CN107084966A - A kind of highly sensitive quantitative detecting method of cardiac muscle troponin I - Google Patents

A kind of highly sensitive quantitative detecting method of cardiac muscle troponin I Download PDF

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CN107084966A
CN107084966A CN201710447631.1A CN201710447631A CN107084966A CN 107084966 A CN107084966 A CN 107084966A CN 201710447631 A CN201710447631 A CN 201710447631A CN 107084966 A CN107084966 A CN 107084966A
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ctni
aptamers
concentration
elisa plate
liquid
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CN107084966B (en
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吕雪飞
李昱
代唯强
李永瑞
邓玉林
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Beijing Institute of Technology BIT
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching

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Abstract

The present invention relates to a kind of highly sensitive quantitative detecting method of cardiac muscle troponin I, belong to technical field of biomedical detection.Detection method of the present invention is that specific recognition, and the rolling circle amplification using nucleic acid and the FRET technology based on graphene oxide are carried out to cardiac muscle troponin I by aptamer, realizes the accurate sensitive quantitative detection to cTnI；The detection method has specific good, sensitivity high, easy to operate and low cost and other advantages, meets the demand of clinical detection.

Description

A kind of highly sensitive quantitative detecting method of cardiac muscle troponin I
Technical field
The present invention relates to a kind of highly sensitive quantitative detecting method of cardiac muscle troponin I, belong to biomedical detection technique Field.
Background technology
Cardiac troponin is the contraction regulatory protein of cardiac muscle, by cardiac muscle troponin I (cTnI), cTnC (cTnC) constituted with three subunits of serum cardiac troponin T (cTnT), wherein cTnI due to the Cardiac-specific with height, into For the preferred mark of clinical diagnosis myocardial infarction.CTnI highly sensitive detection is for improving morning of clinically acute myocardial infarction AMI Phase diagnosis, quick point examine, risk stratification and prognosis evaluation it is significant.
At present common cTnI quantitative detecting methods include radioimmunology (RIA), enzyme linked immunosorbent assay (ELISA), Chemoluminescence method, test strips quick detection etc., these methods are to cTnI detection sensitivity generally in ng/mL levels, it is difficult to full Sufficient clinical diagnosis sets up accurate sensitive cTnI quantitative detecting methods to myocardial infarction to the demands of the highly sensitive detections of cTnI Early diagnosis have important practical significance and clinical value.
In recent years, due to being easily-synthesized of nucleic acid, easily modification and it is amplifiable the characteristics of so that the high sensitivity egg based on nucleic acid White detection method is received significant attention.Many new highly sensitive detection methods are emerged at present, such as immune polymerase chain Formula reaction, immune rolling circle amplification reaction (IRCA), proximity ligation assay etc., the sensitivity of these detection methods and specificity are all bright It is aobvious to be higher than traditional ELISA method.Wherein, rolling circle amplification (RCA) is to use for reference ring-type causal organism DNA molecular in nature The nucleic acid amplification technologies that rolling cycle replication mode is set up, by a primer under the archaeal dna polymerase effect with strand-displacement activity Can trigger along annular DNA template strand displacement synthesis, realize circular DNA template external isothermal linearity amplification, with it is sensitive, Specifically, easy to operate the characteristics of.Common RCA technologies are combined with the immune response of antigen-antibody and generate IRCA methods. In this method, one section of Oligonucleolide primers that can be complementary with circular template is combined on the antibody for recognizing target molecule, with In the presence of the enzyme of strand displacement or unwinding activities, a list being made up of multistage and the complementary repetitive sequence of circular template is amplified Chain DNA.
It is noted that in IRCA methods as probe molecule antibody, easily influenceed by environmental conditions such as pH, temperature And be denatured, and synthesize expensive.And the aptamer come out through index concentration Fas lignand system evolution (SELEX) technology screening Section of DNA/RNA sequence, can and plurality of target material carry out high selectivity, the combination of high specific, widely used In field of biosensors.Compared to antibody, the scope that affinity interaction can occur for aptamer is wider, and cost is lower, it is easy to Modify and property is more stable.Therefore, aptamer has wide development space as probe molecule in Protein Detection field. Up to the present, based on aptamer to cTnI specific recognition, the rolling circle amplification of nucleic acid and FRET The cTnI quantitative detecting methods of technology have no report.
The content of the invention
In view of the deficienciess of the prior art, it is an object of the invention to provide a kind of the highly sensitive of cardiac muscle troponin I Quantitative detecting method, mainly carries out specific recognition to cardiac muscle troponin I, and utilize the rolling of nucleic acid by aptamer Circle amplification technology and the FRET technology based on graphene oxide (GO), are realized to the accurate sensitive fixed of cTnI Amount detection, the detection method has specific good, sensitivity high, easy to operate and low cost and other advantages.
The mesh of the present invention is achieved through the following technical solutions：
A kind of high sensitivity quantitation detection method of cTnI, methods described step is as follows：
(1) the cTnI antibody being coated with after buffer solution dilution is coated with to black ELISA Plate, is then incubated 2h at 37 DEG C；
(2) liquid on black ELISA Plate is discarded, confining liquid, and closing more than the 1h at 4 DEG C is added；
(3) liquid on black ELISA Plate is discarded, black ELISA Plate is washed with PBST 3~5 times, and completes last time and is washed After washing, remove liquid and pat dry；
(4) hole on black ELISA Plate is divided into different experimental groups, by the cTnI of various concentrations and contained respectively CTnI testing sample is added in different experimental groups, is then incubated 1h at 37 DEG C；
(5) operation of repeat step (3)；
(6) the aptamers cyclisation product diluted with screening buffer solution is added into black ELISA Plate, is then incubated at 37 DEG C Educate 1h；
(7) operation of repeat step (3)；
(8) RCA systems, and reaction 12h~15h at 37 DEG C are added into black ELISA Plate；
(9) liquid in black ELISA Plate is discarded, is first washed with PBST 2~5 times, then is washed 2~5 times with PBS, and is completed After last time is washed, empty liquid and pat dry；
(10) fluorescence intensity after detection architecture is added into black ELISA Plate, it is bent to draw standard according to testing result Line；CTnI concentration in testing sample is calculated according to standard curve；Wherein, excitation wavelength is 492nm, and Detection wavelength is 520nm；
In step (1), the constituent of the coating buffer solution and the concentration of each composition are as follows：Na2CO315mM, NaHCO335mM, water is solvent；The volume of cTnI antibody is 1 with the volume ratio for being coated with buffer solution:1000.
In step (2), the confining liquid is made up of BSA (bovine serum albumin(BSA)) and PBST；Wherein, contain in 1mL PBST 1mg BSA, the PBST are containing the phosphate buffer solution that volume fraction is 0.05%Twen-20.
In step (4), the cTnI of more than 6 groups various concentrations is selected in the range of 0~500pg/mL.
In step (6), concentration of the aptamers cyclisation product in screening buffer solution is 50nM；
The composition component of the screening buffer solution and the concentration of each composition are as follows：NaCl 137mM, KCl 2.7mM, Na2HPO4·12H2O 6.5mM, NaH2PO4·2H2O 1.8mM, MgCl21.47mM, water is solvent；
The constituent of the aptamers cyclisation product and the concentration of each composition are as follows：Aptamers primer complex (AP) 0.1 μM, 0.1 μM of padlock probe (LP), T4DNA ligases 10U, T4DNA ligase Buffer, water is solvent；Wherein, aptamers SEQ ID NO.1 of the nucleotide sequence of primer complex in sequence table；The nucleotide sequence of padlock probe is selected from sequence SEQ ID NO.2 in table.
Further, the preparation process of the aptamers cyclisation product is as follows：Aptamers primer complex, locking-type are visited Pin, T4DNA ligases, T4DNA ligases Buffer and deionized water mixing, are subsequently placed at 37 DEG C and react 2h, then boiling water 5min~10min is bathed, aptamers cyclisation product is obtained.
In step (8), the concentration of the constituent of RCA systems and each composition is as follows：Phi 29DNA polymerase 10U, Phi29DNA polymerase buffers, dNTP (deoxyribonucleoside triphosphate) 0.5mM, water is solvent.
In step (10), the concentration of the constituent of detection architecture and each composition is as follows：Graphene oxide 25 μ g/mL, FAM The ssDNA probe 60nM of mark, water is solvent；Wherein, the ssDNA nucleotide sequences in the ssDNA probes of FAM marks are selected from SEQ ID NO.3 in sequence table.
Beneficial effect：
(1) using being easily-synthesized, easily modification, the amplifiable, aptamer being easy to operation and can be specifically bound with cTnI Instead of the antibody commonly used in Protein Detection, Modify to primer is carried out directly in aptamers, experiment difficulty and cost is greatly reduced；
(2) detection to albumen is converted into by the detection to amplified production as signal amplification means using RCA reactions, Greatly improve detection sensitivity；And the FRET technology based on graphene oxide is combined, realize RCA productions The detection of thing, and then accurately sensitively realize cTnI quantitative detection.
Detection method of the present invention has specific good, sensitivity high, easy to operate and low cost and other advantages, meets The demand of clinical detection.
Brief description of the drawings
Fig. 1 is the principle schematic of detection method of the present invention.
Fig. 2 is cyclisation product and affinity sign comparison diagram of the aptamers to cTnI in embodiment 1.
Fig. 3 is that efficiency chart is quenched to FAM label probes in the graphene oxide of various concentrations in embodiment 2.
The standard curve that Fig. 4 quantitatively detects for cTnI in embodiment 3.
Embodiment
With reference to embodiment, the present invention will be further described.
In following examples：
Aptamers primer complex (AP) sequence in nucleotides sequence list described in SEQ ID NO.1 is as follows：
gcctgttgtg agcctcctaa ctacatgttc tcagggttga ggctggatgg cgatggtggc 60
atgcttattc ttgtctccct tttttgtccg tgctagaagg aaacagttac 110
Padlock probe (LP) sequence in nucleotides sequence list described in SEQ ID NO.2 is as follows：
tagcacggac atatatgatg gaccgcagta tgagtatctc ctatcactac taagtggaag 60
aaatgtaact gtttccttc 79
SsDNA probe sequences in nucleotides sequence list described in SEQ ID NO.3 are as follows：
gtttccttct agcac 15
The ssDNA probes of aptamers primer complex, padlock probe and FAM marks give birth to work bioengineering stock by Shanghai The synthesis of part Co., Ltd.
Contain Na in coating buffer solution2CO315mM, NaHCO335mM and the water as solvent；
Confining liquid is made up of BSA and PBST；Wherein, it is containing volume integral containing 1mg BSA, the PBST in 1mL PBST Number is 0.05%Twen-20 phosphate buffer solution；
Screen and contain NaCl 137mM, KCl 2.7mM, Na in buffer solution2HPO4·12H2O 6.5mM, NaH2PO4·2H2O 1.8mM, MgCl21.47mM and the water as solvent；
Multi-function microplate reader：Synergy2, U.S. Bai Teng；The excitation wavelength of fluorescence intensity is 492nm, Detection wavelength For 520nm.
Embodiment 1
In RCA reactions, the success of cyclization is the basis of following amplification reaction, can carry out being cyclized instead by two ways Should：
First method is Cyclization ira situ, i.e., AP, 37 DEG C of incubation 1h are added on transparent 96 orifice plate for securing cTnI, then LP, T4DNA ligase and T4DNA ligases Buffer, 37 DEG C of incubation 1h are added, cyclisation company is carried out on transparent 96 orifice plate Connect.But the method step is relatively complicated, experimental period is longer.
Second method is that aptamers cyclisation product well prepared in advance is added to the hole enzyme of black 96 for having secured cTnI On target, 37 DEG C of incubation 1h.The method is easy to operate, and it is standby disposably to prepare a large amount of aptamers cyclisation products, reduces real Test the time.
Affinity interaction between the HRPO (HRP) modified by the AP and Streptavidin of biotin modification, Color reaction using HRP catalysis substrate tetramethyl benzidines (TMB) investigates aptamers cyclisation product to target protein as foundation CTnI affinity and specificity.
Wherein, the preparation method of aptamers cyclisation product is as follows：Take 1 μM of 5 ' the μ L of AP 5,1 μM of LP 5 of mark biotin The μ L of 0.5 μ L and T4DNA ligases Buffer of μ L, T4DNA ligase 5, then with deionized water polishing to 50 μ L, then will mix Close solution and be placed at 37 DEG C and react 2h, then boiling water bath 10min, enzyme is inactivated, aptamers cyclisation product is obtained, and be put in -20 DEG C Preserve stand-by.
Table 1
Experimental group 1 Experimental group 2 Experimental group 3
Cyclisation product+cTnI The AP+cTnI of mark Cyclisation product+trypsase
Experimental implementation is as follows：
(1) cTnI, trypsase are diluted to 5 μ g/mL respectively with coating buffer solution；Hole on transparent 96 orifice plate is divided It is coated with into after 3 experimental groups, then by the cTnI being coated with after buffer solution dilution into the hole of experimental group 1 and experimental group 2, coating buffering Trypsase after liquid dilution is coated with into the hole of experimental group 3, and 100 μ L, 4 DEG C of coating 10h are added in each hole；
(2) discard and 300 μ L confining liquids are added in the liquid on transparent 96 orifice plate, each hole, 4 DEG C of closing 1h；
(3) liquid on transparent 96 orifice plate is discarded, 300 μ L PBST board-washings are added 3 times in every hole, after last time is rinsed, Empty liquid and pat dry；
(4) with screening buffer solution by after the concentration dilution of prepared aptamers cyclisation product to 60nM, to the He of experimental group 1 100 μ L are added in each hole of experimental group 3, while adding comparable sodium and 5 ' marks of volume into each hole of experimental group 2 The AP of biotin, 37 DEG C of incubation 1h；
(5) repeat step (3)；
(6) added to every hole on transparent 96 orifice plate and press 1:The Streptavidin of the HRP marks of 2000 volume ratio dilution 100 μ L, 37 DEG C of incubation 1h；
(7) liquid on transparent 96 orifice plate is discarded, per hole with 300 μ L PBST board-washings 3 times, then often 300 μ L are added in hole PBST soaks 5min, empties liquid and pats dry, then is cleaned twice with 300 μ L PBS (pH=7.4 phosphate buffer)；
(8) the μ L of TMB nitrite ions 100 are added to every hole on transparent 96 orifice plate, lucifuge colour developing 5min~30min, the moment is seen Solution colour is examined, if blueness is faded, 50 μ L TMB colour developing terminate liquid terminating reactions are added to every hole immediately；Reaction stops Afterwards, ELIASA 450nm reads light absorption value.
It can be seen from Fig. 2 test result, the aptamers cyclisation product in experimental group 1 still has preferably special to cTnI Property and higher affinity, and with 5 in experimental group 2 ' mark biotin AP result compare no significant difference, illustrate fit Cyclic structure in part cyclisation product does not influence AP to target protein cTnI affinity and specificity.Experimental group 3 illustrates Aptamers cyclisation product does not have specific recognition effect for trypsase.
Embodiment 2
Graphene oxide has a series of unique chemical property and structure, has very strong suction-operated to single stranded DNA, Now energy donor and acceptor are close, occur FRET, fluorescent quenching；But when single stranded DNA is complementary to sequence With reference to rear, the double-stranded DNA of formation carries a large amount of negative electrical charges, produces repulsion with graphene oxide, makees the absorption of graphene oxide With dying down, come off from surface, now fluorophor can not be transferred energy to by energy body, and fluorescence recovers, so as to reach detection Purpose.
Graphene oxide is to set up graphene oxide sensor to being quenched for ssDNA probes that marked FAM fluorophors Committed step.When target sequence is not present, graphene oxide sensor is closed, now, and graphene oxide is quenched Efficiency of going out directly influences background signal value during detection, so that the sensitivity to sensor is impacted, therefore, we are to oxygen Analysis is optimized in the concentration of graphite alkene quenching fluorescence probe.
Graphene oxide concentration gradient is set as 0 μ g/mL, 5 μ g/mL, 10 μ g/mL, 25 μ g/mL, 50 μ g/mL and 100 μ g/ ML, the graphene oxide of various concentrations is mixed with the 60nM FAM ssDNA probes marked under the conditions of room temperature lucifuge respectively and incubated 5min is educated, then determines with multi-function microplate reader its fluorescence intensity.According to the graphene oxide pair of test result analysis various concentrations Fluorescent detection probe is quenched efficiency, in detail as shown in Figure 3.As can be seen from Figure 3, with the increase of graphene oxide concentration, to detection Fluorescence probe to be quenched efficiency higher, in 25 μ g/mL concentration, 90% fluorescent quenching can be made, therefore choose in an experiment Concentration carries out subsequent experimental for 25 μ g/mL graphene oxide.
Embodiment 3
The quantitative detecting method based on nucleic acid rolling circle amplification and FRET set up is used for cTnI's In quantitative detection, Cleaning Principle is as shown in figure 1, specific detecting step is as follows：
(1) the cTnI antibody being coated with after buffer solution dilution is coated with to the hole elisa Plates of black 96, and per the μ L of hole 100, so Afterwards 2h is incubated at 37 DEG C；
(2) liquid in the hole elisa Plates of black 96 is discarded, 300 μ L confining liquids are added in every hole, and closed at 4 DEG C 1h；
(3) liquid in the hole elisa Plates of black 96 is discarded, the 300 μ L PBST washing hole elisa Plates 3 of black 96 are added in every hole It is secondary, remove the wash liquid of third time and pat dry the hole elisa Plates of black 96；
(4) hole in the hole elisa Plates of black 96 is divided into 10 experimental groups, respectively by 0pg/mL, 50pg/mL, 75pg/ ML, 100pg/mL, 150pg/mL, 250pg/mL and 500pg/mL cTnI and three group of testing sample be added to 10 it is different In experimental group, and 100 μ L are added in each hole, 1h is incubated at 37 DEG C；
To characterize detection method of the present invention to the detectability of clinical sample, concentration is separately added into human blood sample For 75pg/mL, 150pg/mL and 250pg/mL cTnI standard samples, obtain three groups contain different cTnI concentration treat test sample Product；
(5) operation of repeat step (3)；
(6) the aptamers cyclisation production that 100 μ L screening buffer solutions dilute is added into each hole of the hole elisa Plates of black 96 Thing, is then incubated 1h at 37 DEG C；
(7) operation of repeat step (3)；
(8) 60 μ L RCA systems are added into each hole of the hole elisa Plates of black 96, and 12h is reacted at 37 DEG C；
(9) liquid in the hole elisa Plates of black 96 is discarded, each Kong Xianyong PBST are washed 3 times, then are washed 3 times with PBS, fallen Remove the PBS wash liquids of last time and pat dry the hole elisa Plates of black 96；
(10) 200 μ L detection architectures are added into each hole of the hole elisa Plates of black 96, then on multi-function microplate reader Fluorescence intensity；
According to testing result, using fluorescence intensity as ordinate, using cTnI concentration as abscissa, carry out what linear regression was obtained Calibration curve equation is y=7.177x -66.995, R2=0.983, refer to Fig. 4；
By calculating the standard deviation of blank value, σ=12.12 are obtained；It can be obtained according to the σ of test limit=3,3 σ=36.36, Calibration curve equation is substituted into, the detection that calculating obtains this method is limited to 14.40pg/mL；Similarly, it can be obtained according to the σ of quantitative limit=10, Quantifying for this method is limited to 26.23pg/mL.
(11) according to the standard curve obtained in the fluorescence intensity and step (10) measured to three groups of testing samples, calculate Obtain cTnI concentration in three groups of testing samples.
In step (1), cTnI volume and the volume ratio of coating buffer solution are 1:1000.
In step (6), concentration of the aptamers cyclisation product in screening buffer solution dilution is 50nM；The aptamers cyclisation Contain 0.1 μM of aptamers primer complex, 0.1 μM of padlock probe, T4DNA ligases 10U, T4DNA ligase in product Buffer and the water as solvent.
In step (8), phi 29DNA polymerases 10U, phi29DNA polymerase buffers, dNTP are contained in RCA systems 0.5mM and the water as solvent.
In step (10), ssDNA probes 60nM containing graphene oxide 25 μ g/mL, FAM mark in detection architecture and It is used as the water of solvent.
The concentration of cTnI standard samples by the cTnI concentration surveyed in three groups of testing samples with adding is made to compare, and draws recovery Rate, the results detailed in Table 2.It can be seen from table 2, using the rate of recovery of the method for the invention between 95%~105%, the degree of accuracy It is higher.
Table 2
CTnI concentration (pg/mL) 75 150 250
The rate of recovery (%) 100.2±2.1 97.6±2.7 104.0±2.3
In summary, presently preferred embodiments of the present invention is these are only, is not intended to limit the scope of the present invention. Within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., should be included in the present invention's Within protection domain.
Nucleotides sequence list
<110>Beijing Institute of Technology
<120>A kind of high sensitivity quantitation detection method of cardiac muscle troponin I
<160> 3
<210> 1
<211> 110
<212> DNA
<213>Artificial sequence
<220>
<223>Aptamers primer complex
<400> 1
gcctgttgtg agcctcctaa ctacatgttc tcagggttga ggctggatgg cgatggtggc 60
atgcttattc ttgtctccct tttttgtccg tgctagaagg aaacagttac 110
<210> 2
<211> 79
<212> DNA
<213>Artificial sequence
<220>
<223>Padlock probe
<400> 2
tagcacggac atatatgatg gaccgcagta tgagtatctc ctatcactac taagtggaag 60
aaatgtaact gtttccttc 79
<210> 3
<211> 15
<212> DNA
<213>Artificial sequence
<220>
<223>SsDNA probes
<400> 3
gtttccttct agcac 15

Claims (5)

1. a kind of high sensitivity quantitation detection method of cardiac muscle troponin I, it is characterised in that：Methods described step is as follows：
(1) the cTnI antibody being coated with after buffer solution dilution is coated with to black ELISA Plate, is then incubated 2h at 37 DEG C；
(2) liquid on black ELISA Plate is discarded, confining liquid, and closing more than the 1h at 4 DEG C is added；
(3) liquid on black ELISA Plate is discarded, black ELISA Plate is washed with PBST 3~5 times, and completes last time and is washed Afterwards, remove liquid and pat dry；
(4) hole on black ELISA Plate is divided into different experimental groups, respectively by the cTnI of various concentrations and containing cTnI's Testing sample is added in different experimental groups, is then incubated 1h at 37 DEG C；
(5) operation of repeat step (3)；
(6) the aptamers cyclisation product diluted with screening buffer solution is added into black ELISA Plate, then 1h is incubated at 37 DEG C；
(7) operation of repeat step (3)；
(8) RCA systems, and reaction 12h~15h at 37 DEG C are added into black ELISA Plate；
(9) liquid in black ELISA Plate is discarded, is first washed with PBST 2~5 times, then is washed 2~5 times with PBS, and completes last After once washing, empty liquid and pat dry；
(10) fluorescence intensity after detection architecture is added into black ELISA Plate, standard curve is drawn according to testing result；Root CTnI concentration in testing sample is calculated according to standard curve；Wherein, excitation wavelength is 492nm, and Detection wavelength is 520nm；
In step (1), the constituent of the coating buffer solution and the concentration of each composition are as follows：Na2CO315mM and NaHCO3 35mM, wherein solvent are water；
In step (2), the confining liquid is made up of BSA and PBST；Wherein, it is containing 1mg BSA, the PBST in 1mL PBST Contain the phosphate buffer solution that volume fraction is 0.05%Twen-20；
The composition component of the screening buffer solution and the concentration of each composition are as follows：NaCl 137mM, KCl 2.7mM, Na2HPO4· 12H2O 6.5mM, NaH2PO4·2H2O 1.8mM and MgCl21.47mM, wherein solvent are water；
The constituent of the aptamers cyclisation product and the concentration of each composition are as follows：0.1 μM of aptamers primer complex, locking-type 0.1 μM of probe, wherein T4 DNA ligases 10U, T4 DNA ligase Buffer, solvent are water；The aptamers primer is combined SEQ ID NO.1 of the nucleotide sequence of thing in sequence table, the SEQ of the nucleotide sequence of padlock probe in sequence table ID NO.2；
In step (8), the concentration of the constituent of RCA systems and each composition is as follows：Phi 29 archaeal dna polymerase 10U, phi29 DNA polymerase buffer liquid and dNTP 0.5mM, wherein solvent are water；
In step (10), the concentration of the constituent of detection architecture and each composition is as follows：Graphene oxide 25 μ g/mL and FAM The ssDNA probe 60nM of mark, wherein solvent are water；SsDNA nucleotides sequence column selections in the ssDNA probes of the FAM marks From the SEQ ID NO.3 in sequence table.
2. a kind of high sensitivity quantitation detection method of cardiac muscle troponin I according to claim 1, it is characterised in that： The volume of cTnI antibody is 1 with the volume ratio for being coated with buffer solution:1000.
3. a kind of high sensitivity quantitation detection method of cardiac muscle troponin I according to claim 1, it is characterised in that： In step (4), the cTnI of more than 6 groups various concentrations is selected in the range of 0~500pg/mL.
4. a kind of high sensitivity quantitation detection method of cardiac muscle troponin I according to claim 1, it is characterised in that： In step (6), concentration of the aptamers cyclisation product in screening buffer solution is 50nM.
5. a kind of high sensitivity quantitation detection method of cardiac muscle troponin I according to claim 1, it is characterised in that： The preparation process of the aptamers cyclisation product is as follows：By aptamers primer complex, padlock probe, T4 DNA ligases, T4 DNA ligase Buffer and deionized water mixing, are subsequently placed at 37 DEG C and react 2h, then boiling water bath 5min~10min, obtain Aptamers cyclisation product.
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CN109580958A (en) * 2018-12-05 2019-04-05 中国科学院长春应用化学研究所 The fluorescence and colorimetric dual signal detection kit and detection method of a kind of cardiac muscle troponin I
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CN109580958A (en) * 2018-12-05 2019-04-05 中国科学院长春应用化学研究所 The fluorescence and colorimetric dual signal detection kit and detection method of a kind of cardiac muscle troponin I
CN109444102B (en) * 2018-12-18 2021-03-23 济南大学 Fluorescence biosensor for detecting ochratoxin A and preparation method and application thereof
CN109444102A (en) * 2018-12-18 2019-03-08 济南大学 A kind of biological sensor and its preparation method and application detecting ochratoxin A
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CN110111849B (en) * 2019-05-08 2021-03-26 北京市计算中心 Nucleic acid aptamer computer-assisted screening method based on high-performance computing platform and nucleic acid aptamer
CN110111849A (en) * 2019-05-08 2019-08-09 北京市计算中心 A kind of aptamer computer-aided screening method and aptamer based on high-performance calculation platform
CN110878296A (en) * 2019-10-16 2020-03-13 武汉伊莱瑞特生物科技股份有限公司 High-sensitivity HRP enzyme and preparation method and application thereof
CN110878296B (en) * 2019-10-16 2021-11-30 武汉伊莱瑞特生物科技股份有限公司 High-sensitivity HRP enzyme and preparation method and application thereof
CN110714049A (en) * 2019-11-12 2020-01-21 北京理工大学 Microbial sensor for detecting biomarkers, detection method, culture and detection chip and detection system
CN111273022A (en) * 2020-02-06 2020-06-12 何斌 Rapid myocardial infarction detection method based on nanogold-graphene quantum dots
CN111273022B (en) * 2020-02-06 2023-11-28 上海市胸科医院 Troponin concentration detection method based on nanogold-graphene quantum dots
CN112067823A (en) * 2020-09-14 2020-12-11 华南农业大学 Establishment of chicken cardiac troponin I double-antibody sandwich ELISA detection method
CN113073101A (en) * 2021-03-25 2021-07-06 严鹏科 Oligonucleotide aptamer of cardiac troponin and application thereof
CN113073101B (en) * 2021-03-25 2023-05-16 严鹏科 Oligonucleotide aptamer of cardiac troponin and application thereof
CN115963274A (en) * 2022-09-30 2023-04-14 中国科学院长春应用化学研究所 Detection reagent and method for cardiac troponin I

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