Electrochemical cell sensor for enhancing electron transfer based on cell spreading, preparation and application

Abstract

The invention discloses an electrochemical cell sensor for enhancing electron transfer based on cell spreading, preparation and application. The electrochemical cell sensor comprises a first antibody / RGO (at) Ti3C2Tx cell spreading promoting unit and a FeCN (at) second antibody signal output unit, a second antibody in the FeCN and second antibody signal output unit is covalently connected with FeCN, the FeCN targets the surface of a cell membrane through the second antibody by taking monatomic Fe as a catalytic activity center, and a precipitation type TMB reaction solution is catalyzed by simulating peroxidase activity to generate TMB < + > precipitates around cells; the cell concentration is quantified by quantifying the current generated by the electroactive TMB +. The electrochemical cell sensor can enhance electric signal response by promoting cell height spreading, can be used for detecting cell concentration to realize electric signal output of single cells, and can also be used for detecting cell membrane protein.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, and relates to an electrochemical cell sensor based on cell spreading to enhance electron transfer, its preparation and application. Background Technology

[0002] Rapid and sensitive detection of rare cell abundance and membrane proteins is crucial for early diagnosis, real-time monitoring of disease progression, and guiding personalized clinical decision-making. However, current technologies for rare cell and even single-cell detection fall short of the demands for rapid, high-throughput clinical analysis. Imaging-based methods (such as immunohistochemistry and immunofluorescence) require point-by-point scanning, are time-consuming, and rely on experienced technicians for identification. While flow cytometry can acquire single-cell images, it depends on expensive equipment and complex sample preparation. These limitations collectively hinder the realization of sensitive, rapid, scalable, and cost-effective single-cell detection in clinical samples.

[0003] Electrochemical cell sensing technology, as a promising alternative, offers advantages such as low cost, rapid response, and ease of operation, and possesses strong potential for integration, reuse, and direct analysis in complex biological fluids. Among existing methods, sandwich-type electrochemical cell sensors are the most widely used. Signal amplification strategies have significantly improved cell sensing performance, especially methods that increase the loading of electroactive probes on the cell surface through nucleic acid amplification or highly active nanozymes. However, obtaining detectable current output from a single cell still faces significant challenges. In traditional electrochemical cell sensing technologies, cells are typically spherical, and the influence of highly spread cells on electrical signals has not yet been explored. Cell morphology can be altered through the topological effects of nanomaterials. MXene and RGO, as two-dimensional nanosheet materials, are widely used due to their excellent conductivity and biocompatibility. On the other hand, single-atom nanozymes, by mimicking the atomic-level catalytic sites of biological enzymes, possess catalytic performance comparable to that of biological enzymes, while also exhibiting excellent stability and signal amplification performance.

[0004] In summary, there is an urgent need to develop a novel electrochemical cell sensing technology for rapid and highly sensitive detection of cell abundance or membrane proteins, and to explore the impact of cell spreading on electrical signals, in order to promote the further clinical application of cell detection. This invention aims to construct an electrochemical cell sensor by combining the advantages of RGO@Ti3C2Tx composite materials and functionalized FeCN@antibodies, thereby addressing the problems existing in current technologies. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to combine RGO@Ti3C2T xThe advantages of composite materials and functionalized FeCN single-atom nanozymes provide a method for preparing and applying an accurate, reliable, and rapid-response electrochemical cell sensor that promotes cell spread and enables single-cell response.

[0006] The technical solution of this invention:

[0007] An electrochemical cell sensor based on enhanced electron transfer through cell spreading, comprising a first antibody / RGO@Ti3C2T x Cell spreading unit and FeCN@ secondary antibody signal output unit;

[0008] The first antibody / RGO@Ti3C2Tx cell spreading unit includes a first antibody and RGO@Ti3C2T. x RGO@Ti3C2T x It is composed of GO and Ti3C2T x Layered rough structures, RGO@Ti3C2T, prepared by hydrothermal method x A conductive ink was prepared and modified onto a conductive substrate, after which the first antibody was immobilized on RGO@Ti3C2T. x surface;

[0009] The FeCN@second antibody signal output unit includes a second antibody and FeCN. The FeCN is a single-atom structure characterized by Fe-N4, obtained by multi-step pyrolysis of ZIF-8. The FeCN and the second antibody are covalently linked.

[0010] The first antibody binds to the RGO@Ti3C2T via physical incubation. x The second antibody is attached to the FeCN surface via a bifunctional conjugate.

[0011] The electrochemical cell sensor utilizes a first antibody / RGO@Ti3C2T x The cell spreading unit captures cells and promotes their extensive spreading. Subsequently, the FeCN@second antibody signaling unit couples to the cell surface, forming FeCN@second antibody-CTC-first antibody / RGO@Ti3C2T. x The sandwich-style detection structure;

[0012] The first antibody includes one or more of Epcam, EGFR, HER-2, PD-L1, Vimentin, E-cadherin, N-cadherin, CEA, and CD44.

[0013] The second antibody includes one or more of Epcam, EGFR, HER-2, PD-L1, Vimentin, E-cadherin, N-cadherin, CEA, and CD44.

[0014] The conductive substrate includes one of the following: conductive carbon paper, glassy carbon electrode, gold electrode, ITO, FTO, Al, 316 / 314 stainless steel, Cu electrode, screen-printed electrode, porous foamed nickel, and porous foamed copper.

[0015] The bifunctional coupling agent is centered on polyethylene glycol, with a hydrophobic carbon chain at one end and functional groups such as amino, carboxyl, maleamide, and thiol at the other end.

[0016] A method for preparing an electrochemical cell sensor based on enhanced electron transfer through cell spreading includes the following steps:

[0017] S1. By combining GO and Ti3C2T x The mixture was prepared into a suspension, and ascorbic acid was added. RGO@Ti3C2T was then obtained through a hydrothermal reaction. x ;

[0018] S2. Obtain the RGO@Ti3C2T from step S1. x Homogeneous RGO@Ti3C2T was prepared x Conductive ink, RGO@Ti3C2T x Conductive ink is uniformly drop-coated onto the surface of a conductive substrate to form RGO@Ti3C2T. x / Conductive substrate, with the first antibody and the RGO@Ti3C2T x / Conductive substrate coupling yields the first antibody / RGO@Ti3C2T x Cell spreading unit;

[0019] S3. FeCN is obtained by multi-step pyrolysis of ZIF-8;

[0020] S4. By modifying the FeCN obtained in step S3 with a second antibody, FeCN@second antibody signal output unit is obtained.

[0021] In step S1, GO and Ti3C2T x The mass ratio is 0.5:1 to 3:1, and the hydrothermal reaction is carried out at a constant temperature of 150℃ to 170℃ for no less than 6 hours.

[0022] In step S2, homogeneous RGO@Ti3C2T x The concentration of the conductive ink is 1 mg / mL-50 mg / mL, and RGO@Ti3C2T is used. xConductive ink is uniformly drop-coated onto the surface of a conductive substrate, and after drying, RGO@Ti3C2T is applied. x The nanocomposite layer was placed in a first antibody solution with a concentration of not less than 2 μg / mL and physically incubated to obtain the first antibody / RGO@Ti3C2T. x Cell spreading unit.

[0023] The specific steps of step S3 are as follows: Zn(NO3)2·6H2O and 2-methylimidazole are dissolved in methanol at a molar ratio of 1:2~4. After mixing, the mixture is stirred continuously at room temperature for 2-5 hours. After centrifugation, the precipitate is washed with methanol to obtain ZIF-8. ZIF-8 is dispersed in a 10 vol.% methanol solution, the pH is adjusted to 11, 1-2 times the mass of ZIF-8 CTAB is added, and then 1% of the total solution volume of TEOS is added dropwise. The mixture is stirred at room temperature for 30-60 minutes, and after centrifugation and washing, white ZIF-8@SiO2 is obtained. It is dried overnight at 60°C and then placed in a muffle furnace. The temperature is increased by 5° / min and pyrolyzed at 1000°C under N2 protection for 2 hours. The resulting black powder is etched with NaOH to obtain NC support. NC support and FeCl2 are ground uniformly at a mass ratio of 3:1~1:1 and reacted at 900°C for 2 hours in a muffle furnace under nitrogen protection. The product is washed with HCl to obtain FeCN.

[0024] The specific steps of step S4 are as follows: FeCN and bifunctional conjugate are mixed evenly at a mass ratio of 1:2 to 1:20. After reacting for 1 to 24 hours, the mixture is washed with PBS by centrifugation to remove unbound bifunctional conjugate. A second antibody solution containing 5 to 10 times the mass of FeCN and 1% to 5% TCEP is added, and the mixture is stirred at 4°C for 2 to 12 hours for coupling reaction. The unbound second antibody is washed with PBS to remove it. 1% to 5% BSA solution is added to block the unbound surface sites. After washing, the FeCN@second antibody signal output unit is obtained.

[0025] A method for cell detection based on an electrochemical cell sensor that enhances electron transfer through cell spreading includes adding a primary antibody / RGO@Ti3C2T to a cell suspension to be tested. x Cell spreading unit, followed by washing with primary antibody / RGO@Ti3C2T x The cell spreading unit removes unbound test samples, followed by the addition of the FeCN@second antibody signal output unit for targeted labeling. Then, TMB is generated around the cells via FeCN peroxidase activity in a precipitated TMB single-component reaction solution. + Electroactive precipitation, TMB quantification by CV, DPV or SWV + Oxidation current is used to achieve electrochemical sensing of cells based on the magnitude of the current.

[0026] The cell suspension primary antibody / RGO@Ti3C2T x The incubation time for the cell spreading unit is 30 min to 4 h. The TMB single-component reaction solution is precipitated TMB, which is converted into electroactive TMB through peroxidase catalysis. + Blue crystal precipitate, the reaction time of TMB single-component reaction solution is not less than 3 minutes.

[0027] The beneficial effects of this invention are:

[0028] (1) Able to pass RGO@ Ti3C2T x The physical morphology and topology of the cells promote high cell spreading, which verifies that cell spreading can promote the transmission of sensor electrical signals. The current signal generated by the spread cells is twice that of the unspread cells and 7.6 times that of the signal current of the traditional Au planar electrode and HRP system, which can output the current response of a single cell.

[0029] (2) It can quickly, efficiently and stably detect the number of cells or the abundance of cell membrane proteins in complex systems. Attached Figure Description

[0030] Figure 1 For RGO@Ti3C2T x XRD, Raman, and infrared spectra of (RGM) materials during preparation; where a represents Ti3AlC2, GO, and Ti3C2T. x and RGO@ Ti3C2T x XRD patterns, b represents GO, Ti3C2T x and RGO@ Ti3C2T x Raman plots, cGO, Ti3C2T x and RGO@ Ti3C2T x This is an infrared spectrum.

[0031] Figure 2 Scanning electron microscope (SEM) images and electron transport properties of RGM are shown. Specifically, a is a planar image of RGM from a scanning electron microscope (scale bar: 5 μm); b is a cross-sectional image of RGM (scale bar: 5 μm, RGM thickness: 15 μm); c is a magnified cross-sectional SEM image of RGM (scale bar: 2 μm); d is the cyclic voltammograms of GCE, RGO, RGM, and RGM / nanobody; and e is the electrochemical impedance spectroscopy (EIS) of GCE, RGO, RGM, and RGM / nanobody.

[0032] Figure 3The images show single-atom images of FeCN with spherical aberration lenses; where a is the STEM-HAADF image of FeCN with a scale bar of 50 μm; b is the energy spectrum of FeCN for carbon; c is the energy spectrum of FeCN for nitrogen; d is the energy spectrum of FeCN for iron; e is the energy spectrum of FeCN for mixed elements; and f is the atomic-level iron metal bright spot image of FeCN with a scale bar of 5 nm.

[0033] Figure 4 Images show cell morphology at different incubation times; where a is a confocal morphology image of cells after incubation for 10, 20, 30, and 90 minutes, with red representing the cytoskeleton and blue representing the nucleus, and a scale bar of 5 μm; b is a scanning electron microscope image of cells after incubation for 10, 20, 30, and 90 minutes; c is a statistical graph of the spread area of ​​cells after 10, 20, 30, and 90 minutes; and d is a statistical graph of the number of pseudopodia in cells after 10, 20, 30, and 90 minutes.

[0034] Figure 5 Statistical analysis of DPV current response for the same number of cells with different spreading patterns; where a is a 3D confocal image of cell spreading after 10 min of incubation; b is a 3D confocal image of cell spreading after 20 min of incubation; c is a 3D confocal image of cell spreading after 30 min of incubation; d is a 3D confocal image of cell spreading after 90 min of incubation; e is a comparison of current for the four spreading patterns; f is the number of cells in each field of view for different spreading patterns; g is the concentration of FeCN and HRP in 5*104 A431 cells / mL. -1 Current response of RGM at concentrations 0 and 30 days after storage at 4°C; h = 5 × 10⁴ A431 cells / mL -1 Comparison of current responses generated by RGM and FeCN systems, Au substrate and FeCN system, and Au substrate and HRP system at different concentrations.

[0035] Figure 6 The cell detection benchmark curve and the detection performance of the sensor with a small number of cells are shown in Example 1; where a is the DPV diagram for different cell numbers; b is the sensor's detection benchmark curve; c is the DPV current response diagram for a small number of cells; and d is the detection curve for a small number of cells.

[0036] Figure 7 The EMT detection current for different cell lines at a concentration of 50,000 cells / ml in Example 4 is shown. Detailed Implementation

[0037] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and technical solutions.

[0038] Example 1

[0039] This embodiment uses A431 cells from the Cell Bank of the Chinese Academy of Sciences as the detection target of an electrochemical cell sensor. An electrochemical cell sensor based on cell spreading-enhanced electron transfer is used to detect A431 cell concentration.

[0040] 1) EGFR nanobody @RGO @ Ti3C2T x Preparation of cell spreading unit: 150 mg GO and 110 mg Ti3C2T were mixed. x 200 mg of ascorbic acid was dispersed in 40 mL of deionized water and sonicated for 30 minutes. The solution was transferred to a PTFE-lined stainless steel reactor and reacted at 160 °C for 12 h. After cooling, the product was washed successively with ethanol and deionized water, and finally lyophilized to obtain the RGO@Ti3C2T. x powder.

[0041] RGO@ Ti3C2T x A 10 mg / mL concentration of EGFR nanobody solution was prepared and dispersed in an ethanol / water mixture (volume ratio 7:3). This solution was then uniformly drop-coated onto a conductive carbon substrate, dried under nitrogen, and washed with PBS. Finally, a 1 mg / mL EGFR nanobody solution was applied to the RGO@Ti3C2T substrate. x EGFR nanobodies / RGO@Ti3C2T were obtained using a physical incubation method on a substrate. x Capture electrode.

[0042] 2) Add 2.38 g of Zn(NO3)2 . 6H₂O and 2.76 g of 2-methylimidazole were dissolved in 100 mL and 125 mL of methanol, respectively. The two solutions were mixed and stirred continuously at room temperature for 2 hours. The precipitate was collected by centrifugation, washed with methanol, and dried to obtain ZIF-8. ZIF-8 was then dispersed in 240 mL of 10% methanol aqueous solution, and the pH was adjusted to 11. 0.2 g of hexadecyltrimethylammonium bromide was added, followed by the slow addition of 1.2 mL of tetraethyl orthosilicate. After stirring at room temperature for 30 minutes, the mixture was centrifuged and washed to obtain white ZIF-8@SiO₂ particles, which were dried overnight at 60 °C. Subsequently, the particles were dried under a nitrogen atmosphere at 5 °C·min⁻¹. -1 Carbonization was carried out at 1000℃ for 2 hours. The silica template was removed by etching with 4M NaOH to obtain the NC support. 120 mg of NC support was ground and mixed with 40 mg of FeCl2. The mixture was placed in a ceramic boat and heated at 5℃·min under a nitrogen atmosphere. -1 The product was heated at 900°C for 2 hours. The resulting product was washed with 2M hydrochloric acid to remove residual metal clusters and impurities, ultimately yielding FeCN.

[0043] 3) 100 μg mL-1 FeCN with 1 mg mL -1 C18-PEG4000-MAL was mixed in PBS and then sonicated in an ice bath to ensure uniform dispersion. The mixture was centrifuged at 9000 rpm to remove unreacted C18-PEG4000-MAL and washed three times with PBS. The resulting C18-PEG4000-MAL-modified FeCN was then reacted with 500 μg / mL of a solution containing 1 mM trichloroethylene phosphate. - 1 EGFR nanobody solution was incubated to activate thiol groups. The reaction was carried out at 4°C with gentle stirring for 3 hours. After incubation, the precipitate was centrifuged and thoroughly washed with PBS to remove free EGFR nanobody. To block residual active sites, the product was treated with 1% BSA solution and washed again with PBS to obtain the FeCN@EGFR nanobody signal probe.

[0044] 4) Incubate 1 ml of A431 cell suspension on the EGFR nanobody / RGO@Ti3C2Tx capture electrode at 37°C for 90 min in a 5% CO2 incubator. Wash with PBS to remove unbound cells, then incubate with 2 μg / ml FeCN@EGFR nanobody signal probe for 45 min. After washing with PBS, add 500 μl of precipitated TMB reaction solution and react at 37°C for 6 min. Terminate the reaction with PBS. Quantify TMB in PBS electrolyte using differential pulse voltammetry (DPV). + The oxidation current was measured with a scanning potential range of -0.2 to 0.8 V, a pulse amplitude of 0.05 V, and a pulse width of 0.05 V. The concentration of A431 cells was quantified based on the oxidation current value.

[0045] This embodiment demonstrates the application of EGFR nanobody@RGO@Ti3C2T. x The characterization of the cell spreading unit is as follows:

[0046] like Figure 1 The image shown is of RGO@Ti3C2T. x XRD, FTIR, and Raman data, such as Figure 2 For RGO@ Ti3C2T x SEM, EDS, and cross-sectional images of the conductive substrate, approximately 15 μm thick, with a layered surface structure. CV and EIS images of the series of samples show RGO@Ti3C2T. x Excellent electrical conductivity.

[0047] like Figure 3 The image shown is a spherical aberration lens image of FeCN, where uniformly distributed Fe atom bright spots can be observed, indicating a single-atom structure.

[0048] To examine cell morphology, observations were performed using SEM and confocal microscopy, such as... Figure 4 As shown, the cells extend a large number of sheet-like filamentous pseudopodia within 90 minutes, exhibiting a highly spread-out morphology.

[0049] To compare the current response of spread-out cells and upright cells, four cell detection models with the same number of cells but different spread patterns were established, and DPV (dielectric power generation) was measured. Figure 5 This indicates that the spread-out cells have twice the current response compared to the upright cells.

[0050] To calculate the detection limit of the electrochemical cell sensor for detecting A431 cells, the DPV response was measured for different cell numbers, and a detection standard curve was calculated. The detection limit was 0.17 cells / ml. Figure 6 This indicates that the sensor can also generate an electrical response to a single cell.

[0051] Comparative Example 1

[0052] Conductive ITO was selected as the working electrode. The same number of cell suspensions as in Example 1 were incubated overnight. After cell fixation, the cells were divided into two groups. One group was incubated with rabbit-derived Epcam primary antibody solution and goat anti-rabbit HRP secondary antibody solution, while the other group was incubated with FeCN@EGFR nanobody signal probe. The DPV detection procedure was the same as in Example 1.

[0053] And the use of EGFR nanobody @RGO@Ti3C2T x As a cell spreading unit, the FeCN@EGFR nanobody signal probe was replaced with rabbit-derived Epcam primary antibody solution and goat anti-rabbit HRP secondary antibody solution, and the DPV detection procedure was the same as in Example 1.

[0054] like Figure 5 EGFR nanobody @RGO @ Ti3C2T x Compared with the FeCN@EGFR nanobody signal probe system, the current was increased by 7.6 times compared with the ITO and HRP systems, and by 3.3 times compared with the ITO and FeCN systems. Compared with the EGFR nanobody@RGO@Ti3C2T... x The performance was improved by 1.8 times compared to the HRP system. This is mainly attributed to the excellent electron transport performance of RGM, as well as the nano-effect and excellent peroxidase-mimicking properties of FeCN nanozymes. Combined with RGM, it can promote high cell spreading and expand the contact area between cells and the electrode substrate, resulting in more efficient electron transport compared to unspread cells. These factors combined produce such excellent performance.

[0055] Example 2

[0056] Based on Example 1, the mass ratio of GO to Ti3C2Tx was changed to 0.55:1, and other preparation and testing methods were the same as in Example 1.

[0057] Example 3

[0058] Based on Example 1, the mass ratio of GO to Ti3C2Tx was changed to 3:1, and other preparation and testing methods were the same as in Example 1.

[0059] Example 4

[0060] Based on Example 1, the first and second antibodies were replaced with anti-Epcam antibodies, and the other preparation and testing methods were the same as in Example 1.

[0061] Example 5

[0062] Based on Example 1, the first and second antibodies were replaced with anti-Vimention antibodies, and the other preparation and testing methods were the same as in Example 1.

[0063] Example 6

[0064] Based on Example 1, the homogeneous RGO@Ti3C2Tx conductive ink with a concentration of 50 mg / mL was uniformly drop-coated onto the surface of conductive carbon paper, dried with an infrared lamp, and then coated with a 2 mg / mL EGFR nanobody solution onto the RGO@Ti3C2Tx paper. x The substrate and the remaining steps are the same as in Example 1.

[0065] Example 7

[0066] An electrochemical cell sensor based on enhanced electron transport through cell spreading is used to detect EMT staging in tumor cell lines:

[0067] 1) Capture Unit EGFR Nanobody @RGO @ Ti3C2T x The preparation is the same as in Example 1;

[0068] 2) Preparation of N-Cadherin antibody@FeCN signal probe: Replace the antibody with N-Cadherin antibody, and follow the same steps as in Example 1.

[0069] 3) Incubate 1 ml of cell suspensions from different EMT stages in EGFR nanobody / RGO@Ti3C2T xThe capture electrode was incubated at 37°C in a 5% CO2 incubator for 90 min. Unbound cells were removed by washing with PBS. Immediately afterwards, the electrode was incubated with a 2 μg / ml FeCN@N-Cadherin antibody signal probe for 45 min. After washing with PBS, 500 μL of precipitated TMB reaction solution was added, and the reaction was carried out at 37°C for 6 min. The reaction was then terminated with PBS. The oxidation current of TMB⁺ in the PBS electrolyte was quantified using differential pulse voltammetry (DPV), with a scan potential range of -0.2 to 0.8 V, a pulse amplitude of 0.05 V, and a pulse width of 0.05 V. The EMT stage of the cells was determined based on the oxidation current value.

[0070] Test results as follows Figure 7 As shown, the DPV current trend is consistent with the known EMT staging results.

Claims

1. An electrochemical cell sensor based on enhanced electron transfer through cell spreading, characterized in that, This electrochemical cell sensor, based on cell spreading-enhanced electron transport, includes a first antibody / RGO@Ti3C2T. x Cell spreading unit and FeCN@ secondary antibody signal output unit; The first antibody / RGO@Ti3C2Tx cell spreading unit includes a first antibody and RGO@Ti3C2T. x RGO@Ti3C2T x It is composed of GO and Ti3C2T x Layered rough structures, RGO@Ti3C2T, prepared by hydrothermal method x A conductive ink was prepared and modified onto a conductive substrate, after which the first antibody was immobilized on RGO@Ti3C2T. x surface; The FeCN@second antibody signal output unit includes a second antibody and FeCN. FeCN is a single-atom structure characterized by Fe-N4 obtained by multi-step pyrolysis of ZIF-8. FeCN and the second antibody are covalently linked. The electrochemical cell sensor utilizes a first antibody / RGO@Ti3C2T x The cell spreading unit captures cells and promotes their extensive spreading. Subsequently, the FeCN@second antibody signaling unit couples to the cell surface, forming FeCN@second antibody-CTC-first antibody / RGO@Ti3C2T. x The sandwich-style detection structure.

2. The electrochemical cell sensor based on enhanced electron transfer through cell spreading according to claim 1, characterized in that, The first antibody binds to the RGO@Ti3C2T via physical incubation. x The second antibody is attached to the FeCN surface via a bifunctional conjugate. The first antibody includes one or more of Epcam, EGFR, HER-2, PD-L1, Vimentin, E-cadherin, N-cadherin, CEA, and CD44. The second antibody includes one or more of the following: Epcam, EGFR, HER-2, PD-L1, Vimentin, E-cadherin, N-cadherin, CEA, and CD44; The conductive substrate includes one of the following: conductive carbon paper, glassy carbon electrode, gold electrode, ITO, FTO, Al, 316 / 314 stainless steel, Cu electrode, screen-printed electrode, porous foamed nickel, and porous foamed copper. The bifunctional coupling agent is centered on polyethylene glycol, with a hydrophobic carbon chain at one end and functional groups such as amino, carboxyl, maleamide, and thiol at the other end.

3. A method for preparing an electrochemical cell sensor based on enhanced electron transfer through cell spreading, characterized in that, Includes the following steps: S1. By combining GO and Ti3C2T x The mixture was prepared into a suspension, and ascorbic acid was added. RGO@Ti3C2T was then obtained through a hydrothermal reaction. x ; S2. Obtain the RGO@Ti3C2T from step S1. x Homogeneous RGO@Ti3C2T was prepared x Conductive ink, RGO@Ti3C2T x Conductive ink is uniformly drop-coated onto the surface of a conductive substrate to form RGO@Ti3C2T. x / Conductive substrate, with the first antibody and the RGO@Ti3C2T x / Conductive substrate coupling yields the first antibody / RGO@Ti3C2T x Cell spreading unit; S3. FeCN is obtained by multi-step pyrolysis of ZIF-8; S4. By modifying the FeCN obtained in step S3 with a second antibody, FeCN@second antibody signal output unit is obtained.

4. The preparation method according to claim 3, characterized in that, In step S1, GO and Ti3C2T x The mass ratio is 0.5:1 to 3:1, and the hydrothermal reaction is carried out at a constant temperature of 150℃ to 170℃ for no less than 6 hours.

5. The preparation method according to claim 3, characterized in that, In step S2, homogeneous RGO@Ti3C2T x The concentration of the conductive ink is 1 mg / mL-50 mg / mL, and RGO@Ti3C2T is used. x Conductive ink is uniformly drop-coated onto the surface of a conductive substrate, and after drying, RGO@Ti3C2T is applied. x The nanocomposite layer was placed in a first antibody solution with a concentration of not less than 2 μg / mL and physically incubated to obtain the first antibody / RGO@Ti3C2T. x Cell spreading unit.

6. The preparation method according to claim 3, characterized in that, The specific steps of step S4 are as follows: FeCN and bifunctional conjugate are mixed evenly at a mass ratio of 1:2 to 1:

20. After reacting for 1 to 24 hours, the mixture is washed with PBS by centrifugation to remove unbound bifunctional conjugate. A second antibody solution containing 5 to 10 times the mass of FeCN and 1% to 5% TCEP is added, and the mixture is stirred at 4°C for 2 to 12 hours for coupling reaction. The unbound second antibody is washed with PBS to remove it. 1% to 5% BSA solution is added to block the unbound surface sites. After washing, the FeCN@second antibody signal output unit is obtained.

7. A method for cell detection based on an electrochemical cell sensor that enhances electron transfer through cell spreading, characterized in that, Add the cell suspension to be tested to the primary antibody / RGO@Ti3C2T x Cell spreading unit, followed by washing with primary antibody / RGO@Ti3C2T x The cell spreading unit removes unbound test samples, followed by the addition of the FeCN@second antibody signal output unit for targeted labeling. Then, TMB is generated around the cells via FeCN peroxidase activity in a precipitated TMB single-component reaction solution. + Electroactive precipitation, TMB quantification by CV, DPV or SWV + Oxidation current is used to achieve electrochemical sensing of cells based on the magnitude of the current.

8. The method according to claim 8, characterized in that, The cell suspension primary antibody / RGO@Ti3C2T x The incubation time for the cell spreading unit is 30 min to 4 h. The TMB single-component reaction solution is precipitated TMB, which is converted into electroactive TMB through peroxidase catalysis. + Blue crystal precipitate, the reaction time of TMB single-component reaction solution is not less than 3 minutes.

Images