A fluorescence detection kit for cardiac troponin I and a detection method thereof

Abstract

This invention discloses a fluorescent detection kit for cardiac troponin I and its detection method, belonging to the field of biodetection and in vitro diagnostic technology. The kit includes a biomimetic recognition and trapping unit, a fluorescent labeling unit, a working solution, and a nucleic acid lyase unit. The biomimetic recognition and trapping unit includes a polypropylene (PP) tube with a biomimetic antifouling functional layer modified on its inner wall, cardiac troponin I (cTnI) aptamer A, and auxiliary reagent A. The fluorescent labeling unit includes a tetrahedral framework nucleic acid tFNA linked to cTnI aptamer B, a fluorescently labeled branched hybridization chain reaction (BHCR) probe, and auxiliary reagent B. The kit utilizes the biomimetic recognition and trapping unit and the fluorescent labeling unit to achieve highly specific binding of cTnI in the working solution with aptamer A and aptamer B-tFNA-BHCR probe, immobilizing and labeling it on the PP tube to form a fluorescent sandwich structure. The dual aptamer recognition exhibits high specificity, and the BHCR amplification chain fluorescent probe has high sensitivity. After enzymatic digestion by the nucleic acid lyase, the solution is used for molecular fluorescence measurement, achieving highly sensitive determination of trace cTnI.

Description

Technical Field

[0001] This invention relates to the field of biological detection and in vitro diagnostic technology, and in particular to a cardiac troponin I fluorescence detection kit and its detection method. Background Technology

[0002] Cardiac troponin I (cTnI) is a core specific biomarker for diagnosing acute myocardial infarction and myocardial injury. Changes in its serum concentration can directly reflect the degree of myocardial injury. It is a core detection indicator for clinical emergency and point-of-care testing (POCT) rapid diagnosis. Early micro-detection has important clinical significance in reducing cardiovascular disease mortality.

[0003] Existing mature cTnI fluorescence immunoassay techniques mainly employ time-resolved fluorescence immunoassay (TRFIA), which uses β-diketone ligands to chelate rare earth ions Eu. 3+ / Sm 3+ As a fluorescent probe, it boasts a fluorescence lifetime of hundreds of microseconds. Signal acquisition is achieved through time delay, eliminating interference from serum autofluorescence and stray light background. It primarily utilizes a sandwich immunoassay mode, often combined with magnetic bead separation. However, cTnI fluorescence detection technology suffers from several inherent drawbacks: First, conventional detection tubes lack anti-adsorption modification, making them susceptible to non-specific adsorption by large amounts of albumin and globulin in serum. Second, traditional detection systems rely mainly on immunofluidography, which is qualitative or semi-quantitative. The binding distribution of solid-phase fluorescent reagents to fibers is uneven, resulting in significant batch-to-batch variations. Residual macromolecular impurities continuously interfere with fluorescence readings, leading to poor stability in trace detection. Third, ordinary molecular fluorescent labels produce weak signals, lacking efficient signal amplification and purification mechanisms. Spatial quenching of long-chain nucleic acids after signal amplification leads to insufficient signal release and limited detection sensitivity, making it difficult to meet the detection needs of trace cTnI in ultra-early myocardial injury.

[0004] Based on this, and addressing the shortcomings of existing technologies, this invention innovatively designs a PP ultrafiltration tube immobilized aptamer, combining MPC anti-fouling modification, dual aptamer specific capture, BHCR amplified strand fluorescent labeling, and nuclease cleavage release signal technology into an integrated application. This achieves dual noise reduction through biomimetic anti-interference and dual aptamer recognition, while the BHCR amplified strand and enzyme cleavage release signal synergistically improve sensitivity, enhancing the specificity and sensitivity of myocardial biomarker detection. It is suitable for conventional molecular fluorescence assay instruments, enabling highly sensitive POCT measurement of trace cTnI. Summary of the Invention

[0005] This invention aims to at least partially solve one of the technical problems in the aforementioned technologies. Therefore, the purpose of this invention is to propose a cardiac troponin I fluorescence detection kit and its detection method, solving the technical problems of non-specific adsorption on the tube wall, background interference from residual macromolecular impurities, insufficient sensitivity of ordinary molecular fluorescence signals, and large errors in trace detection of existing cardiac marker cTnI detection methods, thereby achieving highly specific and sensitive molecular fluorescence quantitative detection of cTnI in serum.

[0006] This invention provides a cardiac troponin I fluorescence detection kit, comprising a biomimetic recognition and trapping unit, a fluorescent labeling unit, a working solution, and a nucleic acid lysin unit; the biomimetic recognition and trapping unit comprises a polypropylene (PP) tube with an inner wall modified with a biomimetic antifouling functional layer, a cardiac troponin I (cTnI) aptamer A, and an auxiliary reagent A; the fluorescent labeling unit comprises a tetrahedral framework nucleic acid tFNA connected to a cTnI aptamer B, a fluorescently labeled branched hybridization chain reaction polymer (BHCR) amplification chain probe, and an auxiliary reagent B. The kit uses an MPC / MBA anti-fouling coating to immobilize cTnI aptamers on PP tubes, inhibiting the non-specific adsorption of various proteins and impurities in the blood. Simultaneously, it constructs an aptamer-specific recognition interface to target and capture cTnI from serum in the working solution. Tetrahedral framework nucleic acids with cTnI aptamer B further specifically bind to the captured cTnI, mediating branched hybridization chain reaction on the tube wall for in-situ assembly at the recognition site. Based on the dual aptamer binding, a sandwich-type fluorescently labeled composite structure is formed, precisely immobilizing labeled cTnI. After removing unbound reactants, the nucleic acid components of the composite structure are enzymatically digested using deoxyribonuclease I and auxiliary reagent C, releasing nucleic acid fragments with fluorescent signals. This avoids the technical bottlenecks of traditional techniques, such as spatial quenching of the polymer chain after signal amplification, insufficient solid-phase fluorescence recognition on the PP tube wall, and low sensitivity. It can be used to achieve highly sensitive quantitative fluorescence detection of cTnI.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A cardiac troponin I fluorescence detection kit includes a biomimetic recognition and trapping unit, a fluorescent labeling unit, a working solution, and a nucleic acid lysin unit. The biomimetic recognition and trapping unit comprises a polypropylene (PP) tube with an inner wall modified with a biomimetic antifouling functional layer, cardiac troponin I (cTnI) aptamer A, and an auxiliary reagent A. The fluorescent labeling unit comprises a tetrahedral framework nucleic acid (tFNA) linked to cTnI aptamer B, a fluorescently labeled branched hybridization chain reaction polymer (BHCR) amplification chain probe, and an auxiliary reagent B. The working solution is a Tris-HCl-Mg buffer solution. The nucleic acid lysin unit comprises deoxyribonuclease I and an auxiliary reagent C. The kit utilizes the bispecific recognition and trapping unit and the fluorescent labeling unit to bind cTnI, immobilizing and labeling it on the PP tube to form a fluorescent sandwich structure. After separation, the solution is enzymatically digested with nucleic acid lysin I and auxiliary reagent C to form a fluorescent solution, thereby achieving cTnI fluorescence detection.

[0008] Preferably, the biomimetic antifouling functional layer comprises an antifouling polymer layer modified with 2-methacryloyloxyethyl phosphocholine and N,N-methylenebis(acrylamide) MPC / MBA; the MPC / MBA modified antifouling polymer layer comprises a polymer film formed by copolymerization of alkenyl monomer, mercapto monomer, MPC / MBA monomer, and methanol and N,N-dimethylformamide binary porogen in a PP tube pretreated with benzophenone (BP); the alkenyl monomer is ethylene glycol dimethacrylate (EDMA); the mercapto monomer is pentaerythritol tetrakis(3-mercaptopropionic acid) PETMP; the proportion of each component in the polymerization solution of the MPC / MBA modified antifouling polymer layer, calculated by the sum of mass percentages of 100%, is as follows: the ratio of monomers EDMA:PETMP:MPC:MBA is 15:15:65:5, the ratio of binary porogen methanol-N,N-dimethylformamide is 20:80, and the ratio of total monomers to porogens is 20:80.

[0009] Preferably, the base sequence of the anti-cTnI aptamer A is 5'-SH-C6-CGTGCAGTAC GCCAA CCTTT CTCAT GCGCT GCCCC TCTTA-3'; the anti-cTnI aptamer A is bonded to the surface of the MPC / MBA modified antifouling polymer layer via a mercapto-olefin click chemistry reaction.

[0010] Preferably, the tetrahedral framework nucleic acid is self-assembled using four DNA strands (T1~T4), wherein DNA strand T1 is linked to cTnI aptamer B; the base sequence of cTnI aptamer B is 5′-CGCAT GCCAA ACGTTGCCTC ATAGT TCCCT CCCCG TGTCC-3′; The base sequence of the DNA strand is: T1: CGCAT GCCAA ACGTT GCCTC ATAGT TCCCT CCCCG TGTCCTT TTTTTT TTT ACATTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAGAGC CGC CAT AGT A; T2: ATG TTA GTG GGA GTG GAG GTG AGA TAT CAC CAG GCA GTTGAC AGT GTAGCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C; T3: ATG TTA GTG GGA GTG GAG GTG AGA TCA ACT GCC TGG TGATAA AAC GACACT ACG TGG GAA TCT ACT ATG GCG GCT CTT C; T4: ATG TTA GTG GGA GTG GAG GTG AGA TTC AGA CTT AGG AATGTG CTT CCCACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T.

[0011] Preferably, the branched hybridization chain reaction polymer BHCR amplification probe is assembled through a three-DNA chain (S1~S3) branched hybridization chain reaction and is ligated and fixed onto a tetrahedral framework nucleic acid by complementary pairing with the base sequence "ATG TTA GTG GGA GTG GAGGTG AGA" in the T2~T4 chains; the fluorescent probe is 5'-carboxyfluorescein (5'-FAM); the base sequence of the DNA chains (S1~S3) is: S1: TCT CAC CTC CAC TCC CAC TAA CAT CAA CAT CAG TCT GATAAG CTC TCACCT CGA TTG TGC TTG TTC CTG TGT CGT G; S2: 5'-FAM-ATG TTA GTG GGA GTG GAG GTG AGA CAC GAC ACAGGA ACA AGC ACAATC; S3: 5'-FAM-ATG TTA GTG GGA GTG GAG GTG AGA GAG GTG AGAGCT TAT CAG ACTGAT GTT G.

[0012] Preferably, auxiliary reagent A consists of 10 mmol / L Tris-HCl, 1 mmol / L KCl, 140 mmol / L NaCl, and pH 7.4; auxiliary reagent B consists of 20 mmol / L Tris-HCl, 50 mmol / L MgCl2, and pH 8.0. The Tris-HCl-Mg buffer solution consists of 50 mmol / L Tris-HCl, 2 mmol / L MgCl2, 150 mmol / L NaCl, and pH 7.4; auxiliary reagent C consists of 10 mM Tris-HCl, 50 mM MgCl2, 13 mM CaCl2, and pH 7.5.

[0013] A detection method for cardiac troponin I using a fluorescent detection kit, specifically including the following steps: S1, Preparation of fixation and labeling reagents: (1) Start the biomimetic recognition reaction unit, take the polypropylene (PP) microtube from the kit, add the anti-cTnI aptamer A and auxiliary reagent A, click the chemical reaction to bond the appropriate ligand A on the surface of the polypropylene (PP) tube, and prepare the cTnI fixation reagent; (2) Start the fluorescent labeling unit, take the tetrahedral framework nucleic acid tFNA connected with cTnI aptamer B, the branched hybridization chain reaction polymer BHCR probe and the auxiliary reagent B, and incubate at room temperature to obtain the aptamer B-tFNA-BHCR amplification chain fluorescent probe, and prepare cTnI labeling reagent; S2, Take the sample solution, add it to a PP tube along with the aptamer B-tFNA-BHCR amplification chain fluorescent probe and working solution, and incubate at room temperature to obtain the PP tube-fixed aptamer A-cTnI-aptamer B-tFNA-BHCR amplification chain fluorescently labeled complex. S3, wash the fluorescently labeled complex fixed in the PP tube, add deoxyribonuclease I and auxiliary reagent C for enzymatic digestion; S4, detect the fluorescence signal in the solution and determine cTnI.

[0014] Compared with the prior art, the present invention has the following significant advantages: 1. Dual Anti-interference System: The kit uses an MPC / MBA anti-fouling coating and cTnI aptamers fixed on PP tubes to inhibit the non-specific adsorption of various proteins and impurities in the blood. At the same time, based on the aptamer labeling and binding, a sandwich-type fluorescent labeling composite structure is formed. The dual aptamers target and recognize cTnI in serum, effectively avoiding cross-interference from myocardial homologous markers such as cTnT, CK-MB, and myoglobin, resulting in high recognition specificity.

[0015] 2. Two-stage signal amplification system: Utilizing tetrahedral framework nucleic acids carrying cTnI aptamer B to specifically bind to captured cardiac troponin I, HCR nucleic acid chain amplification of the fluorescent probe is performed simultaneously. Nuclease cleavage releases free fluorescent signals, avoiding the technical bottlenecks of traditional techniques such as spatial quenching of long-chain nucleic acids after signal amplification, insufficient recognition of fluorescent solid phase immobilization on the tube wall, and low sensitivity. This system breaks through the sensitivity limitations of traditional fluorescence detection and can be used to achieve highly sensitive quantitative fluorescence detection of cTnI.

[0016] 3. The fluorescence detection kit system is stable and has strong resistance to matrix interference. It integrates target capture, signal amplification, and molecular fluorescence detection. It is simple to operate, has good molecular fluorescence signal stability, and is easy to promote.

[0017] Other features and advantages of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures particularly pointed out in the written description and the accompanying drawings.

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is a schematic diagram illustrating the measurement principle of a cardiac troponin I fluorescence detection kit according to an embodiment of the present invention; Figure 2 This is a linear fluorescence curve of cardiac troponin I detection kit according to an embodiment of the present invention. Figure 3 shows the fluorescence linearity curve for direct labeling and detection using traditional molecular fluorescent reagents.

[0020] Figure label: For MPC / MBA anti-fouling coating; For aptamer A; It is a fluorescently labeled probe for the aptamer B-BHCR. Detailed Implementation

[0021] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0022] This embodiment provides a cardiac troponin I fluorescence detection kit, including a biomimetic recognition and trapping unit, a fluorescent labeling unit, a working solution, and a nucleic acid lysin unit; the biomimetic recognition and trapping unit includes a polypropylene (PP) tube with a biomimetic antifouling functional layer modified on the inner wall, a cardiac troponin I (cTnI) aptamer A, and an auxiliary reagent A; the fluorescent labeling unit includes a tetrahedral framework nucleic acid tFNA connected to a cTnI aptamer B, a fluorescently labeled branched hybridization chain reaction polymer (BHCR) amplification chain probe, and an auxiliary reagent B. As shown in Figure 1, the kit uses an MPC anti-fouling coating to immobilize cTnI aptamers on PP tubes, inhibiting the non-specific adsorption of various proteins in the blood and simultaneously constructing an aptamer-specific recognition interface to target and capture cTnI in serum. The kit utilizes the specific binding of tetrahedral framework nucleic acids with cTnI aptamer B to the captured cardiac troponin I, mediating the in-situ assembly of branched hybridization reaction chains at the recognition site on the tube wall. Based on the dual aptamer binding, a sandwich-type fluorescently labeled complex structure is formed, accurately recognizing cTnI. Following this, after separating and removing unbound reactants using PP tubes, the fluorescently labeled complex structure is enzymatically digested with deoxyribonuclease I, releasing nucleic acid fragments carrying fluorescent signals. This avoids the technical bottlenecks of traditional techniques, such as spatial quenching of polymer chains after signal amplification, insufficient solid-phase fluorescence recognition, and low sensitivity, and can be used to achieve highly sensitive quantitative fluorescence detection of cTnI.

[0023] Example 1: Reagent Kit Preparation 1. Bionic recognition and capture unit: In this embodiment of the invention, the inner wall of the PP pipe is modified with a biomimetic antifouling functional layer, which includes an antifouling polymer layer modified with 2-methacryloyloxyethyl phosphocholine and N,N-methylenebis(acrylamide) MPC / MBA; the MPC / MBA modified antifouling polymer layer is a polymer film formed by copolymerization of alkenyl monomers, mercapto monomers, MPC / MBA monomers, and methanol and N,N-dimethylformamide binary porogens in a PP pipe pretreated with benzophenone (BP).

[0024] Specific preparation methods include: (1) PP tube surface modification: Prepare a 5 wt% methanol solution of BP, add it to the cleaned PP centrifuge tube, place it in an ultraviolet crosslinker (wavelength 254 nm, power: 6×8 W) for photoinitiation for 40 min, immediately rinse the PP tube with methanol after photoinitiation, and obtain BP modified PP centrifuge tube by blowing it dry with nitrogen.

[0025] (2) Alkenyl grafting: Add 15 wt% EDMA methanol solution to PP centrifuge tube, place it in a UV crosslinker and react for 40 min, rinse with methanol, and blow with nitrogen to obtain PP centrifuge tube with alkenyl monomer grafting uniformly on the inner wall.

[0026] (3) Preparation of MPC / MBA biomimetic antifouling layer: Weigh out the following proportions: ethylene glycol dimethacrylate (EDMA), thiol monomer pentaerythritol tetrakis(3-mercaptopropionic acid) ester (PETMP), and binary porogen methanol-N,N-dimethylformamide. Mix them thoroughly by shaking in a centrifuge tube. The ratio of EDMA:PETMP:MPC:MBA monomers is 15:15:65:5 (based on a total mass percentage of 100%), the ratio of methanol-N,N-dimethylformamide is 20:80, and the ratio of total monomers to porogens is 20:80. The photoinitiator dimethoxy-2-phenylacetophenone (DMPA) accounts for 2.5% of the total monomers. Inject the mixture into the PP tube, seal it with a deoxygenating film, and place it in a UV crosslinker (wavelength 254 nm, power: 6×8 W) for photoinitiation for 7 min. Rinse with methanol to obtain a PP tube modified with an MPC / MBA antifouling polymer layer.

[0027] (4) Aptamer bonding modification: The base sequence of the anti-cTnI aptamer A is 5'-SH-C6-CGTGC AGTACGCCAA CCTTTCTCAT GCGCT GCCCC TCTTA-3'. Take 20 μL (5 μmol / L) of the activated aptamer into a centrifuge tube, add 40 μL of AIBA solution (concentration 10 mmol / L), and dilute with 40 μL of auxiliary reagent A. Auxiliary reagent A consists of 10 mmol / L Tris-HCl, 1 mmol / L KCl, 140 mmol / L NaCl, pH 7.4. Vortex to mix. Add 20 μL of the treated aptamer solution to the above PP tube and react for 1 hour at 60°C. Continue washing the entire column with auxiliary reagent A to remove unfixed nucleic acid aptamers and fix the aptamers in the PP tube.

[0028] 2. Fluorescent labeling unit: The nucleic acid tetrahedral tFNA linked with cTnI aptamer B was hybridized and coupled with a fluorescent label of the BHCR amplification chain to spontaneously assemble a nucleic acid tetrahedral linked tFNA-BHCR amplification chain fluorescent probe.

[0029] (1) The tetrahedral framework nucleic acid is self-assembled by four DNA strands (T1~T4), wherein DNA strand T1 is linked to cTnI aptamer B; the base sequence of the cTnI aptamer B is 5′-CGCAT GCCAA ACGTT GCCTC ATAGTTCCCT CCCCG TGTCC-3′; 1 μL of each of the four single strands T1, T2, T3 and T4 in equimolar mass (100 μmol / L) is mixed in 96 μL of auxiliary reagent B (20 mmol / L Tris, 50 mmol / L MgCl2, pH 8.0), placed in a water bath and incubated at 95°C for 10 min, and then quickly transferred to a 4°C ice-water bath for cooling.

[0030] The base sequence of the DNA strand is: T1: CGCAT GCCAA ACGTT GCCTC ATAGT TCCCT CCCCG TGTCCTT TTTTTT TTT ACATTC CTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAGAGC CGC CAT AGT A; T2: ATG TTA GTG GGA GTG GAG GTG AGA TAT CAC CAG GCA GTT GACAGT GTAGCA AGC TGT AAT AGA TGC GAG GGT CCA ATA C; T3: ATG TTA GTG GGA GTG GAG GTG AGA TCA ACT GCC TGG TGA TAAAAC GACACT ACG TGG GAA TCT ACT ATG GCG GCT CTT C; T4: ATG TTA GTG GGA GTG GAG GTG AGA TTC AGA CTT AGG AAT GTGCTT CCCACG TAG TGT CGT TTG TAT TGG ACC CTC GCA T.

[0031] (2) Preparation of BHCR probe: S1, S2 and S3 (100 μmol / L) in equimolar ratio were dissolved in an auxiliary solution and incubated at 37°C for 1 h to obtain a homogeneous BHCR probe solution; The branched hybridization chain reaction polymer BHCR amplification probe is assembled through a three-strand branched hybridization chain reaction (S1-S3), and is ligated and immobilized onto a tetrahedral framework nucleic acid by complementary pairing with the base sequence "ATG TTA GTG GGA GTG GAG GTGAGA" in the T2-T4 strands; the fluorescent probe is 5'-carboxyfluorescein (5'-FAM); the base sequence of the DNA strands (S1-S3) is as follows: S1: TCT CAC CTC CAC TCC CAC TAA CAT CAA CAT CAG TCT GAT AAGCTC TCACCT CGA TTG TGC TTG TTC CTG TGT CGT G; S2: 5'-FAM-ATG TTA GTG GGA GTG GAG GTG AGA CAC GAC ACA GGAACA AGC ACAATC; S3: 5'-FAM-ATG TTA GTG GGA GTG GAG GTG AGA GAG GTG AGA GCTTAT CAG ACTGAT GTT G.

[0032] (3) Assembly of tFNA-BHCR probe structure: The assembled tFNA tetrahedral structure was dispersed in auxiliary reagent B (20 mmol / L Tris, 50 mmol / L MgCl2, pH 8.0), and then an equimolar amount of BHCR probe was added. The assembly was carried out at room temperature for 1 h to obtain tFNA-BHCR probe solution.

[0033] 3. Preparation of working solution and nucleic acid lysin unit: The working solution is a Tris-HCl-Mg buffer solution with the composition of 50 mmol / L Tris-HCl, 2 mmol / L MgCl2, 150 mmol / L NaCl, and pH 7.4; the nucleic acid lysin unit includes nucleic acid lysin I (2500 U / mL) and auxiliary reagent C, which has the composition of 10 mM Tris·HCl, 50 mM MgCl2, 13 mM CaCl2, and pH 7.5.

[0034] Example 2 Serum cTnI Detection Procedure S1, Preparation of fixation and labeling reagents: (1) Start the biomimetic recognition reaction unit, take the polypropylene (PP) microtube from the kit, add the anti-cTnI aptamer A and auxiliary reagent A, click the chemical reaction to bond the appropriate ligand A on the surface of the polypropylene (PP) tube, and prepare the cTnI fixation reagent; (2) Start the fluorescent labeling unit, take the tetrahedral framework nucleic acid tFNA connected with cTnI aptamer B, the branched hybridization chain reaction polymer BHCR probe and the auxiliary reagent B, and incubate at room temperature to obtain the aptamer B-tFNA-BHCR amplification chain fluorescent probe, and prepare cTnI labeling reagent; S2, add 100 μL of the serum to be tested into a PP tube with an inner functionalized layer. Aptamer A specifically captures serum cTnI, and the MPC / MBA coating inhibits the adsorption of impurity proteins. Wash three times with auxiliary reagent A to remove free impurities from the serum. Add tFNA-BHCR fluorescent molecular probe solution and incubate at 37℃ for 25 min to form a sandwich complex, and obtain the aptamer A-cTnI-aptamer B-tFNA-BHCR amplification chain fluorescent labeling complex fixed in PP tube; S3, thoroughly clean to remove free probe, add nuclease and enzymatically digest at 37℃ for 15 min to lyse long-chain nucleic acid and release small molecule fluorescent fragments; S4. Centrifuge the PP tube at 3000 g for 5 min, transfer the solution and detect the fluorescence intensity. Quantify the cTnI concentration by referring to the standard curve.

[0035] Example 3: Detection Performance Verification The fluorescence solution was detected using a laser-induced fluorescence instrument with an excitation wavelength of 497 nm and an emission wavelength of 512 nm. A series of cTnI solutions were prepared for measurement. As shown in Figure 2, the fluorescence intensity of the system rapidly increased with increasing cTnI concentration. The fluorescence signal intensity (peak area) of the kit of this invention was linearly proportional to the logarithm of the cTnI concentration, with a linear range of 0.05–50 ng / mL and an R² of 0.9779. The lowest detection limit was [not specified in the original text]. ≤0.003 ng / mL; repeatability RSD of different detection experiments (n=3) between the two batches of kits = 1.0~4.5%; compared with the traditional molecular reagent direct labeling detection experiment, as shown in Figure 3, the linear curve of the traditional molecular fluorescent reagent direct labeling has a higher concentration, and the detection... With a concentration of ≤2.0 ng / mL, the reagent kit of this invention improves the detection sensitivity by more than 660 times, enabling sensitive detection of trace amounts of the myocardial disease biomarker cTnI.

[0036] Table 1. Stability of Detection Values The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

[0037] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A cardiac troponin I fluorescence detection kit, characterized in that: The kit includes a biomimetic recognition and trapping unit, a fluorescent labeling unit, a working solution, and a nucleic acid lysin unit. The biomimetic recognition and trapping unit comprises a polypropylene (PP) tube with an inner wall modified with a biomimetic antifouling functional layer, cardiac troponin I (cTnI) aptamer A, and auxiliary reagent A. The fluorescent labeling unit comprises a tetrahedral framework nucleic acid (tFNA) linked to cTnI aptamer B, a fluorescently labeled branched hybridization chain reaction polymer (BHCR) amplification chain probe, and auxiliary reagent B. The working solution is a Tris-HCl-Mg buffer solution. The nucleic acid lysin unit comprises deoxyribonuclease I and auxiliary reagent C. The kit utilizes the bispecific recognition and binding of cTnI by aptamers A and B in the biomimetic recognition and trapping unit and the fluorescent labeling unit to trap, fix, and label cTnI on the PP tube, forming a fluorescent sandwich structure. After separation, the cTnI is enzymatically digested with nucleic acid lysin I to form a fluorescent solution, achieving solution fluorescence detection of cTnI.

2. The cardiac troponin I fluorescence detection kit according to claim 1, characterized in that: The biomimetic antifouling functional layer comprises an antifouling polymer layer modified with 2-methacryloyloxyethyl phosphocholine and N,N-methylenebis(acrylamide) MPC / MBA; the MPC / MBA modified antifouling polymer layer comprises a polymer film formed by copolymerization of alkenyl monomers, mercapto monomers, MPC / MBA monomers, and methanol and N,N-dimethylformamide binary porogens in a PP tube pretreated with benzophenone (BP); the alkenyl monomer is ethylene glycol dimethacrylate (EDMA); the mercapto monomer is pentaerythritol tetrakis(3-mercaptopropionic acid) ester (PETMP); the proportion of each component in the polymerization solution of the MPC / MBA modified antifouling polymer layer, calculated by the sum of mass percentages of 100%, is as follows: the ratio of monomers EDMA:PETMP:MPC:MBA is 15:15:65:5, the ratio of binary porogens methanol and N,N-dimethylformamide is 20:80, and the ratio of total monomers to porogens is 20:

80.

3. The cardiac troponin I fluorescence detection kit according to claim 1, characterized in that: The base sequence of the anti-cTnI aptamer A is 5'-SH-C6-CGTGCAGTAC GCCAA CCTTT CTCAT GCGCTGCCCC TCTTA-3'; the anti-cTnI aptamer A is bonded to the surface of the MPC / MBA modified antifouling polymer layer via a mercapto-olefin click chemistry reaction.

4. The cardiac troponin I fluorescence detection kit according to claim 1, characterized in that: The tetrahedral framework nucleic acid is assembled from four DNA strands (T1~T4), wherein DNA strand T1 is linked to cTnI aptamer B; the base sequence of cTnI aptamer B is 5′-CGCAT GCCAA ACGTT GCCTC ATAGT TCCCT CCCCGTGTCC-3′; The base sequence of the DNA strand is: T1: CGCAT GCCAA ACGTT GCCTC ATAGT TCCCT CCCCG TGTCCTT TTTTTT TTT ACA TTCCTA AGT CTG AAA CAT TAC AGC TTG CTA CAC GAG AAGAGC CGC CAT AGT A; T2: ATG TTA GTG GGA GTG GAG GTG AGA TAT CAC CAG GCA GTTGAC AGT GTA GCAAGC TGT AAT AGA TGC GAG GGT CCA ATA C; T3: ATG TTA GTG GGA GTG GAG GTG AGA TCA ACT GCC TGG TGATAA AAC GAC ACTACG TGG GAA TCT ACT ATG GCG GCT CTT C; T4: ATG TTA GTG GGA GTG GAG GTG AGA TTC AGA CTT AGG AATGTG CTT CCC ACGTAG TGT CGT TTG TAT TGG ACC CTC GCA T.

5. The cardiac troponin I fluorescence detection kit according to claim 1, characterized in that: The branched hybridization chain reaction polymer BHCR amplification chain probe is assembled through a three-strand branched hybridization chain reaction (S1~S3) and is attached to the tetrahedral framework nucleic acid by complementary pairing with the base sequence "ATG TTA GTG GGA GTG GAG GTG AGA" in the T2~T4 strands; the fluorescent probe is 5'-carboxyfluorescein (5'-FAM). The base sequence of the DNA strand (S1~S3) is as follows: S1: TCT CAC CTC CAC TCC CAC TAA CAT CAA CAT CAG TCT GATAAG CTC TCA CCTCGA TTG TGC TTG TTC CTG TGT CGT G; S2: 5'-FAM-ATG TTA GTG GGA GTG GAG GTG AGA CAC GAC ACAGGA ACA AGC ACAATC; S3: 5'-FAM-ATG TTA GTG GGA GTG GAG GTG AGA GAG GTG AGAGCT TAT CAG ACT GATGTT G.

6. The cardiac troponin I fluorescence detection kit according to claim 1, characterized in that: The auxiliary reagent A has the following composition: 10 mmol / L Tris-HCl, 1 mmol / L KCl, 140 mmol / L NaCl, pH 7.4; the auxiliary reagent B has the following composition: 20 mmol / L Tris-HCl, 50 mmol / L MgCl2, pH 8.0; the Tris-HCl-Mg buffer solution consists of 50 mmol / L Tris-HCl, 2 mmol / L MgCl2, 150 mmol / L NaCl, pH 7.4; the auxiliary reagent C consists of 10 mM Tris-HCl, 50 mM MgCl2, 13 mM CaCl2, pH 7.

5.

7. A detection method for cardiac troponin I fluorescent detection kit according to any one of claims 1-6, characterized in that, Includes the following steps: S1, Preparation of fixation and labeling reagents: (1) Start the biomimetic recognition reaction unit, take the polypropylene (PP) microtube from the kit, add the anti-cTnI aptamer A and auxiliary reagent A, click the chemical reaction to bond the appropriate ligand A on the surface of the polypropylene (PP) tube, and prepare the cTnI fixation reagent; (2) Start the fluorescent labeling unit, take the tetrahedral framework nucleic acid tFNA connected with cTnI aptamer B, the branched hybridization chain reaction polymer BHCR probe and the auxiliary reagent B, and incubate at room temperature to obtain the aptamer B-tFNA-BHCR amplification chain fluorescent probe, and prepare cTnI labeling reagent; S2, Take the sample solution, add it to a PP tube along with the aptamer B-tFNA-BHCR amplification chain fluorescent probe and working solution, and incubate at room temperature to obtain the PP tube-fixed aptamer A-cTnI-aptamer B-tFNA-BHCR amplification chain fluorescently labeled complex. S3, wash the fluorescently labeled complex fixed in the PP tube, add deoxyribonuclease I and auxiliary reagent C for enzymatic digestion; S4, detect the fluorescence signal in the solution and determine cTnI.

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