Electrochemiluminescence detection method and detection kit for magnetic bead type cardiac troponin I based on rolling circle amplification

Abstract

The invention belongs to the technical field of protein detection, and relates to an electrochemical luminescence detection method and detection kit for magnetic bead type cardiac troponin I based on rolling circle amplification. The detection method comprises the following steps: S1, synthesizing MBs (at) SA.bio-Ab1; s2, preparing a conjugate Ab2-DNA (Deoxyribonucleic Acid) / pad; s3, mixing the cardiac troponin I solution, the MBs (at) SA.bio-Ab1 and the conjugate Ab2-DNA / pad, and carrying out rolling circle amplification to obtain an amplification product; s4, detecting an ECL value of an amplification product by utilizing an electrochemical luminescence technology; s5, changing the concentration of the cardiac troponin I solution, obtaining a corresponding ECL value, and generating a calibration curve; s6, acquiring an ECL value corresponding to a to-be-detected sample, and substituting the ECL value into the calibration curve to calculate the concentration of cardiac troponin I. The detection method provided by the invention has the characteristics of high sensitivity, simple operation and low cost, and realizes quantitative detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein detection, and relates to an electrochemiluminescence detection method and a detection kit for magnetic bead type cardiac troponin I based on rolling circle amplification. Background Art

[0002] Acute myocardial infarction (AMI) is a major cause of death globally and can be prevented by treating cardiovascular diseases; the rapid and accurate diagnosis and prognosis of AMI have attracted the attention of many researchers. Cardiac troponin I (cTnI) is one of the structural subunits of the cardiac troponin complex and is widely regarded as a biomarker for the early diagnosis of AMI. The concentration of cTnI in human blood varies greatly, from a few ng / L in healthy people to 10 4 ng / L in patients with cardiovascular diseases. In addition, the change of cTnI concentration over time is usually used for clinical prediction rather than the absolute concentration. Since the baseline value of cTnI concentration varies from person to person, the highly sensitive detection of cTnI is crucial for diagnosing this life-threatening disease. Therefore, the highly sensitive detection of cTnI is of great significance for improving the early diagnosis and efficacy evaluation of acute myocardial infarction in clinical practice.

[0003] Currently, there are many common cTnI detection methods, including enzyme-linked immunosorbent assay (ELISA), ECL immunoassay (ECLIA), surface plasmon resonance method (SPR), fluorescence immunoassay (IF), electrochemiluminescence immunoassay (ECIA), and chemiluminescence immunoassay (CLIA), etc. Although ECL immunoassay has been commercialized and applied to in vitro diagnosis, it is still challenging to detect low-abundance biomarkers (such as biomarkers below pg / mL); among them, ELISA is still the most commonly used method for clinical analysis of cTnI. Although the above methods can achieve the detection of cTnI, these methods still have limitations such as long detection time, low sensitivity, high cost, and complex sample processing, which limit their further application, and usually at the ng / mL level, it is difficult to meet the clinical need for highly sensitive detection of cTnI.

[0004] Therefore, establishing an accurate and sensitive quantitative detection method for cardiac troponin I has important practical significance and clinical application value for the early diagnosis of myocardial infarction. Summary of the Invention

[0005] Aiming at the technical problem of low sensitivity in the existing cardiac troponin detection technology, the present invention provides an electrochemiluminescence detection method and a detection kit for magnetic bead type cardiac troponin I based on rolling circle amplification, which have the characteristics of high sensitivity and simple operation, and are used to achieve the quantitative detection of cardiac troponin I.

[0006] An electrochemiluminescence detection method for magnetic bead-based cardiac troponin I based on rolling circle amplification, comprising the following steps:

[0007] S1. Synthesize the magnetic capture probe MBs@SA·bio-Ab1

[0008] Combine the biotinylated antibody biotin-Ab1 with MBs@SA to obtain the magnetic capture probe MBs@SA·bio-Ab1, and resuspend it in a buffer solution for standby;

[0009] S2. Prepare the conjugate Ab2-DNA / pad

[0010] S2.1 Synthesize the recognition probe Ab2-DNA

[0011] Using heterobifunctional sulfo-SMCC as a linker, conjugate the detection antibody Ab2 with ss-DNA to obtain the conjugate Ab2-DNA; the concentration ratio of Ab2 to ss-DNA is (1:4) to (1:10);

[0012] S2.2. Prepare the padlock probe

[0013] Use the RCA template, ss-DNA and T4 DNA ligase to prepare the padlock probe by the template ligation method;

[0014] S2.3. Synthesize the conjugate Ab2-DNA / pad

[0015] Place the padlock probe in step 2.2 and Ab2-DNA in step 2.1 in a buffer solution, incubate and react to obtain the conjugate Ab2-DNA / pad; the final concentration of Ab2-DNA in the conjugate Ab2-DNA / pad is 10 μg / mL;

[0016] S3. Rolling circle amplification

[0017] Mix the cardiac troponin I solution with known concentration, the magnetic capture probe MBs@SA·bio-Ab1 in step S1 and the conjugate Ab2-DNA / pad in step S2, and then obtain the magnetic immune complex through incubation and magnetic separation;

[0018] Add the RCA mixed solution and Ru-DNA to the magnetic immune complex for rolling circle amplification reaction to obtain the amplification product;

[0019] S4. Electrochemiluminescence detection

[0020] Use electrochemiluminescence technology to obtain the ECL value corresponding to the above amplification product;

[0021] S5. Generate a calibration curve

[0022] Change the concentration of the cardiac troponin I solution, and according to steps S3 and S4, obtain the ECL values corresponding to the cardiac troponin I solutions at different concentrations; and generate a calibration curve with the concentration of the cardiac troponin I solution as the abscissa and the ECL value as the ordinate.

[0023] S6. Sample detection

[0024] Collect the sample to be tested, and according to the methods of steps S3 and S4, obtain the ECL value corresponding to the sample to be tested, and then substitute it into the calibration curve to calculate the concentration of cardiac troponin I in the sample to be tested.

[0025] Further defined, in the step S1, the mass ratio of biotin-Ab1 to MBs@SA is 1:40.

[0026] Further defined, in the step S1, biotin-Ab1 and MBs@SA react at 30 °C to 37 °C for 18 min to 30 min to obtain MBs@SA·bio-Ab1.

[0027] Further defined, the steps for synthesizing the padlock probe in the step S2.2 are:

[0028] Anneal the mixture containing the RCA template, ss-DNA, and T4 DNA ligase buffer solution at 95 °C for 5 min and then cool it to room temperature; then add T4 DNA ligase and incubate at 37 °C for 3 h; then add the lysis reaction system and adjust the volume to 150 μL with the buffer solution; finally, incubate at 37 °C for 3 h and at 65 °C for 20 min to obtain the padlock probe.

[0029] Further defined, the sequence of the RCA template is 5’-Phosphate ATA CTA CCT CAC TGG GCC CACCCT CCC CAC CCG GGA CAA CTA TAC AAC-3’;

[0030] The sequence of ss-DNA is 5’-SH-(CH2)6TGA GGT AGT ATG TTG TAT AGT T-3’.

[0031] Further defined, in the steps S2.2 and S2.3, the buffer solution is prepared from NaH2PO4, Na2HPO4, NaCl, and MgCl2, and the pH of the buffer solution is 7.40.

[0032] Further defined, in the step S3, the sequence of Ru-DNA is: 5’-AAC TAT ACA ACA TAC TAC-Ru-3’; the RCA mixed solution is composed of 1 μL of 2 mM dNTP mixture, 4 μL of 10× phi29 polymerase buffer, 4.4 μL of 2 mg / mL BSA, and 0.6 μL of 0.6 U / μL phi29 DNA polymerase.

[0033] Further defined, the rolling circle amplification reaction in the step S3 is specifically: oscillating at 37 °C for 1 h, and then washing the obtained product 2-3 times.

[0034] A bead-based cardiac troponin I detection kit based on rolling circle amplification, comprising biotinylated antibody biotin-Ab1, magnetic beads MBs@SA, recognition probe Ab2-DNA, padlock probe, RCA mixed solution, and Ru-DNA;

[0035] The recognition probe Ab2-DNA is obtained by conjugating the detection antibody Ab2 with ss-DNA, and the concentration ratio of Ab2 to ss-DNA is (1:4) to (1:10);

[0036] The padlock probe is prepared by the template ligation method using an RCA template, ss-DNA, and T4 DNA ligase;

[0037] The sequence of the RCA template is 5’-Phosphate ATA CTA CCT CAC TGG GCC CAC CCT CCCCAC CCG GGA CAA CTA TAC AAC-3’;

[0038] The sequence of the ss-DNA is 5’-SH-(CH2)6TGA GGT AGT ATG TTG TAT AGT T-3’;

[0039] The sequence of the Ru-DNA is: 5’-AAC TAT ACA ACA TAC TAC-Ru-3’;

[0040] The RCA mixed solution is composed of 1 μL of 2 mM dNTP mixture, 4 μL of 10× phi29 polymerase buffer, 4.4 μL of 2 mg / mL BSA, and 0.6 μL of 0.6 U / μL phi29 DNA polymerase.

[0041] The application of the bead-based cardiac troponin I detection kit based on rolling circle amplification in the electrochemical luminescence method for detecting the concentration of cardiac troponin I.

[0042] Compared with the prior art, the beneficial effects of the present invention are:

[0043] 1. The detection method provided by the present invention combines rolling circle amplification reaction (RCA) and electrochemiluminescence immunoassay (ECLIA) to achieve quantitative detection of cardiac troponin I. Specifically, taking the RCA reaction as a signal amplification means, the detection of proteins is transformed into the detection of amplification products, greatly improving the detection sensitivity; moreover, combined with the ECLIA technology, the detection of RCA products is realized, and then the quantitative detection of cardiac troponin I (abbreviation: cTnI) is accurately and sensitively achieved.

[0044] 2. The present invention uses electrochemiluminescence immunoassay (ECLIA) to detect the ECL value corresponding to the amplification product, thereby accelerating the detection speed.

[0045] 3. The detection method adopted by the present invention has the advantages of good specificity, high sensitivity, and automation in operation, meeting the requirements of clinical detection.

[0046] 4. The present invention provides a magnetic bead-based cardiac troponin I detection kit based on rolling circle amplification, which has the characteristics of high sensitivity and simple operation, and can achieve accurate and quantitative detection of cardiac troponin I. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is a schematic diagram of the detection method of cardiac troponin I (abbreviation: cTnI) provided by the present invention;

[0048] Figure 2 is Figure 1 the specific process of the RCA reaction in

[0049] Figure 3 is the ultraviolet absorption spectra of ss-DNA, Ab2 and Ab2-DNA probes;

[0050] Figure 4 is the basic principle of the padlock probe and the analysis results of 12.5% polyacrylamide gel electrophoresis PAGE;

[0051] Figure 5 is the feasibility result of the detection of cardiac troponin I (abbreviation: cTnI) by the detection method of the present invention;

[0052] Figure 6 is the optimization result of the reaction conditions in the detection method of the present invention;

[0053] Figure 7 is the detection analysis of cTnI by the traditional sandwich immunoassay method and the ECLIA-RCA method of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0054] The technical solutions of the present invention will be further described below with reference to the drawings and embodiments.

[0055] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the art to which the present invention pertains.

[0056] Technologies, methods, and devices known to those of ordinary skill in the relevant fields may not be discussed in detail, but where appropriate, such technologies, methods, and devices shall be regarded as part of the specification.

[0057] It should also be understood that the specific embodiments described above are only used to explain the present invention, and the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

[0058] The technical idea of the present invention is: through the specific recognition of antigens and antibodies on the surface of magnetic beads, an ECLIA-RCA detection method combining rolling circle amplification (RCA) and electrochemiluminescence technology (ECLIA) is adopted to achieve accurate and highly sensitive quantitative detection of cardiac troponin I (abbreviated as cTnI). The detection method is as follows.

[0059] First, magnetic beads coated with biotinylated antibody biotin-Ab1 (primary antibody) (MBs@SA·bio-Ab1) are incubated with an antigen (a solution of cardiac troponin I with a known concentration) and a conjugate Ab2-DNA / pad of a detection antibody Ab2 (secondary antibody) to form a sandwich immuno-complex; in order to introduce RCA, a padlock probe formed by annealing an RCA primer (i.e., ss-DNA) with a circular DNA template (RCA template) is used to label each sandwich immuno-complex.

[0060] Then, a rolling circle amplification reaction, i.e., an RCA reaction, is carried out under the action of phi29 DNA polymerase in the RCA reaction solution. At the same time, the generated DNA concatemers are connected to each sandwich immuno-complex, and then hybridized with a large number of complementary ECL probes Ru-DNA to form amplification products. The obtained amplification products are used for electrochemiluminescence detection, i.e., ECL detection, to obtain an electrochemiluminescence signal value (denoted as the ECL value).

[0061] Finally, a series of solutions of cardiac troponin I with known concentrations are prepared, and a series of ECL values are obtained with reference to the above method to construct a calibration curve of the concentration of the cardiac troponin I solution and the ECL value, so as to calculate the concentration of cardiac troponin I in the sample to be measured through the calibration curve.

[0062] The detection method provided by the present invention has the advantages of good specificity, high sensitivity, simple operation, etc., and meets the needs of clinical detection.

[0063] Another technical idea of the present invention is to provide a magnetic bead-based cardiac troponin I detection kit based on rolling circle amplification, which has the characteristics of high sensitivity and simple operation, and can be used for quantitative detection of cardiac troponin I in electrochemiluminescence method.

[0064] The magnetic bead-based cardiac troponin I detection kit provided by the present invention includes biotinylated antibody biotin-Ab1, magnetic beads MBs@SA, recognition probe Ab2-DNA, padlock probe, RCA mixed solution and Ru-DNA.

[0065] Preferably, the recognition probe Ab2-DNA is obtained by conjugating the detection antibody Ab2 with ss-DNA, and the concentration ratio of Ab2 to ss-DNA is (1:4) to (1:10); preferably, the padlock probe is prepared by the template ligation method with an RCA template, ss-DNA and T4 DNA ligase.

[0066] Preferably, the sequence of the RCA template is 5’-Phosphate ATA CTA CCT CAC TGG GCC CAC CCTCCC CAC CCG GGA CAA CTA TAC AAC-3’.

[0067] Preferably, the sequence of ss-DNA is 5’-SH-(CH2)6TGA GGT AGT ATG TTG TAT AGT T-3’.

[0068] Preferably, the sequence of Ru-DNA is: 5’-AAC TAT ACA ACA TAC TAC-Ru-3’;

[0069] Preferably, the RCA mixed solution is composed of 1 μL of 2 mM dNTP mixture, 4 μL of 10×phi29 polymerase buffer, 4.4 μL of 2 mg / mL BSA and 0.6 μL of 0.6 U / μL phi29 DNA polymerase.

[0070] In the present invention, the application of the magnetic bead-based cardiac troponin I detection kit based on rolling circle amplification in the detection of cardiac troponin I by electrochemiluminescence method.

[0071] The following uses specific examples to illustrate the technical solutions provided by the present invention.

[0072] In the following examples, unless otherwise specified, the chemical drugs, reagents, etc. used are all conventional known commercially available products in the art. For example, biotinylated antibody biotin-Ab1, Ab2 and ss-DNA, etc.

[0073] It should be noted that in the following embodiments, mM refers to mmol / L; μM refers to μmol / L; nM refers to nmol / L.

[0074] In the following embodiments, the ss-DNA, RCA template, and Ru-DNA sequences are shown in the following table.

[0075] Table 1 ss-DNA, RCA template, and Ru-DNA sequences

[0076]

[0077] Example 1

[0078] This example provides a magnetic bead-based cardiac troponin I detection kit based on rolling circle amplification, including biotinylated antibody biotin-Ab1, magnetic beads MBs@SA, recognition probe Ab2-DNA, padlock probe, RCA reaction mixture, and Ru-DNA.

[0079] In this example, the recognition probe Ab2-DNA is obtained by conjugating the detection antibody Ab2 with ss-DNA.

[0080] In this example, the concentration ratio of Ab2 to ss-DNA is (1:4) to (1:10).

[0081] In this example, the padlock probe is prepared by the template ligation method using the RCA template, ss-DNA, and T4 DNA ligase.

[0082] In this example, the sequence of the RCA template is 5’-Phosphate ATA CTA CCT CAC TGG GCC CACCCT CCC CAC CCG GGA CAA CTA TAC AAC-3’.

[0083] In this example, the sequence of the ss-DNA is 5’-SH-(CH2)6TGA GGT AGT ATG TTG TAT AGT T-3’.

[0084] In this example, the sequence of the Ru-DNA is: 5’-AAC TAT ACA ACA TAC TAC-Ru-3’;

[0085] In this example, the RCA reaction mixture is composed of 1 μL of 2 mM dNTP mixture, 4 μL of 10× phi29 polymerase buffer, 4.4 μL of 2 mg / mL BSA, and 0.6 μL of 0.6 U / μL phi29 DNA polymerase.

[0086] The detection kit provided in this embodiment can be used for the electrochemiluminescence detection of cardiac troponin I.

[0087] Example 2

[0088] This example mainly uses the detection kit of Example 1 to detect the concentration of cardiac troponin I by electrochemiluminescence method.

[0089] See Figure 1 , the electrochemiluminescence detection method of magnetic bead type cardiac troponin I based on rolling circle amplification provided in this embodiment includes the following steps:

[0090] S1. Synthesis of magnetic capture probe

[0091] The magnetic capture probe (MBs@SA·bio-Ab1) is synthesized by binding biotinylated antibody biotin-Ab1 (i.e., the primary antibody) with MBs@SA.

[0092] Gently vibrate the MBs@SA stock solution (10 mg / mL) in the glass bottle for 30 seconds. Take 100 μL of 10 mg / mL MBs@SA suspension and wash it three times with 500 μL of 10 mM PBS1 buffer solution (pH = 7.40, composed of 8.2 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl and 0.05% Tween 20). Finally, MBs@SA is suspended in 500 μL of solution.

[0093] Add 25 μL of 1 mg / mL biotin-Ab1 prepared with 10 mM PBS2 buffer solution (pH = 7.40, composed of 1.8 mM NaH2PO4, 8.2 mM Na2HPO4, 100 mM NaCl and 5 mM MgCl2) to the above 500 μL of 2 mg / mL MBs@SA solution, and oscillate and react on a temperature-controlled vortex mixer at 37 °C for 30 min. Then, place the centrifuge tube on the magnetic separation rack for 1 min for magnetic separation, and wash it 2 - 3 times by magnetic separation with 200 μL of 10 mM PBS1 buffer solution (pH = 7.40) to obtain MBs@SA·bio-Ab1. Then, wash it three times with PBS1 buffer solution containing 0.1% BSA to remove the conjugated antibody and block any remaining active surface of MBs. Resuspend MBs@SA·bio-Ab1 in 1.0 mL of 10 mM PBS1 buffer solution (pH = 7.40) at a concentration of 1 mg / mL for use.

[0094] S2. Synthesis of Ab2-DNA conjugate

[0095] Using heterobifunctional sulfo-SMCC as a linker, the amino group on the detection antibody Ab2 (secondary antibody) was coupled with the thiol group of thiolated DNA (i.e., ss-DNA) to obtain an Ab2-DNA conjugate.

[0096] Specifically, the synthesis of the Ab2-DNA conjugate is described in detail.

[0097] First, 50 μL of 0.4 mM SMCC (20-fold molar excess) (dissolved in anhydrous DMSO) was added to 500 μL of 0.2 mg / mL Ab2 (dissolved in 10 mM PBS2 buffer solution, pH = 7.40), and the mixture was shaken at room temperature for 2 hours. The activated Ab2 was ultrafiltered and purified using a 10 kDa cut-off membrane (Millipore, 14000 rpm, 3 × 10 min).

[0098] Second, 56 μL of 100 mM dithiothreitol (DTT) and 100 μM ss-DNA prepared with 44.5 μL of 10 mM TE buffer (pH 8.0) were reacted at 37 °C for 1 h to reduce the dimerization of ss-DNA. The reduced ss-DNA was ultrafiltered and purified using a 3 kDa cut-off membrane (Millipore, 14000 rpm, 3 × 10 min).

[0099] In the third step, the reduced ss-DNA (~300 μL) was mixed with the activated Ab2 (~500 μL) prepared above and incubated overnight at 4 °C. Then, the unreacted ss-DNA was removed by ultrafiltration using a 3 kDa cut-off membrane (Millipore, 14000 rpm, 3 × 10 min). The remaining Ab2-DNA conjugate (~500 μL) was stored at 4 °C, which is the recognition probe and ready for use. After diluting the Ab2-DNA by 10-fold, the concentration of Ab2 was 3.2×10 -7 mol / L (0.048 mg / mL), and the concentration of ss-DNA was 3.3×10 -6 mol / L. The average ss-DNA loading per antibody was calculated by measuring the absorbance at 260 nm and 280 nm. The molar absorption coefficients were calculated according to Lambert-Beers law using ss-DNA and Ab2 with standard concentrations: c = A / (ε × 1).

[0100] A 260nm = A 260nm,Ab2 + A 260nm,ss-DNA (1)

[0101] A 280nm = A 280nm,Ab2 + A 280nm,ss-DNA (2)

[0102] A 260nm,Ab2-DNA= ε 260nm,Ab2 C Ab2 + ε 260nm,ss-DNA C ss-DNA (3)

[0103] A 280nm,Ab2-DNA = ε 280nm,Ab2 C Ab2 + ε 280nm,ss-DNA C ss-DNA (4)

[0104] In the above formulas (1) and (2):

[0105] A 260nm 、A 260nm,Ab2 and A 260nm,ss-DNA are the absorbances of the Ab2-DNA conjugate, Ab2, and ss-DNA at 260 nm respectively; A 280nm 、A 280nm,Ab2 and A 280nm,ss-DNA are the absorbances of the Ab2-DNA conjugate, Ab2, and ss-DNA at 280 nm respectively;

[0106] In the above formulas (3) and (4):

[0107] A 260nm,Ab2-DNA is the absorbance of the Ab2-DNA conjugate at 260 nm; ε 260nm,Ab2 is the molar absorption coefficient of Ab2 at 260 nm; C Ab2 is the concentration of Ab2; ε 260nm,ss-DNA is the molar absorption coefficient of ss-DNA at 260 nm; C ss-DNA is the concentration of ss-DNA; A 280nm,Ab2-DNA is the absorbance of the Ab2-DNA conjugate at 280 nm; ε 280nm,Ab2 is the molar absorption coefficient of Ab2 at 280 nm; ε 280nm,ss-DNA is the molar absorption coefficient of ss-DNA at 280 nm.

[0108] Calculated by the above formulas, the concentration ratio of Ab2 to ss-DNA is 1:10.

[0109] S3. Preparation of "padlock probe" and Ab2-DNA / pad conjugate

[0110] The RCA experiment consists of two steps, namely padlock ligation and RCA amplification. Padlock probes are prepared by the template ligation method. The ligation reaction mixture containing 54 μL of 10 μM RCA template, 45 μL of 10 μM ss-DNA, and 26.6 μL of 5×T4 DNA ligase buffer was annealed at 95 °C for 5 min and slowly cooled to room temperature; then 3.75 μL of T4 DNA ligase (400 U / μL) was added to ligate the RCA template and ss-DNA. After incubation at 37 °C for 3 h, a lysis reaction system (600 U Exo III and 1×Exo III buffer) was added, and its volume was adjusted to 150 μL with 10 mM PBS2 buffer solution (pH = 7.4). After incubation at 37 °C for 3 h and 65 °C for 20 min to terminate the reaction; the obtained ligation product (referred to as padlock probe) was stored at 4 °C for later use and used as a template for rolling circle amplification.

[0111] Then, 24 μL of the padlock probe prepared above was mixed with 240 μL of 20 μg / mL (0.02 mg / mL) Ab2-DNA and 216 μL of PBS2, and a coupling reaction was carried out at 37 °C for 1 h. The obtained conjugate (named Ab2-DNA / pad, final concentration 10 μg / mL) was stored at 4 °C for later use without further purification.

[0112] In this step, one-step hybridization method (ECLIA-RCA) was used for amplification, for details see Figure 2 。

[0113] 160 μL of a solution of known concentration of cardiac troponin I (i.e., cTnI) was added to a centrifuge tube containing 20 μL of the above-prepared MBs@SA·bio-Ab1 (0.5 mg / mL) and 20 μL of 0.6 μg / mL synthesized Ab2-DNA / pad, and incubated with shaking on a temperature-controlled vortex mixer at 37 °C for 30 min. The centrifuge tube was placed on a magnetic separation rack for 1 min for magnetic separation, and then the supernatant was discarded. Each time 200 μL of 10 mM PBS1 buffer solution (pH = 7.40) was used, and the washing was repeated 2 - 3 times to obtain a magnetic immune complex.

[0114] Then, 10 μL of the RCA mixed solution and 50 μL of 0.1 nM Ru-DNA were simultaneously added to the above-obtained magnetic immune complex, and the final volume was 60 μL. After shaking at 37 °C for 1 h, the obtained magnetic beads were washed 2 - 3 times with 200 μL of 10 mM PBS1 buffer solution (pH = 7.40) to stop the RCA reaction and obtain the amplification product. Then the amplification product (magnetic beads) was suspended in 200 μL of 10 mM PBS2 buffer solution (pH = 7.40) to obtain the sample to be tested.

[0115] The RCA mixed solution was composed of 1 μL 2 mM dNTP mixture, 4 μL 10× phi29 polymerase buffer, 4.4 μL 2 mg / mL BSA, and 0.6 μL 0.6 U / μL phi29 DNA polymerase.

[0116] S4. Electrochemiluminescence detection

[0117] The ECL value corresponding to the above amplification product is obtained by using electrochemiluminescence technology; specifically, the above samples to be tested are directly placed in the sample cup of the fully automatic ECL instrument in sequence for ECL detection.

[0118] S5. Generate calibration curve

[0119] Changing the concentration of the cardiac troponin I solution, according to step S3 and step S4, obtaining ECL values ​​corresponding to cardiac troponin I solutions with different concentrations; generating a calibration curve with the concentration of the cardiac troponin I solution as the abscissa and the ECL value (i.e., the electrochemiluminescence signal value) as the ordinate;

[0120] S6. Sample testing

[0121] Collect the protein solution sample to be tested, and obtain the ECL value corresponding to the protein solution sample to be tested according to the method of step S3 and step S4, and then substitute it into the calibration curve to calculate the concentration of cardiac troponin I (abbreviated as cTnI) in the protein solution sample to be tested.

[0122] In the above Example 2, in step S1, the temperature of the oscillation reaction can be arbitrarily selected and replaced within 30°C to 37°C; the reaction time can be arbitrarily selected and replaced within 18min to 30min. In step S2, the concentration ratio of Ab2 to ss-DNA can be arbitrarily selected within the range of (1:4) to (1:10).

[0123] The advantages of the electrochemiluminescence detection method and detection kit of magnetic bead-based cardiac troponin I (cTnI for short) based on rolling circle amplification provided by the present invention are verified below.

[0124] Test 1

[0125] The synthesized Ab2-DNA conjugate was characterized by UV-Vis absorption spectroscopy. Figure 3 , where: a is 10 μM ss-DNA, b is 0.048 mg / mL Ab2-DNA conjugate (after the Ab2-DNA stock solution was diluted 10 times), and c is the UV-visible absorption spectrum of 1 mg / mL Ab2.

[0126] Figure 3As shown, the ultraviolet-visible absorption spectrum of the Ab2-DNA conjugate shows an absorption peak at 200 nm and a shoulder peak at 260 nm. The individual Ab2 produces absorption peaks at 210 nm and 280 nm, while single-stranded DNA (ss-DNA) alone shows absorption peaks at 204 nm and 257 nm. After the conjugation of Ab2 and ss-DNA, the maximum wavelengths of the absorption peaks change from 210 nm and 204 nm respectively to 200 nm, indicating the successful synthesis of the Ab2-DNA conjugate. Based on the Lambert-Beer law, by measuring the absorbance at 260 nm and 280 nm, it is calculated that the average number of ss-DNAs loaded per antibody is 1:10. Therefore, preferably, the concentration ratio of Ab2 to ss-DNA is 1:10.

[0127] Experiment 2

[0128] First, the feasibility of the RCA reaction of the present invention was verified by 12.5% non-denaturing polyacrylamide gel electrophoresis (PAGE).

[0129] Specifically: Using primer DNA (ss-DNA) to simulate the Ab2-DNA conjugate, the process is the same as the reaction steps of ECLIA-RCA in the present invention.

[0130] See Figure 4 , (A) The basic principle of the padlock probe. Specifically, ss-DNA hybridizes with the RCA template and is ligated into a circular structure at its 5' and 3' ends under the action of T4 DNA ligase. Subsequently, the hybridized primer ss-DNA is removed by the cleavage action of Exo III to obtain a circular probe product. After further hybridization with ss-DNA, RCA starts to extend at the 3' end of ss-DNA and, with the assistance of the circular probe, is catalyzed to complete the reaction with dNTPs and phi29 DNA polymerase, generating a long single-stranded DNA amplification product.

[0131] See Figure 4, (B) shows the PAGE results of 12.5% of RCA. Lanes 1-5 represent marker, ss-DNA (22 bp, 0.5 μM), RCA template (48 bp, 0.5 μM), padlock probe, and RCA product respectively. It can be seen that lanes 2 and 3 are the single-stranded primer ss-DNA and RCA template respectively, showing independent and distinct bands. After adding T4 DNA ligase and Exo III, the hybridization product of ss-DNA and RCA template formed the characteristic band of circular probe in lane 4. After further adding phi29 DNA polymerase and dNTP, a bright and slow-migrating band appeared in lane 5 for the product hybridized with ss-DNA, indicating that ss-DNA successfully activated the cyclization of RCA template and generated long single-stranded DNA amplification products through RCA reaction. The differences among lanes 2 to 5 further confirmed the successful formation of DNA concatemers through RCA reaction.

[0132] In summary, the amplification can be achieved by the RCA reaction of the present invention, indicating that the detection kit and detection method of the present invention are feasible for the detection of cardiac troponin I.

[0133] Experiment 3

[0134] This experiment mainly studied the feasibility for detecting cTnI.

[0135] In this study, an automated electrochemiluminescence immunoassay analyzer was used to record the ECL signal. During the detection, a voltage of +1.4 V was applied, and the integrated intensity of the ECL signal within 2 seconds was converted into the ECL analysis signal, and the specific operation was based on the experimental procedure of the automated electrochemiluminescence immunoassay analyzer. The ECL signal values of cTnI detection under different conditions obtained on the automated detection platform are shown in Figure 5 in.

[0136] See Figure 5 , the ECL analysis signals under different conditions. (a) In the absence of cTnI, (b) when the cTnI concentration is 10 pg / mL, (c) when the cTnI concentration is 50 pg / mL; (d) without ss-DNA / padlock probe, (e) without padlock probe, (f) 10 pg / mL cTnI (without RCA reaction).

[0137] It can be seen from Figure 5 that in the absence of cTnI, the background ECL signal value (790 a.u., Figure 5 (a)) was obtained. This relatively low ECL signal value may be due to the adsorption of a certain amount of Ru-DNA on the magnetic beads. When the cTnI concentration was 10 pg / mL, a strong ECL signal value (2055 a.u., Figure 5(b)). When the cTnI concentration is further increased to 50 pg / mL, the ECL signal value correspondingly increases to 6342 a.u.( Figure 5 (c)). This phenomenon is attributed to the formation of a sandwich immune complex through the sandwich immunoreaction among the capture probe, cTnI, and Ab2-DNA; after the RCA reaction, Ru-DNA is labeled onto the magnetic beads, and the ECL signal value also increases accordingly. With the increase in cTnI concentration, more Ru-DNA is labeled, resulting in a further increase in the ECL signal value. Through this strategy, local signals can be generated on each magnetic bead carrying a complete sandwich immune complex. As a control, multiple sets of verification experiments were conducted to detect 10 pg / mL of cTnI. When there is no ss-DNA / padlock probe( Figure 5 (d)), padlock probe( Figure 5 (e)), and RCA reaction solution( Figure 5 (f)) in the reaction system, the ECL signal is consistent with the background signal. This indicates that the ECLIA-RCA method provided by the present invention has obvious feasibility in cTnI detection.

[0138] Experiment 4

[0139] The purpose of this experiment is to optimize the detection conditions, mainly including the concentration of Ab2-DNA / pad and the concentration of phi29 DNA polymerase.

[0140] See Figure 6 , (A) Optimization of the dosage of Ab2-DNA / pad. The signal-to-noise ratio S / R corresponding to the ECL signal generated by cTnI was detected at the dosages of Ab2-DNA / pad of 10 pg / mL and 50 pg / mL respectively. (B) Optimization of the dosage of phi29 DNA polymerase. The signal-to-noise ratio S / R corresponding to the ECL signal generated by cTnI was detected at the dosages of phi29 DNA polymerase of 10 pg / mL and 50 pg / mL respectively.

[0141] From Figure 6 the optimization, it can be seen that the optimal conditions during the detection process are: the concentration of Ab2-DNA / pad is 0.6 μg / mL( Figure 6 A), and the concentration of phi29 DNA polymerase is 0.6 U / μL( Figure 6 B).

[0142] Experiment 5

[0143] Under the optimized detection conditions, the analytical performance of the detection method was further studied.

[0144] See Figure 7, (A) Relationship between ECL signal and cTnI concentration (0.01 - 100 pg / mL) in the ECLIA - RCA strategy. The inset shows the calibration curve of cTnI in the range of 0.01 - 50 pg / mL. (B) Relationship between ECL signal and cTnI concentration (0.5 - 200 ng / mL) in the traditional sandwich immunoassay. The inset shows the calibration curve of cTnI in the range of 0.5 - 50 ng / mL. (C) Comparison of ECL signals obtained from detecting cTnI by the traditional sandwich immunoassay (black line) and the ECLIA - RCA method (red line). (D) Relative ECL response values ((I s -I0) / I0) of 50 pg / mL cTnI and other interfering proteins (including 500 ng / mL CEA, 500 ng / mL IgG, 500 ng / mL AFP, and 10 ng / mL EpCAM).

[0145] See Figure 7 , as the cTnI concentration increases in the range of 0 to 100 pg / mL, the ECL intensity increases significantly ( Figure 7 A). When the cTnI concentration is as low as 0.01 pg / mL, the ECL signal generated can be clearly distinguished from the blank sample. In the range of cTnI concentration from 0.01 to 50 pg / mL, the ECL intensity shows a good linear relationship with the cTnI concentration, and the linear regression equation is I = 101C cTnI +1094 (C is in pg / mL, R 2 = 0.9988) ( Figure 7 A). The limit of detection (LOD) is calculated to be 0.003 pg / mL cTnI. This LOD is nearly four orders of magnitude lower than the critical value (70 - 400 pg / mL) of cTnI for liver cancer diagnosis. Thus, it can be seen that the detection kit and detection method proposed in the present invention have very prominent advantages in the detection of cardiac troponin I and can be used in the detection of protein biomarkers.

[0146] In the absence of the RCA reaction, as the cTnI concentration increases in the range of 0 to 200 ng / mL, the ECL signal gradually increases ( Figure 7 B), and the ECL signal shows a good linear relationship with the cTnI concentration in the range of 1 - 50 ng / mL, and its linear regression equation is I = 73C cTnI +896 (C is in ng / mL, R 2 = 0.9995) ( Figure 7 B). The calculated limit of detection is 0.7 ng / mL cTnI (S / N = 3). Compared with the traditional sandwich immunoassay, the sensitivity of the ECLIA - RCA method is four orders of magnitude higher ( Figure 7C). Therefore, the high efficiency and high sensitivity brought by the RCA reaction (rolling circle amplification) to subsequent detection are obvious.

[0147] In addition, to evaluate the selectivity of the detection kit and detection method, some interfering proteins (such as CEA, IgG, AFP, and EpCAM) were analyzed.

[0148] As Figure 7 shown in D, only cTnI can produce a significant ECL response. At the same time, even when the concentration of the interferents increases to 500 ng / mL (CEA, IgG, and AFP) and 10 ng / mL (EpCAM), there is almost no obvious effect on the ECL intensity, and the results are similar to those of the blank control. This indicates that the detection kit and detection method of the present invention have excellent potential for the accurate recognition and monitoring of cTnI in complex systems.

[0149] Experiment 7

[0150] As can be seen from Table 2, in order to verify the applicability of the developed automated ECLIA-RCA method for detecting cTnI in human serum samples, human serum samples provided by the Hospital of Shaanxi Normal University were used for testing.

[0151] First, the cTnI concentrations in two serum samples were detected using the detection kit and detection method (ECLIA-RCA method), and at the same time, the existing chemiluminescence immunoassay was also used for detection. The detection results are shown in Table 2.

[0152] Table 2 Test results corresponding to different detection methods

[0153] Sample ECLIA - RCA method (pg / mL) Chemiluminescent immunoassay (ng / mL) 1 40 0.04 2 165 0.18

[0154] As can be seen from Table 2, the cTnI concentrations calculated using the detection kit and detection method (ECLIA-RCA detection method) proposed in the present invention are 40 pg / mL and 165 pg / mL respectively; the detection results are basically consistent with those of the existing chemiluminescence immunoassay. This indicates that the detection kit and detection method of the present invention are feasible for the detection of cardiac troponin I in serum samples, providing a new approach for the quantitative detection of cardiac troponin I.

[0155] Considering the lower detection limit of the detection kit and detection method (ECLIA-RCA method), the above two serum samples were diluted 5 times and 20 times respectively with 10 mM PBS2 buffer solution (pH = 7.40) for the recovery experiment. The results are shown in Table 3.

[0156] Table 3 Comparison of the determination results of cTnI in human serum specimens by the method of the present invention

[0157]

[0158] As can be seen from Table 3, in the diluted human serum samples, the recovery rate ranges from 93% to 105.00%. This indicates that the detection method and detection kit provided by the present invention also have good applicability for the detection of low-concentration cardiac troponin I.

[0159] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the preferred embodiments. Those of ordinary skill in the art should understand that they can still modify the technical solutions of the present invention or make equivalent replacements, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An electrochemiluminescence detection method for magnetic bead-based cardiac troponin I based on rolling circle amplification, characterized in that: The following steps are involved: S1. Synthesis of magnetic capture probe MBs@SA·bio-Ab1 The biotinylated antibody biotin-Ab1 was combined with MBs@SA to obtain the magnetic capture probe MBs@SA·bio-Ab1, and then resuspended in a buffer solution for later use; S2. Preparation of conjugate Ab2-DNA / pad S2.1 Synthesis of recognition probe Ab2-DNA Using heterobifunctional sulfo-SMCC as a connector, the detection antibody Ab2 is coupled to ss-DNA to obtain a conjugate Ab2-DNA; the concentration ratio of Ab2 to ss-DNA is (1:4) to (1:10); S2.

2. Preparation of padlock probe Padlock probes were prepared by template ligation using RCA template, ss-DNA and T4 DNA ligase; S2.

3. Synthesis of conjugate Ab2-DNA / pad The padlock probe of step 2.2 and the Ab2-DNA of step 2.1 are placed in a buffer solution, and incubated to react to obtain a conjugate Ab2-DNA / pad; the final concentration of Ab2-DNA in the conjugate Ab2-DNA / pad is 10 μg / mL; S3, rolling circle amplification A cardiac troponin I solution of known concentration, the magnetic capture probe MBs@SA·bio-Ab1 of step S1, and the conjugate Ab2-DNA / pad of step S2 are mixed, and then incubated and magnetically separated to obtain a magnetic immune complex; The RCA mixed solution and Ru-DNA are added to the magnetic immune complex to perform a rolling circle amplification reaction to obtain an amplified product; S4. Electrochemiluminescence detection The ECL value corresponding to the above amplification product is obtained by using electrochemiluminescence technology; S5. Generate calibration curve Changing the concentration of the cardiac troponin I solution, according to step S3 and step S4, obtaining the ECL values ​​corresponding to the cardiac troponin I solutions at different concentrations; and generating a calibration curve with the concentration of the cardiac troponin I solution as the abscissa and the ECL value as the ordinate; S6. Sample testing Collect the sample to be tested, and obtain the ECL value corresponding to the sample to be tested according to the method of step S3 and step S4, and then substitute it into the calibration curve to calculate the concentration of cardiac troponin I in the sample to be tested.

2. The electrochemiluminescence detection method of magnetic bead-based cardiac troponin I based on rolling circle amplification according to claim 1, characterized in that: In the step S1, the mass ratio of biotin-Ab1 to MBs@SA is 1:

40.

3. The electrochemiluminescence detection method of magnetic bead-based cardiac troponin I based on rolling circle amplification according to claim 1, characterized in that: In the step S1, biotin-Ab1 and MBs@SA are reacted at 30°C to 37°C for 18min to 30min to obtain MBs@SA·bio-Ab1.

4. The electrochemiluminescence detection method of magnetic bead-based cardiac troponin I based on rolling circle amplification according to claim 1, characterized in that: The steps of synthesizing the padlock probe in step S2.2 are: A mixture containing RCA template, ss-DNA, and T4 DNA ligase buffer solution was annealed at 95°C for 5 minutes and then cooled to room temperature; then T4 DNA ligase was added and incubated at 37°C for 3 hours; then the cleavage reaction system was added and the volume was adjusted to 150 μL with buffer solution; finally, it was incubated at 37°C for 3 hours and 65°C for 20 minutes to obtain a padlock probe.

5. The electrochemiluminescence detection method of magnetic bead-based cardiac troponin I based on rolling circle amplification according to claim 4, characterized in that: The sequence of the RCA template was 5′-Phosphate ATA CTA CCT CAC TGG GCC CAC CCT CCCCAC CCG GGA CAA CTA TAC AAC-3′; The sequence of ss-DNA is 5'-SH-(CH2)6TGA GGT AGT ATG TTG TAT AGT T-3'.

6. The electrochemiluminescence detection method of magnetic bead-based cardiac troponin I based on rolling circle amplification according to claim 5, characterized in that: In the step S2.2 and step S2.3, the buffer solution is prepared from NaH2PO4, Na2HPO4, NaCl and MgCl2, and the pH value of the buffer solution is 7.

40.

7. The electrochemiluminescence detection method of magnetic bead-based cardiac troponin I based on rolling circle amplification according to claim 1, characterized in that: In step S3, the sequence of Ru-DNA is: 5'-AAC TAT ACA ACA TAC TAC-Ru-3'; the RCA mixed solution is composed of 1 μL 2mM dNTP mixture, 4 μL 10×phi29 polymerase buffer, 4.4 μL 2mg / mL BSA and 0.6 μL 0.6U / μL phi29 DNA polymerase.

8. The electrochemiluminescence detection method of magnetic bead-based cardiac troponin I based on rolling circle amplification according to claim 1, characterized in that: The rolling circle amplification reaction in step S3 is specifically: shaking at 37° C. for 1 hour, and then washing the obtained product 2-3 times.

9. A magnetic bead-based cardiac troponin I detection kit based on rolling circle amplification, characterized in that: It includes biotinylated antibody biotin-Ab1, magnetic beads MBs@SA, recognition probe Ab2-DNA, padlock probe, RCA mixed solution and Ru-DNA; The recognition probe Ab2-DNA is obtained by coupling the detection antibody Ab2 with ss-DNA, and the concentration ratio of Ab2 to ss-DNA is (1:4) to (1:10); The padlock probe is prepared by template ligation method using RCA template, ss-DNA and T4 DNA ligase; The sequence of the RCA template is 5′-Phosphate ATACTACCT CAC TGG GCC CAC CCT CCC CAC CCGGGACAACTA TAC AAC-3′; The sequence of the ss-DNA is 5'-SH-(CH2)6TGA GGT AGT ATG TTG TAT AGT T-3'; The sequence of the Ru-DNA is: 5'-AAC TAT ACAACATAC TAC-Ru-3'; The RCA mixed solution is composed of 1 μL of 2 mM dNTP mixture, 4 μL of 10×phi29 polymerase buffer, 4.4 μL of 2 mg / mL BSA and 0.6 μL of 0.6 U / μL phi29 DNA polymerase.

10. Use of the magnetic bead-based cardiac troponin I detection kit based on rolling circle amplification as claimed in claim 9 in detecting the concentration of cardiac troponin I by electrochemiluminescence method.

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