阅读说明：本技术 一种肿瘤细胞快检电化学生物传感器及其制备方法和应用 (Electrochemical biosensor for quickly detecting tumor cells and preparation method and application thereof ) 是由 李奇灵 梁冬鑫 吴春生 杜立萍 于 2021-08-23 设计创作，主要内容包括：本发明公开了肿瘤细胞快检电化学生物传感器,包括柔性丝网印刷电极以及沉积在柔性丝网印刷电极表面的金纳米颗粒,金纳米颗粒的表面修饰有巯基化叶酸分子,且柔性丝网印刷电极表面被牛血清白蛋白封闭；或者包括柔性丝网印刷电极以及修饰在柔性丝网印刷电极表面的金纳米颗粒-巯基化叶酸分子复合物,且柔性丝网印刷电极表面被牛血清白蛋白封闭；本发明还公开了采用滴涂法的制备方法；本发明还公开了在该电化学生物传感器中滴加样品液孵育并采用电化学工作站检测电化学阻抗的应用过程。本发明传感器与肿瘤细胞表面的叶酸受体特异性结合能力较强,实现了对肿瘤细胞的快速检测；本发明的制备方法简单；本发明的应用过程提高了肿瘤细胞的检测效率。(The invention discloses a tumor cell fast detection electrochemical biosensor, which comprises a flexible screen printing electrode and gold nanoparticles deposited on the surface of the flexible screen printing electrode, wherein the surface of the gold nanoparticles is modified with thiolated folic acid molecules, and the surface of the flexible screen printing electrode is sealed by bovine serum albumin; or the gold nanoparticle-thiolated folic acid composite comprises a flexible screen printing electrode and a gold nanoparticle-thiolated folic acid molecule composite modified on the surface of the flexible screen printing electrode, wherein the surface of the flexible screen printing electrode is sealed by bovine serum albumin; the invention also discloses a preparation method adopting the dripping coating method; the invention also discloses an application process of dripping the sample liquid into the electrochemical biosensor for incubation and detecting the electrochemical impedance by adopting the electrochemical workstation. The sensor has stronger specific binding capacity with a folate receptor on the surface of a tumor cell, and realizes the rapid detection of the tumor cell; the preparation method is simple; the application process of the invention improves the detection efficiency of the tumor cells.)

1. The electrochemical biosensor for quickly detecting the tumor cells is characterized by comprising a flexible screen printing electrode and gold nanoparticles deposited on the surface of the flexible screen printing electrode, wherein the surface of each gold nanoparticle is modified with a thiolated folic acid molecule, and the surface of the flexible screen printing electrode is sealed by bovine serum albumin;

or the gold nanoparticle-thiolated folate compound comprises a flexible screen printing electrode and a gold nanoparticle-thiolated folate molecule compound modified on the surface of the flexible screen printing electrode, and the surface of the flexible screen printing electrode is sealed by bovine serum albumin.

2. A method for preparing the electrochemical biosensor for rapid tumor cell detection according to claim 1, comprising the steps of:

step one, dripping a chloroauric acid solution on the surface of a flexible screen printing electrode SPE by adopting an electrodeposition method, and depositing to form gold nanoparticles Au Nps to obtain Au Nps-SPE;

step two, dropwise adding a thiolated folic acid molecule solution on the surface of the Au Nps-SPE obtained in the step one by adopting a dropwise coating method, so that a thiolated folic acid molecule Fa-PEG-SH is connected to the surface of the Au Nps for modification, and the Fa-PEG-SH-Au Nps-SPE is obtained;

and step three, dropwise adding the bovine serum albumin solution on the Fa-PEG-SH-Au Nps-SPE obtained in the step two for sealing, and then cleaning by adopting a phosphate buffer solution to obtain the electrochemical biosensor.

3. A method for preparing the electrochemical biosensor for rapid tumor cell detection according to claim 1, comprising the steps of:

step one, preparing gold nanoparticles Au Nps by a method of reducing chloroauric acid by using sodium citrate, and then adding the Au Nps solution into a thiolated folate molecule Fa-PEG-SH solution to be stirred to obtain a gold nanoparticle-thiolated folate molecule compound Fa-PEG-SH-Au Nps solution;

step two, sequentially adopting ethanol solution with volume fraction of 75% and water to clean the SPE of the flexible screen printing electrode, and drying by adopting nitrogen;

step three, dropwise adding the Fa-PEG-SH-Au Nps solution obtained in the step one onto the surface of the SPE dried in the step two by adopting a dropwise coating method, so that the Fa-PEG-SH-Au Nps is modified on the surface of the SPE to obtain Fa-PEG-SH-Au Nps-SPE;

and step four, dropwise adding the bovine serum albumin solution on the Fa-PEG-SH-Au Nps-SPE obtained in the step three for sealing, and then cleaning by adopting a phosphate buffer solution to obtain the electrochemical biosensor.

4. The method according to claim 2, wherein the concentration of the chloroauric acid solution in the first step is 2.5mmol/L to 3.0 mmol/L.

5. The method according to claim 2, wherein the concentration of the thiolated folate molecule solution in the second step is 50 to 150 μmol/L.

6. The method according to claim 2 or 3, wherein the concentration of the bovine serum albumin solution in step three is 0.01 mg/mL-10 mg/mL, and the blocking time is 0.5 h-1 h.

7. The application of the electrochemical biosensor for rapidly detecting tumor cells according to claim 1, wherein the application comprises the following steps:

step one, dropwise adding a sample solution into a sample adding area of an electrochemical biosensor, and then placing the sample solution into an incubator for incubation;

step two, installing the electrochemical biosensor incubated in the step one into a detection device, and then dropwise adding a phosphate buffer solution containing potassium ferrocyanide and potassium ferricyanide to a sample adding area of the electrochemical biosensor;

and step three, starting an electrochemical workstation matched with the detection device, carrying out electrochemical impedance detection on the sample adding region of the electrochemical biosensor dropwise added with the phosphate buffer solution in the step two, obtaining an electrochemical impedance value, and outputting, recording and storing the electrochemical impedance value.

8. The use of claim 7, wherein the sample solution is added in an amount of 300 μ L in step one; the incubation temperature is 37 ℃, and the incubation time is 0.5-1 h.

9. The use according to claim 7, wherein the concentration of potassium ferrocyanide and potassium ferricyanide in the phosphate buffer solution in the second step is 1mmol/L, and the dropping amount of the phosphate buffer solution is 200. mu.L.

10. The use of claim 7, wherein the electrochemical impedance measurements in step three are performed at a scanning frequency of 10mHz to 100kHz with 5mV AC perturbation applied at open circuit potential.

Technical Field

The invention belongs to the technical field of rapid detection, and particularly relates to a tumor cell rapid detection electrochemical biosensor and a preparation method and application thereof.

Background

Malignant tumors are in an ascending trend all over the world, are usually found late and rapidly, and currently, an effective early detection mode is still lacking. The research focuses on the secondary prevention of tumors, and the detection of precancerous lesions and early malignant tumors through a screening means is beneficial to the implementation of early intervention so as to reduce the morbidity and the mortality. Therefore, the development of rapid and simple screening work is of great significance. In clinical evaluation of diseases by laboratory examination of tumor samples obtained by histological examination or the like, various methods have been proposed in the art for capturing and detecting cancer cells. Considering the high intensity and high load working conditions of clinical laboratories, rapid, efficient and label-free detection methods are the first choice for various biomedical diagnosis and treatment.

The electrochemical impedance spectrum is a label-free detection technology of an electrochemical biosensor, has the characteristics of high sensitivity, strong specificity and quick response, has wide application in the detection of living cells, and is suitable for a quick, simple and convenient clinical detection and analysis method.

The folic acid receptor is a membrane glycoprotein connected with glycosylated phosphatidylinositol, has the molecular weight of 38-40kD, can be combined with exogenous folic acid to take up folic acid, and maintains normal life activities of organisms. Folate receptors are widely distributed in normal and tumor tissues, but their number and activity in tumor tissues is much higher than that of normal cells, sometimes 100-fold higher than that of normal tissues. Three folate receptor isomers have been identified to date, including folate receptor-alpha, folate receptor-beta, and folate receptor-gamma. The expression of the folate receptor-alpha has strong tissue and tumor specificity, the expression level in normal tissues and cells is very low, and once epithelial cells become cancerous, the expression level of the folate receptor-alpha can be greatly increased, for example, the folate receptor-alpha is highly expressed in most malignant cells derived from the epithelial tissues, including cells of endometrial cancer, ovarian cancer, breast cancer, lung cancer, kidney cancer, colon cancer, nasopharyngeal carcinoma and the like. Therefore, based on the expression difference of folate receptors and the high affinity thereof for folate, FR-alpha becomes an attractive target of tumors, and folate molecules become ideal molecules for specifically capturing and detecting folate receptor positive tumor cells.

In various reported biosensors based on folate functionalization, many nanomaterials are used for detecting cancer cells, such as carbon nanotubes, gold nanoparticles, graphene, dendrimers, etc., but most of the folates are covalently immobilized on the electrode surface by acid-amine condensation reaction of EDC/NHS, which is complex in reaction step, long in reaction time, and strict in reaction conditions. From the point of view of commercialization and repeatability, a simple, stable, uniform folate immobilization method will make folate-functionalized based biosensors more easily commercialized.

Disclosure of Invention

The technical problem to be solved by the present invention is to provide an electrochemical biosensor for rapidly detecting tumor cells, which is directed to the above-mentioned deficiencies of the prior art. According to the sensor, gold nanoparticles with large specific surface area are deposited on the surface of the flexible screen-printed electrode and are modified by the thiolated folic acid molecules, or the surface of the flexible screen-printed electrode is modified by the gold nanoparticle-thiolated folic acid molecule compound directly, so that the adsorption capacity of the thiolated folic acid molecules is improved, the specific binding capacity of the sensor and a folic acid receptor on the surface of a tumor cell is enhanced, and the rapid detection of the tumor cell is facilitated.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows: the electrochemical biosensor for quickly detecting the tumor cells is characterized by comprising a flexible screen printing electrode and gold nanoparticles deposited on the surface of the flexible screen printing electrode, wherein the surface of each gold nanoparticle is modified with a thiolated folic acid molecule, and the surface of the flexible screen printing electrode is sealed by bovine serum albumin;

or the gold nanoparticle-thiolated folate compound comprises a flexible screen printing electrode and a gold nanoparticle-thiolated folate molecule compound modified on the surface of the flexible screen printing electrode, and the surface of the flexible screen printing electrode is sealed by bovine serum albumin.

Based on the fact that gold nanoparticles have large specific surface area and good conductivity and biocompatibility, and meanwhile inherent chemical adsorption exists between the gold nanoparticles and thiol molecules, stable gold-sulfur bonds can be formed; or the inherent chemical adsorbability of sulfydryl and gold nanoparticles is firstly utilized to prepare a gold nanoparticle-sulfhydrylated folate molecule compound, and then the compound is modified on the surface of the SPE to construct the folic acid-gold nanoparticle modified electrochemical biosensor, so that the adsorption capacity of the electrochemical biosensor to sulfhydrylated folate molecules is improved, the specific binding capacity of the sensor to folate receptors on the surfaces of tumor cells is further enhanced, the binding speed is improved, particularly the specific binding speed of the sensor to folate receptors in tumor cell membrane proteins is improved, the surface impedance of the SPE in the electrochemical biosensor changes, and the surface impedance is timely converted into an electric signal for detection, and the rapid detection of the tumor cells is favorably realized; meanwhile, the surface of the flexible screen printing electrode in the electrochemical biosensor is sealed by bovine serum albumin, and the SPE surface is covered by the bovine serum albumin to seal the electrode, so that the influence of nonspecific binding on a detection result in a nonspecific binding site in the sensor is avoided, the interference of external factors is reduced, and the specificity and the sensitivity of the sensor are further improved. In addition, the tumor cell fast-detection electrochemical biosensor adopts the disposable flexible screen-printed electrode SPE as a sensing matrix, so that the cost is low, the performance is stable, and the cross contamination is avoided.

In conclusion, the electrochemical biosensor for quickly detecting tumor cells has the advantages of easily available raw materials, simple preparation, low cost, easy commercialization, high sensitivity, strong specificity and quick response, stably realizes the quick and accurate detection of the tumor cells, is favorable for the early diagnosis and early treatment of precancerous lesion and early malignant tumor by accurate detection results, and relieves the current high-strength and high-load working situation of the current clinical laboratory by efficient and quick laboratory detection.

In addition, the invention also provides a method for preparing the electrochemical biosensor chip for rapidly detecting tumor cells, which is characterized by comprising the following steps:

step one, dripping a chloroauric acid solution on the surface of a flexible screen printing electrode SPE by adopting an electrodeposition method, and depositing to form gold nanoparticles Au Nps to obtain Au Nps-SPE;

step two, dropwise adding a thiolated folic acid molecule solution on the surface of the Au Nps-SPE obtained in the step one by adopting a dropwise coating method, so that a thiolated folic acid molecule Fa-PEG-SH is connected to the surface of the Au Nps for modification, and the Fa-PEG-SH-Au Nps-SPE is obtained;

and step three, dropwise adding the bovine serum albumin solution on the Fa-PEG-SH-Au Nps-SPE obtained in the step two for sealing, and then cleaning by adopting a phosphate buffer solution to obtain the electrochemical biosensor.

The method adopts an electrodeposition method to deposit the gold nanoparticles Au Nps on the surface of the flexible screen printing electrode SPE, which is beneficial to promoting the formation of the gold nanoparticles, then adopts a dripping method to connect the thiolated folic acid molecules Fa-PEG-SH on the surface of the Au Nps, improves the adsorption amount of the Fa-PEG-SH on the surface of the Au Nps, and then drips bovine serum albumin for sealing, thereby avoiding the combination interference of non-specific combination sites.

The invention also provides a method for preparing the electrochemical biosensor chip for rapidly detecting tumor cells, which is characterized by comprising the following steps:

step one, preparing gold nanoparticles Au Nps by a method of reducing chloroauric acid by using sodium citrate, and then adding the Au Nps solution into a thiolated folate molecule Fa-PEG-SH solution to be stirred to obtain a gold nanoparticle-thiolated folate molecule compound Fa-PEG-SH-Au Nps solution;

step two, sequentially adopting ethanol solution with volume fraction of 75% and water to clean the SPE of the flexible screen printing electrode, and drying by adopting nitrogen;

step three, dropwise adding the Fa-PEG-SH-Au Nps solution obtained in the step one onto the surface of the SPE dried in the step two by adopting a dropwise coating method, so that the Fa-PEG-SH-Au Nps is modified on the surface of the SPE to obtain Fa-PEG-SH-Au Nps-SPE;

and step four, dropwise adding the bovine serum albumin solution on the Fa-PEG-SH-Au Nps-SPE obtained in the step three for sealing, and then cleaning by adopting a phosphate buffer solution to obtain the electrochemical biosensor.

The invention adopts a reduction method to prepare gold nanoparticles Au Nps, and compounds the gold nanoparticles Au Nps with thiolated folic acid molecules to obtain nanoparticle-thiolated folic acid molecule compounds in advance, and then adopts a dripping method to modify the compounds on the surface of the SPE to obtain Fa-PEG-SH-Au Nps-SPE, thereby ensuring the adsorption capacity of Fa-PEG-SH on the surface of Au Nps, and then dripping bovine serum albumin for sealing, and avoiding the combination interference of non-specific combination sites.

The method is characterized in that the concentration of the chloroauric acid solution in the first step is 2.5mmol/L to 3.0 mmol/L.

The method described above, wherein the concentration of the thiolated folate molecule solution in the second step is 50 μmol/L to 150 μmol/L.

The method is characterized in that the concentration of the bovine serum albumin solution in the step three is 0.01 mg/mL-10 mg/mL, and the sealing time is 0.5 h-1 h.

In addition, the invention also provides an application of the electrochemical biosensor for quickly detecting tumor cells, which is characterized in that the application comprises the following steps:

step one, dropwise adding a sample solution into a sample adding area of an electrochemical biosensor, and then placing the sample solution into an incubator for incubation;

step two, installing the electrochemical biosensor incubated in the step one into a detection device, and then dropwise adding a phosphate buffer solution containing potassium ferrocyanide and potassium ferricyanide to a sample adding area of the electrochemical biosensor;

and step three, starting an electrochemical workstation matched with the detection device, carrying out electrochemical impedance detection on the sample adding region of the electrochemical biosensor dropwise added with the phosphate buffer solution in the step two, obtaining an electrochemical impedance value, and outputting, recording and storing the electrochemical impedance value.

In the application process of the electrochemical biosensor, firstly, a sample liquid is dripped into the sensor, then incubation is carried out, if the sample liquid contains tumor cells, membrane protein folate receptors of the tumor cells are fully and specifically combined with thiolated folate molecules on the surface of the sensor, so that the surface impedance of the SPE is changed, then, after phosphate buffer solution is dripped into a sample area, an electrochemical workstation is started, electrochemical impedance detection is carried out on a solution system in the sample addition area, a signal conditioning and converting circuit arranged in the electrochemical workstation is utilized to analyze and process detection data, so as to obtain the change value of the surface impedance of the SPE, and sound/light/digital signals are respectively output from an alarm device and (or) a display device and are recorded and stored simultaneously, the application process is simple, the incubation time is short, the impedance detection is fast, the electrochemical biosensor responds to the detected sample liquid quickly, the output result is visual and easy to understand, the detection efficiency of the tumor cells is effectively improved, the tumor screening work can be favorably developed in the crowd, and the early discovery, early diagnosis and early treatment strategies of the tumor are realized.

The application is characterized in that the adding amount of the sample liquid in the step one is 300 mu L; the incubation temperature is 37 ℃, and the incubation time is 0.5-1 h.

The application is characterized in that the concentrations of the potassium ferrocyanide and the potassium ferricyanide in the phosphate buffer solution in the step two are both 1mmol/L, and the dropping amount of the phosphate buffer solution is 200 mu L.

The application is characterized in that the scanning frequency of the electrochemical impedance detection in the step three is 10 mHz-100 kHz, and 5mV AC disturbance is applied under the open-circuit potential.

Compared with the prior art, the invention has the following advantages:

1. according to the sensor, gold nanoparticles with large specific surface area are deposited on the surface of the flexible screen-printed electrode and are modified by the thiolated folic acid molecules, or the surface of the flexible screen-printed electrode is modified by the gold nanoparticle-thiolated folic acid molecule compound directly, so that the adsorption capacity of the thiolated folic acid molecules is improved, the specific binding capacity of the sensor and a folate receptor on the surface of a tumor cell is enhanced, the surface impedance change of the flexible screen-printed electrode is converted into an electric signal for detection in time, and the rapid detection of the tumor cell is favorably realized.

2. The sensor disclosed by the invention adopts bovine serum albumin to seal the surface of the flexible screen printing electrode, so that the influence of nonspecific binding between the sensor and tumor cells on a detection result is avoided, and the specificity and the sensitivity of the sensor are further improved.

3. The sensor has the advantages of simple structure, high detection sensitivity, strong specificity and convenient carrying and use, and is beneficial to improving the detection efficiency of tumor cells.

4. The gold nanoparticles are deposited on the surface of the flexible screen printing electrode by adopting an electrodeposition method, so that the gold nanoparticles are formed, the adsorption quantity of the thiolated folic acid molecules on the surface of the gold nanoparticles is improved, the preparation process is simple, and the raw materials are easy to obtain.

5. The sensor has the advantages of simple application process, short incubation time, quick impedance detection, quick response to the detected sample liquid, intuitive and understandable output result, effectively improved detection efficiency of tumor cells, contribution to developing tumor screening work in crowds and realization of early discovery, early diagnosis and early treatment strategies of tumors.

The technical solution of the present invention is further described in detail by the accompanying drawings and examples.

Drawings

FIG. 1 is a schematic diagram of the preparation and application of the electrochemical biosensor for rapid tumor cell detection according to the present invention.

FIG. 2 is a schematic diagram of electrochemical impedance detection in the application process of the electrochemical biosensor for rapid tumor cell detection according to the present invention.

FIG. 3 is a cyclic voltammogram of SPE, Au Nps-SPE, and Fa-PEG-SH-Au Nps-SPE in example 1 of the present invention.

FIG. 4 is a graph showing the results of the impedance system of the SPE, Au Nps-SPE, Fa-PEG-SH-Au Nps-SPE, BSA-Fa-PEG-SH-Au Nps-SPE and the electrochemical biosensor after being specifically bound to tumor cells in example 1 of the present invention.

Detailed Description

In the embodiments 1-2 of the invention, the adopted water is deionized water, the rest reagents and raw materials are analytically pure, and the adopted electrochemical workstation is a German Zazana electrochemical workstation with the model of zennium-E.

Example 1

The electrochemical biosensor for quickly detecting tumor cells comprises a flexible screen-printed electrode and gold nanoparticles deposited on the surface of the flexible screen-printed electrode, wherein thiolated folic acid molecules are modified on the surface of the gold nanoparticles, and the surface of the flexible screen-printed electrode is sealed by bovine serum albumin.

The preparation method of the electrochemical biosensor for rapidly detecting tumor cells comprises the following steps:

step one, sequentially adopting an ethanol solution with a volume fraction of 75% and water to clean a flexible screen printing electrode SPE, adopting nitrogen to blow dry, then adding 2.8mg of chloroauric acid and 13.5 mul of a concentrated sulfuric acid solution with a mass concentration of 98% into 2.5mL of water to obtain a 2.8mmol/L chloroauric acid solution, dropwise adding 200 mul of chloroauric acid solution onto the surface of the flexible screen printing electrode SPE, and adopting a-0.2V precipitation voltage to carry out deposition for 10min to form gold nanoparticles Au Nps, thus obtaining Au Nps-SPE;

step two, adding 10mg of thiolated folic acid molecules into 50mL of water to obtain 100 mu mol/L thiolated folic acid molecule solution, then dropwise adding 30 mu L of thiolated folic acid molecule solution onto the surface of the Au Nps-SPE obtained in the step one by adopting a dropping method, standing overnight at 4 ℃ in a dark place, and drying to enable the thiolated folic acid molecules Fa-PEG-SH to be connected onto the surface of the Au Nps to obtain Fa-PEG-SH-Au Nps-SPE;

and step three, adding 1mg BSA into 10mL of 0.1mol/L phosphate buffer solution to obtain 0.1mg/mL BSA mother solution, then diluting to 0.01mg/mL to obtain BSA solution, dropwise adding 200 mu L of BSA solution onto the Fa-PEG-SH-Au Nps-SPE obtained in the step two, standing at room temperature in a dark place for 30min for sealing to obtain BSA-Fa-PEG-SH-Au Nps-SPE, and then washing for 1h on a shaking table by adopting 0.1mol/L phosphate buffer solution to obtain the electrochemical biosensor.

The application process of the tumor cell rapid detection electrochemical biosensor comprises the following steps:

step one, cleaning an electrochemical biosensor by using 0.1mol/L phosphate buffer solution, drying the electrochemical biosensor by using nitrogen, then dropwise adding 300 mu L of sample liquid into a sample adding area of the electrochemical biosensor, and then placing the electrochemical biosensor in an incubator at 37 ℃ for incubation for 0.5 h;

step two, cleaning the electrochemical biosensor incubated in the step one by using 0.1mol/L phosphate buffer solution, drying by using nitrogen, then installing the electrochemical biosensor into a detection device, and dropwise adding 200 mu L of phosphate buffer solution containing 1mmol/L potassium ferrocyanide and 1mmol/L potassium ferricyanide to a sample adding area of the electrochemical biosensor;

and step three, starting an electrochemical workstation connected with the detection device, carrying out electrochemical impedance detection on the sample adding region of the electrochemical biosensor dropwise added with the phosphate buffer solution in the step two, wherein the scanning frequency adopted by the electrochemical impedance detection is 10 mHz-100 kHz, applying 5mV AC disturbance under open circuit potential to obtain an electrochemical impedance value, and outputting, recording and storing the electrochemical impedance value.

Fig. 1 is a schematic diagram of the preparation and application of the electrochemical biosensor for rapidly detecting tumor cells of the present invention, and as can be seen from fig. 1, the preparation and application processes of the electrochemical biosensor are as follows: depositing Au Nps on SPE, connecting Fa-PEG-SH to Au Nps for modification, then adopting BSA for sealing to obtain an electrochemical biosensor, dropwise adding a sample liquid into the electrochemical biosensor, specifically combining the Fa-PEG-SH with tumor cells (cells in the figure) if the tumor cells exist in the sample liquid, and obtaining data through electrochemical impedance detection and analyzing the data so as to judge the existence of the tumor cells in the sample liquid.

FIG. 2 is a schematic diagram of electrochemical impedance detection in the application process of the electrochemical biosensor for rapidly detecting tumor cells of the present invention, and it can be seen from FIG. 2 that the sensor comprises an electrochemical biosensor and a detection device (including an electrochemical workstation, a signal conditioning and converting circuit, a signal output device and a control device) for detecting the electrochemical biosensor, the electrochemical biosensor is provided with a sample adding region for containing a sample solution, and further comprises a working electrode, a counter electrode and a reference electrode as conductive components, the electrochemical impedance data acquisition, conversion and output are realized by the signal conditioning and conversion circuit and the signal output device (comprising an alarm device, a display device and a recording device), and the control device controls the switch of the sensor.

FIG. 3 is a cyclic voltammogram of the SPE, Au Nps-SPE and Fa-PEG-SH-Au Nps-SPE in this embodiment, and it can be seen from FIG. 3 that the peak current of Au Nps-SPE is significantly increased compared with that of SPE, which indicates that Au Nps has been successfully modified on the SPE surface; the peak current of Fa-PEG-SH-Au Nps-SPE is reduced compared with that of both SPE and Au Nps-SPE, which indicates that Fa-PEG-SH is successfully connected with Au Nps.

FIG. 4 is a graph showing the impedance system results of the SPE, Au Nps-SPE, Fa-PEG-SH-Au Nps-SPE, BSA-Fa-PEG-SH-Au Nps-SPE and the electrochemical biosensor after being specifically combined with tumor cells in example 1 of the present invention, and it can be seen from FIG. 4 that the impedance value of Au Nps-SPE is significantly reduced compared with that of SPE, which indicates that Au Nps has been successfully decorated on the surface of SPE, and the impedance value of Fa-PEG-SH-Au Nps-SPE is further increased compared with that of Au Nps-SPE and SPE, which indicates that Fa-PEG-SH has been successfully connected to Au Nps, and the impedance value of BSA-Fa-PEG-SH-Au Nps-SPE is further increased, which indicates that BSA has completed the sealing of non-specific sites. After the electrochemical biosensor is specifically combined with the tumor cells, the impedance value of the electrochemical biosensor continues to be obviously increased, which shows that the tumor cell rapid detection chemical biosensor has good response to the tumor cells.

Example 2

The electrochemical biosensor for rapidly detecting tumor cells comprises a flexible screen-printed electrode and a gold nanoparticle-thiolated folate molecule compound modified on the surface of the flexible screen-printed electrode, wherein the surface of the flexible screen-printed electrode is sealed by bovine serum albumin.

The preparation method of the electrochemical biosensor for rapidly detecting tumor cells comprises the following steps:

step one, adding 4mg of thiolated folate molecule Fa-PEG-SH into 2mL of water to obtain 2mg/mL of thiolated folate molecule solution, preparing gold nanoparticles Au Nps with the average particle size of 13nm by adopting a method of reducing chloroauric acid by sodium citrate, then adding 0.2mL of Au Nps solution into 2mL of thiolated folate molecule Fa-PEG-SH solution, and stirring for 24h to obtain gold nanoparticle-thiolated folate molecule compound Fa-PEG-SH-Au Nps solution;

step two, sequentially adopting ethanol solution with volume fraction of 75% and water to clean the SPE of the flexible screen printing electrode, and drying by adopting nitrogen;

step three, dripping 30 mu L of Fa-PEG-SH-Au Nps solution obtained in the step one on the surface of the SPE dried in the step two by adopting a dripping method, standing overnight in a dark place at 4 ℃, and drying to enable Fa-PEG-SH-Au Nps to be modified on the surface of the SPE to obtain Fa-PEG-SH-Au Nps-SPE;

step four, adding 1mg BSA into 10mL of 0.1mol/L phosphate buffer solution to obtain 0.1mg/mL BSA mother solution, then diluting to 0.01mg/mL to obtain BSA solution, dropwise adding 200 μ L of BSA solution onto the Fa-PEG-SH-Au Nps-SPE obtained in the step three, standing at room temperature in a dark place for 30min for sealing to obtain BSA-Fa-PEG-SH-Au Nps-SPE, and then washing for 1h on a shaking table by adopting phosphate buffer solution to obtain the electrochemical biosensor.

The application process of the tumor cell rapid detection electrochemical biosensor comprises the following steps:

step one, cleaning an electrochemical biosensor by using 0.1mol/L phosphate buffer solution, drying the electrochemical biosensor by using nitrogen, then dropwise adding 300 mu L of sample liquid into a sample adding area of the electrochemical biosensor, and then placing the electrochemical biosensor in an incubator at 37 ℃ for incubation for 0.5 h;

step two, cleaning the electrochemical biosensor incubated in the step one by using 0.1mol/L phosphate buffer solution, drying by using nitrogen, then installing the electrochemical biosensor into a detection device, and dropwise adding 200 mu L of phosphate buffer solution containing 1mmol/L potassium ferrocyanide and 1mmol/L potassium ferricyanide to a sample adding area of the electrochemical biosensor;

and step three, starting an electrochemical workstation connected with the detection device, carrying out electrochemical impedance detection on the sample adding region of the electrochemical biosensor dropwise added with the phosphate buffer solution in the step two, wherein the scanning frequency adopted by the electrochemical impedance detection is 10 mHz-100 kHz, applying 5mV AC disturbance under open circuit potential to obtain an electrochemical impedance value, and outputting, recording and storing the electrochemical impedance value.

The above description is only for the preferred embodiment of the present invention, and is not intended to limit the present invention in any way. Any simple modification, change and equivalent changes of the above embodiments according to the technical essence of the invention are still within the protection scope of the technical solution of the invention.