Preparation method of an electrochemiluminescence sensor for detecting carcinoembryonic antigen based on electron transfer between Ag-Cys and Cu-MOFs / SOx

Abstract

The present invention relates to a preparation method of an electrochemiluminescence sensor for detecting carcinoembryonic antigen based on the electron transfer between Ag-Cys and Cu-MOFs / SOx, belonging to the technical field of novel sensor construction. Based on the good specificity between antigen and antibody, the sensor uses L-Cys-functionalized Ag hybrid nanomaterial Ag-Cys as the substrate luminescent material, and Cu-MOFs encapsulated with sarcosine oxidase as the quencher, and constructs an electrochemiluminescence sensor through layer-by-layer assembly. The electrochemiluminescence sensor constructed by the present invention has a wide detection range, high sensitivity and low detection limit, and has important significance for the detection of carcinoembryonic antigen.

Description

Field of Technology

[0001] The present invention relates to a preparation method of an electrochemiluminescence sensor for detecting carcinoembryonic antigen based on electron transfer between Ag-Cys and Cu-MOFs / SOx. The present invention uses L-Cys-functionalized Ag hybrid nanomaterial Ag-Cys as the substrate luminescent material, and Cu-MOFs encapsulated with sarcosine oxidase as the quencher. An electrochemiluminescence sensor constructed by layer-by-layer assembly can achieve specific detection of carcinoembryonic antigen, belonging to the field of new sensor construction technology. Background Art

[0002] Carcinoembryonic antigen (CEA) is a tumor-associated antigen first extracted from colon cancer and embryonic tissues. In the past, CEA was used as a specific marker for the early diagnosis of colon cancer and rectal cancer. Studies have found that not only can the CEA value increase in malignant tumors of the gastrointestinal tract, but it can also increase in the sera of breast cancer, lung cancer, and other malignant tumors. Therefore, carcinoembryonic antigen is a broad-spectrum tumor marker and has important clinical value in the differential diagnosis, disease monitoring, and efficacy evaluation of malignant tumors. As an emerging product of the combination of electrochemistry and luminescence technologies, electrochemiluminescence has the advantages of low background noise, wide dynamic range, simple instrument equipment, and high sensitivity, and is favored by many scholars in the fields of biological analysis, food safety analysis, and environmental pollution monitoring.

[0003] The present invention constructs a novel electrochemiluminescence sensor based on nanoscale functional materials for the detection of carcinoembryonic antigen. Using L-Cys-functionalized Ag hybrid nanomaterial Ag-Cys as the substrate luminescent material and Cu-MOFs encapsulated with sarcosine oxidase as the quencher, the detection of carcinoembryonic antigen is achieved. The test results show that the electrochemiluminescence sensor has high sensitivity, low detection limit, and good stability. Based on the above findings, the inventor completed the present invention. Summary of the Invention

[0004] One of the purposes of the present invention is to construct a novel electrochemiluminescence sensor using L-Cys-functionalized Ag hybrid nanomaterial Ag-Cys as the substrate luminescent material and Cu-MOFs encapsulated with sarcosine oxidase as the quencher.

[0005] Another purpose of the present invention is to provide a preparation method of a novel electrochemiluminescence sensor based on electron transfer. The sensor prepared by this method has good stability, selectivity, high sensitivity, and good reproducibility.

[0006] The third purpose of the present invention is to realize the construction of the electrochemiluminescence sensor and effectively detect carcinoembryonic antigen, achieving the use of the electrochemiluminescence sensor in the determination of carcinoembryonic antigen.

[0007] Technical solution of the present invention

[0008] 1. Preparation method of an electrochemiluminescence sensor for detecting carcinoembryonic antigen based on resonance energy transfer between L-Cys-functionalized Ag hybrid nanomaterial Ag-Cys and Cu-MOFs / SOx

[0009] (1) Polish a glassy carbon electrode with a diameter of 4 mm using Al2O3 polishing powder, wash it clean with ultrapure water, and drop 6 μL of a primary antibody marker Ag-Cys-Ab1 solution of L-Cys-functionalized Ag hybrid nanomaterial Ag-Cys combined with carcinoembryonic antigen recognition antibody with a concentration of 0.25 - 2.5 mg mL -1 onto the electrode surface, and dry it into a film at room temperature;

[0010] (2) Drop 3 μL of a BSA solution with a mass fraction of 0.1% onto the electrode surface, wash it with ultrapure water, and dry it at room temperature;

[0011] (3) Drop 6 μL of a series of different concentrations of carcinoembryonic antigen with a concentration of 0.00005 - 50 ng mL -1 onto the electrode surface, incubate for 2 h, rinse with ultrapure water, and dry it at room temperature;

[0012] (4) Drop 6 μL of a secondary antibody marker Cu-MOFs / SOx-Ab2 solution of Cu-MOFs encapsulated with sarcosine oxidase combined with carcinoembryonic antigen recognition antibody, rinse with ultrapure water, and dry it at room temperature to obtain an electrochemiluminescence sensor;

[0013] The preparation steps of the secondary antibody marker Cu-MOFs / SOx-Ab2 solution of Cu-MOFs encapsulated with sarcosine oxidase combined with carcinoembryonic antigen recognition antibody are as follows:

[0014] (1) Preparation of Cu-MOFs

[0015] First, dissolve 0.3 g of Cu(CH3COO)2 and 4 g of C6H5COOH as a regulator in 30 mL of n-butanol to obtain solution A, dissolve 0.8 g of H3BTC in 30 mL of DMF to prepare solution B, slowly add solution B to solution A and stir for 30 min. Observe blue precipitate during the addition of H3BTC. Centrifuge the obtained precipitate and wash it 3 times with ethanol to remove the solvent and remaining unreacted organic acids. Dry the obtained product in vacuum at 60 °C for 12 h to obtain Cu-MOFs;

[0016] (2) Preparation of the secondary antibody marker Cu-MOFs / SOx-Ab2 solution of Cu-MOFs encapsulated with sarcosine oxidase combined with carcinoembryonic antigen recognition antibody

[0017] Disperse 10 mg of the prepared Cu-MOFs in 1 mL of absolute ethanol. Subsequently, mix the Cu-MOFs solution with 6 mL of an aqueous solution containing 0.542 mg mL -1 of EDC and 1.125 mg mL -1 of NHS, react for 30 min under vigorous stirring. Then, while stirring, add 3 mL of an aqueous solution of SOx at 0.67 mg mL -1 dropwise into the above mixture. After stirring for 4 h, centrifuge and wash the obtained Cu-MOFs / SOx three times with water, and collect the precipitate;

[0018] Add 100 μL of the carcinoembryonic antigen recognition antibody Ab2 at 10 μg mL -1 to 1 mL of an aqueous solution of Cu-MOFs / SOx at 1.5 mg mL -1 , incubate at 4 °C for 12 h. Subsequently, add 100 μL of 1% BSA to block non-specific sites and centrifuge to remove unbound antibodies. Disperse the obtained solid in 1 mL of PBS at pH 7.4 to obtain a secondary antibody-labeled solution of Cu-MOFs / SOx-Ab2 with sarcosine oxidase encapsulated and combined with the carcinoembryonic antigen recognition antibody.

[0019] 2. Preparation of the primary antibody-labeled solution of L-Cys-functionalized Ag hybrid nanomaterial Ag-Cys combined with the carcinoembryonic antigen recognition antibody, L-Cys-functionalized Ag hybrid nanomaterial Ag-Cys-Ab1 solution

[0020] (1) Preparation of Ag-Cys

[0021] Mix an aqueous solution of AgNO3 and L-Cys in a molar ratio of 1:2. After that, adjust the pH value to 10 with 1 M NaOH solution. Then, reflux at 95 °C for 2 h. Centrifuge the refluxed product at a speed of 11000 r min -1 and wash with ultrapure water, then freeze-dry to obtain Ag-Cys;

[0022] (2) Preparation of the primary antibody-labeled solution of Ag-Cys combined with the carcinoembryonic antigen recognition antibody, Ag-Cys-Ab1 solution

[0023] Use EDC and NHS to activate the primary antibody-labeled product of the carcinoembryonic antigen capture antibody at 1 - 5 μg mL -1 at 4 °C, and then co-incubate with Ag-Cys for 6 h to obtain the primary antibody-labeled solution Ag-Cys-Ab1.

[0024] 3. Detection of carcinoembryonic antigen

[0025] (1) Ag / AgCl was used as the reference electrode, a platinum wire as the counter electrode, and the prepared electrochemiluminescence sensor as the working electrode, which were correctly connected in the dark box of the chemiluminescence detector. The electrochemical workstation and the chemiluminescence detector were connected together. The high voltage of the photomultiplier tube was set to 800 V, and the test was carried out in a PBS buffer solution with a pH of 7.4 containing 20 - 100 mmol L -1 potassium persulfate;

[0026] (2) The carcinoembryonic antigen standard solution was detected by electrochemiluminescence method, and the voltage test range was -1.8 - 0 V;

[0027] (3) Observe the electrochemiluminescence intensity of the sensor before and after the addition of carcinoembryonic antigen, then record the linear relationship between the electrochemiluminescence intensity value and the carcinoembryonic antigen concentration, and draw a working curve.

[0028] Advantageous effects of the present invention

[0029] (1) The inventor of the present invention applied the L-Cys functionalized Ag hybrid nanomaterial Ag-Cys as the substrate luminescent material and the sarcosine oxidase encapsulated Cu-MOFs as the quencher to the preparation of the electrochemiluminescence sensor. Silver-based nanomaterials are widely used in various applications due to their optical properties and surface plasmon resonance properties, and also have excellent catalytic performance. L-Cys has good biocompatibility and can firmly bind the carcinoembryonic antigen capture antibody, and these molecules can be used as passivators of the interface. The sarcosine oxidase encapsulated Cu-MOFs can increase the immobilization amount of the recognition antibody while having a quenching effect. The characteristics of these materials improve the sensitivity and stability of the sensor.

[0030] (2) The present invention uses a novel donor-acceptor pair with energy level matching leading to electron transfer to construct a sandwich-type electrochemiluminescence sensor and effectively detect carcinoembryonic antigen, and this method is relatively simple to operate.

[0031] (3) The electrochemiluminescence sensor prepared by the present invention is used for the detection of carcinoembryonic antigen. This electrochemiluminescence sensor has high stability, good reproducibility, high sensitivity, and a wide linear range, and can achieve simple, rapid, highly sensitive and specific detection. Specific embodiments

[0032] Example 1 A preparation method of an electrochemiluminescence sensor for detecting carcinoembryonic antigen based on electron transfer between Ag-Cys and Cu-MOFs / SOx

[0033] (1) The glassy carbon electrode with a diameter of 4 mm was polished with Al2O3 polishing powder, cleaned with ultrapure water, and 6 µL of 0.25 mgmL -1The anti - body marker Ag - Cys - Ab1 solution was dropped onto the electrode surface and air - dried to form a film at room temperature;

[0034] (2)Drop 3 μL of 0.1% BSA solution by mass fraction onto the electrode surface, wash it with ultrapure water, and air - dry it at room temperature;

[0035] (3)Drop 6 μL of a series of carcinoembryonic antigens with different concentrations ranging from 0.00005 to 50 ng / mL -1 onto the electrode surface, incubate for 2 h, rinse with ultrapure water, and air - dry at room temperature;

[0036] (4)Drop 6 μL of the secondary antibody marker Cu - MOFs / SOx - Ab2 solution of the carcinoembryonic antigen - recognizing antibody encapsulated with sarcosine oxidase, rinse with ultrapure water, and air - dry at room temperature to obtain an electrochemiluminescence sensor.

[0037] Example 2 Preparation method of an electrochemiluminescence sensor for detecting carcinoembryonic antigen based on electron transfer between Ag - Cys and Cu - MOFs / SOx

[0038] (1)Polish a glassy carbon electrode with a diameter of 4 mm using Al2O3 polishing powder, clean it with ultrapure water, and drop 6 μL of 1.5 mg / mL -1 anti - body marker Ag - Cys - Ab1 solution onto the electrode surface and air - dry it to form a film at room temperature;

[0039] (2)Drop 3 μL of 0.1% BSA solution by mass fraction onto the electrode surface, wash it with ultrapure water, and air - dry it at room temperature;

[0040] (3)Drop 6 μL of a series of carcinoembryonic antigens with different concentrations ranging from 0.00005 to 50 ng / mL -1 onto the electrode surface, incubate for 2 h, rinse with ultrapure water, and air - dry at room temperature;

[0041] (4)Drop 6 μL of the secondary antibody marker Cu - MOFs / SOx - Ab2 solution of the carcinoembryonic antigen - recognizing antibody encapsulated with sarcosine oxidase, rinse with ultrapure water, and air - dry at room temperature to obtain an electrochemiluminescence sensor.

[0042] Example 3 Preparation method of an electrochemiluminescence sensor for detecting carcinoembryonic antigen based on electron transfer between Ag - Cys and Cu - MOFs / SOx

[0043] (1)Polish a glassy carbon electrode with a diameter of 4 mm using Al2O3 polishing powder, clean it with ultrapure water, and drop 6 μL of 2.5 mg / mL -1The anti - body marker Ag - Cys - Ab1 solution was dropped onto the electrode surface and air - dried to form a film at room temperature;

[0044] (2)3 μL of a 0.1% BSA solution was dropped onto the electrode surface, washed with ultrapure water, and air - dried at room temperature;

[0045] (3)6 μL of a series of carcinoembryonic antigen solutions with different concentrations ranging from 0.00005 to 50 ng / mL was dropped onto the electrode surface, incubated for 2 h, rinsed with ultrapure water, and air - dried at room temperature; -1

[0046] (4)6 μL of the secondary - antibody marker Cu - MOFs / SOx - Ab2 solution of the carcinoembryonic - antigen - recognizing antibody encapsulated with sarcosine oxidase was dropped, rinsed with ultrapure water, and air - dried at room temperature to prepare an electrochemiluminescence sensor.

[0047] Example 4 Detection of carcinoembryonic antigen

[0048] (1)Ag / AgCl was used as the reference electrode, a platinum wire was used as the counter electrode, and the prepared electrochemiluminescence sensor was correctly connected to the dark box of the chemiluminescence detector. The electrochemical workstation and the chemiluminescence detector were connected together. The high voltage of the photomultiplier tube was set to 800 V, and the test was carried out in a PBS buffer solution containing 20 mmol / L potassium persulfate; -1

[0049] (2)The carcinoembryonic antigen standard solution was detected by electrochemiluminescence method, and the voltage test range was - 1.8 to 0 V;

[0050] (3)The electrochemiluminescence intensity of the sensor before and after the addition of carcinoembryonic antigen was observed, and then the linear relationship between the electrochemiluminescence intensity value and the carcinoembryonic antigen concentration was recorded to plot the working curve.

[0051] Example 5 Detection of carcinoembryonic antigen

[0052] (1)Ag / AgCl was used as the reference electrode, a platinum wire was used as the counter electrode, and the prepared electrochemiluminescence sensor was correctly connected to the dark box of the chemiluminescence detector. The electrochemical workstation and the chemiluminescence detector were connected together. The high voltage of the photomultiplier tube was set to 800 V, and the test was carried out in a PBS buffer solution containing 40 mmol / L potassium persulfate; -1

[0053] (2)The carcinoembryonic antigen standard solution was detected by electrochemiluminescence method, and the voltage test range was - 1.8 to 0 V;

[0054] ​​​(3) Observe the electrochemiluminescence intensity of the sensor before and after adding carcinoembryonic antigen, then record the linear relationship between the electrochemiluminescence intensity value and the carcinoembryonic antigen concentration, and plot the working curve.

[0055] Example 6 Detection of Carcinoembryonic Antigen

[0056] (1) Use Ag / AgCl as the reference electrode, a platinum wire as the counter electrode, and the prepared electrochemiluminescence sensor as the working electrode, and correctly connect them in the dark box of the chemiluminescence detector. Connect the electrochemical workstation and the chemiluminescence detector together, set the high voltage of the photomultiplier tube to 800 V, and conduct tests in a PBS buffer solution containing 60 mmol L -1 potassium persulfate;

[0057] (2) Detect the carcinoembryonic antigen standard solution by electrochemiluminescence method, and the voltage test range is -1.8 to 0 V;

[0058] (3) Observe the electrochemiluminescence intensity of the sensor before and after adding carcinoembryonic antigen, then record the linear relationship between the electrochemiluminescence intensity value and the carcinoembryonic antigen concentration, and plot the working curve.

[0059] Example 7 Detection of Carcinoembryonic Antigen

[0060] (1) Use Ag / AgCl as the reference electrode, a platinum wire as the counter electrode, and the prepared electrochemiluminescence sensor as the working electrode, and correctly connect them in the dark box of the chemiluminescence detector. Connect the electrochemical workstation and the chemiluminescence detector together, set the high voltage of the photomultiplier tube to 800 V, and conduct tests in a PBS buffer solution containing 80 mmol L -1 potassium persulfate;

[0061] (2) Detect the carcinoembryonic antigen standard solution by electrochemiluminescence method, and the voltage test range is -1.8 to 0 V;

[0062] (3) Observe the electrochemiluminescence intensity of the sensor before and after adding carcinoembryonic antigen, then record the linear relationship between the electrochemiluminescence intensity value and the carcinoembryonic antigen concentration, and plot the working curve.

[0063] Example 8 Detection of Carcinoembryonic Antigen

[0064] (1) Use Ag / AgCl as the reference electrode, a platinum wire as the counter electrode, and the prepared electrochemiluminescence sensor as the working electrode, and correctly connect them in the dark box of the chemiluminescence detector. Connect the electrochemical workstation and the chemiluminescence detector together, set the high voltage of the photomultiplier tube to 800 V, and conduct tests in a PBS buffer solution containing 100 mmol L -1 potassium persulfate;

[0065] (2)Detect the carcinoembryonic antigen standard solution by electrochemiluminescence method, and its voltage test range is -1.8 to 0 V;

[0066] (3)Observe the electrochemiluminescence intensity of the sensor before and after adding carcinoembryonic antigen, then record the linear relationship between the electrochemiluminescence intensity value and the carcinoembryonic antigen concentration, and draw a working curve.

Claims

1. A preparation method of an electrochemiluminescence sensor for detecting carcinoembryonic antigen based on electron transfer between Ag-Cys and Cu-MOFs / SOx, characterized in that, It includes the following steps: (1) Polish a glassy carbon electrode with a diameter of 4 mm using Al2O3 polishing powder, wash it clean with ultrapure water, and drop 6 μL of an Ag-Cys-Ab1 solution of an L-Cys-functionalized Ag hybrid nanomaterial Ag-Cys combined with a primary antibody marker for carcinoembryonic antigen recognition onto the electrode surface. Air-dry it to form a film at room temperature; -1 ​ (2) Drop 3 μL of a 0.1% BSA solution by mass onto the electrode surface, wash it with ultrapure water, and air-dry it at room temperature; (3)Drop 6 μL of a series of carcinoembryonic antigens with different concentrations of 0.00005 - 50 ng mL -1 onto the electrode surface, incubate for 2 h, rinse with ultrapure water, and air-dry at room temperature; (4) Drop 6 μL of the Cu-MOFs encapsulated with sarcosine oxidase and conjugated with the secondary antibody marker Cu-MOFs / SOx-Ab2 of the carcinoembryonic antigen recognition antibody, rinse it with ultrapure water, and air-dry it at room temperature to obtain an electrochemiluminescence sensor; The preparation steps of the Cu-MOFs encapsulated with sarcosine oxidase and conjugated with the secondary antibody marker Cu-MOFs / SOx-Ab2 of the carcinoembryonic antigen recognition antibody are as follows: (1) Preparation of Cu-MOFs First, dissolve 0.3 g of Cu(CH3COO)2 and 4 g of C6H5COOH as a regulator in 30 mL of n-butanol to obtain solution A, dissolve 0.8 g of H3BTC in 30 mL of DMF to prepare solution B, slowly add solution B to solution A and stir for 30 min. Blue precipitate is observed during the addition of H3BTC. Centrifuge the obtained precipitate and wash it 3 times with ethanol to remove the solvent and the remaining unreacted organic acid. Vacuum-dry the obtained product at 60 °C for 12 h to obtain Cu-MOFs; (2) Preparation of the Cu-MOFs encapsulated with sarcosine oxidase and conjugated with the secondary antibody marker Cu-MOFs / SOx-Ab2 of the carcinoembryonic antigen recognition antibody Disperse 10 mg of the prepared Cu-MOFs in 1 mL of absolute ethanol. Subsequently, mix the Cu-MOFs solution with 6 mL of an aqueous solution containing 0.542 mg mL -1 of EDC and 1.125 mg mL -1 of NHS, react for 30 min under vigorous stirring. Then, while stirring, slowly add 3 mL of an aqueous solution of SOx at a concentration of 0.67 mg mL -1 dropwise into the above mixture. After stirring for 4 h, centrifuge and wash the obtained Cu-MOFs / SOx three times with water, and collect the precipitate; Add 100 μL of 10 μg mL -1 carcinoembryonic antigen recognition antibody Ab2 to 1 mL of 1.5 mg mL -1 Cu-MOFs / SOx aqueous solution, incubate at 4 °C for 12 h, then add 100 μL of 1% BSA to block non-specific sites and centrifuge to remove unbound antibodies. Disperse the resulting solid in 1 mL of PBS at pH 7.4 to obtain the secondary antibody-labeled solution of sarcosine oxidase-coated Cu-MOFs conjugated with carcinoembryonic antigen recognition antibody, Cu-MOFs / SOx-Ab2 solution.

2. The preparation method of an electrochemiluminescence sensor for detecting carcinoembryonic antigen based on electron transfer between Ag-Cys and Cu-MOFs / SOx as claimed in claim 1, wherein, The preparation steps of the L-Cys-functionalized Ag hybrid nanomaterial Ag-Cys and conjugated with the primary antibody marker Ag-Cys-Ab1 of the carcinoembryonic antigen recognition antibody are as follows: (1) Preparation of Ag-Cys An aqueous solution of AgNO3 and L-Cys was mixed at a molar ratio of 1:

2. After that, the pH value was adjusted to 10 with 1 M NaOH solution, and the mixture was refluxed at 95 °C for 2 h. Then, the refluxed product was centrifuged at a speed of 11000 r min -1 -1 and washed with ultrapure water, followed by freeze-drying to obtain Ag-Cys; (2) Preparation of the Ag-Cys and conjugated with the primary antibody marker Ag-Cys-Ab1 of the carcinoembryonic antigen recognition antibody Capture the carcinoembryonic antigen antibody with a concentration of 1-5 μg / mL -1 The primary antibody marker of the carcinoembryonic antigen capture antibody was activated with EDC and NHS at 4 °C, and then incubated with Ag-Cys for 6 h to obtain the primary antibody marker Ag-Cys-Ab1 solution.