Analyte detection and quantification

Abstract

The invention features universal methods and components for analyte detection, methods and components for quantifying analytes associated with bladder cancer, and treatment of patients identified as having analyte levels predicted for bladder cancer. The universal methods and components can be used to detect the presence of small amounts of analytes in a sample.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 407,417 filed on September 16, 2022 and U.S. Provisional Application No. 63 / 448,134 filed on February 24, 2023, the disclosures of which are incorporated herein by reference in their entirety.

[0003] Reference to a sequence listing submitted electronically

[0004] The contents of the electronic sequence listing (068469P1WO1.xml; size: 27,355 bytes; and creation date: September 6, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present invention relates to various areas, including general methods and compositions for analyte detection and quantification, methods and compositions for detecting analytes associated with bladder cancer, and bladder cancer treatment. Background Art

[0006] Plasmon sensors can be used to detect the presence or amount of a variety of different analytes, including environmental toxins, polysaccharides, bacteria, viruses, proteins, enzymes, ligands, nucleic acids, substrates, exosomes, cells, drug levels, and tissues. The detection of specific analytes is facilitated by the use of capture molecules that specifically bind to the analyte, thereby facilitating the separation of the target analyte from other analytes. A variety of different plasmon phenomena can be used to measure the captured analytes, including surface plasmon resonance, localized surface plasmon resonance, surface-enhanced Raman scattering, surface-enhanced infrared absorption spectroscopy, and surface-enhanced fluorescence.

[0007] Analyte detection and quantification is useful in many areas, including detection of food contaminants and pathogens, detection of environmental toxins, and detection of biomarkers. Therapeutic and diagnostic applications of surface plasmon resonance-based analyte detection include detection of biomarkers associated with specific diseases or conditions.

[0008] Biomarker detection and quantification can be used to identify subjects who will benefit from therapeutic intervention and guide treatment. For example, surface plasmon resonance-based analyte detection can be used for cancer detection and treatment. Clinical diagnosis is very important in early detection, monitoring tumor progression and treatment response.

[0009] Bellassia et al. 2019 "Front. Chem." 7: 570 mentioned the use of localized surface plasmon resonance and single-stranded DNA for detecting low levels of miR-182, miR-10b, miR-143 and miR-145 associated with bladder cancer.

[0010] U.S. Patent Publication No. 2021 / 0140911 to Habarakada Liyanage et al. includes a description of systems and methods for lateral flow technology that can be used to detect analytes, localized surface plasmon resonance, and various biomarkers, including biomarkers associated with bladder cancer. Summary of the invention

[0011] The invention features general methods and compositions for analyte detection and quantification, methods and compositions for quantifying analytes associated with bladder cancer, and treatment of patients identified as having analyte levels predictive of bladder cancer. The general methods and compositions are described with respect to quantification of various analytes associated with bladder cancer. Combinations of bladder analytes provided may also be detected using other methods.

[0012] Therefore, the first aspect of the present invention relates to an analyte detection signal amplifier. The amplifier comprises (a) an analyte binding molecule; (b) a localized surface plasmon resonance nanostructure; and (c) (i) a bioluminescence resonance energy transfer (BRET) assembly complex, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor, or (ii) a fluorescence resonance energy transfer (FRET) assembly complex, wherein the FRET assembly complex comprises a fluorescence donor conjugated to a fluorophore acceptor.

[0013] Another aspect of the present invention relates to detecting or quantifying an analyte in a sample, comprising the following steps:

[0014] a) contacting a localized surface plasmon resonance sensor with the sample, wherein the sensor comprises an analyte capture molecule;

[0015] b) providing an analyte detection signal amplifier to the sample, wherein the analyte detection signal amplifier comprises a BRET assembly complex and a localized surface plasmon resonance nanostructure and binds to the analyte;

[0016] c) adding luciferase substrate; and

[0017] d) Detection of fluorescence, bioluminescence, localized surface plasmon resonance or surface enhanced Raman scattering.

[0018] Another aspect of the present invention relates to detecting or quantifying an analyte in a sample, comprising the following steps:

[0019] a) contacting a localized surface plasmon resonance sensor with the sample, wherein the sensor comprises an analyte capture molecule;

[0020] b) providing the analyte detection signal amplifier to the sample, wherein the analyte detection signal amplifier comprises a FRET assembly complex and a localized surface plasmon resonance nanostructure and binds to the analyte;

[0021] c) stimulation; and

[0022] d) Detection of fluorescence, localized surface plasmon resonance or surface enhanced Raman scattering.

[0023] Another aspect of the present invention relates to a multi-analyte detection system for detecting an analyte whose presence or amount is associated with bladder cancer. The system comprises:

[0024] a) a first LSAP nanostructure sensor, wherein the first LSAP nanostructure sensor comprises an analyte capture molecule of miR-205;

[0025] b) a second LSAP nanostructure sensor, wherein the second LSAP nanostructure sensor comprises an analyte capture molecule of miR-16-1;

[0026] c) a third LSAP nanostructure sensor, wherein the third LSAP nanostructure sensor comprises an analyte capture molecule of miR-143;

[0027] d) a fourth LSAP nanostructure sensor, wherein the fourth LSAP nanostructure sensor comprises an analyte capture molecule of UCA1 nucleic acid;

[0028] e) a fifth LSAP nanostructure sensor, the fifth LSAP nanostructure sensor comprising an analyte capture molecule of IGF2 or IGF2 nucleic acid; and

[0029] f) A sixth LSAP nanostructure sensor comprising an analyte capture molecule of ANXA10 or ANXA10 nucleic acid; wherein each sensor type may be present on one or more platforms.

[0030] Reference to each sensor type being present on "one or more platforms" means that each platform may have a particular sensor (e.g., multiple sensors of the same type), or that a particular platform may have two or more different types of sensors. In various embodiments, a particular platform has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more than 11 different types of sensors.

[0031] The BRET or FRET assembly complexes and LSPR nanostructures used in different signal amplifier types (e.g., the first, second, third, fourth, fifth, or sixth) can be the same or different; and the LSPR nanostructures used in different sensor types can be the same or different. The use of LSPR nanostructures with different compositions, different sizes, and / or different shapes and different BRET or FRET assembly complexes can be used to facilitate the use of different types of sensors and / or amplifiers on the same platform. In certain embodiments, each platform has the same type or no more than three different types of sensors; each BRET or FRET is the same; each amplifier LSPR nanostructure is the same; and each sensor LSPR nanostructure is the same.

[0032] Another aspect of the present invention relates to a method for determining whether a subject has bladder cancer, the method comprising the step of detecting the presence or amount of miR-205, miR-16-1, miR-143, UCA1 nucleic acid, IGF2 or IGF2 nucleic acid, and ANXA10 or ANXA10 nucleic acid.

[0033] Another aspect of the invention relates to a method of treating bladder cancer in a subject, the method comprising administering to a subject determined to have bladder cancer using the methods described herein a therapeutically effective amount of a bladder cancer therapeutic.

[0034] Other features and advantages of the present invention are apparent from the additional descriptions (including different examples) provided herein. The examples provided illustrate different components and methods that can be used to practice the present invention. Such examples do not limit the claimed invention. Based on this disclosure, those skilled in the art can identify and adopt other components and methods that can be used to practice the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A BRET assembly complex is shown, wherein the BRET assembly complex comprises a luciferase donor (bioluminescence donor) conjugated to a fluorophore acceptor via a Halo tag protein. The Halo tag protein is covalently conjugated to both the luciferase donor and the fluorophore acceptor.

[0036] Figure 2 Examples are provided that illustrate general aspects and embodiments.

[0037] Figure 3A and 3B Calibration plots measuring luminescence and fluorescence of different analytes are shown. Figure 3A CRH, ANAX10, ABL1, IGF2, and UPKB1 luminescence were demonstrated. Figure 3B CRH, ANAX10, ABL1, IGF2, and UPKB1 fluorescence are shown.

[0038] Figure 4A and 4B Calibration plots measuring luminescence and fluorescence of different analytes are shown. Figure 4A Luminescence of miR-200c (200c), miR-16-1 (16-1), miR-205 (205), miR-143 (143) and long noncoding RNA (UCA) was demonstrated. Figure 4B Fluorescence of miR-200c (200c), miR-1 6-1 (16-1), miR-205 (205), miR-143 (143) and long noncoding RNA (UCA) are shown.

[0039] Figure 5A and 5B Calibration plots measuring luminescence and fluorescence of different analytes are shown. Figure 5A NMP22, HCFHrp (hcfHRP), and P53 luminescence are shown. Figure 5B NMP22, HCFHrp (hcfHRP), and P53 fluorescence are shown. DETAILED DESCRIPTION

[0040] The invention features methods and compositions for analyte detection and quantification, methods and compositions for quantifying analytes associated with bladder cancer, and treatment of patients identified as having analyte levels predictive of bladder cancer. As illustrated in the examples provided below, the provided methods and compositions can quantitatively detect small amounts of analytes associated with bladder cancer. Analytes associated with bladder cancer include analytes whose elevated levels characterize bladder cancer and analytes whose decreased levels characterize bladder cancer.

[0041] In certain embodiments, the methods detect an analyte present at a concentration as low as about 0.01 aM, 0.1 aM, or 10 aM. In further embodiments, the detected analyte is present at a concentration less than about 50 aM, 25 aM, 10 aM, 1 aM, or 0.1 aM.

[0042] Different subject areas involving analyte detection include food safety, such as detection of contaminants such as pathogens, mycotoxins, plant and biological marine toxins, toxic chemicals, preservatives, and antioxidants; environmental contaminants such as pesticides, herbicides, aromatic compounds, chemical mixtures, and toxic metal contaminants; human safety, such as detection of explosives and chemical warfare; and health, such as detection of biomarkers associated with specific diseases or conditions, including certain peptides, proteins, DNA, and RNA, and detection of infectious organisms. (See, e.g., Camarca et al., Sensors (Basel) 2021 Jan 29;21(3):906.)

[0043] Therapeutic applications of detecting and quantifying biomarkers associated with specific diseases and sequences can include diagnosis, prognosis, and treatment guidance. Diagnostic applications include early detection and obtaining information about the specific type of disease or condition and the severity or stage of the disease or condition. Prognostic applications include predicting disease outcome and treatment response. Treatment guidance involves the type of treatment selected, monitoring treatment, and adjusting treatment based on the level of a specific analyte.

[0044] Reference to a "subject" refers to mammals, including humans; non-human primates, such as apes, gibbons, gorillas, chimpanzees, orangutans, and macaques; livestock, such as dogs and cats; farm animals, such as poultry, ducks, horses, cattle, goats, sheep, and pigs; and experimental animals, such as mice, rats, rabbits, and guinea pigs. Preferred subjects are humans.

[0045] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to nucleotide polymers, without regard to function or size. Nucleic acids and polynucleotides contain at least two nucleotides and include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA); and in some cases, DNA or RNA derivatives having nitrogenous bases capable of hydrogen bonding with DNA or RNA, wherein the derivatives contain modified nucleobases, sugar moieties, and / or phosphodiester bonds. When discussing nucleic acids and polynucleotides, unless otherwise indicated, the sequences provided are in the 5' to 3' direction.

[0046] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0047] The connecting term "and / or" between multiple listed elements covers both individual options and combined options. For example, where two elements are joined by "and / or", the first option refers to the applicability of the first option without the second option, the second option refers to the applicability of the second option without the first option, and the third option refers to the applicability of the first option and the second option together. Any one of the options is understood to fall within the meaning and thus satisfy the requirements of the term "and / or". The simultaneous applicability of more than one of the options is also understood to fall within the meaning of the term "and / or".

[0048] The terms "or" and "and" have the same meaning as "and / or" unless the context clearly indicates otherwise.

[0049] References to terms followed by various members or examples such as "including," "for example," "eg," "such as," are open-ended descriptions where the listed members or examples are illustrative and additional members or examples may be provided or used.

[0050] The terms "polypeptide", "protein" and "peptide" can be used interchangeably to refer to amino acid sequences without regard to function. Polypeptides and peptides contain at least two amino acids, while proteins contain at least about 10 amino acids. The amino acids provided include naturally occurring amino acids and amino acids provided by cell modification.

[0051] References to "comprise" and variations such as "comprises" and "comprising" used with respect to an element or group of elements are open ended and do not exclude additional, unrecited elements or method steps. Terms such as "including," "containing," and "characterized by" are synonymous with comprising. In the various aspects and embodiments described herein, references to open ended terms such as "comprising" may be replaced by "consisting of" or "consisting essentially of."

[0052] Reference to "consisting of" excludes any element, step or ingredient not specified in the listed claim elements, where such element, step or ingredient is relevant to the claimed invention.

[0053] Reference to "consisting essentially of" limits the scope of a claim to the specified materials or steps and to those materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0054] The term "about" refers to a value within 1 0% (i.e., ± 10%) of the underlying parameter. For example, "about 1:10" includes 1.1:10.1 or 0.9:9.9, and "about 5 hours" includes 4.5 hours or 5.5 hours. The term "about" at the beginning of a string of values ​​modifies each of the values. In one embodiment, the term about refers to a range within 5% of the underlying parameter.

[0055] Unless the context clearly indicates otherwise, all numerical values ​​or numerical ranges include integers within such ranges as well as fractions of numerical values ​​or integers within such ranges. References to integers greater than (more) or greater than or less than include numbers greater than or less than the referenced number, respectively.

[0056] Various references, including articles and patent publications, are cited or described in the Background and throughout the specification. Each of these references is incorporated herein by reference in its entirety. No one reference is admitted to be prior art with respect to any invention disclosed or claimed. In some cases, specific references are indicated to be incorporated herein by reference to highlight the incorporation.

[0057] The definitions provided herein, including definitions in this section and other sections of this application, apply throughout this application.

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0059] The specification has been divided into various chapters and paragraphs, and examples of various embodiments are provided. These separations should not be considered as separating the content of one paragraph or chapter or embodiment from the content of another paragraph or chapter or embodiment. The description provided has a wide range of applications and covers all combinations of various chapters, paragraphs and sentences that can be expected. The discussion of any embodiment is intended to be exemplary only, and is not intended to imply that the scope of the present disclosure, including the claims (unless otherwise provided in the claims), is limited to these examples.

[0060] The present invention is generally disclosed in this article using affirmative language to describe many embodiments of the present invention. The present invention also specifically includes embodiments in which specific themes are excluded in whole or in part, such as substances or materials, method steps and conditions, schemes or procedures. For example, in certain embodiments of the present invention, materials and / or method steps are excluded. Therefore, even if the present invention is generally not expressed in this article according to the content that the present invention does not include, the embodiments that are not clearly excluded in the present invention are still disclosed in this article.

[0061] I. Analyte Detection Signal Amplifier

[0062] The analyte detection signal amplifier comprises (a) an analyte binding molecule; and (b) a localized surface plasmon resonance nanostructure and (i) a bioluminescence resonance energy transfer (BRET) assembly complex, or (ii) a fluorescence resonance energy transfer (FRET) assembly complex.

[0063] Analyte binding molecules specifically bind to the analyte. A variety of different types of analytes can be detected with appropriate binding molecules. Examples of different types of analytes that can be detected include proteins, polypeptides, exosomes, nucleic acids, viruses, cells, tissues, polysaccharides, lipoproteins, lipids, and therapeutic compounds.

[0064] In certain embodiments, the analyte is a protein or polypeptide. Examples of protein and polypeptide analytes that can be detected include enzymes, substrates, antibodies, protein hormones, receptors, and cytokines.

[0065] In certain embodiments, the analyte is a nucleic acid. Examples of nucleic acids that can be detected include RNA and DNA, such as mRNA, tRNA, siRNA, lncRNA, piwiRNA, snoRNA, tircRNA, mitochondrial RNA, non-coding RNA, miRNA, cDNA, genomic DNA, and enzymes (e.g., ribozymes).

[0066] In certain embodiments, the analyte is extracellular RNA, such as extracellular mRNA.

[0067] In certain embodiments, the analyte is an extracellular vesicle, such as an extracellular exosome. Extracellular exosomes may contain cellular components that are different from the cells from which they originate, such as lipids, nucleic acids, and proteins.

[0068] In certain embodiments, the analyte is a virus. Examples of viruses that can be detected include pathogenic viruses that infect a subject, a plant, agricultural product, or a food; or are present in the environment (eg, water supply and ventilation).

[0069] In certain embodiments, the analyte is a cell. Examples of cells that can be detected include bacterial assemblies (e.g., present in the intestine); pathogenic bacteria that infect a subject, a plant, agricultural product, or a food; or present in the environment (e.g., water supply and ventilation).

[0070] In certain embodiments, the analyte is a polysaccharide. Examples of polysaccharides that can be detected include polysaccharides associated with bacteria or a particular disease or condition in a subject.

[0071] In certain embodiments, the analyte is a lipoprotein. Examples of lipoproteins that can be detected include high-density lipoproteins, low-density lipoproteins, and bacterial lipoproteins.

[0072] In certain embodiments, the analyte is a lipid. Examples of lipids that can be detected include cholesterol and hormones.

[0073] In certain embodiments, the analyte is a therapeutic compound. Examples of therapeutic compounds that can be detected include small molecules and macromolecules, such as antibodies and proteins.

[0074] In certain embodiments, the analyte is an environmental pollutant. Examples of pollutants that can be detected include air pollutants, such as nitrogen oxides and sulfur dioxide; water pollutants, such as pesticides, heavy metals, and perfluoroalkyl and polyfluoroalkyl substances (PFAS); and soil pollutants, such as pesticides, heavy metals, and PFAS.

[0075] In certain embodiments, the analyte is an illegal drug.

[0076] Different configurations of analyte binding molecules, LSPR nanostructures, and BRET assembly complexes or (FRET) assembly complexes are possible, such as: (1) both analyte binding molecules and BRET or FRET assembly complexes are linked to LSPR nanostructures; (2) both LSPR nanostructures and BRET or FRET assembly complexes are linked to antigen binding molecules; and (3) both LSPR nanostructures and analyte antigen binding molecules are linked to BRET or FRET assembly complexes.

[0077] IA Analyte Binding Molecules

[0078] Analyte binding molecules are able to specifically bind to the analyte of interest. Specific binding refers to the ability to bind to the analyte based on the specific structure of the analyte and to distinguish (e.g., bind to a significantly greater degree) the targeted analyte from other analytes naturally present in the sample (e.g., biological sample). Although absolute specificity is very helpful for some applications, it may not be necessary. For example, the analyte binding molecule can be used in combination with a capture molecule, both of which have specificity for the same analyte and help detect the presence of the analyte. In different embodiments, the specificity of the binding molecule to the target analyte is at least 10X, at least 100X, or at least 1000X of other analytes present in the sample being tested.

[0079] In certain embodiments, the analyte binding molecule is a single-stranded binding oligonucleotide. The single-stranded binding oligonucleotide comprises a purine or pyrimidine nucleobase or a derivative thereof that is capable of hydrogen bonding to a nucleobase present in DNA or RNA via Watson-Crick hydrogen bonds. Naturally occurring DNA and RNA contain purine (guanine, cytosine, and less commonly hypoxanthine) or pyrimidine (thymine, uracil, or adenine) nucleobases and a backbone consisting of ribose (RNA) or 2′-deoxyribose (DNA) sugars linked together by phosphodiester groups.

[0080] Various modifications can be made to different polynucleotide components to provide modified oligonucleotides that can hydrogen bond with DNA or RNA having a complementary nucleotide sequence. Examples of purine modifications include 2,6-diaminopurine, 3-deaza-adenine, 7-deaza-guanine, and 8-azidoadenine. Examples of pyrimidine modifications include 2-thiothymidine, 5-formamide-uracil, 5-methyl-cytosine, 3-ethynyl-uracil. Examples of phosphodiester modifications include methylphosphonates, thiophosphates, and guanidinopropylphosphoramidates. Examples of phosphate substitutions include triazoles and guanidinium salts. Examples of sugar modifications include 2′-modifications such as 2′-F, 2′-methoxy, 2′-amino, and 2′-azido; locked sugars; 3′ end modifications; and 5′ end modifications. (Ochoa and Milam, Molecules 2000, 25, 4689, which is hereby incorporated by reference in its entirety.)

[0081] Another example of modification includes peptide nucleic acids, in which the sugar-phosphate backbone is replaced by a neutral pseudopeptide backbone. Peptide nucleic acids retain nucleobase complementarity and the ability to hybridize with complementary DNA and RNA. Peptide nucleic acids can be produced, for example, by replacing the phosphodiester backbone with N-(2-aminoethyl)glycine, in which the nucleobase is connected to the backbone by a methylene carbonyl linker. Additional peptide nucleic acid structures and design considerations are provided in the following documents: Brodyagin et al., Journal of Organic Chemistry (J.Org.Chem.) 2021, 17, 1641-1688; and Moccia et al., Artif. DNA: PNA & XNA. 20145 (3): e1107176; each of the documents in the document is hereby incorporated herein by reference in its entirety.

[0082] The size of the oligonucleotide binding molecule and the degree of complementarity with the target DNA or RNA can vary. The degree of complementarity that provides specificity will vary depending on the oligonucleotide structure, for example, purine to pyrimidine nucleobases, and modifications to nucleobases, sugars and / or phosphodiester groups; and reaction conditions. In various embodiments, regarding the size of the binding oligonucleotide molecule, the oligonucleotide comprises at least 10, at least 12, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleobases. In various embodiments, with respect to the degree of complementarity to the target nucleic acid analyte, the oligonucleotide binding molecule comprises a region of 10 or more, 11 or more, 12 or more, 13 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more nucleobases that are at least 90%, at least 95%, or 100% complementary to the target nucleic acid analyte.

[0083] In certain embodiments, the analyte binding molecule is an antibody or a binding fragment comprising an antibody. Antibody binding fragments contain three complementary determining regions in the variable framework region that allow antigen binding. Examples of binding fragments include FAb fragments, single chain variable region fragments (scFV), single domain fragments (dAb), Fv fragments, Camelidae heavy chain variable domains (VHH), mini antibodies and bifunctional antibodies.

[0084] In certain embodiments, the analyte binding molecule is an aptamer. An aptamer is a small single-stranded oligonucleotide with a three-dimensional structure that can specifically bind to a target. An aptamer may be composed of naturally occurring nucleotides or may have one or more modifications. An aptamer may bind to a variety of targets, including bacteria, viruses, proteins, toxins, cells (e.g., cancer cells), and tissues. Initial aptamer selection may be performed using a combinatorial oligonucleotide library by in vitro selection and an iterative process (e.g., systematic evolution of ligands by exponential enrichment (SELEX)). (Kulabhausan et al., Pharmaceutics 2020, 12, 646; and Adachi and Nakamura, Molecules 2019, 24, 4229; both documents are hereby incorporated herein by reference in their entirety.)

[0085] In certain embodiments, the analyte binding molecule is a protein. Different types of proteins can be used to bind to the analyte, such as enzymes for binding to substrates, substrates for binding to enzymes, receptors for binding to ligands, and ligands for binding to receptors.

[0086] In certain embodiments, the analyte binding molecule is a ligand. A ligand may or may not be a protein.

[0087] The analyte binding molecules can be directly anchored to the LSPR nanostructure or can be anchored via a linker. Depending on the LSPR nanostructure, the anchoring can be achieved via electrostatic interactions or covalent bonds.

[0088] Examples of nanoparticle surface immobilization chemistries include: (1) covalent coupling of nanoparticles to linker thiol groups; (2) covalent coupling of active ester-modified nanoparticles to linker amino groups; (3) coupling of maleimide-functionalized nanoparticles to thiol groups; (4) click reaction between dibenzocyclooctyne-modified nanoparticles and azido linkers; (5) electrostatic adsorption of negatively charged nanoparticles and positively charged linker groups; and (6) biotin-neutravidin-mediated attachment.

[0089] The length and composition of the analyte binding molecule linker can vary. The individual atoms of the linker can include atoms such as carbon, nitrogen, silicone, fluorine, oxygen, sulfur, hydrogen and phosphorus.

[0090] Linkers can comprise different types of groups or polymers, such as polyethylene glycol (PEG), polyamino acids, polyacrylamides, polyvinylpyrrolidone, zwitterionic polymers, polysaccharides, poly(N-(2-hydroxypropyl)methacrylamide), poly(oligo(ethylene glycol)methyl ether methacrylate), carboxydextran, hydrocarbon chains, and substituted hydrocarbon chains.

[0091] In certain embodiments, the linker comprises PEG. Different types of PEG can be used, such as linear and branched PEGs of different sizes. In certain embodiments, the PEG is less than about 5K. The examples provided below illustrate PEG-SH (1K) with COOH and NH2 functional groups.

[0092] In certain embodiments, PEG is directly linked to the analyte binding molecule, where PEG serves as both a linker (anchor to the nanoparticle) and a spacer.

[0093] In certain embodiments, PEG is a spacer and is attached to the nanoparticle via another moiety. For example, the linker has a SH moiety that is directly attached to the nanoparticle and is anchored to the nanoparticle via an Au-S bond.

[0094] IBBRET assembly complex

[0095] The BRET assembly complex comprises a luciferase donor connected to an acceptor fluorophore via a luciferase donor-acceptor linker. Upon oxidation of the luciferase substrate, energy is transferred from the luciferase donor to excite the fluorophore acceptor. The emission from the luciferase donor and the fluorophore can be measured. The emission can be characterized by, for example, wavelength, intensity, lifetime, and polarization.

[0096] Energy transfer occurs between the luciferase donor and the fluorophore acceptor when the donor and acceptor are within approximately <10 nm of each other. The closer the donor and acceptor are to each other, the stronger the emission.

[0097] In certain embodiments, the distance between the luciferase donor and the fluorophore acceptor is about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, or about 10 nm.

[0098] Luciferase donor-acceptor linkers can be composed of atoms such as carbon, nitrogen, silicone, oxygen, sulfur, hydrogen, fluorine, and phosphorus.

[0099] Linkers can comprise different types of groups or polymers, such as PEG, polyamino acids, polyacrylamides, polyvinylpyrrolidone, zwitterionic polymers, polysaccharides, poly(N-(2-hydroxypropyl)methacrylamide), poly(oligo(ethylene glycol)methyl ether methacrylate), carboxydextran, hydrocarbon chains, and substituted hydrocarbon chains.

[0100] Linkers provide a stable structure and can be composed of different groups linked together by different types of molecular interactions and can be produced using different techniques. For example, Figure 1 The BRET assembly complex shown in the examples below contains a luciferase molecule connected to a HaloTag protein, wherein the HaloTag protein is connected to a fluorophore molecule. Considering the diameter of the haloTag protein connected to the luciferase donor (about 3.3nm, see, for example, Yazaki et al., "Nucleic Acids Research", Vol. 48, No. 2, January 24, 2020, p. e8) and the 12-atom linker (about 1.2nm) connected to the fluorophore, the overall distance between the luciferase molecule and the fluorophore is about 4.5nm. Alternative techniques for connecting different groups include EDC / NHS coupling, click chemistry, streptavidin-biotin, and complementary oligonucleotides.

[0101] A variety of BRET variants have been developed using different luciferase donors, substates, and fluorophore acceptors. (See, e.g., Dale et al., Front. Bioeng. Biotechno1. 2019, 7, 56, which is hereby incorporated by reference in its entirety.) Examples of different variants are shown in Table 1.

[0102] Table 1

[0103]

[0104] GFP refers to green fluorescent protein, eYFP refers to enhanced yellow fluorescent protein, and RLuc refers to Renilla luciferase. -HT is nanoluciferase, also known as Nluc. Furimazine is 2-furanylmethyl-deoxy-coelenterazine.

[0105] Nluc is derived from the 19kDa subunit of a larger multicomponent luciferase isolated from the deep-sea shrimp Oplophorus gracilirostris. The luminescence of the 19kDa subunit was enhanced by mutagenesis, and a variety of coelenterazine analogs were screened to optimize the substrate. (Dale et al., Frontiers in Bioengineering and Biotechnology, 2019, 7, 56.)

[0106] Examples of additional luciferase donors / substrates include teLuc and diphenyltetrazine. teLuc and diphenyltetrazine are derivatives of Nluc and coelenterazine. (Dale et al., Frontiers in Bioengineering and Biotechnology, 2019, 7, 56.)

[0107] Examples of additional fluorophores and fluorophore systems include boron-dipyrromethene (BODIPY), allylol-tetramethylrhodamine (TAMRA), 4-nitro-7-aminobenzofurazan, Alexa Fluor TM and Fluorophore systems. The system consists of a small halotag protein (33 kDa) fused to a protein of choice and a chloroalkane linker, which is attached to a fluorophore. (Dale et al., Frontiers in Bioengineering and Biotechnology 2019, 7, 56.)

[0108] In certain embodiments, the BRET assembly comprising a luciferase donor conjugated to a fluorophore acceptor is selected from the following combinations: NLuc-HT / HL-Oregon Green, RLuc / YFP, RLuc / fluorescent protein (GFP), RLuc8 / GFP, Firefly luciferase / DsRed, RLuc / ODot, Rluc8 / ODot, and NanoLuc / Halotag-fluorescent ligand.

[0109] ICFRET assembly complex

[0110] The FRET assembly complex comprises a donor fluorophore connected to an acceptor fluorophore via a fluorophore donor-acceptor linker. Upon excitation of the donor fluorophore, energy is transferred from the donor to excite the fluorophore acceptor. The emission from the donor and acceptor fluorophores can be measured. The emission can be characterized by, for example, wavelength, intensity, lifetime, and polarization.

[0111] When the donor and acceptor are within approximately <10 nm of each other, energy transfer occurs between the donor fluorophore and the acceptor fluorophore. (Bajar et al., Sensors (Basel) 2016 Sep 14;16(9):1488.) The closer the donor and acceptor are to each other, the stronger the emission. In various embodiments, the distance is about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, or about 10 nm.

[0112] Fluorophore donor-acceptor linkers can include atoms such as carbon, nitrogen, silicone, oxygen, sulfur, fluorine, hydrogen, and phosphorus.

[0113] Linkers can comprise different types of groups or polymers, such as PEG, polyamino acids, polyacrylamides, polyvinylpyrrolidone, zwitterionic polymers, polysaccharides, poly(N-(2-hydroxypropyl)methacrylamide), poly(oligo(ethylene glycol)methyl ether methacrylate), carboxydextran, hydrocarbon chains, and substituted hydrocarbon chains.

[0114] A variety of combinations of FRET donor and acceptor fluorophores have been developed. Examples of different FRET pairs include: cyan fluorescent protein and yellow fluorescent protein; green fluorescent protein and red fluorescent protein; and far-red fluorescent protein and infrared fluorescent protein. Examples of cyan fluorescent protein and yellow fluorescent protein include Aquamarine, ECFP, mTurquoise2, mCerulean3, mTFP1, EYFP, mVenus, sEYFP, mCitrine, and YPet. Examples of green fluorescent protein and red fluorescent protein include EGFP, NowGFP, Clover, mClover3, mNeonGreen, mRuby2, mRuby3, and mCherry. Examples of far-red fluorescent protein and infrared fluorescent protein include mPlum, eqFP650, and mCardinal. (Bajar et al. "Fluorescent protein (florescent protein)" hypertext: / / doi.org / 10.3390 / s16091488, which is hereby incorporated by reference in its entirety.)

[0115] ID signal amplifier LSPR nanostructure

[0116] The signal amplifier LSPR nanostructure can move in the solution, thereby allowing the analyte binding molecule to contact and bind to the analyte. Preferably, the signal amplifier can be dissolved in the sample to be tested.

[0117] The signal amplifier may contain different plasmonic materials and may have different shapes and sizes. Examples of plasmonic materials include metals such as europium, gold, silver, copper, palladium, and aluminum; and non-metals such as graphene, silicon dioxide, and carbon nanotubes.

[0118] In certain embodiments, the plasmonic material comprises a metal such as rhenium, ruthenium, rhodium, palladium, silver, osmium, iridium, platinum, gold, and aluminum; and combinations of materials. (See, e.g., Park et al. Biosensors (Basel), 2022, 17; 12(3): 180. doi: 10.3390 / bios12030180; and Osborne and Pikramenou (2015) Faraday Diss., 1 85: 291.) Examples of combinations include nickel, gold-silver alloy nanoparticles (Au-Ag), gold-palladium alloy nanoparticles (Au-Pd), gold-copper alloy nanoparticles (Au-Cu), silver-copper alloy nanoparticles (Ag-Cu), gold-silver-copper alloy nanoparticles (Au-Ag-Cu), gold-silver-palladium alloy nanoparticles (Au-Ag-Pd), metal oxide nanoparticles (e.g., titanium dioxide and zinc oxide), and metal-semiconductor nanoparticles (e.g., gold-silicon and silver-silicon).

[0119] The LSPR nanostructure can enhance the BRET and FRET signals. The closer the LSPR nanostructure is to the BRET and FRET assembly complex, the greater the enhancement. Preferably, the LSPR nanostructure is within about 5 nm to about 20 nm of the BRET or FRET assembly complex. In certain embodiments, the LSPR nanostructure is 12 nm, 1 3 nm, 14 nm, 1 5 nm, 16 nm away from the BRET or FRET assembly complex.

[0120] II. Localized Surface Plasmon Resonance Sensors

[0121] The LSPR sensor comprises an analyte capture molecule coupled to a LSPR nanostructure. Binding of the analyte to the sensor produces a detectable change in plasmon resonance. In some embodiments, the sensor LSPR nanostructure may also interact with a BRET assembly complex, a FRET assembly complex, and / or an amplifier LSPR nanostructure.

[0122] II.B. Analyte Capture Molecules

[0123] Analyte capture molecules can be used in combination with analyte binding molecules, where both bind to the same analyte. Examples of different types of analytes are noted in Section I. Like analyte binding molecules, analyte capture binding molecules can bind to a variety of different analytes. Although a particular type of analyte capture molecule (e.g., a polynucleotide or protein) can be the same or different from an analyte binding molecule that binds to the same analyte, the combination of analyte capture molecules and analyte binding molecules should be selected to bind to different parts of the analyte so as not to interfere with each other.

[0124] Analyte capture molecules are able to specifically bind to the analyte of interest. Specific binding refers to the ability to bind to the analyte based on the specific structure of the analyte and to distinguish (e.g., bind to a significantly greater degree) the targeted analyte from other analytes naturally present in the sample (e.g., a biological sample). Although absolute specificity is very helpful for some applications, it may not be necessary. For example, analyte capture molecules can be used in combination with binding molecules, both of which are specific to the analyte and help detect the presence of the analyte. In different embodiments, the specificity of the capture molecule for the target analyte is at least 10X, at least 100X, or at least 1000X of other analytes present in the sample being tested.

[0125] In certain embodiments, the analyte capture molecule is a single-stranded capture oligonucleotide.The single-stranded capture oligonucleotide comprises a purine or pyrimidine nucleobase or a derivative thereof that is capable of hydrogen bonding to nucleobases present in DNA or RNA via Watson-Crick hydrogen bonding.

[0126] Various modifications can be made to different polynucleotide components to provide capture oligonucleotide molecules that can hydrogen bond with RNA and DNA with complementary nucleotide sequences. Examples of purine modifications include 2,6-diaminopurine, 3-deaza-adenine, 7-deaza-guanine, and 8-azidoadenine. Examples of pyrimidine modifications include 2-thiothymidine, 5-formamide-uracil, 5-methyl-cytosine, 3-ethynyl-uracil. Examples of phosphodiester modifications include methylphosphonates, thiophosphates, and guanidinopropylphosphoramidates. Examples of phosphate substitutions include triazoles and guanidinium salts. Examples of sugar modifications include 2′-modifications, such as 2′-F, 2′-methoxy, 2′-amino, and 2′-azido; locked sugars; 3′ end modifications; and 5′ end modifications. (Ochoa and Milam, Molecule 2000, 25, 4689, the document is hereby incorporated by reference in its entirety.)

[0127] Another example of modification is a peptide nucleic acid in which the sugar-phosphate backbone is replaced by a neutral pseudopeptide backbone. Peptide nucleic acids retain nucleobase complementarity and the ability to hybridize with naturally occurring DNA and RNA. Peptide nucleic acids can be produced, for example, by replacing the phosphodiester backbone with N-(2-aminoethyl)glycine, in which the nucleobase is connected to the backbone by a methylene carbonyl linker. Additional peptide nucleic acid structures and design considerations are provided in the following literature: Brodyagin et al., Journal of Organic Chemistry, 2021, 17, 1641-1688; and Moccia et al., Artificial DNA: PNA & XNA, 20145 (3): e1107176; each of the documents is hereby incorporated herein by reference in its entirety.

[0128] The size of the oligonucleotide capture molecule and the degree of complementarity with the target DNA or RNA can vary. The degree of complementarity that provides specificity will vary depending on the oligonucleotide structure, for example, purine versus pyrimidine nucleobases, and modifications to nucleobases, sugars, and / or phosphodiester groups; and reaction conditions. In various embodiments, regarding the capture oligonucleotide molecule size, the oligonucleotide comprises at least 10, at least 12, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleobases. In different embodiments, with respect to the degree of complementarity with the target nucleic acid analyte, the oligonucleotide capture molecule comprises a region of 10 or more, 11 or more, 12 or more, 13 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more nucleobases that are at least 90%, at least 95% or 100% complementary to the target nucleic acid analyte.

[0129] In certain embodiments, the analyte capture molecule is an antibody or a binding fragment comprising an antibody. Antibody binding fragments contain three complementary determining regions in the variable region framework that allow antigen binding. Examples of binding fragments include FAb fragments, single chain variable region fragments (scFV), single domain fragments (dAb), Fv fragments, camelid heavy chain variable domains (VHH), mini antibodies and bifunctional antibodies.

[0130] In certain embodiments, the analyte capture molecule is an aptamer.

[0131] In certain embodiments, the analyte capture molecule is a protein. Different types of proteins can be used to bind to the analyte, such as enzymes for binding to substrates, substrates for binding to enzymes, receptors for binding to ligands, and ligands for binding to receptors.

[0132] In certain embodiments, the analyte capture molecule is a ligand.

[0133] The analyte capture molecules can be directly anchored to the LSPR nanostructure or can be anchored via a linker. Depending on the LSPR nanostructure, the anchoring can be achieved via electrostatic interactions or covalent bonds.

[0134] The length and composition of the analyte capture molecule linker can vary. The individual atoms of the linker can include atoms such as carbon, nitrogen, silicone, oxygen, sulfur, hydrogen, fluorine, and phosphorus.

[0135] Linkers can comprise different types of groups or polymers, such as polyethylene glycol (PEG), polyamino acids, polyacrylamides, polyvinylpyrrolidone, zwitterionic polymers, polysaccharides, poly(N-(2-hydroxypropyl)methacrylamide), poly(oligo(ethylene glycol)methyl ether methacrylate), carboxydextran, hydrocarbon chains, and substituted hydrocarbon chains.

[0136] The linker provides a stable structure and can be composed of different groups connected together by different types of molecular interactions and can be produced using different techniques. Examples of different techniques for connecting different groups include EDC / NHS coupling, click chemistry, streptavidin-biotin and complementary oligonucleotides.

[0137] In certain embodiments, the linker comprises PEG. Different types of PEG can be used, such as linear and branched PEGs of different sizes. In certain embodiments, the PEG is less than about 5K. The examples provided below illustrate mixtures of PEG-SH (1K) with COOH and NH2 functional groups.

[0138] In certain embodiments, PEG is directly attached to the capture molecule, where PEG serves as both a linker (anchor to the nanoparticle) and a spacer.

[0139] In certain embodiments, PEG is a spacer and is attached to the nanoparticle via another moiety. For example, the linker has a C3-SH moiety that is directly attached to the nanoparticle and is fixed to the nanoparticle via an Au-S bond.

[0140] II.C. Sensor LSPR Nanostructure

[0141] Surface plasmon resonances are generated by electron surface waves due to the collective oscillation of free electrons propagating parallel to the interface region. Binding of analytes to the material generates surface plasmon resonances, which produce changes in the refractive index, which can be identified as wavelength shifts or surface plasmon resonance intensity. Different types of plasmon phenomena, such as surface plasmon resonance, localized surface plasmon resonance, and surface-enhanced Raman scattering, can be used for analyte detection. References describing surface plasmon resonance, detection techniques, materials and configurations include Park et al., Biosensors (Basel), 2022, 17;12(3):180, doi:10.3390 / bios12030180; Camarca et al., Sensors 2021, 21, 906, hypertext / / doi.org / / 10.3390 / s2103906; Kim et al., Sensors 2021, 21, 3191, hypertext / / doi.org / / 10.3390 / s21093191; and Liu and Zhang, Micromachines 2021, 12, 826, hypertext / / doi.org / 10.3990 / mil2070826; each of which is hereby incorporated herein in its entirety.

[0142] Plasmons can be classified as bulk, surface and localized surface (nanoparticles). Localized surface plasmons are confined to sub-wavelength sized nanoparticles and are excited at a specific frequency called localized surface plasmon resonance. Localized surface plasmon plasmon nanostructures are affected by the morphology, size, composition and distance between adjacent nanostructures. Examples of different shapes include spheres, prisms, spikes, stars and rods.

[0143] LSPR nanoparticles can be based on different platforms and made of different materials. The nanoparticles can, for example, contain metals such as rhenium, ruthenium, rhodium, palladium, silver, osmium, iridium, platinum, gold, and aluminum; and combinations of materials. (See, e.g., Park et al. Biosensors (Basel), 2022, 1 7;12(3):1 80. doi:10.3390 / bios12030180; and Osborne and Pikramenou (2015) Faraday Discussions, 185:291.) Examples of combinations include nickel, gold-silver alloy nanoparticles (Au-Ag), gold-palladium alloy nanoparticles (Au-Pd), gold-copper alloy nanoparticles (Au-Cu), silver-copper alloy nanoparticles (Ag-Cu), gold-silver-copper alloy nanoparticles (Au-Ag-Cu), gold-silver-palladium alloy nanoparticles (Au-Ag-Pd), metal oxide nanoparticles (e.g., titanium dioxide and zinc oxide), and metal-semiconductor nanoparticles (e.g., gold-silicon and silver-silicon).

[0144] In certain embodiments, the LSPR nanoparticles are provided on substrates such as fibers, fiber arrays or disks and / or patterned plasmon-based sensors such as nanoparticle structure arrays and nanohole or cavity array substrates. The plasmon sensing system can be integrated on a microfluidic platform.

[0145] The substrate can be directly anchored to the LSPR nanostructure or can be anchored through a linker. Depending on the LSPR nanostructure, anchoring can be achieved through electrostatic interactions or covalent bonds. In certain embodiments, the linker attached to the nanoparticle comprises a group provided in Table 2. The compounds of Table 2 that reference "n" provide n=1 to n=10,000.

[0146] Table 2

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153] In certain embodiments, conjugation to nanoparticles is accomplished via thiols, phosphines, amines, polymers, or silica. (See, e.g., Mahota et al., (2019) 3 Biotech 9:57, illustrating functionalization using gold nanoparticles.)

[0154] In certain embodiments, the nanoparticles are functionalized with a silane modifier, such as described in Ahangaran and Navarchian (2020) Advances in Colloid and Interface Science 286:102209.

[0155] Localized surface plasmon resonance can have various uses, such as for confirming the functionalization of nanostructures, amplifying the signal generated by BRET or FRET assembly complexes and / or measuring analyte binding. The amplification of the BRET or FRET signal is affected by the distance between the surface localized plasmon resonance nanostructure and the BRET or FRET assembly complex. Preferably, the distance should be no greater than about 15 nm.

[0156] In some embodiments, (a) sensor LSPR signal or surface enhanced Raman scattering and (b) amplifier LSPR or Raman scattering can enhance each other's signal. In other embodiments, enhancement is achieved when the sensor providing LSPR signal and the amplifier providing LSPR are within about 30 nm.

[0157] III. Analyte Detection Assays

[0158] The amplifiers and sensors described herein can be used to detect a variety of different analytes from different samples. The assay format can vary depending on the sample and the use of the BRET or FRET assembly complex. Various types of samples can be assayed, including biological, food, agricultural, and environmental samples.

[0159] Basic assays involving the use of BRET assembly complexes comprise: (a) capturing an analyte onto a sensor; (b) providing an amplifier; (c) adding a luciferase substrate; and (d) measuring fluorescence, localized surface plasmon resonance, bioluminescence, or surface enhanced Raman scattering. More than one readout may be measured. Unless otherwise indicated, reference to steps (a) and (b) does not provide a particular order. In certain embodiments, step (a) is followed by step (b). In certain embodiments, step (b) is followed by step (a).

[0160] In various embodiments, the analyte is quantified based on two or more different readouts selected from fluorescence, bioluminescence, localized surface plasmon resonance, and surface enhanced Raman scattering; or at least fluorescence and / or bioluminescence are measured. Dual techniques can increase overall accuracy to confirm positive results and are particularly useful in the presence of small amounts of analyte.

[0161] Basic assays involving the use of FRET to assemble complexes comprise: (a) capturing an analyte onto a sensor; (b) providing an amplifier; (c) exciting a fluorophore; and (d) measuring fluorescence or surface enhanced Raman scattering. More than one readout may be measured. Unless otherwise indicated, reference to steps (a) and (b) does not provide a particular order. In certain embodiments, step (a) is followed by step (b). In certain embodiments, step (b) is followed by step (a).

[0162] In certain embodiments, unbound amplifiers are removed prior to the measuring step.

[0163] In certain embodiments, the analyte from a particular sample is purified or not purified prior to being assayed; and / or the analyte from a particular sample is amplified or not amplified prior to being assayed.

[0164] IV. Bladder Cancer Analyte Testing and Treatment

[0165] Bladder cancer is a deadly and highly recurrent disease that requires extensive routine follow-up testing to ensure early detection of progression. Due to its chronic nature, bladder cancer requires extensive routine testing to monitor disease recurrence and progression, especially during the first two years after diagnosis. (Chamie et al., Cancer 2011, 117(23), 5392-401.) Early bladder detection also significantly improves patient outcomes. (Shokeir, BJU Int 2004, 93(2), 216-20.)

[0166] IV.A. Bladder Cancer Analyte Testing

[0167] The following analytes are identified to provide a combination of analytes that can be used for bladder cancer diagnosis and treatment: miR-200C, miR-205, miR-16-1, miR-143, UCA1 nucleic acid, insulin-like growth factor 2 (IGF2) or IGF2 nucleic acid; annexin-10 (ANXA10) or ANXA1 0 nucleic acid, nuclear mitotic apparatus protein (NMP-22) or NMP-22 nucleic acid, human complement factor H-related protein (HCFHrp) or HCFHrp nucleic acid, urine soluble protein (UPK1B) or UPK1B nucleic acid, ABL1 tyrosine kinase (ABL1) or ABL1 nucleic acid, CRH (adrenocorticotropic hormone) or CRH nucleic acid and P53 or P53 nucleic acid. Additional analytes can be tested. Examples of additional analytes are keratin, type I cytoskeleton 17 (KRT17) or KRT17 nucleic acid. Reference to a nucleic acid of a particular analyte provides related nucleic acids and includes different forms (eg, RNA and DNA) and different types of RNA and DNA.

[0168] miR-143 is downregulated in human bladder cancer tissues and cells and is considered a tumor suppressor microRNA. Its functional target is the insulin-like growth factor-1 receptor (IGF-1R). Overexpression of miR-143 inhibits cell proliferation and promotes the chemosensitivity of bladder cancer 5637 cells to gemcitabine. (Wang et al., Oncol. Lett. 2017, 13(1)435-440.)

[0169] MicroRNA 205 is an oncogenic microRNA and plays a role in inhibiting the proliferation, migration and invasion of bladder cancer cells. (Sun et al., Cell Death & Disease 2015, 6(10), e1907-e1907.)

[0170] MicroRNA 200c acts as a tumor suppressor microRNA and negatively regulates angiogenesis in bladder cancer and further negatively regulates the expression of angiogenesis-related proteins such as HIF-1α and VEGF3. (Wu et al., Translational Cancer Research, 2019, 8(8), 2713-2724.)

[0171] MicroRNA 16-1 is downregulated in bladder cancer compared to adjacent normal tissues and functions as a tumor suppressor microRNA. (Jiang et al., Asian Pac J Cancer Prev. 2013; 14(7): 4127-30.)

[0172] Overexpression of lncRNA-UCA1 induces epithelial-mesenchymal transition (EMT) and increases the migration and invasion ability of bladder cancer cells. (Xue et al., Cancer Sci, 2016, 107(1), 18-27.)

[0173] The accession numbers of human IGF2, ANXA10, NMP-22, HCFHrp, UPK1B, ABL1, CRH, P53 and KRT17 are provided in Table 3. CRH is part of a family that includes urocortin proteins (UCN, UCN2, UCN3) homologous to corticotropin-releasing hormone (CRH), its receptors CRHR1 and CRHR2, and corticotropin-releasing hormone binding protein CHRRB. CRH plays a role in the development of human solid cancers. IGF2 is a tumor promoter that drives cancer proliferation through its clients IGF2mRNA and HMGA16mRNA. (Dai et al., eLife 2017, 6, e27155.) ABL1-MS1 is passed to offspring through meiosis according to Mendelian inheritance. The ABL1-MS1 region can affect ABL1 expression in bladder cancer. (Kim et al., BMC Medical Genomics, 2021, 14, 121.) UPK1B is upregulated in bladder cancer and is significantly associated with tumor stage, lymph node metastasis, distant metastasis and poor prognosis of bladder cancer. (Wang et al., Eur Rev Med Pharmacol Sci., 2018 Sep; 22(17): 5471-5480.) ANXA plays a role in regulating cell growth and signal transduction pathways, and downregulating ANXA10 in bladder cancer cell lines can induce increased proliferation and migration. (Munksgaard et al., British Journal of Cancer, 2011, 105.) Nuclear matrix proteins are involved in cell gene expression and response to various xenobiotic stimuli. ( et al., Biomed Res Int 2017, 2017, 9643139-9643139. (See also, Xylinas et al., Urol Oncol 2014, 32(3), 222-9; and Oge et al., Urol Nephrol. 2001; 32(3): 367-70.) Keratin, type I cytoskeletal 17 (KRT17) is overexpressed in some cancers, and increased levels have been shown to be a biomarker for bladder cancer. (U.S. Pat. No. 11,360,092.)

[0174] Table 3 provides different accession numbers for different proteins, as well as examples of protein and nucleic acid accession numbers. There are different variants, such as different protein isoforms and different encoding nucleic acids. The information associated with each of the provided accession numbers is hereby incorporated by reference into this article in its entirety.

[0175] Table 3

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184] The nucleic acid sequences of miR-200C, miR-205, miR-16-1, miR-143, and UCA1 are provided in Table 4 below.

[0185] The level of analytes associated with cancer can be determined using different techniques, such as comparing the test sample with a control sample from a bladder cancer patient and, for example, using information about the level of analytes associated with bladder cancer to assess the level of the analytes. For example, the median NMP22 value for bladder cancer malignancies is 27.8 U / mL (95% confidence interval: 10.5-32.1 U / mL). The median NMP22 value for benign conditions of the bladder is 3.25 U / mL (95% confidence interval: 2.5-3.8 U / mL). Using a reference value of 10.0 U / mL, the sensitivity of NMP22 is 100% and the specificity is 90%. (Zippe et al., Anticancer Research (Anticancer Res.) 1999 July-August; 19 (4A): 2621-3.)

[0186] Patients with transitional cell carcinoma had significantly higher urinary hCFHrp levels compared to healthy volunteers (117.60 U / ml vs. 2.05 U / ml; p<0.001). Using a cutoff of 17.1 U / ml, the sensitivity of hCFHrp was 72.1% and the specificity was 50.5. (Heicappell et al., Eur Urol. 1999 Jan; 35(1): 81-7.)

[0187] In certain embodiments, analyte levels considered predictive of cancer include: NMP22 above 10 ng / mL (approximately 3.8 ng / mL (14.2 pM) present in healthy controls); HCFHrp above about 10 ng / mL; mRNA and microRNA (oncogenic) in the pM range during cancer progression (healthy controls in the fM range); and mRNA and microRNA (tumor suppressors) decreasing to the low pM to fM range after cancer progression (pM ​​range for healthy controls).

[0188] In various embodiments, bladder cancer is assessed by quantifying the presence of: (a) miR-205, miR-16-1, miR-143, UCA1 nucleic acid, IGF2 or IGF2 nucleic acid, and ANXA10 or ANXA nucleic acid; (b) miR-205, miR-16-1, miR-143, UCA1 nucleic acid, mRNA encoding IGF2, and mRNA encoding ANXA10; (c) miR-205, miR-16-1, miR-143, UCA1 nucleic acid, IGF2 or IGF2 nucleic acid, ANXA10 or ANXA nucleic acid. 0 or ANXA nucleic acid, NMP-22 or NMP-22 nucleic acid, HCFHrp or HCFHrp nucleic acid, miR-200C, UPKB or UPKB nucleic acid, ABL1 or ABL1 nucleic acid and CRH or CRH nucleic acid; or (d) miR-205, miR-16-1, miR-143, UCA1 nucleic acid, mRNA encoding IGF2, mRNA encoding ANXA10, mRNA encoding NMP-22, mRNA encoding HCFHrp, miR-200C, mRNA encoding UPKB, mRNA encoding ABL1 and CRH or mRNA encoding CRH. In other embodiments of (a)-(d), (i) quantification of P53 or P53 nucleic acid and / or (ii) quantification of KRT1 7 or KRT17 nucleic acid. In other embodiments, each analyte in the analyte in the provided combination is tested positive (predicting cancer).

[0189] In various embodiments, indicating that a human patient has bladder cancer based on an analyte level compared to the level of the analyte occurring in the general population involves: (a) detecting the amount of miR-205, miR-16-1, miR-143, UCA1 mRNA, IGF2 or IGF2 nucleic acid, ANXA10 or ANXA10 nucleic acid in a biological sample from the subject, wherein an increase in miR-205, UCA1 mRNA, IGF2 or IGF2 nucleic acid, and ANXA10 or ANXA10 nucleic acid compared to the general population is indicative of bladder cancer, and a decrease in miR-143 and miR-16-1 compared to the general population is indicative of bladder cancer; (b) in addition to (a), detecting the amount of NMP-22 or NMP-22 nucleic acid, HCFHrp or HCFHrp nucleic acid, miR-200C, UPKB or UPKB nucleic acid, ABL1 or ABL1 nucleic acid, or CRH or CRH, wherein ... , an increase in NMP-22 or NMP-22 nucleic acid, HCFHrp or HCFHrp nucleic acid, PKB or UPKB nucleic acid, ABL1 or ABL1 nucleic acid or CRH or CRH nucleic acid indicates bladder cancer, and a decrease in miR-200C compared to the general population indicates bladder cancer; (c) in addition to (a) and (b), detecting the amount of P53 or P53 encoding nucleic acid in the biological sample, wherein an increase in P53 or P53 encoding nucleic acid compared to the general population indicates bladder cancer; and (d) in addition to (a), (b) and (c), detecting the amount of KRT17 or KRT17 encoding nucleic acid in the biological sample, wherein an increase in KRT17 or KRT17 encoding nucleic acid compared to the general population indicates bladder cancer. In additional embodiments, for an analyte with increased levels associated with cancer, an analyte level of at least 2-fold, at least 5-fold, at least 10-fold, or at least 25-fold compared to the average amount of the analyte in the general population is indicated as being positive for cancer for the analyte; and an analyte with decreased levels of 20% or less, 40% or less, 50% or less, 75% or less, or 80% or less compared to the average amount of the analyte in the general population is indicated as being positive for cancer.

[0190] Different analytes can be detected using the same or different platforms. Reference to a particular platform refers to a substrate comprising a sensor. For example, a platform can comprise a multi-well plate in which different wells house the same sensor. Alternatively, there can be more than one type of sensor. The use of localized surface plasmon nanostructures of different sizes, shapes and / or materials and different BRET or FRET assembly complexes can be used to facilitate the use of different types of sensors on the same platform.

[0191] In certain embodiments, bladder cancer analytes are detected or quantified using techniques such as those involving quantitative polymerase chain reaction, gas chromatography, and / or flow cytometry.

[0192] In certain embodiments, bladder cancer analytes are detected or quantified using the analyte detection signal amplifiers and sensors described herein.

[0193] In certain embodiments, bladder cancer analytes are quantified from a biological sample without prior analyte purification or amplification. In further embodiments, the biological sample is a urine sample.

[0194] In certain embodiments, the first analyte binding molecule comprising the first energy transfer molecule and the second analyte binding molecule comprising the second energy transfer molecule are used to measure, wherein the first analyte binding molecule or the second analyte binding molecule are connected to the fiber. These two energy transfer molecules provide energy transfer pairs. The site of analyte binding molecule binding and the positioning of energy transfer molecule on the binding molecule should be selected to promote energy transfer. In different embodiments, the analyte binding molecule is connected to independently selected nanoparticles. In other embodiments, each nanoparticle is an independently selected plasma excitation nanoparticle, as provided in Section II.C above.

[0195] In further embodiments, the first energy transfer pair and the second energy transfer pair are FRET or BRET pairs, such as the FRET or BRET pairs provided in Section IB or Section IC above. Preferably, when each binding molecule is bound to the antigen, the distance between the FRET energy transfer pair and the BRET energy transfer pair is within about <10 nm of each other. The closer the donor and the acceptor are to each other, the stronger the emission. In certain embodiments, the distance between the energy transfer pairs is about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, or about 10 nm.

[0196] IV.B. Bladder Cancer Treatment

[0197] Bladder cancer treatment can be performed and adjusted based on the measurement results of the combination of analytes described in Section IV.A. Treatment includes initial treatment of patients diagnosed with bladder cancer and monitoring bladder cancer analytes; adjusting treatment, e.g., increasing the treatment dose, continuing treatment, or interrupting treatment, in view of the bladder cancer analytes; and treatment and monitoring after recurrence.

[0198] Bladder cancer treatments include surgery, chemotherapy, radiotherapy, immunotherapy, and targeted therapy. The choice of a specific treatment regimen takes into account the tumor stage, location, and patient. (Riaz et al., 2021 Expert Opinion on Investigational Drugs, 30: 8837-855, which is hereby incorporated by reference.)

[0199] Approved drugs for bladder cancer treatment include atezolizumab, avelumab, erdafitinib, cisplatin, doxorubicin hydrochloride, enfortumab vedotin-ejfv, erdafitinib, mitomycin, pembrolizumab, nivolumab, sacituzumab govitecan-hziy, and valrubicin. (hypertext / / www.cancer.gov / about-cancer / treatment / drugs / bladder.)

[0200] V. ADDITIONAL ASPECTS AND EMBODIMENTS

[0201] Additional aspects, embodiments, and combinations thereof include the following:

[0202] 1. An analyte detection signal amplifier, comprising (a) an analyte binding molecule; (b) a localized surface plasmon resonance nanostructure; and (c) (i) a bioluminescence resonance energy transfer (BRET) assembly complex, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor, or (ii) a fluorescence resonance energy transfer (FRET) assembly complex, wherein the FRET assembly complex comprises a fluorescence donor conjugated to a fluorophore acceptor.

[0203] 2. The analyte detection signal amplifier according to 1, wherein the analyte binding molecule is a single-stranded polynucleotide, an antibody, an aptamer, a protein, a substrate, a receptor or a ligand.

[0204] 3. The analyte detection signal amplifier according to 1 or 2, wherein the surface localized plasmon resonance nanostructure is a nanorod, and the nanorod comprises a metal.

[0205] 4. The analyte detection signal amplifier according to 3, wherein the nanorods contain gold.

[0206] 5. An analyte detection signal amplifier according to any one of 1 to 4, wherein the surface localized plasmon resonance nanostructure is functionalized with polyethylene glycol (PEG), the BRET assembly complex or the FRET assembly complex is anchored to the localized surface plasmon resonance nanostructure through a first PEG chain, and the analyte binding molecule is anchored to the localized surface plasmon resonance nanostructure through a second PEG chain.

[0207] 6. The analyte detection signal amplifier according to any one of 1 to 5, comprising the BRET assembly complex.

[0208] 7. The analyte detection signal amplifier according to 6, wherein the luciferase donor conjugated to the fluorophore acceptor is selected from the following combinations: NLuc-HT / HL-Oregon Green, RLuc / YFP, RLuc / fluorescent protein (GFP), RLuc8 / GFP, firefly luciferase / DsRed, RLuc / ODot, Rluc8 / ODot or Nano Luc-HT / Halotag-fluorescent ligand.

[0209] 8. A method for detecting or quantifying an analyte in a sample, the method comprising the following steps:

[0210] a) contacting a localized surface plasmon resonance sensor with the sample, wherein the sensor comprises an analyte capture molecule;

[0211] b) providing an analyte detection signal amplifier to the sample, wherein the analyte detection signal amplifier comprises the BRET assembly complex according to any one of 1 to 7 and binds to the analyte;

[0212] c) adding luciferase substrate; and

[0213] d) Detection of fluorescence, bioluminescence, localized surface plasmon resonance or surface enhanced Raman scattering.

[0214] 9. A method for detecting or quantifying an analyte in a sample, the method comprising the following steps:

[0215] a) contacting a localized surface plasmon resonance sensor with the sample, wherein the sensor comprises an analyte capture molecule;

[0216] b) providing an analyte detection signal amplifier to the sample, wherein the analyte detection signal amplifier comprises the FRET assembly complex according to any one of 1 to 5 and binds to the analyte;

[0217] c) exciting the FRET assembly complex; and

[0218] d) Detection of fluorescence, localized surface plasmon resonance or surface enhanced Raman scattering.

[0219] 10. The method according to 8 or 9, wherein the sensor comprises metal.

[0220] 11. The method of 10, wherein the sensor comprises gold.

[0221] 12. The method according to any one of 8 to 11, wherein after step b and before step c, the analyte signal amplifier that is not bound to the analyte is removed.

[0222] 13. The method according to any one of 8 to 12, wherein fluorescence and / or bioluminescence is measured.

[0223] 14. The method according to any one of 8 to 12, wherein surface enhanced Raman scattering is measured.

[0224] 15. A multi-analyte detection system, comprising:

[0225] a) a first LSAP nanostructure sensor, wherein the first LSAP nanostructure sensor comprises an analyte capture molecule of miR-205;

[0226] b) a second LSAP nanostructure sensor, wherein the second LSAP nanostructure sensor comprises an analyte capture molecule of miR-16-1;

[0227] c) a third LSAP nanostructure sensor, wherein the third LSAP nanostructure sensor comprises an analyte capture molecule of miR-143;

[0228] d) a fourth LSAP nanostructure sensor, wherein the fourth LSAP nanostructure sensor comprises an analyte capture molecule of UCA1 nucleic acid;

[0229] e) a fifth LSAP nanostructure sensor, wherein the fifth LSAP nanostructure sensor comprises an analyte capture molecule of IGF2 or IGF2 nucleic acid;

[0230] f) a sixth LSAP nanostructure sensor, wherein the sixth LSAP nanostructure sensor comprises an analyte capture molecule of ANXA10 or ANXA10 nucleic acid;

[0231] Each sensor type can be present on one or more platforms.

[0232] 16. The multi-analyte detection system according to 15, further comprising:

[0233] g) a seventh LSAP nanostructure sensor, wherein the seventh LSAP nanostructure sensor comprises an analyte capture molecule of NMP-22 or NMP-22 nucleic acid;

[0234] h) an eighth LSAP nanostructure sensor, wherein the eighth LSAP nanostructure sensor comprises an analyte capture molecule of HCFHrp or HCFHrp nucleic acid;

[0235] i) a ninth LSPM nanostructure sensor, wherein the ninth LSPM nanostructure sensor comprises an analyte capture molecule of miR-200C;

[0236] j) a tenth LSPM nanostructure sensor, wherein the tenth LSPM nanostructure sensor comprises an analyte capture molecule of UPKB or UPKB nucleic acid;

[0237] k) an eleventh LSAP nanostructure sensor, the eleventh LSAP nanostructure sensor comprising an analyte capture molecule of ABL1 or ABL1 nucleic acid; and

[0238] 1) A twelfth LSAP nanostructure sensor, wherein the twelfth LSAP nanostructure sensor comprises an analyte capture molecule of CRH or CRH nucleic acid; wherein each sensor type may be present on one or more platforms.

[0239] 17. The multi-analyte detection system according to 16, further comprising a thirteenth LSPM nanostructure sensor, wherein the thirteenth LSPM nanostructure sensor comprises an analyte capture molecule of P53 or P53 nucleic acid.

[0240] 18. The multi-analyte detection system according to 16 or 17, further comprising a fourteenth localized surface plasmon nanostructure sensor, wherein the fourteenth localized surface plasmon nanostructure sensor comprises an analyte capture molecule of KRT17 or KRT17 nucleic acid.

[0241] 19. The multi-analyte detection system according to any one of 15 to 18, wherein:

[0242] a) the miR-205 analyte capture molecule is a single-stranded polynucleotide complementary to miR-205;

[0243] b) the miR-16-1 analyte capture molecule is a single-stranded polynucleotide complementary to miR-16-1;

[0244] c) the miR-143 analyte capture molecule is a single-stranded polynucleotide complementary to miR-143;

[0245] d) the analyte capture molecule of the UCA1 nucleic acid is a single-stranded polynucleotide complementary to the UCA1 nucleic acid;

[0246] e) the IGF2 or nucleic acid encoding analyte capture molecule is an antibody specific for mRNA encoding IGF2;

[0247] f) the ANXA10 or nucleic acid encoding analyte capture molecule is an antibody specific for mRNA encoding ANXA10;

[0248] g) the NMP-22 or nucleic acid encoding analyte capture molecule is an NMP-22 specific antibody;

[0249] h) the analyte capture molecule of HCFHrp or encoding nucleic acid is a HCFHrp specific antibody;

[0250] i) the miR-200C analyte capture molecule is a single-stranded polynucleotide complementary to miR-200C;

[0251] j) the UPKB or nucleic acid encoding analyte capture molecule is an antibody specific for mRNA encoding UPKB;

[0252] k) the ABL1 or nucleic acid encoding analyte capture molecule is an antibody specific for mRNA encoding ABL1; and

[0253] 1) The CRH mRNA or nucleic acid encoding analyte capture molecule is an antibody specific for the mRNA encoding CRH; and each sensor type is present on a different platform.

[0254] 20. The multi-analyte detection system according to any one of 15 to 19, wherein each LSPM nanostructure comprises gold.

[0255] 21. The multi-analyte detection system according to any one of 15 to 19, wherein each LSPM nanostructure comprises a gold sphere.

[0256] 22. The multi-analyte detection system according to any one of 15 to 21, wherein each localized surface plasmon nanostructure is functionalized with a polyethylene glycol (PEG) chain, and the analyte binding molecule is conjugated to the PEG chain.

[0257] 23. A method for determining whether a human subject has bladder cancer, the method comprising the steps of:

[0258] a) contacting the multi-analyte detection system according to any one of 15 to 22 with a biological sample from the subject,

[0259] b) providing a detection signal amplifier, wherein:

[0260] i) providing a first detection signal amplifier to a first sensor, wherein the first detection signal amplifier comprises a BRET assembly complex, a localized surface plasmon resonance nanostructure, and an analyte binding molecule of miR-205, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor;

[0261] ii) providing a second detection signal amplifier to a second sensor, wherein the second detection signal amplifier comprises a BRET assembly complex, a localized surface plasmon resonance nanostructure, and an analyte binding molecule of miR-16-1, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor;

[0262] iii) providing a third detection signal amplifier to a third sensor, wherein the third detection signal amplifier comprises a BRET assembly complex, a localized surface plasmon resonance nanostructure, and an analyte binding molecule of miR-143, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor;

[0263] iv) providing a fourth detection signal amplifier to a fourth sensor, wherein the fourth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of a UCA1 nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor;

[0264] v) providing a fifth detection signal amplifier to a fifth sensor, wherein the fifth sensor comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of IGF2 or IGF2 nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor; and

[0265] vi) providing a sixth detection signal amplifier to a sixth sensor, wherein the sixth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of ANXA10 or ANXA10 nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor;

[0266] c) adding luciferase substrate;

[0267] d) detecting fluorescence, localized surface plasmon resonance, bioluminescence or surface enhanced Raman scattering, thereby indicating the presence or amount of an analyte in the sample.

[0268] 24. The method according to 23, wherein step b further comprises:

[0269] vii) providing a seventh signal amplifier to a seventh detection sensor, wherein the seventh detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of NMP-22 or NMP-22 nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor;

[0270] viii) providing an eighth detection signal amplifier to an eighth sensor, wherein the eighth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of HCFHrp or HCFHrp nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor;

[0271] ix) providing a ninth detection signal amplifier to a ninth sensor, wherein the ninth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of a miR-200C nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor;

[0272] x) providing a tenth detection signal amplifier to a tenth sensor, wherein the tenth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of UPKB or UPKB nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor;

[0273] xi) providing an eleventh detection signal amplifier to an eleventh sensor, wherein the eleventh detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of ABL1 or ABL1 nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor; and

[0274] xii) providing a twelfth detection signal amplifier to the twelfth sensor, wherein the twelfth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of CRH or CRH nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor.

[0275] 25. The method according to 24, wherein step b further comprises providing a thirteenth detection signal amplifier to the thirteenth sensor, wherein the thirteenth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex and an analyte binding molecule of P53 or P53 nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor.

[0276] 26. The method according to 24 or 25, wherein step b further comprises providing a fourteenth detection signal amplifier to the fourteenth sensor, wherein the fourteenth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex and an analyte binding molecule of KRT17 or KRT17 nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor.

[0277] 27. The method according to any one of 23 to 26, wherein

[0278] i) the miR-205 analyte binding molecule is a single-stranded polynucleotide complementary to miR-205;

[0279] ii) the analyte binding molecule of miR-16-1 is a single-stranded polynucleotide complementary to miR-16-1;

[0280] iii) the miR-143 analyte binding molecule is a single-stranded polynucleotide complementary to miR-143;

[0281] iv) the analyte binding molecule for the UCA1 nucleic acid is a single-stranded polynucleotide complementary to the UCA1 nucleic acid;

[0282] v) the IGF2 or nucleic acid encoding analyte binding molecule is an antibody specific for mRNA encoding IGF2;

[0283] vi) the analyte binding molecule of ANXA10 or encoding nucleic acid is an antibody specific for mRNA encoding ANXA10;

[0284] vii) the NMP-22 or encoding nucleic acid analyte binding molecule is an NMP-22 specific antibody;

[0285] viii) the analyte binding molecule of HCFHrp or encoding nucleic acid is a HCFHrp specific antibody;

[0286] ix) the analyte binding molecule of miR-200C is a single-stranded polynucleotide complementary to miR-200C;

[0287] x) the UPKB or nucleic acid encoding analyte binding molecule is an antibody specific for mRNA encoding UPKB;

[0288] xi) the ABL1 or nucleic acid encoding analyte binding molecule is an antibody specific for mRNA encoding ABL1; and

[0289] xii) The analyte binding molecule for CRH mRNA or encoding nucleic acid is an antibody specific for mRNA encoding CRH.

[0290] 28. A method for determining whether a subject has bladder cancer, the method comprising the steps of:

[0291] a) contacting the multi-analyte detection system according to any one of 15 to 22 with a biological sample from a human subject,

[0292] b) providing a detection signal amplifier, wherein:

[0293] i) providing a first detection signal amplifier to a first sensor, wherein the first detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of miR-205, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor;

[0294] ii) providing a second detection signal amplifier to a second sensor, wherein the second detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of miR-16-1, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor;

[0295] iii) providing a third detection signal amplifier to a third sensor, wherein the third detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of miR-143 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor;

[0296] iv) providing a fourth detection signal amplifier to a fourth sensor, wherein the fourth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of UCA1 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor;

[0297] v) providing a fifth detection signal amplifier to a fifth sensor, wherein the fifth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of IGF2 or IGF2 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor; and

[0298] vi) providing a sixth detection signal amplifier to a sixth sensor, wherein the sixth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of ANXA10 or ANXA10 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor;

[0299] c) exciting each FRET assembly complex;

[0300] d) detecting fluorescence, localized surface plasmon resonance or surface enhanced Raman scattering, thereby indicating the presence or amount of an analyte in the sample.

[0301] 29. The method according to 28, wherein step b further comprises:

[0302] vii) providing a seventh detection signal amplifier to a seventh sensor, wherein the seventh detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of NMP-22 or NMP-22 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor;

[0303] viii) providing an eighth detection signal amplifier to an eighth sensor, wherein the eighth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of HCFHrp or HCFHrp nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor;

[0304] ix) providing a ninth detection signal amplifier to a ninth sensor, wherein the ninth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of miR-200C nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor;

[0305] x) providing a tenth detection signal amplifier to a tenth sensor, wherein the tenth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of UPKB or UPKB nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor;

[0306] xi) providing an eleventh detection signal amplifier to an eleventh sensor, wherein the eleventh detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of ABL1 or ABL1 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor; and

[0307] xii) providing a twelfth detection signal amplifier to the twelfth sensor, wherein the twelfth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of CRH or CRH nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor.

[0308] 30. The method according to 29, wherein step b further comprises providing a thirteenth detection signal amplifier to the thirteenth sensor, wherein the thirteenth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of P53 or P53 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor.

[0309] 31. The method according to 29 or 30, wherein step b further comprises providing a fourteenth detection signal amplifier to the fourteenth sensor, wherein the fourteenth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex and an analyte binding molecule of KRT17 or KRT17 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor.

[0310] 32. The method according to any one of 28 to 31, wherein

[0311] i) the miR-205 analyte binding molecule is a single-stranded polynucleotide complementary to miR-205;

[0312] ii) the analyte binding molecule of miR-16-1 is a single-stranded polynucleotide complementary to miR-16-1;

[0313] iii) the miR-143 analyte binding molecule is a single-stranded polynucleotide complementary to miR-143;

[0314] iv) the analyte binding molecule for the UCA1 nucleic acid is a single-stranded polynucleotide complementary to the UCA1 nucleic acid;

[0315] v) the IGF2 or nucleic acid encoding analyte binding molecule is an antibody specific for mRNA encoding IGF2;

[0316] vi) the analyte binding molecule of ANXA10 or encoding nucleic acid is an antibody specific for mRNA encoding ANXA10;

[0317] vii) the NMP-22 or encoding nucleic acid analyte binding molecule is an NMP-22 specific antibody;

[0318] viii) the analyte binding molecule of HCFHrp or encoding nucleic acid is a HCFHrp specific antibody;

[0319] ix) the analyte binding molecule of miR-200C is a single-stranded polynucleotide complementary to miR-200C;

[0320] x) the UPKB or nucleic acid encoding analyte binding molecule is an antibody specific for mRNA encoding UPKB;

[0321] xi) the ABL1 or nucleic acid encoding analyte binding molecule is an antibody specific for mRNA encoding ABL1; and

[0322] xii) The analyte binding molecule for CRH mRNA or encoding nucleic acid is an antibody specific for mRNA encoding CRH.

[0323] 33. The method according to 29 further comprises a thirteenth sensor, wherein the thirteenth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex and an analyte binding molecule of P53 or P53 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor.

[0324] 34. The method according to 33 or 32 further comprises providing a fourteenth detection signal amplifier to the fourteenth sensor, wherein the fourteenth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex and an analyte binding molecule of KRT17 or KRT17 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor.

[0325] 35. The method of any one of 23 to 34, wherein the sample is a urine sample.

[0326] 36. The method of 35, wherein the urine sample has not been purified prior to step a.

[0327] 37. A method for determining whether a human subject has bladder cancer, the method comprising the step of detecting the amount of miR-205, miR-16-1, miR-143, UCA1 mRNA, IGF2 or IGF2 nucleic acid, ANXA10 or ANXA10 nucleic acid in a biological sample from the subject.

[0328] 38. The method according to 35, further comprising detecting the amount of NMP-22 or NMP-22 nucleic acid, HCFHrp or HCFHrp nucleic acid, miR-200C, UPKB or UPKB nucleic acid, ABL1 or ABL1 nucleic acid, and CRH or CRH nucleic acid in the biological sample from the subject.

[0329] 39. The method according to 35 or 36, further comprising detecting the amount of P53 or a nucleic acid encoding P53 in the biological sample.

[0330] 40. The method according to any one of 37 to 39, further comprising detecting the amount of KRT17 or a nucleic acid encoding KRT17 in the biological sample.

[0331] 41. A method for treating bladder cancer in a subject, the method comprising (a) detecting the amount of an analyte associated with bladder cancer using a method according to any one of 23 to 40; and (b) administering a therapeutically effective amount of a bladder cancer therapeutic to a subject determined to have bladder cancer based in whole or in part on (a).

[0332] 42. A method of treating bladder cancer in a subject, the method comprising administering to a subject determined to have bladder cancer based in whole or in part on the results of using the method of any one of claims 23 to 40 a therapeutically effective amount of a bladder cancer therapeutic.

[0333] 43. The method of claim 41 or 42, wherein the bladder cancer therapeutic agent is selected from the group consisting of atezolizumab, avelumab, erdafitinib, cisplatin, doxorubicin hydrochloride, ventuumab, erdafitinib, mitomycin, pembrolizumab, nivolumab, gosatuzumab, and valrubicin.

[0334] 44. A method for detecting or quantifying an analyte in a sample, the method comprising the steps of:

[0335] a) contacting a localized surface plasmon resonance sensor with the sample, wherein the sensor comprises an analyte capture molecule;

[0336] b) providing an analyte detection signal amplifier to the sample, wherein the analyte detection signal amplifier comprises the BRET assembly complex according to 1 and binds to the analyte;

[0337] c) adding luciferase substrate; and

[0338] Detect fluorescence, bioluminescence, localized surface plasmon resonance, or surface-enhanced Raman scattering.

[0339] 45. The method of claim 44, wherein the sample is a biological sample from a human patient, and the method comprises using an analyte detection signal amplifier to detect the amount of miR-205, miR-16-1, miR-143, UCA1 mRNA, IGF2 or IGF2 nucleic acid, ANXA10 or ANXA10 nucleic acid in the biological sample.

[0340] 46. ​​The method according to 45, further comprising detecting the amount of NMP-22 or NMP-22 nucleic acid, HCFHrp or HCFHrp nucleic acid, miR-200C, UPKB or UPKB nucleic acid, ABL1 or ABL1 nucleic acid, or CRH or CRH nucleic acid in the biological sample.

[0341] 47. The method according to 44 further comprises detecting the amount of P53 or a nucleic acid encoding P53 in the biological sample.

[0342] 48. The method according to 47, further comprising detecting the amount of KRT17 or a nucleic acid encoding KRT17 in the biological sample.

[0343] 49. A method for treating bladder cancer in a subject, the method comprising (a) detecting the amount of an analyte associated with bladder cancer using a method according to any one of 45 to 48; and (b) administering a therapeutically effective amount of a bladder cancer therapeutic to a subject determined to have bladder cancer based in whole or in part on (a).

[0344] 50. A method of treating bladder cancer in a subject, the method comprising administering a therapeutically effective amount of a bladder cancer therapeutic to a subject determined to have bladder cancer based in whole or in part on the results of using the method of any one of 45 to 48.

[0345] 51. The method of claim 49 or 50, wherein the bladder cancer therapeutic agent is selected from the group consisting of atezolizumab, avelumab, erdafitinib, cisplatin, doxorubicin hydrochloride, ventuumab, erdafitinib, mitomycin, pembrolizumab, nivolumab, gosatuzumab, and valrubicin.

[0346] The examples provided below further illustrate various features of the invention and methods for practicing the invention. The examples provided do not limit the claimed invention.

[0347] Examples

[0348] Example 1: Calibration Curve

[0349] Example 1 demonstrates the generation of a calibration curve using a BRET assembly complex, an amplifier targeting a bladder cancer antigen, and a sensor targeting a bladder cancer antigen. Figure 2 The different components and steps of the overall detection scheme are presented.

[0350] Materials and reagents: HAuCl4, sodium citrate (99.5%), hexadecyltrimethylammonium bromide (CTAB, 99%), D-(+)-glucose (99.5%), sodium borohydride, silver nitrate (99%), L(+)-ascorbic acid (reagent grade), 3-aminopropyl-triethoxysilane (APTES, 94%) were purchased from Sigma-Aldrich. Thiolated polyethylene glycol was purchased from BIOCHEMPEG. 3′-SH-(CH2)3-ssDNA modified with single-stranded oligonucleotides, microRNA and RNase H enzyme were purchased from Integrated DNA Technologies. All chemicals were used without further purification. RNase-free sterile water and PBS buffer (pH 7.2) were obtained from Sigma-Aldrich. Corning 96 multiwell plates were purchased from Sigma-Aldrich.

[0351] The nucleic acids used as ssDNA analyte binding molecules and ssDNA analyte capture molecules are provided in Table 4. CAP1 indicates functionalization as AuNR and used as analyte detection molecule. CAP2 indicates functionalization as Au sphere and used as analyte detection molecule.

[0352] Table 4

[0353]

[0354]

[0355] The microRNA sequences and UCA sequences used as analytes in generating the calibration curves are provided in Table 5.

[0356] Table 5

[0357]

[0358] Thiol-modified-ssDNA, microRNA, mRNA, and proteins were kept at -20°C, and patient samples were stored at -80°C. Protein and Nano-Glo buffer were purchased from Promega Corporation. Washing buffer and blocking buffer were prepared in the laboratory.

[0359] The analytes, binding molecule types, and capture molecule types used in this example are recorded in Table 6. Materials were obtained from commercial sources such as ProSci Inc., Novus Biological, Abbexa, and Sinobiologics.

[0360] Table 6

[0361]

[0362]

[0363] Sensor functionalization. The AuSP (see Example 4 below) incorporated into a chip consisting of a three-dimensional nanostructure was functionalized using polyethylene glycol thiol (1K) and n-hydroxysuccinimide (NHS) to provide SC-PEG-S-Au. The LSPR peak of the PEG (2K)-SH functionalized AuSp chip was displayed at 550 nM. Functionalization was performed using 1 nM AuSP nanoparticles mixed with a 1:2000 molar ratio of PEG-1K at room temperature. The reaction was completed on a medium speed rotary shaker for 24 hours. Excess PEG was removed by ultracentrifugation at 10,000 rpm for 30 minutes. The supernatant was removed and three rounds of washing were continued to remove all excess PEG-SH.

[0364] ssDNA is loaded onto the sensor chip: Ten micromoles of ssDNA disulfide are incubated in 1 mL 10X PBS buffer containing 100 μM tris(2-carboxyethyl)phosphine) (TCEP) at pH 5.2 at room temperature for one hour, while being sonicated for 60 seconds every 15 minutes. The sample is then diluted to the desired volume to obtain 10 μμM ssDNA disulfide. The sensor chip is positioned at the bottom of each well of a 96-well white plate and loaded with 200 μμL 10 μμM ssDNA disulfide. The plate is sealed and placed in the dark at room temperature for 12 hours. After 12 hours, 4 μμL 3M NaCl solution is added five times at one-hour intervals. During each addition, the solution is sonicated for 10 seconds. After the final sonication, the wells are sealed and incubated for another 12 hours. The blocking buffer (StartingBlock from Thermo Fisher) is then added. TM (PBS) blocking buffer) was added to the chip to avoid nonspecific binding, the functionalized chip was incubated for 30 minutes, and then the chip was centered and dried at 25°C for 2 hours before use in analytical applications.

[0365] Antibody loading onto sensor chip: Sensor chip containing AuSP functionalized with C-PEG-SH (1K) was subjected to EDC / NHS coupling in MES buffer at pH 6.0 to provide COOH activation, and then conjugate buffer containing 20ug / mL antibody solution was added. The reaction was continued for eight hours, and then the chip was washed with washing buffer to remove unbound antibody from the surface. After removing unbound antibody, the chip surface was blocked with blocking buffer.

[0366] Gold nanorod preparation: AuNR synthesis was performed in two different steps. The seed solution was prepared by adding 250 μL of 10 mM HAuCl4 solution to 7.5 mL of 100 mM CTAB solution in a 20 mL glass vial at room temperature and stirring at medium speed. After five minutes, 600 μL of 10 mM NaBH4 solution was injected and allowed to react for 15 minutes. NaBH4 was used as a reducing agent, and after the addition of NaBH4, a color change from yellow to light appeared.

[0367] To prepare 250 mL of AuNR solution, 250 mL of 0.055 M CTAB solution and 17.86 mL of 10 mM HAuCl4 solution were combined and mixed thoroughly. 3.57 mL of 10 mM AgNO3 solution was added, followed by 1.96 mL of 10 mM L-ascorbic acid. The solution was mixed thoroughly. The reduction of HAuCl4 was confirmed by the formation of a colorless solution.

[0368] Initially, 342 μL of seed solution was injected into the AuNR solution, and the reaction was allowed to complete for at least four hours. The color changed from colorless light wine red to brown-black, indicating the formation of AuNR. After AuNR formation, excess CTAB was removed by centrifugation twice. The wavelength of the obtained AuNR was about 680 nm and the average size was about 40 nm.

[0369] AuNR PEG functionalization: AuNR was redissolved in Tris buffer at pH 3.0, combined with 1 mM / 1 OD SC-PEG(1K)-SH: NH2-PEG(1K)-SH 1:1 solution and allowed to react for 3 hours. PEG-functionalized AuNR was extracted into chloroform using a solvent-solvent extraction method, after which the chloroform was completely evaporated.

[0370] Nucleic acid loading onto AuNRs: Reactions were performed in 20 mL scintillation glass vials washed with 12 M NaOH. 10 μM ssDNA CAP2 solution was mixed with 1 mL PBS buffer at pH 5.2 containing 100 μM TCEP solution for one hour. After 1 hour, the mixture was diluted in PBS 10X buffer at pH 8.0 containing 1 OD SC-PEG-SH:NH2-PEG-SH (1K) functionalized AuNR solution. The reaction was continued for 12 hours, and then 20 μL 3M NaCl was added dropwise five times at one hour intervals (total addition amount was 1 ml). After each addition, the solution was sonicated for 10 seconds. After the NaCl addition was completed, the reaction was placed in the dark for 24 hours before use.

[0371] Antibody loading on AuNR: SH-PEG-SC-1K and NH2-PEG-SH functionalized AuNR were coupled with maleimide (MA)-PEG-NH2 in PBS buffer 10X at pH 8.0 for 4 hours. The isolated AuNR was reactivated using EDC / NHS coupling and 20 μg / mL antibody solution was added. The reaction was continued at room temperature in conjugation buffer for eight hours, and the AuNR was purified by centrifugation.

[0372] ssDNA is loaded onto AuNR: PEG-cleaved (SC-PEG-SH(1K):NH2-PEG-SH(1K))AuNR is ligand exchanged with single-stranded oligonucleotide (ssDNA). Single-stranded oligonucleotides in disulfide form are obtained, and disulfide bonds are reduced using TCEP before ligand exchange. 10 μM ssDNA disulfide is reduced using acetate buffer (500 mM, pH 5.2) containing 100 μtM TCEP at room temperature for 1 hour, and 60 seconds of ultrasonic treatment is performed every 15 minutes. After the disulfide is reduced to thiol, the solution is added to a mixture of a solution containing 1 OD / 1 mL AuNR in 10 mM PBS at pH 8.0 in a 12 M NaOH-treated glass vial. After gently shaking by hand, the reaction is allowed to complete for another 12 hours.

[0373] After complete reaction, 20 μl of 3M NaCl solution was added dropwise to each vial with gentle manual shaking, followed by 10 seconds of sonication. NaCl helps to reduce the repulsive forces between the two strands, thereby increasing the loading of ssDNA onto the nanoparticles. The vials were stored in a drawer (dark room temperature) for at least 24 hours before use.

[0374] BRET assembly: Mix nine microliters of 9 μg / μl Nluc-HT enzyme solution with 0.6 μl of 1 mM Oregon Green dye in the presence of 150 μl of 10% CA-630 was reacted in 2 mL 1X PBS buffer in a dark 20 mL scintillation vial. The reaction was continued on a medium speed rotary shaker in a cold room for 16 hours. The Nluc-HT-HL-Oregon labeled molecules were used directly without further purification.

[0375] Nluc-HT-Oregon BRET molecule preparation: After conjugation of ssDNA / mRNA / protein antibody to AuNR, AuNR was coupled to PBS buffer 10X containing MA-PEG-NH2 at pH 8.0 for 4 hours. AuNR was separated by centrifugation, blocked on the surface using blocking buffer, and purified again by centrifugation. 100 μtL of 1 OD / mL solution of Nluc-HT-HL-Oregon BRET pre-assembly was injected into HEPES buffer containing AuNR at pH 8.0, and the reaction was continued at 4°C for 3 hours. After 3 hours, the AuNR-coupled Nluc-HT-HL-Oregon assembly was purified using a 100 MW cutoff filter.

[0376] Detection Assay: The detection assay was evaluated using different amounts of analyte added to urine. The assay was performed using a 96-well plate. Each well was prepared to detect a specific biomarker. Calibration was performed using amounts of analyte ranging from 100 ng / mL to 100 ag / mL.

[0377] The AuSP-assembled pores were functionalized with ssDNA CAP1 to capture nucleic acids or with antibodies to capture proteins, followed by blocking of free substrates to avoid nonspecific binding. 100 μL of human urine sample (centrifuged at 3000 rpm for 15 minutes to remove all large cell debris) was then mixed with 100 μL of hybridization / coupling buffer and then added directly to each well and incubated for 4 hours. The sensor capture molecules capture specific biomarkers directly from urine without any purification or amplification. Nonspecific molecular binding was eliminated using polyethylene glycol thiol as a spacer and blocking agent. The 96-well plate was washed with wash buffer and dried under N2 gas, 200 μL of blocking buffer was added again to avoid further nonspecific attachment, and it was allowed to stand at room temperature for 30 minutes.

[0378] 100 μtL 1OD amplifier solution was added to the target well where the target analyte had been captured. The amplifier contained a BRET assembly carrying the AuNR solution with ssDNA CAP2 for nucleic acid determination or antibodies for protein determination. The reaction was allowed to proceed for 4 hours in hybridization or coupling buffer. Excess AuNR was removed and the well was washed with wash buffer to remove all unbound AuNR from the solution.

[0379] Finally, the plate was placed in a GLOMAX Discover microplate reader and 100 μl_ of reaction buffer containing furimazine (3 μl_:10 mL) was injected into each well, the plate was then shaken for 20 seconds, and the generated luminescence and fluorescence were reported.

[0380] Results: Detection of analytes in the test range of 100 ng / ml to 100 ag / ml was mostly linear. Figure 3A , 3B , 4A, 4B, 5A and 5B show calibration graphs for measuring luminescence and fluorescence of different analytes. Figure 3A CRH, ANAX10, ABL1, IGF2, and UPKB1 luminescence were demonstrated. Figure 3B CRH, ANAX10, ABL1, IGF2, and UPKB1 fluorescence are shown. Figure 4A Luminescence of miR-200c (200c), miR-16-1 (16-1), miR-205 (205), miR-143 (143) and long noncoding RNA (UCA) was demonstrated. Figure 4BFluorescence of miR-200c (200c), miR-16-1 (16-1), miR-205 (205), miR-143 (143), and long noncoding RNA (UCA) are shown. Figure 5A NMP22, HCFHrp (hcfHRP), and P53 luminescence are shown. Figure 5B NMP22, HCFHrp (hcfHRP), and P53 fluorescence are shown.

[0381] Tables 7-12 provide the individual data points along with the standard deviations.

[0382] Table 7

[0383] Concentration (nm) CRH std ANXA std IGF2 std ABL1 Std UPKB1 std 100 16500 1240 15600 1140 17800 1184 14670 1200 17800 1120 1 13980 895 12800 1275 15300 275 12800 980 15300 775 0.01 10200 945 9850 945 10200 945 9800 850 10200 945 1.00E-04 8700 1020 6350 725 8750 720 7300 680 8750 720 1.00E-06 6430 488 4220 625 6520 688 5900 420 6520 688 1.00E-08 3700 720 3100 472 4200 820 3200 475 4200 820

[0384] Row intensity values ​​obtained for mRNA luminescence assay.

[0385] Table 8

[0386] Concentration(nm) CRH std ANXA std IGF2 std ABL1 Std UPKB1 std 100 7520 275 7300 320 8270 320 6230 470 8270 320 1 4920 295 4175 420 5400 420 5200 340 5400 420 0.01 3100 320 3900 580 4100 580 4180 230 4100 580 1.00E-04 1980 422 2800 225 2900 225 2015 120 2900 225 1.00E-06 1025 280 1285 180 1980 180 1300 102 1980 180 1.00E-08 680 295 875 175 1020 175 870 78 1020 175

[0387] Row intensity values ​​obtained for mRNA fluorescence assay

[0388] Table 9

[0389] Concentration (nm) 200C std 16-1 std 205 std 143 std UCA std 100 15600 675 12780 555 14920 565 13800 480 17800 1120 1 12800 820 10900 248 11300 290 10800 750 15300 775 0.01 9700 740 8720 752 9520 520 9300 425 10200 945 1E-4 7650 275 6480 624 6490 725 7500 275 8750 720 1E-6 5420 498 5240 325 5340 240 5400 478 6520 688 1E-8 3100 620 2810 397 1970 225 3200 590 4200 820

[0390] Row intensity values ​​obtained for mRNA luminescence assay.

[0391] Table 10

[0392]

[0393]

[0394] Row intensity values ​​obtained for microRNA and lncRNA fluorescence assays.

[0395] Table 11

[0396] Concentration (nm) NMP22 std hcfHrp std P53 std 100 15800 1040 15725 1175 14285 1120 1 13800 1175 13785 1257 11200 775 0.01 10800 845 10820 945 9800 945 1E-4 8300 625 8600 758 7450 820 1E-6 6420 525 6520 428 5275 720 1E-8 3800 372 3200 472 3800 480

[0397] Row intensity values ​​obtained for protein luminescence assay.

[0398] Table 12

[0399] Concentration (nm) NMP22 std hcfHrp std P53 std 100 5800 220 5900 420 7430 320 1 4575 230 4675 520 5325 420 0.01 3600 154 3500 680 4025 580 1E-4 2100 105 2275 275 2800 225 1E-6 1085 132 980 200 1055 180 1E-8 640 120 612 108 687 175

[0400] Row intensity values ​​obtained for protein fluorescence assay.

[0401] Table 13 summarizes the equations of the calibration graphs for samples containing different amounts of analytes in urine. The LOD is calculated by measuring the relative intensity of the blank sample and then calculating the Z (mean + 3σ) value. The Z value is then converted into a relative concentration using the calibration curve. The blank sample consists of nanoparticles functionalized with a spacer molecule (SC-PEG (1K) -SH), but with the presence of capture molecules. A signal amplifier solution is added, and after four hours, the solution is removed and washed with wash buffer, and luminescence and fluorescence intensity are obtained by adding a substrate. The first group (left side) of Table 13 provides bioluminescence, and the second group (right side) provides fluorescence.

[0402] Table 13

[0403]

[0404]

[0405] Example 3: 3D assembly of AuSP particles

[0406] Different types of AuSP particles can be produced, including AuSPs incorporated into three-dimensional frameworks and used as sensors. An alternative to using three-dimensional AuSP particle chips is to use three-dimensional assemblies of AuSP particles. An example of a technique that can be used to produce three-dimensional assemblies of AuSPs involves activating the AuSPs by treating them with 1:1 HCl:MeOH for 1 hour. 96-well flat clear glass bottom white polystyrene wells. The plate was then washed with copious amounts of Milli-Q water and dried in a vacuum oven at 75°C for 24 hours. The glass bottom was then treated with 300 μL of 40% n-octyltrimethoxysilane in 200 proof alcohol for 1 hour and washed 5 times with ethanol to remove unbound or aggregated particles. The plate was sonicated at medium speed for 10 minutes each time. The 96-well plate was then dried in a vacuum oven at 75°C for 24 hours.

[0407] The n-octyltrimethoxysilane functionalized plates were treated by adding 100 μL of SC-PEG-SH functionalized AuSP at 100 nM concentration in IPA to each well and allowing the solution to evaporate slowly for 24 h. After 24 h, the particles were washed and incubated in running buffer for 12 h to remove any unbound nanoparticles from the assembly.

[0408] Example 4: Three-dimensional array

[0409] Example 4 demonstrates techniques that can be used to produce three-dimensional arrays using gold nanoparticles and glass fibers. For example, other types of nanoparticles, other types of fibers, and alternative chemistries with different sizes and shapes can be used throughout the application. Nanoparticles are produced based on procedures provided by the following document: Masterson et al., (2020) Anal. Chem. 92, 13, 9295-9304, which is hereby incorporated by reference herein.

[0410] AuSNP-citrate: AuSNP (spherical) was synthesized using a slightly modified public procedure (Masterson et al., (2020) Analytical Chemistry 92, 13, 9295-9304) to provide a larger volume. Briefly, 10 ml of HAuCl4H2O solution (4 mg / mL) was added to 390 mL of H2O in a 2L round-bottom flask on a heating mantle. The temperature was then slowly increased until the solution began to boil, followed by a rapid injection of 3 mL of sodium citrate aqueous solution (10 mg / mL). The reaction was allowed to proceed for 5 minutes while stirring, and then the heat source was removed and the red solution was cooled.

[0411] AuNR-CTAB: The initial step of AuNR (nanorod) seed solution generation was to add 250 μL of 10 mM HAuCl4 solution to 7.5 mL of 100 mM hexadecyltrimethylammonium bromide ("CTAB") solution in a 20 mL glass vial at room temperature and under moderate stirring. Five minutes later, 600 μL of 10 mM NaBH4 solution was injected, and the reaction was continued for another 15 minutes. NaBH4 was used as a reducing agent, and after the addition of NaBH4, a yellow to light color change occurred (seed solution).

[0412] AuNR solution was prepared by adding 2 mL of 10 mM HAuCl4 solution to 28 ml of 100 mM CTAB solution, mixing well, adding 0.4 ml of 10 mM AgNO3 and adding 0.22 ml of 100 mM L-ascorbic acid and mixing well. Reduction of HAuCl4 was confirmed by the formation of a colorless solution.

[0413] Finally, 48 μL of the seed solution was injected into the AuNR solution, and the reaction was allowed to complete for at least four hours.

[0414] AuTNP-TEA / CTAB: AuTNP (triangle) was chemically synthesized using a previously developed procedure with minor modifications. Briefly, Et3PAu(I)Cl (18.0 mg, 0.05 mmol) was dissolved in 40 mL CH3CN and stirred at room temperature for 10 minutes. Next, 0.038 mL triethylamine ("TEA") was injected into the solution, and the temperature was gradually increased. At 40°C, 600 μL of poly(methylhydrogensiloxane) ("PMHS") with a MW of 1700 to 3200 Daltons was added, the reaction was carried out under slow magnetic stirring, and the temperature was increased to 50°C. During the reaction, the color of the solution changed from colorless to pink, purple, and light blue. The reaction stopped at the 690 nm UV peak.

[0415] Array preparation: Three-dimensional microfilms were obtained using glass fiber sheets and nitrocellulose sheets with or without binders. Sheet parameters were basis weight 75 g / m 2 , diameter 0.43mm, wicking rate 5 (s / 2cm) and water absorption rate 79mg / cm 2 The substrate was cut into the desired shape using a carbon steel hollow punch set and / or custom die to accommodate the orifice plate diameters as shown in Table 14.

[0416] Table 14

[0417] Multi-well plate Micropore diameter (mm) Tool size (mm) 96-well plate 5 4.7625mm 48-well plate 6 5.7625mm 24-well plate 10，13 9.7625, 12.7625mm 12-well plate 10，14 9.7625, 13.7625mm 6-well plate 10，14，20 9.7625, 13.7625, 19.7625mm

[0418] After obtaining a specific size, the cut was washed with isopropanol and methanol, sonicated at medium speed for 1 hour, and dried in a vacuum oven at 80° C. to remove the organic solvent. This step helps to remove excess microfibers and clean the surface of the glass fiber before functionalization.

[0419] The substrate was then reacted with 40% 3-aminopropyl-triethoxysilane ("APTES") in isopropyl alcohol ("IPA") / ethanol in a sealed container on a rotary shaker for 5 hours. Excess APTES was removed, and the substrate was washed five times in IPA / ethanol with 10-minute shaking intervals. After removing excess APTES, the fiber array was dried in a vacuum oven at 120°C.

[0420] Nanoparticles functionalized with polyethylene glycol thiol (PEG(lk)-SH): AuSP-citrate, AuTNP-TEA / CTAB, AuNRS-CTAB, and AuNST-CTAB were ligand exchanged with PEG-SH, respectively. Thus, 10 mM-PEG-SC (N-hydroxysuccinimide)-SH / 1 optical density (OD) nanoparticle solution (NPS) was dissolved in Tri-HCl buffer at pH 3.0, and the reaction was continued for 3 hours. The PEG-functionalized nanoparticles were isolated by solvent extraction using CHCl3.

[0421] Self-assembly: PEGylated nanoparticles (20 mL 3OD solution) were added to 100 fiber arrays in a 50 mL centrifuge tube and placed on a medium speed rotary shaker for 12 hours. The chip was separated and then washed with a large amount of chloroform. During the final wash, the fiber array was sonicated in a cold bath at medium speed for one hour to remove loosely bound nanoparticles. The 2D nanoparticles on the 3D glass fiber array were then ready for use.

[0422] Although the present invention has been described and illustrated with reference to certain specific embodiments thereof, those skilled in the art will recognize that various adaptations, changes, modifications, substitutions, deletions or additions may be made to the procedures and schemes without departing from the spirit and scope of the present invention.

Claims

1. An analyte detection signal amplifier, comprising (a) an analyte binding molecule; (b) a localized surface plasmon resonance nanostructure; and (c) (i) a bioluminescence resonance energy transfer (BRET) assembly complex, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor, or (ii) a fluorescence resonance energy transfer (FRET) assembly complex, wherein the FRET assembly complex comprises a fluorescence donor conjugated to a fluorophore acceptor. 2 . The analyte detection signal amplifier according to claim 1 , wherein the analyte binding molecule is a single-stranded polynucleotide, an antibody, an aptamer, a protein, a substrate, a receptor or a ligand. 3 . The analyte detection signal amplifier according to claim 1 , wherein the surface localized plasmon resonance nanostructure is a nanorod comprising a metal. The analyte detection signal amplifier according to claim 3 , wherein the nanorods comprise gold.

5. The analyte detection signal amplifier according to any one of claims 1 to 4, wherein the surface localized plasmon resonance nanostructure is functionalized with polyethylene glycol (PEG), the BRET assembly complex or the FRET assembly complex is anchored to the localized surface plasmon resonance nanostructure through a first PEG chain, and the analyte binding molecule is anchored to the localized surface plasmon resonance nanostructure through a second PEG chain. The analyte detection signal amplifier according to any one of claims 1 to 5, comprising the BRET assembly complex.

7. The analyte detection signal amplifier according to claim 6, wherein the luciferase donor conjugated to the fluorophore acceptor is selected from the following combinations: NLuc-HT / HL-Oregon Green, RLuc / YFP, RLuc / fluorescent protein (GFP), RLuc8 / GFP, firefly luciferase / DsRed, RLuc / ODot, Rluc8 / ODot or Nano Luc-HT / Halotag-fluorescent ligand.

8. A method for detecting or quantifying an analyte in a sample, the method comprising the following steps: a) contacting a localized surface plasmon resonance sensor with the sample, wherein the sensor comprises an analyte capture molecule; b) providing an analyte detection signal amplifier to the sample, wherein the analyte detection signal amplifier comprises the BRET assembly complex according to any one of claims 1 to 7 and binds to the analyte; c) adding luciferase substrate; and d) Detection of fluorescence, bioluminescence, localized surface plasmon resonance or surface enhanced Raman scattering.

9. A method for detecting or quantifying an analyte in a sample, the method comprising the following steps: a) contacting a localized surface plasmon resonance sensor with the sample, wherein the sensor comprises an analyte capture molecule; b) providing an analyte detection signal amplifier to the sample, wherein the analyte detection signal amplifier comprises the FRET assembly complex according to any one of claims 1 to 5 and binds to the analyte; c) exciting the FRET assembly complex; and d) Detection of fluorescence, localized surface plasmon resonance or surface enhanced Raman scattering.

10. The method of claim 8 or 9, wherein the sensor comprises metal. The method of claim 10 , wherein the sensor comprises gold.

12. The method according to any one of claims 8 to 11, wherein after step b and before step c, the analyte signal amplifier not bound to the analyte is removed.

13. The method according to any one of claims 8 to 12, wherein fluorescence and / or bioluminescence is measured.

14. The method according to any one of claims 8 to 12, wherein surface enhanced Raman scattering is measured.

15. A multi-analyte detection system, comprising: a) a first LSAP nanostructure sensor, wherein the first LSAP nanostructure sensor comprises an analyte capture molecule of miR-205; b) a second LSAP nanostructure sensor, wherein the second LSAP nanostructure sensor comprises an analyte capture molecule of miR-16-1; c) a third LSAP nanostructure sensor, wherein the third LSAP nanostructure sensor comprises an analyte capture molecule of miR-143; d) a fourth LSAP nanostructure sensor, wherein the fourth LSAP nanostructure sensor comprises an analyte capture molecule of UCA1 nucleic acid; e) a fifth LSAP nanostructure sensor, wherein the fifth LSAP nanostructure sensor comprises an analyte capture molecule of IGF2 or IGF2 nucleic acid; f) a sixth LSAP nanostructure sensor, wherein the sixth LSAP nanostructure sensor comprises an analyte capture molecule of ANXA10 or ANXA10 nucleic acid; Each sensor type can be present on one or more platforms.

16. The multi-analyte detection system of claim 15, further comprising: g) a seventh LSAP nanostructure sensor, wherein the seventh LSAP nanostructure sensor comprises an analyte capture molecule of NMP-22 or NMP-22 nucleic acid; h) an eighth LSAP nanostructure sensor, wherein the eighth LSAP nanostructure sensor comprises an analyte capture molecule of HCFHrp or HCFHrp nucleic acid; i) a ninth LSPM nanostructure sensor, wherein the ninth LSPM nanostructure sensor comprises an analyte capture molecule of miR-200C; j) a tenth LSPM nanostructure sensor, wherein the tenth LSPM nanostructure sensor comprises an analyte capture molecule of UPKB or UPKB nucleic acid; k) an eleventh LSPM nanostructure sensor, wherein the eleventh LSPM nanostructure sensor comprises an analyte capture molecule of ABL1 or ABL1 nucleic acid; as well as 1) a twelfth localized surface plasmon nanostructure sensor, wherein the twelfth localized surface plasmon nanostructure sensor comprises an analyte capture molecule of CRH or CRH nucleic acid; Each sensor type can be present on one or more platforms. 17 . The multi-analyte detection system according to claim 16 , further comprising a thirteenth LSAP nanostructure sensor, wherein the thirteenth LSAP nanostructure sensor comprises an analyte capture molecule of P53 or P53 nucleic acid.

18. The multi-analyte detection system according to claim 16 or 17, further comprising a fourteenth LSPP nanostructure sensor, wherein the fourteenth LSPP nanostructure sensor comprises an analyte capture molecule of KRT17 or KRT17 nucleic acid.

19. The multi-analyte detection system according to any one of claims 15 to 18, wherein: a) the miR-205 analyte capture molecule is a single-stranded polynucleotide complementary to miR-205; b) the miR-16-1 analyte capture molecule is a single-stranded polynucleotide complementary to miR-16-1; c) the miR-143 analyte capture molecule is a single-stranded polynucleotide complementary to miR-143; d) the analyte capture molecule of the UCA1 nucleic acid is a single-stranded polynucleotide complementary to the UCA1 nucleic acid; e) the IGF2 or nucleic acid encoding analyte capture molecule is an antibody specific for mRNA encoding IGF2; f) the ANXA10 or nucleic acid encoding analyte capture molecule is an antibody specific for mRNA encoding ANXA10; g) the NMP-22 or nucleic acid encoding analyte capture molecule is an NMP-22 specific antibody; h) the analyte capture molecule of HCFHrp or encoding nucleic acid is a HCFHrp specific antibody; i) the miR-200C analyte capture molecule is a single-stranded polynucleotide complementary to miR-200C; j) the UPKB or nucleic acid encoding analyte capture molecule is an antibody specific for mRNA encoding UPKB; k) the ABL1 or nucleic acid encoding analyte capture molecule is an antibody specific for mRNA encoding ABL1; and 1) The CRH mRNA or nucleic acid encoding analyte capture molecule is an antibody specific for the mRNA encoding CRH; and each sensor type is present on a different platform.

20. The multi-analyte detection system of any one of claims 15 to 19, wherein each LSPM nanostructure comprises gold.

21. The multi-analyte detection system according to any one of claims 15 to 19, wherein each LSPM nanostructure comprises a gold sphere.

22. The multi-analyte detection system according to any one of claims 15 to 21, wherein each LSPM nanostructure is functionalized with a polyethylene glycol (PEG) chain, and the analyte binding molecule is conjugated to the PEG chain.

23. A method for determining whether a human subject has bladder cancer, the method comprising the steps of: a) contacting the multi-analyte detection system according to any one of claims 15 to 22 with a biological sample from the subject, b) providing a detection signal amplifier, wherein: i) providing a first detection signal amplifier to a first sensor, wherein the first detection signal amplifier comprises a BRET assembly complex, a localized surface plasmon resonance nanostructure, and an analyte binding molecule of miR-205, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor; ii) providing a second detection signal amplifier to a second sensor, wherein the second detection signal amplifier comprises a BRET assembly complex, a localized surface plasmon resonance nanostructure, and an analyte binding molecule of miR-16-1, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor; iii) providing a third detection signal amplifier to a third sensor, wherein the third detection signal amplifier comprises a BRET assembly complex, a localized surface plasmon resonance nanostructure, and an analyte binding molecule of miR-143, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor; iv) providing a fourth detection signal amplifier to a fourth sensor, wherein the fourth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of a UCA1 nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor; v) providing a fifth detection signal amplifier to a fifth sensor, wherein the fifth sensor comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of IGF2 or IGF2 nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor; and vi) providing a sixth detection signal amplifier to a sixth sensor, wherein the sixth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of ANXA10 or ANXA10 nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor; c) adding luciferase substrate; d) detecting fluorescence, localized surface plasmon resonance, bioluminescence or surface enhanced Raman scattering, thereby indicating the presence or amount of an analyte in the sample.

24. The method according to claim 23, wherein step b further comprises: vii) providing a seventh signal amplifier to a seventh detection sensor, wherein the seventh detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of NMP-22 or NMP-22 nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor; viii) providing an eighth detection signal amplifier to an eighth sensor, wherein the eighth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of HCFHrp or HCFHrp nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor; ix) providing a ninth detection signal amplifier to a ninth sensor, wherein the ninth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of a miR-200C nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor; x) providing a tenth detection signal amplifier to a tenth sensor, wherein the tenth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of UPKB or UPKB nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor; xi) providing an eleventh detection signal amplifier to an eleventh sensor, wherein the eleventh detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of ABL1 or ABL1 nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor; and xii) providing a twelfth detection signal amplifier to the twelfth sensor, wherein the twelfth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of CRH or CRH nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor.

25. The method according to claim 24, wherein step b further comprises providing a thirteenth detection signal amplifier to the thirteenth sensor, wherein the thirteenth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex and an analyte binding molecule of P53 or P53 nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor.

26. The method according to claim 24 or 25, wherein step b further comprises providing a fourteenth detection signal amplifier to the fourteenth sensor, wherein the fourteenth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a BRET assembly complex, and an analyte binding molecule of KRT17 or KRT17 nucleic acid, wherein the BRET assembly complex comprises a luciferase donor conjugated to a fluorophore acceptor.

27. The method according to any one of claims 23 to 26, wherein i) the miR-205 analyte binding molecule is a single-stranded polynucleotide complementary to miR-205; ii) the analyte binding molecule of miR-16-1 is a single-stranded polynucleotide complementary to miR-16-1; iii) the miR-143 analyte binding molecule is a single-stranded polynucleotide complementary to miR-143; iv) the analyte binding molecule for the UCA1 nucleic acid is a single-stranded polynucleotide complementary to the UCA1 nucleic acid; v) the IGF2 or nucleic acid encoding analyte binding molecule is an antibody specific for mRNA encoding IGF2; vi) the analyte binding molecule of ANXA10 or encoding nucleic acid is an antibody specific for mRNA encoding ANXA10; vii) the NMP-22 or encoding nucleic acid analyte binding molecule is an NMP-22 specific antibody; viii) the analyte binding molecule of HCFHrp or encoding nucleic acid is a HCFHrp specific antibody; ix) the analyte binding molecule of miR-200C is a single-stranded polynucleotide complementary to miR-200C; x) the UPKB or nucleic acid encoding analyte binding molecule is an antibody specific for mRNA encoding UPKB; xi) the ABL1 or nucleic acid encoding analyte binding molecule is an antibody specific for mRNA encoding ABL1; and xii) The analyte binding molecule for CRH mRNA or encoding nucleic acid is an antibody specific for mRNA encoding CRH.

28. A method for determining whether a subject has bladder cancer, the method comprising the steps of: a) contacting a multi-analyte detection system according to any one of claims 15 to 22 with a biological sample from a human subject, b) providing a detection signal amplifier, wherein: i) providing a first detection signal amplifier to a first sensor, wherein the first detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of miR-205, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor; ii) providing a second detection signal amplifier to a second sensor, wherein the second detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of miR-16-1, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor; iii) providing a third detection signal amplifier to a third sensor, wherein the third detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of miR-143 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor; iv) providing a fourth detection signal amplifier to a fourth sensor, wherein the fourth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of UCA1 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor; v) providing a fifth detection signal amplifier to a fifth sensor, wherein the fifth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of IGF2 or IGF2 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor; and vi) providing a sixth detection signal amplifier to a sixth sensor, wherein the sixth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of ANXA10 or ANXA10 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor; c) exciting each FRET assembly complex; d) detecting fluorescence, localized surface plasmon resonance or surface enhanced Raman scattering, thereby indicating the presence or amount of an analyte in the sample.

29. The method according to claim 28, wherein step b further comprises: vii) providing a seventh detection signal amplifier to a seventh sensor, wherein the seventh detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of NMP-22 or NMP-22 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor; viii) providing an eighth detection signal amplifier to an eighth sensor, wherein the eighth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of HCFHrp or HCFHrp nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor; ix) providing a ninth detection signal amplifier to a ninth sensor, wherein the ninth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of miR-200C nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor; x) providing a tenth detection signal amplifier to a tenth sensor, wherein the tenth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of UPKB or UPKB nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor; xi) providing an eleventh detection signal amplifier to an eleventh sensor, wherein the eleventh detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of ABL1 or ABL1 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor; and xii) providing a twelfth detection signal amplifier to the twelfth sensor, wherein the twelfth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of CRH or CRH nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor.

30. The method according to claim 29, wherein step b further comprises providing a thirteenth detection signal amplifier to the thirteenth sensor, wherein the thirteenth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex, and an analyte binding molecule of P53 or P53 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor.

31. The method according to claim 29 or 30, wherein step b further comprises providing a fourteenth detection signal amplifier to the fourteenth sensor, wherein the fourteenth detection signal amplifier comprises a localized surface plasmon resonance nanostructure, a FRET assembly complex and an analyte binding molecule of KRT17 or KRT17 nucleic acid, wherein the FRET assembly complex comprises a fluorophore donor conjugated to a fluorophore acceptor.

32. The method according to any one of claims 28 to 31, wherein i) the miR-205 analyte binding molecule is a single-stranded polynucleotide complementary to miR-205; ii) the analyte binding molecule of miR-16-1 is a single-stranded polynucleotide complementary to miR-16-1; iii) the miR-143 analyte binding molecule is a single-stranded polynucleotide complementary to miR-143; iv) the analyte binding molecule for the UCA1 nucleic acid is a single-stranded polynucleotide complementary to the UCA1 nucleic acid; v) the IGF2 or nucleic acid encoding analyte binding molecule is an antibody specific for mRNA encoding IGF2; vi) the analyte binding molecule of ANXA10 or encoding nucleic acid is an antibody specific for mRNA encoding ANXA10; vii) the NMP-22 or encoding nucleic acid analyte binding molecule is an NMP-22 specific antibody; viii) the analyte binding molecule of HCFHrp or encoding nucleic acid is a HCFHrp specific antibody; ix) the analyte binding molecule of miR-200C is a single-stranded polynucleotide complementary to miR-200C; x) the UPKB or nucleic acid encoding analyte binding molecule is an antibody specific for mRNA encoding UPKB; xi) the ABL1 or nucleic acid encoding analyte binding molecule is an antibody specific for mRNA encoding ABL1; and xii) The analyte binding molecule for CRH mRNA or encoding nucleic acid is an antibody specific for mRNA encoding CRH.

33. The method of any one of claims 23 to 32, wherein the sample is a urine sample.

34. The method of claim 33, wherein the urine sample has not been subjected to purification prior to step a.

35. A method for determining whether a human subject has bladder cancer, the method comprising the step of detecting the amount of miR-205, miR-16-1, miR-143, UCA1 mRNA, IGF2 or IGF2 nucleic acid, ANXA10 or ANXA10 nucleic acid in a biological sample from the subject.

36. The method of claim 35, further comprising detecting the amount of NMP-22 or NMP-22 nucleic acid, HCFHrp or HCFHrp nucleic acid, miR-200C, UPKB or UPKB nucleic acid, ABL1 or ABL1 nucleic acid, and CRH or CRH nucleic acid in the biological sample from the subject.

37. The method according to claim 35 or 36, further comprising detecting the amount of P53 or a nucleic acid encoding P53 in the biological sample.

38. The method of any one of claims 35 to 37, further comprising detecting the amount of KRT17 or a nucleic acid encoding KRT17 in the biological sample.

39. A method for treating bladder cancer in a subject, the method comprising (a) detecting the amount of an analyte associated with bladder cancer using a method according to any one of claims 23 to 38; and (b) administering a therapeutically effective amount of a bladder cancer therapeutic to a subject determined to have bladder cancer based in whole or in part on (a).

40. A method of treating bladder cancer in a subject, the method comprising administering to a subject determined to have bladder cancer based in whole or in part on the results of using the method of any one of claims 23 to 38 a therapeutically effective amount of a bladder cancer therapeutic.

41. The method of claim 39 or 40, wherein the bladder cancer therapeutic agent is selected from the group consisting of atezolizumab, avelumab, erdafitinib, cisplatin, doxorubicin hydrochloride, enfortumab vedotin-ejfv, erdafitinib, mitomycin, pembrolizumab, nivolumab, sacituzumab govitecan-hziy, and valrubicin.

42. A method for detecting or quantifying an analyte in a sample, the method comprising the steps of: a) contacting a localized surface plasmon resonance sensor with the sample, wherein the sensor comprises an analyte capture molecule; b) providing an analyte detection signal amplifier to the sample, wherein the analyte detection signal amplifier comprises the BRET assembly complex according to claim 1 and binds to the analyte; c) adding luciferase substrate; and Detect fluorescence, bioluminescence, localized surface plasmon resonance, or surface-enhanced Raman scattering.

43. The method of claim 42, wherein the sample is a biological sample from a human patient, and the method comprises using an analyte detection signal amplifier to detect the amount of miR-205, miR-16-1, miR-143, UCA1 mRNA, IGF2 or IGF2 nucleic acid, ANXA10 or ANXA10 nucleic acid in the biological sample.

44. The method of claim 43, further comprising detecting the amount of NMP-22 or NMP-22 nucleic acid, HCFHrp or HCFHrp nucleic acid, miR-200C, UPKB or UPKB nucleic acid, ABL1 or ABL1 nucleic acid, or CRH or CRH nucleic acid in the biological sample.

45. The method of claim 44, further comprising detecting the amount of P53 or a nucleic acid encoding P53 in the biological sample.

46. ​​The method of claim 45, further comprising detecting the amount of KRT17 or a nucleic acid encoding KRT17 in the biological sample.

47. A method for treating bladder cancer in a subject, the method comprising (a) detecting the amount of an analyte associated with bladder cancer using a method according to any one of claims 43 to 46; and (b) administering a therapeutically effective amount of a bladder cancer therapeutic to a subject determined to have bladder cancer based in whole or in part on (a).

48. A method of treating bladder cancer in a subject, the method comprising administering to a subject determined to have bladder cancer based in whole or in part on the results of using the method of any one of claims 43 to 46 a therapeutically effective amount of a bladder cancer therapeutic.

49. The method of claim 47, wherein the bladder cancer therapeutic is selected from the group consisting of atezolizumab, avelumab, erdafitinib, cisplatin, doxorubicin hydrochloride, ventuumab, erdafitinib, mitomycin, pembrolizumab, nivolumab, gosatuzumab, and valrubicin.

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