Reagent combination for biosensor, biosensor and application of biosensor in cancer marker detection

Abstract

The invention provides a reagent combination for a biosensor, the biosensor and application of the biosensor in cancer marker detection, the reagent combination for the biosensor comprises a substrate material and a platinum-coated gold nanorod probe, and the substrate material comprises NH2-MXene (at) AuNPs and biosynthetic quantum dots. The reagent combination for the biosensor, the biosensor and the application of the biosensor in cancer marker detection are used for detecting target protein, can effectively improve the detection sensitivity, accuracy and speed and effectively reduce the detection difficulty, and have important significance in deep application of the biosensor in medical clinical diagnosis.

Description

Technical Field

[0001] The present application relates to the technical field of biosensors, and in particular to a reagent combination for biosensors, biosensors and their application in the detection of cancer markers. Background Art

[0002] Biosensors are analytical devices used to detect analytes. They can convert the type and concentration of analytes into quantitative data that is easily accepted by people through a series of reactions, which is convenient for subsequent analysis. They are widely used in many fields such as biomedical research, drug synthesis screening, environmental monitoring and protection, health quarantine, forensic identification, and biomarker detection. However, the detection sensitivity of existing biosensors is limited, and it is difficult to achieve the low picogram / milliliter (pg / mL) sensitivity required for clinical applications. Therefore, the development of a more sensitive biosensor is of great significance for the further application of sensors in multiple industries. Summary of the invention

[0003] In view of this, the purpose of the present application is to propose a reagent combination for a biosensor, a biosensor and its application in cancer marker detection, so as to solve the above-mentioned technical problems.

[0004] In a first aspect of the present application, a reagent combination for a biosensor is provided, comprising a substrate material and a platinum-coated gold nanorod probe, wherein the substrate material comprises NH2-MXene@AuNPs and biosynthesized quantum dots.

[0005] In a second aspect of the present application, a biosensor is provided for detecting a target protein, comprising a sensing substrate and a platinum-coated gold nanorod probe, wherein the sensing substrate is prepared from NH2-MXene@AuNPs and biosynthetic quantum dots.

[0006] Furthermore, the biosensor is an electrochemical biosensor; the sensing substrate of the electrochemical biosensor is prepared by the following method:

[0007] NH2-MXene@AuNPs, biosynthetic quantum dots, a first antibody of a target protein, and bovine serum albumin are sequentially applied to a glassy carbon electrode to obtain a sensing substrate of the electrochemical biosensor.

[0008] Furthermore, the biosensor is a colorimetric biosensor; the sensing substrate of the colorimetric biosensor is prepared by the following method:

[0009] NH2-MXene@AuNPs, biosynthetic quantum dots, the first antibody of the target protein, and bovine serum albumin are sequentially added dropwise to the well plate to obtain the sensing substrate of the colorimetric biosensor.

[0010] Furthermore, the platinum-coated gold nanorod probe is prepared by the following method:

[0011] The gold nanorods were mixed with CTAB solution, AgNO3 solution and ascorbic acid solution, and then HCl and H2PtCl6 solution were added to react to obtain platinum-coated gold nanorods;

[0012] Bovine serum albumin is added to the platinum-coated gold nanorod solution for incubation, and after the incubation is completed, a second antibody of the target protein is added for reaction to obtain the platinum-coated gold nanorod probe.

[0013] Furthermore, the gold nanorods are prepared by the following method:

[0014] Mixing the HAuCl4 solution and the CTAB solution to obtain a first mixed solution;

[0015] Adding NaBH4 solution to the first mixed solution, stirring and standing to obtain a seed solution;

[0016] Completely dissolving CTAB and NaOL in deionized water to obtain a second mixed solution; sequentially adding AgNO3 solution, HAuCl4 solution and acrylic acid solution to the second mixed solution to obtain a growth solution;

[0017] The seed solution is added into the growth solution to generate the gold nanorods through reaction.

[0018] Furthermore, the NH2-MXene@AuNPs are prepared by the following method:

[0019] Adding Ti2CTx-Mxene to an ethanol solution and performing ultrasonic treatment to obtain a Ti2CTx-Mxene-ethanol solution;

[0020] APTES solution was added to Ti2CTx-Mxene-ethanol solution to obtain NH2-MXene;

[0021] Ultrasonic mixing of NH2-MXene and gold nanoparticles to obtain the NH2-MXene@AuNPs;

[0022] Wherein, the gold nanoparticles are prepared by the following method:

[0023] trisodium citrate is added to deionized water, heated to boiling, and then HAuCl4 solution is added to obtain a third mixed solution;

[0024] The trisodium citrate solution and the HAuCl4 solution are added to the third mixed solution in batches for multiple times to obtain the gold nanoparticles.

[0025] Furthermore, the biosynthetic quantum dots are prepared by the following method:

[0026] Staphylococcus aureus is activated in NB liquid culture medium, and then a portion of the bacterial solution is transferred to LB liquid culture medium for culture, and CdCl2 and sterile water are added to the LB liquid culture medium and incubated to synthesize the biosynthetic quantum dots.

[0027] Furthermore, the target proteins include metaphase protein, alpha-fetoprotein, carcinoembryonic antigen and prostate specific antigen.

[0028] The third aspect of the present application provides an application of a biosensor in the detection of cancer markers, wherein the concentration of metaphase protein is detected using a biosensor according to any one of the second aspects.

[0029] As can be seen from the above, the present application provides a reagent combination for biosensors, biosensors and their applications in cancer marker detection, including NH2-MXene@AuNPs, biosynthetic quantum dots and platinum-coated gold nanorod probes. NH2-MXene@AuNPs have good electrical conductivity, so the electrical conductivity of the sensing surface can be ensured, and the large number of amino groups introduced can promote the connection with BQDs. BQDs can be effectively fixed on the NH2-MXene@AuNPs material through their own carboxyl groups. Since BQDs are obtained by biosynthesis and have protein A, when used for the detection of target proteins, they can be accurately connected to the Fc fragment of the first antibody of the target protein, thereby playing a role in directional fixation and efficient recognition of the first antibody. The platinum-coated gold nanorod probe has excellent electrochemical conductivity and unique catalytic properties for H2O2 and TMB (3,3',5,5'-tetramethylbenzidine). Whether applied to electrochemical biosensors or colorimetric biosensors, it can amplify the signal and effectively reduce false positive and false negative results during the test process, thereby improving the detection sensitivity and accuracy. The reagent combination for biosensors, biosensors and their application in cancer marker detection provided by the present disclosure can effectively improve the detection sensitivity, accuracy and speed, effectively reduce the difficulty of detection, and are of great significance to the in-depth application of biosensors in medical clinical diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the present application or related technologies, the drawings required for use in the embodiments or related technical descriptions are briefly introduced below. Obviously, the drawings described below are only embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0031] Figure 1Ti2CT x -Mxene, NH2-MXene, AuNPs, NH2-MXene morphology characterization diagram, where A is Ti2CT x -MXene, B is the SEM image of NH2-MXene, C is the TEM image of AuNPs, and D is the TEM image of NH2-MXene@AuNPs.

[0032] Figure 2 This is a magnified TEM image of a single spherical gold nanoparticle.

[0033] Figure 3 The morphology characterization, size and element distribution diagram of BQDs, where A is the laser scanning confocal image of BQDs, B is the TEM image of BQDs, C is the size distribution diagram of BQDs (the horizontal axis is the size), and D is the elemental composition distribution diagram of BQDs (the horizontal axis is the element, and the vertical axis is the atomic ratio).

[0034] Figure 4 The morphology characterization diagrams of AuNR and Au@Pt NRs, where A and B are TEM images of AuNR at different magnifications, and C and D are TEM images of Au@Pt NRs at different magnifications.

[0035] Figure 5 This is the EDX spectrum of Au@Pt NRs (the horizontal axis is the radiation energy and the vertical axis is the signal intensity).

[0036] Figure 6Figure 1 is a schematic diagram of the conductive properties of GCE, NH2-MXene / GCE, NH2-MXene@AuNPs / GCE, BQDs / NH2-MXene@AuNPs / GCE, and MF-Probe, as well as a schematic diagram of the conductive properties of each stage in the manufacturing process of the electrochemical biosensor; A is a CV curve diagram of GCE, NH2-MXene / GCE, and NH2-MXene@AuNPs / GCE, in which a is the curve of GCE, b is the curve of NH2-MXene / GCE, and c is the curve of NH2-MXene@AuNPs / GCE, the horizontal axis is voltage, and the vertical axis is current; B is the CV curve diagram of NH2-MXene@AuNPs / GCE before and after antibody incubation. DPV curves of Ps / GCE and BQDs / NH2-MXene@AuNPs / GCE, where a is the DPV curve of BQDs / NH2-MXene@AuNPs / GCE after antibody incubation, b is the DPV curve of BQDs / NH2-MXene@AuNPs / GCE before antibody incubation, c is the DPV curve of NH2-MXene@AuNPs / GCE after antibody incubation, and d is the DPV curve of NH2-MXene@AuNPs / GCE before antibody incubation. The horizontal axis is voltage and the vertical axis is current; C is the DPV curve of MF-Probe, where the horizontal axis is voltage and the vertical axis is current. Without MF-probe is the result without MF-probe, and With MF-probe is the result with MF-probe; D is the DPV curve of NH2-MXene@AuNPs and Au@Pt Schematic diagram of the catalytic ability of NRs for TMB, where the horizontal axis is wavelength, the vertical axis is absorbance, and Control is the curve of the control group; E is the EIS curve of the electrode at each stage in the preparation process of the electrochemical biosensor; F is the DPV curve of the electrode at each stage in the preparation process of the electrochemical biosensor, where the horizontal axis is voltage and the vertical axis is current; among them, in Figures E and F, a~g are the curves of GCE, NH2-MXene@AuNPs / GCE, BQDs / NH2-MXene@AuNPs / GCE, Ab1 / BQDs / NH2-MXene@AuNPs / GCE, BSA / Ab1 / BQDs / NH2-MXene@AuNPs / GCE, MDK / BSA / Ab1 / BQDs / NH2-MXene@AuNPs / GCE, and MF-Probe / MDK / BSA / Ab1 / BQDs / NH2-MXene@AuNPs / GCE, respectively.

[0037] Figure 7Figure 1 is a schematic diagram of the results of MDK detection by the biosensor; A is a DPV curve diagram of the electrochemical biosensor detecting MDK at different concentrations, in which a~i are the curves of MDK at concentrations of 5fg / mL, 10fg / mL, 50fg / mL, 100fg / mL, 1pg / mL, 10pg / mL, 100pg / mL, 1ng / mL, and 10ng / mL, respectively, with the horizontal axis being the voltage and the vertical axis being the current; B is a calibration curve of the electrochemical biosensor detecting MDK at different concentrations, in which the horizontal axis is the logarithm of the concentration and the vertical axis is the relative peak current; C is a UV-visible absorption spectrum diagram of the colorimetric biosensor detecting MDK at different concentrations, in which a~i are 5pg / mL, 10pg / mL, 100fg / mL, 1ng / mL, 5ng / mL, respectively. L is the curve of MDK with concentrations of 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL, with the horizontal axis being the wavelength and the vertical axis being the absorbance; D is the calibration curve of the colorimetric biosensor detecting MDK with different concentrations, with the horizontal axis being the logarithm of the concentration and the vertical axis being the absorbance; E is a schematic diagram of the color change of MDK with different concentrations detected by the colorimetric biosensor under sunlight, with the horizontal axis being the logarithm of the concentration and the vertical axis being the absorbance from left to right corresponding to MDK with concentrations of 5 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, and 100 ng / mL respectively; F is the linear relationship between the RGB value of the color in Figure E and the MDK concentration, with the horizontal axis being the logarithm of the concentration and the vertical axis being the normalized RGB intensity.

[0038] Figure 8Figure 1 is a schematic diagram of the results of using the biosensor to detect the MDK concentration in the serum of healthy donors and thyroid cancer patients; A is a heat map of signal changes in the detection of MDK concentration in serum using electrochemical biosensors, colorimetric biosensors and ELISA equipment, wherein Electrochemical Mode is the electrochemical mode, i.e., the detection result corresponding to the electrochemical biosensor, and the color bar label Current represents the current; Colorimetric Mode is the colorimetric mode, i.e., the detection result corresponding to the colorimetric biosensor, and the color bar label Absorbance represents the absorbance; the color bar label OD value in the ELISA heat map represents the OD value; B is a rectangular diagram of MDK concentration in the serum of healthy donors and thyroid cancer patients (the horizontal axis Healthy represents the healthy donor, the horizontal axis Thyroid represents the cancer represents thyroid cancer patients, and the ordinate is MDK concentration); C is a scatter plot of MDK concentration in the serum of healthy donors and thyroid cancer patients (***p<0.001, ****p<0.0001, t test), where Normal represents normal healthy donors and Patients represents thyroid cancer patients; D is a ROC curve diagram for clinical diagnosis of thyroid cancer using biosensors, and AUC in the figure is the area under the curve. DETAILED DESCRIPTION

[0039] In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0040] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present application should be the usual meanings understood by people with ordinary skills in the field to which the present application belongs. The "first", "second" and similar words used in the embodiments of the present application do not represent any order, quantity or importance, but are only used to distinguish different components. "Including" or "comprising" and similar words mean that the elements or objects appearing in front of the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects. "Connect" or "connected" and similar words are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0041] A biosensor is an analytical device used to detect analytes. It can convert the type, concentration and other properties of the analyte into quantitative data that is easily accepted by people through a series of reactions, which is convenient for subsequent analysis. It has a wide range of applications in biomedical research, drug synthesis screening, environmental monitoring and protection, health quarantine, forensic identification, biomarker detection and many other fields.

[0042] Midkine (MDK), also known as mid-heparin-related growth factor, is a soluble protein. Its expression level increases significantly when cancer occurs (including lung cancer, thyroid cancer, etc.), and it has been shown to be detectable in urine and blood. In recent years, it has been widely regarded as a cancer marker with great potential. Normally, MDK is at a very low level (less than 600pg / mL) in the serum of healthy adults. When cancer develops, the MDK level expression level far exceeds 1ng / mL. Many reports have pointed out that overexpression of MDK is also one of the important indicators of impaired prognosis in lung cancer patients. At present, MDK is one of the most promising cancer markers. In addition to Enzyme-Linked Immunosorbent Assay (ELISA), there is no other reliable method for quantitative analysis of MDK. However, this method has limited sensitivity and a narrow linear range, and it is usually difficult to achieve the low picogram / milliliter (pg / mL) sensitivity required for clinical applications. Therefore, the development of a more sensitive and faster MDK analysis method is of great significance for early diagnosis, prognosis assessment, and monitoring of cancer.

[0043] Based on the above situation, the present disclosure provides a reagent combination for a biosensor, which can be used to detect a variety of target proteins including MDK, and has the characteristics of high detection sensitivity, more convenient operation, and faster detection. The reagent combination includes NH2-MXene@AuNPs, biosynthetic quantum dots and platinum-coated gold nanorod probes.

[0044] MXene is a two-dimensional transition metal carbide / carbonitride material. It is a new type of two-dimensional nanomaterial obtained by etching the MAX phase of layered ceramic materials to remove the A element. It has a unique multilayer structure and excellent electrical conductivity, mechanical properties and surface functionalization capabilities. It is also environmentally friendly and has unique application potential in energy storage, catalysis, electromagnetic shielding, sensing, biomedicine and other fields. After being modified with functional groups or different nanomaterials, MXene can have more outstanding performance in terms of electron transmission ability, biocompatibility, and exposure of more binding sites.

[0045] NH2-MXene material is an amino-modified MXene material. A large number of amino groups are introduced on the surface of the MXene material, making the surface smoother and the area larger.

[0046] AuNPs are gold nanoparticles. Metal nanoparticles are aggregates of metal atoms with a size of 1 to 100 nanometers, which is smaller than the wavelength of light. Due to their small size, they will produce quantum confinement effects, and increasing or decreasing the number of metal atoms will cause significant changes in their structure, electronic and optical properties. Therefore, unlike macroscopic metal materials, the size, morphology and element distribution of metal nanoparticles determine their mechanical behavior, surface adsorption, transport, catalytic activity and photoelectric properties, and they have broad application prospects in the construction of biosensors, research on electrochemical catalysis, photoelectricity, and physicochemical properties.

[0047] NH2-MXene@AuNPs is an amino-modified MXene material modified with gold nanoparticles. Anchoring gold nanoparticles on the amino-modified MXene material can effectively increase the conductivity of the sensing surface. When used in biosensors to detect and analyze the analytes, it can effectively improve the sensitivity and speed of the biosensor.

[0048] Quantum dots are nanoscale semiconductor materials that have been widely used in fields such as biosensors and solar cells. Biosynthesized quantum dots (BQDs) are quantum dots synthesized using biosynthetic methods of microorganisms. Traditionally, quantum dots are chemically synthesized, but microbial synthesis methods have been developed as an alternative strategy. In microbial synthesis methods, quantum dots synthesized by microorganisms have better biocompatibility than chemically synthesized quantum dots because proteins and peptides are attached to their surfaces.

[0049] Platinum-coated gold nanorod probes (MF-Probe) can be used as multifunctional signal probes with excellent peroxidase-like activity. The core-shell bimetallic nanomaterials provide excellent biocompatibility for antibody fixation through Pt-N bonds, and also show significant ability in improving electrochemical conductivity. They also have unique catalytic properties for H2O2 and TMB (3,3',5,5'-tetramethylbenzidine). Whether used in electrochemical biosensors or colorimetric biosensors, they can amplify signals and thus effectively improve detection sensitivity.

[0050] In some embodiments, the substrate material further comprises a first antibody against a target protein and bovine serum albumin.

[0051] In some embodiments, the platinum-coated gold nanorod probe is a probe modified with a secondary antibody against a target protein.

[0052] When the reagent combination disclosed in the present invention is used to detect the target protein, NH2-MXene@AuNPs, biosynthetic quantum dots, the first antibody of the target protein, and bovine serum albumin are first applied to the carrier in sequence to complete the preparation of the sensing substrate of the biosensor, and then the target protein and platinum-coated gold nanorod probes are added to achieve the determination of the target protein. The reagent combination for biosensors disclosed in the present invention can be used in electrochemical biosensors and can also be used in colorimetric biosensors. When applied to electrochemical biosensors, the carrier is a glassy carbon electrode, and when applied to colorimetric biosensors, the carrier is a well plate. It should be noted that those skilled in the art can apply the reagent combination disclosed in the present invention to other sensors as needed, and only need to replace the corresponding carrier as needed, without specific limitation.

[0053] The NH2-MXene@AuNPs disclosed in the present invention have good electrical conductivity, so the electrical conductivity of the sensing surface can be ensured, and the large number of amino groups introduced can promote the connection with BQDs. BQDs are effectively fixed on the NH2-MXene@AuNPs material through their own carboxyl groups. Since BQDs are obtained by biosynthesis and have protein A, they can be accurately connected to the Fc fragment of the first antibody of the target protein, thereby playing a role in directional fixation and efficient recognition of the first antibody. Platinum-coated gold nanorod probes (MF-Probes) can be used as multifunctional signal probes with excellent peroxidase-like activity. Its core-shell bimetallic nanomaterials provide excellent biocompatibility for antibody fixation through Pt-N bonds, so the second antibody of the target protein can be effectively fixed. MF-Probe has shown remarkable ability in improving electrochemical conductivity and has unique catalytic properties for H2O2 and TMB (3,3',5,5'-tetramethylbenzidine). Whether applied to electrochemical biosensors or colorimetric biosensors, it can amplify signals and effectively reduce false positive and false negative results during the test process, thereby improving detection sensitivity and accuracy. Compared with existing biosensor reagents, the reagent combination for biosensors provided by the present disclosure can effectively improve detection sensitivity, accuracy and speed, effectively reduce detection difficulty, and is of great significance to the in-depth application of biosensors in medical clinical diagnosis.

[0054] The present disclosure also provides a biosensor for detecting a target protein, comprising a sensing substrate and a platinum-coated gold nanorod probe, wherein the sensing substrate is prepared from NH2-MXene@AuNPs and biosynthetic quantum dots.

[0055] The biosensor disclosed in the present invention includes a sensing substrate and a platinum-coated gold nanorod probe. The sensing substrate is made of NH2-MXene@AuNPs and BQDs. When the sensing substrate is used to detect the target protein, NH2-MXene@AuNPs, as a bottom conductive material, has excellent electrochemical activity; BQDs are anchored in NH2-MXene@AuNPs, which can promote the efficient recognition and directional fixation of antibodies; the platinum-coated gold nanorod probe can further amplify the electrical signal and provide excellent biocompatibility for antibody fixation. Through the cooperation of NH2-MXene@AuNPs, BQDs and MF-Probe, the sensitivity and accuracy of the biosensor can be effectively improved, and the efficient detection of the target protein can be achieved.

[0056] The biosensor disclosed in the present invention may be an electrochemical biosensor or a colorimetric biosensor. Regardless of the specific type of biosensor, it has the characteristics of high sensitivity, high accuracy, and convenience.

[0057] In some embodiments, the biosensor is an electrochemical biosensor; the sensing substrate of the electrochemical biosensor is prepared by the following method:

[0058] NH2-MXene@AuNPs, biosynthetic quantum dots, a first antibody of a target protein, and bovine serum albumin are sequentially applied to a glassy carbon electrode to obtain a sensing substrate of the electrochemical biosensor.

[0059] When the prepared biosensor is an electrochemical biosensor, the NH2-MXene@AuNPs solution is firstly coated onto a purified glassy carbon electrode. The nanostructured carbon electrode (N-MXene@AuNPs / GCE) was prepared by applying a BQDs solution to the NH2-MXene@AuNPs / GCE to obtain a biosynthesized quantum dot-modified N-MXene@AuNPs / GCE glassy carbon electrode (BQDs / NH2-MXene@AuNPs / GCE). The first antibody (Ab1) of the target protein was applied to the surface of BQDs / NH2-MXene@AuNPs / GCE for recognition and connection to obtain Ab1 / BQDs / NH2-MXene@AuNPs / GCE. Bovine serum albumin (BSA) was then used to debride the nonspecific binding sites and seal the electrode surface to obtain the sensing substrate of the electrochemical biosensor (BSA / Ab1 / BQDs / NH2-MXene@AuNPs / GCE).

[0060] In some embodiments, the biosensor is a colorimetric biosensor; the sensing substrate of the colorimetric biosensor is prepared by the following method:

[0061] NH2-MXene@AuNPs, biosynthetic quantum dots, the first antibody of the target protein, and bovine serum albumin are sequentially added dropwise to the well plate to obtain the sensing substrate of the colorimetric biosensor.

[0062] When the prepared biosensor is a colorimetric biosensor, the NH2-MXene@AuNPs solution is first dripped onto the well plate, and then the BQDs and the first antibody of the target protein are dripped onto the well plate in sequence, and then BSA is added to clean the nonspecific binding sites to obtain the final sensing substrate of the colorimetric biosensor. The sensing substrate of the electrochemical biosensor and the sensing substrate of the colorimetric biosensor prepared by the above method have excellent conductive properties, and under the action of BQDs, more first antibodies of the target protein can be directionally identified and efficiently fixed, thereby improving the sensitivity and accuracy of the detection. When the electrochemical biosensor is used to detect the target protein, the sensing substrate of the electrochemical biosensor is placed in the target protein to be detected, and then the MF-Probe is added to complete the detection of the target protein. When the colorimetric biosensor is used to detect the target protein, the target protein is dripped into the sensing substrate of the colorimetric biosensor, and then the MF-Probe is added to complete the detection of the target protein, and the entire detection process is more efficient and convenient.

[0063] In some embodiments, before using the biosynthetic quantum dots to prepare a biosensor, they need to be activated. The activation process is: mixing the biosynthetic quantum dots with tris-hydroxymethylaminomethane hydrochloride (Tris-HCl), 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) and stirring to achieve the activation of the biosynthetic quantum dots. Whether it is preparing a sensing substrate for a colorimetric biosensor or a sensing substrate for an electrochemical biosensor, the biosynthetic quantum dots need to be activated first and then used to prepare the sensing substrate of the corresponding biosensor.

[0064] In some embodiments, the platinum-coated gold nanorod probe is prepared by the following method:

[0065] The gold nanorods were mixed with CTAB solution, AgNO3 solution and ascorbic acid solution, and then HCl and H2PtCl6 solution were added to react to obtain platinum-coated gold nanorods;

[0066] Bovine serum albumin is added to the platinum-coated gold nanorod solution for incubation, and after the incubation is completed, a second antibody of the target protein is added for reaction to obtain the platinum-coated gold nanorod probe.

[0067] In order to synthesize MF-Probe, platinum-coated gold nanorods (Au@Pt NRs) need to be prepared first, and then Au@Pt NRs are used to prepare MF-Probe. The synthesis process of Au@Pt NRs is as follows: first, gold nanorods (AuNR) are diluted in a three-necked flask, and then cetyltrimethylammonium bromide (CTAB) solution, silver nitrate (AgNO3) solution and ascorbic acid solution are added to the three-necked flask and stirred; then hydrochloric acid (HCl) and chloroplatinic acid (H2PtCl6) solution are added to the three-necked flask and stirred continuously, and Au@Pt NRs are obtained after the reaction. In order to obtain MF-Probe, Au@Pt NRs need to be incubated. First, Au@Pt NRs are added to BSA, incubated for a certain period of time, and then the second antibody of the target protein is added. After a certain period of reaction, MF-Probe is obtained. The platinum-coated gold nanorod probes prepared in this way can effectively improve the electrochemical conductivity, and at the same time have unique catalytic properties for H2O2 and TMB. They can be successfully used in electrochemical biosensors and colorimetric biosensors to amplify signals, thereby further improving the detection sensitivity and accuracy of biosensors.

[0068] In some embodiments, the gold nanorods are prepared by the following method:

[0069] Mixing the HAuCl4 solution and the CTAB solution to obtain a first mixed solution;

[0070] Adding NaBH4 solution to the first mixed solution, stirring and standing to obtain a seed solution;

[0071] Completely dissolving CTAB and NaOL in deionized water to obtain a second mixed solution;

[0072] Adding AgNO3 solution, HAuCl4 solution and acrylic acid solution to the second mixed solution in sequence to obtain a growth solution;

[0073] The seed solution is added into the growth solution to generate the gold nanorods through reaction.

[0074] The specific synthesis steps of gold nanorods (AuNR) are as follows: first, a tetrachloroauric acid (HAuCl4) solution and a hexadecyltrimethylammonium bromide (CTAB) solution are mixed in a flask to obtain a first mixed solution; then sodium borohydride (NaBH4) is added dropwise to the flask (i.e., added dropwise to the first mixed solution), and stirred at the same time, and the color of the solution changes from yellow to brown; then the mixed solution in the flask is placed at room temperature for passivation to obtain a seed solution. Subsequently, CTAB and sodium oleate (NaOL) are completely dissolved in a beaker containing deionized water to obtain a second mixed solution; after the second mixed solution is cooled, an AgNO3 solution and a HAuCl4 solution are added to the beaker (i.e., the second mixed solution), and stirred for a certain period of time, during which the color changes from yellow to colorless and transparent, and stirring is continued; then an acrylic acid solution (AA) is added, and vigorously stirred to obtain a growth solution; the seed solution is added to the growth solution, and after shaking and stirring, it is allowed to stand for a period of time to react and generate AuNR.

[0075] In some embodiments, the NH2-MXene@AuNPs are prepared by the following method:

[0076] Adding Ti2CTx-Mxene to an ethanol solution and performing ultrasonic treatment to obtain a Ti2CTx-Mxene-ethanol solution;

[0077] APTES solution was added to Ti2CTx-Mxene-ethanol solution to obtain NH2-MXene;

[0078] Ultrasonic mixing of NH2-MXene and gold nanoparticles to obtain the NH2-MXene@AuNPs;

[0079] Wherein, the gold nanoparticles are prepared by the following method:

[0080] trisodium citrate is added to deionized water, heated to boiling, and then HAuCl4 solution is added to obtain a third mixed solution;

[0081] The trisodium citrate solution and the HAuCl4 solution are added to the third mixed solution in batches for multiple times to obtain the gold nanoparticles.

[0082] In order to obtain NH2-MXene@AuNPs, it is first necessary to prepare gold nanoparticles (AuNPs) and NH2-MXene separately.

[0083] The specific preparation method of NH2-MXene is as follows: titanium carbide-based MXene (Ti2CTx-Mxene) is ultrasonically treated with an ethanol solution to obtain a Ti2CTx-Mxene-ethanol solution; 3-aminopropyltriethoxysilane (APTES) is slowly added to the obtained Ti2CTx-Mxene-ethanol solution, and the reaction is stirred to obtain a reaction product, the reaction product is centrifuged, and washed with deionized water to remove unreacted APTES, and then dried in a vacuum dryer to obtain the final NH2-MXene.

[0084] The specific preparation method of AuNPs is as follows: trisodium citrate is added to deionized water, and then the solution is placed in a three-necked flask, stirred vigorously, and heated to boiling; subsequently, HAuCl4 is slowly added to the three-necked flask, and the color of the solution changes from light yellow to pink within 10 minutes, indicating that gold seeds are synthesized to obtain a third mixed solution; after the third mixed solution is cooled to 90°C, trisodium citrate solution and HAuCl4 solution are added dropwise, and the addition of trisodium citrate solution and HAuCl4 solution is repeated many times to react to obtain gold nanoparticles.

[0085] The preparation process of NH2-MXene@AuNPs is as follows: NH2-MXene is ultrasonically mixed with AuNPs to obtain NH2-MXene@AuNPs. Due to the presence of -NH2 groups in NH2-MXene, it stably connects AuNPs through Au-N covalent interactions, thereby achieving the successful preparation of NH2-MXene@AuNPs.

[0086] In some embodiments, the biosynthetic quantum dots are prepared by the following method:

[0087] Staphylococcus aureus is activated in NB liquid culture medium, and then a portion of the bacterial solution is transferred to LB liquid culture medium for culture, and CdCl2 and sterile water are added to the LB liquid culture medium and incubated to synthesize the biosynthetic quantum dots.

[0088] In order to synthesize biosynthetic quantum dots, it is necessary to prepare nutrient broth (NB) liquid culture medium and lysogenic broth (LB) liquid culture medium first. Beef extract, peptone and sodium chloride are dissolved in distilled water to obtain NB liquid culture medium. Yeast extract, peptone and sodium chloride are dissolved in distilled water to obtain LB liquid culture medium. Before using NB liquid culture medium and LB liquid culture medium, they need to be sterilized. Staphylococcus aureus is activated in NB liquid culture medium for a certain period of time, and then part of the bacterial solution is transferred to LB culture medium and cultured for a certain period of time to keep the cells in the stationary phase; sterile water containing cadmium chloride (CdCl2) is added to the culture medium and incubated for a certain period of time to allow intracellular biosynthesis to obtain BQDs.

[0089] In some embodiments, the target protein includes mid-stage protein, alpha-fetoprotein, carcinoembryonic antigen and prostate-specific antigen. The biosensor disclosed in the present invention can be used not only to detect MDK, but also to detect alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA), with high detection sensitivity and rapidity, which is of great significance for early diagnosis, prognosis evaluation and monitoring of cancer.

[0090] The present disclosure also provides an application of a biosensor in the detection of cancer markers, using a biosensor according to any one of the above embodiments to detect the concentration of metaphase protein.

[0091] Hereinafter, MDK will be taken as an example to specifically illustrate the technical effects of the present disclosure.

[0092] Before detecting MDK, it is necessary to prepare the corresponding sensor. In this embodiment, an electrochemical biosensor and a colorimetric biosensor are prepared respectively. Before preparing the corresponding biosensor, it is also necessary to prepare a reagent combination for the biosensor. The complete preparation process will be described in detail below.

[0093] (1) Synthesis of NH2-MXene@AuNPs

[0094] (① Preparation of NH2-MXene materials

[0095] 30 mg of Ti2CTx-Mxene was ultrasonically treated with 15 mL of ethanol solution for 10 minutes to obtain a Ti2CTx-Mxene-ethanol solution; then 200 μL of APTES solution was slowly added to the Ti2CTx-Mxene-ethanol solution and stirred at 800 rpm for 24 hours to react to obtain NH2-MXene. The reaction product was centrifuged at 4000 rpm and washed with deionized water three times to remove unreacted APTES, and then dried in a 50 ° C vacuum dryer for 12 hours to obtain the final NH2-MXene product. NH2-MXene was resuspended in deionized water to a concentration of 2 mg / mL and then stored at 4 ° C until use.

[0096] ②Preparation of AuNPs

[0097] 51.76 mg of trisodium citrate was added to 80 mL of deionized water and mixed, and then the solution was placed in a three-necked flask and stirred vigorously, and heated to boiling for 15 minutes; then 533 μL of 25 mM HAuCl4 was slowly added to the three-necked flask, and the color of the solution changed from light yellow to pink within 10 minutes, indicating that gold seeds were synthesized, and the third mixed solution was obtained; after the third mixed solution was cooled to 90°C, 553 μL of 60 mM trisodium citrate solution and 533 μL of 25 mM HAuCl4 solution were added dropwise, and the addition of trisodium citrate solution and HAuCl4 solution was repeated 7 times to react to generate AuNPs. The reaction product was centrifuged, resuspended in deionized water, cooled to room temperature, and stored at 4°C until use.

[0098] (③NH 2- Preparation of MXene@AuNPs

[0099] 2 mL of NH2-MXene solution was ultrasonically mixed with 1 mL of AuNPs solution for 2 h to generate NH2-MXene@AuNPs. The reaction product was centrifuged at 3000 rpm and washed 3 times to obtain the final NH2-MXene@AuNPs, which were stored at 4 °C for later use.

[0100] (2) Synthesis of BQDs

[0101] Preparation of NB liquid medium: Dissolve 5 g of beef extract, 10 g of peptone and 5 g of sodium chloride in 1 L of distilled water to obtain NB liquid medium.

[0102] Preparation of LB liquid medium: Dissolve 3 g of yeast extract, 10 g of peptone and 5 g of sodium chloride in 1 L of distilled water to obtain LB liquid medium.

[0103] The NB liquid medium and the LB liquid medium were sterilized. Staphylococcus aureus was activated in the sterilized NB liquid medium for 24 hours, and then 1 mL of the bacterial solution was transferred to the sterilized new LB medium and cultured for 12 hours (140 rpm, 37 ° C) to keep the cells in the stationary phase. Then, 500 μL of sterile water containing 11.4 mg CdCl2 was added to the LB medium and incubated for 12 hours, and BQDs were obtained by intracellular biosynthesis. The synthesized product was centrifuged at 4000 rpm, washed three times with Tris-HCl buffer (pH = 8, 0.1 M), and resuspended in the solvent. The optical density (OD) value was measured to determine the product concentration. The BQDs of the measured concentration were heated in a water bath at 65 ° C for 30 min to kill Staphylococcus aureus and terminate the reaction, and then stored at 4 ° C until use.

[0104] (3) Preparation of MF-Probe

[0105] (①Preparation of Au@Pt NRs

[0106] Step 1: Synthesis of AuNR

[0107] Mix 5 mL of 0.5 mM HAuCl4 and 5 mL of 0.2 mM CTAB in a 20 mL flask to obtain a first mixed solution; add 1 mL of 6 mM NaBH4 solution dropwise to the first mixed solution while stirring at 1200 rpm, and the color of the solution changes from yellow to brown; then place the mixed solution in the flask at room temperature for 30 minutes for passivation to obtain a seed solution. 9g CTAB and 1.234g NaOL were completely dissolved in a beaker containing 250mL deionized water to obtain a second mixed solution; after the second mixed solution was cooled to 30°C, 18mL of 4mM AgNO3 solution was added to the beaker (i.e., the second mixed solution), mixed evenly, and then 250mL of 1mM HAuCl4 solution was added, and the mixture was allowed to stand at 30°C for 15min; then, the above solution was stirred at 700rpm for 90min, the temperature was kept constant, and the color of the solution was observed to change from yellow to colorless and transparent. After slowly stirring at 400rpm for 15min, 1.25mL of 0.064M AA was added, and the mixture was vigorously stirred for 30s to obtain a growth solution. Then 400μL of seed solution was added to the growth solution, gently shaken to mix, and allowed to stand at 30°C for 12h to generate AuNR. Finally, the obtained AuNR was washed three times by centrifugation at 7000rpm deionized water, resuspended in 20mL deionized water, and placed in a 4°C refrigerator for standby use.

[0108] Step 2: Synthesis of Au@Pt NRs

[0109] 250 μL of AuNR solution was diluted to 5 mL in a three-necked flask, and then 10 mL of 0.05 M CTAB solution, 500 μL of 20 mM AgNO3 solution and 500 μL of 100 mM ascorbic acid solution were added to the three-necked flask and stirred at 70 ° C for 1 hour; then 480 μL of 0.1 M HCl and 440 μL of 2 mM H2PtCl6 were added to the three-necked flask and stirred at 70 ° C for 4 hours to obtain Au@Pt NRs. The reaction product was centrifuged at 8000 rpm for 10 min and washed twice, and resuspended in 1 mL of deionized water for use.

[0110] (② Preparation of MF-Probe

[0111] 5 μL BSA solution (1%) was added to 200 μL Au@Pt NRs solution and incubated at 4°C for 30 minutes, then 50 μL of the second antibody solution of mid-term protein (10 μg / mL) was added dropwise and reacted at 4°C for 12 hours to prepare MF-Probe. The MF-Probe was washed twice by centrifugation at 7000 rpm with pH 7.0 phosphate buffer solution (PBS solution) and resuspended in 200 μL deionized water for use.

[0112] (4) Preparation of sensing substrate for electrochemical biosensor

[0113] GCE purification: After GCE was polished with nano-alumina powder and ultrasonically treated in deionized water and ethanol to remove surface adsorbed impurities, the GCE surface was dried with a nitrogen flow and stored at 4 °C.

[0114] Activation of biosynthesized quantum dots: 200 μL BQDs, 400 μL Tris-HCl, 0.2 M EDC, and 0.05 M NHS were mixed and stirred on a shaker at 26 °C for 30 min to achieve activation of biosynthesized quantum dots.

[0115] Preparation of sensing substrate:

[0116] 10 μL NH2-MXene@AuNPs solution was drop-coated onto purified GCE and dried in air at room temperature to obtain NH2-MXene@AuNPs / GCE; 6 μL activated BQDs solution was applied to NH2-MXene@AuNPs / GCE at 37 °C for 30 min to obtain BQDs / NH2-MXene@AuNPs / GCE; then 10 μL of the first antibody (Ab1) solution (20 μg / mL) of mid-term protein was applied to the surface of BQDs / NH2-MXene@AuNPs / GCE for recognition and connection; 1% BSA solution was used to debride nonspecific binding sites and block the electrode surface to obtain the sensing substrate of the electrochemical biosensor (BSA / Ab1 / BQDs / NH2-Mxene@AuNPs / GCE).

[0117] (5) Preparation of Sensing Substrate for Colorimetric Biosensor

[0118] A 96-well plate was selected as the well plate of the colorimetric biobed sensor. 10 μL NH2-MXene@AuNPs, 6 μL activated BQDs, 10 μL first antibody (Ab1) solution (20 μg / mL) of mid-phase protein, and 1% BSA solution were sequentially added to the 96-well plate to obtain the sensing substrate of the colorimetric biosensor.

[0119] Below, some experiments are conducted to study the characteristics of NH2-MXene@AuNPs, BQDs, MF-Probe and biosensors to further demonstrate the technical effects of the present disclosure.

[0120] (1) Morphological characterization of Ti2CTx-MXene, NH2-MXene, AuNPs and NH2-MXene@AuNPs

[0121] The morphologies of Ti2CTx-MXene, NH2-MXene, AuNPs and NH2-MXene@AuNPs were revealed by scanning electron microscope (SEM) and transmission electron microscope (TEM). Figure 1 shown.

[0122] from Figure 1 As can be seen in A, the layered Ti2CTx-MXene presents a loosely stacked accordion shape, while the NH2-MXene material modified by APTES, such as Figure 1 As shown in B, its surface is smoother, the area is effectively enlarged, and a large number of amino groups are introduced to promote the subsequent connection of BQDs.

[0123] from Figure 1 As can be seen from Figure C, well-dispersed single spherical nanoparticles are formed with a uniform diameter of 10 nanometers. From the enlarged view of the single spherical nanoparticle (i.e. Figure 2 ), which further confirmed the single crystal form of AuNPs. Figure 1 As shown in D, AuNPs are uniformly integrated on the surface of NH2-MXene, demonstrating the successful synthesis of NH2-MXene@AuNPs.

[0124] (2) Morphological characterization of BQDs

[0125] The morphology of BQDs was characterized by laser scanning confocal microscopy and TEM, and the size and elemental composition distribution of BQDs were analyzed. Figure 3 As shown. Figure 3 As shown in A, small spherical particles with green fluorescence in Staphylococcus aureus can be observed under laser scanning confocal microscopy, confirming the synthesis of BQDs with good dispersion and relatively uniform size. Figure 3The unique morphology of Staphylococcus aureus can be seen in B and 3C, with a diameter of about 0.71 μm. The elemental composition of CdS4Se quantum dots isolated from Staphylococcus aureus was analyzed by X-ray photoelectron spectroscopy (XPS), such as Figure 3 As shown in D, the existence of the three elements Cd, S and Se was confirmed in a ratio of approximately 1:4:2, further proving that quantum dots were synthesized inside Staphylococcus aureus and the synthesized quantum dot material was CdS4Se2.

[0126] (3) Characterization of morphology and element distribution of AuNR and Au@Pt NRs

[0127] The morphology and element distribution of AuNR and Au@Pt NRs were characterized by TEM. Figure 4 As shown. Figure 4 As shown in A and 4B, the gold nanorods are well dispersed, presenting a smooth and obvious rod-like structure with a uniform size of about 50 nm. Figure 4 The inset of B shows the clear lattice fringes of the gold nanorods, indicating that they are well crystalline with a lattice spacing of 0.23 nm, corresponding to the Au(gold) (111) crystal plane. These results indicate that the AuNRs synthesized by the seed growth method can be used as templates for the synthesis of Au@Pt NRs. Figure 4 As shown in Figures C and 4D, Pt (platinum) particles with a size of about 3-4 nm are evenly distributed on the surface of AuNR, forming a non-dense core-shell structure according to the Volmer-Weber growth model. Figure 4 In the inset of D, clear lattice fringes can be observed, and the lattice spacing is determined to be 0.241 nm, corresponding to the Pt(111) crystal plane.

[0128] The element distribution of Au@Pt NRs was characterized by EDX, such as Figure 5 As shown, it is shown that the nanocomposite material is composed of Au and Pt elements.

[0129] (4) Study on the electrical conductivity of GCE, NH2-MXene / GCE, and NH2-MXene@AuNPs / GCE

[0130] Containing 10mM [Fe(CN)6] 3- The conductivity properties of GCE, NH2-MXene / GCE, and NH2-MXene@AuNPs / GCE were evaluated by cyclic voltammetry (CV) with a voltage range of -0.2 V to 0.6 V and a scan rate of 50 mV / s. Figure 6As shown in A. Since NH2-MXene has excellent electrical conductivity, it can effectively enhance the current signal after combining with GCE. Figure 6 It can also be seen in A that compared with bare GCE (curve a), NH2-MXene / GCE (curve b) shows a significantly enhanced current signal. NH2-MXene@AuNPs / GCE (curve c) has the highest current response, with an Ipa value of 98.65μA, indicating that NH2-MXene can further enhance the current signal after combining with gold nanoparticles, and is more sensitive when used in biosensors to detect target proteins.

[0131] The electroactive surface area of ​​different materials can be quantitatively estimated using the Randles-Sevcik equation (Li et al. 2021):

[0132] I pa =2.69×10 5 n 2 / 3 A eff D 1 / 2 v 1 / 2 C0

[0133] Among them I pa represents the anode peak current, n represents the number of transferred electrons, A eff Electroactive surface area (cm 2 ), D is [Fe(CN)6] 3- The diffusion coefficient of the redox probe (equal to 1) is (6.70±0.02)×10 -6 cm 2 / s, v is the scanning rate, C0 is the concentration of the redox probe (10 mmol / cm -3 ). According to the above formula, the surface area of ​​NH2-MXene@AuNPs / GCE is 6.4×10 -2 cm 2 , which are 1.58 and 2.02 times of those of NH2-MXene / GCE and bare GCE, respectively. The combined modification of gold nanoparticles and -NH2 groups effectively increases the active surface area, making NH2-MXene@AuNPs have excellent electrochemical activity, and it can be used as a bottom conductive material for biosensors.

[0134] (5) Study on the antibody capture ability of BQDs

[0135] In order to study the antibody capture ability of BQDs, electrodes modified with NH2-MXene@AuNPs and BQDs / NH2-MXene@AuNPs composite materials, namely NH2-MXene@AuNPs / GCE and BQDs / NH2-MXene@AuNPs / GCE, were prepared. Under the same conditions, the electrodes modified with NH2-MXene@AuNPs and BQDs / NH2-MXene@AuNPs were incubated with 20 μg / mL MDK antibody at 37 °C for 1 h. After incubation, the 10 mM [Fe(CN)6] 3- The peak current difference (ΔI / μA) of the two composite materials before and after binding with MDK antibody was measured by differential pulse voltammetry (DPV) in 0.1M KCl solution. The results are as follows: Figure 6 B. After MDK antibody incubation, the ΔI on NH2-MXene@AuNPs / GCE (curve b, d) is 32.70μA, while the ΔI on BQDs / NH2-MXene@AuNPs / GCE (curve a, c) drops to 50.45μA, the current drops significantly, and the resistance increases significantly, indicating that more MDK antibodies are fixed on BQDs / NH2-MXene@AuNPs / GCE after antibody incubation, which further proves that BQDs can promote the immobilization of antibodies and improve the capture ability of antibodies. When applied to biosensors, it can effectively improve the capture ability of biosensors for antibodies, thereby improving the detection sensitivity and speed.

[0136] (6) Study on the electrochemical performance of MF-Probe

[0137] The electrochemical performance of MF-Probe was studied using DPV. The electrochemical biosensor was used to detect 0.1 ng / mL MDK in a PBS buffer solution containing 5 mM H2O2. The control group only used the sensing substrate of the electrochemical biosensor to detect MDK without adding MF-Probe. The results are shown in Figure 2. Figure 6 As shown in C. Figure 6 It can be observed in C that in the absence of MF-Probe, the current is relatively small, only 9.66μA. In the presence of MF-Probe, the attraction and catalytic ability of Au@Pt NRs lead to rapid redox of H2O2, increasing the current signal to 18.46μA, which is 1.91 times higher than that of the counterpart without MF-Probe, effectively proving that MF-Probe has excellent signal amplification ability. Therefore, the biosensor prepared by using it can effectively amplify the signal and improve the detection sensitivity and accuracy.

[0138] (7) Potential application of MF-Probe in colorimetric detection

[0139] In order to further study the potential application of MF-Probe in colorimetric biosensor detection and eliminate the influence of substrate materials on the colorimetric mode, a two-component TMB colorimetric substrate (containing TMB and H2O2) was used. Figure 6 As shown in Figure D, it can be observed that the control group (deionized water) and NH2-MXene@AuNPs show the smallest absorption peak, while MF-Probe shows an obvious absorption peak at 650nm. The results show that Au@Pt NRs have peroxidase-like activity and can oxidize H2O2 in the TMB two-component reagent, causing TMB color change. Therefore, MF-Probe can also be used as a signal probe for MDK detection in the colorimetric mode. The colorimetric biosensor prepared using it also has the characteristics of sensitive detection and high accuracy.

[0140] (8) Research on the manufacturing process of electrochemical biosensors

[0141] Electrochemical impedance spectroscopy (EIS) and DPV were used to verify the electrochemical biosensor fabrication process. The results are shown in Figure 2. Figure 6 As shown in Figures 6E and 6F, it can be seen that the Rct value of the electrode modified with NH2-MXene@AuNPs and BQDs (curves b and c) is significantly lower than that of the unmodified bare GCE (curve a), indicating that NH2-MXene@AuNPs and BQDs can effectively promote the electron transfer rate on the electrode surface; after modification with the first antibody of MDK (curve d), BSA (curve e), MDK (curve f) and MF-Probe (curve g), higher impedance can be observed, and the gradual increase in resistance indicates that the non-conductive bioactive substances are successfully modified layer by layer, indicating that the antigen and antibody are successfully attached to the electrode, which further indicates that the electrochemical biosensor is successfully prepared and can be successfully applied to the detection of MDK.

[0142] (9) Research on the application of biosensors in MDK detection

[0143] ① Detection of MDK concentration using electrochemical biosensor

[0144] Prepare MDK solutions with concentrations of 5fg / mL, 10fg / mL, 50fg / mL, 100fg / mL, 1pg / mL, 10pg / mL, 100pg / mL, 1ng / mL, and 10ng / mL, place the sensing substrate (BSA / Ab1 / BQDs / NH2-MXene@AuNPs / GCE) of the electrochemical biosensor in a container containing the MDK solutions of the above concentrations, and then add MF-Probe, and the final configuration is

[0145] MF-Probe / MDK / BSA / Ab1 / BQDs / NH2-MXene@AuNPs / GCE. During each modification process, the sensing substrate of the electrochemical biosensor was gradually washed with 0.1 M PBS buffer solution (pH 7.4) to remove the unattached molecules on the electrode surface at 4 °C until the next electrochemical measurement. The test results are shown in Figure 7 A. At the same time, the detection results of the electrochemical biosensor were calibrated to obtain a calibration curve, where the error bar = SD (n = 3), such as Figure 7 As shown in B.

[0146] ② Detection of MDK concentration using colorimetric biosensor

[0147] Prepare 5pg / mL, 10pg / mL, 100fg / mL, 1ng / mL, 5ng / mL, 10ng / mL, 20ng / mL, 50ng / mL, 100ng / mL MDK solution, add the above concentration of MDK solution to the sensing substrate of the colorimetric biosensor, then add MF-Probe, and add 150μL TMB two-component colorimetric solution, incubate at 37℃ in the dark for 15 minutes, add 75μL 5mM H2SO4 solution to terminate the reaction. Use a multiplate reader to scan the sample at 1nm intervals in the wavelength range of 300-600nm to obtain the absorption spectrum of each well. The results are as follows: Figure 7 C; the measured absorbance was calibrated to obtain the corresponding calibration curve, where the error bar = SD (n = 3), and the results are as follows Figure 7 D. In addition, photos were taken with a smartphone within 15 minutes, and the results are shown in Figure 7 E and read using ImageJ Figure 7 The RGB values ​​of the colors in E were used to obtain a linear relationship between the RGB values ​​and the MDK concentration, where the error bars = SD (n = 3), as shown in Figure 7 As shown in F.

[0148] exist Figure 7In A, it can be seen that the electrochemical signal intensity increases with the increase of MDK concentration, and the relative peak current ΔI (ΔI = I-I0, where I represents the current response corresponding to different concentrations of midkine, and I0 represents the current response at 0 fg / mL) has a good linear relationship with the logarithmic value of MDK in the range of 5 fg / mL to 10 ng / mL (e.g. Figure 7 B), the linear regression equation is ΔI = 1.688 × lg c +9.758(R 2 =0.994). The detection limit (LOD) can be calculated as LOD=KS_D / m, where K is the confidence concentration related constant, usually K=3, SD is the relative standard deviation of 10 blank values, and m is the slope of the calibration curve within the concentration range. After calculation, the detection limit is 1.620fg / mL (signal-to-noise ratio is 3).

[0149] Due to the special properties of MF-Probe, MDK can also be detected by UV-visible spectroscopy (such as Figure 7 C, D) and mobile phones (such as Figure 7 E, F) to achieve. Figure 7 As shown in C and 7E, with the increase of MDK, the absorption peak at 450nm rises sharply, and the gray value reading of RGB color also increases significantly. Figure 7 D) It can be seen that the sensor exhibits two linear ranges. In the MDK concentration range of 5 pg / mL to 5 ng / mL, the linear regression equation is I = 0.300 × lg c +1.239(R 2 =0.996), in the range of 5ng / mL to 100ng / mL, the linear regression equation is I = 1.213 × lg c +0.629(R 2 =0.992), the LOD determined by colorimetry was 2.295 pg / mL; the linear regression equation obtained in Image 7F was y=0.301×lg c +0.037(R 2 =0.993), the linear range was wide, from 10pg / mL to 100ng / mL, and the LOD was low, at 1.930pg / mL.

[0150] It can be seen that the biosensor proposed in the present disclosure can not only select a mode according to specific detection requirements, but also form a self-calibration mechanism through signals between different modes, making MDK detection more convenient, rapid and accurate.

[0151] (10) Research on the application of biosensors in clinical diagnosis of cancer

[0152] Description of test samples: The test samples for this test were collected from Tianjin Medical University and Tianjin Medical University Cancer Research Institute and Hospital. The test samples include healthy donors and donors from thyroid cancer patients. After all test samples are collected, they need to be pretreated. The pretreatment process includes: slightly inverting the collected blood samples 10 times, and then placing them at 37°C for 2 hours until separation; then centrifuging at 3000rpm for 10 minutes and obtaining serum from the separated blood samples, and storing them at -80°C; during the test, in order to reduce the influence of other impurities in the serum, the serum was diluted 32 times as the final test sample.

[0153] The electrochemical biosensor, colorimetric biosensor and ELISA device disclosed in the present invention were used to detect the MDK concentration in the serum of healthy donors and donors of thyroid cancer patients. The results are as follows: Figure 8 As shown. Figure 8 As can be seen from Figure A, the detection results of the electrochemical biosensor and the colorimetric biosensor are consistent with the signal changes of the commercial ELISA detection, and for the same sample, the signal intensity of the biosensor proposed in the present disclosure is higher than that of its ELISA counterpart, which effectively shows that the biosensor of the present disclosure has higher sensitivity. Figure 8 As shown in B, the MDK level in thyroid cancer patients was much higher than that in healthy volunteers. The t-test revealed a significant difference in the mean MDK level between thyroid cancer patients and healthy donors (P < 0.0001), indicating that MDK is an ideal biomarker for clinical diagnosis (e.g. Figure 8 C). In addition, the receiver operating characteristic (ROC) curve showed that the proposed biosensor achieved high-precision cancer diagnosis (AUC = 1) and successfully distinguished thyroid cancer patients from healthy individuals (e.g. Figure 8 D). This effectively demonstrates that the biosensor disclosed in the present invention can be used for clinical diagnosis of thyroid cancer, and also indirectly proves the potential of midkine as a biomarker for cancer diagnosis.

[0154] The present disclosure provides a reagent combination for a biosensor, a biosensor, and its application in cancer marker detection, wherein AuNPs are anchored on the aminated MXene by covalent bonding to ensure the conductivity of the sensing surface. Since AuNPs can covalently bind to the -SH and -NH2 groups in the first antibody of the target protein through Au-S or Au-N bonds, a certain amount of the first antibody of the target protein can be randomly fixed. At the same time, compared with the random binding of AuNPs to the first antibody of the target protein in NH2-MXene@AuNPs, the introduced BQDs have a high density of protein A and carboxyl groups on its surface, and the carboxyl groups can tightly connect the BQDs to the NH2-MXene@AuNPs through amide bonds, while protein A can specifically bind to the Fc region of the first antibody of the target protein, thereby increasing the capture of the first antibody and more exposure of the active binding sites, thereby effectively improving the detection sensitivity, accuracy and speed.

[0155] On this basis, the platinum-coated gold nanorod probe (MF-Probe) has the function of amplifying signals and is used as a multifunctional signal probe with excellent peroxidase-like activity. The core-shell bimetallic nanomaterial of the platinum-coated gold nanorod provides excellent biocompatibility for antibody fixation through Pt-N bonds, exhibits significant ability in improving electrochemical conductivity, and has unique catalytic properties for H2O2 and TMB. When the midkine level is determined by electrochemical and colorimetric methods, false positive and false negative results can be minimized, further improving the detection sensitivity and accuracy.

[0156] The biosensor disclosed in the present invention achieves a wide detection range from 5fg / mL to 100ng / mL, with a detection limit (LOD) of 1.620fg / mL. It can be detected using an electrochemical workstation, a microplate reader, or even a mobile phone. Compared with the existing ELISA, it not only has a lower LOD, but also responds more sensitively to trace MDK, and can distinguish subtle changes in MDK levels in the serum of healthy individuals and thyroid cancer patients with high precision. The biosensor disclosed in the present invention can be used to evaluate trace MDK levels in complex matrices, which is helpful for in-depth research on the role of MDK in cancer screening and prognosis evaluation, and is of great significance for the clinical diagnosis and prognosis evaluation of cancer.

[0157] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present application (including the claims) is limited to these examples. In line with the concept of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0158] In addition, when details are set forth to describe exemplary embodiments of the present application, it is obvious to those skilled in the art that the present application embodiments can be implemented without these details or with changes in these details. Therefore, these descriptions should be considered illustrative rather than restrictive.

[0159] For simplifying explanation and discussion, and in order not to make the embodiment of the present application difficult to understand, the known power supply / ground connection with other parts may or may not be shown in the provided drawings. In addition, the device can be shown in the form of a block diagram to avoid making the embodiment of the present application difficult to understand, and this also takes into account the following fact, that is, the details of the implementation method of these block diagram devices are highly dependent on the platform to be implemented in the embodiment of the present application (that is, these details should be completely within the scope of understanding of those skilled in the art). In the case of elaborating specific details to describe the exemplary embodiments of the present application, it is obvious to those skilled in the art that the embodiment of the present application can be implemented without these specific details or when these specific details are changed. Therefore, these descriptions should be considered to be illustrative rather than restrictive.

[0160] Although the present application has been described in conjunction with the specific embodiments of the present application, many replacements, modifications and variations of these embodiments will be apparent to those of ordinary skill in the art based on the foregoing description. The present application embodiments are intended to encompass all such replacements, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application embodiments should be included in the protection scope of the present application.

Claims

1. A reagent combination for a biosensor, characterized in that: It includes a base material and a platinum-coated gold nanorod probe, wherein the base material includes NH2-MXene@AuNPs and biosynthesized quantum dots.

2. A biosensor, characterized in that: Used to detect target proteins, including a sensing substrate and a platinum-coated gold nanorod probe, wherein the sensing substrate is prepared by NH2-MXene@AuNPs and biosynthetic quantum dots.

3. The biosensor according to claim 2, characterized in that The biosensor is an electrochemical biosensor; the sensing substrate of the electrochemical biosensor is prepared by the following method: NH2-MXene@AuNPs, biosynthetic quantum dots, a first antibody of a target protein, and bovine serum albumin are sequentially applied to a glassy carbon electrode to obtain a sensing substrate of the electrochemical biosensor.

4. The biosensor according to claim 2, characterized in that The biosensor is a colorimetric biosensor; the sensing substrate of the colorimetric biosensor is prepared by the following method: NH2-MXene@AuNPs, biosynthetic quantum dots, the first antibody of the target protein, and bovine serum albumin are sequentially added dropwise to the well plate to obtain the sensing substrate of the colorimetric biosensor.

5. The biosensor according to claim 2, characterized in that The platinum-coated gold nanorod probe is prepared by the following method: The gold nanorods were mixed with CTAB solution, AgNO3 solution and ascorbic acid solution, and then HCl and H2PtCl6 solution were added to react to obtain platinum-coated gold nanorods; Bovine serum albumin is added to the platinum-coated gold nanorod solution for incubation, and after the incubation is completed, a second antibody of the target protein is added for reaction to obtain the platinum-coated gold nanorod probe.

6. The biosensor according to claim 5, characterized in that The gold nanorods are prepared by the following method: Mixing the HAuCl4 solution and the CTAB solution to obtain a first mixed solution; Adding NaBH4 solution to the first mixed solution, stirring and standing to obtain a seed solution; Completely dissolving CTAB and NaOL in deionized water to obtain a second mixed solution; sequentially adding AgNO3 solution, HAuCl4 solution and acrylic acid solution to the second mixed solution to obtain a growth solution; The seed solution is added into the growth solution to generate the gold nanorods through reaction.

7. The biosensor according to claim 2, characterized in that: The NH2-MXene@AuNPs were prepared by the following method: Adding Ti2CTx-Mxene to an ethanol solution and performing ultrasonic treatment to obtain a Ti2CTx-Mxene-ethanol solution; APTES solution was added to Ti2CTx-Mxene-ethanol solution to obtain NH2-MXene; Ultrasonic mixing of NH2-MXene and gold nanoparticles to obtain the NH2-MXene@AuNPs; Wherein, the gold nanoparticles are prepared by the following method: trisodium citrate is added to deionized water, heated to boiling, and then HAuCl4 solution is added to obtain a third mixed solution; The trisodium citrate solution and the HAuCl4 solution are added to the third mixed solution in batches for multiple times to obtain the gold nanoparticles.

8. The biosensor according to claim 2, characterized in that The biosynthetic quantum dots are prepared by the following method: Staphylococcus aureus is activated in NB liquid culture medium, and then a portion of the bacterial solution is transferred to LB liquid culture medium for culture, and CdCl2 and sterile water are added to the LB liquid culture medium and incubated to synthesize the biosynthetic quantum dots.

9. The biosensor according to claim 2, characterized in that: The target proteins include metaphase protein, alpha-fetoprotein, carcinoembryonic antigen and prostate specific antigen.

10. Application of a biosensor in cancer marker detection, characterized in that: A biosensor according to any one of claims 2 to 9 is used to detect the concentration of metaphase protein.

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