A method for constructing an electrochemical biosensor based on a double conductive antifouling hydrogel and application thereof

Abstract

The application discloses a construction method and application of an electrochemical biosensor based on a double-conducting antifouling hydrogel, and belongs to the fields of biochemical analysis, functional nanomaterials and life science technologies.In the application, an antifouling conductive hydrogel is designed and synthesized by taking MXene as a conductive framework.The inherent surface hydrophilicity of the hydrogel endows the hydrogel with good antifouling capacity.Meanwhile, the introduction of gamma-methacryloxypropyl trimethoxysilane and poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid improves the stability and conductivity of the hydrogel.In addition, a large number of [Ru(NH3)6] 3+ can be loaded on MXene nanosheets, realizing effective integration of antifouling and internal standard to improve the detection precision of the biosensor.Based on this, the electrochemical antifouling biosensor can realize sensitive detection of carcinoembryonic antigen in a wide linear range, and the detection limit is as low as 0.41 pg / mL.

Description

Technical Field

[0001] The invention discloses a construction method of an electrochemical biosensor based on a double-conductive anti-sewage gel and its application in carcinoembryonic antigen detection, belonging to the fields of biochemical analysis, functional nanomaterials and life science technology. Background Art

[0002] Electrochemical biosensors have attracted widespread attention for early disease diagnosis due to their advantages, including low background signal, fast response, wide linear range, and simple instrumentation. In complex biological environments, the adsorption of nonspecific substances such as proteins, polysaccharides, and lipids on the sensing interface reduces the detection sensitivity and accuracy of biosensors, posing a challenge to the practical application of electrochemical analysis. Carcinoembryonic antigen (CEA), as a broad-spectrum tumor marker, has important clinical value in the differential diagnosis of malignant tumors, disease monitoring, and therapeutic efficacy evaluation. Therefore, it is of great significance to construct an efficient electrochemical antifouling biosensor for the sensitive and accurate detection of CEA in serum.

[0003] In order to solve the problem of biological fouling at the electrochemical sensing interface, a variety of antifouling materials have been developed. Among them, bovine serum albumin (BSA) can be regarded as the most primitive antifouling material as a blocking agent to prevent nonspecific adsorption. However, BSA is highly sensitive to the modification interface and detection environment, and is prone to protein hydrolysis and denaturation under certain conditions, which limits its application in electrochemical antifouling systems. With the development of antifouling systems, polyethylene glycol (PEG), peptides, zwitterions and hydrogels have been found to have antifouling properties. However, the oxidizability of PEG greatly affects its antifouling ability; the synthesis and modification of zwitterions are relatively difficult, which also limits its application as an antifouling material; the hydrolysis and denaturation of proteins affect the stability of antifouling peptides; the modification of hydrogels at the limited electrode interface will hinder the electron transfer between the electrode and the signal probe, thereby reducing the output signal of the electrochemical sensor. These problems have hindered the further development of antifouling interfaces.

[0004] Given the impact of anti-sewage gel on the output signal of the electrochemical sensing system, it is very important to develop hydrogels with excellent conductivity. So far, the development of conductive hydrogels has gradually matured. The introduction of conductive polymers, nanoparticles and conductive ions gives hydrogels good conductive properties. On this basis, in order to promote the application of anti-sewage gel in microelectrodes, it is necessary to integrate the above-mentioned conductive materials and develop dual or multiple conductive hydrogels. In addition, flexible conductive hydrogels are easily delaminated from the substrate, thereby affecting their electrochemical stability and hydrophilicity, which also poses a challenge to their application in electrochemical analysis. To meet the above challenges, the present invention introduces MXene as a conductive element, whose unique sheet structure provides a larger active surface area and stronger conductivity. In addition, γ-methacryloxypropyltrimethoxysilane (KH570) and poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid (PEDOT:PSS) are introduced to avoid the aggregation of MXene, thereby improving the stability and conductivity of the anti-sewage gel.

[0005] In electrochemical analysis, the detection results are often interfered with by background signals, instrument errors, and environmental factors, thus affecting the detection accuracy of the sensor. The ratio sensing strategy introduces an internal standard as a correction factor, and evaluates the detection results by comparing two independent signals, effectively reducing the adverse effects of the above interferences. Based on interface antifouling and ratio detection technology, the present invention constructs a ratio-type electrochemical biosensor based on a dual-conductive anti-fouling gel, which has good detection sensitivity and accuracy, and a detection limit as low as 0.41pg / mL, which better meets the needs of clinical precision detection of CEA. Summary of the Invention

[0006] One of the technical tasks of the present invention is to make up for the shortcomings of the existing technology and prepare a highly efficient dual-conductive anti-sewage gel for electrochemical biological analysis, which greatly improves the specificity and service life of electrochemical biosensors.

[0007] The second technical task of the present invention is to use interface anti-fouling and ratio detection technology to construct an electrochemical biosensor based on double conductive anti-fouling gel, which improves the detection sensitivity and accuracy, and the raw materials used are low in cost, the preparation process is simple, and the operation is safe.

[0008] The third technical task of the present invention is to provide the use of the electrochemical biosensor based on the dual conductive anti-fouling gel constructed by the construction method, namely, for the sensitive and accurate detection of carcinoembryonic antigen; the constructed electrochemical anti-fouling biosensor has a low detection limit, a wide detection range and good specificity for CEA detection, and can be used for the differential diagnosis, disease monitoring and efficacy evaluation of various malignant tumors, and has certain industrial application prospects.

[0009] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] 1. A method for constructing an electrochemical biosensor based on a dual-conductive anti-sewage gel

[0011] The glassy carbon electrode was polished to a mirror surface with Al2O3 slurry and washed alternately with ethanol and ultrapure water. 3-7 μL of pregel solution was quickly drop-coated on the polished electrode surface to form an antifouling interface. Subsequently, the modified electrode was incubated in a 50 μM complementary chain H1 solution for 1 h. Next, the electrode was incubated in a 50 μM ferrocene-binding aptamer chain H2 solution for 1 h. Finally, the modified electrode was incubated in different concentrations of CEA solution for 50 min to construct a dual-conductor-based electrochemical device. The electrochemical biosensor of the anti-sewage gel is prepared by dissolving 20 mg of carboxylated MXene in 10 mL of ultrapure water and ultrasonically dispersing it uniformly; adding 20 μL of a mixture of 0.1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.4 M N-hydroxysuccinimide (NHS) and reacting for 3 h to activate the carboxyl group; adding 48.5 mL of [Ru(NH3)6] 3+ , and then the resulting mixture was centrifuged and washed three times, and vacuum dried at 25 ° C for 8 h to obtain MXene-Ru; 62 mg of MXene-Ru was uniformly dispersed in 10 mL of ultrapure water and ultrasonically treated for 30 min; then 15.7 μL of KH570 was added and stirred at room temperature for 48 h; the mixture was centrifuged and washed three times, and vacuum dried at 25 ° C for 2 h; 12.5 mg of the dried product was dispersed in 5 mL of ultrapure water and ultrasonically treated for 30 min; 0.5 mL of PEDOT:PSS was added and stirred in an ice bath under nitrogen protection for 2 h; next, 84.5 mg of N-isopropylacrylamide monomer, 3 mg of N,N'-methylenebisacrylamide, and 13.5 mg of K2S2O8 were added in sequence under stirring; finally, 2 μL of N,N,N',N'-tetramethylethylenediamine was added to obtain a pre-gel solution;

[0012] The carboxylated MXene is prepared by dissolving 1.6 g of LiF in 20 mL of HCl and stirring at 45° C. for 48 h; slowly adding 1 g of Ti3AlC2 to the solution to etch away Al; then, centrifuging the etched product at 4000 rpm, washing with ultrapure water, and vacuum drying at 45° C. for 12 h to obtain MXene nanosheets; then uniformly dispersing 60 mg of MXene in 40 mL of ultrapure water and ultrasonically treating for 30 min, followed by adding 0.51 g of ClCH2COOH and stirring at 0° C. for 40 min; finally, adding 1.92 mL of NaOH and stirring at 60° C. for 4 h; the resulting solution is centrifuged and washed, and then vacuum dried at 60° C. for 12 h to obtain the carboxylated MXene;

[0013] The complementary chain H1 solution is obtained by uniformly dispersing the complementary chain H1 in a TE buffer solution having a pH of 7.4;

[0014] The ferrocene-binding aptamer chain H2 solution is prepared by uniformly dispersing the aptamer chain H2 in a TE buffer solution having a pH of 7.4, followed by sequentially adding 2 mg of carboxylated ferrocene and 10 μL of a mixed solution of 0.1 M EDC and 0.4 M NHS, and reacting for 5 hours to obtain the ferrocene-binding aptamer chain H2 solution; the CEA solutions of different concentrations are prepared by uniformly dispersing CEA at concentrations of 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1 μg / mL in a phosphate buffered saline solution (PBS) having a pH of 7.4 to obtain CEA solutions of different concentrations;

[0015] Each step of electrode interface modification requires gentle rinsing with ultrapure water to remove incompletely bound reagents. 2. Application of the electrochemical biosensor based on the dual conductive anti-sewage gel constructed by the construction method for the detection of a broad-spectrum tumor marker CEA

[0016] Differential pulse voltammetry (DPV) testing was performed in PBS with a pH of 7.0-8.0 using a three-electrode system using the constructed electrochemical biosensor as the working electrode, a platinum electrode as the counter electrode, and a calomel electrode as the reference electrode. The scanning potential range applied in the experiment was -0.6-0.6 V, the pulse width was 0.05 s, and the amplitude was 50 mV. A linear curve was drawn based on the DPV test results, and the applicable detection range of the constructed electrochemical biosensor was 1 pg / mL-1 μg / mL, with a detection limit as low as 0.41 pg / mL. In addition, the test results showed that the constructed electrochemical biosensor had excellent sensitivity, precision, electrochemical stability, storage stability, specificity, and reproducibility, and was suitable for trace detection of CEA in serum.

[0017] Beneficial technical effects of the present invention:

[0018] 1. The present invention prepares a highly efficient dual-conductive anti-fouling gel for electrochemical analysis. Using MXene as the conductive backbone, KH570 and the conductive polymer PEDOT:PSS are introduced to enhance electrochemical stability, promoting the application of conductive hydrogels in microelectrode analysis. Furthermore, the superhydrophilicity of the hydrogel imparts excellent anti-fouling capabilities to the sensing interface, and the introduction of KH570 enhances the structural stability of the hydrogel, thereby increasing the service life of the hydrogel's anti-fouling interface.

[0019] 2. The present invention constructs an electrochemical biosensor based on a dual-conductive anti-fouling gel; the combination of interface anti-fouling and ratio analysis technology improves the detection sensitivity and accuracy of the electrochemical biosensor; at the same time, the encapsulation of the internal standard factor in the anti-fouling gel avoids secondary electrode modification, effectively eliminates operational errors, and simplifies the construction process of the electrochemical biosensor.

[0020] 3. The electrochemical biosensor constructed in the present invention based on the dual-conductive anti-sewage gel has a low detection limit, a wide detection range, and excellent detection precision, electrochemical stability, storage stability, specificity, and reproducibility for CEA detection. It is suitable for the differential diagnosis, disease monitoring, and efficacy evaluation of various malignant tumors. The constructed electrochemical biosensor has a simple preparation process and low cost, and has certain prospects for industrial application. DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. Any changes made to the technical solution of the present invention by professionals in this field should fall within the protection scope of the present invention.

[0022] Example 1 A method for constructing an electrochemical biosensor based on a dual-conductive anti-fouling gel: A glassy carbon electrode was polished to a mirror surface with Al2O3 slurry and washed alternately with ethanol and ultrapure water; 3 μL of pre-gel solution was quickly drop-coated onto the polished electrode surface to form an anti-fouling interface; then, the modified electrode was incubated in a 50 μM solution of complementary chain H1 for 1 hour; next, the electrode was incubated in a 50 μM solution of ferrocene-binding aptamer chain H2 for 1 hour; finally, the modified electrode was incubated in CEA solutions of different concentrations for 50 minutes to construct an electrochemical biosensor based on a dual-conductive anti-fouling gel;

[0023] The pre-gel solution is prepared by dissolving 20 mg of carboxylated MXene in 10 mL of ultrapure water and ultrasonically dispersing it uniformly; adding 20 μL of a mixture of 0.1 M EDC and 0.4 M NHS and reacting for 3 h to activate the carboxyl groups; adding 48.5 mL of [Ru(NH3)6] 3+ , and then the resulting mixture was centrifuged and washed three times, and vacuum dried at 25 ° C for 8 h to obtain MXene-Ru; 62 mg of MXene-Ru was uniformly dispersed in 10 mL of ultrapure water and ultrasonically treated for 30 min; then 15.7 μL of KH570 was added and stirred at room temperature for 48 h; the mixture was centrifuged and washed three times, and vacuum dried at 25 ° C for 2 h; 12.5 mg of the dried product was dispersed in 5 mL of ultrapure water and ultrasonically treated for 30 min; 0.5 mL of PEDOT:PSS was added and stirred in an ice bath under nitrogen protection for 2 h; next, 84.5 mg of N-isopropylacrylamide monomer, 3 mg of N,N'-methylenebisacrylamide, and 13.5 mg of K2S2O8 were added in sequence under stirring; finally, 2 μL of N,N,N',N'-tetramethylethylenediamine was added to obtain a pre-gel solution;

[0024] The carboxylated MXene is prepared by dissolving 1.6 g of LiF in 20 mL of HCl and stirring at 45° C. for 48 h; slowly adding 1 g of Ti3AlC2 to the solution to etch away Al; then, centrifuging the etched product at 4000 rpm, washing with ultrapure water, and vacuum drying at 45° C. for 12 h to obtain MXene nanosheets; then uniformly dispersing 60 mg of MXene in 40 mL of ultrapure water and ultrasonically treating for 30 min, followed by adding 0.51 g of ClCH2COOH and stirring at 0° C. for 40 min; finally, adding 1.92 mL of NaOH and stirring at 60° C. for 4 h; the resulting solution is centrifuged and washed, and then vacuum dried at 60° C. for 12 h to obtain the carboxylated MXene;

[0025] The complementary chain H1 solution is obtained by uniformly dispersing the complementary chain H1 in a TE buffer solution having a pH of 7.4;

[0026] The ferrocene-binding aptamer chain H2 solution is prepared by uniformly dispersing the aptamer chain H2 in a TE buffer solution having a pH of 7.4, followed by sequentially adding 2 mg of carboxylated ferrocene and 10 μL of a mixed solution of 0.1 M EDC and 0.4 M NHS, and reacting for 5 hours to obtain the ferrocene-binding aptamer chain H2 solution; the CEA solutions of different concentrations are prepared by uniformly dispersing CEA at concentrations of 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1 μg / mL in PBS having a pH of 7.4 to obtain CEA solutions of different concentrations;

[0027] Each step of modification of the electrode interface requires gentle rinsing with ultrapure water to remove incompletely bound reagents. Example 2 A method for constructing an electrochemical biosensor based on a dual-conductive anti-fouling gel: A glassy carbon electrode is polished to a mirror surface with Al2O3 slurry and washed alternately with ethanol and ultrapure water; 5 μL of pre-gel solution is quickly drop-coated on the polished electrode surface to form an anti-fouling interface; then, the modified electrode is incubated in a 50 μM complementary chain H1 solution for 1 hour; next, the electrode is incubated in a 50 μM ferrocene-binding aptamer chain H2 solution for 1 hour; finally, the modified electrode is incubated in CEA solutions of different concentrations for 50 minutes to construct an electrochemical biosensor based on a dual-conductive anti-fouling gel;

[0028] The pre-gel solution is prepared by dissolving 20 mg of carboxylated MXene in 10 mL of ultrapure water and ultrasonically dispersing it uniformly; adding 20 μL of a mixture of 0.1 M EDC and 0.4 M NHS and reacting for 3 h to activate the carboxyl groups; adding 48.5 mL of [Ru(NH3)6] 3+ , and then the resulting mixture was centrifuged and washed three times, and vacuum dried at 25 ° C for 8 h to obtain MXene-Ru; 62 mg of MXene-Ru was uniformly dispersed in 10 mL of ultrapure water and ultrasonically treated for 30 min; then 15.7 μL of KH570 was added and stirred at room temperature for 48 h; the mixture was centrifuged and washed three times, and vacuum dried at 25 ° C for 2 h; 12.5 mg of the dried product was dispersed in 5 mL of ultrapure water and ultrasonically treated for 30 min; 0.5 mL of PEDOT:PSS was added and stirred in an ice bath under nitrogen protection for 2 h; next, 84.5 mg of N-isopropylacrylamide monomer, 3 mg of N,N'-methylenebisacrylamide, and 13.5 mg of K2S2O8 were added in sequence under stirring; finally, 2 μL of N,N,N',N'-tetramethylethylenediamine was added to obtain a pre-gel solution;

[0029] The carboxylated MXene is prepared by dissolving 1.6 g of LiF in 20 mL of HCl and stirring at 45° C. for 48 h; slowly adding 1 g of Ti3AlC2 to the solution to etch away Al; then, centrifuging the etched product at 4000 rpm, washing with ultrapure water, and vacuum drying at 45° C. for 12 h to obtain MXene nanosheets; then uniformly dispersing 60 mg of MXene in 40 mL of ultrapure water and ultrasonically treating for 30 min, followed by adding 0.51 g of ClCH2COOH and stirring at 0° C. for 40 min; finally, adding 1.92 mL of NaOH and stirring at 60° C. for 4 h; the resulting solution is centrifuged and washed, and then vacuum dried at 60° C. for 12 h to obtain the carboxylated MXene;

[0030] The complementary chain H1 solution is obtained by uniformly dispersing the complementary chain H1 in a TE buffer solution having a pH of 7.4;

[0031] The ferrocene-binding aptamer chain H2 solution is prepared by uniformly dispersing the aptamer chain H2 in a TE buffer solution having a pH of 7.4, followed by sequentially adding 2 mg of carboxylated ferrocene and 10 μL of a mixed solution of 0.1 M EDC and 0.4 M NHS, and reacting for 5 hours to obtain the ferrocene-binding aptamer chain H2 solution; the CEA solutions of different concentrations are prepared by uniformly dispersing CEA at concentrations of 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1 μg / mL in PBS having a pH of 7.4 to obtain CEA solutions of different concentrations;

[0032] Each step of modification of the electrode interface requires gentle rinsing with ultrapure water to remove incompletely bound reagents. Example 3 A method for constructing an electrochemical biosensor based on a dual-conductive anti-fouling gel: A glassy carbon electrode is polished to a mirror surface with Al2O3 slurry and washed alternately with ethanol and ultrapure water; 7 μL of pre-gel solution is quickly drop-coated on the polished electrode surface to form an anti-fouling interface; then, the modified electrode is incubated in a 50 μM complementary chain H1 solution for 1 hour; next, the electrode is incubated in a 50 μM ferrocene-binding aptamer chain H2 solution for 1 hour; finally, the modified electrode is incubated in different concentrations of CEA solutions for 50 minutes to construct an electrochemical biosensor based on a dual-conductive anti-fouling gel;

[0033] The pre-gel solution is prepared by dissolving 20 mg of carboxylated MXene in 10 mL of ultrapure water and ultrasonically dispersing it uniformly; adding 20 μL of a mixture of 0.1 M EDC and 0.4 M NHS and reacting for 3 h to activate the carboxyl groups; adding 48.5 mL of [Ru(NH3)6] 3+, and then the resulting mixture was centrifuged and washed three times, and vacuum dried at 25 ° C for 8 h to obtain MXene-Ru; 62 mg of MXene-Ru was uniformly dispersed in 10 mL of ultrapure water and ultrasonically treated for 30 min; then 15.7 μL of KH570 was added and stirred at room temperature for 48 h; the mixture was centrifuged and washed three times, and vacuum dried at 25 ° C for 2 h; 12.5 mg of the dried product was dispersed in 5 mL of ultrapure water and ultrasonically treated for 30 min; 0.5 mL of PEDOT:PSS was added and stirred in an ice bath under nitrogen protection for 2 h; next, 84.5 mg of N-isopropylacrylamide monomer, 3 mg of N,N'-methylenebisacrylamide, and 13.5 mg of K2S2O8 were added in sequence under stirring; finally, 2 μL of N,N,N',N'-tetramethylethylenediamine was added to obtain a pre-gel solution;

[0034] The carboxylated MXene is prepared by dissolving 1.6 g of LiF in 20 mL of HCl and stirring at 45° C. for 48 h; slowly adding 1 g of Ti3AlC2 to the solution to etch away Al; then, centrifuging the etched product at 4000 rpm, washing with ultrapure water, and vacuum drying at 45° C. for 12 h to obtain MXene nanosheets; then uniformly dispersing 60 mg of MXene in 40 mL of ultrapure water and ultrasonically treating for 30 min, followed by adding 0.51 g of ClCH2COOH and stirring at 0° C. for 40 min; finally, adding 1.92 mL of NaOH and stirring at 60° C. for 4 h; the resulting solution is centrifuged and washed, and then vacuum dried at 60° C. for 12 h to obtain the carboxylated MXene;

[0035] The complementary chain H1 solution is obtained by uniformly dispersing the complementary chain H1 in a TE buffer solution having a pH of 7.4;

[0036] The ferrocene-binding aptamer chain H2 solution is prepared by uniformly dispersing the aptamer chain H2 in a TE buffer solution having a pH of 7.4, followed by sequentially adding 2 mg of carboxylated ferrocene and 10 μL of a mixed solution of 0.1 M EDC and 0.4 M NHS, and reacting for 5 hours to obtain the ferrocene-binding aptamer chain H2 solution; the CEA solutions of different concentrations are prepared by uniformly dispersing CEA at concentrations of 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1 μg / mL in PBS having a pH of 7.4 to obtain CEA solutions of different concentrations;

[0037] Each step of electrode interface modification requires gentle rinsing with ultrapure water to remove incompletely bound reagents. Example 4: Application of the electrochemical biosensor based on the dual conductive anti-sewage gel constructed by the construction methods described in Examples 1, 2, and 3 for the detection of a broad-spectrum tumor marker, CEA.

[0038] The constructed electrochemical biosensor was used as the working electrode, the platinum electrode as the counter electrode, and the calomel electrode as the reference electrode. A three-electrode system was used to perform a DPV test in PBS with a pH value of 7.0. The scanning potential range applied in the experiment was -0.6 to 0.6 V, the pulse width was 0.05 s, and the amplitude was 50 mV. A linear curve was drawn based on the DPV test results, and the applicable detection range of the constructed electrochemical biosensor was 1 pg / mL to 1 μg / mL, with a detection limit as low as 0.41 pg / mL. In addition, the test results showed that the constructed electrochemical biosensor had excellent sensitivity, precision, electrochemical stability, storage stability, specificity, and reproducibility, and was suitable for trace detection of CEA in serum.

[0039] Example 5 Application of the electrochemical biosensor based on the dual conductive anti-sewage gel constructed by the construction methods described in Examples 1, 2 and 3 for the detection of the broad-spectrum tumor marker CEA

[0040] The constructed electrochemical biosensor was used as the working electrode, the platinum electrode as the counter electrode, and the calomel electrode as the reference electrode. A three-electrode system was used to perform a DPV test in PBS with a pH value of 7.4. The scanning potential range applied in the experiment was -0.6 to 0.6 V, the pulse width was 0.05 s, and the amplitude was 50 mV. A linear curve was drawn based on the DPV test results, and the applicable detection range of the constructed electrochemical biosensor was 1 pg / mL to 1 μg / mL, with a detection limit as low as 0.41 pg / mL. In addition, the test results showed that the constructed electrochemical biosensor had excellent sensitivity, precision, electrochemical stability, storage stability, specificity, and reproducibility, and was suitable for trace detection of CEA in serum.

[0041] Example 6 Application of the electrochemical biosensor based on the dual conductive anti-sewage gel constructed by the construction methods described in Examples 1, 2 and 3 for the detection of the broad-spectrum tumor marker CEA

[0042] The constructed electrochemical biosensor was used as the working electrode, the platinum electrode as the counter electrode, and the calomel electrode as the reference electrode. A three-electrode system was used to perform a DPV test in PBS with a pH value of 8.0. The scanning potential range applied in the experiment was -0.6 to 0.6 V, the pulse width was 0.05 s, and the amplitude was 50 mV. A linear curve was drawn based on the DPV test results, and the applicable detection range of the constructed electrochemical biosensor was 1 pg / mL to 1 μg / mL, with a detection limit as low as 0.41 pg / mL. In addition, the test results showed that the constructed electrochemical biosensor had excellent sensitivity, precision, electrochemical stability, storage stability, specificity, and reproducibility, and was suitable for trace detection of CEA in serum. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Flowchart of the construction method of the electrochemical biosensor based on the dual conductive anti-fouling gel (glassy carbon electrode: GCE; dual conductive hydrogel: KMPPH; complementary chain: H1; aptamer chain: H2; ferrocene: Fc; carcinoembryonic antigen: CEA; γ-methacryloxypropyltrimethoxysilane: KH570; poly(3,4-ethylenedioxythiophene)-polystyrenesulfonic acid: PEDOT:PSS; N,N'-methylenebisacrylamide: BIS; N-isopropylacrylamide monomer: BIPAM).

[0044] Figure 2 X-ray diffraction spectra of Ti3AlC2, MXene and K-MXene.

[0045] Figure 3 Transmission electron microscope image of MXene.

[0046] Figure 4 Infrared spectra of carboxylated MXene and K-MXene.

[0047] Figure 5 (A) Physical image and (B) elemental mapping of KMPPH.

[0048] Figure 6 Static water contact angle diagrams of (A) bare GCE and (B) KMPPH / GCE.

[0049] Figure 7 Schematic representation of the construction process of the electrochemical biosensor based on the dual-conductive, anti-sewage gel, as measured by (A) cyclic voltammetry, (B) differential pulse voltammetry, and (C) electrochemical impedance spectroscopy. (Inset: corresponding equivalent circuit diagram).

[0050] Figure 8Differential pulse voltammetry curves of (A) bare electrode and (B) KMPPH modified electrode incubated in different concentrations of fetal bovine serum; and differential pulse voltammetry curves of (C) bare electrode and (D) KMPPH modified electrode incubated in different concentrations of human serum.

[0051] Figure 9 Fluorescence imaging of the bare electrode and KMPPH modified electrode after incubation in fluorescein-conjugated bovine serum albumin solution for 90 min.

[0052] Figure 10 These are the differential pulse voltammetry curves of GCE, KMPPH / GCE, Fc-dsDNA / KMPPH / GCE, and CEA / Fc-dsDNA / KMPPH / GCE in phosphate-buffered saline.

[0053] Figure 11 This is a gel electrophoresis representation of the DNA hybridization process.

[0054] Figure 12 Condition optimization diagram of the electrochemical biosensor based on the dual-conductive anti-sewage gel, including: (A) H2-Fc concentration optimization result diagram; (B) CEA incubation time optimization result diagram.

[0055] Figure 13 (A) DPV curves of the electrochemical biosensor based on the dual-conductive anti-sewage gel after incubation with different concentrations of CEA and (B) the corresponding calibration curve; and (C) DPV curves of the control electrochemical biosensor without internal standard after incubation with different concentrations of CEA and (D) the corresponding calibration curve; where a to h represent the concentrations of 0 pg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1 μg / mL, respectively.

[0056] Figure 14 (A) Signal stability, (B) storage stability, (C) specificity, and (D) reproducibility of the electrochemical biosensor based on the dual-conductive anti-fouling gel (immunoglobulin G: IgG; prostate-specific antigen: PSA; carbohydrate antigen 125: CA125; alpha-fetoprotein: AFP).

Claims

1. A method for constructing an electrochemical biosensor based on a dual-conductive anti-sewage gel, characterized in that: The glassy carbon electrode was polished to a mirror surface with Al2O3 slurry and washed alternately with ethanol and ultrapure water. 3-7 μL of pre-gel solution was quickly drop-coated onto the polished electrode surface to form an antifouling interface. Subsequently, the modified electrode was incubated in a 50 μM solution of complementary chain H1 for 1 h. Next, the electrode was incubated in a 50 μM solution of ferrocene-binding aptamer chain H2 for 1 h. Finally, the modified electrode was incubated in different concentrations of carcinoembryonic antigen solutions for 50 min to construct an electrochemical biosensor based on a dual conductive antifouling gel. The pre-gel solution was prepared by dissolving 20 mg of carboxylated MXene in 10 mL of ultrapure water and sonicating it for uniform dispersion. 20 μL of a mixture of 0.1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.4 M N-hydroxysuccinimide was added and reacted for 3 h to activate the carboxyl groups. 48.5 mL of [Ru(NH3)6] 3+ , then the resulting mixture was centrifuged and washed three times, and vacuum dried at 25°C for 8h to obtain MXene-Ru; 62mg of MXene-Ru was uniformly dispersed in 10mL of ultrapure water and ultrasonically treated for 30min; then 15.7μL of γ-methacryloxypropyltrimethoxysilane was added and stirred at room temperature for 48h; the mixture was centrifuged and washed three times, and vacuum dried at 25°C for 2h; 12.5mg of the dry product was dispersed in 5mL of ultrapure water and ultrasonically treated for 30min; 0.5mL of poly(3,4-ethylenedioxythiophene)-polystyrenesulfonic acid was added and stirred in an ice bath under nitrogen protection for 2h; next, 8 4.5 mg of N-isopropylacrylamide monomer, 3 mg of N,N'-methylenebisacrylamide and 13.5 mg of K2S2O8; finally, 2 μL of N,N,N',N'-tetramethylethylenediamine was added to obtain a pregel solution; the different concentrations of carcinoembryonic antigen were obtained by uniformly dispersing the carcinoembryonic antigen in a phosphate buffered saline solution with a pH of 7.4 at concentrations of 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL and 1 μg / mL respectively; each step of electrode interface modification required gentle rinsing with ultrapure water to remove incompletely bound reagents.

2. The method for constructing an electrochemical biosensor based on a dual conductive anti-sewage gel according to claim 1, wherein: The carboxylated MXene is prepared by dissolving 1.6 g of LiF in 20 mL of HCl and stirring at 45°C for 48 h; slowly adding 1 g of Ti3AlC2 to the above solution to etch away Al; next, the resulting etched product is centrifuged at 4000 rpm, washed with ultrapure water, and vacuum-dried at 45°C for 12 h to obtain MXene nanosheets; 60 mg of MXene is uniformly dispersed in 40 mL of ultrapure water, ultrasonically treated for 30 min, and then 0.51 g of ClCH2COOH is added and stirred at 0°C for 40 min; finally, 1.92 mL of NaOH is added and stirred at 60°C for 4 h; the resulting solution is centrifuged and washed, and then vacuum-dried at 60°C for 12 h to obtain the carboxylated MXene.

3. The method for constructing an electrochemical biosensor based on a dual conductive anti-sewage gel according to claim 1, wherein: The complementary chain H1 solution is obtained by uniformly dispersing the complementary chain H1 in a TE buffer solution having a pH of 7.

4.

4. The method for constructing an electrochemical biosensor based on a dual conductive anti-sewage gel according to claim 1, wherein: The ferrocene-binding aptamer chain H2 solution is prepared by uniformly dispersing the aptamer chain H2 in a TE buffer solution having a pH of 7.4, followed by sequentially adding 2 mg of carboxylated ferrocene and 10 μL of a mixed solution of 0.1 M 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.4 M N-hydroxysuccinimide, and reacting for 5 hours to obtain the ferrocene-binding aptamer chain H2 solution.

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