A chemiluminescence rapid detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor

Abstract

The present invention provides a rapid chemiluminescence detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor. In this method, oligonucleic acid strands and aptamers are used to construct self-assembled DNA nucleic acid double strands. In the presence of one or more target objects (different ligands), the strong binding force between the aptamer and the ligand causes the self-assembled DNA nucleic acid double strands to disintegrate, releasing the corresponding single-stranded nucleic acid strands to bind to the complementary DNA single strands on the magnetic microsphere carriers. Through magnetic separation, different chemiluminescence probes are used for chemiluminescence detection of the corresponding target objects. The self-assembled DNA nucleic acid double strands can detect multiple target objects in the same initial system, reducing the detection time and cost for practical applications. Moreover, the self-assembled DNA nucleic acid strands can be replaced with aptamer strands for other target objects or used to detect two or more target objects, having broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemical medical detection technology, in combination with nucleic acid aptamer technology, and in particular to a chemiluminescence rapid detection method for multiple tumor markers based on a self-assembled DNA nucleic acid chain sensor. Background Art

[0002] Alphafetoprotein (AFP) is a 70kDa glycoprotein containing about 4% carbohydrates, which is mainly produced by the fetal liver during pregnancy. Under normal circumstances, the serum concentration of alpha-fetoprotein drops sharply after the birth of a child, and its synthesis is greatly inhibited in adulthood. However, more than 70% of liver cancer patients have high serum concentrations of AFP excreted by tumors, so AFP is clinically used as a tumor marker for liver cancer and other cancers. Therefore, early screening of serum AFP plays a very important role in the early clinical diagnosis and prognosis of tumor patients.

[0003] Adenosine triphosphate (ATP), as the main energy molecule of cell metabolism, plays an irreplaceable role in regulating various life activities. A large number of studies have shown that the concentration of ATP in organisms is closely related to many diseases, such as hypoglycemia, cardiovascular disease and some malignant tumors. In addition, ATP can be used as an effective indicator of cell metabolism to monitor the course of some tumor patients in real time. Therefore, sensitive and reliable detection of ATP is the key to pathological diagnosis.

[0004] Aptamer (Apt) technology has shown outstanding technical advantages in analytical chemistry, especially in the quantitative detection of trace substances. Aptamers are oligonucleotide fragments that can specifically bind to target ligands that are screened in vitro by SELEX technology. With its unique high affinity and specificity, it can be used as a recognition probe and biosensor. Compared with protein antibodies, aptamers can not only efficiently and specifically recognize and bind ligands, but also have the advantages of easy labeling, easy synthesis, and stable properties. However, in order to obtain a feasible detection method, it is key to establish an aptamer sensor that is efficient and can detect multiple ligands simultaneously. First of all, the aptamer sensor should meet the requirements of the luminescence system selected in the experiment at the molecular level. What kind of marker is used and the method of connecting the marker to the sensor need to be designed in a targeted manner; in addition, the selection of what kind of separation carrier and capture probe can completely collect the signal substance and realize the luminescence reaction that is regular with the content of the signal substance, which also needs to be designed according to the properties of the luminescence system, marker, aptamer and other substances; in addition, the choice of the luminescence system is related to the relationship between the final detection result and the actual content of the substance being detected, so it is also an important factor affecting the detection effect.

[0005] Currently, fluorescence, ultraviolet and other methods are mostly used in clinical practice to detect tumor markers, which have problems such as poor sensitivity and unsatisfactory application effects. In addition, there is also the chemiluminescence method (CL) for detecting tumor markers, but most of them can only be used for the detection of single tumor markers and cannot achieve simultaneous detection of multiple components, and there are still problems such as long time consumption and insufficient sensitivity. Summary of the Invention

[0006] Aiming at the deficiencies and problems in the prior art, the present invention aims to provide a rapid chemiluminescence detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor. By skillfully combining the aptamer technology with the "homogeneous reaction and heterogeneous separation" characteristics of magnetic microspheres, a chemiluminescence biosensor based on self-assembled aptamers and DNA nucleic acid strands is created, which can achieve simultaneous detection of two or even multiple tumor markers. The present invention realizes a rapid chemiluminescence detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor, which can quickly and efficiently detect AFP and ATP simultaneously, provides a new idea for medical detection, and is of great significance for the research and determination of multiple components, and is more expected to further realize fully automatic large-scale detection.

[0007] A rapid chemiluminescence detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor, which involves adenosine triphosphate aptamer (ATP aptamer, Apt1), alpha-fetoprotein aptamer (AFP aptamer, Apt2), signal DNA strand 1 (Signal DNA 1, S1), bridge DNA strand (Bridge-DNA, B), signal DNA strand 2 (Signal DNA 2, S2), DNA strand complementary to S1 (Capture-DNA complementary to S1, C1), and DNA strand complementary to S2 (Capture-DNA complementary to S2, C2). Among them, ATP can specifically recognize Apt1, AFP can specifically recognize Apt2, S1 can base-pair complementarily with C1, S2 can base-pair complementarily with C2, and B is mainly used to assist in constructing the self-assembled DNA nucleic acid strand. The amino groups at the ends of C1 and C2 can bind to carboxyl-modified magnetic microspheres through amide bonds. During the process, horseradish peroxidase-labeled streptavidin (HRP-SA) cross-linked by streptavidin (SA) and horseradish peroxidase (HRP) and the chemiluminescence reagent phenylglyoxal (PG) are also used.

[0008] The specific technical solution is as follows:

[0009] 1) Mix Apt1, Apt2, S1, B, and S2 evenly and react fully;

[0010] 2) Mix the product obtained in step 1) with AFP and ATP evenly and react fully;

[0011] 3) Fix amino-modified C1 on the surface of carboxyl magnetic microspheres through aminocarboxylic acid reaction to obtain magnetic microsphere-C1 complex;

[0012] 4) Mix the product obtained in step 2) with an excessive amount of magnetic microsphere-C1 complex evenly, and take the solid phase after the reaction;

[0013] 5) Mix the solid phase obtained in step 4) with SA-HRP evenly and react fully to obtain a chemiluminescent biosensor for detecting AFP;

[0014] 6) Mix the chemiluminescent biosensor for detecting AFP with luminol-H 2 O 2 reagent evenly, and then detect the chemiluminescent CL value;

[0015] 7) Fix amino-modified C2 on the surface of carboxyl magnetic microspheres through aminocarboxylic acid reaction to obtain magnetic microsphere-C2 complex;

[0016] 8) Mix the remaining solution of the reaction in step 4) with an excessive amount of magnetic microsphere-C2 complex evenly, and take the solid phase after the reaction to obtain a chemiluminescent biosensor for detecting ATP;

[0017] 9) Mix the chemiluminescent biosensor for detecting ATP with PG reagent evenly, and then detect the chemiluminescent CL value.

[0018] Preferably, step 1) includes the following steps: respectively take 6 μL of 100 μM Aptl, Apt2, S1, B, and S2 in a 1.5 mL centrifuge tube, then add 20 μL of BA buffer solution, mix evenly and incubate in a water bath at 80-100 °C for 4-6 min, and finally place it in a 4 °C refrigerator overnight for standby.

[0019] Preferably, step 2) includes the following steps: take 10 μL of 10 -8 μM AFP and 10 μL of 10 -8 μM ATP into the solution obtained in step 1), mix evenly and react with shaking at 35-39 °C for 40-60 min.

[0020] Preferably, step 3) includes the following steps: Take magnetic microspheres, wash them with 0.05 - 0.2 M imidazole buffer solution, then take the solid phase and resuspend it in imidazole buffer containing EDC, incubate with shaking at 35 - 39 °C for 15 - 25 min, and then add amino-modified C1 at a ratio of the amount of magnetic microspheres to amino-modified C1 of 4:(1 - 2) (μg:pmol), and react with shaking at 35 - 39 °C for 40 - 60 min.

[0021] Preferably, step 3) further includes the following steps: After reacting with shaking at 35 - 39 °C for 40 - 60 min, take the solid phase and wash it with PBST buffer solution.

[0022] Preferably, step 3) further includes the following steps: After washing with PBST buffer solution, take the solid phase and add 8 - 12% BSA solution, shake at 35 - 39 °C for 40 - 60 min, and then take the solid phase and wash it with PBST buffer solution to obtain the magnetic microsphere-C1 complex.

[0023] Preferably, step 4) includes the following steps: Mix the product obtained in step 2) with the magnetic microsphere-C1 complex, react with shaking at 35 - 39 °C for 40 - 60 min, and then take the solid phase and wash it with PBST buffer solution.

[0024] Preferably, step 5) includes the following steps: Mix 100 μL SA-HRP with a concentration of 1:8000 - 1:6000 with the solid phase obtained in step 4), react with shaking at 35 - 39 °C for 40 - 60 min, and then take the solid phase and wash it with PBST buffer solution.

[0025] Preferably, step 6) includes the following steps: Mix the product obtained in step 5) with 50 μL luminol solution, transfer it to a measuring bottle, and then add 50 μL hydrogen peroxide solution to detect the chemiluminescence CL value.

[0026] Preferably, step 7) includes the following steps: Take magnetic microspheres, wash them with 0.05 - 0.2 M imidazole buffer solution, then take the solid phase and resuspend it in imidazole buffer containing EDC, incubate with shaking at 35 - 39 °C for 15 - 25 min, and then add amino-modified C2 at a ratio of the amount of magnetic microspheres to amino-modified C2 of 2:(1 - 2) (μg:pmol), and react with shaking at 35 - 39 °C for 40 - 60 min.

[0027] Preferably, step 7) further includes the following steps: After reacting with shaking at 35 - 39 °C for 40 - 60 min, take the solid phase and wash it with PBST buffer solution.

[0028] Preferably, step 7) further includes the following steps: after washing with PBST buffer solution, take the solid phase and add it to an 8-12% BSA solution, shake it at 35-39°C for 40-60 min, and then take the solid phase and wash it with PBST buffer solution to obtain the magnetic microsphere-C2 complex.

[0029] Preferably, step 8) includes the following steps: mix the remaining solution from the reaction in step 4) with the magnetic microsphere-C2 complex, shake and react at 35-39°C for 40-60 min, and then take the solid phase and wash it with PBST buffer solution.

[0030] Preferably, step 9) includes the following steps: mix the product obtained in step 8) with 10 μL of tetrabutyl solution, transfer it to a measuring flask, and then add 90 μL of PG reagent to detect the chemiluminescence CL value.

[0031] Preferably, in the above technical solutions, taking the solid phase is achieved by magnetically separating and removing the supernatant.

[0032] Preferably, detecting the chemiluminescence signal CL value is achieved by using a weak chemiluminescence instrument BPCL.

[0033] In the above technical solutions, the BA buffer solution, imidazole buffer solution, and PBST buffer solution can be selected according to the general technical knowledge in the art to implement the present invention with conventional formulations.

[0034] In the above technical solutions, the reporting sequence of Apt1 is as follows: 5’-AGAGAACCTGGGGGAGTATTGCGGAGGAAGGT-3’. The reporting sequence of Apt2 is as follows: 5’-GTGACGCTCCTAACGCTGACTCAGGTGCAGTTCTCGACTCGGTCTTGATGTGGGTCCTGTCCGTCCGAACCAATC-3’. The reporting sequence of S1 is as follows: 5’-Biotin-GATTGGTTCGGACGG-3’. The reporting sequence of B is as follows: 5’-ACCCACATCAAGACCGAGTCGAGAACTGCACCTGAGTCAGCGTTCGGAGCGTCACAAAAAACCTTCCTCCGC-3’. The reporting sequence of S2 is as follows: 5’-TCCCCCAGGTTCTCTGGGGGGGGGG-3’. The reporting sequence of C1 is as follows: 5’-NH 3 -CCGTCCGAACCAATC-3’. The reporting sequence of C2 is as follows: 5’-AGAGAACCTGGGGGA-NH 3 -3’. The above reporting sequences can be customized and purchased from reagent sales companies.

[0035] The product obtained in step 1) refers to a self-assembled DNA nucleic acid strand formed by base complementary pairing of Apt1, Apt2, S1, B, and S2 respectively.

[0036] The sufficient reaction in step 2) means that Aptl specifically binds to ATP, Apt2 specifically binds to AFP, and S1, B, and S2 are dispersed in the solution.

[0037] The solid phase obtained in step 4) refers to the solid phase formed by base complementary pairing of S1 with C1 of the magnetic microsphere-C1 complex.

[0038] In the remaining solution of the reaction in step 4), there are double strands formed by base complementary pairing of Apt1 with ATP, double strands formed by base complementary pairing of Apt2 with AFP, and dispersed S2 and B.

[0039] The chemiluminescent biosensor for detecting ATP in step 8) is formed by base complementary pairing of S2 with C2 in the magnetic microsphere-C2 complex.

[0040] The excessive magnetic microsphere-C1 complex and magnetic microsphere-C2 complex mean that when C1 and C2 immobilized on the magnetic microspheres specifically bind to S1 and S2 respectively, the amount of the magnetic microsphere-C1 complex or the magnetic microsphere-C2 complex is excessive, so there are still unbound magnetic microsphere-C1 complexes without S1 or unbound magnetic microsphere-C2 complexes without S2 remaining free in the system after the binding reaction is completed; specifically, the amount of the magnetic microsphere-C1 complex or the magnetic microsphere-C2 complex can be determined according to the estimation of the content of S1 or S2 in the sample to be measured. Of course, the present invention can also be implemented with reference to the amount of the magnetic microsphere-C1 complex or the magnetic microsphere-C2 complex in the embodiments of the present invention.

[0041] When performing detection using the method of the present invention, a group of AFP and ATP standard solutions with concentration gradients and known concentrations can be detected first using this method to draw the linear relationship between the AFP and ATP concentrations and the CL value of the luminescence signal, and then the sample to be measured can be detected, and the detection result can be brought into the above linear relationship to obtain the actual detection value. The drawing of the calibration curve is performed using the method of the present invention, and the selection of the injection concentration gradient, the selection of the chart mode, the correction of errors, etc. can be determined according to the general technical knowledge in the art.

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

[0043] The present invention provides a rapid chemiluminescence detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor. In this method, an oligonucleic acid strand and an aptamer are used to construct a self-assembled DNA nucleic acid strand. In the presence of target substances alpha-fetoprotein (AFP) and adenosine triphosphate (ATP), the self-assembled DNA nucleic acid strand can be disassembled into single-stranded structures. The released single-stranded nucleic acid strands bind to complementary DNA strands and are separated magnetically, and the target substances are detected separately by chemiluminescence. There are two chemiluminescence detection mechanisms in the present invention: one is the chemiluminescence reaction generated by SA-HRP bound to C1 and luminol-H 2 O 2 reagent, and the other is the chemiluminescence signal generated by the instantaneous derivatization reaction of guanine base (G) on C2 and PG reagent. This self-assembled DNA nucleic acid strand can detect two target substances in the same initial system, reducing the detection time and cost for practical applications. Moreover, this self-assembled DNA nucleic acid strand can be replaced with aptamer strands for other target substances or detect two or more target substances, having broad application prospects. The lowest detection limits of this method for detecting AFP and ATP are 1.8 pg / mL and 0.5 nM respectively, and it has high specificity through selective investigation. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 is a schematic diagram of the principle of the detection method of the present invention.

[0045] Figure 2 is the standard curve graph of AFP and ATP in Example 1 of the present invention.

[0046] Figure 3 is the experimental result of the specificity investigation of the detection method for AFP and ATP in Example 1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0047] The following will describe the specific embodiments of the present invention in detail. To avoid excessive unnecessary details, the well-known structures or functions will not be described in detail in the following embodiments.

[0048] The following approximate language used in the embodiments can be used for quantitative expression, indicating that a certain variation in quantity is allowed without changing the basic function. Therefore, the numerical values modified by language such as "about" and "around" are not limited to the exact numerical values themselves. In some embodiments, "about" means that the modified numerical value is allowed to vary within an accurate range of plus or minus ten percent (10%) of the exact value. For example, "about 100" can represent any value between 90 and 110. In addition, in the expression of "about the first numerical value to the second numerical value", both the first and second numerical values are modified approximately. In some cases, the approximate language may be related to the accuracy of the measuring instrument.

[0049] Unless otherwise defined, the technical and scientific terms used in the following examples have the same meaning as commonly understood by those skilled in the art to which this invention belongs.

[0050] The test reagents and consumables used in the following examples are all conventional biochemical reagents unless otherwise specified; the experimental methods are all conventional methods unless otherwise specified; the quantitative experiments in the following examples are all set up with three repeated experiments, and the results are averaged; the % in the following examples is the mass percentage unless otherwise specified.

[0051] Example 1

[0052] 1 Experimental principle

[0053] The schematic diagram of the principle of the detection method of the present invention is as Figure 1 shown. First, the present invention constructs a self-assembled DNA nucleic acid strand formed by base complementary pairing of Apt1, Apt2, S1, B, and S2, and then adds ATP and AFP to specifically recognize Apt1 and Apt2 in the self-assembled DNA nucleic acid strand, causing the self-assembled DNA nucleic acid strand to disintegrate. Using C1 and C2 as the stationary phase, they are fixed on the surface of magnetic microspheres to form magnetic microsphere-C1 complex and magnetic microsphere-C2 complex. Then, the magnetic microsphere-C1 complex is used to specifically recognize S1 with biotin in the self-assembled DNA nucleic acid strand disintegration solution, and the magnetic microsphere solid phase is taken, and then SA-HRP is added to bind it to biotin to construct a chemiluminescent biosensor for detecting AFP. Similarly, the magnetic microsphere-C2 complex is used to specifically recognize S2 in the remaining self-assembled DNA nucleic acid strand disintegration solution to construct a chemiluminescent biosensor for detecting ATP. Finally, the chemiluminescence generated by SA-HRP and luminol and the chemiluminescence generated by the G base on S2 and PG reagent are used to measure the CL signal.

[0054] 2 Experimental method

[0055] 2.1 Preparation of self-assembled DNA nucleic acid strand

[0056] Equal amounts of 6 μL, 100 μM of Apt1, Apt2, S1, B, and S2 are taken in a 1.5 mL centrifuge tube, then 20 μL of BA buffer is added, mixed well, incubated in a water bath at 80-100 °C for 4-6 min, and finally placed in a 4 °C refrigerator overnight for standby.

[0057] 2.2 Disintegration of self-assembled DNA nucleic acid strand

[0058] Take 10 μL, 10 -8 μM AFP and 10 μL, 10 -8 μM ATP and add them to the self-assembled DNA nucleic acid strand, mix well, and react with shaking at 35-39 °C for 40-60 min.

[0059] Preparation of Chemiluminescent Biosensor for Detecting AFP

[0060] Take magnetic microspheres, wash them with 0.05 - 0.2 M imidazole buffer solution, and then resuspend the solid phase in imidazole buffer containing EDC. Incubate with shaking at 35 - 39 °C for 15 min. Then add amino-modified C1 at a ratio of 4:(1 - 2) (μg:pmol) of magnetic microspheres to amino-modified C1, and react with shaking at 35 - 39 °C for 40 - 60 min. Then wash the solid phase with PBST buffer solution. After washing, add 8 - 12% BSA solution to the solid phase, shake at 35 - 39 °C for 40 - 60 min, and then wash the solid phase with PBST buffer solution to obtain magnetic microsphere-C1 complex. Add the disassembly solution of self-assembled DNA nucleic acid chain to the magnetic microsphere-C1 complex. After reacting with shaking at 35 - 39 °C for 40 - 60 min, wash the solid phase with PBST buffer solution. Take 100 μL of SA-HRP with a concentration of 1:8000 - 1:6000 and mix it evenly. After reacting with shaking at 35 - 39 °C for 40 - 60 min, wash the solid phase with PBST buffer solution.

[0061] 2.4 Chemiluminescent Detection of AFP

[0062] Mix the chemiluminescent biosensor for detecting AFP with 50 μL of luminol solution, transfer it to a measuring bottle, then add 50 μL of hydrogen peroxide solution, mix well and immediately put it into a chemiluminescent detector for determination. The chemiluminescent signal is detected by a BPCL ultra-weak chemiluminescence analyzer, displayed and recorded by the computer terminal connected to the instrument, and the chemiluminescent intensity is quantified by the peak value of the output signal.

[0063] 2.5 Performance Evaluation of Chemiluminescent Biosensor for Detecting AFP

[0064] We first carried out a standard addition recovery experiment on AFP in human serum samples to evaluate the accuracy and precision of this method. Add different concentrations of AFP (0.1 ng / mL, 5 ng / mL, 10 ng / mL) to human serum samples respectively. Under optimized conditions, the experimental steps are the same as above, and each group of experiments is carried out in parallel three times to calculate the recovery rate of the spiked samples. Secondly, in order to test the specific selectivity of this experimental method, we selected several substances similar to AFP (CEA, IgG, PSA, and BSA) for chemiluminescent detection. The concentrations of AFP, CEA, IgG, PSA, and BSA are all 10 μM, and other conditions remain unchanged to explore the influence of different substances on the chemiluminescent signal intensity.

[0065] Preparation of Chemiluminescent Biosensor for Detecting ATP

[0066] Take magnetic microspheres and wash them with 0.05 - 0.2 M imidazole buffer solution. Then take the solid phase and resuspend it in imidazole buffer solution containing EDC, incubate it with shaking at 35 - 39 °C for 15 - 25 min. Then add amino-modified C2 in a ratio of 2:(1 - 2) (μg:pmol) of magnetic microspheres to the amount of amino-modified C2, and react with shaking at 35 - 39 °C for 40 - 60 min. After reacting with shaking at 35 - 39 °C for 40 - 60 min, take the solid phase and wash it with PBST buffer solution. After washing with PBST buffer solution, take the solid phase and add 8 - 12% BSA solution, shake it at 35 - 39 °C for 40 - 60 min, then take the solid phase and wash it with PBST buffer solution to obtain magnetic microsphere-C2 complex. Take the remaining self-assembled DNA nucleic acid strand dissociation solution after the reaction in step 2.3 and mix it with the magnetic microsphere-C2 complex, react with shaking at 35 - 39 °C for 40 - 60 min, then take the solid phase and wash it with PBST buffer solution.

[0067] 2.7 Chemiluminescence detection of ATP

[0068] Take 10 μL of tetrabutyl solution and mix it with the chemiluminescent biosensor for detecting ATP, transfer it to a measuring bottle, then add 90 μL of PG reagent, and immediately put it into a chemiluminescence detector for determination. The chemiluminescence signal is detected by a BPCL weak chemiluminescence instrument, displayed and recorded by the computer terminal connected to the instrument, and the chemiluminescence intensity is quantified by the peak value of the output signal.

[0069] 2.8 Performance evaluation of the chemiluminescent biosensor for detecting ATP

[0070] We first carried out a standard addition recovery experiment on ATP in human serum samples to evaluate the accuracy and precision of this method. Add different concentrations of ATP (5 ng / mL, 50 ng / mL, 100 ng / mL) to human serum samples respectively. Under the optimized conditions, the experimental steps are the same as above, and each group of experiments is carried out in parallel three times to calculate the recovery rate of the spiked samples. Secondly, in order to test the specific selectivity of this experimental method, we selected several substances similar to ATP (UTP, CTP, and GTP) for chemiluminescence detection. Among them, the concentrations of ATP, UTP, CTP, and GTP are all 10 μM, and other conditions remain unchanged to explore the influence of different substances on the chemiluminescence signal intensity.

[0071] 3 Experimental results

[0072] 3.1 Establishment of AFP standard curve

[0073] Under the optimized experimental conditions (4 μL magnetic beads, 1.5 μL C1, dilution factor 1:7000 SA-HRP), a series of AFP with different concentrations was detected by chemiluminescence using this sensor, and a chemiluminescence standard curve was established as shown in Figure 2 A in -1 ~10 ng·mL -1 . The results showed that in the range of AFP concentration from 0.01 ng·mL -1 to 10 ng·mL

[0074] 3.2 Performance evaluation of the chemiluminescent biosensor for detecting AFP

[0075] Under the optimized experimental conditions (4 μL magnetic beads, 1.5 μL C1, dilution factor 1:7000 SA-HRP), four analytes, CEA, IgG, PSA, and BSA, were selected to investigate the specificity of the chemiluminescent biosensor for detecting AFP. In the experiment, the above four analytes at a level of 10 ng / mL AFP were added to the chemiluminescent biosensor for detecting AFP respectively, and then chemiluminescence detection was carried out. As shown in Figure 3 A of

[0076] the results showed that the ΔCL signal value of the control group with added AFP increased significantly; while the signal values of the groups with added CEA, IgG, PSA, and BSA remained basically unchanged, and the ΔCL signal value was almost zero. It was speculated that the AFP aptamer could not recognize and specifically bind these four analytes. At the same time, when these interfering analogues were mixed with AFP, the ΔCL signal value could also increase significantly. It can be seen that the chemiluminescent biosensor for detecting AFP designed in the present invention has good selectivity for AFP.

[0077] Table 1 Results of the recovery test of spiked AFP in samples

[0078]

[0079] 3.3 Establishment of the ATP standard curve

[0080] Under the optimized experimental conditions (2 μL magnetic beads, 1.5 μL C2), a series of ATP with different concentrations were detected by chemiluminescence using this sensor, and a chemiluminescence standard curve was established as shown in Figure 2 B in. The results showed that in the range of ATP concentration from 5 nM to 100 nM, the ΔCL signal value had a very good linear relationship with the logarithm of ATP concentration, and the lowest detection limit was 0.5 nM (S / N = 3).

[0081] 3.4 Performance evaluation of the chemiluminescent biosensor for detecting ATP

[0082] Under the optimized experimental conditions (2 μL magnetic beads, 1.5 μL C2), three analytes, UTP, CTP, and GTP, were selected to investigate the specificity of the chemiluminescent biosensor for detecting ATP. In the experiment, the above three analytes at a level of 10 ng / mL AFP were added to the chemiluminescent biosensor for detecting ATP respectively, and then chemiluminescence detection was carried out. As shown in Figure 3 The results in B showed that the ΔCL signal value of the control group with added ATP increased significantly; while the signal values of the groups with added UTP, CTP, and GTP remained basically unchanged, and the ΔCL signal value was almost zero. It was speculated that the ATP aptamer could not recognize and specifically bind these three analytes. At the same time, when these interfering analogs were mixed with ATP, the ΔCL signal value could also increase significantly. Thus, it can be seen that the chemiluminescent biosensor for detecting ATP designed in the present invention has good selectivity for ATP.

[0083] On the other hand, a standard addition recovery experiment of ATP in human serum samples was carried out in the present invention to evaluate the precision of the chemiluminescent biosensor for detecting ATP. In the experiment, three different concentrations of ATP were added to the human serum samples diluted 50 times respectively, and then chemiluminescence detection was carried out by the chemiluminescent biosensor for detecting ATP. The results are shown in Table 2. The average recovery rate was between 98.56% and 100.24%, and the relative standard deviation was between 3.4% and 6.3% after three repeated determinations. Thus, it can be seen that the chemiluminescent biosensor for detecting ATP designed in the present invention has good precision and strong feasibility, and can be used for the quantitative analysis of ATP in actual samples.

[0084] Table 2 Results of the standard addition recovery test of ATP in the samples

[0085]

[0086] 4 Experimental conclusions

[0087] The present invention designs a rapid chemiluminescence detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor through nucleic acid aptamer technology and magnetic separation technology. For the detection of AFP, under optimized experimental conditions (4 μL magnetic beads, 1.5 μL Cl, dilution factor 1:7000 SA-HRP), within the range of 0.01 ng·mL to 10 ng·mL -1 there is a good linear relationship between the increase in chemiluminescence signal (ΔCL signal value, Y) and the measured AFP concentration (X), and the lowest detection limit is 1.8 pg·mL -1 (S / N = 3). The average spiked recovery rate of the samples is between 90.00% and 100.40%, and after three repeated measurements, the relative standard deviation is between 2.1% and 5.0%. For the detection of ATP, under optimized experimental conditions (2 μL magnetic beads, 1.5 μL C2), within the range of 5 nM to 100 nM, there is a good linear relationship between the increase in chemiluminescence signal (ΔCL signal value, Y) and the measured ATP concentration (X), and the lowest detection limit is 0.5 nM (S / N = 3). The average spiked recovery rate of the samples is between 98.56% and 100.24%, and after three repeated measurements, the relative standard deviation is between 3.4% and 6.3%. This method has high precision and high specificity, and can be used for the synchronous and highly sensitive rapid detection of AFP and ATP in actual samples. The present invention provides a new idea for the analysis and detection of AFP and ATP, and is of great significance for realizing the research and determination of multiple components.

[0088] Example 2

[0089] A rapid chemiluminescence detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor, comprising the following steps:

[0090] 1) Mix Apt1, Apt2, S1, B, and S2 evenly and react fully;

[0091] 2) Mix the product obtained in step 1) with AFP and ATP evenly and react fully;

[0092] 3) Fix amino-modified C1 on the surface of carboxyl magnetic microspheres through aminocarboxylic acid reaction to obtain a magnetic microsphere-C1 complex;

[0093] 4) Mix the product obtained in step 2) with the magnetic microsphere-C1 complex evenly, and take the solid phase after the reaction;

[0094] 5) Mix the solid phase obtained in step 4) with SA-HRP evenly and react fully to obtain a chemiluminescent biosensor for detecting AFP;

[0095] 6) Mix the chemiluminescent biosensor for detecting AFP with luminol-H 2 O2 Mix the reagents, and then detect the chemiluminescence CL value;

[0096] 7) Fix the amino-modified C2 on the surface of carboxyl magnetic microspheres through an aminocarboxylic reaction to obtain a magnetic microsphere-C2 complex;

[0097] 8) Mix the remaining solution from the reaction in step 4) with the magnetic microsphere-C2 complex, and after the reaction, take the solid phase to obtain the chemiluminescent biosensor for detecting ATP;

[0098] 9) Mix the chemiluminescent biosensor for detecting ATP with the PG reagent, and then detect the chemiluminescence CL value.

[0099] Based on the above technical solutions, the following conditions are met:

[0100] Step 1) includes the following steps: respectively take 6 μL of 100 μM Apt1, Apt2, S1, B, and S2 in a 1.5 mL centrifuge tube, then add 20 μL of BA buffer solution, mix well and incubate in a water bath at 80 - 100 °C for 4 - 6 min, and finally place it in a 4 °C refrigerator overnight for standby.

[0101] Step 2) includes the following steps: take 10 μL of 10 -8 μM AFP and 10 μL of 10 -8 μM ATP into the solution obtained in step 1), mix well and react with shaking at 35 - 39 °C for 40 - 60 min.

[0102] Step 3) includes the following steps: take magnetic microspheres, wash them with 0.05 - 0.2 M imidazole buffer solution, then take the solid phase and resuspend it in imidazole buffer solution containing EDC, incubate with shaking at 35 - 39 °C for 15 - 25 min, and then add amino-modified C1 at a ratio of 4:(1 - 2) (μg:pmol) of the amount of magnetic microspheres to amino-modified C1, and react with shaking at 35 - 39 °C for 40 - 60 min.

[0103] Step 3) also includes the following steps: after reacting with shaking at 35 - 39 °C for 40 - 60 min, take the solid phase and wash it with PBST buffer solution.

[0104] Step 3) also includes the following steps: after washing with PBST buffer solution, take the solid phase and add 8 - 12% BSA solution, shake at 35 - 39 °C for 40 - 60 min, and then take the solid phase and wash it with PBST buffer solution to obtain the magnetic microsphere-C1 complex.

[0105] Step 4) includes the following steps: Mix the product obtained in Step 2) with the magnetic microsphere-C1 complex, and after shaking and reacting at 35-39 °C for 40-60 min, take the solid phase and wash it with PBST buffer solution.

[0106] Step 5) includes the following steps: Mix 100 μL of SA-HRP with the product obtained in Step 4), and after shaking and reacting at 35-39 °C for 40-60 min, take the solid phase and wash it with PBST buffer solution to obtain the chemiluminescent biosensor for detecting AFP.

[0107] Step 6) includes the following steps: Mix the product obtained in Step 5) with 50 μL of luminol solution, transfer it to a measuring bottle, and then add 50 μL of hydrogen peroxide solution to detect the chemiluminescent CL value.

[0108] Step 7) includes the following steps: Take the magnetic microspheres, wash them with 0.05-0.2 M imidazole buffer solution, then take the solid phase and resuspend it in the imidazole buffer solution containing EDC, incubate it with shaking at 35-39 °C for 15-25 min, and then add amino-modified C2 in a ratio of 2:(1-2) (μg:pmol) of the amount of magnetic microspheres to amino-modified C2, and react with shaking at 35-39 °C for 40-60 min.

[0109] Step 7) further includes the following steps: After reacting with shaking at 35-39 °C for 40-60 min, take the solid phase and wash it with PBST buffer solution.

[0110] Step 7) further includes the following steps: After washing with PBST buffer solution, take the solid phase and add 8-12% BSA solution, shake it at 35-39 °C for 40-60 min, and then take the solid phase and wash it with PBST buffer solution to obtain the magnetic microsphere-C2 complex.

[0111] Step 8) includes the following steps: Mix the remaining solution of the reaction in Step 4) with the magnetic microsphere-C2 complex, and after reacting with shaking at 35-39 °C for 40-60 min, take the solid phase and wash it with PBST buffer solution.

[0112] Step 9) includes the following steps: Mix the product obtained in Step 8) with 10 μL of tetrabutyl solution, transfer it to a measuring bottle, and then add 90 μL of PG reagent to detect the chemiluminescent CL value.

[0113] Example 3

[0114] A chemiluminescent rapid detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor includes the following steps:

[0115] 1) Mix single-stranded Apt1, Apt2, S1, B, and S2 evenly and react fully;

[0116] 2) Mix the product obtained in step 1) with AFP and ATP, and react fully.

[0117] 4) Fix the amino-modified C1 on the surface of the carboxyl magnetic microspheres through aminocarboxylic acid reaction to obtain a magnetic microsphere-C1 complex.

[0118] 7) Mix the product obtained in step 2) with the magnetic microsphere-C1 complex, and take the solid phase after the reaction.

[0119] 10) Mix the solid phase obtained in step 4) with SA-HRP, and react fully to obtain a chemiluminescent biosensor for detecting AFP.

[0120] 13) Mix the chemiluminescent biosensor for detecting AFP with luminol-H 2 O 2 reagent, and then detect the chemiluminescent CL value.

[0121] 20) Fix the amino-modified C2 on the surface of the carboxyl magnetic microspheres through aminocarboxylic acid reaction to obtain a magnetic microsphere-C2 complex.

[0122] 23) Mix the remaining solution of the reaction in step 4) with the magnetic microsphere-C2 complex, and take the solid phase after the reaction to obtain a chemiluminescent biosensor for detecting ATP.

[0123] 26) Mix the chemiluminescent biosensor for detecting ATP with PG reagent, and then detect the chemiluminescent CL value.

[0124] Based on the above technical solutions, the following conditions are met:

[0125] Step 1) includes the following steps: Take 6 μL of 100 μM Apt1, Apt2, S1, B, and S2 in a 1.5 mL centrifuge tube respectively, then add 20 μL of BA buffer solution, mix well and incubate in a water bath at 80 - 100 °C for 4 - 6 min, and finally place it in a 4 °C refrigerator overnight for standby.

[0126] Step 2) includes the following steps: Take 10 μL of 10 -8 μM AFP and 10 μL of 10 -8 μM ATP into the solution obtained in step 1), mix well and react with shaking at 35 - 39 °C for 40 - 60 min.

[0127] Step 3) includes the following steps: Take magnetic microspheres, wash them with 0.05 - 0.2 M imidazole buffer solution, then take the solid phase and resuspend it in imidazole buffer solution containing EDC, incubate with shaking at 35 - 39 °C for 15 - 25 min, and then add amino-modified C1 at a ratio of the amount of magnetic microspheres to amino-modified C1 of 4:(1 - 2) (μg:pmol), and react with shaking at 35 - 39 °C for 40 - 60 min.

[0128] Step 3) also includes the following steps: After reacting with shaking at 35 - 39 °C for 40 - 60 min, take the solid phase and wash it with PBST buffer solution.

[0129] Step 3) also includes the following steps: After washing with PBST buffer solution, take the solid phase and add 8 - 12% BSA solution, shake at 35 - 39 °C for 40 - 60 min, and then take the solid phase and wash it with PBST buffer solution to obtain the magnetic microsphere-C1 complex.

[0130] Step 4) includes the following steps: Mix the product obtained in Step 2) with the magnetic microsphere-C1 complex, react with shaking at 35 - 39 °C for 40 - 60 min, and then take the solid phase and wash it with PBST buffer solution.

[0131] Step 5) includes the following steps: Mix 100 μL SA-HRP with the product obtained in Step 4), react with shaking at 35 - 39 °C for 40 - 60 min, and then take the solid phase and wash it with PBST buffer solution to obtain the chemiluminescent biosensor for detecting AFP.

[0132] Step 6) includes the following steps: Mix the product obtained in Step 5) with 50 μL luminol solution, transfer it to a measuring bottle, and then add 50 μL hydrogen peroxide solution to detect the chemiluminescent CL value.

[0133] Step 7) includes the following steps: Take magnetic microspheres, wash them with 0.05 - 0.2 M imidazole buffer solution, then take the solid phase and resuspend it in imidazole buffer solution containing EDC, incubate with shaking at 35 - 39 °C for 15 - 25 min, and then add amino-modified C2 at a ratio of the amount of magnetic microspheres to amino-modified C2 of 2:(1 - 2) (μg:pmol), and react with shaking at 35 - 39 °C for 40 - 60 min.

[0134] Step 7) also includes the following steps: After reacting with shaking at 35 - 39 °C for 40 - 60 min, take the solid phase and wash it with PBST buffer solution.

[0135] Step 7) further includes the following steps: After washing with PBST buffer solution, take the solid phase and add it to a 8-12% BSA solution, shake at 35-39 °C for 40-60 min, and then take the solid phase and wash it with PBST buffer solution to obtain the magnetic microsphere-C2 complex.

[0136] Step 8) includes the following steps: Mix the remaining solution from the reaction in Step 4) with the magnetic microsphere-C2 complex, shake and react at 35-39 °C for 40-60 min, and then take the solid phase and wash it with PBST buffer solution.

[0137] Step 9) includes the following steps: Mix the product obtained in Step 8) with 10 μL of tetrabutyl solution, transfer it to a measuring flask, and then add 90 μL of PG reagent to detect the chemiluminescence CL value.

[0138] Example 4

[0139] A chemiluminescence rapid detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor includes the following steps:

[0140] 1) Mix Apt1, Apt2, S1, B, and S2 evenly and react fully;

[0141] 2) Mix the product obtained in Step 1) with AFP and ATP evenly and react fully;

[0142] 3) Fix the amino-modified C1 on the surface of the carboxyl magnetic microspheres through aminocarboxylic acid reaction to obtain the magnetic microsphere-C1 complex;

[0143] 4) Mix the product obtained in Step 2) with the magnetic microsphere-C1 complex, and take the solid phase after the reaction;

[0144] 5) Mix the solid phase obtained in Step 4) with SA-HRP evenly and react fully to obtain the chemiluminescence biosensor for detecting AFP;

[0145] 6) Mix the chemiluminescence biosensor for detecting AFP with luminol-H 2 O 2 reagent evenly, and then detect the chemiluminescence CL value;

[0146] 7) Fix the amino-modified C2 on the surface of the carboxyl magnetic microspheres through aminocarboxylic acid reaction to obtain the magnetic microsphere-C2 complex;

[0147] 8) Mix the remaining solution from the reaction in Step 4) with the magnetic microsphere-C2 complex, and take the solid phase after the reaction to obtain the chemiluminescence biosensor for detecting ATP;

[0148] 9) Mix the chemiluminescence biosensor for detecting ATP with PG reagent evenly, and then detect the chemiluminescence CL value.

[0149] On the basis of the above technical solutions, the following conditions are satisfied:

[0150] Step 1) includes the following steps: Take 6 μL of 100 μM Apt1, Apt2, S1, B, and S2 in a 1.5 mL centrifuge tube, then add 20 μL of BA buffer solution, mix well, incubate in a water bath at 80 - 100 °C for 4 - 6 min, and finally place it in a 4 °C refrigerator overnight for standby.

[0151] Step 2) includes the following steps: Take 10 μL of 10 -8 μM AFP and 10 μL of 10 -8 μM ATP into the solution obtained in Step 1), mix well, and react with shaking at 35 - 39 °C for 40 - 60 min.

[0152] Step 3) includes the following steps: Take magnetic microspheres, wash them with 0.05 - 0.2 M imidazole buffer solution, then take the solid phase and resuspend it in imidazole buffer solution containing EDC, incubate with shaking at 35 - 39 °C for 15 - 25 min, and then add amino-modified C1 at a ratio of the amount of magnetic microspheres to amino-modified C1 of 4∶(1 - 2) (μg∶pmol), and react with shaking at 35 - 39 °C for 40 - 60 min.

[0153] Step 3) also includes the following steps: After reacting with shaking at 35 - 39 °C for 40 - 60 min, take the solid phase and wash it with PBST buffer solution.

[0154] Step 3) also includes the following steps: After washing with PBST buffer solution, take the solid phase and add 8 - 12% BSA solution, shake at 35 - 39 °C for 40 - 60 min, then take the solid phase and wash it with PBST buffer solution to obtain the magnetic microsphere - C1 complex.

[0155] Step 4) includes the following steps: Mix the product obtained in Step 2) with the magnetic microsphere - C1 complex, react with shaking at 35 - 39 °C for 40 - 60 min, then take the solid phase and wash it with PBST buffer solution.

[0156] Step 5) includes the following steps: Mix 100 μL of SA - HRP with the product obtained in Step 4), react with shaking at 35 - 39 °C for 40 - 60 min, then take the solid phase and wash it with PBST buffer solution to obtain the chemiluminescent biosensor for detecting AFP.

[0157] Step 6) includes the following steps: Mix the product obtained in Step 5) with 50 μL of luminol solution, transfer it to a measuring bottle, then add 50 μL of hydrogen peroxide solution, and detect the chemiluminescence CL value.

[0158] Step 7) includes the following steps: Take magnetic microspheres, wash them with 0.05 - 0.2 M imidazole buffer solution, then take the solid phase and resuspend it in imidazole buffer solution containing EDC, incubate with shaking at 35 - 39 °C for 15 - 25 min, and then add amino-modified C2 at a ratio of the amount of magnetic microspheres to amino-modified C2 of 2:(1 - 2) (μg:pmol), and react with shaking at 35 - 39 °C for 40 - 60 min.

[0159] Step 7) further includes the following steps: After reacting with shaking at 35 - 39 °C for 40 - 60 min, take the solid phase and wash it with PBST buffer solution.

[0160] Step 7) further includes the following steps: After washing with PBST buffer solution, take the solid phase and add 8 - 12% BSA solution, shake at 35 - 39 °C for 40 - 60 min, and then take the solid phase and wash it with PBST buffer solution to obtain the magnetic microsphere-C2 complex.

[0161] Step 8) includes the following steps: Mix the remaining solution from the reaction in Step 4) with the magnetic microsphere-C2 complex, react with shaking at 35 - 39 °C for 40 - 60 min, and then take the solid phase and wash it with PBST buffer solution.

[0162] Step 9) includes the following steps: Mix the product obtained in Step 8) with 10 μL of tetrabutyl solution, transfer it to a measuring flask, and then add 90 μL of PG reagent to detect the chemiluminescence CL value.

[0163] Example 5

[0164] A chemiluminescence rapid detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor, which can be further extended to other detection methods, includes the following steps:

[0165] 1) Mix Apt1, Apt2, S1, B, and S2 evenly and react fully;

[0166] 2) Mix the product obtained in Step 1) with AFP and ATP evenly and react fully;

[0167] 3) Fix amino-modified C1 on the surface of carboxyl magnetic microspheres through aminocarboxylic acid reaction to obtain a magnetic microsphere-C1 complex;

[0168] 4) Mix the product obtained in Step 2) with the magnetic microsphere-C1 complex, and take the solid phase after the reaction;

[0169] 5) Mix the solid phase obtained in Step 4) with SA-Au evenly and react fully to obtain a chemiluminescent biosensor for detecting AFP;

[0170] 6) The chemiluminescent biosensor for detecting AFP and luminol-H2 O 2 Mix the reagents evenly and then detect the chemiluminescence CL value;

[0171] 7) Fix the amino-modified C2 on the surface of carboxyl magnetic microspheres through the aminocarboxylic acid reaction to obtain a magnetic microsphere-C2 complex;

[0172] 8) Mix the remaining solution from the reaction in step 4) with the magnetic microsphere-C2 complex, take the solid phase after the reaction, and thus obtain a chemiluminescent biosensor for detecting ATP;

[0173] 9) Mix the chemiluminescent biosensor for detecting ATP with the PG reagent and then detect the chemiluminescence CL value.

[0174] Based on the above technical solutions, the following conditions are satisfied:

[0175] Step 1) includes the following steps: respectively take 6 μL of 100 μM Apt1, Apt2, S1, B, and S2 in a 1.5 mL centrifuge tube, then add 20 μL of BA buffer solution, mix evenly and incubate in a water bath at 80-100 °C for 4-6 min, and finally place it in a 4 °C refrigerator overnight for standby.

[0176] Step 2) includes the following steps: take 10 μL of 10 -8 μM AFP and 10 μL of 10 -8 μM ATP into the solution obtained in step 1), mix evenly and react with shaking at 35-39 °C for 40-60 min.

[0177] Step 3) includes the following steps: take magnetic microspheres, wash them with 0.05-0.2 M imidazole buffer solution, then take the solid phase and resuspend it in imidazole buffer solution containing EDC, incubate with shaking at 35-39 °C for 15-25 min, and then add amino-modified C1 at a ratio of 4:(1-2) (μg:pmol) of the amount of magnetic microspheres to amino-modified C1, and react with shaking at 35-39 °C for 40-60 min.

[0178] Step 3) also includes the following steps: after reacting with shaking at 35-39 °C for 40-60 min, take the solid phase and wash it with PBST buffer solution.

[0179] Step 3) also includes the following steps: after washing with PBST buffer solution, take the solid phase and add 8-12% BSA solution, shake at 35-39 °C for 40-60 min, and then take the solid phase and wash it with PBST buffer solution to obtain a magnetic microsphere-C1 complex.

[0180] Step 4) includes the following steps: Mix the product obtained in step 2) with the magnetic microsphere-C1 complex, and after oscillating and reacting at 35-39 °C for 40-60 min, take the solid phase and wash it with PBST buffer solution.

[0181] Step 5) includes the following steps: Mix 100 μL of SA-Au with the product obtained in step 4), and after oscillating and reacting at 35-39 °C for 40-60 min, take the solid phase and wash it with PBST buffer solution, thus obtaining the chemiluminescent biosensor for detecting AFP.

[0182] Step 6) includes the following steps: Mix the product obtained in step 5) with 50 μL of luminol solution, transfer it to a measuring flask, and then add 50 μL of hydrogen peroxide solution to detect the chemiluminescent CL value.

[0183] Step 7) includes the following steps: Take the magnetic microspheres, wash them with 0.05-0.2 M imidazole buffer solution, then take the solid phase and resuspend it in the imidazole buffer solution containing EDC, oscillate and incubate at 35-39 °C for 15-25 min, and then add amino-modified C2 in a ratio of 2:(1-2) (μg:pmol) of the amount of magnetic microspheres to amino-modified C2, and oscillate and react at 35-39 °C for 40-60 min.

[0184] Step 7) further includes the following steps: After oscillating and reacting at 35-39 °C for 40-60 min, take the solid phase and wash it with PBST buffer solution.

[0185] Step 7) further includes the following steps: After washing with PBST buffer solution, take the solid phase and add 8-12% BSA solution, oscillate at 35-39 °C for 40-60 min, and then take the solid phase and wash it with PBST buffer solution to obtain the magnetic microsphere-C2 complex.

[0186] Step 8) includes the following steps: Mix the remaining solution of the reaction in step 4) with the magnetic microsphere-C2 complex, and after oscillating and reacting at 35-39 °C for 40-60 min, take the solid phase and wash it with PBST buffer solution.

[0187] Step 9) includes the following steps: Mix the product obtained in step 8) with 10 μL of tetrabutyl solution, transfer it to a measuring flask, and then add 90 μL of PG reagent to detect the chemiluminescent CL value.

[0188] Example 6

[0189] A chemiluminescent rapid detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor, including the following steps:

[0190] 1) Mix Apt1, Apt2, S1, B, and S2 evenly and react fully;

[0191] 2) Mix the product obtained in step 1) with AFP and ATP, and react fully.

[0192] 3) Fix the amino-modified C1 on the surface of the carboxyl magnetic microspheres through an aminocarboxylic reaction to obtain a magnetic microsphere-C1 complex.

[0193] 4) Mix the product obtained in step 2) with the magnetic microsphere-C1 complex, and take the solid phase after the reaction.

[0194] 5) Mix the solid phase obtained in step 4) with SA-HRP and react fully to obtain a chemiluminescent biosensor for detecting AFP.

[0195] 6) Mix the chemiluminescent biosensor for detecting AFP with luminol-H 2 O 2 reagent, and then detect the chemiluminescence CL value.

[0196] 7) Fix the amino-modified C2 on the surface of the carboxyl magnetic microspheres through an aminocarboxylic reaction to obtain a magnetic microsphere-C2 complex.

[0197] 8) Mix the remaining solution from the reaction in step 4) with the magnetic microsphere-C2 complex, and take the solid phase after the reaction to obtain a chemiluminescent biosensor for detecting ATP.

[0198] 9) Mix the chemiluminescent biosensor for detecting ATP with PG reagent, and then detect the chemiluminescence CL value.

[0199] The above has described the embodiments of the present invention in detail, but the content described is only the preferred embodiments of the present invention and is not used to limit the present invention. Any modifications, equivalent replacements, and improvements made within the scope of the application of the present invention shall be included within the protection scope of the present invention.

Claims

1. A rapid chemiluminescence detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor, characterized in that, it involves adenosine triphosphate aptamer Apt1, alpha-fetoprotein aptamer Apt2, signal DNA strand 1 S1, bridge DNA strand B, signal DNA strand 2 S2, DNA strand C1 complementary to S1, and DNA strand C2 complementary to S2; among them, ATP can specifically recognize Apt1, AFP can specifically recognize Apt2, S1 can base-complementary pair with C1, S2 can base-complementary pair with C2, and B is mainly used to assist in constructing the self-assembled DNA nucleic acid strand. The amino groups at the ends of C1 and C2 can bind to the carboxyl-modified magnetic microspheres through amide bonds; during the process, streptavidin SA and horseradish peroxidase HRP-crosslinked horseradish peroxidase-labeled streptavidin SA-HRP and chemiluminescence reagent benzoylformaldehyde PG are also used; this method includes the following steps: 1) Mix Apt1, Apt2, S1, B, and S2 evenly and react fully; 2) Mix the product obtained in step 1) with AFP and ATP evenly and react fully; 3) Fix the amino-modified C1 on the surface of the carboxyl magnetic microspheres through an aminocarboxylic reaction to obtain a magnetic microsphere-C1 complex; 4) Mix the product obtained in step 2) with an excessive amount of magnetic microsphere-C1 complex evenly, and take the solid phase after the reaction; 5) Mix the solid phase obtained in step 4) with SA-HRP evenly and react fully to obtain a chemiluminescent biosensor for detecting AFP; 6) Mix the chemiluminescent biosensor for detecting AFP with luminol-H 2 O 2 reagent, and then detect the chemiluminescence CL value; 7) Fix the amino-modified C2 on the surface of the carboxyl magnetic microspheres through an aminocarboxylic reaction to obtain a magnetic microsphere-C2 complex; 8) Mix the remaining solution from the reaction in step 4) with an excessive amount of magnetic microsphere-C2 complex evenly, and take the solid phase after the reaction to obtain a chemiluminescent biosensor for detecting ATP; 9) Mix the chemiluminescent biosensor for detecting ATP with the PG reagent, and then detect the chemiluminescence CL value; Step 1) includes the following steps: respectively take equal amounts of 6 µL and 100 µmol / L of Apt1, Apt2, S1, B, and S2 in a 1.5 mL centrifuge tube, then add 20 µL of BA buffer solution, mix evenly and incubate in a water bath at 80-100 °C for 4-6 min; Step 2) includes the following steps: Take 10 µL of 10 -8 µmol / L AFP and 10 µL of 10 -8 µmol / L ATP and add them to the solution obtained in step 1). After mixing, react with shaking at 35-39 °C for 40-60 min; Step 3) includes the following steps: take magnetic microspheres, wash them with 0.05-0.2 M imidazole buffer solution, then take the solid phase and resuspend it in imidazole buffer solution containing EDC, shake and incubate at 35-39 °C for 15-25 min, and then add the amino-modified C1 in a ratio of 4 µg:(1-2) pmol of the amount of magnetic microspheres to amino-modified C1, shake and react at 35-39 °C for 40-60 min; after shaking and reacting at 35-39 °C for 40-60 min, take the solid phase and wash it with PBST buffer solution; after washing with PBST buffer solution, take the solid phase and add 8-12% BSA solution, shake at 35-39 °C for 40-60 min, and then take the solid phase and wash it with PBST buffer solution to obtain the magnetic microsphere-C1 complex.

2. A rapid chemiluminescence detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor according to claim 1, characterized in that, Step 4) includes the following steps: Mix the product obtained in Step 2) with the magnetic microsphere-C1 complex, shake and react at 35-39 °C for 40-60 min, and then take the solid phase and wash it with PBST buffer solution.

3. A rapid chemiluminescence detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor according to claim 1, characterized in that, Step 5) includes the following steps: Mix 100 µL of SA-HRP with a concentration of 1:8000-1:6000 with the solid phase obtained in Step 4), shake and react at 35-39 °C for 40-60 min, and then take the solid phase and wash it with PBST buffer solution.

4. A rapid chemiluminescence detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor according to claim 1, characterized in that, Step 6) includes the following steps: Mix the product obtained in Step 5) with 50 µL of luminol solution, transfer it to a measuring flask, and then add 50 µL of hydrogen peroxide solution to detect the chemiluminescence CL value.

5. A rapid chemiluminescence detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor according to claim 1, characterized in that, Step 7) includes the following steps: Take magnetic microspheres, wash them with 0.05-0.2 M imidazole buffer solution, and then resuspend the solid phase in imidazole buffer solution containing EDC, shake and incubate at 35-39 °C for 15-25 min, and then add amino-modified C2 in a ratio of 2 µg:(1-2) pmol of magnetic microspheres to amino-modified C2, shake and react at 35-39 °C for 40-60 min; after shaking and reacting at 35-39 °C for 40-60 min, take the solid phase and wash it with PBST buffer solution; after washing with PBST buffer solution, take the solid phase and add 8-12% BSA solution, shake at 35-39 °C for 40-60 min, and then take the solid phase and wash it with PBST buffer solution to obtain the magnetic microsphere-C2 complex.

6. A rapid chemiluminescence detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor according to claim 1, characterized in that, Step 8) includes the following steps: Mix the remaining solution from the reaction in Step 4) with the magnetic microsphere-C2 complex, shake and react at 35-39 °C for 40-60 min, and then take the solid phase and wash it with PBST buffer solution.

7. A rapid chemiluminescence detection method for multiple tumor markers based on a self-assembled DNA nucleic acid strand sensor according to claim 1, characterized in that, Step 9) includes the following steps: Mix the product obtained in Step 8) with 10 µL of tetrabutyl solution, transfer it to a measuring flask, and then add 90 µL of PG reagent to detect the chemiluminescence CL value.

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