CN107084966B - 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 PDFInfo
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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"
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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"
- G01N2021/6432—Quenching
Abstract
The present invention relates to a kind of highly sensitive quantitative detecting methods of cardiac muscle troponin I, belong to technical field of biomedical detection.Detection method of the present invention is to carry out specific recognition to cardiac muscle troponin I by aptamer, and using the rolling circle amplification of nucleic acid and the fluorescence resonance energy transfer technology based on graphene oxide, realize the accurate sensitive quantitative detection to cTnI；The detection method has many advantages, such as the good, high sensitivity of specificity, easy to operate and at low cost, meets the demand of clinical detection.
Description
Technical field
The present invention relates to a kind of highly sensitive quantitative detecting methods of cardiac muscle troponin I, belong to biomedical detection technique
Field.
Background technique
Cardiac troponin is the contraction regulatory protein of cardiac muscle, by cardiac muscle troponin I (cTnI), cTnC
(cTnC) it is constituted with three subunits of serum cardiac troponin T (cTnT), wherein cTnI is due to the Cardiac-specific with height, at
For the preferred marker of clinical diagnosis myocardial infarction.The highly sensitive detection of cTnI is for improving morning of clinically acute myocardial infarction AMI
Phase diagnosis, quickly point examine, risk stratification and prognosis evaluation are of great significance.
At present common cTnI quantitative detecting method include radioimmunology (RIA), enzyme linked immunosorbent assay (ELISA),
Chemoluminescence method, test strips quickly detect, these methods are to the detection sensitivity of cTnI usually in ng/mL level, it is difficult to full
Demand of the sufficient clinical diagnosis to the highly sensitive detection of cTnI, therefore accurate sensitive cTnI quantitative detecting method is established to myocardial infarction
Early diagnosis have important practical significance and clinical value.
In recent years, due to being easily-synthesized of nucleic acid, easily modification and amplifiable feature so that the highly sensitive egg based on nucleic acid
White detection method receives significant attention.Many novel 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 cyclic annular causal organism DNA molecular in nature
The nucleic acid amplification technologies that rolling cycle replication mode is established, by a primer under the archaeal dna polymerase effect with strand-displacement activity
Can cause along annular DNA template strand displacement synthesis, realize circular DNA template external isothermal linearity amplification, have it is sensitive,
Specifically, feature easy to operate.Common RCA technology is combined with the immune response of antigen-antibody and produces IRCA method.?
In this method, identifies and combine one section of Oligonucleolide primers that can be complementary with circular template on the antibody of target molecule, have
Under the action of the enzyme of strand displacement or unwinding activities, the list that a repetitive sequence complementary with circular template by multistage is constituted is amplified
Chain DNA.
It is noted that the influence as the antibody of probe molecule in IRCA method, vulnerable to environmental conditions such as pH, temperature
And it is denaturalized, 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 substance carry out highly selective, high specific combination, widely used
In field of biosensors.Compared to antibody, the range that affinity interaction can occur for aptamer is wider, and cost is lower, is easy to
It modifies and property is more stable.Therefore, aptamer has wide development space in Protein Detection field as probe molecule.
Up to the present, based on aptamer to the specific recognition of cTnI, the rolling circle amplification of nucleic acid and fluorescence resonance energy transfer
The cTnI quantitative detecting method of technology has not been reported.
Summary of the invention
In view of the deficiencies of the prior art, the present invention intends 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 by aptamer, and utilizes the rolling of nucleic acid
Circle amplification technology and the fluorescence resonance energy transfer technology for being based on graphene oxide (GO) are realized to the accurate sensitive fixed of cTnI
Amount detection, the detection method have many advantages, such as specific good, high sensitivity, easy to operate and at low cost.
Mesh of the invention is achieved through the following technical solutions:
A kind of high sensitivity quantitation detection method of cTnI, the method comprises the following steps:
(1) the cTnI antibody after buffer dilutes will be coated with to be coated with to black ELISA Plate, is then incubated for 2h at 37 DEG C；
(2) liquid on black ELISA Plate is discarded, confining liquid is added, and closes 1h or more at 4 DEG C；
(3) liquid on black ELISA Plate is discarded, is washed black ELISA Plate 3~5 times with PBST, and completes last time and washes
After washing, removes liquid and pat dry；
(4) hole on black ELISA Plate is divided into different experimental groups, by the cTnI of various concentration and is contained respectively
The sample to be tested of cTnI is added in different experimental groups, is then incubated for 1h at 37 DEG C；
(5) operation of step (3) is repeated；
(6) it is added into black ELISA Plate with the diluted aptamers cyclisation product of buffer is screened, is then incubated at 37 DEG C
Educate 1h；
(7) operation of step (3) is repeated；
(8) RCA system is added into black ELISA Plate, and reacts 12h~15h at 37 DEG C；
(9) liquid in black ELISA Plate is discarded, is first washed 2~5 times with PBST, then is washed 2~5 times with PBS, and is completed
After last time is washed, empties 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 sample to be tested is calculated according to standard curve；Wherein, excitation wavelength 492nm, Detection wavelength 520nm；
In step (1), the constituent of the coating buffer and the concentration of each ingredient are as follows: Na2CO315mM,
NaHCO335mM, water are solvent；The volume of cTnI antibody and the volume ratio of coating buffer are 1:1000.
In step (2), the confining liquid is made of BSA (bovine serum albumin(BSA)) and PBST；Wherein, contain in 1mL PBST
1mg BSA, the PBST are the phosphate buffer solutions for being 0.05%Twen-20 containing volume fraction.
In step (4), the cTnI of 6 groups or more various concentrations is selected within the scope of 0~500pg/mL.
In step (6), concentration of the aptamers cyclisation product in screening buffer is 50nM；
The constituent component of the screening buffer and the concentration of each ingredient are as follows: NaCl 137mM, KCl 2.7mM,
Na2HPO4·12H2O 6.5mM, NaH2PO4·2H2O 1.8mM, MgCl21.47mM, water are solvent；
The constituent of the aptamers cyclisation product and the concentration of each ingredient are as follows: aptamers primer complex (AP)
0.1 μM, 0.1 μM of padlock probe (LP), T4DNA ligase 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 step of the aptamers cyclisation product is as follows: aptamers primer complex, locking-type are visited
Needle, T4DNA ligase, T4DNA ligase 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 system and each ingredient is as follows: phi 29DNA polymerase 10U,
Phi29DNA polymerase buffer, dNTP (deoxyribonucleoside triphosphate) 0.5mM, water is solvent.
In step (10), the concentration of the constituent of detection architecture and each ingredient is as follows: graphene oxide 25 μ g/mL, FAM
The ssDNA probe 60nM of label, water is solvent；Wherein, the ssDNA nucleotide sequence in the ssDNA probe of FAM label is selected from
SEQ ID NO.3 in sequence table.
The utility model has the advantages that
(1) aptamer be easily-synthesized, easily modify, is amplifiable, convenient for operating and can specifically bind with cTnI is used
Instead of antibody common in Protein Detection, Modify to primer is carried out directly in aptamers, greatly reduces experiment difficulty and cost；
(2) signal amplification means are used as using RCA reaction, the detection to amplified production will be converted into the detection of albumen,
Greatly improve detection sensitivity；And the fluorescence resonance energy transfer technology based on graphene oxide is combined, realize RCA production
The detection of object, and then accurately sensitively realize the quantitative detection of cTnI.
Detection method of the present invention has many advantages, such as the good, high sensitivity of specificity, easy to operate and at low cost, meets
The demand of clinical detection.
Detailed description of the invention
Fig. 1 is the schematic illustration of detection method of the present invention.
Fig. 2 is that cyclisation product and aptamers characterize comparison diagram to the affinity of cTnI in embodiment 1.
Fig. 3 is that efficiency chart is quenched to FAM label probe in the graphene oxide of various concentration in embodiment 2.
Fig. 4 is the standard curve of cTnI quantitative detection in embodiment 3.
Specific embodiment
The present invention will be further described With reference to embodiment.
In following embodiment:
Aptamers primer complex (AP) sequence described in SEQ ID NO.1 is as follows in nucleotides sequence list:
gcctgttgtg agcctcctaa ctacatgttc tcagggttga ggctggatgg cgatggtggc 60
atgcttattc ttgtctccct tttttgtccg tgctagaagg aaacagttac 110
Padlock probe (LP) sequence described in SEQ ID NO.2 is as follows in nucleotides sequence list:
tagcacggac atatatgatg gaccgcagta tgagtatctc ctatcactac taagtggaag 60
aaatgtaact gtttccttc 79
SsDNA probe sequence described in SEQ ID NO.3 is as follows in nucleotides sequence list:
gtttccttct agcac 15
The ssDNA probe of aptamers primer complex, padlock probe and FAM label is by the raw work bioengineering stock in Shanghai
The synthesis of part Co., Ltd.
Contain Na in coating buffer2CO315mM, NaHCO335mM and water as solvent；
Confining liquid is made of BSA and PBST；Wherein, 1mg BSA is contained in 1mL PBST, the PBST is containing volume point
Number is the phosphate buffer solution of 0.05%Twen-20；
It screens and contains NaCl 137mM, KCl 2.7mM, Na in buffer2HPO4·12H2O 6.5mM, NaH2PO4·2H2O
1.8mM, MgCl21.47mM and water as solvent；
Multi-function microplate reader: Synergy2, U.S. Bai Teng；The excitation wavelength of fluorescence intensity is 492nm, Detection wavelength
For 520nm.
In RCA reaction, the success of cyclization is the basis of following amplification reaction, can carry out being cyclized by two ways anti-
It answers:
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 is added and T4DNA ligase Buffer, 37 DEG C of incubation 1h carry out cyclisation company on transparent 96 orifice plate
It connects.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 96 hole enzyme of black for having secured cTnI
On target, 37 DEG C of incubation 1h.The method is easy to operate, and it is spare disposably to prepare a large amount of aptamers cyclisation products, reduces real
Test the time.
The affinity interaction between horseradish peroxidase (HRP) modified by the AP and Streptavidin of biotin modification,
Using the color reaction of HRP catalysis substrate tetramethyl benzidine (TMB) as foundation, aptamers cyclisation product is investigated to target protein
The affinity and specificity of cTnI.
Wherein, aptamers cyclisation product the preparation method is as follows: take 1 μM 5 ' label biotin 5 μ L of AP, 1 μM of LP 5
μ L, 0.5 μ L and T4DNA ligase Buffer of T4DNA ligase, 5 μ L, then then will be mixed with deionized water polishing to 50 μ L
It closes solution and is placed at 37 DEG C and react 2h, then boiling water bath 10min, inactivate enzyme, obtain aptamers cyclisation product, and be put in -20 DEG C
It saves stand-by.
Table 1
| |
Experimental group 2 | Experimental group 3 |
| Cyclisation product+cTnI | The AP+cTnI of label | Cyclisation product+trypsase |
Experimental implementation is as follows:
(1) cTnI, trypsase are diluted to 5 μ g/mL respectively with coating buffer；Hole on transparent 96 orifice plate is divided
It is coated with after 3 experimental groups, then by the cTnI after buffer dilutes is coated with 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) liquid on transparent 96 orifice plate is discarded, 300 μ L confining liquids, 4 DEG C of closing 1h are added in each hole；
(3) liquid on transparent 96 orifice plate is discarded, 300 μ L PBST is added in every hole board-washing 3 times, after last time is rinsed,
It empties liquid and pats dry；
(4) with screening buffer by after the concentration dilution to 60nM of prepared aptamers cyclisation product, to 1 He of experimental group
100 μ L are added in each hole of experimental group 3, while 5 ' labels of comparable sodium and volume being added into each hole of experimental group 2
The AP of biotin, 37 DEG C of incubation 1h；
(5) step (3) are repeated；
(6) Streptavidin of the diluted HRP label of volume ratio by 1:2000 is added in every hole on transparent 96 orifice plate
100 μ L, 37 DEG C of incubation 1h；
(7) liquid on transparent 96 orifice plate is discarded, then 300 μ L are added with 300 μ L PBST board-washing 3 times in every hole in every hole
PBST impregnates 5min, empties liquid and pats dry, then is cleaned twice with 300 μ L PBS (phosphate buffer of pH=7.4)；
(8) 100 μ L of TMB developing solution is added in every hole on transparent 96 orifice plate, is protected from light colour developing 5min~30min, and the moment is seen
Solution colour is examined, if blue is faded, 50 μ L TMB colour developing terminate liquid is added to every hole immediately and terminates reaction；Reaction stops
Afterwards, microplate reader 450nm reads light absorption value.
Test result according to fig. 2 is it is found that the aptamers cyclisation product in experimental group 1 still has cTnI preferably specifically
Property and higher affinity, and with 5 in experimental group 2 ' label biotin AP result compare no significant difference, illustrate fit
Cyclic structure in ligand cyclisation product does not influence AP to the affinity and specificity of target protein cTnI.3 explanation of experimental group
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,
Energy donor and receptor are close at this time, and fluorescence resonance energy transfer, fluorescent quenching occurs；But when single stranded DNA is complementary sequence
In conjunction with rear, the double-stranded DNA of formation has a large amount of negative electrical charges, generates repulsion with graphene oxide, makees the absorption of graphene oxide
It with dying down, falls off from surface, fluorophor can not be transferred energy to by energy body at this time, and fluorescence restores, to reach detection
Purpose.
Graphene oxide is to establish graphene oxide sensor to being quenched for the ssDNA probe that FAM fluorophor is marked
Committed step.In the absence of target sequence, graphene oxide sensor is in close state, at this point, graphene oxide is quenched
Efficiency of going out directly influences background signal value when detection, so that the sensitivity to sensor impacts, 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/
The graphene oxide of various concentration is mixed under the conditions of room temperature is protected from light with the ssDNA probe of 60nM FAM label respectively and is incubated by mL
5min is educated, then measures its fluorescence intensity with multi-function microplate reader.According to the graphene oxide pair of test result analysis various concentration
Efficiency is quenched in fluorescent detection probe, 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
The graphene oxide that concentration is 25 μ g/mL carries out subsequent experimental.
Embodiment 3
The quantitative detecting method based on nucleic acid rolling circle amplification and fluorescence resonance energy transfer established is used for cTnI's
In quantitative detection, testing principle is as shown in Figure 1, specific detecting step is as follows:
(1) the cTnI antibody after buffer dilutes will be coated with to be coated with to 96 hole elisa Plates of black, and 100 μ L of every hole, so
2h is incubated at 37 DEG C afterwards；
(2) liquid in 96 hole elisa Plates of black is discarded, 300 μ L confining liquids are added in every hole, and close at 4 DEG C
1h；
(3) liquid in 96 hole elisa Plates of black is discarded, 300 μ L PBST washing 96 hole elisa Plates 3 of black are added in every hole
It is secondary, it removes the wash liquid of third time and pats dry 96 hole elisa Plates of black；
(4) hole in 96 hole elisa Plates of black is divided into 10 experimental groups, respectively by 0pg/mL, 50pg/mL, 75pg/
CTnI and three group of sample to be tested of mL, 100pg/mL, 150pg/mL, 250pg/mL and 500pg/mL be added to 10 it is different
100 μ L are added in experimental group, and in each hole, are incubated for 1h 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 sheet
For the cTnI standard sample of 75pg/mL, 150pg/mL and 250pg/mL, obtain three groups contain different cTnI concentration to test sample
Product；
(5) operation of step (3) is repeated；
(6) 100 μ L are added into each hole of 96 hole elisa Plates of black to be produced with the diluted aptamers cyclisation of screening buffer
Then object is incubated for 1h at 37 DEG C；
(7) operation of step (3) is repeated；
(8) 60 μ L RCA systems are added into each hole of 96 hole elisa Plates of black, and react 12h at 37 DEG C；
(9) liquid in 96 hole elisa Plates of black is discarded, each Kong Xianyong PBST is washed 3 times, then washs 3 times with PBS,
It removes the PBS wash liquid of last time and pats dry 96 hole elisa Plates of black；
(10) 200 μ L detection architectures are added into each hole of 96 hole elisa Plates of black, 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 obtained
Calibration curve equation is y=7.177x -66.995, R2=0.983, it is detailed in Fig. 4；
By calculating the standard deviation of blank value, σ=12.12 are obtained；It can be obtained according to=3 σ of detection limit, 3 σ=36.36,
Calibration curve equation is substituted into, the detection that this method is calculated is limited to 14.40pg/mL；Similarly, it can be obtained according to quantitative limit=10 σ,
Quantifying for this method is limited to 26.23pg/mL.
(11) standard curve according to obtained in the fluorescence intensity and step (10) that measure to three groups of samples to be tested calculates
Obtain cTnI concentration in three groups of samples to be tested.
In step (1), the volume of cTnI and the volume ratio of coating buffer are 1:1000.
In step (6), concentration of the aptamers cyclisation product in screening buffer dilution is 50nM；The aptamers cyclisation
In product contain 0.1 μM of aptamers primer complex, 0.1 μM of padlock probe, T4DNA ligase 10U, T4DNA ligase
Buffer and water as solvent.
In step (8), phi 29DNA polymerase 10U, phi29DNA polymerase buffer, dNTP are contained in RCA system
0.5mM and water as solvent.
In step (10), containing 25 μ g/mL of graphene oxide in detection architecture, the ssDNA probe 60nM of FAM label and
Water as solvent.
The concentration of the cTnI standard sample of the cTnI concentration and addition surveyed in three groups of samples to be tested is made into ratio, obtains recycling
Rate, as a result see Table 2 for details.According to table 2, using the rate of recovery of the method for the invention between 95%~105%, 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 conclusion the above is merely preferred embodiments of the present invention, being not intended to limit the scope of the present invention.
All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention
Within protection scope.
Nucleotides sequence list
<110>Beijing Institute of Technology
<120>the high sensitivity quantitation detection method of a kind 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 probe
<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: the method comprises the following steps:
(1) the cTnI antibody after buffer dilutes will be coated with to be coated with to black ELISA Plate, is then incubated for 2h at 37 DEG C；
(2) liquid on black ELISA Plate is discarded, confining liquid is added, and closes 1h or more at 4 DEG C；
(3) liquid on black ELISA Plate is discarded, is washed black ELISA Plate 3~5 times with PBST, and completes last time and washs
Afterwards, it removes liquid and pats dry；
(4) hole on black ELISA Plate is divided into different experimental groups, respectively by the cTnI of various concentration and containing cTnI's
Sample to be tested is added in different experimental groups, is then incubated for 1h at 37 DEG C；
(5) operation of step (3) is repeated；
(6) it is added into black ELISA Plate with the diluted aptamers cyclisation product of buffer is screened, is then incubated for 1h at 37 DEG C；
(7) operation of step (3) is repeated；
(8) RCA system is added into black ELISA Plate, and reacts 12h~15h at 37 DEG C；
(9) liquid in black ELISA Plate is discarded, is first washed 2~5 times with PBST, then is washed 2~5 times with PBS, and is completed last
After once washing, empties liquid and pat dry；
(10) into black ELISA Plate be added detection architecture after fluorescence intensity, draw standard curve according to testing result；Root
CTnI concentration in sample to be tested is calculated according to standard curve；Wherein, excitation wavelength 492nm, Detection wavelength 520nm；
In step (1), the constituent of the coating buffer and the concentration of each ingredient are as follows: Na2CO315mM and NaHCO3
35mM, wherein solvent is water；
In step (2), the confining liquid is made of BSA and PBST；Wherein, 1mg BSA is contained in 1mL PBST, the PBST is
The phosphate buffer solution for being 0.05%Twen-20 containing volume fraction；
The constituent component of the screening buffer and the concentration of each ingredient are as follows: NaCl 137mM, KCl 2.7mM, Na2HPO4·
12H2O 6.5mM, NaH2PO4·2H2O 1.8mM and MgCl21.47mM, wherein solvent is water；
The constituent of the aptamers cyclisation product and the concentration of each ingredient are as follows: 0.1 μM of aptamers primer complex, locking-type
0.1 μM of probe, T4 DNA ligase 10U, T4 DNA ligase Buffer, wherein solvent is water；The aptamers primer is compound
The nucleotide sequence of object the SEQ ID NO.1 in sequence table, SEQ of the nucleotide sequence of padlock probe in sequence table
ID NO.2；
In step (8), the concentration of the constituent of RCA system and each ingredient is as follows: phi 29 archaeal dna polymerase 10U, phi29
DNA polymerase buffer liquid and dNTP 0.5mM, wherein solvent is water；
In step (10), the concentration of the constituent of detection architecture and each ingredient is as follows: graphene oxide 25 μ g/mL and FAM
The ssDNA probe 60nM of label, wherein solvent is water；SsDNA nucleotides sequence column selection in the ssDNA probe of the FAM label
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 and the volume ratio of coating buffer are 1:1000.
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 6 groups or more various concentrations is selected within the scope 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 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 step of the aptamers cyclisation product is as follows: by aptamers primer complex, padlock probe, T4 DNA ligase, 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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|---|---|---|---|---|
| CN109580958B (en) * | 2018-12-05 | 2022-02-01 | 中国科学院长春应用化学研究所 | Fluorescence and colorimetric dual-signal detection kit and detection method for cardiac 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 |
| CN111662909B (en) * | 2019-03-05 | 2022-07-15 | 中国科学技术大学 | Cardiac troponin I specific nucleic acid aptamer and application thereof |
| 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 |
| 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 |
| CN110714049B (en) * | 2019-11-12 | 2021-07-02 | 北京理工大学 | Microbial sensor for detecting biomarkers, detection method, culture and detection chip and detection system |
| 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 |
| CN113073101B (en) * | 2021-03-25 | 2023-05-16 | 严鹏科 | Oligonucleotide aptamer of cardiac troponin and application thereof |
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2017
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