica. For example, the one or more oligonucleotides may bind to a sequence present in multiple strains of Salmonella enterica, thereby indicating the presence of at least one of the Salmonella strains.

[0027] To detect a specific target, one or more oligonucleotides can be used in combination.For example, in a given mixture, there can be multiple oligonucleotides with different sequences.In some cases, one or more oligonucleotides comprise a first oligonucleotide that is identical to the sequence of SEQ ID NO:9, or comprises 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 a second oligonucleotide that is identical to the sequence of SEQ ID NO:10, or comprises 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 comprise a first oligonucleotide that is identical to the sequence of SEQ ID NO:9, and a second oligonucleotide that is identical to the sequence of SEQ ID NO:10. In some cases, the one or more oligonucleotides comprise a first oligonucleotide comprising a sequence identical to, 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 to, or complementary to, the sequence of SEQ ID NO: 11, and a second oligonucleotide comprising a sequence identical to, 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 to, or complementary to, the sequence of SEQ ID NO: 12. In some cases, the one or more oligonucleotides comprise a first oligonucleotide comprising a sequence identical to SEQ ID NO: 11, and a second oligonucleotide comprising a sequence identical to SEQ ID NO: 12.In some cases, the one or more oligonucleotides comprise a first oligonucleotide comprising a sequence identical to, 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 to, or complementary to, the sequence of SEQ ID NO: 13, and a second oligonucleotide comprising a sequence identical to, 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 to, or complementary to, the sequence of SEQ ID NO: 14. In some cases, the one or more oligonucleotides comprise a first oligonucleotide comprising a sequence identical to SEQ ID NO: 13, and a second oligonucleotide comprising a sequence identical to SEQ ID NO: 14. The one or more oligonucleotides comprise a first oligonucleotide comprising a sequence identical to, 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 to, or complementary to, the sequence of SEQ ID NO: 15, and a second oligonucleotide comprising a sequence identical to, 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 to, or complementary to, the sequence of SEQ ID NO: 16. In some cases, the one or more oligonucleotides comprise a first oligonucleotide comprising a sequence identical to SEQ ID NO: 15 and a second oligonucleotide comprising a sequence identical to SEQ ID NO: 16.In some cases, the one or more oligonucleotides include a first oligonucleotide that is identical to, 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 to, or complementary to, the sequence of SEQ ID NO: 17, and a second oligonucleotide that is identical to, 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 to, or complementary to, the sequence of SEQ ID NO: 18. In some cases, the one or more oligonucleotides include a first oligonucleotide that is identical to, or a second oligonucleotide that is identical to, SEQ ID NO: 17, and a second oligonucleotide that is identical to, SEQ ID NO: 18. The combination 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 containing multiple nanoparticles can be formed, which can enable the generation of a matrix or colloidal solution. One or more oligonucleotides can be bound to nanoparticles, so that multiple nanoparticles can be bound to a single target, and similarly, multiple targets can be bound to a single nanoparticle that has multiple conjugated oligonucleotides.The binding event can produce a matrix formed by nanoparticles, target nucleic acid, and one or more oligonucleotides.This matrix can have optical properties different from that of the solution without this matrix, and therefore, the formation of the matrix can be detected by observing the optical properties of the solution.

[0028] The optical properties of the matrix can be adjusted or depend on the distance between adjacent nanoparticles. The distance between adjacent nanoparticles can be adjusted through the length of the oligonucleotides. The distance can be adjusted by the number of nucleotides separating the target sequences on a given nucleic acid target. For example, as described herein, multiple oligonucleotides can be bound to a nucleic acid target. A first oligonucleotide can bind to a first sequence that is multiple nucleotides away from the binding position of a second oligonucleotide. The first oligonucleotide can bind to a sequence that is at least 30 nucleotides away from the sequence to which the second oligonucleotide binds. The first oligonucleotide can bind to a sequence that is 30 nucleotides or less away from the sequence to which the second oligonucleotide binds. The first oligonucleotide may 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 away from the sequence to which the second oligonucleotide binds. The first oligonucleotide may bind to a sequence that is 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 fewer nucleotides or more away from the sequence to which the second oligonucleotide binds. The first oligonucleotide may bind to a sequence that is 50-70 nucleotides away from the sequence to which the second oligonucleotide binds. The length of the oligonucleotides can alter the ability of a matrix to form based on the properties of the nanoparticles and oligonucleotides.For example, too short oligonucleotides or insufficient spacing between adjacent binding sites may cause adjacent nanoparticles to come into direct contact or spatially inhibit the formation of a matrix.Similarly, the size of nanoparticles may be related to the distance that nanoparticles are spaced apart.For example, smaller nanoparticles may allow for a smaller distance between adjacent nanoparticles while still generating a matrix upon binding of target nucleic acid.Therefore, regardless of the specific oligonucleotide length and nucleotide spacing, the generation of a matrix upon binding of target nucleic acid may be sufficient to detect the presence of target nucleic acid.

[0029] The one or more oligonucleotides can comprise a length or number of nucleotides. For example, the 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 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, or 50 nucleotides in length or less. The oligonucleotides can be about 16 to about 24 nucleotides in length. The oligonucleotides can be about 10 to about 20 nucleotides in length. The oligonucleotides can be about 20 to about 30 nucleotides in length.

[0030] The systems, methods, and compositions of the present disclosure may include nanoparticles. The nanoparticles may be used to detect the presence of target nucleic acids. The nanoparticles may include or otherwise incorporate one or more oligonucleotides. Incorporated may mean that the nanoparticle and the oligonucleotide are conjugated to each other. Incorporated may mean that the nanoparticle and the oligonucleotide are connected to each other. In some embodiments, one nanoparticle of the one or more nanoparticles incorporates one, two, three, four, five, or six oligonucleotides. In some embodiments, one nanoparticle of the one or more nanoparticles incorporates at least one, two, three, four, five, or six or more oligonucleotides. One or more nanoparticles may each (e.g., independently) incorporate one, two, three, four, five, or six oligonucleotides. For example, a first nanoparticle can include a first oligonucleotide and a second oligonucleotide, where the first and second oligonucleotides comprise different sequences. For example, a first nanoparticle can include a first oligonucleotide and a second nanoparticle can include a second oligonucleotide, where the first and second oligonucleotides include different sequences. In another example, a first nanoparticle can include a first oligonucleotide and a second nanoparticle can include a second oligonucleotide, where the first and second oligonucleotides include the same sequence.

[0031] The 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 reflectance, absorption, or transmission of specific wavelengths. The nanoparticles can comprise materials with optical properties, such as reflectance, transmittance, etc. The optical properties of the nanoparticles can allow the particles to be detected in solution. The optical properties can change in the presence of target nucleic acid. For example, in the presence of target nucleic acid, the nanoparticles can form a matrix of nanoparticles with optical properties different from a single nanoparticle.

[0032] Various compositions may include the use of gold nanoparticles. Given its inherent stability as a pure metal, gold is a practical element for use in nanoscience. Gold nanoparticles are recognized for their optical and electronic properties, such as those described at https: / / www.sigmaaldrich.com / BR / pt / technical-documents / technical-article / materials-science-and-engineering / biosensors-and-imaging / gold-nanoparticles. Gold possesses exceptional optical properties, including a high extinction coefficient, chemical stability, water solubility, localized surface plasmon resonance, and inherent photostability. The interaction or discrepancy between the chemical bond between the nanoparticle and the oligonucleotide allows for the identification of positive or negative results. Observations evidenced by a deviation from the original color indicate a negative result. The strength of the bond between the conjugate and the nucleic acid being investigated can determine the sensitivity and specificity of the method.

[0033] The characteristic reddish appearance of gold nanoparticles is related to their reduced size and the large number of electrons on their surface. According to theory, the luminescence signal (plasmon resonance) generated by the incidence of light on the surface of the nanoparticles in the conduction band propagates with a wave associated with the receiving characteristic. Surface resonance bands in the visible range can be observed, resulting from the vibration of electrons in the region relative to the lattice of metal ions. Therefore, a color change from red to blue can be observed in the presence of particle aggregation or when the diameter increases.

[0034] The optical and electronic properties of gold nanoparticles can be tuned by varying their size, shape, surface chemistry, or state of aggregation. Optical interactions and generation can be determined by their size and dimensions. A beam of light propagating in close proximity to colloidal nanoparticles can interact with free electrons and induce oscillations of charge signals. This phenomenon is directly related to the frequency of visible light. Short gold nanoparticles (approximately 30 nm) have absorption wavelengths in the blue-green portion of the spectrum (450 nm), while red wavelengths in the spectrum are reflected at 700 nm. When red is absorbed, blue is reflected, resulting in a blue or purple solution. As particle size increases toward the mass limit, the surface plasmon resonance wavelength shifts, and more visible wavelengths are reflected, giving the nanoparticles a bright or translucent color. The surface plasmon resonance is flexible and can change depending on the application, size, or shape. Chemical interactions of gold nanoparticles in contact with saline solution or excess salt result in a neutral reaction and cause the nanoparticles to aggregate. This results in a change from their original red color to blue. This problem can be rectified by desalting or can be protected by coating with polymers, small molecules, and specific biological recognition molecules.

[0035] Gold nanoparticles (AuNPs) can contain surface citrate molecules in solution. These surface citrate molecules can be replaced with differentially thiol-functionalized oligonucleotides, such as antisense oligonucleotides (ASOs). The unique optical properties of AuNPs, related to the targeting ability of oligonucleotides, can be used to develop selective detection platforms. The reactivity of oligonucleotides to targets can be correlated with surface resonance in the visible range without the need for any expensive instrumentation. Optical changes can be observed with the naked eye or using a camera or other image analysis techniques, as opposed to requiring observation of specific wavelengths with more expensive equipment. Furthermore, the ratio of oligonucleotides (e.g., 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 using a defined concentration of the target analyte (DNA / RNA) by varying the incubation temperature, knowing that optimal sensitivity is achieved at 37 °C. Observing the relative sensitivity of ASO-coated gold nanoparticles is accompanied by a relative increase in absorbance at 660 nm throughout the monitoring. This makes it possible to evaluate different colloid distribution profiles in the analyzed range.

[0036] The one or more nanoparticles may be characterized by size. The one or more nanoparticles may be characterized by an average size of about 10 nanometers (nm). The one or more nanoparticles may 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, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm. The one or more nanoparticles may 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, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm, or more. The one or more nanoparticles may be characterized by an average size of 20 nm or less, 30 nm or less, 40 nm or less, 50 nm or less, 60 nm or less, 70 nm or less, 80 nm or less, 90 nm or less, 100 nm or less, 110 nm or less, 120 nm or less, 130 nm or less, 140 nm or less, 150 nm or less, 160 nm or less, 170 nm or less, 180 nm or less, 190 nm or less, or 200 nm or less, or a smaller average size. The one or more nanoparticles may 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, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm. The one or more nanoparticles may 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, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm, or more.The one or more nanoparticles may be characterized by a size of about 10 nm or less, 20 nm or less, 30 nm or less, 40 nm or less, 50 nm or less, 60 nm or less, 70 nm or less, 80 nm or less, 90 nm or less, 100 nm or less, 110 nm or less, 120 nm or less, 130 nm or less, 140 nm or less, 150 nm or less, 160 nm or less, 170 nm or less, 180 nm or less, 190 nm or less, or 200 nm or less, or smaller.

[0037] 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 feces sample, a saliva 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 of animal origin or derived from an animal. The sample can be of mammalian origin or derived from a mammal. The sample can be of plant origin or derived from a plant. The sample can be of human origin or derived from a human. The sample can comprise nucleic acid.

[0038] nucleic acid target

[0039] The nucleic acid targets of the present disclosure can be derived from a sample. The biological sample can be a sample derived from or from a subject. The sample can include any number of macromolecules, e.g., cellular macromolecules. The sample can include a plurality of cells. The sample can be a tissue sample, such as a biopsy, core biopsy, needle aspirate, or fine needle aspirate. The sample can be a tumor sample. The sample can be a fluid sample, such as a blood sample, plasma sample, urine sample, or saliva sample. The sample can be a skin sample. The biological sample can be a buccal swab. The sample can be a plasma or serum sample. The sample can include one or more cells. The one or more cells can be derived from or from a tumor. The biological sample can be, for example, blood, plasma, serum, urine, saliva, mucosal excretion, sputum, feces, or tears.

[0040] The nucleic acid target may be derived from one or more cells. The nucleic acid target may comprise deoxyribonucleic acid (DNA). The DNA may be any type of DNA, including genomic DNA. The nucleic acid target may be viral DNA. The nucleic acid target may comprise ribonucleic acid (RNA). The RNA may be any type of RNA, including messenger RNA, transfer RNA, ribosomal RNA, and microRNA. The RNA may be viral RNA. The nucleic acid may comprise a human genome sequence. The nucleic acid may comprise an animal genome sequence. The nucleic acid may comprise a plant genome sequence. The nucleic acid may comprise a fungal genome sequence. The nucleic acid may comprise an archaeal genome sequence. The nucleic acid may comprise a pathogen-associated sequence. The nucleic acid may comprise a wild-type sequence. The nucleic acid may comprise a mutant sequence.

[0041] The target nucleic acid or nucleic acids can be of any length, for example, up to 1, 2, 3, 4, 5, 10, 20, 50, 100, 500, 1000, 5000, 10,000, 50,000, or 100,000 nucleotides or more.

[0042] In some examples, the target nucleic acid may include a gene or a portion thereof. The nucleic acid target may include a gene whose detection may be useful for diagnosing one or more diseases. The gene may be a viral gene or a bacterial gene, whose detection may be useful in identifying the presence or absence of a pathogen in a subject. In some cases, the method of the present disclosure is useful for detecting the presence or absence of one or more infectious agents (e.g., viruses, bacteria, fungi) in a subject. The nucleic acid target may be a human gene.

[0043] The nucleic acid target may be 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 an infectious pathogen or a nucleic acid derived from an infectious pathogen. For example, the nucleic acid target may include a sequence from or derived from a human papillomavirus (HPV) gene. The nucleic acid target may include a sequence indicative of the presence of HPV in a subject. The nucleic acid target may include a sequence derived from an HPV L1 capsid gene, an HPV L2 capsid gene, an HPV E6 gene, an HPV E7 gene, or a fragment thereof. The nucleic acid target may encode a protein or polypeptide. For example, the nucleic acid target may encode an HPV L1 capsid protein, an HPV L2 capsid protein, an HPV E6 protein, an HPV E7 protein, or a fragment thereof. The nucleic acid target may include a sequence from a Salmonella species or a sequence derived from a Salmonella species. The nucleic acid target may include a sequence from or derived from Salmonella enterica. The nucleic acid target may include a sequence from or derived from different species or serovars of Salmonella. For example, the nucleic acid target may include a sequence from or derived from Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, or Salmonella prorum. The nucleic acid target may include a sequence shared between two or more different species or serovars of Salmonella. For example, the nucleic acid target may include a sequence shared by Salmonella gallinarum and Salmonella prorum. In another example, the nucleic acid may include a sequence shared between multiple members of the Salmonella genus or multiple strains of Salmonella enterica. For example, one or more oligonucleotides may bind to a sequence present in multiple strains of Salmonella enterica, thereby indicating the presence of at least one strain of Salmonella.

[0044] In some cases, the method can be carried out by using a composition 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 include a nucleic acid and hybridize with another nucleic acid. The method can include inducing or causing 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 condensing agent. The solution (e.g., a condensation solution) and / or condensing agent can cause aggregation of the nanoparticles. For example, the presence of a salt can induce aggregation of the nanoparticles. The solution can cause the formation of a nanoparticle matrix. The aggregation or formation of the nanoparticle matrix can depend on structures or molecules associated with the nanoparticles. The nanoparticles can include one or more oligonucleotides that can interact to form an aggregate or matrix. In the presence of a molecule (e.g., a complementary sequence) that binds to the one or more oligonucleotides, aggregation or matrix formation can be prevented or inhibited. The condensing agent can include magnesium chloride.

[0045] The formation of nanoparticle aggregates can be detected relative to the formation of a nanoparticle matrix and can be used to detect one or more target nucleic acids. A solution can be added to the mixture to aggregate the nanoparticles or form a nanoparticle matrix. The formation of nanoparticle aggregates versus matrix formation can depend on the presence of the target nucleic acid. In the absence of target nucleic acid, the nanoparticles can form aggregates, but in the presence of target nucleic acid, the nanoparticles can form a matrix. The aggregates and matrix can have different optical properties and therefore can be detectable and distinguishable based on these optical properties. Because aggregation or matrix formation depends on the presence of the target nucleic acid, detection of the aggregates or matrix can indicate the presence of the target nucleic acid.

[0046] The method may include detecting or determining an optical parameter of the solution or composition. The detecting or determining step may include using a sensor to detect wavelengths. The detecting or determining step may include using a camera. The detecting or determining step may include image analysis techniques. The optical parameter may include the absorption, transmission, scattering, or reflection of light or other waves at a wavelength or a range of wavelengths. The optical parameter may include a brightness parameter (e.g., color brightness), a saturation parameter (e.g., color intensity), or a hue parameter (e.g., tint). The determining step may include the use of color space analysis. The optical parameter may be binned based on a wavelength or a range of wavelengths. For example, the amount of red or green in a color may be parameterized. Based on color theory, a color need not be simultaneously red and green, allowing a single parameter to be generated based on a red-green scale. The parameterization may assign a single value to the redness or greenness value, where the amount of red is parameterized as a positive number and the amount of green is parameterized as a positive number. Similarly, the amount of yellow or blue in a color can be parameterized. Red-green and yellow-blue analysis can indicate a specific parameterized color, which can be used for further analysis. The luminosity or brightness of a solution can also be parameterized. The optical parameter can be optical density, for example, optical density at 520 nm or 560 nm. By observing the optical density of a sample, the sample can be called positive or negative. A negative control sample, a biological control sample, or a positive control can be used to calibrate the method or to enable sample normalization or referencing. For example, an optical parameter can be observed to be significantly (e.g., statistically significant) lower or higher than the negative control. This significant difference can be used to determine whether the sample is a negative or positive sample. Various wavelengths can be detected using the methods of the present disclosure. For example, detection may be at about 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,410nm, 415nm, 420nm, 425nm, 430nm, 435nm, 440nm, 445nm, 450nm, 455nm, 460nm, 465nm, 470n m, 475nm, 480nm, 485nm, 490nm, 495nm, 500nm, 505nm, 510nm, 515nm, 520nm, 525nm, 530nm, 535 nm, 540nm, 545nm, 550nm, 555nm, 560nm, 565nm, 570nm, 575nm, 580nm, 585nm, 590nm, 595nm, 6 00nm, 605nm, 610nm, 615nm, 620nm, 625nm, 630nm, 635nm, 640nm, 645nm, 650nm, 655nm, 660nm, 665nm, 670nm, 675, nm, 680nm, 685nm, 690nm, 695nm, 700nm, 705nm, 710nm, 715nm, 720nm, 725 nm, 730nm, 735nm, 740nm, 745nm, 750nm, 755nm, 760nm, 765nm, 770nm, 775nm, 780nm, 785nm, 79 The method may be performed using wavelengths of 0 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, detection may be performed at wavelengths of 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, 450nm, 455nm, 460nm, 465nm, 470nm, 475,nm, 480nm, 485nm, 490nm, 495nm, 500nm, 505nm, 510nm, 515nm, 520nm, 525nm, 530nm, 535nm, 540nm, 545nm, 550nm, 555nm, 560nm, 565nm, 570nm, 575,nm, 580nm, 585nm, 590nm, 595nm, 600nm, 605nm,610nm, 615nm, 620nm, 625nm, 630nm, 635nm, 640nm, 645nm, 650nm, 655nm, 660nm, 665nm, 670nm, 675,nm, 680nm, 685nm, 690nm, 695nm, 700nm, 705nm, 710nm, 715nm, 720nm, 725nm, 730nm, 735nm, 740nm, 745nm, 750nm, 755nm, 760nm, 765nm, 77 The wavelengths may be 700nm, 775nm, 780nm, 785nm, 790nm, 795nm, 800nm, 805nm, 810nm, 815nm, 820nm, 825nm, 830nm, 835nm, 840nm, 845nm, 850nm, 855nm, 860nm, 865nm, 870nm, 875nm, 880nm, 885nm, 890nm, 895nm, or 900nm, or higher. For example, detection is below 300nm, below 305nm, below 310nm, below 315nm, below 320nm, below 325nm, below 330nm, below 335nm, 340nm m or less, 345nm or less, 350nm or less, 355nm or less, 360nm or less, 365nm or less, 370nm or less, 375,nm or less, 380nm or less, 385nm or less , 390nm or less, 395nm or less, 400nm or less, 405nm or less, 410nm or less, 415nm or less, 420nm or less, 425nm or less, 430nm or less, 435 Below nm, below 440nm, below 445nm, below 450nm, below 455nm, below 460nm, below 465nm, below 470nm, below 475,nm, below 480nm , 485nm or less, 490nm or less, 495nm or less, 500nm or less, 505nm or less, 510nm or less, 515nm or less, 520nm or less, 525nm or less, 530 below nm, below 535 nm, below 540 nm, below 545 nm, below 550 nm, below 555 nm, below 560 nm, below 565 nm, below 570 nm, below 575, nm , 580nm or less, 585nm or less, 590nm or less, 595nm or less, 600nm or less, 605nm or less, 610nm or less, 615nm or less, 620nm or less, 625 nm or less, 630nm or less, 635nm or less, 640nm or less, 645nm or less, 650nm or less, 655nm or less, 660nm or less, 665nm or less, 670nm or less,675,nm or less, 680nm or less, 685nm or less, 690nm or less, 695nm or less, 700nm or less, 705nm or less, 710nm or less, 715nm or less, 720nm or less, 725nm or less, 730nm or less, 735n m or less, 740nm or less, 745nm or less, 750nm or less, 755nm or less, 760nm or less, 765nm or less, 770nm or less, 775,nm or less, 780nm or less, 785nm or less, 790nm or less, 795nm or less, Detection can be performed using wavelengths of 800 nm or less, 805 nm or less, 810 nm or less, 815 nm or less, 820 nm or less, 825 nm or less, 830 nm or less, 835 nm or less, 840 nm or less, 845 nm or less, 850 nm or less, 855 nm or less, 860 nm or less, 865 nm or less, 870 nm or less, 875 nm or less, 880 nm or less, 885 nm or less, 890 nm or less, 895 nm or less, or 900 nm or less, or wavelengths less than these. Detection can be performed by obtaining a spectrum from 300 nm to 900 nm, or any subset of the wavelength range. For example, detection can be performed from 450 nm to 700 nm.

[0047] Detection can be performed using a plate reader, spectrophotometer, or other device capable of detecting light or UV radiation. For example, the device can be equipped with a detector that can quantify the amount of light received. The device can use a monochromator to direct specific wavelengths to the sample or observe a given wavelength from the sample.

[0048] The method may include comparing the optical parameters of the test solution with a reference sample or control solution. The optical parameters of the test solution and the reference solution may be determined and compared. Detection of the target nucleic acid may be determined based on the comparison between the test solution and the reference solution. For example, the reference solution may be a positive control solution and may contain the target nucleic acid. This reference solution may contain optical parameters. The test solution may also be analyzed to determine whether the solution contains the target nucleic acid, and the optical parameters of the test solution and the reference solution may be the same (or substantially similar). The reference solution may also be a negative control-type solution in which the target nucleic acid is absent or another known sequence other than the target sequence is present. The optical parameters of the test solution and the reference solution may be compared, and a determination may be made based on the similarity of the optical parameters.

[0049] Agglomerated nanoparticles or nanoparticle matrices may have optical parameters that are not the same as one or more nanoparticles in solution. Because the optical parameters may be different when comparing aggregated nanoparticles (or nanoparticle matrices) with nanoparticles in solution, it may be possible to detect the presence of aggregated nanoparticles or nanoparticle matrices and distinguish a solution with aggregated particles (or nanoparticle matrices) from a solution with unagglomerated nanoparticles or single nanoparticles in solution. Target nucleic acid detection can use this difference to determine whether the target nucleic acid is present. The target nucleic acid may inhibit the formation of aggregates or matrices, such that the absence of aggregates or matrices indicates the presence of the target nucleic acid.

[0050] For example, a biosensor homogeneous dispersion (AuNP-ASO; no target nucleic acid; no condensing agent) can be characterized as the greatest distance from the AuNP-ASO, with minimal particle aggregation observed. Reactivity can be visualized by a partial change in color to reddish. A positive heterogeneous dispersion (AuNP-ASO + target RNA / DNA + MgCl2) can be characterized as controlled separation of the AuNP-ASO via bridges formed upon recognition of the genetic material (RNA / DNA). The annealing distance can involve a minimum number of bases (60 ± 10 nucleotides). Partial particle aggregation in the presence of MgCl2 can be observed, associated with the visualization of an opaque gray / purple color. A negative heterogeneous dispersion (AuNP-ASO + MgCl2) can be characterized as high aggregation of the AuNP-ASO in the presence of MgCl2, associated with the visualization of a color change from reddish to gray / translucent blue. Based on the color of the solution, which is based at least on agglutination, different dispersion patterns can be distinguished and the presence of the target nucleic acid can be determined.

[0051] The method can determine the presence or absence of one or more target nucleic acids in a body 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 body 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 body sample with a specificity of at least 95%, 96%, 97%, 98%, or 99%. The method can determine the presence or absence of one or more target nucleic acids in a body sample with an accuracy of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0052] The method can be carried out using a device that allows the method to be carried out. Figure 5 shows an exemplary device. An inlet to the device is available for adding a sample and a lysis solution. The sample solution then flows into a mixing module, a heating chamber, and a filter so that the sample solution contains extracted nucleic acids. The sample flows into a pool to which a biosensor (e.g., AuNPs containing oligonucleotides) is added. This is mixed in the mixing module and then flows into a new pool to which a developer solution (e.g., a condensation solution) is added. This solution is then mixed in the mixing module and then flows into a new pool to perform an optical reading, such as an RGB reading, or to measure another optical parameter, such as those described elsewhere herein.

[0053] The method can be carried out in a plate containing one or more wells. For example, the method can use a 96-well plate. For example, multiple assays can be carried out simultaneously, with a given sample present in each well. A plate reader can then be used to analyze the wells and provide an output for each sample. Thus, the assay allows for rapid and efficient multiplexing, which can reduce waiting time and improve throughput. The method can also be carried out using a cuvette or tube, or other container. For example, the method can be carried out by mixing the sample in a tube (e.g., a centrifuge tube or a microcentrifuge tube). The sample can then be added to the cuvette and read via a spectrophotometer or other device capable of detecting optical properties.

[0054] The ability of the methods and compositions to identify the presence of a target nucleic acid can be evaluated, for example, in terms of assay accuracy, assay sensitivity, assay specificity, assay positive predictive value (PPV), assay negative predictive value (NPV), or "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 proportion of incorrectly classified samples. Accuracy can be calculated, for example, as the total number of correctly classified samples divided by the total number of samples in the test population. Sensitivity is a measure of "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 "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. 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 stronger 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 more. 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 more. Other useful measures of a test's usefulness include the "positive predictive value," which is the percentage of actual positives that test positive, and the "negative predictive value," which is the percentage of actual negatives that test negative.In some embodiments, the methods 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 more. In some embodiments, the methods 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 more. In some embodiments, the methods described herein exhibit an accuracy of at least about 75%, e.g., at least about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, or about 100%. For example, the method can identify the presence of a target nucleic acid with an area 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 more. In other embodiments, the method can identify the presence of a target nucleic acid with a specificity of at least about 75%, e.g., at least about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, or about 100%. For example, the method can identify the presence of a target nucleic acid with a specificity 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 more. In some embodiments, the method is capable of identifying the presence of a target nucleic acid with a specificity of at least about 75%, e.g., a sensitivity of at least about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, or about 100%.For example, the method can identify the presence of a target nucleic acid with a sensitivity 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 more. In other embodiments, the method can identify the presence of a target nucleic acid with a specificity and sensitivity of at least about 75%, respectively, e.g., at least about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, or about 100% (e.g., at least about 80% specificity and at least about 80% sensitivity, or, e.g., at least about 80% specificity and at least about 95% sensitivity).

[0055] The disclosed method can be performed in a short time and may be faster than other methods with similar accuracy metrics. For example, the disclosed method can be performed in 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. Methods starting with pre-loaded nanoparticles (e.g., one or more nanoparticles loaded with one or more oligonucleotides) can be performed in less than 60 minutes. The disclosed method starting with pre-loaded nanoparticles (e.g., one or more nanoparticles loaded with one or more oligonucleotides) can be performed in 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, or less. The present methods, starting with one or more nanoparticles that are not incorporated with one or more oligonucleotides, can be performed in 60 minutes or less, 55 minutes or less, 50 minutes or less, 45 minutes or less, 40 minutes or less, 35 minutes or less, 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or less.

[0056] kit The present disclosure provides a kit for carrying out the method of the present disclosure.The present disclosure also provides a kit containing the composition described in the present disclosure.The kit may include nanoparticles and oligonucleotides as described elsewhere herein.For example, the kit may include nanoparticles incorporated into one or more oligonucleotides.The kit may include a condensation solution.The kit may include a tube containing nanoparticles.

[0057] The kit may include instructions for using any of the foregoing in the methods described herein. The kit may include solutions or other components that can be used as standards, negative or positive controls. For example, the kit may include standards that can be used as baselines for color analysis or optical parameter determination. The kit may include swabs or other instruments for collecting samples from subjects. The kit may include devices for adding solutions or otherwise performing the methods of the present disclosure.

[0058] Computer Control System The present disclosure provides a computer control system programmed to implement the methods of the present disclosure. Figure 12 shows a computer system (1201) programmed or otherwise configured to implement some of the methods disclosed elsewhere herein. The computer system (1201) can control various aspects of the present disclosure, such as processing images of a sample, determining and processing optical parameters, receiving images from a user, processing images so that the colors and color differences of the sample and reference colors can be identified, processing images using an edge detector algorithm, and outputting results to the user regarding the presence of nucleic acids. The computer system (1201) can be a user's electronic device or a computer system located remotely from the electronic device. The electronic device can be a mobile electronic device.

[0059] The computer system (1201) includes a central processing unit (CPU, also referred to herein as a "processor" and a "computer processor") (1205), which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system (1201) also includes memory or storage locations (1210) (e.g., random access memory, read-only memory, flash memory), electronic storage devices (1215) (e.g., hard disks), communication interfaces (1220) (e.g., network adapters) for communicating with one or more other systems, and peripheral devices (1225), such as cache, other memory, data storage devices, and / or electronic display adapters. The memory (1210), storage units (1215), interfaces (1220), and peripheral devices (1225) communicate with the CPU (1205) via a communication bus (solid lines), such as a motherboard. The storage device (1215) may be a data storage device (or data repository) for storing data. The computer system 1201 may be operatively connected to a computer network ("network") 1230 with the aid of a communication interface 1220. The network 1230 may be the Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. The network 1230 may, in some cases, be a telecommunications and / or data network. The network 1230 may include one or more computer servers, which may enable distributed computing, such as cloud computing. The network 1230 may, in some cases, with the aid of the computer system 1201, implement a peer-to-peer network, which may enable devices coupled to the computer system 1201 to act as clients or servers.

[0060] The CPU (1205) can execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory (1210). The instructions may be directed to the CPU (1205), which can then be programmed or otherwise configured to implement the methods of the present disclosure. Examples of operations performed by the CPU (1205) may include fetch, decode, execute, and writeback.

[0061] The CPU 1205 may be part of a circuit, such as an integrated circuit. One or more other components of the system 1201 may also be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC).

[0062] The storage device (1215) can store files such as drivers, libraries, and saved programs. The storage device (1215) can store user data, such as user personalization settings and user programs. The computer system (1201) may optionally include one or more additional data storage devices external to the computer system (1201), such as located on a remote server in communication with the computer system (1201) over an intranet or the Internet.

[0063] 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 a remote computer system of a provider or a user (e.g., a service provider). Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., an Apple® iPad, a Samsung® Galaxy Tab), a telephone, a smartphone (e.g., an Apple® iPhone, an Android-enabled device, a Blackberry®), or a personal digital assistant. A user can access the computer system (1201) via the network (1230).

[0064] The methods described herein can be performed by machine (e.g., computer processor) executable code stored in an electronic storage location of the computer system (1201), such as, for example, on memory (1210) or electronic storage (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 storage (1215) and stored in memory (1210) for immediate access by the processor (1205). In some situations, the electronic storage (1215) can be eliminated, and the machine-executable instructions are stored in memory (1210).

[0065] The code may be pre-compiled and configured for use with a machine having a processor adapted to execute the code, or may be compiled at run-time. The code may be provided in a programming language that can be selected to enable the code to be executed in a pre-compiled or on-the-fly compiled manner.

[0066] Aspects of the systems and methods provided herein, such as the computer system (1201), may be embodied in programming. Various aspects of the technology may be thought of as "products" or "articles of manufacture," typically in the form of machine- (or processor-) executable code and / or associated data executed or embodied on a type of machine-readable medium. The machine-executable code may be stored in electronic storage devices such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. "Storage" type media may include any or all of the tangible memory of a computer or processor, or its associated modules, such as various semiconductor memories, tape drives, disk drives, etc., which may provide non-transitory recording media at any time for programming the software. All or portions of the software may sometimes be communicated via the Internet or various other telecommunications networks. Such communication may enable loading of the software from one computer or processor to another, for example, from a management server or host computer to an application server computer platform. Thus, other types of media that may carry software elements include optical, electrical, and electromagnetic waves, such as those used over wired and terrestrial optical communication networks between local devices and various air links. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered media that carry software. As used herein, unless limited to non-transitory tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0067] Thus, a machine-readable medium such as a computer-executable code may take many forms, including, but not limited to, a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include optical or magnetic disks, such as any of the storage devices in any computer(s), such as those that may be used to implement the databases shown in the figures. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those 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 tape, other magnetic media, CD-ROMs, DVDs or DVD-ROMs, other optical media, punch cards, paper tapes, other physical storage media with patterns of holes, RAM, ROM, PROMs and EPROMs, FLASH-EPROMs, other memory chips or cartridges, carrier waves that transport data or instructions, cables or links that transmit such carrier waves, or other media from which a computer can read programming code and / or data. Many forms of computer-readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0068] The computer system (1201) may include or be in communication with an electronic display (1235) that includes a user interface (UI) (1240), for example, to provide method results, optical parameters, or images of a solution. Examples of UIs include, without limitation, graphical user interfaces (GUIs) and web-based user interfaces.

[0069] The methods and systems of the present disclosure can be implemented by one or more algorithms. The algorithms can be implemented by software when executed by the central processing unit (1205). The algorithms can, for example, determine optical parameters of solutions through image analysis, perform optical parameter comparisons, and normalize optical parameters to a baseline or control.

[0070] List of Embodiments Embodiment 1. A method for processing or analyzing a sample, comprising: (a) contacting a sample with a composition comprising one or more nanoparticles having one or more oligonucleotides incorporated therein to provide a test composition, wherein the one or more oligonucleotides hybridize to one or more target nucleic acids, if present, in the sample; (b) forming a nanoparticle matrix from one or more nanoparticles hybridized to one or more target nucleic acids in the presence of one or more target nucleic acids; (c) determining an optical parameter of the test composition that is indicative of the presence or absence of one or more nucleic acids in the sample. Embodiment 2. The method of embodiment 1, wherein the optical parameters are determined by color space analysis. Embodiment 3. The method of embodiment 1 or 2, wherein the optical parameter comprises absorption, transmission, scattering, or reflection of light at a wavelength or range of wavelengths. Embodiment 4. The method of any one of embodiments 1 to 3, wherein the optical parameter comprises a luminosity parameter (e.g., color brightness), a saturation parameter (e.g., color intensity), or a color tone parameter (e.g., hue). Embodiment 5. The method of any one of embodiments 1-4, wherein (c) further comprises comparing the optical parameters of the test composition with corresponding optical parameters determined from a corresponding reference composition. Embodiment 6. The method of any one of embodiments 1 to 5, wherein the method determines the presence or absence of one or more target nucleic acids in a sample with a sensitivity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Embodiment 7. The method of any one of embodiments 1 to 6, wherein the method determines the presence or absence of one or more target nucleic acids in a sample with a specificity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Embodiment 8. The method of any one of embodiments 1 to 7, wherein the method determines the presence or absence of one or more target nucleic acids in a sample with an accuracy of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Embodiment 9. The method of any one of embodiments 1-8, 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, and a tissue sample. Embodiment 10. The method of any one of embodiments 1 to 9, wherein the sample is derived from a mammal (e.g., a human). Embodiment 11. The method of any one of embodiments 1 to 10, wherein the sample is derived from an animal. Embodiment 12. The method of any one of embodiments 1 to 11, wherein the sample is derived from a plant. Embodiment 13. The method of any one of embodiments 1 to 12, wherein the sample comprises a lysis solution. Embodiment 14. The method of any one of embodiments 1 to 13, comprising the step of: (b) contacting the test composition with a nanoparticle condensing agent and / or a salt. Embodiment 15 The method of embodiment 14, wherein the condensing agent comprises magnesium chloride. Embodiment 16 The method of any one of embodiments 1 to 15, wherein at least about 40% of the nucleotides in the one or more oligonucleotides are guanine or cytosine. Embodiment 17. The method of any one of embodiments 1 to 15, wherein about 40% to about 60% of the nucleotides in the one or more oligonucleotides are guanine or cytosine. Embodiment 18 The method of any one of embodiments 1 to 17, wherein the one or more oligonucleotides are characterized by a melting temperature (Tm) of at least about 65°C (°C). Embodiment 19. The method of any one of embodiments 1 to 17, wherein the one or more oligonucleotides are characterized by a Tm of about 65°C to about 75°C. Embodiment 20. The method of any one of embodiments 1 to 19, wherein one or more oligonucleotides comprises a conjugate moiety at the 5' end. Embodiment 21 The method of embodiment 20, wherein the conjugate moiety is a 5'-thiol. Embodiment 22 The method of embodiment 21, wherein the 5'-thiol comprises a thioalkyl, such as thiohexyl. Embodiment 23 The method of any one of embodiments 1 to 22, wherein one nanoparticle of the one or more nanoparticles incorporates 1, 2, 3, 4, 5, or 6 oligonucleotides. Embodiment 24 The method of any one of embodiments 1 to 23, wherein one or more nanoparticles each (e.g., independently) incorporate 1, 2, 3, 4, 5, or 6 oligonucleotides. Embodiment 25. The method of any one of embodiments 1 to 24, wherein the one or more nanoparticles comprise gold. Embodiment 26. The method of any one of embodiments 1 to 25, wherein the one or more nanoparticles are characterized by an average size of about 10 nanometers (nm) to about 200 nm. Embodiment 27. The method of any one of embodiments 1 to 26, wherein the one or more oligonucleotides are 16 to 24 nucleotides in length. Embodiment 28. The method of any one of embodiments 1 to 27, wherein the one or more oligonucleotides are associated 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. Embodiment 29. The method of any one of embodiments 1 to 28, wherein the one or more oligonucleotides comprise two oligonucleotides, a first oligonucleotide that hybridizes to a first region of the target nucleic acid and a second oligonucleotide that hybridizes to a second region of the target nucleic acid. Embodiment 30. The method of embodiment 29, wherein the distance between the first region and the second region of the target nucleic acid is about 50 to 70 nucleotides. Embodiment 31. The method of any one of embodiments 1 to 30, wherein one or more oligonucleotides hybridize to 10 to 30 nucleotides of one or more target nucleic acids. Embodiment 32 The method of any one of embodiments 1 to 31, wherein the one or more nanoparticles form aggregates in the absence of the one or more target nucleic acids. Embodiment 33 The method of any one of embodiments 1 to 32, wherein the one or more target nucleic acids are derived from one or more viruses or one or more bacteria. Embodiment 34 The method of embodiment 33, wherein the one or more target nucleic acids are not derived from a coronavirus. Embodiment 35. The method of embodiment 33, wherein the one or more target nucleic acids are not derived from SARS-CoV-2 or one or more mutants thereof. Embodiment 36 The method of any one of embodiments 33-35, wherein the one or more viruses comprises an influenza virus or a human papillomavirus. Embodiment 37 The method of any one of embodiments 33 to 36, wherein the one or more bacteria comprises Salmonella. Embodiment 38 The method of embodiment 37, wherein the Salmonella comprises Salmonella enterica. Embodiment 39. The method of embodiment 37, wherein the Salmonella comprises one or more strains or serotypes of Salmonella. Embodiment 40. The method of embodiment 39, wherein the one or more Salmonella strains or serotypes comprise one or more members selected from the group consisting of Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella prorum. Embodiment 41. The method of any one of embodiments 1 to 40, wherein the one or more target nucleic acids are associated with one or more diseases or disorders. Embodiment 42 The method of embodiment 41, wherein the one or more diseases or conditions include an infectious disease, cancer, or a degenerative disease. Embodiment 43 The method of any one of embodiments 1 to 42, wherein the one or more target nucleic acids encode a polypeptide or protein. Embodiment 44. The method of any one of embodiments 1 to 43, wherein the one or more target nucleic acids comprise DNA or RNA. Embodiment 45 The method of embodiment 44, wherein the DNA is genomic DNA. Embodiment 46 The method of embodiment 44, wherein the RNA is genomic RNA. Embodiment 47 The method of embodiment 44, wherein the RNA is double-stranded RNA or single-stranded RNA. Embodiment 48 The method of embodiment 44, wherein the RNA is double-stranded DNA or single-stranded DNA. Embodiment 49. The method of any one of embodiments 1 to 48, wherein the one or more target nucleic acids are derived from a human papillomavirus (HPV) or one or more variants thereof. Embodiment 50. The method of any one of embodiments 1 to 49, wherein the one or more target nucleic acids comprise one or more members selected from an HPV L1 capsid protein, an HPV L2 capsid protein, an HPV E6 protein, an HPV E7 protein, and fragments of any thereof. Embodiment 51. The method of any one of embodiments 1 to 50, wherein the one or more target nucleic acids are from Salmonella. Embodiment 52 The method of embodiment 51, wherein the Salmonella comprises Salmonella enterica. Embodiment 53 The method of embodiment 51, wherein the Salmonella comprises one or more strains or serotypes of Salmonella. Embodiment 54. The method of embodiment 53, wherein the one or more Salmonella strains or serotypes comprise one or more members selected from the group consisting of Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella prorum. Embodiment 55. The method of any one of embodiments 1 to 54, wherein the one or more oligonucleotides comprise a sequence selected from SEQ ID NOs: 1 to 18. Embodiment 56. A composition for detecting one or more target nucleic acids, comprising: one or more nanoparticles incorporating one or more oligonucleotides, wherein the one or more oligonucleotides are complementary to one or more target nucleic acids; the one or more nanoparticles form a nanoparticle matrix in the presence of the one or more target nucleic acids, the nanoparticle matrix having different optical parameters compared to a solution containing the corresponding nanoparticles not in the nanoparticle matrix; A composition comprising: Embodiment 57. A composition for detecting a target nucleic acid, comprising: one or more nanoparticles incorporating one or more oligonucleotides, wherein a first oligonucleotide of the one or more oligonucleotides is complementary to the target nucleic acid and a second oligonucleotide of the one or more oligonucleotides is complementary to the target nucleic acid in a second sequence, and wherein the one or more nanoparticles comprise gold; forming a nanoparticle matrix from one or more nanoparticles in the presence of one or more target nucleic acids; A composition comprising: Embodiment 58. The composition of any one of embodiments 56 or 57, wherein at least about 40% (e.g., about 40% to about 60%) of the nucleotides in one or more oligonucleotides are guanine or cytosine. Embodiment 59. The composition of any one of embodiments 56 to 58, wherein one or more oligonucleotides are characterized by a melting temperature (Tm) of at least about 65 degrees Celsius (°C) (e.g., about 65°C to about 75°C). Embodiment 60. The composition of any one of embodiments 56 to 59, wherein one or more oligonucleotides comprises a conjugate moiety at the 5' end. Embodiment 61 The composition of embodiment 60, wherein the conjugate moiety is a 5'-thiol. Embodiment 62 The composition of embodiment 61, wherein the 5' thiol is a thioalkyl group. Embodiment 63 The composition of embodiment 62, wherein the thioalkyl is a thiohexyl group. Embodiment 64. The composition of any one of embodiments 56 to 63, wherein one nanoparticle of the one or more nanoparticles incorporates 1, 2, 3, 4, 5, or 6 oligonucleotides. Embodiment 65. The composition of any one of embodiments 56-64, wherein one or more nanoparticles each (e.g., independently) incorporate 1, 2, 3, 4, 5, or 6 oligonucleotides. Embodiment 66. The composition of any one of embodiments 56-65, wherein the one or more nanoparticles comprise gold. Embodiment 67. The composition of any one of embodiments 56 to 66, wherein the one or more nanoparticles are characterized by an average size of about 10 nanometers (nm) to about 200 nm. Embodiment 68. The composition of any one of embodiments 56 to 67, wherein one or more oligonucleotides are 16 to 24 nucleotides in length. Embodiment 69. The composition of any one of embodiments 56 to 68, wherein the one or more oligonucleotides are associated 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. Embodiment 70. The composition of any one of embodiments 56 to 69, wherein the one or more oligonucleotides comprise two oligonucleotides, a first oligonucleotide that hybridizes to a first region of the target nucleic acid and a second oligonucleotide that hybridizes to a second region of the target nucleic acid. Embodiment 71. The composition of embodiment 70, wherein the distance between the first region and the second region of the target nucleic acid is about 50 to 70 nucleotides. Embodiment 72. The composition of any one of embodiments 56 to 69, wherein the one or more target nucleic acids are derived from one or more viruses or one or more bacteria. Embodiment 73. The composition of embodiment 72, wherein the one or more target nucleic acids are not derived from a coronavirus. Embodiment 74. The composition of embodiment 73, wherein the one or more target nucleic acids are not derived from SARS-CoV-2 or one or more mutants thereof. Embodiment 75. The composition of any one of embodiments 68-74, wherein the one or more viruses comprises an influenza virus or a human papillomavirus. Embodiment 76 The composition of embodiment 73, wherein the one or more bacteria comprises Salmonella. Embodiment 77. The composition of embodiment 76, wherein the Salmonella comprises Salmonella enterica. Embodiment 78. The composition of embodiment 77, wherein the Salmonella comprises one or more strains or serotypes of Salmonella. Embodiment 79. The composition of embodiment 78, wherein the one or more Salmonella strains or serotypes comprise one or more members selected from the group consisting of Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella prorum. Embodiment 80. The composition of any one of embodiments 56-79, wherein the one or more target nucleic acids are associated with one or more diseases or disorders. Embodiment 81. The composition of embodiment 80, wherein the one or more diseases or conditions include an infectious disease, cancer, or a degenerative disease. Embodiment 82. The composition of any one of embodiments 56 to 81, wherein the one or more target nucleic acids encode a polypeptide or protein. Embodiment 83. The composition of any one of embodiments 56 to 82, wherein the one or more target nucleic acids comprise DNA or RNA. Embodiment 84 The composition of embodiment 83, wherein the DNA is genomic DNA. Embodiment 85. The composition of embodiment 83, wherein the RNA is genomic RNA. Embodiment 86 The composition of embodiment 83, wherein the RNA is double-stranded RNA or single-stranded RNA. Embodiment 87 The composition of embodiment 83, wherein the RNA is double-stranded DNA or single-stranded DNA. Embodiment 88. The composition of any one of embodiments 56 to 87, wherein the one or more target nucleic acids are derived from a human papillomavirus (HPV) or one or more variants thereof. Embodiment 89. The composition of any one of embodiments 56 to 88, wherein the one or more target nucleic acids comprise one or more members selected from HPV L1 capsid protein, HPV L2 capsid protein, HPV E6 protein, HPV E7 protein, and fragments of any thereof. Embodiment 90. The composition of any one of embodiments 56 to 89, wherein the one or more target nucleic acids are from Salmonella. Embodiment 91. The composition of embodiment 90, wherein the Salmonella comprises Salmonella enterica. Embodiment 92 The composition of embodiment 90, wherein the Salmonella comprises one or more strains or serotypes of Salmonella. Embodiment 93. The composition of embodiment 92, wherein the one or more Salmonella strains or serotypes comprise one or more members selected from the group consisting of Salmonella typhimurium, Salmonella enteritidis, Salmonella gallinarum, and Salmonella prorum. Embodiment 94. The composition of any one of embodiments 56 to 93, wherein one or more oligonucleotides comprise a sequence selected from SEQ ID NOs: 1 to 18. Embodiment 95. A kit for identifying the presence of a target nucleic acid, comprising: (i) one or more gold nanoparticles having one or more oligonucleotides incorporated therein; (ii) a condensation solution; and (iii) instructions for using the one or more gold nanoparticles having one or more oligonucleotides incorporated therein. Embodiment 96. A kit for identifying the presence of a target nucleic acid, comprising: (i) the composition of any one of embodiments 56 to 94; (ii) a condensation solution; and (iii) instructions for using the one or more gold nanoparticles having one or more oligonucleotides incorporated therein. [Example]

[0071] Example 1: Production of nanoparticles containing oligonucleotides. Gold nanoparticles containing antisense oligonucleotides (ASOs) were generated by conjugating nucleic acids onto the nanoparticle surface. The binding of nucleic acid sequences to the nanoparticle surface occurs through covalent attachment of ASOs to the stabilizing citrate groups. The conjugation step was performed by ligating oligonucleotides to gold particles. Each ASO molecule was able to form a covalent bond via the thiol group on only a single gold particle.

[0072] First, gold nanoparticles were combined with ASO by adding the nanoparticles and ASO to citric acid and hydrochloric acid. The solution was homogenized by stirring to allow conjugation to occur. The solution was then centrifuged to remove free ASO molecules (e.g., ASO that was not bound to the gold particles). After centrifugation, the supernatant was discarded and the pellet was resuspended in HEPES buffer. The final product should be free of free ASO molecules and all gold should be bound to ASO to increase signal sensitivity.

[0073] The conjugation process involved using 2 mL of a suspension of gold nanoparticles (AuNPs) with a concentration of 1 mM Au atoms and an average size of 20 nm, and adding 36.6 μL of ASO at a concentration of 50 μM, resulting in 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) were added to adjust the pH to 3.0. After this step, the solution was stirred at 350 rpm for 20 minutes and then centrifuged at 14,000 rpm for 15 minutes. The supernatant was discarded, and the pellet was resuspended in 2 mL of HEPES buffer (10 mM).

[0074] Figure 4 shows multiple UV-visible curves for nanoparticles conjugated with ASO. The nanoparticles conjugated with ASO exhibit a large peak in the UV-visible spectrum, whereas the nanoparticles alone without ASO exhibit no UV-visible spectrum. Notably, this can also be observed with the naked eye; in this case, the AuNP-ASO solution is reddish in color, while the solution of unconjugated nanoparticles is generally clear and colorless.

[0075] Figure 3 shows an exemplary schematic of a nanoparticle-ASO that can be fabricated using this method. As shown, a single nanoparticle can have multiple different oligonucleotides attached to it (a "single-nanomultiplex"). Furthermore, multiple oligonucleotides can be attached to different nanoparticles (a "multi-nanomultiplex"), where multiple nanoparticles can be attached to oligonucleotides with the same or different sequences.

[0076] Example 2: Nucleic acid detection. Two samples are analyzed by RT-PCR, using AuNPs as the detection agent. AuNPs containing oligonucleotides are incubated with two different solutions: one containing the target analyte (and detectable via RT-PCR) and the other lacking the target nucleic acid. After incubation of the AuNPs with the sample solution, a color developer solution containing magnesium chloride is added. Furthermore, a standard solution is generated to serve as a baseline for color measurements. Figures 1A-1B show an exemplary schematic of this process. In Figure 1A, Tube 1 shows a sample tube containing a solution of AuNPs (circular) and oligonucleotides (spiral). Tube 2 shows that DNA / RNA from a sample is added to Tube 1. The DNA / RNA is obtained by nucleic acid digestion or other extraction from other DNA / RNA, including viruses or biological material. The DNA / RNA interacts with and potentially anneals to the oligonucleotides. In Tube 3, a color developer solution is added to Tube 2 to induce an observable change in the tube, such as promoting condensation of nucleic acid structures, in response to the presence of the target nucleic acid. Tube schematic 4 in Figure 1B shows the results from tube 3, where the target nucleic acid is absent. The tube becomes transparent / clear, indicating a negative result. Tube schematic 5, on the other hand, shows the results from tube 3, where the target nucleic acid is present. The tube becomes opaque, indicating a positive result. Figures 2A-2B show exemplary structural diagrams of positive versus negative samples. In a negative sample, the developing solution aggregates the particles, turning the solution a translucent grayish color (Figure 2A). On the other hand, if the oligonucleotides are able to bind to the target nucleic acid, the developing solution induces the nucleic acid and nanoparticles to form a particle matrix, which is a cloudy reddish color. Figure 2C shows a series of cuvettes containing solutions; the one on the left shows a negative signal, which is particle aggregation, and the one on the right shows a positive signal, indicated by the reddish color of the solution.

[0077] First, a standard is defined through the identification of the primary colors red, green, and blue, respectively. From this, the CIELAB color space is determined, which is a system for evaluating the elements of luminosity or transparency, tone or hue, and chroma or chromaticity. * ) is the luminance (Y-axis) value that varies approximately from white to black, assuming a value of 0 (zero) for absolute black and a value of 100 for full white. Hues are represented by primary colors, ranging from green to red. * and b, which can vary from blue to yellow. * They are distinguished by the value of L and are expressed in a way similar to the perception of color. * a * b * The color space is created using inverse color theory, where no two colors can be simultaneously green and red, or simultaneously yellow and blue. * indicates brightness, and a * and b * is the color coordinate. * =brightness;a * = red / green coordinates (+a indicates red, -a indicates green);b * = yellow / blue coordinate (+b indicates yellow and -b indicates blue); color difference is defined by a numerical comparison between the sample and the standard. The absolute difference in color coordinate between the sample and the target is known as delta (Δ). L * (ΔL), a * (Δa) and b * The delta of (Δb) can be negative (-) or positive (+). However, the total difference delta E (ΔE) is always positive. These are the * = difference between light and dark (+=light, -=dark); Δa * = difference between red and green (+=more red, -=more green); Δb * = difference between yellow and blue (+=more yellow, -=more blue); ΔE * = total color difference. To calculate the total color difference between the three coordinates, use the following formula: ΔE * =[(ΔL * )2+(Δa * )2+(Δb *)2]1 / 2. When comparing RT-PCR positive and RT-PCR negative samples, each delta coordinate is significantly different, demonstrating that the color analysis can distinguish between positive and negative samples.

[0078] Example 3: Detection of Nucleic Acids in Patient Samples A sample from a patient is collected using a sterile nasopharyngeal swab and immediately placed in a tube containing PBS medium. The sample is homogenized to disaggregate cells from the swab. 500 μL of the sample is transferred from the collection tube to a tube containing the same volume (v / v) of lysis solution; the sample is homogenized and then allowed to stand for 5 minutes to allow the extraction reaction to occur. The entire volume from the microtube is then drawn into a syringe, a 0.22 μm filter is attached to the syringe, and the sample is applied dropwise directly to the biosensor; 40 μL of developer solution is then added. After waiting 1 minute, the test is read. Three potential results are possible: if the target nucleic acid is present, the displayed color will be dark pink / red; if the target reagent is absent, the displayed color will be blue / light gray. If none of the above colors are displayed, or if the color does not match the color on the printed control color chart, the test is considered invalid. This result may be due to insufficient sample or incorrect execution of the procedure; the test may be repeated to obtain a valid result. Negative and positive control swabs can also be obtained, and similar steps can be performed as quality controls. Figure 5 shows an exemplary system for detection used in this example. As shown, the sample is added to the inlet, mixed and homogenized for 5 minutes, then transferred to a heated chamber and passed through a filter. A biosensor solution containing nanoparticles incorporated into oligonucleotides is then added through a port and allowed to mix with the sample. After mixing with the biosensor, a revealing solution is then added to the port and allowed to mix with the sample (and biosensor). This resulting solution is then forced into a new reservoir, where an RGB reading (or reading of another optical parameter) can be taken. The system can use a pump (such as a syringe pump) to move the solution through the system.

[0079] Example 4: Nanoparticle production and HPV detection

[0080] Nanoparticles were produced based on the protocol described in Example 1. Multiple types of nanoparticles were produced, including nanoparticles with different ASOs. The protocol was first performed using ASO number 1 (SEQ ID NO: 1) to produce nanoparticles with ASO number 1. This process was then repeated for ASO numbers 3, 4, and 6 (SEQ ID NOs: 3, 4, and 6, respectively). Four vials contained 2 mL of suspension, each containing one of the ASOs. Equal volumes of the four suspensions, 2 mL each, were mixed to produce a total of 8 mL containing a mixture of particles conjugated with the four ASOs to produce the biosensor solution. The biosensors were stored in a refrigerator at 4-8 °C until use with clinical samples.

[0081] The biosensor solution had a clear, reddish appearance and a maximum UV-visible absorption peak at 530 nm. Furthermore, a control sequence ("PROBE") corresponding to the HPV viral sequence was used to verify the hybridization of the ASO to the complementary sequence. After homogenization, the solution was subjected to agarose gel electrophoresis and tested with the free sample, the ASO, and the PROBE without AuNPs. PCR, used for signal amplification and differentiation of the ASO, was performed at two concentrations to confirm binding. The tested ASO had 20 bases, and the PROBE had 100 bases.

[0082] Figure 6 shows data obtained by detecting the presence of HPV RNA using a biosensor solution. HPV RNA was expressed in the genome of CasKi (squamous cell carcinoma) cells, and the biosensor solution (i.e., a solution containing AuNP-ASO) was applied to the cells. L929 cells (connective mouse tissue) without HPV RNA were also grown. RNA from CasKi and L929 cells was extracted and assayed using the biosensor. A PBS sample was also assayed as a negative control / background signal. The spectral data of the samples was read, parameterized, and normalized using an algorithm to generate spectral scores. As shown in Figure 6, samples containing extracted CasKi RNA ("CasKi-20ng," "CasKi-10ng," and "CasKi-5ng") exhibited significantly higher spectral scores than samples containing extracted L929 RNA ("929-20ng," "929-10ng," and "929-5ng"), which exhibited spectra similar to those of samples containing only PBS ("negative control"). Thus, samples positive for HPV were distinguishable from samples negative for HPV RNA. In addition, assays against nucleic acids different from the target nucleic acid produced results consistent with negative samples (e.g., PBS alone), demonstrating specificity in the recognition of the target sequence.

[0083] Example 5. Detection of nucleic acids using optical density.

[0084] First, bacterial samples were generated by growing cultures at 37°C for ~16 hours (e.g., overnight) in a BOD (Biochemical Oxygen Demand) chamber to an optical density of 0.5. Samples were first diluted between 100-fold and 100,000-fold using 0.1% peptone water, followed by a serial dilution curve. The diluted samples were then incubated at 95°C for 5 minutes in a dry heat bath. After incubation, the samples were placed on ice to stop boiling. The samples were then vortexed for 10 seconds and then placed on ice. The samples were then added to a 96-well plate by pipetting 100 μl of sample. Along with the test samples, negative controls and biosensor-only samples were also generated according to the following table:

[0085] [Table 2]

[0086] The various samples were added to the plate and incubated at room temperature for 5 minutes. A developing solution was created by mixing 0.79 g of MgCl2 in water to create a total volume of 50 mL. 10 μl of developing solution was added to the wells, incubated at room temperature for 2-4 minutes, and then read using an absorbance reading at 520 nm. The optical density at 520 nm was then obtained and can be analyzed using the following formula:

[0087]

number

[0088] After calculations were performed, a cutoff was determined (2 standard deviations below the negative control). Readings above the cutoff were considered negative and readings below the cutoff were considered positive.

[0089] Example 6: Detection of HPV using optical density measurements

[0090] HPV samples were prepared similarly to those described in Example 6. Using three biological replicates, samples of the Caski cell line plus the HeLa cell line harboring HPV-16 and HPV-18 were diluted to form a serial dilution curve. The AuNP-ASO biosensor was then added to the solution and used to detect the presence of HPV at a statistically significant level relative to the negative control of the biosensor alone (i.e., the absence of target nucleic acid). A reference sample was also used to evaluate the colloidal dispersion and determine the uncertainty range. Figure 7A shows the optical density (OD) at 560 nm used to detect the signal. 560 ) and Figure 7B shows the results of an assay for the detection of HPV using CasKi cells, a reference sensor, and a biosensor-only sample. 560 Figure 7C shows the individual values of OD of biological replicates of HeLa cells, reference sensor, and biosensor-only samples. 560 Table 3 shows the analytical performance of this assay.

[0091] Table 3. Analytical performance of the HPV assay

[0092] [Table 3]

[0093] Figures 8A and 8B show the ROC (Receiver Operating Characteristic) curves for dilutions of the CasKi cell line with HPV and the HeLa cell line with HPV, where for the assay using the CasKi cell line (~400 copies of HPV / cell), the AUC (area under the curve) is 1, and for the HeLa cell line (~40 copies of HPV / cell), the AUC is 0.9750.

[0094] Example 7. Detection of Salmonella

[0095] The nanoparticles were produced based on the protocol described in Example 1. Additionally, a reference sample was prepared. The dilutions were mixed with AuNP-ASO, and the optical density of the sample was read. Figure 9 shows the optical densities normalized to the reference samples of 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 10 or more Salmonella samples experience the prozone phenomenon, thus producing distinct and multiple signals. Figure 10 shows a graph of colony forming units per milliliter ("UFC / ml") plotted against the processed optical density parameter. As shown, 10 6 Salmonella samples with dilutions below cfu / ml were below the cutoff (cutoff = 2 times the standard deviation of the negative control), while E. coli samples were above the cutoff. Figure 11 shows the ROC curve analysis with an AUC of 1. Table 4 shows the analytical performance of the assay for Salmonella.

[0096] Table 4. Analytical performance of the Salmonella assay

[0097] [Table 4]

[0098] The method can be performed on a food sample to detect the presence of Salmonella in the food sample. The method can also determine the amount of Salmonella in the food sample.

[0099] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided within the specification. While the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it will be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It will be appreciated that various alternatives to the embodiments of the present invention described herein are available for practicing the present invention. It is therefore contemplated that the present invention shall cover any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

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

2. The method of claim 1 , wherein the optical parameters are determined by color space analysis.

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

4. The method of any one of claims 1 to 3, wherein the optical parameter comprises a luminosity parameter (e.g., color lightness), a saturation parameter (e.g., color intensity), or a hue parameter (e.g., color tint).

5. 5. The method of claim 1, wherein (c) further comprises comparing the optical parameters of the test composition with corresponding optical parameters determined from a corresponding reference composition.

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

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

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

10. 10. The method of any one of claims 1 to 9, wherein the one or more oligonucleotides have a Tm of about 65°C to about 75°C.

11. 11. The method of any one of claims 1 to 10, wherein one nanoparticle of said one or more nanoparticles incorporates 1, 2, 3, 4, 5, or 6 oligonucleotides, optionally each of said oligonucleotides being the same or different.

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

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

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

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

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

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

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

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

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

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

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

25. 25. The method of any one of claims 1 to 24, wherein the presence of said one or more target nucleic acids is associated with one or more diseases or disorders in a subject containing said one or more target nucleic acids.

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

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

28. 28. The method of any one of claims 1 to 27, wherein the one or more oligonucleotides comprise a sequence selected from SEQ ID NOs: 1 to 18, or a sequence at least 90% identical to a sequence selected from SEQ ID NOs: 1 to 18.

29. 1. A composition for detecting one or more target nucleic acids, comprising: a. one or more nanoparticles incorporating one or more oligonucleotides, wherein the one or more oligonucleotides are complementary to the one or more target nucleic acids; and b. 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, the nanoparticle matrix comprising different optical parameters compared to a solution comprising corresponding nanoparticles not bound to the one or more target nucleic acids; A composition comprising:

30. 1. A composition for detecting a target nucleic acid, comprising: a. one or more nanoparticles incorporating one or more oligonucleotides, wherein a first oligonucleotide of the one or more oligonucleotides is complementary to a target nucleic acid at a first sequence of the target nucleic acid and a second oligonucleotide of the one or more oligonucleotides is complementary to the target nucleic acid at a second sequence of the target nucleic acid, and the one or more nanoparticles comprise gold; b. the one or more nanoparticles forming a nanoparticle matrix in the presence of the one or more target nucleic acids; A composition comprising:

31. 31. The composition of any one of claims 29 to 30, wherein one nanoparticle of the one or more nanoparticles incorporates 1, 2, 3, 4, 5, or 6 oligonucleotides.

32. 32. The composition of any one of claims 29-31, wherein the one or more nanoparticles each (e.g., independently) incorporate 1, 2, 3, 4, 5, or 6 oligonucleotides.

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

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

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

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

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

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

39. 37. The composition of claim 36, wherein the one or more target nucleic acids comprise bacterial nucleic acids, and the bacterial nucleic acids comprise Salmonella nucleic acids.

40. 40. The composition of any one of claims 29 to 39, wherein the one or more oligonucleotides comprise a sequence selected from SEQ ID NOs: 1 to 18, or a sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 1 to 18.

41. 1. A kit for identifying the presence of a target nucleic acid, comprising: (i) one or more gold nanoparticles having one or more oligonucleotides incorporated therein; (ii) a condensation solution; and (iii) instructions for using the one or more gold nanoparticles having one or more oligonucleotides incorporated therein.

42. 41. A kit for identifying the presence of a target nucleic acid, comprising: (i) the composition of any one of claims 29-40; (ii) a condensation solution; and (iii) instructions for using one or more gold nanoparticles having one or more oligonucleotides incorporated therein.

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