WO2024059153A2 - Target recycling amplification process (trap) - Google Patents

Abstract

The current disclosure provides an improved sensitivity assay for detecting and quantifying nucleic acids in a sample. The assay utilizes target recycling amplification process (TRAP) for the digital detection of nucleic acids in tandem with photonic resonator absorption microscopy (PRAM) and utilizes toehold-mediated DNA strand displacement reactions. The disclosure also provides a system and method for use of the assay.

Description

TARGET RECYCLING AMPLIFICATION PROCESS (TRAP)

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to United States Provisional Patent Application No. 63/406,540, filed September 14, 2022, which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under award number 1R01EB029805-01 awarded by the National Institute of Health. The government has certain rights in the invention.

BACKGROUND

Exosomal microRNAs (miRNAs) sequestered within extracellular vesicles play diverse roles in biological processes, including cell-cell communication, cell proliferation, and inflammatory response. miRNAs participate in post-transcriptional regulation of gene expression and inappropriate miRNA release from exosomes can result in the development of cardiovascular diseases and cancers. On this basis, exosomal miRNAs are recognized as pivotal biomarkers for diagnosing cancer development and monitoring the progression of disease. Additionally, exosomal miRNAs concentrations have been found to be correlated with therapeutic effectiveness. However, exosomal miRNAs may be present in extremely low concentrations, which is a current barrier to utilizing exosomal miRNAs as biomarkers. For exosomes isolated from cells or plasma, there can be far less than a single miRNA per exosome on average, even for the most abundant target sequences

Traditional methods such as quantitative reverse transcription polymerase chain reaction (qRT-PCR) for miRNA have been considered as the gold standard for miRNA quantification with femtomolar limits of detection. However, the traditional qRT-PCR requires complicated enzymatic amplification and complicated primer designs. Other quantification methods such as Northern blots and oligonucleotide microarrays are performed on cell lysates; where fluorescent reporters are constructed to obtain enhanced fluorescence signals for profiling miRNA in cells.

As such, current methods have not been adopted for clinical use for exosomal miRNA detection without enzymatic target amplification. Therefore, there is an unmet need in the art for an ultrasensitive and highly selective diagnostic approach without enzymatic amplification to effectively detect and quantify exosomal miRNAs. The disclosure herein addresses limitations of low-level miRNA detection. The system, method, and assay disclosed herein can be used to determine health status; early disease and medical condition diagnosis; identification of biomarkers; evaluation of therapeutic efficiency; and longitudinal monitoring of disease progression.

SUMMARY

In one aspect, example embodiments provide a system for detecting nucleic acids in a sample, comprising: a biosensor, comprising a photonic crystal (PC), wherein the PC is immobilized to a nucleic acid capture strand sequence; a nucleic acid linker strand 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 imaging platform, wherein the capture strand is pre-treated with the linker-protector complex thereby binding the linkerprotector complex to the capture strand and creating a first toehold; target RNA in the sample is capable of binding the first toehold thereby displacing the protector strand from the linkerprotector complex; the AuNP probe tethers to the linker strand at a second toehold region, thereby displacing the target RNA; and wherein the imaging platform is configured to quantify the displaced target RNA in the sample by measuring the tethered AuNP.

In a further aspect, example embodiments provide a biologic assay comprising: a biosensor containing a capture strand oligonucleotide sequence and a linker strand-protector strand oligonucleotide complex; a reaction solution; oligonucleotide strands; and a population of nanoparticles; wherein the nanoparticles are bound to the surface of the biosensor using the oligonucleotide strands, and wherein the nucleotide strands are comprised of random nucleic acid sequences.

In another aspect, example embodiments provide a method for detecting nucleic acids in a sample, comprising the steps of: immobilizing a oligonucleotide capture strand to the surface of a biosensor, thereby creating an assay surface; pretreating the capture strand with a oligonucleotide linker strand - oligonucleotide protector strand complex; adding an assay medium to the assay surface, wherein the assay medium comprises a biological sample that may contain the target RNA, wherein the target RNA is capable of binding to a first free toehold region on the linker strand thereby replacing the protector strand; adding a tethered nanoparticle probe capable of binding to a second toehold region thereby creating releasing the target RNA; and quantifying the number of nanoparticles bound to the second toehold region using an imaging platform. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic of miRNA detection workflow. Figure la demonstrates extracted exosomal miRNAs placed in a PDMS reservoir applied to the PC surface along with probes linked to AuNPs. Figure lb is a schematic showing DNA linker pre-annealed with a partially complementary protector and hybridized with DNA capture. Figure 1c demonstrates Target Recycling Amplification Process (TRAP) for digital resolution detection of microRNAs on a PC biosensor surface in which target miRNA displaces a protector strand on the PC-immobilized capture molecule to reveal a linker sequence by a strand displacement reaction

Figure 2 demonstrates kinetic discrimination of miRNA-375 using TRAP. Figure 2a demonstrates dose-response TRAP images at single particle resolution at 10 mins and 20 mins. Figure 2b demonstrates quantification of particle counts as a function of miRNA-375 concentration for three trials with standard error shown. Blank represents reaction without miRNA target present.

Figure 3 demonstrates selectivity of TRAP in detecting single nucleotide variants (SNVs) of miRNA-375. Figure 3a demonstrates TRAP images. Figure 3b demonstrates counted numbers of bound nanoparticle on PC surface in the presence of miRNA-375 or 5 different SNVs at the 1st, 5th, 12th, 18th and 22nd from 5’ terminal of miRNA. Approximately 6000: 1 selectivity is demonstrated for detection of the target sequence against single base mismatched targets at each location investigated.

Figure 4 demonstrates multiplexed miRNA detection performed in different reservoirs of one PC biosensor. Figure 4a demonstrates utilization 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 demonstrates the sequences of target miRNAs. Figure 4c demonstrates PRAM images and Figure 4d demonstrates particle counts on the PC surface with or without 1 fM miRNA targets.

Figure 5 demonstrates a comparison of limit of detection. Figure 5a is a comparison of the limit of detection for exosomal miRNA-375 and miRNA-21 using TRAP and qRT-PCR methods. Figure 5b is relative expressive levels of exosomal miRNA-375 and miRNA-21 derived from MCF-7 and DU145 cells resolved with TRAP and qRT-PCR. The expressive level of miRNA-375 in MCF-7 cells was defined as one. The relative expressive level was compared to miRNA-375 in MCF-7 cells.

Figure 6 demonstrates TEM imaging. Figure 6a demonstrates TEM imaging of NanoUrchin AuNPs. Figure 6b demonstrates a conjugate product of NanoUrchin AuNPs with probe DNA and m-PEGlK.

Figure 7 demonstrates near-field intensity. Figure 7a demonstrates near-field profile of PC- AuNPs hybrid under resonant condition (X = 620 nm at normal incidence). Figure 7b is representative PC resonant reflected spectrum. Figure 7c is a close-up view of the reflected spectrum for the PC surface without or with attached AuNP. The reflected resonant intensity was obtained under a 20A~ lens.

Figure 8 is a native PAGE imaging of different length linker strands tested in the reaction.

Figure 9 demonstrates optimization of linker strand length. TRAP image panel demonstrates particle count for different lengths of linker strand, captured at a reaction time of 30 minutes.

Figure 10 demonstrates optimization of linker strand concentration imaged at 90 minutes. TRAP image panel demonstrates particle count of various linker strand concentrations.

Figure 11 demonstrates digital resolution. Figure Ila is a TRAP image panel with digital resolution of captured AuNPs as a function of target concentration (rows) with 10 minutes and 20 minutes. Figure 11b is a linear calibration curve of the miR-375 detection assay in buffer. Figure 11c demonstrates a linear regression used to plot the dose response, where x is the concentration of target sequence, y is the AuNPs counts. The dashed horizontal line indicates the threshold (blank signal + 3 standard derivations). The error bars represent the standard deviation of three independent assays.

Figure 12 demonstrates TRAP images. Figure 12a demonstrates a TRAP image panel with digital resolution of captured AuNPs as a function of target concentration (rows) with 10 minutes and 20 minutes. Figure 12b demonstrates quantification of particle count as a function of target concentration at 10 and 20 minutes. The dashed horizontal line indicates the threshold (blank signal + 3 standard derivations). The error bars represent the standard deviation of three independent assays.

Figure 13 demonstrates a 12% PAGE. Within 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 demonstrates linear calibration curves. Figure 14a demonstrates a linear calibration curve of the miR-375 and miR-21 detection assay in buffer by using qRTPCR. Figure 14b is a linear regression model used to portrait the dose response, where x is the concentration of target sequence, y is the Cq. Figure 14c demonstrates linear regression of a miR-21 response case.

Figure 15 demonstrates kinetic discrimination of miR-375 concentration in MCF-7 cell exosomes using TRAP. Figure 15a demonstrates dose-response plot for detection of miRNA-375 by TRAP, with detection shown at 10 and 20 minutes in a room temperature assay protocol. Figure 15b demonstrates quantification of particle count as a function of miR-375 concentration in MCF-7 cell exosomes for three trials. The blank represents the notarget control.

Figure 16 is a kinetic discrimination of miR-375 concentration in DU145 cell exosomes using TRAP. Figure 16a demonstrates a dose-response plot for detection of miRNA-375 by TRAP, with detection shown at 10 and 20 minutes in a room temperature assay protocol. Figure 16b demonstrates quantification of particle count as a function of miR-375 concentration in DU145 cell exosomes for three trials.

Figure 17 demonstrates kinetic discrimination of miR-21 concentration in MCF-7 cell exosomes using TRAP. Figure 17a demonstrates dose-response plot for detection of miRNA-21 by TRAP, with detection shown at 10 and 20 minutes in a room temperature assay protocol. Figure 17b demonstrates quantification of particle count as a function of miR-375 concentration in MCF-7 cell exosomes for three trials. DETAILED DESCRIPTION

It is to be understood that the particular aspects of the specification are described herein are not limited to specific embodiments presented and can vary. It also will be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting. Moreover, particular embodiments disclosed herein can be combined with other embodiments disclosed herein, as would be recognized by a skilled person, without limitation.

Throughout this specification, unless the context specifically indicates otherwise, the terms “comprise” and “include” and variations thereof (e.g., “comprises,” “comprising,” “includes,” and “including”) will be understood to indicate the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other component, feature, element, or step or group of components, features, elements, or steps. Any of the terms "comprising", "consisting essentially of', and "consisting of' may be replaced with either of the other two terms, while retaining their ordinary meanings.

As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise.

Unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values herein that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

As used herein and in the drawings, ranges and amounts can be expressed as “about” a particular 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 value or range of values. Therefore, about 5% also means 4.5% - 5.5%, for example.

“Sample” as used herein refers to any type of sample, containing a nucleotide sequence and encompasses biological sample. “Biological sample” refers to a sample of body tissue, including but not limited to an organ punch or tissue biopsy, or fluid, including but not limited to blood, cerebrospinal fluid, plasma, or saliva from a warm-blooded animal such as a mammal, preferably a human, which is afflicted with, or has the potential to be afflicted with one or more diseases and/or disorders described herein. A biological sample can also refer to tissue or blood samples obtained from non-human mammals and other animals. In an alternative embodiment the sample is from a non-mammal host, which may contain the target nucleotide sequence.

In view of the present disclosure, the methods and compositions described herein can be configured by the person of ordinary skill in the art to meet the desired need.

1. Overview

The current disclosure provides an improved sensitivity assay for detecting and quantifying nucleic acids in a sample. The disclosure also provides a system and method for use of the assay. The assay utilizes target recycling amplification process (TRAP) for the digital detection of nucleic acids in tandem with photonic resonator absorption microscopy (PRAM) and utilizes toehold-mediated DNA strand displacement reactions. The disclosed assay, system, and methods provide enhanced sensitivity to detect and quantify nucleic acids in a biological sample and can be used in numerous settings including health status, early disease and medical condition diagnosis, identification of biomarkers, evaluation of therapeutic efficiency, and longitudinal monitoring of disease progression. The PRAM instrument is described in U.S. Patent Application No. 16/170,111 while 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.

Exosomal microRNAs (miRNAs) sequestered within extracellular vesicles play diverse roles in biological processes, including cell-cell communication, cell proliferation, and inflammatory response. miRNAs participate in post-transcriptional regulation of gene expressions. As a result, inappropriate miRNA release from exosomes can result in the development of diseases and cancers. On this basis, exosomal miRNAs are recognized as pivotal biomarkers for diagnosing cancer and disease development and monitoring the progression of diseases. Additionally, exosomal miRNAs concentrations have been found to be correlated with therapeutic effectiveness. Nucleic acid alterations including alternations in RNA, DNA, and PNA can be indicative of disease onset, disease progression, or therapeutic effectiveness. However, nucleic acids, including exosomal miRNAs may be present in extremely low concentrations, which is a current barrier to utilizing exosomal miRNAs or other nucleic acids as biomarkers. Exosomes isolated from cells or plasma may be less than a single miRNA per exosome on average, even for the most abundant target sequences. As such, traditional methods such as quantitative reverse transcription polymerase chain reaction (qRT-PCR), the gold standard for quantification of nucleic acids, such as miRNA, is considered the gold standard for miRNA quantification with femtomolar limits of detection. However, traditional qRT-PCR requires complicated enzymatic amplification and complicated primer designs which negatively impact detection limits. Alternative quantification methods such as Northern blots and oligonucleotide microarrays are performed on cell lysate and often include the construction of fluorescent reporters to obtain enhanced fluorescence signals for profiling miRNA in cells. Constrained by the detection limit, none of these methods have been adopted for clinical use for exosomal miRNA detection without enzymatic target amplification. As a result, there is an unmet need to develop an ultrasensitive and highly selective diagnostic approach without enzymatic amplification to effectively detect and quantify exosomal miRNAs and other nucleic acids.

2. Target Recycling Amplification Process (TRAP)

As used herein “Target Recycling Amplification process (TRAP)” refers to a method that allows for amplification of a nucleic acid signal to increase the sensitivity of biosensors, assays for biomarker detection, and other types of nucleic acid detection. In an initial step of the current disclosure, miRNA is extracted from exosomes isolated from cell culture media (Figure la) using a variety of commonly known protocols in the art. A photonic crystal (PC) surface is then prepared with an immobilized capture DNA or RNA through any DNA or RNA mobilization process. Examples of RNA and DNA mobilization processes include, but are not limited to covalent bonding methods, streptavidin-biotin methods, PEG linkers, and self-assembled monolayer linkers. The photonic crystal has previously been 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.

Following immobilization of the capture DNA the PC is pretreated with a linkerprotector complex consisting of a protector DNA and a linker DNA. The linker-protector complex is designed with free unhybridized regions at both 5' and 3' ends of the linker strand, resulting in formation of toehold- 1 and toehold-2 for miRNA or other nucleic acid binding and probe DNA binding, respectively.

As used herein “protector DNA or protector” refers to a a nucleic acid or nucleic acid sequence that is not functional in the system but keeps unintended DNA from reacting. One of skill in the art will readily understand the role of “protector DNA”. As used herein “linker DNA” or “substrate” or “bridge” refer to a DNA sequence used to link the captured label to the detection substrate. In some embodiments, the linker works via hybridization of both the capture and protector DNAs.

As used herein “Toehold Mediated Strand Displacement (SDR) or toehold-mediated branch migration reaction or entropy-driven toehold SDR” refers to an enzyme-free molecular method by which one strand of a nucleic acid (output) is exchanged with another nucleic acid strand (input). More particularly, the terms refer to any system where a duplex nucleic acid initially has a short toehold sequence of 4-8 nucleotides. In one embodiment the sequence is single stranded. An 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 wherin toehold is hidden until exposed by another reaction, such as in TRAP, are called "toehold sequestering” and can also be part of the disclosure herein. In an exemplary embodiment the toehold reactions occur via addition of reaction solutions, commonly known in the art, to a Polydimethylsiloxane (PDMS) reservoir attached to the PC surface. The reservoirs can also be comprised of a wide range of materials including, but not limited to, plastic (such as acrylic, polycarbonate, polyester), glass.

In a preferred embodiment of the current disclosure, the exosomal extraction samples are added to reaction wells resulting in binding of the miRNA or target nucleic acid to the free toehold- 1 region on the linker strand thereby replacing the protector strands through a DNA strand displacement. The displacement allows a probe DNA conjugated to a gold nanoparticle (AuNP) to invade the toehold-2 on linker strand and release the target miRNA with a second DNA strand displacement reaction resulting in the exposure of an additional DNA linker sequence (toehold-2). Probe-modified AuNPs hybridize with the DNA linker sequence and the tethered AuNPs are imaged by the reduction of reflected light intensity from the PC surface (See Figures 1 A-1C and 6) in location where an AuNP bound. The current disclosure has the improved technical effect that the bound AuNPs on the surface can be enumerated using an automated image processing algorithm that identifies image pixels with reduced reflected intensity compared to the background, using a MATLAB script. Further inventive is that the released target nucleic acid or miRNA can then participate in additional TRAP reactions, resulting in increased nanoparticle binding and amplified signal. Further inventive, in an alternative embodiment, the TRAP method is capable of multiplexing, with sample volume requirements of less than < 20 pL thereby allowing for application in frequent patient monitoring. The assay has been applied to the detection of biological exosomal miRNAs and results have been validated with qRT-PCR.

II. Nucleic Acid Detection System and Assay

The system of the current disclosure is comprised of biosensor, comprising a photonic crystal (PC), wherein the PC is immobilized to a nucleic acid capture strand sequence; a nucleic acid linker strand annealed to a nucleic acid protector strand to form a linkerprotector complex a reaction solution, a probe strand, a gold nanoparticle (AuNP); a sample; and an imaging platform.

As disclosed herein, the capture strand is pre-treated with the linker-protector complex thereby binding the linker-protector complex to the capture strand and creating a first toehold. In a preferred embodiment of the disclosure, the linker-protector sequence is added in excess to the reaction solution.

The target nucleic acid in the sample is capable of binding the first toehold thereby displacing the protector strand from the linker-protector complex. The gold nanoparticle probe (AuNP) tethers to the linker strand at a 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 tethered AuNP. The system disclosed herein allows for the detection of the presence of a nucleotide sequence whose sequence is a biomarker for health, disease, the presence of a viral pathogen, or the presence of a bacterial pathogen. In a preferred embodiment of the current disclosure the nucleic acid is miRNA. Alternatively, the nucleic acid can be comprised of one or more alternative RNAs, including but not limited to miRNA, tRNA, rRNA, snRNA, long non-coding RNA (IncRNDA), circular RNA (circRNA), short interfering RNA (siRNA), or messenger RNA (mRNA). In the preferred embodiment the RNA is micro RNA (miRNA). In alternate embodiment, the nucleic acid can also be DNA, LNA, or PNA.

As used herein, “sample” refers to any type of sample, containing a nucleotide sequence and encompasses biological sample. “Biological sample” refers to a sample of body tissue, including but not limited to an organ punch or tissue biopsy, or fluid, including but not limited to blood, cerebrospinal fluid, plasma, or saliva from a warm-blooded animal such as a mammal, preferably a human, which is afflicted with, or has the potential to be afflicted with one or more diseases and/or disorders described herein. A biological sample can also refer to tissue or blood samples obtained from non-human mammals and other animals. In view of the present disclosure, the methods and compositions described herein can be configured by the person of ordinary skill in the art to meet the desired need.

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

In a preferred embodiment of the current disclosure the nanoparticle probe is a gold nanoparticle. In an alternate embodiment the nanoparticles, quantum dots, metal -based nanoparticles, magnetic nanoparticles, fluorophores, nanodiamonds, plasmonic fluors or nanoparticles comprised of dielectric materials such as SiCh or TiCh. In a preferred embodiment the linker strand binding to AuNP is reversible, allowing for RNA binding to additional AuNP resulting in target recycling and signal amplification. The tethered nanoparticle, such as a AuNP, at a given location on the photonic crystal (PC) results in a reduction in reflected light intensity from the PC surface at that location. The nanoparticles of the current disclosure are tethered to the surface of the biosensor using nucleotide tethers comprised of a non-specific nucleotide sequence. The nucleotide tethers can be homogenous or heterogenous in sequence and of a non-specific length. In some embodiments the nucleotide tethers are about 5 to 200 nucleotides in length. In some embodiments the tethers are about 5-50, about 51-100, about 101-150, or about 151-200 nucleotides in length. In yet further embodiments the nucleotide tethers are 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 system described herein has a detection limit in the attomloar range down to a single RNA strand.

In a preferred embodiment of the current disclosure the biosensor is a photonic crystal. In a preferred embodiment the photonic crystal is immobilized with the nucleic acid capture strand using a salinization process and amine-terminated capture DNA. In an alternate embodiment the biosensor can also be a whispering gallery mode biosensor that is a ring resonator, microtoroid, or microsphere. In further embodiments the biosensor is a waveguide structure through which light travels laterally, an acoustic biosensor, or a photoacoustic biosensor.

As disclosed herein, in the preferred embodiment, the imaging platform of the current disclosure comprises a light source that is 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 a resonant peak intensity value measured on a pixel-by- pixel basis across the photonic crystal. Alternatively, the imaging platform can be surface plasmon resonance imaging. In another embodiment the imaging platform is dark field microscopy. In yet another embodiment the imaging platform is interferometric intensity imaging. In an alternate embodiment the imaging platform comprises a non-imaging detection instrument.

The current disclosure also provides an assay comprising a biosensor containing a capture strand oligonucleotide sequence and a linker strand-protector strand oligonucleotide complex; a reaction solution; oligonucleotide strands; and a population of nanoparticles; wherein the nanoparticles are bound to the surface of the biosensor using the oligonucleotide strands, and wherein the nucleotide strands are comprised of random nucleic acid sequences. A sample or biological sample is added to the assay and wherein RNA within the sample is capable of binding a free toehold region of the linker-protector complex. The biological assay of the current disclosure includes a population of nanoparticles are tethered to an oligonucleotide sequence wherein the population of nanoparticles binds a toehold region.

The assay, as disclosed herein can be used to detect the presence of nucleic acids, preferably RNA. In a preferred embodiment the RNA is microRNA (miRNA) indicative of health status, early disease and medical condition diagnosis, identification of biomarkers, evaluation of therapeutic efficiency, and longitudinal monitoring of disease progression. In alternative embodiments the RNA is microRNA (miRNA), transfer RNA (tRNA), ribosomal (rRNA), small nuclear RNA (snRNA), long non-coding RNA (IncRNDA), circular RNA (circRNA), short interfering RNA (siRNA), or messenger RNA (mRNA). In a preferred embodiment the nanoparticles are gold nanoparticles but can alternatively be quantum dots, metal-based nanoparticles, magnetic nanoparticles, fluorophores, nanodiamonds, plasmonic fluors or nanoparticles comprised of dielectric materials such as SiCh or TiCh. In a preferred embodiment the biosensor of the assay comprises a photonic crystal, further comprising an imaging platform configured to quantify a resonant peak intensity value measured on a pixel- by-pixel basis across the photonic crystal. However, the biosensor can also comprise a non- imaging detection instrument, a whispering gallery mode biosensor that is a ring resonator, microtoroid, or microsphere. The biosensor can alternatively comprise a a waveguide structure through which light travels laterally. In yet another embodiment the biosensor is an acoustic biosensor, wherein the biosensor is a photoacoustic biosensor. The assay of the current disclosure is advantageous in that the assay is stable at room temperature.

The current disclosure also provides a method for detecting nucleic acid sequences of interest in a sample, such as nucleic acids sequences associated with a health state, early disease and medical condition diagnosis, identification of biomarkers, evaluation of therapeutic efficiency, and longitudinal monitoring of disease progression. As used herein “medical condition” refers to a pathophysiology and its associated progression. Examples of medical conditions include any disease or dysfunction of the integumentary System, Skeletal System, Muscular System, Nervous System, Endocrine System, Cardiovascular System, Lymphatic System, Respiratory System, Digestive System, Urinary System, and Reproductive System. More specifically the pathophysiology. More specifically, medical conditions include cardiovascular disease; CNS diseases such as but limited to Alzheimer’s disease, Parkinson’s disease, and stroke; muscular dystrophy, lupus, cancer, lung pathophysiology, and tumors (malignant and benign).

The method disclosed herein is for detecting nucleic acids in a sample, comprising the steps of immobilizing a oligonucleotide capture strand to the surface of a biosensor, thereby creating an assay surface; pretreating the capture strand with a oligonucleotide linker strand - oligonucleotide protector strand complex; adding an assay medium to the assay surface, wherein the assay medium comprises a biological sample that may contain the target RNA, wherein the target RNA is capable of binding to a first free toehold region on the linker strand thereby replacing the protector strand; adding a tethered nanoparticle probe capable of binding to a second toehold region thereby creating releasing the target RNA; and quantifying the number of nanoparticles bound to the second toehold region using an imaging platform. The method can be used to detect a nucleotide including RNA such as transfer RNA (tRNA), ribosomal (rRNA), small nuclear RNA (snRNA), long non-coding RNA (IncRNDA), 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. In a preferred method, disclosed herein quantitative differences in reflected light intensity from the biosensor surface at each nanoparticle location is indicative of RNA copy number. The method allows for quantitative limits in the attomolar range, alternatively a single nucleotide sequence copy. In a preferred embodiment the biosensor comprises a photonic crystal, wherein 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. In a preferred embodiment the imaging platform is configured to quantify a resonant peak intensity value measured on a pixel-by-pixel basis across the photonic crystal.

In a preferred embodiment of the method, the nanoparticles are gold nanoparticles but can alternatively be quantum dots, metal-based nanoparticles, magnetic nanoparticles, fluorophores, nanodiamonds, plasmonic fluors or nanoparticles comprised of dielectric materials such as SiCh or TiCh. In a preferred embodiment the biosensor of the assay comprises a photonic crystal, further comprising an imaging platform configured to quantify a resonant peak intensity value measured on a pixel-by-pixel basis across the photonic crystal. However, the biosensor can also be a non-imaging detection instrument, a whispering gallery mode biosensor that is a ring resonator, microtoroid, or microsphere. The biosensor can alternatively comprise a a waveguide structure through which light travels laterally. In yet another embodiment the biosensor is an acoustic biosensor, wherein the biosensor is a photoacoustic biosensor. In a preferred embodiment of the method, after quantification of the tethered nanoparticles the nanoparticles are removed from the biosensor surface by replacing the assay buffer, by agitation of the assay buffer without replacement of the assay buffer, or wherein the nanoparticle is removed from the biosensor surface by application of a magnetic field.

One of skill in the art will understand that the system, assay, and methods disclosed herein may also contain a sample medium comprising components required for performing the assay and methods disclosed herein, as well as for harvesting, storing or preserving the collected samples and/or biological samples.

The TRAP system, assay, and method as disclosed herein exhibit multiple technical advantages over the prior art. The TRAP system is able to achieve single copy to attomolar nucleic acid concentration detection levels. The present disclosure achieves this by minimizing nonspecific AuNP binding resulting in enhanced target-triggered signaling. More particularly, in the preferred embodiment of the current disclosure toehold- 1 participates in the target miRNA-triggered strand displacement reaction, while toehold-2 allows for invasion of the probe-annealed AuNP. Further, advantageous, the toehold-1 and linker lengths have been optimized and are technically advantageous over the prior art. In the preferred embodiment of the current disclosure, toehold-1 was designed with five bases. In alternate embodiments the toehold-a base could be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases. The Toehold-2 can be comprised of any number of bases, with an increase in the number of bases achieving faster kinetics and recycling of the target miRNAs. However, when the protector strand length is held constant a longer toehold-2 will introduce more uncovered bases on the linker strand terminal and may be associated with non-specific binding in the absence of target miRNA. The current disclosure overcomes this limitation by using linker strands containing 2 or 3 uncovered bases in the toehold-2 region to produce a distinct capture-linker- probe (C-L-P) complex in the presence of the target but not in the absence of the target strand. In the preferred embodiment of the current disclosure a linker strand containing only two initial uncovered bases was utilized as it demonstrates the unexpected technical effect of a 120-fold enhanced signal-to-noise versus three free bases (See Figure 8). However, a linker of 0-50 uncovered bases could be used. In some emodiments a linker length 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 uncovered bases could be used. Further, the preferred embodiment of the current disclosure reveals that a linker strand concentration of 1 pM to 20pM maximizes AnNP capture, but at a linker concentration of 50 pM the AuNPs captured on the PC surface decreased.

Further, the system, assay, and method of the current disclosure have the technical effect of being stable at room temperature. This overcomes a significant limitation of current assays and systems, which require cold storage. Further, the PRAM system, assay, and method provided herein have the additional surprising technical effect of providing a result in less than twenty minutes allowing for rapid assessment of health status, early disease and medical condition diagnosis, identification of biomarkers, evaluation of therapeutic efficiency, and longitudinal monitoring of disease progression. As such the system, assay, and method described herein can be used at the point of care. III. Photonic Resonator Absorption Microscopy (PRAM) Working principle

The PRAM biosensing platform is illustrated in Figure 19. PRAM can visualize individual gold nanoparticle (AuNPs) tags on a photonic crystal (PC) surface through resonance coupling, in addition to the alternative tags disclosed herein. The detection principle of PRAM utilizes the resonant PC reflection at a wavelength of = 625 nm to provide a high reflected intensity from collimated low intensity LED illumination of the same wavelength into a webcam-variety image sensor. More specifically, Port 1 is coupled to a fiber-coupled 617nm LED light source (M617F2, Thorlabs), and a lens group (F810SMA- 635, Thorlabs) is first utilized to collimate the output beam. A zero-order half-wave plate (WPH10M-633, Thorlabs) rotates the polarization of the collimated beam in order to excite the TM resonance 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 plan-fluorite objective 20*/0.5 numerical aperture (NA) objective, from which a collimated beam impinges onto the PC surface at normal incidence. A manual three-axis stage (PT3, Thorlabs) is used to secure the PC sample at the focal plane of the objective. The reflected light from the PC resonator is the 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, at a particular resonant wavelength and incident angle, complete interference occurs and no light is transmitted, resulting in nearly 100% reflection efficiency. The resonant reflectance magnitude is dramatically reduced (Figure 19C) by the addition of absorbing AuNPs upon the PC surface, resulting in the ability to observe each AuNP by illuminating with light from an LED and making images of the reflected intensity (Figure 19B).

By measuring the resonant peak intensity value (PIV) on a pixel-by-pixel basis across the PC using a microscopy, the output of PRAM is PIV images of attached AuNPs. The images may be gathered by illuminating the structure with collimated broadband light through the transparent substrate, while the front surface of the PC is immersed in aqueous media. The AuNPs are strategically selected to provide strong absorption by localized surface plasmon resonance at the same wavelength. Thus, each surface-bound AuNP registers in the PC reflected image as a location with reduced intensity, compared to the surrounding regions without AuNPs. By immobilizing target-activated AuNP probes on a PC surface, PRAM has been used to quantify nucleic acids and proteins with single-particle resolution. Previous work focused on detecting chemically synthetic miRNAs with similar detection limits to qRT-PCR methods, in which each detected miRNA molecule was associated with one AuNP tag. The current disclosure adds the technical effect of providing an amplification mechanism that further reduces detection limits, as the target nucleic acid molecule is consumed by the detection process. This is achieved through use of DNA-fueled molecular machines, which are cascade DNA circuits that are driven by fuel strands (in TRAP, for example, the miRNA target and the probe sequence act as a fuel) where the whole process cycles due to entropy. The DNA-fueled molecular machines include a series of toehold- mediated DNA strand displacement reactions involving target recycling, as tools for building switchable nanodevices, controlled nanoparticle assembly, mediated gene expression, and programmed DNA computation. This results in the technical effects of a >400 fold reduction in miRNA detection limits, into sub-attomolar concentrations and decreasing the assay time to 20 minutes using PRAM detection in conjunction with target recycling by a DNA-fueled molecular machine. Furthermore, PRAM couple with TRAP offers the additional technical effects of a single step, room temperature, single vessel reaction requiring only synthetic nucleic acids with a low total cost per sample tested.

EXAMPLES

The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only and should not be construed as limiting the scope of the disclosure in any way.

MATERIALS AND METHODS

Photonic crystals

The photonic crystals (PCs) used herein are comprised of a low refractive index periodic grating structure that is coated with a higher refractive index material (TiO2). PCs are fabricated on glass 8-inch diameter glass wafers deposited with a 10 nm etch stop layer of AI2O3. The periodic grating patterns are constructed by depositing a layer of SiO2 followed by large area ultraviolet interference lithography. Finally, a thin layer of TiO2 approximately 100 nm thick is deposited on the etched wafers. The resulting PCs are diced into smaller chips of 1 A~ 1.2 cm². The PC is designed to function as a narrowband optical resonator that optimally reflects = 625 nm with nearly 100% efficiency under water immersion.1

DNA sequence design

Random sequences composed of only A, T, G, and C were designed using NUPACK software to reduce secondary structures and interactions. Domain sequences were verified with NUPACK to ensure selected domain sequences possessed minimal secondary structure and crosstalk. For alkanethiol modification sequences, a 10 poly-A spacer was chosen due to its known low interaction with the gold interface to improve sample stability and coverage. Toehold probes were designed to be robust for changes in temperature, concentration, and salinity by considering AAG° = AGG°(SSSS) - AGG°(XXXX) = 0, where SC is spurious target-linker complex, and XC is the correct target. The branch migration reaction of the toehold strand displacement reaction additionally ensures selectivity, as a single base mismatch causes a AGG° increase of +1.83 to +5.9 kcal/mol.2 AGG° values were also examined in NUPACK. Nucleic acids

Oligonucleotides used in the TRAP capture system were purchased were designed with standard purification. Concentrations of the oligonucleotides 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 as shown in Table 1.

Table 1: Oligonucleotide Sequences

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

Preparation of double stranded linker-protector (LP)

The protector oligonucleotide strand was annealed to the linker sequence with a stoichiometric ratio of 1 :2 with the protector in excess. For the annealing process, the oligos were heated to 95°C for ten minutes then were allowed to cool to room temperature. Annealed linker-protector was diluted in a IxTE, 12.5 mM MgCh and 0.025% Tween20 buffer, then the product was stored at 4 °C until use.

Polyacrylamide gel electrophoresis

A 12% polyacrylamide gel was employed for the verification of the formation of the TRAP nucleic acid complexes and products. Reaction products were loaded on the 1.5 mm thin gel. Electrophoresis was carried out at 165 V for 40 min at room temperature in 1 A-TBE buffer. After separation, the gel was stained by gel red and imaged with the Bio-red fluorescence gel imaging system.

Nanoparticle conjugation

NanoUrchin AuNPs (ImL 80-nm diameter) were functionalized with thiolmodified DNA via gold-sulfur chemistry. Thiol-modified DNA was activated with two equivalents of tris(2-carboxyethyl) phosphine hydrochloride (TCEP). NanoUrchin AuNPs (80-nm) were functionalized by mixing deprotected alkanethiol oligonucleotides with aqueous nanoparticle solution (particle concentration 1 OD) to a final probe strand concentration of 100 nM, then add m-PEGIK to a final concentration of 80 pg/mL. After ~48 h, the solution was centrifuged at 800 ref for 10 min to remove excess thiol -DNA and the supernatant removed using a micropipette. Deposited DNA- AuNPs were rinsed with an equal volume of 10 mM TE (0.025% Tween20, pH 7.4) and the centrifuging/rinsing procedure repeated two times. The final deposition was resuspended in stock solution (10 mM TE 0.025% Tween20, pH 7.4). and stored at 4°C.

Cell culture and exosomal total RNA isolation

MCF-7 and DU145 cells were cultured in Eagle’s Minimum Essential Medium (EMEM) with 10% exosome-depleted fetal bovine serum . Cells werw incubated at 37 °C in 5% CO2 for 72 hours to 90% confluency. Exosomes from cell culture supernatant were isolated with Total Exosome Isolation Reagent via overnight incubation at 2°C to 8°C. After centrifugation at 10,000 x g for 60 min, the exosome pellet was resuspended in PBS. Total RNA was extracted from exosomes. miRNA quantification by qPCR miR-21 and miR-375 were quantified by qPCR with Pre-designed TaqMan primers for miR-21 and miR-375, respectively. Complementary DNAs (cDNAs) were synthesized and the cDNA product diluted 20-fold and 2 pl of diluted cDNA mixed with TaqMan Universal Master Mix II, 1 pl of primers, and RNase-free water to a final volume of 20 pl. The reaction was performed and monitored by a realtime PCR detection system using the following conditions: pre-denature at 95°C for 10 min and 40 cycles of denature (94°C for 40s) and anneal (60°C for 30s). For absolute quantification of miR-375 and miR-21 in MCF-7 and DU145 cells a standard curve was established using synthetic miR-375 AND miR-21 with different concentrations ranging from IfM to 100 nM.

Example 1: Optimization of toehold length and concentration of linker strand

Achievement of ultrasensitive limits of detection using the TRAP system, it is necessary to minimize nonspecific AuNP binding and to demonstrate enhanced target- triggered signaling. In the TRAP design of the current disclosure (Figure 1), toehold-1 participates in the target miRNA-triggered strand displacement reaction, while toehold-2 allows for invasion of the probe-functionalized AuNP. Toehold- 1 was designed with 5 bases and a longer toehold-2 results in faster kinetics and recycling of the target miRNAs. However, with a constant protector strand length, the longer toehold-introduces more uncovered bases on the linker strand terminal and results in non-specific binding in the absence of target miRNA. The design was optimized using four sets of linker sequences with different toehold-2 lengths.

The linker strand was pre-hybridized with the protector strand and mixed with probe DNA and capture strands. The binding characteristics of the different linker sequences were analyzed with native PAGE (Figure 8). Ffour linkers with different sequence options were tested to ensure formation of a stable capture-linker-protector structures and to test that the capture-linker-probe (C-L-P) complex, which mimics a bound AuNP with a probe DNA, was only formed in the presence of the miRNA target. Presence of a C-L-P band in the absence of the miRNA target (Figure 8 Lanes 2, Lane 5, Lane 8, and Lane 11), indicates the length of toehold-2 allows for non-specific binding. The results for the longest linker sequence (L4 with a 4-nt initial toehold) demonstrate that the C-L-P band formed even when no target was present, as a result of non-specific reactivity. In contrast, the shortest linker sequence (LI with 1-nt initial toehold) did not form the C-L-P complex when the miRNA target was added, indicating that the target could not remove the protector strand. As shown in Figure 8, the linker strands (L2 and L3) containing 2 or 3 uncovered bases in the toehold-2 region produced a distinct C-L-P band in the presence of the target but not in the absence of the target strand produce.

The toehold length of the linker strand was further optimized by analyzing the L2, L3 and L4 linkers with two, three, and four initial uncovered bases in the toehold-2 region, respectively with the TRAP system. As shown in Figure 9, in the absence of target miRNA the longest linker strand results in exceedingly high background signal that is consistent with the results from the PAGE experiments in Figure 8. However, after addition of a 10 pM concentration of target miRNA, the linker strand with only two initial uncovered bases on the toehold-2 region demonstrated enhanced signal-to-noise of 142, a 120-fold enhanced ratio versus the three-free base linker. The design of two free bases for the initial uncovered linker terminal toehold (L2) was selected and used in all subsequent experiments.

The TRAP system of the current disclosure the DNA-probe functionalized AuNP and PC tethered capture strand are bridged by the linker strand. After optimizing the uncovered toehold length of the linker strand, the relationship between linker strand concentration and TRAP nucleic acid system sensitivity was evaluated. Concentrations of the linker strands ranging from 0 to 50 pM were evaluated, and the bound AuNPs on the PC surface were counted (Figure 10). The captured AuNPs gradually increased from 174 to 500 when the linker concentrations were increased from 1 pM to 20 pM. However, the AuNPs on the PC surface decreased to 311 as the linker concentration was increased to 50 pM. The optimal concentrations are related to the constant numbers of probe DNA on AuNP and capture strands on PC. In the presence of excess linker strands, both probe DNA and capture strands can hybridize with linker strands individually, resulting in fewer probe DNA and capture strands that are connected by the linker strands. As a result, a 20 pM concentration of the linker-protector complex was determined to be optimal, with the protector in two times excess.

Example 2: Quantification of the Sensitivity of TRAP

A constant reaction volume of 20 pL solution, including target miRNA, the linkerprotector complex, and probe DNA modified AuNPs, was added into a PDMS reservoir. Separate wells were used for different concentrations of miRNAs ranging from 0.1 aM to 1 pM. Reactions were performed at room temperature and imaged after 10 min and 20 min. An increased number of AuNPs were on PC surface (32, 82, 276, to 538 AuNPs) with increasing concentrations of miRNA-375 (0 aM, 1 aM, 1 fM to IpM) at 10 mins (Figure 2a, additional PRAM images are shown in Fig. 11; calibration curve R²-0.957 from 0.1 aM and 1 pM for miRNA-375, Figure 2b) at 10 minutes. With a longer reaction time of 20 minutes (R²= 0.999), the tethered AuNPs on PC increased by approximately 1.5-fold (Figure 3a, and Figure 11) and the linear relationship between the particle counts and miRNA-375 concentrations increased. A 20-minute reaction time provided a higher density of captured AuNPs, slightly better sensitivity and better signal-to-noise for miR-375. On this basis, the limit of detection (LOD; concentration at a signal threshold of three standard deviations (3c) above background noise (3c+blank)), was calculated as 0.24 aM for miRNA-375 at 20 minutes (Figures 2b and 11). The robustness and universality of TRAP nucleic acid design was confirmed with another target miRNA-21 that indicated a LOD of 0.356 aM (Fig. 12)

Example 3: Quantification of the Sensitivity of TRAP

Dose-dependent immobilized AuNPs on the PC surface under various concentrations of target miRNA-375 were used to determine the sensitivity of TRAP. Target miRNA, the linker-protector complex, and probe DNA modified AuNPs (20 pL), was added into a PDMS reservoir. Separate wells were used for different concentrations of miRNAs ranging from 0.1 aM to 1 pM. Reactions were performed at room temperature and imaged after 10 min and 20 min. An increased number of AuNPs on PC surface (32, 82, 276, to 538) was observed with higher concentrations of miRNA-375 (0 aM, 1 aM, 1 fM to IpM) at 10 mins (Figures 2a and 11). A linear calibration (Figure 2b) from 0.1 aM and 1 pM for miRNA-375 demonstrated an R² = 0.957) at 10 minutes. A 20 minute reaction time (R2= 0.999), resulted in the tethered AuNPs on PC increaseing by approximately 1.5-fold (Figures 3a and 11) with an increased linear relationship between the particle counts and miRNA-375 concentrations. A 20-minute reaction time provided a higher density of captured AuNPs, better sensitivity and better signal-to-noise for miR-375. On this basis, the limit of detection (LOD) was calculated as 0.24 aM for miRNA-375 at 20 minutes (Figures 2b and 11). The robustness and universality of TRAP nucleic acid design, was demonstrated using a second target miRNA-21 was investigated and a LOD of 0.356 aM was observed (Fig. 17)

Example 4: Selectivity of TRAP at single-base precision

The selectivity of TRAP at single-base precision as determined at five different single nucleotide variants (SNVs) of miRNA-375, with the mismatch position at the 1st, 5th, 12th, 18th and 22nd from the 5' end tested in TRAP. The wild type of miRNA-375 target was tested at concentration of 1 fM, and the mismatched SNVs were tested at 1 pM. The TRAP images of wild type of miRNA-375 target showed -302 nanoparticles while the mismatched SNVs demonstrated a background signal of 72 nanoparticles or fewer on PC (Figure 3). When mismatch positions are located away from the initial toehold and using a miRNA with a 1000-fold mismatch (1 pM) of the correct target, the resulting signal was still less than 25% of the signal of the correct target at IfM. This demonstrates that the TRAP system keeps high selectivity at single-base precision for miRNA detection. The branch migration reaction of the toehold strand displacement reaction additionally ensures selectivity, as a single base mismatch causes a AGG° increase of +1.83 to +5.9 kcal/mol.

Example 5: Multiplexing miRNA detection with one-batch TRAP

The TRAP system can be readily adapted to detect any miRNA sequence, and by measuring separate sub-volumes of a test sample in independent wells on the same PC biosensor, multiple assays can be performed in parallel. This capability was demonstrated using three DNA strands designed and optimized using the same principles and steps as detailed herein, above, with the feasibility of the DNA probes confirmed using PAGE analysis (Figure 13). In the multiplex TRAP assays (Figure 4a), the same capture strands were used in every reservoir, and the only differences between the wells were the target miRNAs (Figure 9b), nanoparticle probes, and the specially designed linker-protector complexes. PRAM images were recorded after a 20 minute incubation and bound AuNP counts calculated (Figure 9d). In the presence of 1 f miRNAs, elevated counts of nanoparticles are observed upon the PC surface while the blank controls (with no target) displayed weak signals. Data for all five miRNA targets tested was similar demonstrating that TRAP is applicable to a variety of miRNA targets that can be quantified simultaneously.

Example 6: Monitoring miRNA-375 and miRNA-21 expression 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 affecting tumorigenesis and metastasis. TRAP was used to diagnose cancer through monitoring the miRNAs expression in exosomes, miRNA-375 and miRNA-21 were chosen as the models.

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

Dilutions of the exosomal miRNA extract, dose-responsive curves were obtained (Figures 15-18) and the amount of miRNA-21 in MCF-7 cell-derived exosomes was higher (up to 2535x) than miRNA-375 and the amount of exosomal miRNA-21 in DU145 cell- derived exosomes was higher (up to 2412x) than that of miRNA-375. The amount of miRNA-375 and miRNA-21 in DU145 cell-derived exosomes was similar to that in MCF-7. As shown in Figure 5b, the results obtained by the two methods were consistent, which proved the accuracy and reliability of the TRAP method.

The detection limit of TRAP for miRNA-375 and miRNA-21 in cancer cell exosomes derived from MCF-7 and DU145 cultures are 1.2 copies/pL (2 aM of miRNA-375 and miRNA-21 in MCF-7 and DU145, respectively), which is 31-fold and 61-fold lower than that from qRT-PCR, respectively. Example 7: Coupling Behavior of AuNPs with PC

The coupling behavior of AuNPs with the PC, was achieved using a finite element method (FEM) simulation to investigate the near-field intensity distribution of the PC bound AuNP (Figure 12a). Optical absorption of PC-coupled AuNPs was further analyzed by measuring the PC resonant reflected spectrum (Figures 12b and 12c). AuNP binding resulted in a localized quenching of the PC reflection intensity AI/I of approximately 12%, which is used as the contrast mechanism for image-based detection of single PC-attached nanoparticles.

Conclusion

In conclusion, a target recycling amplification process (TRAP) detection method was developed using photonic resonator absorption microscopy, which has utility in monitoring exosomal miRNAs with ultra-sensitivity and single-base mismatch selectivity. The approach is a single-step, wash-free, enzyme-free, isothermal, 20-minute, room temperature process that can be readily adapted toward any miRNA target through different DNA probe designs. The AuNP-probe complex is designed to be universally used for all targets. The TRAP method achieves cancer biomarker miRNA detection with a detection limit of a single copy of nucleic acid to 0.24 aM for miRNA-375 and 0.356 aM of miRNA-21. The target miRNA concentration can be measured over a broad range from 1 aM to 1 pM with single-base precision, through digital counting of bound AuNPs on photonic crystal surfaces.

TRAP can be performed with multiplexed miRNA detection in a single batch. Compared with traditional qRT-PCR methods, TRAP showed similar accuracy in profiling exosomal miRNAs derived from cancer cells, but also exhibited at least 31-fold and 61-fold enhancement in the limits of miRNA-375 and miRNA-21 detection, respectively.

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Claims

CLAIMS What is claimed is:

1. A system for detecting nucleic acids in a sample, comprising: a biosensor, comprising a photonic crystal (PC), wherein the PC is immobilized to a nucleic acid capture strand sequence; a nucleic acid linker strand 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 imaging platform, wherein: the capture strand is pre-treated with the linker-protector complex thereby binding the linker-protector complex to the capture strand and creating a first toehold; target RNA in the sample is capable of binding the first toehold thereby displacing the protector strand from the linker-protector complex; the AuNP probe tethers to the linker strand at a second toehold region, thereby displacing the target RNA; and wherein the imaging platform is configured to quantify the displaced target RNA in the sample by measuring the tethered AuNP.

2. The system of claim 1, wherein the binding of the linker-protector complex to the capture strand creates the first toehold region on the linker strand.

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

4. The system of claim 3, wherein the binding of the target RNA to the first toehold region displaces the protector strand and exposes the second toehold region.

5. The system of claim 4, wherein the AuNP probe binds to the 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 probe is imaged using photonic resonator absorption microscopy (PRAM).

7. The system of claim 7, wherein the tethered AuNP at a given location on the PC results in a reduction in reflected light intensity from the PC surface at that location.

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

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

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

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

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

13. The system of claim 1, wherein 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.

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

15. The system of claim 1, wherein the nucleic acids are any nucleic acids that form complementary base pairs.

16. The system of claim 1, wherein the nucleic acids are DNA.

17. The system of claim 1, wherein the nucleic acids are RNA.

18. The system of claim 1, wherein the nucleic acids are peptide nucleic acids (PNA).

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

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

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

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

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

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

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

26. The system of claim 1, wherein the biosensor comprises a waveguide structure through which light travels 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. A biologic assay comprising: a biosensor containing a capture strand oligonucleotide sequence and a linker strandprotector strand oligonucleotide complex; a reaction solution; oligonucleotide strands; and a population of nanoparticles; wherein the nanoparticles are bound to the surface of the biosensor using the oligonucleotide strands, and wherein the nucleotide strands are comprised of random nucleic acid sequences.

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

31. The biological assay of claim 29, wherein the population of nanoparticles are tethered to an oligonucleotide sequence.

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

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

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

35. The biologic assay of claim 29, wherein the nanoparticles are gold nanoparticles, quantum dots, metal-based nanoparticles, magnetic nanoparticles, fluorophores, nanodiamonds, plasmonic fluors or nanoparticles comprised of dielectric materials such as SiCh or TiCh.

36. The biologic assay of claim 21, wherein the biosensor comprises a photonic crystal, further comprising an imaging platform configured to quantify a resonant peak intensity value measured on a pixel-by-pixel basis across the photonic crystal.

37. The biologic assay of claim 29, wherein the biosensor comprises a non-imaging detection instrument.

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

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

40. The biologic assay of claim 29, wherein the biosensor comprises a waveguide structure through which light travels laterally.

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

42. The biologic assay of claim 29, wherein the biosensor is a photoacoustic biosensor.

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

44. A method for detecting nucleic acids in a sample, comprising the steps of: immobilizing a oligonucleotide capture strand to the surface of a biosensor, thereby creating an assay surface; pretreating the capture strand with a oligonucleotide linker strand - oligonucleotide protector strand complex; adding an assay medium to the assay surface, wherein the assay medium comprises a biological sample that may contain the target RNA, wherein the target RNA is capable of binding to a first free toehold region on the linker strand thereby replacing the protector strand; adding a tethered nanoparticle probe capable of binding to a second toehold region thereby creating releasing the target RNA; and quantifying the number of nanoparticles bound to the second toehold region using an imaging platform.

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

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

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

48. The method of claim 44, wherein the biosensor comprises a photonic crystal, and wherein 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. The method of claim 48, wherein the imaging platform is configured to quantify a resonant peak intensity value measured on a pixel-by-pixel basis across the photonic crystal.

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

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

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

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

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

55. The method of claim 44, wherein the biosensor is a photoacoustic biosensor.

56. The method of claim 44, wherein the nanoparticles are gold nanoparticles, quantum dots, metal-based nanoparticles, magnetic nanoparticles, or nanoparticles comprised of dielectric materials such as SiCh or TiCh.

57. The method of claim 44, wherein after quantification of the tethered nanoparticles the nanoparticles are removed from the biosensor surface.

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

59. The method of claim 57, wherein the nanoparticle is removed from the biosensor surface by agitation of the assay buffer without replacement of the assay buffer.

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