Probe group for detecting soluble PD-L1 in peripheral blood and application thereof

Abstract

The invention relates to a probe set for detecting soluble PD-L1 in peripheral blood and application thereof.The probe set comprises a recognition probe and a substrate probe, the recognition probe is formed by hybridization of an aptamer and an activator ssDNA, the substrate probe comprises modified carboxyl magnetic beads and streptomycin avidin modified horse radish peroxidase, and the substrate probe is formed by hybridization of the aptamer and the activator ssDNA. The modified carboxyl magnetic beads are obtained by modifying carboxyl magnetic beads by ssDNA modified with amino and biotin. The HRP-ssDNA signal probe is innovatively connected to the surface of the modified carboxyl magnetic bead, a large number of HRP-ssDNA signal report probes are loaded on the surface of the modified carboxyl magnetic bead, the HRP-ssDNA signal probes on the surface of the modified carboxyl magnetic bead are circularly sheared by activated Cas12a enzyme, the concentration of the report probes in a limited range can be greatly improved by the modified carboxyl magnetic bead, and the detection sensitivity is high. The problem of low reaction efficiency caused by free diffusion is solved, the shearing efficiency of the Cas12a enzyme can be remarkably improved, and single target input and mass product output are realized. The chemiluminiscence sensor prepared from the probe is used for detecting sPD-L1 in serum, is high in sensitivity and specificity, and overcomes the defect of sensitivity in the prior art.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a probe set for detecting soluble PD-L1 in peripheral blood and its application. Background Art

[0002] The rise of tumor immunotherapy, especially immune checkpoint (ICB) therapy, has significantly improved the treatment prospects for patients with advanced tumors. To date, anti-programmed cell death ligand 1 (PD-L1) immunohistochemistry (IHC) on tissue sections is the only verified companion diagnostic method in first-line immunotherapy for advanced and metastatic cancers, especially non-small cell lung cancer (NSCLC). However, this method has limitations such as invasiveness, tumor tissue heterogeneity, and difficulty in dynamic monitoring. Therefore, exploring new biomarkers and detection technologies is crucial to achieve accurate prediction of personalized immunotherapy.

[0003] Soluble PD-L1 (sPD-L1) in peripheral blood is derived from metalloproteinase cleavage and splice variants of membrane-bound PD-L1, retains the PD-1 binding domain, has immunosuppressive function, can interfere with PD-1 on T cells, inhibit immune response, and promote immune escape of tumor cells. In addition, sPD-L1 can also act as a "bait" to mediate resistance to PD-L1 blocking antibodies, and is therefore considered a key biomarker for predicting the efficacy of immune checkpoint inhibitors. Studies have shown that compared with healthy individuals, sPD-L1 levels are significantly increased in patients with various tumors, and are associated with poor survival rates in a variety of tumor types (such as malignant melanoma, gastric cancer, lung cancer, pancreatic cancer, liver cancer, breast cancer, biliary tract cancer, renal cell carcinoma, and colorectal cancer).

[0004] In the related art, the ELISA method is generally used to detect soluble PD-L1 (sPD-L1) in peripheral blood. However, the ELISA method for detecting soluble PD-L1 (sPD-L1) in peripheral blood has the following defects: the detection limit is at the ng / mL level, which makes it difficult to detect low-abundance sPD-L1 in the early clinical stage or during the medication remission period. At the same time, the antibodies used in the ELISA method are expensive and not conducive to preservation. Summary of the invention

[0005] In view of the shortcomings of the prior art described above, the present invention provides a probe for detecting soluble PD-L1 in peripheral blood, and a preparation method and application thereof, in order to solve the technical problems that the detection limit of the ELISA method for detecting soluble PD-L1 (sPD-L1) in peripheral blood is at the ng / mL level, which makes it difficult to detect low-abundance sPD-L1 in the early clinical stage or during the medication remission period, and the antibodies used in the ELISA method are expensive and not conducive to storage.

[0006] To achieve the above objectives, the scheme of this application is as follows:

[0007] In the first aspect, the present invention provides a probe group for detecting soluble PD-L1 in peripheral blood, wherein the probe group includes a recognition probe and a substrate probe (MB@ssDNA-HRP), wherein the recognition probe is formed by hybridization of an aptamer and an activator ssDNA, and the substrate probe includes modified carboxyl magnetic beads and horseradish peroxidase (HorseradishPeroxidase, HRP) modified with streptavidin, and the modified carboxyl magnetic beads are obtained by modifying the carboxyl magnetic beads with ssDNA modified with amino groups (-NH2) and biotin.

[0008] Optionally, the amino group is modified at the 5' end of the ssDNA, and the amino group of the ssDNA reacts and binds to the carboxyl magnetic beads.

[0009] Optionally, the biotin is modified at the 3' end of the ssDNA.

[0010] Optionally, the aptamer can specifically recognize soluble anti-programmed death ligand 1 (sPD-L1).

[0011] Optionally, the activator ssDNA can activate the Cas12a enzyme.

[0012] Optionally, the nucleotide sequence of the aptamer is shown as SEQ ID NO.1, and the nucleotide sequence of the activator ssDNA is shown as SEQ ID NO.2.

[0013] Optionally, the nucleotide sequence of the ssDNA is shown in SEQ ID NO.4.

[0014] Optionally, the recognition probe is prepared by a method comprising the following steps:

[0015] The aptamer is mixed with the activator ssDNA and a buffer, and then incubated together, and cooled to obtain the recognition probe;

[0016] Optionally, the molar ratio of the aptamer to the activator ssDNA is 1-2:1-2.

[0017] Optionally, the buffer is a PBS buffer containing magnesium chloride and having a pH of 7.2-7.5.

[0018] Optionally, in the PBS buffer, the concentration of magnesium chloride is 0.5-0.6 mM.

[0019] Optionally, the co-incubation temperature is 90-100° C., and the co-incubation time is 4-6 min.

[0020] Optionally, the temperature is lowered to room temperature at a cooling rate of 0.05-0.2°C / min.

[0021] Optionally, the substrate probe is prepared by a method comprising the following steps:

[0022] A carbodiimide hydrochloride solution and an N-hydroxysuccinimide (NHS) solution are added to a carboxyl magnetic bead solution, activated, and magnetically separated. A biotinylation reagent is added to the obtained precipitate, wherein the biotinylation reagent includes an amino group and biotin. The reaction is performed, and magnetic separation is performed for a second time. A blocking solution is added to the obtained precipitate, and the mixture is incubated and magnetically separated for a third time. Streptavidin coupled with horseradish peroxidase is added to the obtained precipitate, and the reaction is performed for a second time. The substrate probe is prepared.

[0023] Optionally, the carbodiimide hydrochloride is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

[0024] Optionally, the mass ratio of the carbodiimide hydrochloride to the N-hydroxysuccinimide is 8-12:8-12.

[0025] Optionally, the activation temperature is 20-30° C., and the activation time is 0.5-1.5 h.

[0026] Optionally, during the activation process, the rotation speed is 500-1000 rpm.

[0027] Optionally, the mass ratio of the carboxyl magnetic beads to the biotinylation reagent is 8-12; 0.05-0.06.

[0028] Optionally, the mass ratio of the carboxyl magnetic beads to the streptavidin coupled with horseradish peroxidase is 8-12:0.0005-0.001.

[0029] Optionally, the reaction temperature is 20-30° C., and the reaction time is 0.5-1.5 h.

[0030] Optionally, during the reaction process, the rotation speed is 500-1000 rpm.

[0031] Optionally, the incubation temperature is 20-30° C., and the incubation time is 0.5-1.5 h.

[0032] Optionally, the temperature of the secondary reaction is 20-30° C., and the duration of the secondary reaction is 20-40 min.

[0033] Optionally, during the secondary reaction, the rotation speed is 500-1000 rpm.

[0034] In a second aspect, the present application also provides a chemiluminescent sensor for detecting soluble anti-programmed cell death ligand 1 in peripheral blood, wherein the chemiluminescent sensor comprises the probe set as described above.

[0035] Optionally, the chemiluminescent sensor also includes a Cas12a enzyme.

[0036] In a third aspect, the present application also provides use of the probe set as described above or the chemiluminescent sensor as described above in the preparation of a kit for detecting soluble anti-programmed cell death ligand 1 in peripheral blood.

[0037] In a fourth aspect, the present application also provides a kit for detecting soluble anti-programmed cell death ligand 1 in peripheral blood, wherein the kit comprises the probe set as described above or the chemiluminescent sensor as described above.

[0038] As described above, the probe set for detecting soluble PD-L1 in peripheral blood and its application of the present application have the following beneficial effects:

[0039] The present application designs a probe set for detecting soluble anti-programmed cell death ligand 1 (sPD-L1) in peripheral blood, the probe set comprising a substrate probe (MB@ssDNA-HRP) and a recognition probe, the substrate probe comprising modified carboxyl magnetic beads and streptavidin-modified horseradish peroxidase (Horseradish Peroxidase, HRP), wherein the modified carboxyl magnetic beads are obtained by modifying the carboxyl magnetic beads with ssDNA modified with amino (-NH2) and biotin (Biotin), and the substrate probe can be used as a non-specific cleavage substrate of Cas12a protein (i.e., Cas12a enzyme); the recognition probe is a nucleic acid probe capable of recognizing soluble PD-L1, which is composed of a nucleic acid aptamer (Apt) capable of specifically recognizing soluble PD-L1 and an "activator" single-stranded nucleic acid (activator) of the Cas12a protein. MBs@ssDNA-HRP is complementary to ssDNA. When soluble (i.e., Soluble) PD-L1 is present, Apt recognizes sPD-L1 and undergoes conformational changes, releasing "activator ssDNA". The "activator ssDNA" complementarily binds to the CRISPR RNA (crRNA) of the Cas12a protein, activates the nonspecific cleavage activity of Cas12a, shears the ssDNA on MBs@ssDNA-HRP, and releases horseradish peroxidase (HRP) into the free homogeneous solution. After magnetic separation, luminol (i.e., Luminol) and hydrogen peroxide solution are added to the supernatant, and the generated chemiluminescent signal is detected. The soluble PD-L1 in the solution can be quantitatively detected through the output of the chemiluminescent signal.

[0040] The present application innovatively connects HRP-ssDNA signal probes to the surface of modified carboxyl magnetic beads, loads a large number of HRP-ssDNA signal reporter probes on the surface of modified carboxyl magnetic beads, and the activated Cas12a enzyme cyclically shears the HRP-ssDNA signal probes on the surface of the magnetic beads. At the same time, the modified carboxyl magnetic beads can greatly increase the concentration of reporter probes in the confined area, overcome the problem of low reaction efficiency caused by free diffusion, and significantly improve the shearing efficiency of the Cas12a enzyme, so as to simply realize single target input and large-scale product output.

[0041] The chemiluminescent sensor made of the probe of the present application is used to detect sPD-L1 in serum, which has high sensitivity and strong specificity, making up for the shortcomings of the sensitivity of the existing detection technology. It not only helps the individualized treatment of tumor patients, but also can effectively monitor the prognosis of immunotherapy.

[0042] This application uses a recognition probe composed of a nucleic acid aptamer and a Cas12a "activator" to identify the target protein. Compared with antibodies, nucleic acid aptamers are not only cheap, easy to modify, and easy to store, but also can recognize targets with high specificity and high affinity.

[0043] The substrate probe MB@ssDNA-HRP of the present application concentrates the output signal in a limited range, and the activated Cas12a protein can circulate and shear ssDNA around the probe, releasing more HRP, so that the output signal is further enhanced.

[0044] Different from detecting PD-L1 on tissue sections, the chemiluminescent sensor made of the probe of the present application is used to detect sPD-L1 in serum, which is non-invasive / minimally invasive and has higher patient acceptance. At the same time, due to the easy availability of blood samples, effective dynamic monitoring can be performed during the treatment process.

[0045] In the prior art, the ELSIA method is used to detect sPD-L1 in serum, and the detection time is about 165 minutes. However, the chemiluminescence sensor made of the probe of the present application is used to detect sPD-L1 in serum, and the detection time is about 50 minutes. The present application significantly shortens the reaction time and improves the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a schematic diagram of the principle of this application;

[0047] Figure 2 This is a process flow chart for preparing the substrate probe of the present application;

[0048] Figure 3 This is a colorimetric characterization result diagram of the substrate probe of the present application;

[0049] Figure 4Schematic diagram of the recognition probe in the chemiluminescent sensor of the present application, MFE proxy structure represents the minimum free energy proxy structure, and Free energy of the secondary structure represents the free energy of the secondary structure;

[0050] Figure 5 This is the chemiluminescent signal diagram in feasibility verification. Fold means multiple, Without means no, With means yes, and CL intensity means the intensity of chemiluminescent signal, the same below;

[0051] Figure 6 This is the result of chemiluminescence signal analysis of samples with different concentrations in the sensitivity test. Wavelength indicates wavelength.

[0052] Figure 7 It is a calibration curve diagram of different concentrations of sPD-L1 relative to the chemiluminescence signal value (i.e., linear fitting curve);

[0053] Figure 8 This is a graph of specificity test results, and Blank represents the blank group. DETAILED DESCRIPTION

[0054] The present invention is further described below through specific examples, but it should be pointed out that the specific material ratios, process conditions and results described in the embodiments of the present invention are only used to illustrate the present invention and cannot be used to limit the scope of protection of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

[0055] like Figure 1 As shown, the principle of the chemiluminescent sensor of the present application is:

[0056] The present application designs a probe set for detecting soluble anti-programmed cell death ligand 1 (sPD-L1) in peripheral blood, the probe set comprising a substrate probe (MB@ssDNA-HRP) and a recognition probe, the substrate probe comprising modified carboxyl magnetic beads and streptavidin-modified horseradish peroxidase (Horseradish Peroxidase, HRP), wherein the modified carboxyl magnetic beads are obtained by modifying the carboxyl magnetic beads with ssDNA modified with amino groups (-NH2) and biotin (Biotin), and the substrate probe can be used as a non-specific cleavage substrate for Cas12a protein (i.e., Cas12a enzyme); the recognition probe is a nucleic acid probe capable of recognizing soluble PD-L1, and is composed of a nucleic acid aptamer (Apt) that can specifically recognize soluble PD-L1 and an "activator" (activator ssDNA) of the Cas12a protein. When soluble (i.e., Soluble) PD-L1 is present, Apt recognizes sPD-L1 and undergoes a conformational change, releasing the "activator The "activator ssDNA" is complementary to the CRISPR RNA (crRNA) of the Cas12a protein, activates the non-specific cleavage activity of Cas12a, shears the ssDNA on the MBs@ssDNA-HRP, releases horseradish peroxidase (HRP) into the free homogeneous solution, and after magnetic separation, adds luminol (i.e., Luminol) and hydrogen peroxide solution to the supernatant to detect the generated chemiluminescent signal. The soluble PD-L1 in the solution can be quantitatively detected through the output of the chemiluminescent signal.

[0057] The present invention is described in detail below by specific examples. It should also be understood that the following examples are only used to specifically illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention belong to the scope of protection of the present invention. The specific process parameters and the like in the following examples are also only examples within a suitable range, that is, those skilled in the art can make a selection within a suitable range through the description herein, and are not limited to the specific values ​​​​exemplified below.

[0058] (I) Construction of MB@ssDNA-HRP (i.e., substrate probe)

[0059] according to Figure 2 The process shown is to construct MB@ssDNA-HRP (i.e., substrate probe), and the specific steps are:

[0060] 1.1 Use 200 μL MEST solution (commercially available, containing 100 mM MES and 0.05% Tween-20, pH 5.0) to wash 100 μL carboxyl magnetic beads (i.e., MB-COOH) solution (commercially available, concentration 1 μM) twice, and use a magnetic stand for magnetic separation for 1 min each time, and then discard the supernatant;

[0061] 1.2 Add 100 μL of 10 mg / mL EDC (i.e., 1-ethyl-(3-dimethylaminopropyl)) solution (prepared using MEST solution) and 100 μL of 10 mg / mL NHS (i.e., N-hydroxysuccinimide) solution (prepared using MEST solution) to the washed carboxyl magnetic beads, and activate them in a metal bath at 25°C and 800 rpm for 1 hour;

[0062] 1.3 After 1 min of magnetic separation, the supernatant was discarded, and 55 μL of 100 μM 5'NH2C6-ssDNA-3'Biotin (commercially available) solution was added to the resulting precipitate, and the reaction was carried out in a metal bath at 25°C and 800 rpm for 1 h;

[0063] 1.4 After magnetic separation for 1 min, the supernatant was discarded, 200 μL of blocking solution (specifically PBS buffer containing 1% BSA) was added to the obtained precipitate, and incubated at 25°C for 1 h to obtain a mixture;

[0064] 1.5 After the mixture was magnetically separated for 1 min, the supernatant was discarded, and 40 μL of 2 μg / mL HRP-coupled streptavidin (i.e., streptavidin-modified horseradish peroxidase) solution was added to the resulting precipitate, and a secondary reaction was performed in a metal bath at 25°C and 800 rpm for 30 min;

[0065] 1.6 After magnetic separation for 1 min, the supernatant was discarded and the precipitate was washed 8 times with 200 μL PBST (PBS, 0.05% Tween-20) to obtain MB@ssDNA-HRP;

[0066] 1.7 Resuspend MB@ssDNA-HRP in 1 mL storage solution (0.5 mM Tris-HCl, pH 8.0) and store at 4°C for later use.

[0067] (II) Colorimetric characterization of probe MB@ssDNA-HRP

[0068] 2.1 Take 5uL MB-COOH (commercially available, concentration of 10mg / mL) solution, 5uL supernatant obtained by magnetic separation in step 1.6, namely MB@ssDNA-HRP separation supernatant (MB@ssDNA-HRP supernatant), and 5uL MB@ssDNA-HRP liquid (MB@ssDNA-HRP) in step 1.7, and add the three liquids into enzyme-free EP tubes respectively;

[0069] 2.2 Add 35uL PBS buffer (0.01M, pH 7.4) to each tube to make up to 40uL, then add 40uLTMB (3,3',5,5'-tetramethylbenzidine) colorimetric solution (commercially available) to each tube, react for 15min in the dark, observe with the naked eye and take pictures. The results are as follows: Figure 3 shown.

[0070] like Figure 3 As shown, after 15 minutes of reaction in the dark, the MB@ssDNA-HRP liquid showed obvious color. This result shows that the MB@ssDNA-HRP was successfully constructed in this application, and the introduced HRP still has strong catalytic activity.

[0071] (III) Construction of hybridization probes

[0072] Table 1 Nucleotide sequence table

[0073]

[0074] Note: All nucleotide sequences were synthesized by biological companies.

[0075] 2 μL of 100 nM Apt solution (nucleotide sequence as shown in Table 1, prepared with enzyme-free PBS buffer), 2 μL of 100 nM activator ssDNA solution (nucleotide sequence as shown in Table 1, prepared with enzyme-free PBS buffer) and 16 μL of hybridization buffer (specifically PBS buffer containing 0.55 mM MgC2 and pH 7.4) were mixed evenly, and then incubated in a 95°C water bath for 5 min, and then slowly cooled to room temperature at a cooling rate of 0.1°C / s to form a hybridization probe. The prepared hybridization probe (as shown in Figure 4 Store at 4°C until use.

[0076] (IV) Feasibility Verification

[0077] 4.1 Prepare sPD-L1(-) system and sPD-L1(+) system, wherein;

[0078] The sPD-L1(-) system is as follows: 10 μL of a hybridization probe solution having a concentration of 10 nM and 10 μL of a hybridization buffer (same as above) are mixed evenly to obtain the sPD-L1(-) system;

[0079] The sPD-L1(+) system is as follows: 10 μL of a 10 nM hybridization probe solution and 10 μL of a 1 ng / mL sPD-L1 solution are mixed evenly to obtain a sPD-L1(+) system;

[0080] 4.2 The sPD-L1(-) system and the sPD-L1(+) system were incubated at 37°C for 20 min to obtain incubation products;

[0081] 4.3 Mix 40 μL of 10 μM Cas12a protein solution, 40 μL of 10 μM crRNA solution (nucleotide sequence is shown in Table 1, prepared using enzyme-free PBS buffer) and 920 μL of DEPC water, and then incubate at 37 ° C for 30 min to obtain an incubation product;

[0082] 4.4 Mix 40 μL of the liquid obtained in step 1.7, 20 μL of the incubation product obtained from the sPD-L1(-) system in step 4.2, 10 μL of the incubation product obtained in step 4.3, 10 μL of NEBuffer r2.1 (10×) and 60 μL of DEPC water, and then incubate at 37°C for 30 minutes to activate the trans-cleavage activity of cas12a, thereby cleaving the MB@ssDNA-HRP substrate to obtain an incubation product;

[0083] 4.5 Mix 40 μL of the liquid obtained in step 1.7, 20 μL of the sPD-L1(+) system in step 4.2, 10 μL of the incubation product obtained in step 4.3, 10 μL of NEBuffer r2.1 (10×) and 60 μL of DEPC water, and then incubate at 37°C for 30 min to activate the trans-cleavage activity of cas12a, thereby cleaving the MB@ssDNA-HRP substrate to obtain an incubation product;

[0084] 4.6 After the incubation products obtained in step 4.4 and step 4.5 were magnetically separated for 1 min using a magnetic stand, 80 μL of the supernatant was transferred to a 96-well black ELISA plate. The chemiluminescence detection was set on the SpectraMax iD3 multi-function microplate reader with a wavelength of 430 nm. The signal value was detected every 40 s for a total of 4 min. After setting the program and position, luminol solution (final concentration of 5 mM) and hydrogen peroxide solution (final concentration of 75 mM) were added to the supernatant, and the chemiluminescence signal value was immediately detected on the SpectraMax iD3 multi-function microplate reader. The results are as follows: Figure 5 shown.

[0085] like Figure 5 As shown, when the target is present, a significantly amplified chemiluminescent signal appears, and when the target is not present, the chemiluminescent signal is almost negligible. This result shows that the chemiluminescent sensor made by the probe set of the present application is feasible for detecting soluble anti-programmed cell death ligand 1 in peripheral blood.

[0086] (V) Sensitivity test

[0087] Six experimental groups were set up, and the concentrations of sPD-L1 solutions in each group were 0, 2pg / mL, 5pg / mL, 10pg / mL, 100pg / mL and 1000pg / mL, respectively. The sPD-L1(+) system in (IV) was treated in the same manner and the chemiluminescence signal values ​​were detected. The results are shown in Figure 6 As shown; Prism software was used for linear fitting, and the detection limit was calculated according to the blank mean + 3 times the blank standard deviation. The results are shown in Figure 7 shown.

[0088] like Figure 6 As shown, in the range of 0-1000 pg / mL, as the target concentration increases, the chemiluminescence intensity also gradually increases.

[0089] like Figure 7 As shown, the chemiluminescent signal and the logarithmic value of the sPD-L1 concentration are strongly linearly correlated, ranging from 2pg / mL to 1000pg / mL, and the fitted linear equation is Y=5716IgC-116.2 (R2=0.989), where Y is the chemiluminescent intensity signal and C is the concentration of sPD-L1. By repeating the blank sample for 3 times, the detection limit was calculated to be 1.46pg / mL. This result shows that the chemiluminescent sensor made of the probe set of the present application has high sensitivity in detecting soluble anti-programmed cell death ligand 1 in peripheral blood.

[0090] (VI) Specificity test

[0091] Four experimental groups were set up, and each group detected common proteins and target proteins in serum, respectively, and was divided into the following four groups: Group A was a blank group, in which the target protein was replaced by an equal volume of PBS buffer; Group B's target protein was tumor necrosis factor-α (TNF-α, 1 ng / mL); Group C's target protein was myeloperoxidase (myeloperoxidase, 1 ng / mL); Group D's target protein was sPD-L1 (1 ng / mL), which was treated in the same way as the sPD-L1(+) system in (IV), and the chemiluminescence signal value was detected. The results are shown in Figure 2. Figure 8 shown.

[0092] like Figure 8As shown, the chemiluminescent signals generated by other proteins are comparable to the blank control results and can be ignored. This result shows that the chemiluminescent sensor made by the probe set of the present application detects soluble anti-programmed cell death ligand 1 in peripheral blood, is not interfered by other common proteins, has good anti-interference ability, and can specifically detect sPD-L1.

[0093] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A probe set for detecting soluble anti-programmed cell death ligand 1 in peripheral blood, characterized in that: The probe group includes a recognition probe and a substrate probe, wherein the recognition probe is formed by hybridization of an aptamer and an activator ssDNA, and the substrate probe includes modified carboxyl magnetic beads and horseradish peroxidase modified with streptavidin, and the modified carboxyl magnetic beads are obtained by modifying the carboxyl magnetic beads with ssDNA modified with amino groups and biotin.

2. The probe set according to claim 1, characterized in that: The amino group is modified at the 5' end of the ssDNA, and the amino group of the ssDNA reacts and binds to the carboxyl magnetic beads; And / or, the biotin is modified at the 3' end of the ssDNA.

3. The probe set according to claim 1, wherein The nucleotide sequence of the aptamer is shown in SEQ ID NO.1, and the nucleotide sequence of the ssDNA is shown in SEQ ID NO.2; And / or, the nucleotide sequence of the ssDNA is shown as SEQ ID NO.

4.

4. The probe set according to claim 1, characterized in that: The recognition probe is prepared by a method comprising the following steps: The aptamer is mixed with the activator ssDNA and a buffer, and then incubated together, and cooled to obtain the recognition probe; And / or, the substrate probe is prepared by a method comprising the following steps: A carbodiimide hydrochloride solution and an N-hydroxysuccinimide solution are added to a carboxyl magnetic bead solution for activation and magnetic separation, and then a biotinylation reagent is added to the obtained precipitate, wherein the biotinylation reagent includes an amino group and biotin, and then a reaction is performed, and magnetic separation is performed twice. A blocking solution is added to the obtained precipitate, and the mixture is incubated and magnetically separated three times. Streptavidin coupled with horseradish peroxidase is added to the obtained precipitate, and the reaction is continued and magnetic separation is performed four times to obtain the substrate probe.

5. The probe set according to claim 4, characterized in that: The molar ratio of the aptamer to the activator ssDNA is 1-2:1-2; And / or, the buffer solution is a PBS buffer solution containing magnesium chloride and having a pH of 7.2-7.5; And / or, the co-incubation temperature is 90-100° C., and the co-incubation time is 4-6 min; and / or, cooling to room temperature at a cooling rate of 0.05-0.2°C / min.

6. The probe set according to claim 4, characterized in that: The carbodiimide hydrochloride is selected from 1-ethyl-(3-dimethylaminopropyl)carbodiimide; and / or, the mass ratio of the carbodiimide hydrochloride to the N-hydroxysuccinimide is 8-12:8-12; And / or, the activation temperature is 20-30° C., and the activation time is 0.5-1.5 h; And / or, the mass ratio of the carbodiimide hydrochloride to the biotinylation reagent is 8-12: 0.05-0.06； and / or, the mass ratio of the carbodiimide hydrochloride to the streptavidin coupled with horseradish peroxidase is 8-12::0.0005-0.001; And / or, the reaction temperature is 20-30°C, and the reaction time is 0.5-1.5h; And / or, the incubation temperature is 20-30° C., and the incubation time is 0.5-1.5 h; And / or, the temperature of the secondary reaction is 20-30° C., and the duration of the secondary reaction is 20-40 min.

7. A chemiluminescent sensor for detecting soluble anti-programmed cell death ligand 1 in peripheral blood, characterized in that: The chemiluminescent sensor comprises the probe set according to any one of claims 1 to 6.

8. The chemiluminescent sensor according to claim 7, characterized in that The chemiluminescent sensor also includes a Cas12a enzyme.

9. Use of the probe set according to any one of claims 1 to 6 or the chemiluminescent sensor according to claim 7 or 8 in the preparation of a kit for detecting soluble anti-programmed cell death ligand 1 in peripheral blood.

10. A kit for detecting soluble anti-programmed cell death ligand 1 in peripheral blood, characterized in that: The kit comprises the probe set according to any one of claims 1 to 6 or the chemiluminescent sensor according to claim 7 or 8.

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