A method for efficient and sensitive quantitative detection of PD-L1 positive tumor cells based on steric hindrance

Abstract

The application discloses a method for high-efficiency and sensitive quantitative detection of PD-L1 positive tumor cells based on steric hindrance, and comprises the following steps: (1) preparing a working electrode: a DNA single strand containing a PD-L1 aptamer and a primer exchange reaction (PER) primer sequence is modified on the surface of a gold electrode; and (2) detecting the number of PD-L1 positive tumor cells in co-incubated cells. The application realizes convenient and sensitive detection of PD-L1 positive tumor cells, and provides a new means for judging whether a patient adopts an immunotherapy in a clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomarker detection, and particularly relates to a method for efficiently and sensitively quantitatively detecting PD-L1 positive tumor cells based on steric hindrance. BACKGROUND

[0002] Cancer is one of the main causes of human death at present, and seriously threatens the life and health of the public. Traditional treatment measures mainly include surgery, chemotherapy and radiotherapy. In recent years, new methods such as immunotherapy, targeted therapy and intervention have emerged, providing new treatment approaches for cancer patients. Tumor immunotherapy refers to enhancing the immune function of the body to achieve the purpose of clearing tumor cells. The immune checkpoint therapy, which won the Nobel Prize in Physiology or Medicine in 2018, is a hot spot in the field of tumor treatment research.

[0003] PD-L1 (programmed-death ligand 1) is one of the main immune checkpoints, and is an important target in the field of tumor diagnosis and treatment research in recent years. PD-L1 is highly expressed on the surface of tumor cells, and by combining with PD-1 (programmed cell death protein 1) on the surface of CD8+ T cells, it can escape T cell-mediated cell killing, thereby helping tumor cells to achieve immune escape. Blocking the interaction between PD-1 and PD-L1 can restore the immune system's attack on tumor cells, thereby achieving the purpose of treating tumors.

[0004] FDA (U.S. Food and Drug Administration) has approved the marketing of PD-1 blockers such as pembrolizumab and nivolumab for the treatment of advanced melanoma and non-small cell lung cancer. However, only 20-40% of patients respond to this therapy, and the proportion of patients who can be cured for a long time is even smaller. Studies have shown that the degree of patient response to this therapy is positively correlated with the expression level of PD-L1 in the body, and patients with low expression have poorer treatment effects. Considering the high price of immunotherapy drugs and the potential serious side effects, early assessment of the PD-L1 level in the body of patients has a certain guiding effect on patient classification and subsequent use of this immunotherapy.

[0005] The current method for evaluating the expression level of PD-L1 is immunohistochemistry, which is a protein quantification using antibodies. There are two disadvantages: first, sampling is required, and due to the small sample volume and the characteristics of tumor heterogeneity, false negative results are easily produced; second, the glycosylation side chain modification of PD-L1 hinders the contact between large molecules of antibodies and proteins, resulting in false negatives and poor treatment effects. Therefore, there is an urgent need for a more simple and accurate method for quantifying PD-L1.

[0006] The research found that the expression level of PD-L1 on the surface of circulating tumor cells in peripheral blood is consistent with the expression level of tumor tissue, suggesting that the expression of PD-L1 on the surface of CTCs (circulating tumor cells) can be used as an index for detection. Previous studies have screened PD-L1 aptamers, which are only one quarter the size of antibodies, can directly bind to proteins through glycosyl side chains, and make up for the shortcomings of antibodies. Nucleic acid aptamers have been applied to the field of tumor treatment.

[0007] Due to the small number of CTCs in peripheral blood, generally 1-10 / 10ml, it is necessary to amplify the detection signal in order to obtain accurate and high sensitivity detection results. The PER (primer exchange reaction) signal amplification method uses only one short nucleic acid chain and a hairpin structure. Under the action of DNA polymerase, by limiting the supply of specific dNTPs, a long chain of DNA containing multiple tandem repeat units can be amplified. By modifying fluorescent groups or electrical signal molecules on the repeat units, the purpose of signal amplification is achieved. This method only needs to provide dNTPs, and can independently generate single-stranded DNA of any specified sequence as needed.

[0008] In summary, nucleic acid aptamers are used to recognize target CTCs, and PD-L1 aptamers are used to capture cells expressing PD-L1 protein. Due to the steric hindrance of the captured cells, further signal amplification reaction on the electrode surface is hindered, and finally an electrical signal output is achieved in inverse proportion to the number of captured cells, so that PD-L1 can be sensitively and accurately quantitatively detected. This study is expected to provide guidance for grading and drug use for tumor patients who need PD-1 blocker drugs for immunotherapy, and has important clinical application value. SUMMARY

[0009] In view of the above defects, the present application provides an electrochemical method for high-sensitivity detection of PD-L1 positive tumor cells.

[0010] In order to achieve the above-mentioned purpose of the application, the technical scheme adopted by the present application is as follows: a novel electrochemical analysis method for detecting PD-L1 positive tumor cells, comprising the following steps: (1) preparing a working electrode: modifying a DNA single strand containing a PD-L1 aptamer and a primer exchange reaction (PER) primer sequence on the surface of a gold electrode;

[0011] (2) Detecting the number of PD-L1 positive tumor cells in co-incubated cells: the working electrode is co-incubated with the cells to be tested, followed by primer exchange reaction PER, after the reaction product is hybridized with the complementary strand, HRP is modified to the electrode surface through the interaction of biotin and streptavidin-labeled horseradish peroxidase (SA-HRP), and the substrate tetramethylbenzidine is catalyzed to generate an electrical signal.

[0012] Further, in step (1), Step 1 co-incubates Primer-SH with 3mM TCEP at room temperature for 0.5-1h, 10μL of the above Primer-SH of different concentrations is added dropwise on the electrode surface, and incubated at room temperature for 1-2h;

[0013] Step 2 adds 10-20μL of 1mM MCH dropwise on the electrode surface, and incubates at room temperature for 0.5-1h to prevent non-specific adsorption on the electrode surface;

[0014] In step (2), Step 1 immerses the electrode into a solution containing different concentrations of cells, respectively, and incubates at 37℃ for 2h;

[0015] Step 2 takes out the electrode, and then immerses the electrode into an amplification solution, respectively, and reacts at 37℃;

[0016] Step 3 takes out the electrode, and then adds 10μL of 1μM Bio dropwise on the electrode surface, respectively, and incubates at 37℃ for 0.5-1h;

[0017] Step 4 immerses the electrode into a solution containing different concentrations of SA-HRP, respectively.

[0018] Further, in step (1), Step 1 co-incubates Primer-SH with 3mM TCEP at room temperature for 0.5-1h, 10μL of the above Primer-SH of different concentrations is added dropwise on the electrode surface, and incubated at room temperature for 1-2h.

[0019] Further, in step (1), the nucleotide sequence of the primer Primer-SH is as shown in SEQ ID NO: 1, the nucleotide sequence of the primer Bio is as shown in SEQ ID NO: 2, the nucleotide sequence of the primer Hairpin is as shown in SEQ ID NO: 3, the nucleotide sequence of the primer clean G is as shown in SEQ ID NO: 4, the nucleotide sequence of the primer PD-Apt-FAM is as shown in SEQ ID NO: 5, and the nucleotide sequence of the primer RAM-FAM is as shown in SEQ ID NO: 6.

[0020] Further, in step (1), Step 2 drops 10-20 μL of 1 mM MCH on the electrode surface, and incubates at room temperature for 0.5-1 h to prevent non-specific adsorption on the electrode surface.

[0021] Further, in step (2), Step 1 immerses the electrode into a solution containing different concentrations of cells, and incubates at 37℃ for 2 h.

[0022] Further, in step (2), the amplification solution used in Step 2 is 1 μM Hairpin, 1 mM (dATP, dTTP, dCTP), 1x Reaction buffer, and 0.8 U / μL Bst DNA polymerase.

[0023] Further, in step (2), after Step 3 removes the electrode, 10 μL of 1 μM primer Bio is dropped on the electrode surface, and incubated at 37℃ for 0.5-1 h.

[0024] Further, in step (2), Step 4 immerses the electrode into a solution containing different concentrations of SA-HRP, and incubates at room temperature for 0.5 h. Electrochemical testing is performed in TMB color developing solution, and the i-t curve is scanned with the following parameters: starting voltage -0.1 V, sensitivity 1e-5, and operation time 100 s. The measured i-t curve reflects the change in the concentration gradient of the solution near the electrode surface, and the current size is proportional to the concentration gradient on the electrode surface. Therefore, the final stable current value after applying voltage is proportional to the adsorbed HRP concentration on the electrode, and the stable current value is used as the electrical signal output.

[0025] Beneficial effects: The detection method of the present application has high sensitivity, and the detection limit is lower than 10 cells / mL. This method can realize the detection of PD-L1 level in patients, and is expected to provide a new technical means for patient grading and drug guidance for immunotherapy.

[0026] Compared with the traditional method, the present application has the following advantages: (1) The present application can directly detect in peripheral blood, which is a non-invasive detection method; (2) The method of the present application is simple and fast, which only needs to modify the PD-L1 containing aptamer sequence on the electrode, and ingeniously uses the spatial steric hindrance generated after the electrode surface captures PD-L1 positive cells, and combines a new type of PER signal amplification method to realize high sensitivity detection effect.

[0027] (3) The application of PD-L1 positive tumor cell detection based on this method mainly includes: a) detecting the PD-L1 level in patients and grading; b) determining whether to use anti-PD-L1 immunotherapy drugs according to the PD-L1 level of the patient. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Schematic diagram of electrochemical method for high-sensitivity detection of PD-L1 positive tumor cells of the present application.

[0029] Figure 2 Schematic diagram of fluorescence imaging of HepG2 cells co-incubated with different sequences. (A) and (B) are the corresponding blank control groups of (C) and (D). (C) HepG2 cells co-incubated with FAM-modified PD-L1 aptamer. (D) HepG2 cells co-incubated with FAM-modified random sequence.

[0030] Figure 3 Non-denaturing polyacrylamide gel electrophoresis (PAGE) image of different samples of the present application. Lane 1: 1 μM Primer-SH; Lane 2: 1 μM Hairpin; Lane 3: 1 μM Primer-SH + 1 μM Hairpin; Lane 4: 1 μM Hairpin + 0.8 U / μL Bst DNA Polymerase + 1 μM clean G + 10 μM (dATP + dTTP + dCTP); Lane 5: 1 μM Primer-SH + Lane 4; Lane 6: 1 μM Bio + Lane 5; Lane 7: 1 μM Bio.

[0031] Figure 4 Feasibility schematic diagram of the present application. Figure 4 A is the EIS graph of each step of electrode surface modification. Figure 4 B is the i-t curve under different conditions.

[0032] Figure 5 Primer-SH concentration optimization graph of the method of the present application.

[0033] Figure 6 Hairpin concentration optimization graph of the method of the present application.

[0034] Figure 7 Time optimization graph of PER amplification of the method of the present application.

[0035] Figure 8 HRP concentration optimization graph of the method of the present application.

[0036] Figure 9 Sensitivity analysis graph of the sensor of the present application. Figure 9 A is the i-t curve of the present application when detecting different numbers of HepG2 cells in PBS buffer. Figure 9 B is the corresponding relationship between current signal response value and cell concentration. The inset is the calibration curve of current value and LogCcell.

[0037] Figure 10Specificity analysis chart of the sensor of the present application. The current signal response value when the present application is incubated with different cells respectively: blank group; L-02; Hela; MDA-MB-231; HepG2; mixed group of HepG2, Hela and L-02; mixed group of MDA-MB-231, Hela and L-02.

[0038] Figure 11 Analysis chart of the method proposed by the present application in a complex system. The current response value when the present application is in PBS, DMEM and serum system respectively with or without target. DETAILED DESCRIPTION

[0039] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the accompanying drawings, which show several embodiments of the present application. However, the present application can be realized in different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the disclosure of the present application more thorough and comprehensive.

[0040] Example 1

[0041] The method for high-efficiency and sensitive quantitative detection of PD-L1 positive tumor cells based on steric hindrance of the present application comprises the following steps: (1) preparing a working electrode: a DNA single strand containing a PD-L1 aptamer and a primer exchange reaction (PER) primer sequence is modified on the surface of a gold electrode; Step 1: incubate Primer-SH and 3mM TCEP at room temperature for 0.8h, and drop 10μL of different concentrations of the above Primer-SH on the electrode surface, and incubate at room temperature for 2h. Step 2: drop 15μL of 1mM MCH on the electrode surface, and incubate at room temperature for 0.5h to prevent non-specific adsorption on the electrode surface.

[0042] The nucleotide sequence of the primer Primer-SH is as shown in SEQ ID NO: 1, the nucleotide sequence of the primer Bio is as shown in SEQ ID NO: 2, the nucleotide sequence of the primer Hairpin is as shown in SEQ ID NO: 3, the nucleotide sequence of the primer clean G is as shown in SEQ ID NO: 4, the nucleotide sequence of the primer PD-Apt-FAM is as shown in SEQ ID NO: 5, and the nucleotide sequence of the primer RAM-FAM is as shown in SEQ ID NO: 6.

[0043] (2) Detect the number of PD-L1 positive tumor cells in the co-incubated cells: co-incubate the working electrode with the cells to be tested, then perform the primer exchange reaction PER, and after the reaction product hybridizes with the complementary strand, HRP is modified to the electrode surface through the interaction of biotin and streptavidin (HRP), which catalyzes the production of an electrical signal by the substrate tetramethylbenzidine.

[0044] Step 1: Immersing the electrode in a solution containing different concentrations of cells at 37℃ for 2h.

[0045] Step 2: Taking out the electrode and immersing the electrode in an amplification solution (1 μM Hairpin, 1 mM (dATP, dTTP, dCTP), 1x Reaction buffer, 0.8 U / μL Bst DNA polymerase) at 37℃ for reaction.

[0046] Step 3: After taking out the electrode, 10 μL of 1 μM Bio was added to the electrode surface and incubated at 37℃ for 0.5h.

[0047] Step 4: Immersing the electrode in a solution containing different concentrations of SA-HRP at room temperature for 0.5h. Electrochemical testing was performed in TMB developing solution, and the i-t curve was scanned with the following parameters: starting voltage -0.1V, sensitivity 1e-5, and operation time 100s. The measured i-t curve reflects the change in the concentration gradient of the solution near the electrode surface, and the current size is proportional to the concentration gradient on the electrode surface. Therefore, the final stable current value after applying voltage is proportional to the concentration of HRP adsorbed on the electrode, and the stable current value is taken as the electrical signal output.

[0048] Example 2

[0049] The present application is a high-efficiency and sensitive method for quantitatively detecting PD-L1 positive tumor cells based on steric hindrance, which comprises the following steps: In step (1), the working electrode is prepared: a DNA single strand containing PD-L1 aptamer and primer exchange reaction PER primer sequence is modified on the surface of a gold electrode; Step 1: Primer-SH is co-incubated with 3mM TCEP at room temperature for 0.5h, and 10 μL of different concentrations of the above Primer-SH is added to the electrode surface, and incubated at room temperature for 1.5h. Step 2: 10 μL of 1 mM MCH is added to the electrode surface and incubated at room temperature for 0.5h to prevent non-specific adsorption on the electrode surface.

[0050] In step (2), Step 3: After taking out the electrode, 10 μL of 1 μM Bio was added to the electrode surface and incubated at 37℃ for 0.8h.

[0051] Example 3

[0052] A method for high-efficiency and sensitive quantitative detection of PD-L1 positive tumor cells based on steric hindrance, comprising the following steps: in step (1), preparing a working electrode: modifying a DNA single strand containing a PD-L1 aptamer and a primer exchange reaction (PER) primer sequence on the surface of a gold electrode; Step 1: incubate Primer-SH with 3mM TCEP at room temperature for 1h, and drop 15μL of the above Primer-SH with different concentrations on the electrode surface, and incubate at room temperature for 1h. Step 2: drop 20μL of 1mM MCH on the electrode surface, and incubate at room temperature for 0.5-1h to prevent non-specific adsorption on the electrode surface.

[0053] In step (2), Step 3: after taking out the electrode, drop 10μL of 1μM Bio on the electrode surface, and incubate at 37℃ for 1h.

[0054] Test Example 1

[0055] Experimental part of the present application

[0056] 1.1 Instruments

[0057] Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), amperometric i-t curve (i-t), and chronocoulometry (CC) were all completed using a three-electrode CHI 660C electrochemical workstation. EIS was measured in 2mL of 2.5mM [Fe(CN)6]3- / 4-solution containing 1M KNO3. Fluorescence imaging was performed using an inverted fluorescence microscope (Olympus IX73-Camera DP80). Cell counting was performed using a cell counter (Invitrogen Countess). The heating device was a ThermoStat plus constant temperature metal bath (Eppendorf, Germany).

[0058] 1.2 Materials and reagents

[0059] All oligonucleotide sequences used in the application are listed in Table 1, synthesized by Shenguo Biotechnology Co., Ltd., and purified by HPLC. Tris(2-carboxyethyl)phosphine (TCEP) was purchased from Shanghai McLean Biotech Co., Ltd. Mercaptohexanol (MCH) was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. Deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), horseradish peroxidase-labeled streptavidin (SA-HRP), DMEM (high glucose) medium were purchased from Shanghai Shenguo Biotechnology Co., Ltd. 10x Reaction Buffer, Bst DNA polymerase were purchased from New England Biolabs, Inc. 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution was purchased from Shanghai Biyun Tian Biotechnology Co., Ltd. Hydrogen peroxide (H2O2) was purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd. Fetal bovine serum (FBS) was purchased from Yingaojie (Shanghai) Trade Co., Ltd. Ruthenium hexamine (RuHex) was purchased from Sigma-Aldrich. All nucleic acid sequences used in the application are shown in Table 1:

[0060] Table 1

[0061]

[0062]

[0063] 1.3 Preparation of working electrode

[0064] Primer-SH was incubated with 3mM TCEP at room temperature for 0.5-1h, and the electrode was dried with nitrogen. Then 10μL of Primer-SH with different concentrations was added to the surface of the electrode, and incubated at room temperature for 1-2h. Then 10-20μL of 1mM MCH was added to the surface of the electrode, and incubated at room temperature for 0.5-1h to prevent non-specific adsorption on the surface of the electrode.

[0065] 1.4 Detection of the number of PD-L1 positive tumor cells in co-incubated cells

[0066] The electrodes were incubated in the solution containing different concentrations of cells at 37 °C for 2 h. The electrodes were taken out and then were incubated in the amplification solution (1 μΜ Hairpin, 1 mM (dATP, dTTP, dCTP), 1 x Reaction buffer, 0.8 U / μL Bst DNA polymerase) at 37 °C. 10 μL 1 μΜ Bio was added on the surface of the electrodes, respectively, and was incubated at 37 °C for 0.5-1 h. Then the electrodes were incubated in the solution containing different concentrations of SA-HRP at room temperature for 0.5 h. Since HRP can catalyze the transfer of electrons of TMB, the electrochemical test was finally carried out in the TMB color developing solution, and the i-t curve was scanned with the parameters set as: initial voltage-0.1 V, sensitivity 1e-5, and operation time 100 s. The i-t curve measured reflects the change of the concentration gradient of the solution near the surface of the electrode, and the current size is proportional to the concentration gradient on the surface of the electrode. Therefore, the final stable current value after applying voltage is proportional to the HRP concentration adsorbed on the electrode, and the stable current value is taken as the electrical signal output.

[0067] Test Example 2

[0068] 1.5 Analysis in complex system

[0069] HepG2, MDA-MB-231, and Hela cells were cultured in DMEM medium containing 10% FBS and 1% streptavidin-penicillin solution, and L-02 cells were cultured in RPMI1640 medium containing 10% FBS and 1% streptavidin-penicillin solution. All the cells were cultured in a cell incubator at 37 °C containing 5% CO2. According to the regulations of the local ethics committee, the whole blood of healthy people was obtained from the First Affiliated Hospital of Nanjing Medical University (Nanjing, China), and after centrifugation at 3000 rpm, the serum in the upper layer of the test tube was collected for subsequent experiments.

[0070] In the present application, in order to evaluate the potential of the method in actual biological samples, the system of the method was replaced by the complex environment DMEM and human serum sample that may be faced in clinical practical application, and the detection analysis with or without target was carried out.

[0071] The liver cancer tissues were provided by the First Affiliated Hospital of Nanjing Medical University. The present application was approved by the Ethics Committee of Nanjing University and Nanjing Medical University.

[0072] 1.6 Results and discussion

[0073] 1.6.1 Design of sensor

[0074] In order to realize the detection of PD-L1 positive tumor cells, as shown in FIG. 1, the sensor was designed as follows: Figure 1As shown, a DNA strand, Primer-SH, containing the PD-L1 aptamer sequence was modified on the electrode surface. When no PD-L1-expressing cells were present, the Primer-SH contained primers for subsequent PER amplification. When the PER system was supplied with hairpin, Bst DNA polymerase, Clean G, and three specific dNTPs, the primers were amplified to a long DNA chain containing multiple tandem repeats under the action of the polymerase through cleverly designed nucleic acid sequences. Next, biotin-modified PF-bio, complementary to the short fragment units, was added to the electrode and co-incubated with the amplification product. Multiple PF-bio molecules then bound to the long DNA chain, achieving signal amplification. Subsequently, streptavidin-modified HRP bound to the electrode surface through the interaction between streptavidin and biotin. HRP possesses catalytic activity, enabling enzymatic reactions of TMB substrates in the electrolyte to generate electrical signals. When PD-L1-expressing cells are present, they are captured by the PD-L1 aptamer. Due to the large cell size, PER raw materials such as hairpin have difficulty accessing the primer chains, hindering subsequent amplification reactions and resulting in only a weak electrical signal output. Ultimately, a signal-off electrical signal is generated that is inversely proportional to the number of captured cells. Thus, the steric hindrance effect generated after cell capture enables the quantification of PD-L1-expressing cells.

[0075] 1.6.2 Feasibility Analysis of Sensors

[0076] Figure 2 This is a schematic diagram of fluorescence imaging of HepG2 cells co-incubated with different sequences. (A) and (B) are the corresponding blank control groups for (C) and (D). (C) HepG2 cells co-incubated with FAM-modified PD-L1 aptamers. (D) HepG2 cells co-incubated with FAM-modified random sequences.

[0077] To verify the feasibility of the constructed method for quantitative detection of PD-L1-positive tumor cells based on steric hindrance, several steps involved were validated sequentially. First, to verify the capture of PD-L1-highly expressing cells by the selected aptamers, the PD-L1 aptamer sequence PD-Apt-FAM modified with the FAM fluorescent group and the random sequence RAM-FAM were co-incubated with HepG2 cells, respectively. Figure 2 The results observed by fluorescence inverted microscopy showed that fluorescence could only be observed on the cell surface when HepG2 was co-incubated with the aptamer, proving that the aptamer can successfully bind to cells that highly express PD-L1.

[0078] Figure 3Non-denaturing polyacrylamide gel electrophoresis (PAGE) images of different samples of the present application. Lane 1: 1 μΜ Primer-SH; Lane 2: 1 μΜ Hairpin; Lane 3: 1 μΜ Primer-SH + 1 μΜ Hairpin; Lane 4: 1 μΜ Hairpin + 0.8 U / μL Bst DNA Polymerase + 1 μΜ clean G + 10 μΜ (dATP + dTTP + dCTP); Lane 5: 1 μΜ Primer-SH + Lane 4; Lane 6: 1 μΜ Bio + Lane 5; Lane 7: 1 μΜ Bio.

[0079] Next, in order to verify the function of signal amplification that PER can achieve, the feasibility of PER was verified by PAGE. As shown in Figure 3 When Primer-SH and Hairpin coexist, under the action of Bst polymerase, ladder-shaped products are obtained by amplification, as mentioned in the literature, which indicates that a plurality of long nucleic acid chains containing different numbers of short fragment repeat units are obtained. Moreover, after the amplification product is incubated with PF-bio at 37°C, as shown in lane 7, the mobility of the incubation product is slower than that of lane 7, proving the successful combination of PF-bio and the amplification product. However, if Primer-SH is not present, the product obtained by the reaction is not ladder-shaped, which indicates that the PER amplification reaction is not initiated at this time.

[0080] Figure 4 Schematic diagram of the feasibility of the present application. Figure 4 A is the EIS diagram of each step of modification of the electrode surface. Figure 4 B is the i-t curve under different conditions.

[0081] Then, electrochemical technology EIS was used to characterize the progress of each step of the reaction, and i-t was used to verify the feasibility of the scheme. As shown in Figure 4 A shows that after Primer-SH is modified to the electrode, the electron transfer is hindered, and the charge transfer resistance (Rct) increases rapidly, indicating that Primer-SH is successfully modified to the electrode. Then the PER amplification reaction is carried out, at which time Rct increases significantly, which is due to the long chain obtained by amplification further hindering the electron transfer. Then PF-bio is incubated on the electrode surface, at which time Rct increases significantly compared to the previous one, which is due to the combination of multiple PF-bio to the long chain to produce greater hindrance. When Primer-SH is directly incubated with HepG2 cells, Rct increases sharply compared to other modifications on the electrode, which is due to the larger steric hindrance caused by the cells captured by the aptamer. The EIS results obtained above successfully characterize each step of modification on the electrode.

[0082] To further confirm the design of the scheme, i-t was used to detect the current signal generated by HRP catalyzing the substrate TMB on the electrode under different conditions, such as Figure 4 The obtained i-t curve shows that when only Primer-SH is modified, a certain amount of HRP is bound on the electrode, and a current signal output is generated, because one PF-bio can be complementary to one Primer-SH. Then, when the PER amplification reaction is carried out without HepG2 cells, the current signal value generated is significantly increased, because the long chain containing multiple tandem repeat units obtained by amplification can bind to multiple PF-bios, and then more HRP can be bound. When the PER amplification is carried out in the presence of HepG2 cells, the current signal value is slightly lower than when only Primer-SH is modified, because after Primer-SH binds to HepG2, the large volume of the cell hinders the approach of the subsequent PER raw materials, thereby inhibiting the occurrence of PER; at the same time, the steric hindrance generated by the cell also blocks the approach of PF-bio. The above two factors result in that the number of HRP bound on the electrode surface is less than that in other cases, and thus the lowest electrical signal output is generated. In summary, the experimental results obtained can clearly prove the feasibility of the method for electrochemical analysis of cancer cell surface PD-L1 level based on steric hindrance.

[0083] 1.6.3 Analysis of the effect of different conditions on the sensor

[0084] Figure 5 The figure for optimization of the concentration of Primer-SH in the method of the application.

[0085] In order to make the method present the best detection effect and achieve the best detection performance, several key conditions that may interfere with the experimental results are optimized: the concentration of Primer-SH, the concentration of Hairpin, the PER amplification time, and the concentration of HRP. First, the concentration of Primer-SH modified on the electrode surface is optimized. Primer-SH contains PD-L1 aptamer and PER primer sequence. When too much is modified on the electrode, because Primer-SH can be complementary to one PF-bio, even when cells are present, the excess Primer-SH that is not captured by cells will directly hybridize with PF-bio, thereby generating a strong background signal. When too little is modified on the electrode, even when no cells are present, only limited DNA long chains can be amplified, and thus limited current signal values are generated, which may result in that the signal-to-noise ratio of the experimental results does not meet the requirements. For example, Figure 5As shown, the signal-to-noise ratio gradually increased with the increase of the Primer-SH concentration, and reached the maximum value when the concentration was 1 μM. After that, the signal-to-noise ratio decreased with the increase of the concentration. Therefore, the concentration of Primer-SH was 1 μM in the subsequent experiments.

[0086] Figure 6 The figure for the optimization of the Hairpin concentration in the method of the present application. Then, the concentration of Hairpin was optimized. Hairpin is the raw material for the PER reaction, and the concentration ratio of Hairpin to Primer-SH has an important influence on the amplification efficiency and effect of PER. As shown, Figure 6 As shown, the signal-to-noise ratio gradually increased with the increase of the Primer-SH concentration, and reached the maximum value when the concentration was 1 μM. After that, the signal-to-noise ratio decreased with the increase of the concentration. Therefore, the concentration of Primer-SH was 1 μM in the subsequent experiments. Figure 7 The figure for the optimization of the PER amplification time in the method of the present application. Then, the PER amplification time was optimized, which is closely related to the number of tandem repeat units contained in the amplified long chain, i.e. the number of subsequent HRP binding. As shown, Figure 7 As shown, the signal-to-noise ratio gradually increased with the increase of the Primer-SH concentration, and reached the maximum value when the concentration was 1 μM. After that, the signal-to-noise ratio decreased with the increase of the concentration. Therefore, the concentration of Primer-SH was 1 μM in the subsequent experiments. Figure 8 The figure for the optimization of the HRP concentration in the method of the present application. Finally, the HRP concentration was optimized, which is directly related to the strength of the signal output. As shown, Figure 8 As shown, the signal-to-noise ratio gradually increased with the increase of the Primer-SH concentration, and reached the maximum value when the concentration was 1 μM. After that, the signal-to-noise ratio decreased with the increase of the concentration. Therefore, the concentration of Primer-SH was 1 μM in the subsequent experiments.

[0087] 1.6.4 Performance analysis of the sensor

[0088] Figure 9 The figure for the sensitivity analysis of the sensor of the present application. Figure 9 A is the i-t curve of the present application when detecting different numbers of HepG2 cells in PBS buffer. Figure 9 B is the corresponding relationship between the current signal response value and the cell concentration. The inset is the calibration curve of the current value and LogCcell.

[0089] After determining the feasibility of the method, the application further invents its analysis performance from the aspects of sensitivity and specificity. Quantitative experiments are performed on HepG2 cells expressing PD-L1 using the method of the application. The HepG2 cells resuspended in PBS are gradient diluted and then detected by the method. Specifically, the cells of different concentrations (ranging from 0 to 106 cells / ml) are respectively incubated with the electrodes modified with Primer-SH, and the subsequent PER amplification experiments are performed to detect the current signal values. As shown in Figure 9 , with the increase of the cell concentration, the measured current signal gradually decreases, and the current signal value has a good linear negative correlation with the logarithmic value logCcell of the cell concentration. The linear response range is 102-106 cells / mL, and the detection limit is lower than 10 cells / mL.

[0090] Figure 10 The specificity analysis chart of the sensor of the application. The current signal response values of the application when incubated with different cells: blank group; L-02; Hela; MDA-MB-231; HepG2; mixed group of HepG2, Hela and L-02; mixed group of MDA-MB-231, Hela and L-02.

[0091] In order to explore whether the scheme can detect the high and low levels of PD-L1 expressed by different cells, several different cells (HepG2, MDA-MB-231, Hela, L-02) are detected by using this strategy. Previous studies have shown that PD-L1 is highly expressed in HepG2 and MDA-MB-231 cells, and is lowly expressed in Hela cells. From the measured results Figure 10 , it can be seen that only when the working electrode is incubated with HepG2 and MDA-MB-231 cells, the current signal value decreases obviously; and when incubated with Hela and normal liver cells L-02, the measured electric signal has almost no difference compared with the blank control group. This also verifies the difference in the expression level of PD-L1 on the surface of different cells, and proves that the method has good specificity.

[0092] 1.6.5 Analysis of the sensor in complex systems

[0093] Figure 11 The analysis chart of the proposed method in complex systems. The current response values of the application in PBS, DMEM and serum systems with and without targets.

[0094] To further explore the possibility of this method for future clinical applications, the complex environment that may be encountered in the clinic was simulated and experiments were carried out in such a system using this method. In actual clinical practice, cells are generally in DMEM culture medium or blood samples, so different concentrations of HepG2 cells were resuspended in DMEM culture medium and human serum samples instead of PBS buffer and incubated with the working electrode to carry out subsequent detection experiments. As shown in Figure 11 The measured electrical signal values in the DMEM medium or human serum system of this method only have a slight difference from the values measured in the buffer; although the electrical signal values measured in the human serum system of this method have decreased compared with the buffer, they can still distinguish whether there are PD-L1-expressing tumor cells, and the signal decrease may be caused by other interfering components such as proteins and other substances in the serum. The above experimental results show that this method has good anti-interference performance and has the potential for further application in human actual samples.

[0095] 1.7 Summary

[0096] The present application proposes a method for efficient and sensitive quantitative detection of PD-L1 positive tumor cells based on steric hindrance. This method mainly utilizes the steric hindrance generated by the cell volume. When there is no cell, the electrode surface can smoothly carry out chain amplification reaction, and through the ingenious design of the primer sequence of the amplification reaction, the obtained amplification product is a long chain containing multiple tandem repeat units, and then multiple HRP are combined on each repeat unit through complementary pairing between chains and other actions, realizing the function of signal amplification. Finally, the current signal generated by HRP catalyzing TMB is used as the signal output mode. When there are PD-L1-expressing cells, the modified PD-L1 aptamer sequence on the electrode captures the cells, at this time the large steric hindrance of the cells hinders the approach of the chain amplification reaction raw material chain, inhibits the occurrence of the PER reaction, greatly reduces the number of HRP combined on the electrode, and finally generates an electrochemical signal output that is inversely related to the number of captured cells. This method simply modifies the PD-L1 aptamer sequence on the electrode surface, ingeniously utilizes the steric hindrance effect generated after cell capture, uses the novel isothermal chain amplification method PER reaction, and successfully realizes the high-sensitivity detection of the PD-L1 level on the surface of tumor cells. Moreover, this method shows good detection performance in complex systems such as culture medium and human serum, which also shows the potential of this method for future clinical applications. In summary, this method can realize the detection of the PD-L1 level in patients, and is expected to provide new technical means for patient grading and drug guidance for immunotherapy.

[0097] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are merely exemplary of the principles and application of the present application. Numerous modifications and changes can be made by those skilled in the art without departing from the spirit and scope of the application. The scope of the application is not to be limited by the embodiments shown and described but only by the claims and their equivalents.

[0098] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods and should be within the scope of the present application.

Claims

1. A method for efficient sensitive quantification of PD-L1 positive tumor cells based on steric hindrance, characterized by The method comprises the following steps: (1) preparing a working electrode: a DNA single strand Primer-SH containing a PD-L1 aptamer and a primer exchange reaction (PER) primer sequence is modified on the surface of a gold electrode; (2) detecting the number of PD-L1 positive tumor cells in co-incubated cells: the working electrode is co-incubated with the cells to be detected, and then a primer exchange reaction (PER) is performed, the nucleotide sequence of the primer Primer-SH is shown in SEQ ID NO: 1, the nucleotide sequence of the primer Bio is shown in SEQ ID NO: 2, the nucleotide sequence of the primer Hairpin is shown in SEQ ID NO: 3, the nucleotide sequence of the primer clean G is shown in SEQ ID NO: 4, the nucleotide sequence of the primer PD-Apt-FAM is shown in SEQ ID NO: 5, and the nucleotide sequence of the primer RAM-FAM is shown in SEQ ID NO: 6; after the reaction product is hybridized with a complementary strand, HRP is modified on the surface of the electrode through the interaction of biotin and streptavidin-labeled horseradish peroxidase (SA-HRP), and an electrical signal is generated by the catalysis of a substrate tetramethylbenzidine.

2. The method according to claim 1, wherein in step (1), Step 1 co-incubates Primer-SH with 3 mM TCEP at room temperature for 0.5-1 h, 10 μL of the above Primer-SH with different concentrations is added dropwise on the surface of the electrode, and the room temperature incubation time is 1-2 h; Step 2 adds 10-20 μL of 1 mM mercaptohexanol (MCH) dropwise on the surface of the electrode, and the room temperature incubation time is 0.5-1 h to prevent non-specific adsorption on the surface of the electrode. In step (2), Step 1 immerses the electrode in a solution containing different concentrations of cells, and incubates at 37°C for 2 h; Step 2 takes out the electrode and immerses the electrode in an amplification solution, and reacts at 37°C; Step 3 takes out the electrode and adds 10 μL of 1 μM biotin-modified complementary strand Bio dropwise on the surface of the electrode, and incubates at 37°C for 0.5-1 h; Step 4 immerses the electrode in a solution containing different concentrations of SA-HRP.

3. The method according to claim 1, wherein in step (1), Step 1 co-incubates Primer-SH with 3 mM tris (2-carboxyethyl) phosphine (TCEP) at room temperature for 0.5-1 h, 10 μL of the above Primer-SH with different concentrations is added dropwise on the surface of the electrode, and the room temperature incubation time is 1-2 h. In step (2), Step 1 immerses the electrode in a solution containing different concentrations of cells, and incubates at 37°C for 2 h. ​ ​ ​ ​ ​ 4. The method for high efficient sensitive quantification of PD-L1 positive tumor cells based on steric hindrance according to claim 3, characterized in that: ​ 5. The method for high efficient sensitive quantification of PD-L1 positive tumor cells based on steric hindrance according to claim 2, characterized in that: In step (2), the amplification solution described in Step2 is 1 µM Hairpin1, 1 mM dATP, dTTP and dCTP, 1× enzyme reaction buffer, and 0.8 U / µL Bst DNA polymerase.

6. The method for high efficient sensitive quantification of PD-L1 positive tumor cells based on steric hindrance according to claim 5, characterized in that: In step (2), after Step3, 10 µL of 1 µM primer Bio was added to the surface of the electrode, and incubated at 37°C for 0.5-1 h.

7. The method of claim 6, wherein the method is a high-efficiency sensitive quantitative detection of PD-L1 positive tumor cells based on steric hindrance. In step (2), Step4, the electrode was immersed in a solution containing different concentrations of SA-HRP, and incubated at room temperature for 0.5 h. Electrochemical testing was performed in TMB color developing solution, and the i-t curve was scanned with the following parameters: starting voltage -0.1 V, sensitivity 1e-5, and operation time 100 s. The measured i-t curve reflects the change in the concentration gradient of the solution near the electrode surface, and the current size is proportional to the concentration gradient on the electrode surface. Therefore, the final stable current value after applying voltage is proportional to the concentration of HRP adsorbed on the electrode, and the stable value of the current is used as the electrical signal output.

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