Protein molecular marking method for constructing high-performance electrochemical sensor

Abstract

The invention belongs to the field of electrochemical biosensors, and particularly relates to a protein molecular marking method for constructing a high-performance electrochemical sensor. Organic ligands on the surfaces of sulfide colloidal quantum dots are replaced with water-soluble ligands through liquid-phase ligand replacement, then the water-soluble ligands are mixed with carboxyl activating agents to activate carboxyl of the water-soluble ligands, then the water-soluble ligands are uniformly mixed with protein molecules, and the protein molecules are effectively marked on the quantum dots. A working electrode of a planar three-electrode is coated with the quantum dots with the surfaces combined with the water-soluble ligands and marked with the protein molecules through a one-step method, and after drying, the electrochemical biosensor which is wide in detection range, high in response sensitivity and high in anti-interference performance is prepared.

Description

Technical Field

[0001] The present application belongs to the field of electrochemical biosensors, and more specifically, relates to a protein molecule labeling method for constructing a high-performance electrochemical sensor. Background Art

[0002] Quantum dots (QD) have unique optical properties, such as broad absorption peaks, narrow emission peaks, good photostability, and fluorescence intensity, which make them widely used in physics, chemistry, electronics, and biology. Studies have shown that using quantum dots to label glucose oxidase and then coating it on electrodes can be used to prepare glucose electrochemical biosensors, which are more sensitive than traditional methods of detecting glucose. However, the protein molecules (glucose oxidase) adsorbed on the electrode surface are easy to fall off, and the luminescence effect of quantum dots is not stable in electrochemical biosensors. The detection sensitivity and detection response speed of electrochemical biosensors need to be further improved.

[0003] The prior art can fix the protein molecules adsorbed on the electrode surface by coating the electrode surface with thin film deposition stabilizers such as chitosan and Nafion, but the electron transfer between the enzyme and the substrate is affected, which seriously hinders the charge transfer. Patent document CN114705740A discloses a platinum nanoparticle-bismuth sulfide complex glucose electrochemical sensor, which uses an embedding method to fix glucose oxidase on a platinum nanomaterial-hedgehog-shaped bismuth sulfide composite material and modify a glassy carbon electrode, and then drips a Nafion solution after drying, and low-temperature drying to obtain a glucose electrochemical biosensor. The preparation steps of this sensor are complicated, requiring layers of superimposed modification layers, and the linear range of glucose detection is narrow and the detection limit is high, with a detection range of 0.003mM~1.9mM, and it is impossible to effectively detect micromole and millimole glucose solutions.

[0004] Patent document CN114813877A discloses a sensor for detecting glucose, in which a ternary composite material solution of sulfide colloidal quantum dots and gold nanosphere particles and glucose oxidase with oleic acid on the surface is coated on a planar three-electrode working electrode to prepare a sensor for detecting glucose. However, the labeling efficiency of glucose oxidase in the above-mentioned ternary composite material is low, which further leads to less glucose oxidase coupled to the sensor, so that the range of glucose detection by the sensor is limited to 100nmol / L~10mmol / L, and it is impossible to accurately and effectively detect high-concentration glucose solutions, and the response sensitivity is only 0.238μA / dec, which cannot detect changes in glucose concentration sensitively, quickly and accurately. In addition, the preparation cost of the sensor is high, and it is necessary to use precious metal (gold) nanoparticles to stably bind glucose oxidase and accelerate electron transfer in the enzyme catalysis process, which is not conducive to large-scale production.

[0005] Therefore, providing a method for efficiently labeling protein molecules and providing a high-performance electrochemical biosensor constructed based on this method have important application value. Summary of the invention

[0006] In view of the defects of the prior art, the purpose of the present application is to provide a protein molecule labeling method for constructing a high-performance electrochemical sensor, aiming to solve the problems that the existing protein molecules cannot be stably labeled on quantum dots, and the preparation of electrochemical biosensors based on the existing methods has complicated operating steps, requires layer-by-layer modification, requires the additional use of cross-linking agents, precious metal nanoparticles, etc., and has high preparation costs. In addition, the prepared electrochemical biosensors have low response sensitivity, limited detection range, and cannot respond quickly and accurately.

[0007] To achieve the above objectives, in a first aspect, the present application provides a protein molecule labeling method for constructing a high-performance electrochemical sensor, comprising the following steps: S1. After mixing the sulfide colloidal quantum dots with organic ligands bound to the surface and a weakly polar organic solvent or a non-polar organic solvent, the mixture is evenly mixed with a solution containing a water-soluble ligand, and the liquid phase ligand is replaced, followed by centrifugation, washing, and drying to obtain sulfide colloidal quantum dots with the water-soluble ligands bound to the surface; The water-soluble ligand is a water-soluble ligand containing a thiol group and a carboxyl group; S2. Evenly mix the sulfide colloidal quantum dots with the water-soluble ligands bound to the surface, a carboxyl activator and protein molecules, activate the carboxyl groups of the water-soluble ligands to label the protein molecules, and obtain the sulfide colloidal quantum dots with the water-soluble ligands bound to the surface and labeling the protein molecules.

[0008] Preferably, in step S1, the weakly polar organic solvent is an organic solvent with a dielectric constant ≤5.

[0009] Preferably, in step S1, the organic ligand is one or more of fatty acid, fatty amine, trioctylphosphine and trioctylphosphine oxide.

[0010] Preferably, in step S1, the sulfide colloidal quantum dots are one or more of lead sulfide colloidal quantum dots, zinc sulfide colloidal quantum dots and bismuth sulfide colloidal quantum dots.

[0011] Preferably, in step S1, the water-soluble ligand is one or more of glutathione, mercaptopropionic acid and thioglycolic acid.

[0012] Preferably, in step S1, the mass ratio of the sulfide colloidal quantum dots to the water-soluble ligand is 1:(0.5-2).

[0013] Preferably, in step S2, the carboxyl activating agent comprises EDC and / or NHS.

[0014] Preferably, in step S2, the protein molecule is one or more of glucose oxidase, catalase, cholesterol oxidase, triglyceride oxidase, urease, alkaline phosphatase, lactate dehydrogenase, antigen and antibody.

[0015] Preferably, in step S2, the ratio of the sulfide colloidal quantum dots with surface-bound water-soluble ligands to the carboxyl activator is 10 mg: (100-500) μmol.

[0016] Preferably, in step S2, the mass ratio of the sulfide colloidal quantum dots with surface-bound water-soluble ligands to the protein molecules is 1:(1-5).

[0017] In a second aspect, the present application provides a sulfide colloidal quantum dot with a water-soluble ligand bound to its surface and labeling protein molecules, which is prepared using the above-mentioned protein molecule labeling method.

[0018] In a third aspect, the present application provides a method for constructing a high-performance electrochemical biosensor using the above-mentioned sulfide colloidal quantum dots, comprising the following steps: The dispersion of the sulfide colloidal quantum dots is coated on the working electrode of the planar three-electrode, and after drying to form a film, an electrochemical biosensor is prepared.

[0019] Preferably, the coating method is one or more of spin coating, drop coating, spray coating and electrospray printing.

[0020] Preferably, the working electrode is a gold electrode.

[0021] In a fourth aspect, the present application provides a high-performance electrochemical biosensor prepared using the above method.

[0022] In general, the above technical solutions conceived by this application have the following technical advantages compared with the prior art: (1) The protein molecule labeling method for constructing a high-performance electrochemical sensor provided in this application replaces the organic ligand on the surface of the sulfide colloidal quantum dots with a water-soluble ligand through liquid-phase ligand replacement, and then mixes with a carboxyl activator to activate the carboxyl group of the water-soluble ligand, and then mixes with the protein molecules evenly, so that the protein molecules are effectively labeled on the quantum dots. The quantum dots prepared in this application, which are bound to the surface of the water-soluble ligand and label the protein molecules, are coated on the working electrode of the planar three-electrode by a one-step method, and after drying, an electrochemical biosensor with a wide detection range, high response sensitivity, and strong anti-interference can be prepared.

[0023] (2) The present application regulates the ratio between sulfide colloidal quantum dots with water-soluble ligands bound to the surface, carboxyl activators and protein molecules during the protein molecule labeling process, thereby effectively labeling the protein molecules on the quantum dots. At the same time, the sulfide colloidal quantum dots with water-soluble ligands bound to the surface and labeling the protein molecules can be stably modified on the working electrode. On the basis of the protein molecules specifically recognizing specific biological molecules, the small size effect of the quantum dots is synergistic, so that the active center of the protein molecule coupled on the working electrode directly interacts with the specific biological molecule, and the biological molecule recognition signal is better converted into an electrical signal output, thereby being able to highly sensitively detect the concentration of the specific biological molecule in the sample to be tested.

[0024] (3) The electrochemical biosensor for detecting glucose constructed in the present application is prepared by effectively labeling glucose oxidase onto quantum dots and modifying them onto the working electrode. The sensor can detect glucose solutions in the range of 0.1 μmol / L to 100 mmol / L, and the response sensitivity can reach 1.68 μA / dec.

[0025] (4) Compared with existing electrochemical biosensors, the high-performance electrochemical biosensor provided in the present application can stably attach sulfide colloidal quantum dots that bind water-soluble ligands to the modified surface and label protein molecules to the electrode through a simple one-step coating. There is no need to use precious metal nanomaterials, no need to use additional coating film deposition stabilizers, and no need for repeated coating, thus avoiding complex sensor construction processes. It has the advantages of simple preparation steps and low cost, and is suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a flow chart of a protein molecule labeling method for constructing a high-performance electrochemical sensor provided in an embodiment of the present application; Figure 2 This is a diagram showing the phenomenon of replacing the oleic acid ligand of the lead sulfide colloidal quantum dots with a glutathione ligand provided in Example 1 of the present application; Figure 3 It is a Fourier transform infrared spectrum (FTIR) of lead sulfide colloidal quantum dots with oleic acid ligands bound to the surface (PbS CQDs-OA), lead sulfide colloidal quantum dots with glutathione ligands bound to the surface (PbS CQDs-GSH), quantum dots with glutathione ligands bound to the surface and labeled with protein molecules (PbS CQDs-GSH-GOx), and glucose oxidase (GOx) prepared in Example 1 of the present application; Figure 4The UV-Vis-NIR spectra of lead sulfide colloidal quantum dots with oleic acid ligands bound to the surface (PbS CQDs-OA), lead sulfide colloidal quantum dots with glutathione ligands bound to the surface (PbS CQDs-GSH), quantum dots with glutathione ligands bound to the surface and labeled with protein molecules (PbS CQDs-GSH-GOx), and glucose oxidase (GOx) prepared in Example 1 of the present application, wherein Content A is the UV-Vis-NIR spectra of PbS CQDs-OA, and Content B is the UV-Vis-NIR spectra of PbS CQDs-GSH, PbS CQDs-GSH-GOx, and GOx; Figure 5 It is a differential pulse voltammetry curve of the electrochemical biosensor modified with PbS CQDs-GSH-GOx prepared in Example 1 of the present application for detecting glucose test solutions of different concentrations; Figure 6 It is a linear fitting curve of the current peak value around 0 mV of the DPV test curve corresponding to the PbS CQDs-GSH-GOx modified electrochemical biosensor prepared in Example 1 of the present application for detecting glucose test solutions of different concentrations; Figure 7 The responsiveness of the electrochemical biosensor modified with PbS CQDs-GSH-GOx prepared in Example 1 of the present application to detect KCl solution, NaCl solution, glycine solution, ascorbic acid solution, uric acid solution, and glucose solution; Figure 8 It is a Fourier transform infrared spectrum (FTIR) graph of the quantum dots (CQDs) prepared in Comparative Example 1 of the present application and the quantum dots (CQDs / GOx) with glucose oxidase (GOx) labeled on the surface. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0028] In the description of this application, it should be understood that the term "and / or" is a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. The symbol " / " herein indicates that the associated objects are in an or relationship, for example, A / B means A or B.

[0029] In the description of the embodiments of the present application, words such as "exemplary" or "for example" are used to indicate examples, illustrations or descriptions. Any embodiment or design described as "exemplary" or "for example" in the embodiments of the present application should not be interpreted as being more preferred or more advantageous than other embodiments or designs. Specifically, the use of words such as "exemplary" or "for example" is intended to present related concepts in a specific way.

[0030] In the description of the embodiments of the present application, unless otherwise specified, “plurality” means two or more than two.

[0031] Liquid-phase ligand replacement is performed using quantum dots with organic ligands bound to the surface and protein molecules, so that the organic ligands on the surface of the quantum dots can be replaced by protein molecules, but the replacement efficiency is low, and only a small number of protein molecules can be labeled on the quantum dots. The DPV current signal of the prepared electrochemical biosensor is small when the quantum dots with surface-labeled protein molecules are modified onto the working electrode of the planar three-electrode. Patent document CN114813877A adds gold nanosphere particles to improve the signal-to-noise ratio of the sensor, accelerate electron transfer during enzyme catalysis, and enhance the sensitivity of the sensor, but the detection range of the sensor is still small, the response sensitivity is low, and it is unable to detect quickly and accurately. Based on this, the present application provides a protein molecule labeling method for constructing a high-performance electrochemical sensor, such as Figure 1 As shown, the following steps are included: S1. After mixing the sulfide colloidal quantum dots with organic ligands bound to the surface and a weakly polar organic solvent or a non-polar organic solvent, the mixture is evenly mixed with a solution containing a water-soluble ligand, and liquid-phase ligand replacement is performed, followed by centrifugation, washing, and drying to obtain sulfide colloidal quantum dots with water-soluble ligands bound to the surface; The water-soluble ligand is a water-soluble ligand containing a thiol group and a carboxyl group; S2. Evenly mix the sulfide colloidal quantum dots with the water-soluble ligands bound to the surface, a carboxyl activator and protein molecules, activate the carboxyl groups of the water-soluble ligands to label the protein molecules, and obtain the sulfide colloidal quantum dots with the water-soluble ligands bound to the surface and labeling the protein molecules.

[0032] The method of using quantum dots to label protein molecules provided in the present application is to replace the organic ligands of the sulfide colloidal quantum dots with water-soluble ligands, then mix them evenly with a carboxyl activator to activate the carboxyl groups of the water-soluble ligands, and finally mix them evenly with protein molecules, so that the protein molecules are effectively labeled on the sulfide colloidal quantum dots.

[0033] In the present application, the weakly polar organic solvent or non-polar organic solvent can dissolve the organic ligand and provide sufficient solvation effect to support the liquid phase ligand replacement reaction. In some embodiments, in step S1, the weakly polar organic solvent is an organic solvent with a dielectric constant ≤ 5, selected from one or more of chloroform, toluene, xylene, and hexane.

[0034] In some embodiments, in step S1, the solution containing water-soluble ligands is prepared by mixing water-soluble ligands with deionized water. By mixing sulfide colloidal quantum dots with organic ligands bound to their surfaces with a weakly polar organic solvent or a non-polar organic solvent, and then mixing the mixture evenly with the solution containing water-soluble ligands, the quantum dot solution with or without ligand replacement can be quickly separated.

[0035] In some embodiments, in step S1, the organic ligand is one or more of fatty acid, fatty amine, trioctylphosphine, and trioctylphosphine oxide.

[0036] In some embodiments, the above-mentioned fatty acids include but are not limited to one or more of lauric acid, palmitic acid, oleic acid, stearic acid, myristic acid, elaidic acid, arachidic acid, heneicosanoic acid, tricosanoic acid, docosanoic acid, tetracosanoic acid, hexacosanoic acid, heptacosanoic acid, octacosanoic acid and cis-13-docosaenoic acid.

[0037] In some embodiments, the above-mentioned fatty amines include but are not limited to one or more of decylamine, didecylamine, undecylamine, tridecylamine, tetradecylamine, pentadecylamine, hexadecylamine, octadecylamine, didodecylamine, tri-dodecylamine, cyclododecylamine, didodecylamine, trioctadecylamine, trioctylamine, N,N-dimethyldecylamine, N,N-dimethylundecylamine, N,N-dimethyldodecylamine, N,N-dimethyltridecylamine, N,N-dimethyltetradecylamine and N,N-dimethylhexadecylamine.

[0038] In some embodiments, in step S1, the sulfide colloidal quantum dots are one or more of lead sulfide colloidal quantum dots, zinc sulfide colloidal quantum dots and bismuth sulfide colloidal quantum dots. It is to be understood that the present application does not limit the source of the sulfide colloidal quantum dots, which can be purchased from commercial products or prepared in the laboratory. Exemplary, the preparation method of lead sulfide colloidal quantum dots whose organic ligand is oleic acid can be to synthesize surface-bound oleic acid lead sulfide colloidal quantum dots by the instantaneous nucleation reaction of lead oleate and bis(trimethylsilyl) sulfide using a hot injection method.

[0039] In some embodiments, in step S1, the water-soluble ligand is one or more of glutathione, mercaptopropionic acid and thioglycolic acid.

[0040] In some embodiments, in step S1, the mass ratio of the sulfide colloidal quantum dots to the water-soluble ligands is 1:(0.5~2), preferably 1:(0.8~2), which can replace all the organic ligands bound to the surface of the sulfide colloidal quantum dots with water-soluble ligands.

[0041] In some embodiments, in order to make the carboxyl activation reaction of the water-soluble ligand in step S2 more stable and more fully proceed, the sulfide colloidal quantum dots with surface-bound water-soluble ligands and the carboxyl activator can be first mixed evenly to form an active intermediate, and then mixed evenly with the protein molecules so that the protein molecules react with the active intermediate and are labeled on the sulfide colloidal quantum dots, thereby preparing sulfide colloidal quantum dots with surface-bound water-soluble ligands and labeled protein molecules.

[0042] In some embodiments, in step S2, the mixing is performed at a rotation speed of 100-300 rpm and for a time of 0.5 h-2 h.

[0043] In some embodiments, in step S2, the carboxyl activator includes but is not limited to one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). In the present application, the carboxyl activator can react with the carboxyl group of the water-soluble ligand on the surface of the sulfide colloidal quantum dots to activate the carboxyl group by forming an activated intermediate. The carboxyl activator EDC can react with the carboxyl group of the water-soluble ligand to form a property-active intermediate, and NHS can react with the intermediate to form a stable NHS ester, reducing the possibility of hydrolysis of the intermediate, so that the carboxyl activation reaction can remain effective for a longer time, while avoiding the occurrence of side reactions.

[0044] In the present application, the sulfide colloidal quantum dots with water-soluble ligands bound to the surface can form amide bonds with protein molecules after carboxyl activation treatment, so that the protein molecules are labeled on the sulfide colloidal quantum dots. By carrying out the corresponding protein molecule labeling, the concentration of specific biological molecules in the sample to be tested can be detected. Exemplarily, by labeling glucose oxidase on sulfide colloidal quantum dots, the concentration of glucose in the sample to be tested can be detected; by labeling a certain antigen on sulfide colloidal quantum dots, the concentration of the antibody corresponding to the antigen in the sample to be tested can be detected. In some embodiments, in step S2, the above-mentioned protein molecule can be but not limited to glucose oxidase, catalase, cholesterol oxidase, triglyceride oxidase, urease, alkaline phosphatase, lactate dehydrogenase, antigen, antibody, one or more. It can be understood that the present application does not specifically limit the types of the above-mentioned antigens and antibodies. In practical applications, the corresponding types of antigens or antibodies can be labeled on quantum dots and detected according to the detection requirements.

[0045] In some embodiments, in step S2, the ratio of the sulfide colloidal quantum dots with surface-bound water-soluble ligands to the carboxyl activator is 10 mg: (100-500) μmol, which can properly activate the carboxyl groups of the water-soluble ligands on the surface of the quantum dots, and when it is used to prepare an electrochemical biosensor, it will not affect the conductivity of the electrochemical biosensor.

[0046] In some embodiments, in step S2, the mass ratio of the sulfide colloidal quantum dots with surface-bound water-soluble ligands to the protein molecules is 1:(1~5), which can successfully label the protein molecules on the quantum dots, and prepare sulfide colloidal quantum dots with surface-bound water-soluble ligands and labeled with protein molecules. The dispersion containing the quantum dots is coated on the working electrode of the planar three-electrode, and after drying, an electrochemical biosensor with a wide detection range, a low detection limit, and a high response sensitivity can be prepared, thereby avoiding the complex and time-consuming modification process of traditional electrochemical biosensors.

[0047] On the other hand, the present application also provides a sulfide colloidal quantum dot with a water-soluble ligand bound to the surface and labeled with a protein molecule, and the quantum dot is prepared using the above method.

[0048] The present application also provides a method for constructing a high-performance electrochemical biosensor using the above-mentioned quantum dots with water-soluble ligands bound to the surface and labeled with protein molecules, comprising the following steps: The dispersion of the sulfide colloidal quantum dots with water-soluble ligands bound to the surface and labeled with protein molecules is coated on the working electrode of the planar three-electrode, and the electrochemical biosensor is prepared after drying to form a film.

[0049] In some embodiments, the coating method is one or more of spin coating, drop coating, spray coating and electrospray printing.

[0050] In some embodiments, the working electrode is a gold electrode.

[0051] Based on this, the present application also provides a high-performance electrochemical biosensor, which is prepared using the above method.

[0052] The electrochemical biosensor provided in this application is prepared by coating a dispersion containing quantum dots on a three-electrode plane in a simple one-step method, and after drying, a high-performance electrochemical biosensor can be prepared, which can respond quickly and sensitively to the object to be detected, and has a wide detection range and high detection accuracy. Compared with existing electrochemical biosensors, this application has the advantages of simple preparation steps and low preparation cost, and is suitable for large-scale batch production.

[0053] It should be understood that materials of the same or similar type, model, quality, nature or function as the reagents and instruments used in the following examples can be used to implement the present application. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0054] The following are examples and comparative examples: Example 1 The protein molecule labeling method provided in this embodiment comprises the following steps: (1) Synthesis of lead sulfide colloidal quantum dots with oleic acid ligands bound to the surface 1.8 g of lead oxide (PbO), 6 mL of oleic acid (OA) and 20 mL of octadecene (ODE) were mixed by high-speed stirring at 90° C. in a vacuum environment to prepare a lead precursor, and the lead precursor was heated to 120° C.

[0055] In the glove box, use a pipette to add 280 μL of bis(trimethylsilyl) sulfide (C 6 H 18 SSi 2 ) and 10 mL of octadecene (ODE) were mixed to prepare the sulfur precursor.

[0056] In a nitrogen environment, the sulfur precursor was quickly injected into the lead precursor, and then cooled in cold water for 30 seconds. The precipitate was collected and washed several times with toluene and acetone, and the precipitate was collected by centrifugation and vacuum dried to prepare lead sulfide colloidal quantum dots with oleic acid ligands on the surface, namely PbS CQDs-OA.

[0057] (2) Ligand replacement The above 0.05 g of PbS CQDs-OA was mixed with 10 mL of chloroform solution to prepare a quantum dot dispersion with a concentration of 5 mg / mL of surface-bound oleic acid ligands.

[0058] 0.46 g of glutathione (GSH) and 10 mL of deionized water were mixed to prepare a glutathione solution with a concentration of 0.15 mol / L.

[0059] The quantum dot dispersion with oleic acid ligands bound to the surface and the glutathione solution were mixed in a volume ratio of 1:1 (e.g. Figure 2 As shown in the figure on the left), the liquid phase ligand replacement reaction was carried out at room temperature with magnetic stirring at 200 rpm for 0.5 h, using the GSH ligand to replace the OA ligand. After the reaction, a stratification phenomenon (such as Figure 2As shown in the figure on the right, the upper solution turns black and the lower solution turns transparent. This is because the quantum dots after ligand replacement exist in the upper deionized water with a lower density. Then take out the upper quantum dot aqueous solution, add chloroform solution and mix thoroughly, centrifuge to remove the upper clear liquid and collect the precipitate. Then mix the precipitate and deionized water thoroughly to dissolve the precipitate, then add ethanol to wash several times, centrifuge to collect the precipitate, and after vacuum drying, prepare the lead sulfide colloidal quantum dots with glutathione ligands bound to the surface, namely PbS CQDs-GSH.

[0060] (3) Activation of the carboxyl group of glutathione ligand bound to the quantum dot surface 0.1 g of the above-mentioned PbS CQDs-GSH was mixed with 10 mL of PBS to prepare a quantum dot dispersion with a surface-bound glutathione ligand having a concentration of 10 mg / mL.

[0061] Prepare a carboxyl activation solution: mix 100 μmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) with 1 mL of PBS to obtain an EDC solution with a concentration of 0.1 mol / L; mix 100 μmol of N-hydroxysuccinimide (NHS) with 1 mL of PBS to obtain an NHS solution with a concentration of 0.1 mol / L, and then mix the EDC solution and the NHS solution to obtain a carboxyl activation solution.

[0062] The quantum dot dispersion with glutathione ligand bound to the surface and the carboxyl activation solution were mixed in a volume ratio of 1:2, and magnetically stirred at 200 rpm for 30 minutes at 25°C to activate the carboxyl group on the glutathione ligand to obtain a quantum dot dispersion with active water-soluble ligand properties.

[0063] (4) Labeling protein molecules onto quantum dots 3 mL of the above-mentioned water-soluble ligand-active quantum dot dispersion was mixed with 1 mL of 50 mg / mL glucose oxidase (GOx) solution, and magnetically stirred at 200 rpm for 1.5 h at 25 ° C. The supernatant was removed by centrifugation and the precipitate was collected. The precipitate was then washed several times with PBS, collected by centrifugation, and vacuum dried to prepare quantum dots with glutathione ligands bound to the surface and labeled with protein molecules, namely PbS CQDs-GSH-GOx. PbS CQDs-GSH-GOx was characterized by FTIR and UV-Vis-NIR characterization methods.

[0064] Figure 3The following are Fourier transform infrared spectra (FTIR) of lead sulfide colloidal quantum dots with oleic acid ligands on the surface (PbS CQDs-OA), lead sulfide colloidal quantum dots with glutathione ligands on the surface (PbS CQDs-GSH), quantum dots with glutathione ligands on the surface and labeled with protein molecules (PbS CQDs-GSH-GOx), and glucose oxidase (GOx). It can be seen that PbS CQDs-OA quantum dots have obvious CH stretching vibration peaks at wave numbers of 2850~2950 cm⁻¹; there is a C=C double bond absorption peak near the wave number of 1600 cm⁻¹; there is a CH bending vibration near the wave number of 1397 cm⁻¹, indicating that oleic acid ligands (OA) exist on the surface of lead sulfide colloidal quantum dots. The CH stretching vibration peak of PbS CQDs-GSH quantum dots at the wave number 2850~2950 cm⁻¹ is weakened; there is an obvious SH stretching vibration near 2539 cm-1, which originates from the -SH of glutathione ligand; there is an NH stretching vibration peak near the wave number 3249 cm⁻¹; there is a related absorption peak of the peptide bond near the wave number 1336 cm⁻¹, indicating the reduction of oleic acid ligands (OA) on the surface of lead sulfide colloidal quantum dots and the presence of glutathione (GSH) ligands. The PbS CQDs-GSH-GOx quantum dots have an NH stretching vibration peak near the wave number 3249 cm⁻¹. There is a broad absorption peak around the wave number 1540 cm⁻¹, which originates from the absorption peaks of the aromatic ring of tryptophan and the benzene ring of tyrosine, which are unique to glucose oxidase. There is an obvious peptide bond-related absorption peak near the wave number 1336 cm⁻¹, indicating that the surface of lead sulfide colloidal quantum dots is bound to glutathione (GSH) ligands and glucose oxidase (GOx) is successfully labeled.

[0065] Figure 4 The UV-Vis-NIR spectra of lead sulfide colloidal quantum dots with oleic acid ligands on the surface (PbS CQDs-OA), lead sulfide colloidal quantum dots with glutathione ligands on the surface (PbS CQDs-GSH), quantum dots with glutathione ligands on the surface and labeled with protein molecules (PbS CQDs-GSH-GOx), and glucose oxidase (GOx) are shown. It can be seen that PbS CQDs-OA quantum dots have an obvious absorption peak at a wavelength of 958nm ( Figure 4Content A), the average size of quantum dots is 3nm. The absorption peak of PbS CQDs-GSH quantum dots at 200~300nm is obviously different from that of PbS CQDs-OA quantum dots, showing a slight absorption peak around 976nm, which proves the reduction of oleic acid ligand (OA) and the increase of glutathione ligand (GSH). FAD is a key cofactor in glucose oxidase and plays an important role in the enzyme catalysis process. The flavin part in FAD has strong absorption properties, especially in the UV-visible region. Its absorption peak usually appears in the range of 450~475nm, which is caused by Figure 4 It can be seen from content B that glucose oxidase (GOx) and PbS CQDs-GSH-GOx quantum dots both show the same absorption peak at a wavelength of 466 nm, which is related to the absorption characteristics of the FAD (flavin adenine dinucleotide) conjugated system in the enzyme molecule, indicating the successful labeling of glucose oxidase (GOx).

[0066] (5) Preparation of electrochemical biosensors The above-mentioned PbS CQDs-GSH-GOx and PBS were evenly mixed to prepare a dispersion with a concentration of 10 mg / mL, and then 10 μL of the dispersion was evenly spin-coated on the working electrode of the screen-printed gold three-electrode. The spin coating was only performed once, and after drying at room temperature, a PbS CQDs-GSH-GOx-modified electrochemical biosensor was obtained.

[0067] 10 μL of phosphate buffer was dripped on the surface of the working electrode of the prepared electrochemical biosensor as a blank control group, and then the differential pulse voltammetry (DPV) detection mode was selected by the electrochemical workstation, and the pulse width was set to 50 ms, the pulse amplitude was set to 50 mV, and the potential increment was set to 4 mV to test the differential pulse voltammetric characteristics of the working electrode surface. Glucose test solutions of different concentrations (0, 0.1 μmol / L, 1 μmol / L, 10 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 500 μmol / L, 1 mmol / L, 2 mmol / L, 5 mmol / L, 10 mmol / L, 100 mmol / L) were dripped on the surface of the working electrode of the prepared electrochemical biosensor, and the differential pulse voltammetric characteristics of the working electrode surface were tested by the electrochemical workstation. The peak current size in the differential pulse voltammetric curve during the test process can be used to directly detect different concentrations of glucose.

[0068] Figure 5The figure shows the differential pulse voltammetry curves of the electrochemical biosensor modified with PbS CQDs-GSH-GOx for detecting glucose test solutions of different concentrations. It can be seen that the current peak value around 0 mV increases significantly with the increase of the detected glucose concentration (0.1 μmol / L~100 mmol / L), and the current peak value is as high as 20 μA, which indicates that the electrochemical biosensor prepared in this embodiment can detect glucose concentration with high sensitivity.

[0069] Figure 6 Shown is the linear fitting curve of the DPV test curve corresponding to the current peak around 0mV of the electrochemical biosensor modified with PbS CQDs-GSH-GOx to detect glucose test solutions of different concentrations. It is calculated that the response sensitivity of the electrochemical biosensor for detecting glucose is 1.68μA / dec, that is, for every order of magnitude (10 times) change in glucose concentration, the change in the electrical signal output by the detection sensor is 1.68μA. It is more sensitive to glucose and can detect changes in glucose concentration more quickly and accurately.

[0070] (6) Selectivity of electrochemical biosensors Common interfering substances in the glucose detection process (KCl, NaCl, glycine, ascorbic acid, uric acid) were prepared into solutions with a concentration of 500 μmol / L, and then dripped onto the working electrode surface of the electrochemical biosensor. The current value of the electrochemical biosensor before and after the addition of the interfering substances was recorded, where A 0 is the initial current without adding any interfering substances, and A is the response current measured after adding different interfering substances, which is used to evaluate the selectivity of the electrochemical biosensor.

[0071] Figure 7 The figure shows the responsiveness of the electrochemical biosensor modified by PbS CQDs-GSH-GOx to detect KCl solution, NaCl solution, glycine solution, ascorbic acid solution, uric acid solution, and glucose solution. It can be seen that at the same concentration, compared with the above-mentioned interfering substances, the electrochemical biosensor has the largest current difference in detecting glucose, has high responsiveness and excellent selectivity to glucose, and can be effectively used in the detection of glucose.

[0072] In summary, the PbS CQDs-GSH-GOx modified electrochemical biosensor prepared in this application can achieve high-sensitivity detection of glucose, while effectively avoiding or reducing interference triggered by non-target factors, and has high detection accuracy in complex real samples. In addition, the above electrochemical biosensor has a wide detection range and has excellent detection effects on glucose in the concentration range of 0.1μmol / L~100mmol / L.

[0073] Comparative Example 1 (without ligand replacement) (1) Synthesis of lead sulfide colloidal quantum dots with oleic acid ligands bound to the surface According to the method provided in Example 1, lead sulfide colloidal quantum dots with oleic acid ligands bound to the surface were synthesized and recorded as CQDs.

[0074] (2) Labeling protein molecules onto quantum dots 0.1 g of CQDs and 3 mL of n-octane were mixed to obtain a CQDs quantum dot dispersion. The CQDs quantum dot dispersion was mixed with 1 mL of 50 mg / mL glucose oxidase (GOx) solution and magnetically stirred at 25°C for 1.5 h to prepare quantum dots labeled with protein molecules (glucose oxidase, GOx), namely CQDs / GOx.

[0075] Figure 8 The following are Fourier transform infrared spectra (FTIR) of lead sulfide colloidal quantum dots (CQDs) with oleic acid ligands bound to the surface and quantum dots (CQDs / GOx) labeled with glucose oxidase on the surface. It can be seen that CQDs / GOx quantum dots have a peptide bond-related absorption peak near the wave number 1646 cm⁻¹, but the intensity is weak, indicating that only a small part of GOx is labeled on the lead sulfide colloidal quantum dots.

[0076] (3) Preparation of electrochemical biosensors The above-mentioned PbS CQDs-GOx and PBS were evenly mixed to prepare a solution with a concentration of 10 mg / mL, and then 10 μL of the solution was evenly spin-coated on the working electrode of the screen-printed gold three-electrode. After drying at room temperature, a PbS CQDs-GOx-modified electrochemical biosensor was obtained.

[0077] Then, the method provided in Example 1 was used to detect glucose test solutions of different concentrations (0, 0.1 μmol / L, 1 μmol / L, 10 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 500 μmol / L, 1 mmol / L, 2 mmol / L, 5 mmol / L, 10 mmol / L, 100 mmol / L) using the electrochemical biosensor.

[0078] The experiment found that the DPV signal of the PbS CQDs-GOx modified electrochemical biosensor prepared in this comparative example for detecting glucose was significantly lower than that in Example 1. When the electrochemical biosensor modified with PbS CQDs-GOx detected glucose solutions of different concentrations, a linear fit was performed at the current peak value of about 0 mV, and the response sensitivity of the electrochemical biosensor for detecting glucose was calculated to be 0.31 μA / dec, which was significantly lower than that in Example 1. In addition, when the glucose concentration was greater than 1 mmol / L, the current peak value of the DPV curve at about 0 mV did not change significantly.

[0079] Comparative Example 2 (the carboxyl group of the glutathione ligand bound to the surface of the quantum dot was not activated) (1) Synthesis of lead sulfide colloidal quantum dots with glutathione ligands bound to the surface According to the method provided in step (1) and step (2) of Example 1, quantum dots PbSCQDs-OA with oleic acid ligands bound to the surface were synthesized, and then ligand replacement was performed to prepare quantum dots with glutathione ligands bound to the surface, namely PbSCQDs-GSH.

[0080] (2) Labeling protein molecules onto quantum dots 0.1 g of PbS CQDs-GSH and 3 mL of PBS were mixed to obtain a PbS CQDs-GSH quantum dot dispersion. The above PbS CQDs-GSH quantum dot dispersion was mixed with 1 mL of 50 mg / mL glucose oxidase (GOx) solution, magnetically stirred at 25 ° C for 1.5 h, centrifuged to remove the supernatant, and collected the precipitate. The precipitate was then washed several times with PBS, centrifuged to collect the precipitate, and vacuum dried to prepare quantum dots labeled with protein molecules.

[0081] (3) Preparation of electrochemical biosensors The quantum dots labeled with protein molecules and PBS were mixed evenly to prepare a solution with a concentration of 10 mg / mL, and then 10 μL of the solution was spin-coated evenly on the working electrode of the screen-printed gold three-electrode. After drying at room temperature, an electrochemical biosensor was obtained.

[0082] Then, the method provided in Example 1 was used to detect glucose test solutions of different concentrations (0, 0.1 μmol / L, 1 μmol / L, 10 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 500 μmol / L, 1 mmol / L, 2 mmol / L, 5 mmol / L, 10 mmol / L, 100 mmol / L) using the electrochemical biosensor.

[0083] The experiment found that when the electrochemical biosensor prepared in this comparative example detected glucose solutions of different concentrations, when the glucose concentration was 0.1μmol / L~5mmol / L, the current peak value of the DPV curve at about 0mV increased with the increase of the glucose solution concentration; when the glucose concentration continued to increase, the current peak value of the DPV curve at about 0mV did not continue to increase. The reason for this may be that the efficiency of protein molecules (glucose oxidase GOx) labeled on quantum dots (PbS CQDs-GSH) is low, which makes the electrochemical biosensor very easy to reach saturation when detecting glucose solutions, and the range of glucose detection becomes narrower.

[0084] Comparative Example 3 (1) Synthesis of lead sulfide colloidal quantum dots with glutathione ligands bound to the surface According to the method provided in step (1) and step (2) of Example 1, quantum dots PbSCQDs-OA with oleic acid ligands bound to the surface were synthesized, and then ligand replacement was performed to prepare quantum dots with glutathione ligands bound to the surface, namely PbSCQDs-GSH.

[0085] (2) Activation of the carboxyl group of the glutathione ligand bound to the quantum dot surface According to the method provided in step (3) of Example 1, a quantum dot dispersion liquid with surface-bound glutathione ligands and a carboxyl activation solution were prepared.

[0086] Then, a dispersion of quantum dots (PbS CQDs-GSH) with glutathione ligands bound to the surface and a carboxyl activation solution were added, wherein the volume ratio of the PbS CQDs-GSH dispersion to the carboxyl activation solution was 1:6, and other parameters were the same as those in Example 1, to obtain a dispersion of quantum dots with active water-soluble ligands, which was recorded as PbS CQDs-GSH A1 .

[0087] In addition, a dispersion of quantum dots with glutathione ligands bound to the surface (PbS CQDs-GSH) and a carboxyl activation solution were added, wherein the volume ratio of the PbS CQDs-GSH dispersion to the carboxyl activation solution was 2:1, and the other parameters were the same as those in Example 1, to obtain a water-soluble quantum dot dispersion with active ligand properties, denoted as PbS CQDs-GSH A2 .

[0088] (3) Labeling protein molecules onto quantum dots According to the method provided in step (4) of Example 1, 3 mL of the above-mentioned water-soluble quantum dot dispersion PbS CQDs-GSH A1 、PbS CQDs-GSH A2and 1 mL of 50 mg / mL glucose oxidase (GOx) solution, and then dried under vacuum to prepare quantum dots with glutathione ligands bound to the surface and labeled with protein molecules, which were denoted as PbS CQDs-GSH A1 -GOx, PbS CQDs-GSH A2 -GOx.

[0089] (4) Preparation of electrochemical biosensors According to the method provided in step (5) of Example 1, the above-mentioned quantum dots (PbS CQDs-GSH A1 -GOx, PbS CQDs-GSH A2 -GOx) and PBS were mixed evenly to prepare a solution with a concentration of 10 mg / mL, and then 10 μL of the solution was evenly spin-coated on the working electrode of the screen-printed gold three-electrode. After drying at room temperature, PbS CQDs-GSH A1 -GOx, PbS CQDs-GSH A2 -GOx-modified electrochemical biosensor.

[0090] Then, the method provided in Example 1 was used to detect glucose test solutions of different concentrations (0, 0.1 μmol / L, 1 μmol / L, 10 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 500 μmol / L, 1 mmol / L, 2 mmol / L, 5 mmol / L, 10 mmol / L, 100 mmol / L) using the electrochemical biosensor.

[0091] Experimental results show that according to PbS CQDs-GSH A1 When the electrochemical biosensor modified with -GOx detected glucose solutions of different concentrations, a linear fit was performed at the current peak of about 0 mV, and the response sensitivity of the electrochemical biosensor for detecting glucose was calculated to be 0.56 μA / dec, which was significantly lower than that in Example 1. This may be due to the excessive amount of carboxyl activation solution used, which affected the quantum dots transducing biomolecule-related signals, resulting in a decrease in the conductivity of the biomolecule-quantum dot-electrode interface, thereby reducing the response sensitivity of the electrochemical biosensor and reducing the detection accuracy.

[0092] Using PbS CQDs-GSH A2-GOx-modified electrochemical biosensor was used for detection. When the glucose concentration was 0.1μmol / L~10mmol / L, the current peak value of the DPV curve at around 0mV increased with the increase of the glucose solution concentration; when the glucose concentration continued to increase, the current peak value of the DPV curve at around 0mV did not continue to increase. This may be due to the small amount of carboxyl activation solution used, resulting in low carboxyl activation of glutathione ligands on the surface of quantum dots (PbS CQDs-GSH), which reduced the efficiency of protein molecules (glucose oxidase GOx) labeled on quantum dots, and ultimately limited the detection range of the electrochemical biosensor.

[0093] Comparative Example 4 (1) Synthesis of lead sulfide colloidal quantum dots with glutathione ligands bound to the surface According to the method provided in step (1) and step (2) of Example 1, quantum dots PbSCQDs-OA with oleic acid ligands bound to the surface were synthesized, and then ligand replacement was performed to prepare quantum dots with glutathione ligands bound to the surface, namely PbSCQDs-GSH.

[0094] (2) Activation of the carboxyl group of the glutathione ligand bound to the quantum dot surface According to the method provided in step (3) of Example 1, a quantum dot dispersion liquid with glutathione ligands bound to the surface and a carboxyl activation solution were prepared. Then, the quantum dot dispersion liquid with glutathione ligands bound to the surface and the carboxyl activation solution were mixed in a volume ratio of 1:2, and magnetically stirred for 30 minutes at 25°C to activate the carboxyl groups on the glutathione ligands, thereby obtaining a quantum dot dispersion liquid with active water-soluble ligand properties.

[0095] (3) Labeling protein molecules onto quantum dots According to the method provided in step (4) of Example 1, 3 mL of the above-mentioned water-soluble quantum dot dispersion with active ligand properties was mixed with 1 mL of 5 mg / mL and 1 mL of 70 mg / mL glucose oxidase (GOx) solution, respectively, and after vacuum drying, quantum dots with glutathione ligands bound to the surface and labeled with protein molecules were prepared, which were respectively denoted as PbS CQDs-GSH B1 -GOx, PbS CQDs-GSH B2 -GOx.

[0096] (4) Preparation of electrochemical biosensors According to the method provided in step (5) of Example 1, the above quantum dots PbS CQDs-GSH B1 -GOx, PbS CQDs-GSH B2-GOx and PBS were mixed evenly to prepare a solution with a concentration of 10 mg / mL, and then 10 μL of the solution was evenly spin-coated on the working electrode of the screen-printed gold three-electrode. After drying at room temperature, PbS CQDs-GSH B1 -GOx, PbS CQDs-GSH B2 -GOx-modified electrochemical biosensor.

[0097] Then, the method provided in Example 1 was used to detect glucose test solutions of different concentrations (0, 0.1 μmol / L, 1 μmol / L, 10 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 500 μmol / L, 1 mmol / L, 2 mmol / L, 5 mmol / L, 10 mmol / L, 100 mmol / L) using the electrochemical biosensor.

[0098] Experiments have shown that PbS CQDs-GSH B1 -GOx-modified electrochemical biosensor detects glucose. When the glucose concentration is 0.1μmol / L~1mmol / L, the current peak of the DPV curve at around 0mV increases with the increase of the glucose solution concentration; when the glucose concentration is greater than 1mmol / L, the current peak of the DPV curve at around 0mV does not continue to increase. This may be due to the fact that there are too few protein molecules (glucose oxidase GOx) labeled on the quantum dots (PbS CQDs-GSH), which limits the detection range.

[0099] PbS CQDs-GSH B2 -GOx-modified electrochemical biosensor detects glucose test solutions of different concentrations. The linear fitting curve of the DPV test curve corresponding to the current peak around 0mV shows a response sensitivity of 0.2μA / dec. This may be due to the excessive number of protein molecules (glucose oxidase GOx) labeled on the quantum dots (PbS CQDs-GSH), which hinders the transduction of biological signals and leads to a decrease in detection sensitivity.

[0100] Example 2 (1) Synthesis of lead sulfide colloidal quantum dots with glutathione ligands bound to the surface According to the method provided in step (1) and step (2) of Example 1, quantum dots PbSCQDs-OA with oleic acid ligands bound to the surface were synthesized, and then ligand replacement was performed to prepare quantum dots with glutathione ligands bound to the surface, namely PbSCQDs-GSH.

[0101] (2) Activation of the carboxyl group of the glutathione ligand bound to the quantum dot surface According to the method provided in step (3) of Example 1, a dispersion of quantum dots (PbSCQDs-GSH) with surface bound glutathione ligands and a carboxyl activation solution were prepared.

[0102] Then, the PbS CQDs-GSH dispersion and the carboxyl activation solution were mixed in a volume ratio of 1:4 and magnetically stirred at 25°C for 30 min to activate the carboxyl groups on the glutathione ligands to obtain a quantum dot dispersion with active water-soluble ligand properties.

[0103] (3) Labeling protein molecules onto quantum dots According to the method provided in step (4) of Example 1, 5 mL of the above-mentioned water-soluble ligand-active quantum dot dispersion and 1 mL of a 20 mg / mL glucose oxidase (GOx) solution were mixed and vacuum dried to prepare quantum dots (PbS CQDs-GSH-GOx) with glutathione ligands bound to the surface and labeled with protein molecules.

[0104] (4) Preparation of electrochemical biosensors According to the method provided in step (5) of Example 1, the above-mentioned PbS CQDs-GSH-GOx and PBS were mixed evenly to prepare a solution with a concentration of 10 mg / mL, and then 10 μL of the solution was evenly spin-coated on the working electrode of the screen-printed gold three-electrode. After drying at room temperature, a PbS CQDs-GSH-GOx modified electrochemical biosensor was obtained.

[0105] Then, the method provided in Example 1 was used to detect glucose test solutions of different concentrations (0, 0.1 μmol / L, 1 μmol / L, 10 μmol / L, 50 μmol / L, 100 μmol / L, 200 μmol / L, 500 μmol / L, 1 mmol / L, 2 mmol / L, 5 mmol / L, 10 mmol / L, 100 mmol / L) using the electrochemical biosensor.

[0106] The experiment found that when the electrochemical biosensor modified with PbS CQDs-GSH-GOx detected glucose solutions of different concentrations, the current peak at about 0mV increased significantly with the increase of the detected glucose concentration (0.1μmol / L ~100mmol / L), and the current peak reached 18μA. The current peak of the above DPV test curve at about 0mV was linearly fitted, and the response sensitivity of the electrochemical biosensor for detecting glucose was calculated to be 1.62μA / dec, which can achieve rapid response and high sensitivity detection of glucose and improve the accuracy of detection.

[0107] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A protein molecule labeling method for constructing a high-performance electrochemical sensor, characterized in that: The steps include: S1. After mixing the sulfide colloidal quantum dots with organic ligands bound to the surface and a weakly polar organic solvent or a non-polar organic solvent, the mixture is evenly mixed with a solution containing a water-soluble ligand, and liquid-phase ligand replacement is performed, followed by centrifugation, washing, and drying to obtain sulfide colloidal quantum dots with the water-soluble ligands bound to the surface; The water-soluble ligand is a water-soluble ligand containing a thiol group and a carboxyl group; S2. Evenly mix the sulfide colloidal quantum dots with the water-soluble ligands bound to the surface, a carboxyl activator, and protein molecules, activate the carboxyl groups of the water-soluble ligands to label the protein molecules, and obtain the sulfide colloidal quantum dots with the water-soluble ligands bound to the surface and labeling the protein molecules.

2. The protein molecule labeling method according to claim 1, characterized in that: In step S1, the weakly polar organic solvent is an organic solvent with a dielectric constant ≤ 5; and / or, The organic ligand is one or more of fatty acid, fatty amine, trioctylphosphine and trioctylphosphine oxide; and / or, The sulfide colloidal quantum dots are one or more of lead sulfide colloidal quantum dots, zinc sulfide colloidal quantum dots and bismuth sulfide colloidal quantum dots; and / or, The water-soluble ligand is one or more of glutathione, mercaptopropionic acid and thioglycolic acid.

3. The protein molecule labeling method according to claim 1 or 2, characterized in that: In step S1, the mass ratio of the sulfide colloidal quantum dots to the water-soluble ligand is 1:(0.5~2).

4. The protein molecule labeling method according to claim 1, characterized in that: In step S2, the carboxyl activating agent includes EDC and / or NHS; and / or, The protein molecule is one or more of glucose oxidase, catalase, cholesterol oxidase, triglyceride oxidase, urease, alkaline phosphatase, lactate dehydrogenase, antigen and antibody.

5. The protein molecule labeling method according to claim 1 or 4, characterized in that: In step S2, the ratio of the sulfide colloidal quantum dots with surface-bound water-soluble ligands to the carboxyl activator is 10 mg: (100-500) μmol.

6. The protein molecule labeling method according to claim 1 or 4, characterized in that: In step S2, the mass ratio of the sulfide colloidal quantum dots with surface-bound water-soluble ligands to the protein molecules is 1:(1-5).

7. A sulfide colloidal quantum dot with a water-soluble ligand bound to its surface and labeled with a protein molecule, characterized in that: The protein molecule is prepared by the protein molecule labeling method according to any one of claims 1 to 6.

8. A method for constructing a high-performance electrochemical biosensor using the sulfide colloidal quantum dots according to claim 7, characterized in that: The steps include: The dispersion of the sulfide colloidal quantum dots is coated on the working electrode of the planar three-electrode, and after drying to form a film, an electrochemical biosensor is prepared.

9. The method according to claim 8, characterized in that The coating method is one or more of spin coating, drop coating, spray coating and electrospray printing; and / or, The working electrode is a gold electrode.

10. A high performance electrochemical biosensor, characterized in that: The method is prepared by the method as claimed in claim 8 or 9.

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