Target Recycling Amplification Process (TRAP)

Abstract

The present disclosure provides an assay with improved sensitivity for detecting and quantifying nucleic acids in a sample. The assay utilizes a target recycling amplification process (TRAP) for digital detection of nucleic acids in combination with photon resonator absorption microscopy (PRAM), and a toehold-mediated DNA strand displacement reaction. The present disclosure also provides systems and methods for using the assay.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 406,540, filed September 14, 2022, which is incorporated herein by reference.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under Grant No. 1R01EB029805-01 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] Exosomal microRNAs (miRNAs), encapsulated within extracellular vesicles, play diverse roles in biological processes, including intercellular communication, cell proliferation, and inflammatory responses. miRNAs are involved in the post-transcriptional regulation of gene expression, and inappropriate release of miRNAs from exosomes may contribute to the development of cardiovascular disease and cancer. For this reason, exosomal miRNAs have been recognized as important biomarkers for cancer diagnosis and disease progression monitoring. Furthermore, exosomal miRNA concentrations have been found to correlate with therapeutic efficacy. However, exosomal miRNAs can be present at extremely low concentrations, which currently poses a barrier to their use as biomarkers. For exosomes isolated from cells or plasma, even the most abundant target sequences may be present at an average of less than one miRNA per exosome.

[0004] Traditional methods such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) have been the gold standard for quantifying miRNAs with femtomolar detection limits. However, traditional qRT-PCR requires complex enzymatic amplification and complex primer design. Other quantification methods, such as Northern blots and oligonucleotide microarrays, are performed on cell lysates; intracellular miRNA profiling requires the construction of fluorescent reporters to obtain enhanced fluorescence signals. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, current methods have not been adopted for clinical use for detecting exosomal miRNA without enzymatic target amplification.Therefore, there is a need in the art for an ultrasensitive and highly selective diagnostic approach that does not involve enzymatic amplification to effectively detect and quantify exosomal miRNA.The present disclosure addresses the limitations of low-level miRNA detection.The systems, methods, and assays disclosed herein can be used to determine health status; early disease and pathology diagnosis; identify biomarkers; evaluate treatment efficacy; and monitor disease progression over time. [Means for solving the problem]

[0006] In one aspect, an exemplary embodiment provides a system for detecting nucleic acids in a sample, the system comprising: a biosensor including a photonic crystal (PC), wherein the PC is immobilized to a nucleic acid capture strand sequence; a nucleic acid linker strand that is annealed to a nucleic acid protector strand to form a linker-protector complex; a reaction solution; a probe strand; gold nanoparticles (AuNPs); a sample; and an imaging platform, wherein the capture strand is pre-treated with the linker-protector complex, whereby the linker-protector complex binds to the capture strand and forms a first toehold; target RNA in the sample is capable of binding to the first toehold, thereby displacing the protector strand from the linker-protector complex; an AuNP probe binds to the linker strand at a second toehold region, thereby displacing the target RNA; and the imaging platform is configured to quantify the displaced target RNA in the sample by measuring the bound AuNPs.

[0007] In a further aspect, an exemplary embodiment provides a biological assay comprising: a biosensor comprising a capture strand oligonucleotide sequence and a linker strand-protector strand oligonucleotide conjugate; a reaction solution; an oligonucleotide strand; and a population of nanoparticles; the nanoparticles are attached to the surface of the biosensor using the oligonucleotide strand, and the nucleotide strand is composed of a random nucleic acid sequence.

[0008] In another aspect, an exemplary embodiment provides a method for detecting nucleic acids in a sample, the method comprising: immobilizing an oligonucleotide capture strand on a surface of a biosensor, thereby creating an assay surface; pre-treating the capture strand with an oligonucleotide linker strand-oligonucleotide protector strand complex; adding an assay medium to the assay surface, the assay medium including a biological sample that may contain a target RNA, the target RNA being capable of binding to a first free toe-hold region on the linker strand, thereby displacing the protector strand; adding bound nanoparticle probes capable of binding to a second toe-hold region, thereby creating and releasing the target RNA; and quantifying the number of nanoparticles bound to the second toe-hold region using an imaging platform. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows a schematic of the miRNA detection workflow. Figure 1a shows extracted exosomal miRNA placed in a PDMS reservoir and applied to a PC surface along with AuNP-linked probes. Figure 1b is a schematic showing a DNA linker pre-annealed with a partially complementary protector and hybridized to a DNA capture. Figure 1c shows the target recycling amplification process (TRAP) for digital resolution detection of microRNAs on a PC biosensor surface, in which the target miRNA displaces the protector strand on the PC-immobilized capture molecule, revealing the linker sequence through a strand displacement reaction. [Figure 2] Figure 2 shows the kinetic identification of miRNA-375 using TRAP. Figure 2a shows dose-response TRAP images at single particle resolution at 10 and 20 minutes. Figure 2b shows quantification of particle number as a function of miRNA-375 concentration in triplicate experiments, along with standard errors. The blank represents a reaction in which no miRNA target is present. [Figure 3]Figure 3 shows the selectivity of TRAP in detecting single-nucleotide variants (SNVs) of miRNA-375. Figure 3a shows a TRAP image. Figure 3b shows the number of nanoparticles bound to the PC surface in the presence of five different SNVs at positions 1, 5, 12, 18, and 22 from the 5' end of miRNA-375 or miRNA. A selectivity of approximately 6000:1 is demonstrated for detecting the target sequence relative to single-base mismatched targets at each position investigated. [Figure 4] Figure 4 shows multiplexed miRNA detection performed in different reservoirs on a single PC biosensor. Figure 4a shows the use of a 6-well PDMS gasket applied to a single PC for simultaneous measurement of three negative controls and three separate miRNA targets. Figure 4b shows the array of target miRNAs. Figure 4c shows a PRAM image, and Figure 4d shows particle counts on the PC surface in the presence and absence of 1 fM miRNA targets. [Figure 5] Figure 5 shows a comparison of detection limits. Figure 5a shows a comparison of detection limits for exosomal miRNA-375 and miRNA-21 using TRAP and qRT-PCR. Figure 5b shows the relative expression levels of exosomal miRNA-375 and miRNA-21 from MCF-7 cells and DU145 cells analyzed by TRAP and qRT-PCR. The expression level of miRNA-375 in MCF-7 cells was set to 1. The relative expression levels were compared to miRNA-375 in MCF-7 cells. [Figure 6] Figure 6 shows TEM imaging. Figure 6a shows TEM imaging of NanoUrchin AuNPs. Figure 6b shows the conjugation product of NanoUrchin AuNPs with probe DNA and m-PEG1K. [Figure 7] Figure 7 shows the near-field intensity. Figure 7a shows the near-field profile of a PC-AuNP hybrid under resonant conditions (λ = 620 nm at normal incidence). Figure 7b shows a representative PC resonant reflection spectrum. Figure 7c shows an enlarged view of the reflection spectra of the PC surface with and without AuNPs attached. The reflection resonant intensity was obtained under a 20 Å lens. [Figure 8] FIG. 8 shows native PAGE imaging of linker strands of various lengths tested in the reactions. [Figure 9] Figure 9 shows the optimization of linker chain length. The TRAP image panel shows the number of particles captured for linker chains of various lengths over a 30-minute reaction time. [Figure 10] Figure 10 shows the optimization of linker chain concentration imaged at 90 minutes. The TRAP image panel shows particle counts for various linker chain concentrations. [Figure 11] Figure 11 shows the digital resolution. Figure 11a is a TRAP image panel showing the digital resolution of captured AuNPs as a function of target concentration (rows) at 10 and 20 minutes. Figure 11b is a linear calibration curve for the miR-375 detection assay in buffer. Figure 11c shows the linear regression used to plot the dose-response, where x is the concentration of the target sequence and y is the number of AuNPs. The dashed horizontal line indicates the threshold (blank signal + 3 standard deviations). Error bars represent the standard deviation of three independent assays. [Figure 12] Figure 12 shows TRAP images. Figure 12a shows a TRAP image panel showing the digital resolution of captured AuNPs as a function of target concentration (rows) at 10 and 20 minutes. Figure 12b shows the quantification of particle number as a function of label concentration at 10 and 20 minutes. The dashed horizontal line indicates the threshold (blank signal + 3 standard deviations). Error bars represent the standard deviation of three independent assays. [Figure 13] Figure 13 shows a 12% PAGE. In each panel: Lane 1: capture + linker + probe; Lane 2: capture + linker + miRNA; Lane 3: capture + linker-protector duplex + probe; Lane 4: capture + linker-protector duplex + probe + miRNA. [Figure 14]Figure 14 shows linear calibration curves. Figure 14a shows linear calibration curves for miR-375 and miR-21 detection assays in buffer using qRTPCR. Figure 14b shows the linear regression model used to describe the dose response, where x is the concentration of the target sequence and y is Cq. Figure 14c shows the linear regression of the miR-21 response example. [Figure 15] Figure 15 shows the kinetics of miR-375 concentration in MCF-7 cell exosomes using TRAP. Figure 15a shows a dose-response plot for the detection of miRNA-375 by TRAP, showing the detection results after 10 and 20 minutes in a room temperature assay protocol. Figure 15b shows the quantification of particle number as a function of miR-375 concentration in MCF-7 cell exosomes in triplicate. Blank represents a non-target control. [Figure 16] Figure 16 shows the kinetics of miR-375 concentration in DU145 cell exosomes using TRAP. Figure 16a shows a dose-response plot for the detection of miRNA-375 by TRAP, showing the detection results after 10 and 20 minutes in a room temperature assay protocol. Figure 16b shows the quantification of particle number as a function of miR-375 concentration in DU145 cell exosomes in triplicate. [Figure 17] Figure 17 shows the kinetics of miR-21 concentration in MCF-7 cell exosomes using TRAP. Figure 17a shows a dose-response plot for the detection of miRNA-21 by TRAP, showing detection results after 10 and 20 minutes in a room temperature assay protocol. Figure 17b shows quantification of particle number as a function of miR-375 concentration in MCF-7 cell exosomes in triplicate. DETAILED DESCRIPTION OF THE INVENTION

[0010] It is to be understood that the specific aspects of the present invention described herein are not limited to the specific embodiments presented and may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting unless expressly defined herein. Furthermore, specific embodiments disclosed herein can be combined with other embodiments disclosed herein without limitation, as will be recognized by one of ordinary skill in the art.

[0011] Throughout this specification, unless the context specifically indicates otherwise, the words "comprise", "include", and variations thereof (e.g., "comprises", "comprising", "includes" and "including") will be understood to indicate the inclusion of a stated component, function, element, or step, or group of components, functions, elements, or steps, but not the exclusion of other components, functions, elements, or steps, or group of components, functions, elements, or steps. Any of the terms "comprising", "consisting essentially of", and "consisting of" may be substituted for either of the other two terms while retaining their ordinary meaning.

[0012] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0013] Unless otherwise indicated or apparent from the context and the understanding of one of ordinary skill in the art, numerical values ​​expressed herein as ranges can, in different embodiments of this disclosure, assume subranges down to one-tenth of the unit of the lower limit of the range, unless any particular numerical value within the stated range or the context clearly dictates otherwise.

[0014] As used in this specification and drawings, ranges and amounts can be expressed as "about" a particular numerical value or range. "About" also includes the exact amount. For example, "about 5%" means "about 5%" and also "5%." The term "about" can also refer to ±10% of a given numerical value or range of numerical values. Thus, about 5% means, for example, 4.5% to 5.5%.

[0015] As used herein, "sample" refers to any type of sample containing a nucleic acid sequence, including a biological sample. "Biological sample" refers to a sample of bodily tissue, such as, but not limited to, an organ punch or tissue biopsy, or bodily fluid, such as, but not limited to, blood, cerebrospinal fluid, plasma, or saliva, from a warm-blooded animal, such as a mammal, preferably a human, that is suffering from or may be suffering from one or more of the diseases and / or disorders described herein. Biological samples can also refer to tissue or blood samples obtained from non-human mammals and other animals. In alternative embodiments, the sample is obtained from a non-mammalian host and may contain the target nucleotide sequence.

[0016] In accordance with the present disclosure, the methods and compositions described herein can be adapted by one of skill in the art to meet desired needs.

[0017] 1. Overview The present disclosure provides an assay with improved sensitivity for detecting and quantifying nucleic acids in a sample. The disclosure also provides systems and methods for using the assay. The assay utilizes a target recycling amplification process (TRAP) for digital detection of nucleic acids in conjunction with photon resonator absorption microscopy (PRAM), utilizing a toehold-mediated DNA strand displacement reaction. The disclosed assays, systems, and methods improve the sensitivity of detecting and quantifying nucleic acids in biological samples and can be used in a variety of contexts, including health status, early disease and pathology diagnosis, biomarker identification, evaluation of treatment efficacy, and longitudinal monitoring of disease progression. PRAM devices are described in U.S. Patent Application No. 16 / 170,111, and various aspects of photonic crystal (PC) biosensors are described in U.S. Patent Nos. 7,479,404, 7,521,769, 7,531,786, 7,737,392, 7,742,662, and 7,968,836, all of which are incorporated herein by reference.

[0018] Exosomal microRNAs (miRNAs), encapsulated within extracellular vesicles, play diverse roles in biological processes, including intercellular communication, cell proliferation, and inflammatory responses. miRNAs are involved in the post-transcriptional regulation of gene expression. Consequently, inappropriate release of miRNAs from exosomes can lead to the development of disease and cancer. For this reason, exosomal miRNAs have been recognized as important biomarkers for diagnosing cancer and disease onset and monitoring disease progression. Furthermore, exosomal miRNA concentrations have been found to correlate with therapeutic efficacy. Changes in nucleic acids, including RNA, DNA, and PNA, can indicate disease onset, disease progression, or therapeutic efficacy. However, nucleic acids, including exosomal miRNAs, can be present at extremely low concentrations, presenting a current barrier to the use of exosomal miRNAs or other nucleic acids as biomarkers. In exosomes isolated from cells or plasma, even the most abundant target sequences may be present at an average of less than one miRNA per exosome. Therefore, conventional methods such as quantitative reverse transcription-polymerase chain reaction (qRT-PCR), which is the gold standard for quantifying nucleic acids such as miRNAs, are considered the gold standard for quantifying miRNAs with femtomolar detection limits. However, conventional qRT-PCR requires complex enzymatic amplification and complex primer design, which negatively impacts the detection limit. Alternative quantification methods, such as Northern blots and oligonucleotide microarrays, are performed on cell lysates and often involve the construction of fluorescent reporters to enhance the fluorescent signal for intracellular miRNA profiling. Due to their limited detection limits, none of these methods have been adopted for clinical use for exosomal miRNA detection without enzymatic target amplification. Consequently, there is a need for the development of ultrasensitive and highly selective diagnostic approaches for effectively detecting and quantifying exosomal miRNAs and other nucleic acids without enzymatic amplification.

[0019] 2. Target Recycling Amplification Process (TRAP) As used herein, the term "target recycling amplification process (TRAP)" refers to a method that enables amplification of nucleic acid signals to enhance the sensitivity of biosensors, biomarker detection assays, and other types of nucleic acid detection. In the initial step of this disclosure, miRNA is extracted from exosomes isolated from cell culture media using various protocols commonly known in the art (Figure 1a). Next, a photonic crystal (PC) surface is prepared with capture DNA or RNA immobilized by any DNA or RNA transfer process. Examples of RNA and DNA transfer processes include, but are not limited to, covalent linkage, streptavidin-biotin, PEG linkers, and self-assembled monolayer linkers. Photonic crystals have been previously described in U.S. Patent Nos. 7,479,404, 7,521,769, 7,531,786, 7,737,392, 7,742,662, and 7,968,836, all of which are incorporated herein by reference.

[0020] After the capture DNA is immobilized, the PC is pretreated with a linker-protector complex consisting of a protector DNA and a linker DNA. The linker-protector complex has free, unhybridized regions at both the 5' and 3' ends of the linker strand, forming Toehold-1 and Toehold-2 for miRNA or other nucleic acid binding and probe DNA binding, respectively.

[0021] As used herein, "protector DNA or protector" refers to a nucleic acid or nucleic acid sequence that does not function in the system but prevents unintended DNA from reacting. Those skilled in the art will easily understand the role of "protector DNA".

[0022] As used herein, "linker DNA" or "substrate" or "bridge" refers to a DNA sequence used to link a captured label to a detection substrate. In some embodiments, the linker functions through hybridization of both the capture DNA and the protector DNA.

[0023] As used herein, "toehold-mediated strand displacement reaction (SDR) or toehold-mediated branch migration reaction or entropy-driven toehold SDR" refers to an enzyme-free molecular method in which one strand of nucleic acid (output) is exchanged with another nucleic acid strand (input). More specifically, this term refers to any system in which a double-stranded nucleic acid initially has a short toehold sequence of 4-8 nucleotides. In one embodiment, this sequence is single-stranded. The invading strand binds to the toehold, and a branch migration reaction occurs between the three strands, allowing the invading strand to displace the protector strand. Systems in which the toehold is hidden until exposed by another reaction, such as the TRAP method, are referred to as "towhold sequestration" and may be part of the present disclosure. In an exemplary embodiment, the toehold reaction occurs by adding a reaction solution commonly known in the art to a polydimethylsiloxane (PDMS) reservoir attached to a PC surface. The reservoir may also be constructed from a wide variety of materials, including, but not limited to, plastics (e.g., acrylic, polycarbonate, polyester), glass, etc.

[0024] In a preferred embodiment of the present disclosure, an exosome extract sample is added to a reaction well, whereby miRNA or target nucleic acid binds to the free toehold-1 region on the linker strand, displacing the protector strand through a DNA strand displacement reaction. This displacement allows a probe DNA conjugated to gold nanoparticles (AuNPs) to invade toehold-2 on the linker strand, releasing the target miRNA through a second DNA strand displacement reaction and exposing an additional DNA linker sequence (toehold-2). The probe-modified AuNPs hybridize with the DNA linker sequence, and the bound AuNPs are imaged by the reduction in reflected light intensity from the PC surface at the location of AuNP binding (see Figures 1A-1C and 6). The present disclosure has the improved technical effect of being able to count surface-bound AuNPs using an automated image processing algorithm that uses a MATLAB script to identify image pixels with reduced reflected light intensity compared to the background. A further invention is that the released target nucleic acid or miRNA can participate in an additional TRAP reaction, resulting in increased nanoparticle binding and signal amplification. In an alternative embodiment, the TRAP method is multiplexable and requires less than 20 μL of sample, making it suitable for frequent patient monitoring. The assay has been applied to the detection of biological exosomal miRNA, and the results have been validated by qRT-PCR.

[0025] II. Nucleic Acid Detection Systems and Assays The system of the present disclosure includes a biosensor including a photonic crystal (PC), where the PC is immobilized to a nucleic acid capture strand sequence; a nucleic acid linker strand that is annealed to a nucleic acid protector strand to form a linker-protector complex, a reaction solution, a probe strand, a gold nanoparticle (AuNP); a sample; and an imaging platform.

[0026] As disclosed herein, the capture strand is pretreated with a linker-protector complex, which then binds to the capture strand to form a first toehold. In a preferred embodiment of the present disclosure, the linker-protector sequence is added in excess to the reaction solution.

[0027] A target nucleic acid in a sample can bind to the first toehold, thereby displacing the protector strand from the linker-protector complex. A gold nanoparticle probe (AuNP) binds to the linker strand at the second toehold region, thereby displacing the target nucleic acid, and the imaging platform is configured to quantify the displaced target nucleic acid in the sample by measuring the bound AuNP. The systems disclosed herein can detect the presence of nucleotide sequences that are biomarkers for health conditions, disease, the presence of viral pathogens, or the presence of bacterial pathogens. In preferred embodiments of the present disclosure, the nucleic acid is miRNA. Alternatively, the nucleic acid can comprise one or more alternative RNAs, including, but not limited to, miRNA, tRNA, rRNA, snRNA, long non-coding RNA (lncRNA), circular RNA (circRNA), short interfering RNA (siRNA), or messenger RNA (mRNA). In a preferred embodiment, the RNA is microRNA (miRNA). In alternative embodiments, the nucleic acid may be DNA, LNA, or PNA.

[0028] As used herein, "sample" refers to any type of sample containing a nucleotide sequence, including biological samples. A "biological sample" refers to a sample of bodily tissue, such as, but not limited to, an organ punch or tissue biopsy, or a bodily fluid, such as, but not limited to, blood, cerebrospinal fluid, plasma, or saliva, from a warm-blooded animal, such as a mammal, preferably a human, that is suffering from or may be suffering from one or more of the diseases and / or disorders described herein. A biological sample may also refer to tissue or blood samples obtained from non-human mammals and other animals. Given the present disclosure, the methods and compositions described herein can be adapted by those skilled in the art to meet desired needs.

[0029] In a preferred embodiment of the system, binding of the linker-protector complex to the capture strand forms a first toe-hold region on the linker strand. Binding of the target RNA to the first toe-hold region on the linker strand displaces the protector strand in a first displacement reaction. The target RNA then binds to the first toe-hold region, thereby displacing the protector strand and exposing a second toe-hold region. A nanoparticle probe binds to the linker at the second toe-hold region, thereby displacing the target RNA in a second displacement reaction, and the bound nanoparticle probe is imaged using photon resonator absorption microscopy (PRAM).

[0030] In a preferred embodiment of the present disclosure, the nanoparticle probe is a gold nanoparticle. Alternative embodiments include nanoparticles, quantum dots, metal-based nanoparticles, magnetic nanoparticles, fluorescent materials, nanodiamonds, plasmonic fluorophores, or nanoparticles containing dielectric materials such as SiO2 or TiO2. In a preferred embodiment, the linker chain attachment to the AuNP is reversible, allowing RNA to bind to additional AuNPs, resulting in target recycling and signal amplification. Bound nanoparticles, such as AuNPs, at a predetermined location on a photonic crystal (PC) reduce the intensity of reflected light from the PC surface at that location. The nanoparticles of the present disclosure are attached to the surface of a biosensor using nucleotide tethers containing nonspecific nucleotide sequences. The nucleotide tethers can be uniform or nonuniform in sequence and nonspecific in length. In some embodiments, the length of the nucleotide tether is about 5-200 nucleotides. In some embodiments, the length of the tether is about 5-50, about 51-100, about 101-150, or about 151-200 nucleotides. In yet other embodiments, the nucleotide tether is about 5-25, about 26-50, about 51-75, about 76-100, about 101-125, about 126-150, about 151-175, or about 176-200 nucleotides in length. The systems described herein have detection limits in the attomole range, down to single RNA strands.

[0031] In a preferred embodiment of the present disclosure, the biosensor is a photonic crystal. In a preferred embodiment, the photonic crystal is immobilized with a nucleic acid capture strand using a saltation process and amine-terminated capture DNA. In an alternative embodiment, the biosensor may be a whispering gallery mode biosensor, which is a ring resonator, a microtoroid, or a microsphere. In a further embodiment, the biosensor is a waveguide structure in which light propagates laterally, an acoustic biosensor, or a photoacoustic biosensor.

[0032] As disclosed herein, in a preferred embodiment, the imaging platform of the present disclosure includes a light source configured to excite a resonance of the PC and a detector configured to detect light reflected from the photonic crystal. The imaging platform is configured to quantify measured resonance peak intensity values ​​pixel by pixel across the photonic crystal. Alternatively, the imaging platform may be surface plasmon resonance imaging. In other embodiments, the imaging platform is a dark-field microscope. In yet other embodiments, the imaging platform is an interferometric intensity imaging platform. In alternative embodiments, the imaging platform includes a non-imaging detection device.

[0033] The present disclosure also provides an assay comprising a biosensor comprising a capture strand oligonucleotide sequence and a linker strand-protector strand oligonucleotide complex; a reaction solution; an oligonucleotide strand; and a population of nanoparticles, wherein the nanoparticles are bound to the surface of the biosensor using the oligonucleotide strand, and the nucleotide strand is composed of a random nucleic acid sequence. A sample or biological sample is added to the assay, and RNA in the sample can bind to the free toe-hold region of the linker-protector complex. The biological assay of the present disclosure comprises a population of nanoparticles bound to the oligonucleotide sequence, and the population of nanoparticles binds to the toe-hold region.

[0034] The assays disclosed herein can be used to detect the presence of nucleic acids, preferably RNA. In preferred embodiments, the RNA is microRNA (miRNA), which is indicative of health status, early disease and pathology diagnosis, biomarker identification, evaluation of treatment efficacy, and longitudinal monitoring of disease progression. In alternative embodiments, the RNA is microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), long non-coding RNA (lncRNA), circular RNA (circRNA), short interfering RNA (siRNA), or messenger RNA (mRNA). In preferred embodiments, the nanoparticles are gold nanoparticles, but may alternatively be quantum dots, metal-based nanoparticles, magnetic nanoparticles, fluorescent materials, nanodiamonds, plasmonic fluorophores, or nanoparticles comprising dielectric materials such as SiO2 or TiO2. In preferred embodiments, the biosensor of the assay includes a photonic crystal and further includes an imaging platform configured to quantify the resonant peak intensity values ​​measured pixel-by-pixel across the photonic crystal. However, the biosensor can also include non-imaging detection devices, whispering gallery mode biosensors that are ring resonators, microtoroids, or microspheres. Alternatively, the biosensor can include a waveguide structure in which light propagates laterally. In yet other embodiments, the biosensor is an acoustic biosensor, and the biosensor is an optoacoustic biosensor. The assays of the present disclosure are advantageous in that they are stable at room temperature.

[0035] The present disclosure also provides a method for detecting nucleic acid sequences of interest in a sample, such as nucleic acid sequences related to health status, early disease and disease diagnosis, biomarker identification, evaluation of treatment efficacy, and longitudinal monitoring of disease progression. As used herein, "disease" refers to pathophysiology and its progression. Examples of disease states include any disease or dysfunction of the skin, skeletal, muscular, nervous, endocrine, cardiovascular, lymphatic, respiratory, digestive, urinary, and reproductive systems. More specifically, pathophysiology. More specifically, disease states include cardiovascular disease; central nervous system diseases, including but not limited to Alzheimer's disease, Parkinson's disease, and stroke; muscular dystrophy, lupus, cancer, pulmonary pathophysiology, and tumors (malignant and benign).

[0036] The method disclosed herein is for detecting nucleic acids in a sample and includes the steps of immobilizing an oligonucleotide capture strand on a biosensor surface, thereby creating an assay surface; pretreating the capture strand with an oligonucleotide linker strand-oligonucleotide protector strand complex; adding an assay medium to the assay surface, the assay medium containing a biological sample that may contain a target RNA, where the target RNA is capable of binding to a first free toe-hold region on the linker strand, thereby displacing the protector strand; adding a nanoparticle probe bound to a second toe-hold region, thereby releasing the target RNA; and quantifying the number of nanoparticles bound to the second toe-hold region using an imaging platform. This method can be used to detect nucleotides, including RNAs such as transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), long non-coding RNA (lncRNA), circular RNA (circRNA), short interfering RNA (siRNA), or messenger RNA (mRNA). In a preferred embodiment, the RNA is microRNA (miRNA). In alternative embodiments, the nucleic acid is DNA or PNA.

[0037] In a preferred method, as disclosed herein, the quantitative difference in reflected light intensity from the biosensor surface at each nanoparticle location represents the RNA copy number. This method allows for quantitative limits in the attomole range, or even single nucleotide sequence copies. In a preferred embodiment, the biosensor includes a photonic crystal, and the imaging platform includes a light source configured to excite the resonance of the photonic crystal and a detector configured to detect light reflected from the photonic crystal. In a preferred embodiment, the imaging platform is configured to quantify the measured resonant peak intensity values ​​for each pixel across the photonic crystal.

[0038] In a preferred embodiment of the method, the nanoparticles are gold nanoparticles, but may alternatively be quantum dots, metal-based nanoparticles, magnetic nanoparticles, fluorescent materials, nanodiamonds, plasmonic phosphors, or nanoparticles comprising dielectric materials such as SiO2 or TiO2. In a preferred embodiment, the biosensor of the assay includes a photonic crystal and further includes an imaging platform configured to quantify the resonant peak intensity values ​​measured pixel-by-pixel across the photonic crystal. However, the biosensor may also be a non-imaging detection device, such as a whispering gallery mode biosensor, a ring resonator, a microtoroid, or a microsphere. Alternatively, the biosensor may include a waveguide structure in which light propagates laterally. In yet other embodiments, the biosensor is an acoustic biosensor, and the biosensor is a photoacoustic biosensor. In a preferred embodiment of the method, after quantifying the bound nanoparticles, the nanoparticles are removed from the biosensor surface by exchanging the assay buffer, by agitation without exchanging the assay buffer, or by applying a magnetic field.

[0039] Those skilled in the art will understand that the systems, assays, and methods disclosed herein may also include sample media containing components necessary for performing the assays and methods disclosed herein, as well as for collecting, storing, or preserving collected and / or biological samples.

[0040] The TRAP system, assay, and method disclosed herein offer numerous technical advantages over conventional techniques. The TRAP system can achieve single-copy detection down to attomole nucleic acid concentrations. The present disclosure achieves this by minimizing nonspecific AuNP binding, enhancing the target trigger signal. More specifically, in preferred embodiments of the present disclosure, toehold-1 participates in the target miRNA-triggered strand displacement reaction, while toehold-2 enables the entry of the probe-annealed AuNP. Furthermore, advantageously, the lengths of toehold-1 and the linker are optimized, providing technical advantages over conventional techniques. In preferred embodiments of the present disclosure, toehold-1 is designed with five bases. In alternative embodiments, toehold-a bases can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases. Toehold-2 can contain any number of bases; increasing the number of bases achieves faster kinetics and recycling of the target miRNA. However, for a given length of protector strand, a longer toehold-2 introduces more exposed bases at the end of the linker strand, potentially leading to nonspecific binding in the absence of target miRNA. The present disclosure overcomes this limitation by using a linker strand containing two or three exposed bases in the toehold-2 region, generating distinct capture-linker-probe (CLP) complexes in the presence of target strand but not in its absence. In a preferred embodiment of the present disclosure, a linker strand containing only two initial exposed bases was utilized, as this demonstrated the unexpected technical benefit of a 120-fold enhanced signal-to-noise ratio compared to three free bases (see Figure 8). However, linkers with 0 to 50 exposed bases may also be used. In some embodiments, linkers of 0, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 35, about 40, about 45, or about 50 exposed bases in length can be used.Furthermore, preferred embodiments of the present disclosure reveal that linker chain concentrations of 1 pM to 20 pM maximize the capture of AuNPs, whereas when the linker concentration was 50 pM, the captured AuNPs on the PC surface decreased.

[0041] Furthermore, the disclosed systems, assays, and methods have the technical advantage of being stable at room temperature, overcoming the significant limitations of current assays and systems that require cryogenic storage.Furthermore, the PRAM systems, assays, and methods provided herein have the surprising additional technical advantage of providing results in less than 20 minutes, enabling rapid assessment of health status, early diagnosis of disease and pathology, identification of biomarkers, evaluation of treatment efficacy, and longitudinal monitoring of disease progression.As such, the systems, assays, and methods described herein can be used in clinical settings.

[0042] III. Photon Resonator Absorption Microscopy (PRAM) Operation Principle The PRAM biosensing platform is shown in Figure 19. In addition to the alternative tags disclosed herein, PRAM can visualize individual gold nanoparticle (AuNP) tags on a photonic crystal (PC) surface through resonant coupling. The PRAM's detection principle utilizes resonant PC reflection at wavelength λ = 625 nm, providing a webcam-type image sensor with high reflection intensity from collimated low-intensity LED illumination of the same wavelength. More specifically, port 1 is coupled to a fiber-coupled 617 nm LED light source (M617F2, Thorlabs), and a lens group (F810SMA-635, Thorlabs) is used to first collimate the output beam. A zero-order half-wave plate (WPH10M-633, Thorlabs) rotates the polarization of the collimated light beam, exciting the TM resonant mode of the PC cavity. A plano-convex lens (LA1509-A-ML, Thorlabs) then focuses the beam onto the back focal plane of an Olympus 20x / 0.5 numerical aperture (NA) objective, from which a collimated beam is perpendicularly incident on the PC surface. A manual three-axis stage (PT3, Thorlabs) is used to fix the PC sample at the focal plane of the objective. The reflected light from the PC resonator is collected by the same objective and redirected by a 50 / 50 non-polarizing beam splitter (CCM1-BS013, Thorlabs). A doublet (AC254-200-A-ML, Thorlabs) projects the image plane onto a charge-coupled device (CCD) camera (GS3-U3-51S5M-C, Point Grey) with a resolution of 177 nm / pixel. As shown in Figure 19C, perfect interference occurs at a specific resonant wavelength and angle of incidence, resulting in no light transmission and nearly 100% reflection efficiency. By adding absorbing AuNPs to the PC surface, the magnitude of the resonant reflectance is dramatically reduced (Figure 19C), and as a result, each AuNP can be observed by illuminating it with light from an LED and creating an image of the reflected intensity (Figure 19B).

[0043] By measuring the peak intensity values ​​(PIV) for each pixel across the entire PC using a microscope, the output of PRAM is a PIV image of the attached AuNPs. Images can be collected by illuminating the structure with collimated broadband light through a transparent substrate and immersing the front surface of the PC in an aqueous medium. The AuNPs are strategically selected to provide strong absorption at the same wavelength due to localized surface plasmon resonance. Therefore, each AuNP bound to the surface is recorded as a position of reduced intensity in the reflected image of the PC compared to surrounding areas without AuNPs. By immobilizing target-activated AuNP probes on the PC surface, PRAM has been used to quantify nucleic acids and proteins with single-particle resolution. Previous studies have focused on detecting chemically synthesized miRNAs with detection limits similar to those of qRT-PCR, where each detected miRNA molecule is associated with a single AuNP tag. In this disclosure, we add the technical effect of providing an amplification mechanism to further reduce the detection limit because the target nucleic acid molecule is consumed in the detection process. This is achieved by using a DNA fuel molecular machine, a cascade DNA circuit driven by a fuel chain (e.g., in TRAP, miRNA target and probe sequences act as fuel). The DNA fuel molecular machine involves a series of toehold-mediated DNA strand displacement reactions with target recycling as a tool for building switchable nanodevices, controlled nanoparticle assembly, mediated gene expression, and programmed DNA computation. Consequently, combining target recycling with PRAM detection by the DNA fuel molecular machine achieves the technical advantage of reducing the miRNA detection limit to below 400-fold attomolar concentrations and shortening the assay time to 20 minutes. Furthermore, combining PRAM with TRAP offers the additional technical advantage of a single-step, room-temperature, single-vessel reaction requiring only synthetic nucleic acids, resulting in a lower overall cost per test sample. [Example]

[0044] The following examples illustrate specific embodiments of the present invention and various applications thereof, and are presented for illustrative purposes only and should not be construed as limiting the scope of the disclosure in any way.

[0045] material and method photonic crystal As used herein, photonic crystals (PCs) contain periodic diffractive structures with a low refractive index coated with a high refractive index material (TiO). PCs are fabricated on 8-inch diameter glass wafers on which a 10 nm etch-stop layer of AlO is deposited. The periodic diffractive pattern is constructed by depositing a SiO layer followed by large-area ultraviolet interference lithography. Finally, a thin layer of TiO, approximately 100 nm thick, is deposited on the etched wafer. The resulting PCs have a surface area of ​​1 Å to 1.2 cm. 2 The PC is designed to function as a narrowband optical cavity, optimally reflecting λ=625 nm with nearly 100% efficiency when immersed in water.

[0046] DNA sequence design To reduce secondary structure and interactions, random sequences consisting only of A, T, G, and C were designed using the NUPACK software. The domain sequences were validated in NUPACK to ensure the selected domain sequences had minimal secondary structure and crosstalk. For the alkanethiol-modified sequences, a 10-mer polyA spacer, known for its low interaction with gold interfaces, was selected to improve sample stability and coverage. The toehold probe was designed to tolerate changes in temperature, concentration, and salt concentration by considering ΔΔG° = ΔGG°(SSSS) - ΔGG°(XXXX) = 0, where SC is the false target-linker complex; XC is the correct target. Branch migration reactions in the toehold strand displacement reaction further ensure selectivity, as a single base mismatch increases ΔGG° by +1.83 to +5.9 kcal / mol. The ΔGG° values ​​were also validated in NUPACK.

[0047] nucleic acid The oligonucleotides used in the TRAP capture system were purchased and designed with standard purification. The oligonucleotide concentrations were calculated based on the molar extinction coefficient of single-stranded DNA. The same PC capture DNA sequence was used for all five DNA sequences. The probe sequence was terminated with a 5' dithiol group. The capture sequence was terminated with a 3' amine group. The sequences of the oligonucleotides used are shown in Table 1.

[0048] [Table 1-1] [Table 1-2]

[0049] The above sequences are exemplary, and one of skill in the art will readily recognize that the miRNA sequence, including the target, linker, protector, and probe sequences, can target any nucleotide of interest and is not limited to the sequences disclosed herein.

[0050] Preparation of linker-protector (LP) duplexes The protector oligonucleotide strand was annealed to the linker sequence at a 1:2 stoichiometry, ensuring an excess of protector. The annealing process involved heating the oligos to 95°C for 10 minutes and then cooling to room temperature. The annealed linker-protector was diluted with 1x TE, 12.5 mM MgCl2, and 0.025% Tween 20 buffer and stored at 4°C until use.

[0051] Polyacrylamide gel electrophoresis To confirm the formation of TRAP nucleic acid complexes and products, a 12% polyacrylamide gel was used. Reaction products were loaded onto a 1.5 mm thick gel. Electrophoresis was performed in 1 Å TBE buffer at 165 V for 40 minutes at room temperature. After separation, the gel was stained with Gel Red and imaged using a Bio-red fluorescent gel imaging system.

[0052] Nanoparticle binding NanoUrchin AuNPs (1 mL, 80 nm diameter) were functionalized with thiol-modified DNA via gold-sulfur chemistry. The thiol-modified DNA was activated using two equivalents of tris(2-carboxyethyl)phosphine hydrochloride (TCEP). NanoUrchin AuNPs (80 nm) were functionalized by mixing deprotected alkanethiol oligonucleotides with an aqueous nanoparticle solution (particle concentration 1 OD) to a final probe strand concentration of 100 nM, followed by the addition of m-PEG1K to a final concentration of 80 μg / mL. After approximately 48 h, the solution was centrifuged at 800 rcf for 10 min to remove excess thiol-DNA, and the supernatant was removed with a micropipette. The precipitated DNA-AuNPs were washed with an equal volume of 10 mM TE (0.025% Tween 20, pH 7.4), and the centrifugation / washing procedure was repeated twice. The final pellet was resuspended in stock solution (10 mM TE 0.025% Tween 20, pH 7.4) and stored at 4°C.

[0053] Cell culture and exosomal total RNA isolation MCF-7 and DU145 cells were cultured in Eagle's Minimum Essential Medium (EMEM) containing 10% exosome-depleted fetal bovine serum. Cells were incubated at 37°C in 5% CO2 for 72 hours until 90% confluent. Exosomes obtained from cell culture supernatants were isolated using Total Exosome Isolation Reagent after overnight incubation at 2°C–8°C. After centrifugation at 10,000 × g for 60 minutes, the exosome pellet was resuspended in PBS. Total RNA was extracted from the exosomes.

[0054] Quantification of miRNAs by qPCR miR-21 and miR-375 were quantified by qPCR using predesigned TaqMan primers for miR-21 and miR-375, respectively. Complementary DNA (cDNA) was synthesized, and 2 μl of the 20-fold diluted cDNA product was mixed with TaqMan Universal Master Mix II, 1 μl of primer, and RNase-free water to a final volume of 20 μl. The reaction was performed using a real-time PCR detection system under the following conditions: Pre-denaturation at 95°C for 10 min, followed by 40 cycles of denaturation (94°C for 40 s) Annealing (30 s at 60°C) was performed and monitored. For absolute quantification of miR-375 and miR-21 in MCF-7 and DU145 cells, standard curves were generated using different concentrations of synthetic miR-375 and miR-21 ranging from 1 fM to 100 nM. [Example]

[0055] Optimization of linker chain toehold length and concentration To achieve ultrasensitive detection limits using the TRAP system, it is necessary to minimize nonspecific AuNP binding and enhance target-triggered signaling. In the disclosed TRAP design (Figure 1), toehold-1 is involved in the strand displacement reaction triggered by the target miRNA, while toehold-2 allows the entry of probe-functionalized AuNPs. Toehold-1 is designed with five bases, and a longer toehold-2 results in faster kinetics and recycling of the target miRNA. However, for a constant protector strand length, a longer toehold introduces more exposed bases at the end of the linker strand, resulting in nonspecific binding in the absence of target miRNA. This design was optimized using four sets of linker sequences with different toehold-2 lengths.

[0056] The linker strand was prehybridized with the protector strand and then mixed with the probe DNA and capture strand. The binding characteristics of different linker sequences were analyzed by native PAGE (Figure 8). Four linkers with different sequence options were tested to confirm the formation of stable capture-linker-protector structures. We confirmed that capture-linker-probe (CLP) complexes, which mimic AuNPs bound to the probe DNA, only formed in the presence of the miRNA target. The presence of a CLP band in the absence of the miRNA target (Figure 8, lanes 2, 5, 8, and 11), indicates that the length of the toehold-2 allows nonspecific binding. The results with the longest linker sequence (L4, 4-nt initial toehold) indicate that a CLP band formed even in the absence of the target, resulting from a nonspecific reaction. On the other hand, the shortest linker sequence (L1, 1-nt initial toehold) did not form a CLP complex when the miRNA target was added, indicating that the target was unable to remove the protector strand. As shown in Figure 8, linker strands containing two or three exposed bases in the toehold-2 region (L2 and L3) produced clear CLP bands in the presence of target but not in the absence of target strand.

[0057] The toehold length of the linker strand was further optimized by analyzing L2, L3, and L4 linkers with two, three, and four initially exposed bases, respectively, using the TRAP system. As shown in Figure 9, in the absence of target miRNA, the longest linker strand produced a very high background signal, consistent with the results of the PAGE experiment in Figure 8. However, after adding target miRNA at a concentration of 10 pM, a linker strand with only the first two exposed bases in the toehold 2 region exhibited an improved signal-to-noise ratio of 142, a 120-fold improvement compared to the three free-base linkers. Two free bases were designed for the first exposed linker terminal toehold (L2) and used in all subsequent experiments.

[0058] In the disclosed TRAP system, DNA probe-functionalized gold nanoparticles and PC-bound capture strands are crosslinked by linker strands. After optimizing the exposed toehold length of the linker strands, we evaluated the relationship between linker strand concentration and the sensitivity of the TRAP nucleic acid system by counting the AuNPs bound to the PC surface (Figure 10). Increasing the linker concentration from 1 pM to 20 pM gradually increased the number of captured AuNPs from 174 to 500. However, increasing the linker concentration to 50 pM reduced the number of AuNPs on the PC surface to 311. The optimal concentration is related to the constant ratio of probe DNA on the AuNPs to capture strands on the PC. With excess linker strands, both the probe DNA and capture strands hybridize with the linker strands, respectively, resulting in fewer probe DNAs and capture strands bound by the linker strands. As a result, a 20 pM concentration of the linker-protector complex, with a two-fold excess of protector, was found to be optimal. [Example]

[0059] Quantifying TRAP sensitivity A constant reaction volume of 20 μL of solution containing target miRNA, linker-protector complex, and probe DNA-modified AuNPs was added to a PDMS reservoir. Separate wells were used for different concentrations of miRNA ranging from 0.1 aM to 1 pM. The reaction was carried out at room temperature and imaged after 10 and 20 min. Increasing the concentration of miRNA-375 (0 aM, 1 aM, 1 fM to 1 pM) also increased the number of AuNPs on the PC surface at 10 min (32, 82, 276 to 538 AuNPs) (Figure 2a; additional PDMS images are shown in Figure 11; calibration curve R for miRNA-375 from 0.1 aM to 1 pM). 2 -0.957, Figure 2b). When the reaction time was extended to 20 min (R 2= 0.999), the bound AuNPs on the PC increased approximately 1.5-fold (Figure 3a and Figure 11), further strengthening the linear relationship between particle number and miRNA-375 concentration. A 20-minute reaction time resulted in a higher density of captured AuNPs, slightly improving miR-375 sensitivity and signal-to-noise ratio. Based on these results, the limit of detection (LOD; the concentration at which the signal threshold is three standard deviations (3σ) above background noise (3σ + blank)) was calculated to be 0.24 aM for miRNA-375 after 20 minutes (Figures 2b and 11). The robustness and versatility of the TRAP nucleic acid design were confirmed with another target, miRNA-21, with an LOD of 0.356 aM (Figure 12). [Example]

[0060] Quantifying TRAP sensitivity The sensitivity of TRAP was determined using AuNPs immobilized on a PC surface in a dose-dependent manner for various concentrations of target miRNA-375. The target miRNA, linker-protector complex, and probe DNA-modified AuNPs (20 μL) were added to a PDMS reservoir. Separate wells were used for different concentrations of miRNA ranging from 0.1 aM to 1 pM. The reaction was carried out at room temperature and imaged after 10 and 20 min. At 10 min, increasing concentrations of miRNA-375 (0 aM, 1 aM, 1 fM to 1 pM) resulted in an increase in the number of AuNPs on the PC surface (32, 82, 276 to 538) (Figures 2a and 11). Linear calibration from 0.1 aM and 1 pM for miRNA-375 (Figure 2b) showed a linear correlation coefficient of 10 min. 2 = 0.957. After 20 minutes of reaction time (R 2At a pH of 0.999, the bound AuNPs on the PC increased approximately 1.5-fold (Figures 3a and 11), demonstrating a linear relationship between particle number and miRNA-375 concentration. A 20-minute reaction time resulted in a higher density of captured AuNPs, improving the sensitivity and signal-to-noise ratio of miR-375. Based on these results, the limit of detection (LOD) for miRNA-375 at 20 minutes was calculated to be 0.24 aM (Figures 2b and 11). The robustness and versatility of the TRAP nucleic acid design were demonstrated in the investigation of a second target, miRNA-21, where an LOD of 0.356 aM was observed (Figure 17). [Example]

[0061] TRAP selectivity at single-base accuracy We determined the selectivity of TRAP with single-base precision for five different single-nucleotide variants (SNVs) of miRNA-375, with mismatch positions at positions 1, 5, 12, 18, and 22 from the 5' end. The wild-type miRNA-375 target was tested at a concentration of 1 fM, and the mismatch SNVs were tested at 1 pM. TRAP images of the wild-type miRNA-375 target detected approximately 302 nanoparticles, while the mismatch SNVs showed background signals of less than 72 nanoparticles on the PC (Figure 3). When the mismatch position was far from the initial toehold and a miRNA with a 1000-fold mismatch (1 pM) with the correct target was used, the resulting signal was still less than 25% of that of the correct target at 1 fM. This demonstrates that the TRAP system maintains high selectivity for miRNA detection with single-base precision. Furthermore, the branch migration reaction of the toehold transfer reaction ensures selectivity, since a single base mismatch increases ΔGG° by +1.83 to +5.9 kcal / mol. [Example]

[0062] Multiplexing miRNA detection with one batch of TRAP The TRAP system can be easily adapted to detect any miRNA sequence, allowing multiple assays to be performed in parallel by measuring separate subvolumes of the test sample in independent wells on the same PC biosensor. This performance was demonstrated using three DNA strands designed and optimized using the same principles and procedures detailed above, and the feasibility of the DNA probes was confirmed by PAGE analysis (Figure 13). In the multiplex TRAP assay (Figure 4a), the same capture strand was used for all reservoirs; the only differences between wells were the target miRNA (Figure 9b), nanoparticle probe, and specially designed linker-protector complex. After 20 min of incubation, PRAM images were recorded and the number of bound AuNPs was calculated (Figure 9d). In the presence of 1 fM miRNA, an increased number of nanoparticles was observed on the PC surface, while the blank control (no target) showed only a weak signal. The data for all five miRNA targets tested were similar, demonstrating the applicability of the TRAP method to simultaneously quantify a variety of miRNA targets. [Example]

[0063] Monitoring the expression of miRNA-375 and miRNA-21 in exosomes for cancer diagnosis miRNA-375 and miRNA-21 have been identified as important biomarkers for breast cancer, while circulating miRNA-375 is significantly overexpressed in the blood of prostate cancer patients and is involved in several processes that affect tumorigenesis and metastasis. TRAP was used to diagnose cancer by monitoring the expression of miRNAs in exosomes, and miRNA-375 and miRNA-21 were selected as models.

[0064] Total RNA was extracted from exosomes of breast cancer cell lines (MCF-7) and human prostate cancer cell lines (DU145). The extracted samples were used to test the expression of miRNA-375 and miRNA-21 in the TRAP system (Figure 1). As a gold standard for validating the accuracy of TRAP, qRT-PCR quantification was simultaneously performed (Figure 14). The detection limits of TRAP for miRNA-375 and miRNA-21 in buffer were 0.15 copies / µL (0.24 aM for miRNA-375) and 0.21 copies / µL (0.304 aM for miRNA-21), which were 259- and 372-fold lower than those obtained by qRT-PCR for the same targets (Figure 10a).

[0065] Dilutions of exosomal miRNA extracts were performed, and dose-response curves (Figures 15-18) were obtained. The amount of miRNA-21 in exosomes derived from MCF-7 cells was higher than that of miRNA-375 (up to 2535-fold), while the amount of exosomal miRNA-21 in exosomes derived from DU145 cells was higher than that of miRNA-375 (up to 2412-fold). The amounts of miRNA-375 and miRNA-21 in exosomes derived from DU145 cells were similar to those in MCF-7 cells. As shown in Figure 5b, the results obtained by the two methods were consistent, demonstrating the accuracy and reliability of the TRAP method.

[0066] The detection limits of TRAP for miRNA-375 and miRNA-21 in cancer cell exosomes obtained from MCF-7 and DU145 cultures were 1.2 copies / μL (2 aM for miRNA-375 and miRNA-21 in MCF-7 and DU145, respectively), which are 31- and 61-fold lower than the detection limits of qRT-PCR. [Example]

[0067] Binding behavior of AuNPs and PC The binding behavior of AuNPs and PC was achieved using finite element method (FEM) simulations to investigate the near-field intensity distribution of PC-bound AuNPs (Figure 12a). The optical absorption of PC-bound AuNPs was further analyzed by measuring the PC resonant reflectance spectrum (Figures 12b and 12c). Upon AuNP binding, the PC reflection intensity ΔI / I was locally reduced to approximately 12%, which can be used as a contrast mechanism for image-based detection of single PC-bound nanoparticles.

[0068] conclusion In conclusion, a target recycling amplification process (TRAP) detection method using photon resonator absorption microscopy has been developed, which is useful for monitoring exosomal miRNAs with ultrahigh sensitivity and single-base mismatch selectivity. This approach is a single-step, wash-free, enzyme-free, isothermal, 20-minute room-temperature process that can be easily adapted to any miRNA target using different DNA probe designs. The AuNP-probe complexes are designed to be universal for all targets. The TRAP method achieves cancer biomarker miRNA detection with detection limits of 0.24 aM for miRNA-375 and 0.356 aM for miRNA-21 per nucleic acid copy. By digitally counting the bound AuNPs on the photonic crystal surface, target miRNA concentrations can be measured with single-base precision over a wide range from 1 aM to 1 pM.

[0069] TRAP can perform multiplexed miRNA detection in a single batch. Compared with conventional qRT-PCR, TRAP showed comparable accuracy in profiling cancer cell-derived exosomal miRNAs, but showed at least 31-fold and 61-fold improvements in the detection limits for miRNA-375 and miRNA-21, respectively.

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Claims

1. 1. A system for detecting nucleic acids in a sample, comprising: a biosensor comprising a photonic crystal (PC), wherein the PC is immobilized on a nucleic acid capture strand sequence; a nucleic acid linker strand that is annealed to the nucleic acid protector strand to form a linker-protector complex; Reaction solution Probe strand Gold nanoparticles (AuNPs), Samples; and Includes an imaging platform the capture strand is pretreated with the linker-protector conjugate, whereby the linker-protector conjugate binds to the capture strand and forms a first toehold; the target RNA in the sample is capable of binding to the first toehold, thereby displacing the protector strand from the linker-protector complex; The AuNP probe binds to the linker strand at the second toehold region, thereby displacing the target RNA; The imaging platform is configured to quantify displaced target RNA in a sample by measuring the bound AuNPs.

2. 2. The system of claim 1, wherein binding of the linker-protector complex to the capture strand forms a first toe-hold region on the linker strand.

3. The system of claim 2 , wherein the target RNA binds to a first toe-hold region on the linker strand, thereby displacing the protector strand in a first displacement reaction.

4. 4. The system of claim 3, wherein binding of the target RNA to the first toe-hold region displaces the protector strand, exposing the second toe-hold region.

5. The system of claim 4 , wherein the AuNP probe is bound to a linker at the second toehold region, thereby displacing the target RNA in a second displacement reaction.

6. The system of claim 1 , wherein the bound AuNP probes are imaged using photon cavity absorption microscopy (PRAM).

7. The system of claim 7 , wherein the bound AuNPs at a given location on the PC reduce the reflected light intensity from the PC surface at that location.

8. The system of claim 1 , wherein the binding of the linker chain to the AuNP is reversible.

9. The system of claim 8, wherein the reversible binding allows RNA binding to additional AuNPs, resulting in target recycling and signal amplification.

10. The system of claim 1 , wherein the detection limit is a single RNA strand.

11. 2. The system of claim 1, wherein the RNA is a microRNA (miRNA), tRNA, rRNA, snRNA, long non-coding RNA (lncRNP), circular RNA (circRNA), short interfering RNA (siRNA), or messenger RNA (mRNA).

12. The system according to claim 1 , wherein the linker-protector sequence is added to the reaction solution in excess.

13. The system of claim 1 , wherein the imaging platform includes a light source configured to excite a resonance of a photonic crystal and a detector configured to detect light reflected from the photonic crystal.

14. The system of claim 1 , wherein the imaging platform is configured to quantify resonant peak intensity values ​​measured on a pixel-by-pixel basis across the photonic crystal.

15. The system of claim 1 , wherein the nucleic acid is any nucleic acid that forms complementary base pairs.

16. The system of claim 1 , wherein the nucleic acid is DNA.

17. The system of claim 1 , wherein the nucleic acid is RNA.

18. The system of claim 1 , wherein the nucleic acid is a peptide nucleic acid (PNA).

19. The system of claim 1 , wherein the imaging platform is a surface plasmon resonance imaging platform.

20. The system of claim 1 , wherein the imaging platform is a dark field microscope.

21. The system of claim 1 , wherein the imaging platform is a coherent intensity imaging platform.

22. The system of claim 1 , wherein the photonic crystal is immobilized with a nucleic acid capture strand using a salification process and amine-terminated capture DNA.

23. The system of claim 1 , wherein the imaging platform includes a non-imaging detection device.

24. The system of claim 1 , wherein the biosensor is a whispering gallery mode biosensor.

25. 17. The system of claim 16, wherein the whispering gallery biosensor is a ring resonator, a microtoroid, or a microsphere.

26. The system of claim 1 , wherein the biosensor includes a waveguide structure through which light propagates laterally.

27. The system of claim 1 , wherein the biosensor is an acoustic biosensor.

28. The system of claim 1 , wherein the biosensor is a photoacoustic biosensor.

29. 1. A biological assay comprising: a biosensor comprising a capture strand oligonucleotide sequence and a linker strand-protector strand oligonucleotide complex; Reaction solution; an oligonucleotide chain; and comprising a population of nanoparticles; The assay, wherein the nanoparticles are attached to the surface of the biosensor using the oligonucleotide strands, the nucleotide strands being composed of random nucleic acid sequences.

30. 30. The biological assay of claim 29, wherein a biological sample is added to the assay and RNA within the sample is capable of binding to the free toehold region of the linker-protector complex.

31. 30. The biological assay of claim 29, wherein the population of nanoparticles is bound to an oligonucleotide sequence.

32. 32. The biological assay of claim 31, wherein the population of nanoparticles is bound to a toehold region.

33. 30. The biological assay of claim 29, wherein the RNA for detection is a microRNA (miRNA).

34. 30. The biological assay of claim 29, wherein the RNA for detection is microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), long non-coding RNA (lncRNA), circular RNA (circRNA), short interfering RNA (siRNA), or messenger RNA (mRNA).

35. The nanoparticles may be gold nanoparticles, quantum dots, metal-based nanoparticles, magnetic nanoparticles, fluorescent materials, nanodiamonds, plasmonic phosphors, or SiO ２ or TiO ２ 30. The biological assay of claim 29, wherein the nanoparticles comprise a dielectric material such as:

36. 22. The biological assay of claim 21, wherein the biosensor comprises a photonic crystal and further comprises an imaging platform configured to quantify resonant peak intensity values ​​measured pixel by pixel across the photonic crystal.

37. 30. The biological assay of claim 29, wherein the biosensor comprises a non-imaging detection device.

38. 30. The biological assay of claim 29, wherein the biosensor is a whispering gallery mode biosensor.

39. 39. The biological assay of claim 38, wherein the whispering gallery mode biosensor is a ring resonator, a microtoroid, or a microsphere.

40. 30. The biological assay of claim 29, wherein the biosensor comprises a waveguide structure through which light propagates laterally.

41. 30. The biological assay of claim 29, wherein the biosensor is an acoustic biosensor.

42. 30. The biological assay of claim 29, wherein the biosensor is an optoacoustic biosensor.

43. 30. The biological assay of claim 29, wherein the biological assay is stable at room temperature.

44. 1. A method for detecting a nucleic acid in a sample, comprising: immobilizing oligonucleotide capture strands onto the surface of a biosensor, thereby creating an assay surface; pre-treating the capture strand with an oligonucleotide linker strand-oligonucleotide protector strand complex; adding an assay medium to the assay surface, the assay medium comprising a biological sample that may contain a target RNA, the target RNA being capable of binding to a first free toe-hold region on the linker strand, thereby displacing the protector strand; adding a bound nanoparticle probe capable of binding to a second toehold region, thereby creating and liberating said target RNA; and using an imaging platform to quantify the number of nanoparticles bound to the second toe-hold region.

45. 45. The method of claim 44, wherein the RNA for detection is microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), long non-coding RNA (lncRNA), circular RNA (circRNA), short interfering RNA (siRNA), or messenger RNA (mRNA).

46. 45. The method of claim 44, wherein the quantitative difference in reflected light intensity from the biosensor surface at the location of each nanoparticle represents the RNA copy number.

47. 45. The method of claim 44, wherein the quantitative limit of detection is a single RNA copy.

48. 45. The method of claim 44, wherein the biosensor comprises a photonic crystal and the imaging platform comprises a light source configured to excite a resonance of the photonic crystal and a detector configured to detect light reflected from the photonic crystal.

49. 49. The method of claim 48, wherein the imaging platform is configured to quantify resonant peak intensity values ​​measured on a pixel-by-pixel basis across the photonic crystal.

50. 45. The method of claim 44, wherein the imaging platform comprises a non-imaging detection device.

51. 45. The method of claim 44, wherein the biosensor is a whispering gallery mode biosensor.

52. 45. The method of claim 44, wherein the whispering gallery biosensor is a ring resonator, a microtoroid, or a microsphere.

53. 45. The method of claim 44, wherein the biosensor comprises a waveguide structure through which light propagates laterally.

54. 45. The method of claim 44, wherein the biosensor is an acoustic biosensor.

55. 45. The method of claim 44, wherein the biosensor is an optoacoustic biosensor.

56. The nanoparticles may be gold nanoparticles, quantum dots, metal-based nanoparticles, magnetic nanoparticles, or SiO ２ or TiO ２ 45. The method of claim 44, wherein the nanoparticles comprise a dielectric material such as

57. 45. The method of claim 44, wherein after quantifying the bound nanoparticles, the nanoparticles are removed from the biosensor surface.

58. 58. The method of claim 57, wherein the nanoparticles are removed from the biosensor surface by exchanging the assay buffer.

59. 58. The method of claim 57, wherein the nanoparticles are removed from the biosensor surface by agitating the assay buffer without replacing the assay buffer.

60. 58. The method of claim 57, wherein if the nanoparticles are magnetic nanoparticles, the nanoparticles are removed from the biosensor surface by application of a magnetic field.

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