A protein molecular labeling method for constructing high-performance electrochemical sensors

Abstract

The application belongs to the field of electrochemical biosensors, and more particularly relates to a protein molecule labeling method for constructing a high-performance electrochemical sensor. In the application, organic ligands on the surface of sulfide colloidal quantum dots are replaced with water-soluble ligands through liquid-phase ligand replacement, and then mixed with a carboxyl activator to activate the carboxyl groups of the water-soluble ligands, followed by uniform mixing with protein molecules, so that the protein molecules are effectively labeled on the quantum dots. The quantum dots prepared in the application, which have water-soluble ligands on the surface and are labeled with protein molecules, are coated on the working electrode of a planar three-electrode system through a one-step method, and after drying, an electrochemical biosensor with a wide detection range, high response sensitivity and strong anti-interference performance is prepared.

Description

TECHNICAL FIELD

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

[0002] Quantum dots (QDs) have unique optical properties, such as wide absorption peaks, narrow emission peaks, good light stability, 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 an electrode can be used to prepare a glucose electrochemical biosensor, which is more sensitive than traditional methods of detecting glucose. However, the protein molecules (glucose oxidase) adsorbed on the surface of the electrode are prone to falling off, and the luminescence effect of quantum dots is not stable enough in the electrochemical biosensor. The detection sensitivity and response speed of the electrochemical biosensor need to be further improved.

[0003] The prior art can fix the protein molecules adsorbed on the surface of the electrode by coating a thin film of chitosan, Nafion, or other deposition stabilizers on the surface of the electrode, 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 composite glucose electrochemical sensor, which uses an embedding method to fix glucose oxidase on a platinum nano-material-hedgehog bismuth sulfide composite material and modifies a glassy carbon electrode. After drying, Nafion solution is dropped and coated, and a glucose electrochemical biosensor is obtained after low-temperature drying. The preparation steps of this sensor are complex, requiring one layer after another of modification layers, and the linear range of glucose detection is narrow and the lower limit of detection is high, with a detection range of 0.003mM~1.9mM, which cannot effectively detect micromolar and millimolar glucose solutions.

[0004] Patent document CN114813877A discloses a sensor for detecting glucose, which coats a ternary composite material solution of surface-bound sulfide colloidal quantum dots and gold nanospheres, and glucose oxidase 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 a small amount of glucose oxidase coupled on the sensor, limiting the range of glucose detection by the sensor, with a detection range of 100nmol / L~10mmol / L, which cannot accurately and effectively detect high-concentration glucose solutions, and the response sensitivity is only 0.238μA / dec, which cannot sensitively, quickly, and accurately detect changes in glucose concentration. In addition, the preparation cost of this sensor is high, as it needs to use noble metal (gold) nanoparticles to stably bind glucose oxidase and accelerate the electron transfer in the enzyme catalysis process, which is not conducive to large-scale production and manufacturing.

[0005] Therefore, a method for efficiently labeling protein molecules is provided, and a high-performance electrochemical biosensor constructed based on the method has important application value. SUMMARY

[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 that the operation steps of the electrochemical biosensor prepared based on the existing method are complex, additional cross-linking agents, noble metal nanoparticles, etc. are needed, the preparation cost is high, and the response sensitivity of the prepared electrochemical biosensor is low, the detection range is limited, and the electrochemical biosensor cannot respond quickly and accurately.

[0007] To achieve the above-mentioned purpose, in a first aspect, the present application provides a protein molecule labeling method for constructing a high-performance electrochemical sensor, comprising the following steps:

[0008] S1, mixing sulfide colloidal quantum dots with surface-bound organic ligands and a weakly polar organic solvent or a non-polar organic solvent, then uniformly mixing with a solution containing water-soluble ligands, performing liquid phase ligand replacement, then centrifuging, washing, and drying to obtain sulfide colloidal quantum dots with the above-mentioned water-soluble ligands bound to the surface;

[0009] The above-mentioned water-soluble ligand is a water-soluble ligand containing a thiol group and a carboxyl group;

[0010] S2, uniformly mixing the above-mentioned sulfide colloidal quantum dots with surface-bound water-soluble ligands with a carboxyl activator and a protein molecule, activating the carboxyl group of the above-mentioned water-soluble ligand to label the protein molecule, to obtain sulfide colloidal quantum dots with the above-mentioned water-soluble ligands bound to the surface and the above-mentioned protein molecule labeled.

[0011] Preferably, in step S1, the weakly polar organic solvent is an organic solvent with a dielectric constant ≤ 5.

[0012] Preferably, in step S1, the organic ligand is one or more of a fatty acid, a fatty amine, trioctylphosphine, and trioctylphosphine oxide.

[0013] 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.

[0014] Preferably, in step S1, the water-soluble ligand is one or more of glutathione, mercaptopropionic acid, and mercaptoacetic acid.

[0015] Preferably, in step S1, the mass ratio of the above sulfide colloidal quantum dots and the above water-soluble ligand is 1:(0.5-2).

[0016] Preferably, in step S2, the above carboxyl activator comprises EDC and / or NHS.

[0017] Preferably, in step S2, the above protein molecule is one or more of glucose oxidase, catalase, cholesterol oxidase, triglyceride oxidase, urease, alkaline phosphatase, lactate dehydrogenase, antigen and antibody.

[0018] Preferably, in step S2, the ratio of the above sulfide colloidal quantum dots with surface-bound water-soluble ligand and the above carboxyl activator is 10 mg:(100-500) μmol.

[0019] Preferably, in step S2, the mass ratio of the above sulfide colloidal quantum dots with surface-bound water-soluble ligand and the above protein molecule is 1:(1-5).

[0020] In a second aspect, the present application provides a sulfide colloidal quantum dot with surface-bound water-soluble ligand and labeled protein molecule, which is prepared by the above protein molecule labeling method.

[0021] In a third aspect, the present application provides a method for constructing a high-performance electrochemical biosensor using the above sulfide colloidal quantum dots, comprising the following steps:

[0022] The dispersion liquid of the above sulfide colloidal quantum dots is coated on the working electrode of a planar three-electrode, and after drying into a film, an electrochemical biosensor is prepared.

[0023] Preferably, the coating method is one or more of spin coating, drop coating, spray coating and electro-spray printing.

[0024] Preferably, the working electrode is a gold electrode.

[0025] In a fourth aspect, the present application provides a high-performance electrochemical biosensor, which is prepared by the above method.

[0026] Overall, compared with the prior art, the above technical solutions conceived by the present application mainly have the following technical advantages:

[0027] (1) The protein molecule marking method for constructing a high-performance electrochemical sensor provided by the application replaces the organic ligand on the surface of the sulfide colloidal quantum dots with a water-soluble ligand through liquid phase ligand replacement, then mixes the water-soluble ligand with a carboxyl activator to activate the carboxyl group of the water-soluble ligand, and then mixes the water-soluble ligand with a protein molecule to mark the protein molecule on the quantum dots. The quantum dots prepared by the application, which have water-soluble ligands on the surface and are marked with protein molecules, are coated on the working electrode of a planar three-electrode system by a one-step method, and an electrochemical biosensor with a wide detection range, high response sensitivity and strong anti-interference ability can be prepared after drying.

[0028] (2) The application adjusts the ratio among the sulfide colloidal quantum dots having water-soluble ligands on the surface, the carboxyl activator and the protein molecule in the protein molecule marking process, so that the protein molecule is effectively marked on the quantum dots. The sulfide colloidal quantum dots having water-soluble ligands on the surface and marked with protein molecules can be stably modified on the working electrode. On the basis of the specific recognition of the protein molecule to specific biomolecules and the small size effect of the quantum dots, the active center of the protein molecule coupled on the working electrode directly reacts with the specific biomolecules, and the biomolecule recognition signal is better converted into an electrical signal output, so that the concentration of the specific biomolecule in the sample to be tested can be detected with high sensitivity.

[0029] (3) The electrochemical biosensor for detecting glucose constructed by the application can effectively mark the glucose oxidase on the quantum dots and modify the quantum dots on the working electrode. The prepared sensor can detect a glucose solution in the range of 0.1 μmol / L to 100 mmol / L, and the response sensitivity can reach 1.68 μA / dec.

[0030] (4) Compared with the existing electrochemical biosensor, the high-performance electrochemical biosensor provided by the application can stably adhere the modified sulfide colloidal quantum dots having water-soluble ligands on the surface and marked with protein molecules on the electrode by a simple one-step coating, without using noble metal nanomaterials, without using additional coating film deposition stabilizers, and without repeated coating. The application avoids the complex sensor construction process, has the advantages of simple preparation steps and low cost, and is suitable for large-scale production and manufacturing. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is a flow chart of the protein molecule marking method for constructing a high-performance electrochemical sensor provided by the embodiment of the application;

[0032] Figure 2 is a phenomenon diagram of replacing the oleic acid ligand of lead sulfide colloidal quantum dots with glutathione ligand provided by Example 1 of the application;

[0033] Figure 3are Fourier transform infrared spectroscopy (FTIR) diagrams of PbS CQDs-OA, PbS CQDs-GSH, PbS CQDs-GSH-GOx and glucose oxidase (GOx) prepared in Example 1 of the present application, wherein content A is the FTIR diagram of PbS CQDs-OA, and content B is the FTIR diagram of PbS CQDs-GSH, PbS CQDs-GSH-GOx and GOx;

[0034] Figure 4 are UV-Vis-NIR spectrograms of PbS CQDs-OA, PbS CQDs-GSH, PbS CQDs-GSH-GOx and glucose oxidase (GOx) prepared in Example 1 of the present application, wherein content A is the UV-Vis-NIR spectrogram of PbS CQDs-OA, and content B is the UV-Vis-NIR spectrogram of PbS CQDs-GSH, PbS CQDs-GSH-GOx and GOx;

[0035] Figure 5 are differential pulse voltammetry curves of the PbS CQDs-GSH-GOx modified electrochemical biosensor prepared in Example 1 of the present application for detecting different concentrations of glucose test solutions;

[0036] Figure 6 are linear fitting curves of the DPV test curves of the PbS CQDs-GSH-GOx modified electrochemical biosensor prepared in Example 1 of the present application for detecting different concentrations of glucose test solutions at the current peak value of about 0 mV;

[0037] Figure 7 are responses of the PbS CQDs-GSH-GOx modified electrochemical biosensor prepared in Example 1 of the present application to KCl solution, NaCl solution, glycine solution, ascorbic acid solution, uric acid solution and glucose solution.

[0038] Figure 8 are Fourier transform infrared spectroscopy (FTIR) diagrams of quantum dots (CQDs) and quantum dots (CQDs / GOx) surface-labeled with glucose oxidase (GOx) prepared in Comparative Example 1 of the present application. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application, and are not used to limit the present application.

[0040] In the description of the present application, it should be understood that the term "and / or" is a description of an associated relationship with the associated object, which means that there can be three relationships, for example, A and / or B can represent the existence of A alone, the existence of A and B at the same time, and the existence of B alone. In this paper, the symbol " / " represents the relationship of or, for example, A / B represents A or B.

[0041] In the description of the embodiments of the present application, the words "exemplary" or "for example" are used to mean serving as an example, instance, or illustration. Any embodiment or design described as "exemplary" or "for example" in the embodiments of the present application should not be construed as being more preferred or advantageous than other embodiments or designs. Rather, the use of the words "exemplary" or "for example" is intended to present concepts in a concrete manner.

[0042] In the description of the embodiments of the present application, unless otherwise specified, "a plurality of" means two or more.

[0043] Liquid phase ligand replacement is performed on quantum dots and protein molecules with surface-bound organic ligands, which can replace the organic ligands on the surface of the quantum dots with protein molecules, but the replacement efficiency is low, and only a small part of the protein molecules can be labeled on the quantum dots. The quantum dots with surface-labeled protein molecules are modified to the working electrode of a planar three-electrode, and the DPV current signal of the electrochemical biosensor prepared is small. The patent document CN114813877A adds gold nanoballs to improve the signal-to-noise ratio of the sensor, accelerate the electron transfer in the enzyme catalysis process, and improve the sensitivity of the sensor, but the detection range of the sensor is still small, the response sensitivity is low, and the detection cannot be quickly and accurately performed. Based on this, the present application provides a protein molecule labeling method for constructing a high-performance electrochemical sensor, as shown in Figure 1 The method comprises the following steps:

[0044] S1, mixing sulfide colloidal quantum dots with surface-bound organic ligands and weakly polar or non-polar organic solvents, then uniformly mixing with a solution containing water-soluble ligands to perform liquid phase ligand replacement, and then centrifuging, washing and drying to obtain sulfide colloidal quantum dots with surface-bound water-soluble ligands;

[0045] The water-soluble ligand is a water-soluble ligand containing a thiol group and a carboxyl group;

[0046] S2, uniformly mixing the above-mentioned sulfide colloidal quantum dots with surface-bound water-soluble ligands and carboxyl activators and protein molecules, activating the carboxyl group of the water-soluble ligand to label the protein molecules, and obtaining sulfide colloidal quantum dots with surface-bound water-soluble ligands and labeled protein molecules.

[0047] The method for labeling protein molecules with quantum dots provided in the present application comprises the following steps: replacing the organic ligand of the sulfide colloidal quantum dots with water-soluble ligand, then mixing the water-soluble ligand with carboxyl activator to activate the carboxyl group of the water-soluble ligand, and finally mixing the protein molecules with the activated water-soluble ligand, so that the protein molecules are effectively labeled on the sulfide colloidal quantum dots.

[0048] 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 has a dielectric constant ≤ 5 and is selected from one or more of chloroform, toluene, xylene, and hexane.

[0049] In some embodiments, in step S1, the solution containing water-soluble ligand is prepared by mixing water-soluble ligand with deionized water. By mixing the sulfide colloidal quantum dots with surface-bound organic ligand and weakly polar organic solvent or non-polar organic solvent, and then mixing with the solution containing water-soluble ligand, the quantum dot solution with completed / unfinished ligand replacement can be quickly separated.

[0050] In some embodiments, in step S1, the organic ligand is one or more of fatty acid, fatty amine, trioctylphosphine, and trioctylphosphine oxide.

[0051] In some embodiments, the fatty acid includes, but is not limited to, one or more of lauric acid, palmitic acid, oleic acid, stearic acid, myristic acid, elaidic acid, arachidic acid, heneicosylic acid, tricosylic acid, docosylic acid, tetracosylic acid, hexacosylic acid, heptacosylic acid, octacosylic acid, and cis-13-docosenoic acid.

[0052] In some embodiments, the fatty amine includes, but is not limited to, one or more of decylamine, didodecylamine, undecylamine, tridecylamine, tetradecylamine, pentadecylamine, hexadecylamine, octadecylamine, didodecylamine, tridodecylamine, cyclododecylamine, dioctadecylamine, trioctylamine, N,N-dimethyldecylamine, N,N-dimethylundecylamine, N,N-dimethyl-dodecylamine, N,N-dimethyltridecylamine, N,N-dimethyltetradecylamine, and N,N-dimethylhexadecylamine.

[0053] 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 can be understood that the source of the above-mentioned sulfide colloidal quantum dots is not limited in the present application, which can be purchased from commercial products or prepared in the laboratory. For example, the preparation method of lead sulfide colloidal quantum dots with oleic acid as the organic ligand can be as follows: using hot injection method, the lead sulfide colloidal quantum dots with surface-bound oleic acid are synthesized by instantaneous nucleation reaction of lead oleate and bis(trimethylsilyl) sulfide.

[0054] In some embodiments, in step S1, the water-soluble ligand is one or more of glutathione, mercaptopropionic acid, and mercaptoacetic acid.

[0055] In some embodiments, in step S1, the mass ratio of the sulfide colloidal quantum dots and the water-soluble ligand is 1: (0.5-2), preferably 1: (0.8-2), which can enable the organic ligand on the surface of the sulfide colloidal quantum dots to be completely replaced by the water-soluble ligand.

[0056] In some embodiments, in order to enable the carboxyl activation reaction of the water-soluble ligand in step S2 to be more stable and more fully carried out, the sulfide colloidal quantum dots with the surface-bound water-soluble ligand and the carboxyl activation agent can be mixed uniformly to form a reactive intermediate, and then mixed uniformly with the protein molecules, so that the protein molecules react with the reactive intermediate, thereby being labeled on the sulfide colloidal quantum dots, and sulfide colloidal quantum dots with the surface-bound water-soluble ligand and the labeled protein molecules can be prepared.

[0057] In some embodiments, in step S2, the mixing speed is 100-300 rpm, and the mixing time is 0.5-2 h.

[0058] In some embodiments, in step S2, the carboxyl activation agent 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 activation agent 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 activation agent EDC can react with the carboxyl group of the water-soluble ligand to form an active intermediate, and the 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 period of time, while avoiding the generation of side reactions.

[0059] The carboxyl-activated surface-bound water-soluble ligand sulfide colloidal quantum dots in the present application can form amide bonds with protein molecules, so that the protein molecules are labeled on the sulfide colloidal quantum dots. By performing corresponding protein molecule labeling, the concentration of specific biomolecules in the sample to be detected can be detected. For example, by labeling glucose oxidase on the sulfide colloidal quantum dots, the concentration of glucose in the sample to be detected can be detected; by labeling an antigen on the sulfide colloidal quantum dots, the concentration of the antibody corresponding to the antigen in the sample to be detected can be detected. In some embodiments, the protein molecules in step S2 can be, but are not limited to, one or more of glucose oxidase, catalase, cholesterol oxidase, triglyceride oxidase, urease, alkaline phosphatase, lactate dehydrogenase, antigen, and antibody. It can be understood that the present application does not specially limit the types of the above-mentioned antigens and antibodies, and in actual application, the corresponding types of antigens or antibodies can be labeled on the quantum dots according to the detection requirements and detection is performed.

[0060] In some embodiments, the ratio of the surface-bound water-soluble ligand sulfide colloidal quantum dots to the carboxyl-activated agent in step S2 is 10 mg:(100-500) μmol, which can appropriately activate the carboxyl groups of the water-soluble ligands on the surface of the quantum dots, and when used for preparing an electrochemical biosensor, will not affect the conductivity of the electrochemical biosensor.

[0061] In some embodiments, the mass ratio of the surface-bound water-soluble ligand sulfide colloidal quantum dots to the protein molecules in step S2 is 1:(1-5), which can successfully label the protein molecules on the quantum dots, and prepare the surface-bound water-soluble ligand sulfide colloidal quantum dots labeled with protein molecules. After the dispersion liquid containing the quantum dots is coated on the working electrode of the planar three-electrode, and dried, an electrochemical biosensor with a wide detection range, a low lower limit of detection, and a high response sensitivity can be prepared, avoiding the complex and time-consuming modification process of the traditional electrochemical biosensor.

[0062] On the other hand, the present application also provides a surface-bound water-soluble ligand sulfide colloidal quantum dot labeled with a protein molecule, which is prepared by the above method.

[0063] The present application also provides a method for constructing a high-performance electrochemical biosensor using the above-mentioned surface-bound water-soluble ligand quantum dots labeled with a protein molecule, comprising the following steps:

[0064] The dispersion liquid of the above-mentioned surface-bound water-soluble ligand sulfide colloidal quantum dots labeled with a protein molecule is coated on the working electrode of the planar three-electrode, and after drying to form a film, an electrochemical biosensor is prepared.

[0065] In some embodiments, the coating is one or more of spin coating, drop coating, spray coating, and electrospray printing.

[0066] In some embodiments, the working electrode is a gold electrode.

[0067] Based on this, the application further provides a high-performance electrochemical biosensor prepared by the above method.

[0068] The electrochemical biosensor provided by the application can be prepared by simply coating a dispersion liquid containing quantum dots on a planar three-electrode through a simple one-step method, and a high-performance electrochemical biosensor can be prepared after drying. The electrochemical biosensor can quickly and sensitively respond to the to-be-detected substance, has a wide detection range, and has high detection precision. Compared with existing electrochemical biosensors, the application has the advantages of simple preparation steps, low preparation cost, and is suitable for large-scale batch production.

[0069] It should be understood that materials similar to or the same as the types, models, qualities, properties, or functions of the reagents and instruments used in the following examples can be used to implement the application. Unless otherwise specified, 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 channels.

[0070] The following are examples and comparative examples:

[0071] Example 1

[0072] The protein molecule labeling method provided in this embodiment includes the following steps:

[0073] (1) Synthesis of lead sulfide colloidal quantum dots with surface-bound oleic acid ligand

[0074] After 1.8 g of lead oxide (PbO), 6 mL of oleic acid (OA), and 20 mL of octadecene (ODE) are mixed at high speed under vacuum at 90°C, a lead precursor is prepared, and the lead precursor is heated to 120°C.

[0075] In a glove box, 280 μL of bis(trimethylsilyl) sulfide (C6H 18 SSi2) and 10 mL of octadecene (ODE) are mixed to prepare a sulfur precursor.

[0076] The sulfur precursor is quickly injected into the lead precursor under a nitrogen environment, and then cooled in cold water for 30 s. The precipitate is collected and washed several times with toluene and acetone, centrifuged to collect the precipitate, and vacuum dried to prepare lead sulfide colloidal quantum dots with surface-bound oleic acid ligand, i.e., PbS CQDs-OA.

[0077] (2) Ligand replacement

[0078] The above 0.05 g of PbS CQDs-OA and 10 mL of chloroform solution were mixed to prepare a quantum dot dispersion solution with a concentration of 5 mg / mL of surface-bound oleic acid ligand.

[0079] 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.

[0080] The quantum dot dispersion solution with surface-bound oleic acid ligand and the glutathione solution were mixed in a volume ratio of 1:1 (e.g. Figure 2 The liquid phase ligand replacement reaction was carried out at room temperature under 200 rpm magnetic stirring for 0.5 h, and the OA ligand was replaced by the GSH ligand, as shown in the left side of the figure. Figure 2 After the reaction was completed, the upper layer solution became black and the lower layer solution became transparent, because the quantum dots after ligand replacement existed in the upper layer deionized water with lighter density. Then the upper layer quantum dot aqueous solution was taken out, chloroform solution was added for mixing, the supernatant was removed by centrifugation, and the precipitate was collected. Then the precipitate was mixed with deionized water to dissolve the precipitate, and then washed several times with ethanol, and the precipitate was collected by centrifugation, and vacuum dried to prepare lead sulfide colloidal quantum dots with surface-bound glutathione ligand, i.e. PbS CQDs-GSH.

[0081] (3) Activation of the carboxyl group of the glutathione ligand bound to the surface of the quantum dots

[0082] The above 0.1 g of PbS CQDs-GSH and 10 mL of PBS were mixed to prepare a quantum dot dispersion solution with a concentration of 10 mg / mL of surface-bound glutathione ligand.

[0083] A carboxyl activation solution was prepared: 100 μmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 1 mL of PBS were mixed to obtain an EDC solution with a concentration of 0.1 mol / L; 100 μmol of N-hydroxysuccinimide (NHS) and 1 mL of PBS were mixed to obtain an NHS solution with a concentration of 0.1 mol / L, and then the EDC solution and the NHS solution were all mixed to obtain the carboxyl activation solution.

[0084] The quantum dot dispersion solution with surface-bound glutathione ligand and the carboxyl activation solution were mixed in a volume ratio of 1:2, and the carboxyl group on the glutathione ligand was activated under the condition of 200 rpm magnetic stirring at 25°C for 30 min to obtain a quantum dot dispersion solution with water-soluble ligands with active properties.

[0085] (4) Labeling protein molecules to quantum dots

[0086] The 3 mL of the above water-soluble ligand property active quantum dot dispersion liquid and 1 mL of glucose oxidase (GOx) solution with a concentration of 50 mg / mL were mixed, and 200 rpm magnetic stirring was carried out at 25°C for 1.5 h. The supernatant was removed by centrifugation, and the precipitate was collected. Then the precipitate was washed several times with PBS, and the precipitate was collected by centrifugation. After vacuum drying, the quantum dots with surface-bound glutathione ligand and labeled with protein molecules, namely PbS CQDs-GSH-GOx, were prepared. PbS CQDs-GSH-GOx was characterized by FTIR and UV-Vis-NIR characterization means.

[0087] Figure 3 The Fourier transform infrared spectra (FTIR) of the lead sulfide colloidal quantum dots (PbS CQDs-OA) with surface-bound oleic acid ligand, the lead sulfide colloidal quantum dots (PbS CQDs-GSH) with surface-bound glutathione ligand, the quantum dots (PbS CQDs-GSH-GOx) with surface-bound glutathione ligand and labeled with protein molecules, and the glucose oxidase (GOx) are shown. It can be seen that the PbS CQDs-OA quantum dots have obvious C-H stretching vibration peaks at a wave number of 2850~2950 cm⁻¹; there is a C=C double bond absorption peak near a wave number of 1600 cm⁻¹; there is a C-H bending vibration near a wave number of 1397 cm⁻¹, indicating that there is an oleic acid ligand (OA) on the surface of the lead sulfide colloidal quantum dots. The PbS CQDs-GSH quantum dots have weakened C-H stretching vibration peaks at a wave number of 2850~2950 cm⁻¹; there is an obvious S-H stretching vibration near a wave number of 2539 cm⁻¹, which is derived from the -SH of the glutathione ligand; there is an N-H stretching vibration peak near a wave number of 3249 cm⁻¹; there is a related absorption peak of the peptide bond near a wave number of 1336 cm⁻¹, indicating the reduction of the oleic acid ligand (OA) on the surface of the lead sulfide colloidal quantum dots and the presence of the glutathione (GSH) ligand. The PbS CQDs-GSH-GOx quantum dots have an N-H stretching vibration peak near a wave number of 3249 cm⁻¹; there is a relatively wide absorption peak near a wave number of 1540 cm⁻¹, which is derived from the aromatic ring of the specific amino acid tryptophan of the glucose oxidase and the benzene ring absorption peak of tyrosine; there is an obvious peptide bond related absorption peak near a wave number of 1336 cm⁻¹, indicating that the glutathione (GSH) ligand is bound to the surface of the lead sulfide colloidal quantum dots, and the glucose oxidase (GOx) is successfully labeled.

[0088] Figure 4UV-Vis-NIR spectra of PbS CQDs-OA with surface-bound oleic acid ligand, PbS CQDs-GSH with surface-bound glutathione ligand, PbS CQDs-GSH-GOx with surface-bound glutathione ligand and labeled with protein molecules, and glucose oxidase (GOx). It can be seen that the PbS CQDs-OA quantum dots have a clear absorption peak at a wavelength of 958 nm Figure 4 Content A), and the average size of the quantum dots is 3 nm. The absorption peak of the PbS CQDs-GSH quantum dots at 200-300 nm is significantly different from that of the PbS CQDs-OA quantum dots, and a small absorption peak is shown at about 976 nm, which proves the reduction of the oleic acid ligand (OA) and the increase of the glutathione ligand (GSH). FAD is a key cofactor in glucose oxidase and plays an important role in enzyme catalysis. The flavin part in FAD has strong absorption properties, especially in the ultraviolet-visible light region, and its absorption peak is usually in the range of 450-475 nm, which is Figure 4 Content B) It can be seen that the glucose oxidase (GOx) and the PbS CQDs-GSH-GOx quantum dots both have 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 molecules, indicating the successful labeling of the glucose oxidase (GOx).

[0089] (5) Preparation of electrochemical biosensor

[0090] The above PbS CQDs-GSH-GOx and PBS were mixed uniformly to prepare a dispersion liquid with a concentration of 10 mg / mL, and then 10 μL of the dispersion liquid was uniformly spin-coated on the working electrode of the screen-printed gold three-electrode, only once. After drying at room temperature, a PbS CQDs-GSH-GOx modified electrochemical biosensor was obtained.

[0091] 10 μL phosphate buffer solution was added on the surface of the prepared working electrode of the electrochemical biosensor as a blank control group, then differential pulse voltammetry (DPV) detection mode was selected by using an electrochemical workstation, pulse width was set as 50 ms, pulse amplitude was set as 50 mV, and potential increment was set as 4 mV, and the differential pulse voltammetry characteristics of the surface of the working electrode were tested. 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) of glucose test solutions were added on the surface of the prepared working electrode of the electrochemical biosensor, and the differential pulse voltammetry characteristics of the surface of the working electrode were tested by using an electrochemical workstation, and the detection of different concentrations of glucose can be directly realized by the peak current in the differential pulse voltammetry curve in the testing process.

[0092] Figure 5 The differential pulse voltammetry curve of the PbS CQDs-GSH-GOx modified electrochemical biosensor for detecting different concentrations of glucose test solutions is shown, and it can be seen that the current peak around 0 mV is obviously increased with the increase of the detected glucose concentration (0.1 μmol / L~100 mmol / L), and the current peak is as high as 20 μA, which indicates that the electrochemical biosensor prepared in this embodiment can sensitively detect the glucose concentration.

[0093] Figure 6 The linear fitting curve of the DPV test curve of the PbS CQDs-GSH-GOx modified electrochemical biosensor for detecting different concentrations of glucose test solutions around the current peak of 0 mV is shown, and the response sensitivity of the electrochemical biosensor for detecting glucose is calculated as 1.68 μA / dec, that is, the output of the detection sensor is 1.68 μA for each change of 10 times of the glucose concentration, which is more sensitive to glucose and can more quickly and accurately detect the change of the glucose concentration.

[0094] (6) Selectivity of the electrochemical biosensor

[0095] Common interfering substances (KCl, NaCl, glycine, ascorbic acid, and uric acid) in the glucose detection process were respectively configured into a solution with a concentration of 500 μmol / L, and then were added on the surface of the working electrode of the electrochemical biosensor, and the current value of the electrochemical biosensor before and after adding the interfering substances was recorded, wherein A0 is the initial current without adding any interfering substance, and A is the response current measured after adding different interfering substances, and the selectivity of the electrochemical biosensor is evaluated.

[0096] Figure 7 The response of the PbS CQDs-GSH-GOx modified electrochemical biosensor to KCl solution, NaCl solution, glycine solution, ascorbic acid solution, uric acid solution, and glucose solution is shown. It can be seen that the current difference of the electrochemical biosensor for detecting glucose is the largest at the same concentration compared with the above-mentioned interfering substances, and the electrochemical biosensor has high responsiveness and excellent selectivity to glucose, and can be effectively applied to the detection of glucose.

[0097] In summary, the PbS CQDs-GSH-GOx modified electrochemical biosensor prepared in the application can realize high-sensitivity detection of glucose, and can effectively avoid or reduce the interference triggered by non-target factors, and has high detection accuracy in complex real samples. In addition, the above-mentioned electrochemical biosensor has a wide detection range, and has excellent detection effect on glucose in the concentration range of 0.1 μmol / L to 100 mmol / L.

[0098] Comparative Example 1 (without ligand replacement)

[0099] (1) Synthesis of lead sulfide colloidal quantum dots combined with oleic acid ligand

[0100] According to the method provided in Example 1, lead sulfide colloidal quantum dots combined with oleic acid ligand were synthesized, which were denoted as CQDs.

[0101] (2) Labeling protein molecules to quantum dots

[0102] 0.1 g of CQDs and 3 mL of n-octane were mixed to obtain a CQDs quantum dot dispersion. The above CQDs quantum dot dispersion and 1 mL of glucose oxidase (GOx) solution with a concentration of 50 mg / mL were mixed, and magnetically stirred at 25°C for 1.5 h to prepare quantum dots labeled with protein molecules (glucose oxidase, GOx), i.e. CQDs / GOx.

[0103] Figure 8 The Fourier transform infrared spectrum (FTIR) of lead sulfide colloidal quantum dots combined with oleic acid ligand (CQDs) and quantum dots labeled with glucose oxidase (CQDs / GOx) is shown. It can be seen that the 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.

[0104] (3) Preparation of electrochemical biosensor

[0105] The PbS CQDs-GOx and PBS were mixed uniformly to prepare a solution with a concentration of 10 mg / mL, and then 10 μL of the solution was uniformly spin-coated on the working electrode of the screen-printed gold three-electrode, and after drying at room temperature, a PbS CQDs-GOx modified electrochemical biosensor was obtained.

[0106] Then, the electrochemical biosensor was used to detect 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) of glucose test solutions by using the method provided in Example 1.

[0107] It was found that the DPV signal of the PbS CQDs-GOx modified electrochemical biosensor prepared in the present comparative example for detecting glucose was significantly lower than that in Example 1. According to the linear fitting of the current peak value at about 0 mV when the PbS CQDs-GOx modified electrochemical biosensor detected different concentrations of glucose solutions, it was calculated that the response sensitivity of the electrochemical biosensor for detecting glucose was 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 at about 0 mV of the DPV curve did not change significantly.

[0108] Comparative Example 2 (carboxyl of glutathione ligand on surface of quantum dots was not activated)

[0109] (1) Synthesis of lead sulfide colloidal quantum dots with glutathione ligand on surface

[0110] According to the methods provided in steps (1) and (2) of Example 1, quantum dots PbSCQDs-OA with oleic acid ligand on surface were synthesized, and then ligand replacement was performed to prepare quantum dots with glutathione ligand on surface, i.e. PbS CQDs-GSH.

[0111] (2) Labeling protein molecules on quantum dots

[0112] 0.1 g of PbS CQDs-GSH and 3 mL of PBS were mixed to obtain a PbS CQDs-GSH quantum dot dispersion. The PbS CQDs-GSH quantum dot dispersion and 1 mL of glucose oxidase (GOx) solution with a concentration of 50 mg / mL were mixed, and magnetically stirred at 25°C for 1.5 h. The supernatant was removed by centrifugation, and the precipitate was collected. Then the precipitate was washed several times with PBS, and the precipitate was collected by centrifugation. After vacuum drying, quantum dots labeled with protein molecules were prepared.

[0113] (3) Preparation of the electrochemical biosensor

[0114] The quantum dots labeled with the protein molecules and PBS were mixed uniformly to prepare a solution with a concentration of 10 mg / mL, and then 10 μL of the solution was uniformly spin-coated on the working electrode of the screen-printed gold three-electrode, and after drying at room temperature, the electrochemical biosensor was obtained.

[0115] Then, the electrochemical biosensor was used to detect 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) of the glucose test solution by using the method provided in Example 1.

[0116] It was found that when the electrochemical biosensor prepared in the present comparative example was used to detect glucose solutions with different concentrations, when the glucose concentration was 0.1 μmol / L to 5 mmol / L, the current peak value of the DPV curve at about 0 mV 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 0 mV did not continue to increase. The reason was analyzed to be that the efficiency of the protein molecules (glucose oxidase GOx) labeled on the quantum dots (PbS CQDs-GSH) was low, which led to that the electrochemical biosensor was easily saturated when detecting the glucose solution, and the range of detecting glucose was narrowed.

[0117] Comparative Example 3

[0118] (1) Synthesis of lead sulfide colloidal quantum dots with surface-bound glutathione ligand

[0119] According to the methods provided in steps (1) and (2) of Example 1, the quantum dots with surface-bound oleic acid ligand PbSCQDs-OA were synthesized, and then ligand replacement was performed to prepare the quantum dots with surface-bound glutathione ligand, i.e., PbS CQDs-GSH.

[0120] (2) Activation of the carboxyl group of the glutathione ligand bound on the surface of the quantum dots

[0121] According to the method provided in step (3) of Example 1, the quantum dot dispersion liquid with surface-bound glutathione ligand and the carboxyl activation solution were prepared.

[0122] Then the quantum dots (PbS CQDs-GSH) dispersion liquid with surface-bound glutathione ligand, a carboxyl activation solution, wherein the volume ratio of the PbS CQDs-GSH dispersion liquid and the carboxyl activation solution is 1:6, and other parameters are the same as in Example 1, are obtained to obtain a water-soluble ligand active quantum dot dispersion liquid, denoted as PbS CQDs-GSH A1 .

[0123] In addition, the quantum dots (PbS CQDs-GSH) dispersion liquid with surface-bound glutathione ligand, a carboxyl activation solution, wherein the volume ratio of the PbS CQDs-GSH dispersion liquid and the carboxyl activation solution is 2:1, and other parameters are the same as in Example 1, are obtained to obtain a water-soluble ligand active quantum dot dispersion liquid, denoted as PbS CQDs-GSH A2 .

[0124] (3) Labeling protein molecules to quantum dots

[0125] According to the method provided in step (4) of Example 1, 3 mL of the above water-soluble ligand active quantum dot dispersion liquid PbS CQDs-GSH A1 , PbS CQDs-GSH A2 and 1 mL of glucose oxidase (GOx) solution with a concentration of 50 mg / mL are mixed, and vacuum drying is performed to prepare quantum dots with surface-bound glutathione ligand and labeled with protein molecules, denoted as PbS CQDs-GSH A1 -GOx, PbS CQDs-GSH A2 -GOx, respectively.

[0126] (4) Preparation of electrochemical biosensor

[0127] According to the method provided in step (5) of Example 1, the above quantum dots with surface-bound glutathione ligand and labeled with protein molecules (PbS CQDs-GSH A1 -GOx, PbS CQDs-GSH A2 -GOx) and PBS are mixed uniformly to prepare a solution with a concentration of 10 mg / mL, and then 10 μL of the solution is uniformly spin-coated on the working electrode of a screen-printed gold three-electrode, and after drying at room temperature, PbS CQDs-GSH A1 -GOx, PbS CQDs-GSH A2 -GOx modified electrochemical biosensor are prepared, respectively.

[0128] Then the method provided in Example 1 is used to detect 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) of glucose test solution by using the above electrochemical biosensor.

[0129] It is found in the experiment that when the electrochemical biosensor modified by PbS CQDs-GSH A1 -GOx is used to detect glucose solutions with different concentrations, the current peak at about 0 mV is linearly fitted, and the response sensitivity of the electrochemical biosensor for detecting glucose is calculated to be 0.56 μA / dec, which is significantly lower than that in Example 1. This may be due to the excessive amount of carboxyl activation solution, which affects the signal of the quantum dot transduction biomolecule, resulting in a decrease in the conductivity of the biomolecule-quantum dot-electrode interface, thereby reducing the response sensitivity of the electrochemical biosensor and the detection accuracy.

[0130] The electrochemical biosensor modified by PbS CQDs-GSH A2 -GOx is used for detection, and when the glucose concentration is 0.1 μmol / L-10 mmol / L, the current peak of the DPV curve at about 0 mV increases with the increase of the glucose solution concentration; when the glucose concentration continues to increase, the current peak of the DPV curve at about 0 mV does not continue to increase, which may be due to the insufficient amount of carboxyl activation solution, resulting in a low carboxyl activation of the glutathione ligand on the surface of the quantum dots (PbS CQDs-GSH), reducing the efficiency of the protein molecules (glucose oxidase GOx) labeled on the quantum dots, and ultimately limiting the detection range of the electrochemical biosensor.

[0131] Comparative Example 4

[0132] (1) Synthesis of lead sulfide colloidal quantum dots with surface-bound glutathione ligand

[0133] According to the methods provided in steps (1) and (2) of Example 1, quantum dots with surface-bound oleic acid ligand PbSCQDs-OA are synthesized, and then ligand replacement is performed to prepare quantum dots with surface-bound glutathione ligand, i.e., PbS CQDs-GSH.

[0134] (2) Activation of the carboxyl group of the glutathione ligand on the surface of the quantum dots

[0135] The quantum dot dispersion liquid with surface-bound glutathione ligand and the carboxyl-activated solution were prepared according to the method provided in step (3) of Example 1. Then the quantum dot dispersion liquid with surface-bound glutathione ligand and the carboxyl-activated solution were mixed in a volume ratio of 1:2, and magnetically stirred at 25°C for 30 min to activate the carboxyl group on the glutathione ligand, thereby obtaining a water-soluble ligand active quantum dot dispersion liquid.

[0136] (3) Labeling protein molecules to quantum dots

[0137] According to the method provided in step (4) of Example 1, 3 mL of the above water-soluble ligand active quantum dot dispersion liquid was mixed with 1 mL of glucose oxidase (GOx) solution with a concentration of 5 mg / mL and 1 mL of glucose oxidase (GOx) solution with a concentration of 70 mg / mL, respectively. After vacuum drying, quantum dots with surface-bound glutathione ligand and labeled protein molecules were prepared, which were denoted as PbS CQDs-GSH B1 -GOx, PbS CQDs-GSH B2 -GOx.

[0138] (4) Preparation of electrochemical biosensor

[0139] According to the method provided in step (5) of Example 1, the above quantum dots with surface-bound glutathione ligand and labeled protein molecules PbS CQDs-GSH B1 -GOx, PbS CQDs-GSH B2 -GOx were mixed with PBS uniformly to prepare a solution with a concentration of 10 mg / mL. Then 10 μL of the solution was uniformly spin-coated on the working electrode of a screen-printed gold three-electrode, and after drying at room temperature, PbS CQDs-GSH B1 -GOx, PbS CQDs-GSH B2 -GOx modified electrochemical biosensor.

[0140] Then, the above electrochemical biosensor was used to detect glucose test solutions with 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) according to the method provided in Example 1.

[0141] It was found that PbS CQDs-GSH B1- When the GOx modified electrochemical biosensor detects glucose, the current peak value of the DPV curve at about 0 mV increases with the increase of the concentration of the glucose solution when the glucose concentration is 0.1 μmol / L to 1 mmol / L; when the glucose concentration is greater than 1 mmol / L, the current peak value of the DPV curve at about 0 mV does not continue to increase, which may be due to the fact that the protein molecules (glucose oxidase GOx) labeled on the quantum dots (PbS CQDs-GSH) are too few, thereby limiting the detection range.

[0142] PbS CQDs-GSH B2 - The linear fitting curve of the current peak value of the DPV test curve at about 0 mV corresponding to the GOx modified electrochemical biosensor detecting different concentrations of glucose test solution is calculated to obtain a response sensitivity of 0.2 μA / dec, which may be due to the fact that the protein molecules (glucose oxidase GOx) labeled on the quantum dots (PbS CQDs-GSH) are too many, thereby hindering the biological signal conduction and causing the detection sensitivity to decrease.

[0143] Example 2

[0144] (1) Synthesis of lead sulfide colloidal quantum dots with surface-bound glutathione ligands

[0145] According to the method provided in steps (1) and (2) of Example 1, quantum dots with surface-bound oleic acid ligands, i.e., PbSCQDs-OA, are synthesized, and then ligand replacement is performed to prepare quantum dots with surface-bound glutathione ligands, i.e., PbS CQDs-GSH.

[0146] (2) Activation of the carboxyl group of the glutathione ligand bound to the surface of the quantum dots

[0147] According to the method provided in step (3) of Example 1, a dispersion of quantum dots with surface-bound glutathione ligands (PbSCQDs-GSH) and a carboxyl activation solution are prepared.

[0148] Then, the dispersion of PbS CQDs-GSH and the carboxyl activation solution are mixed at a volume ratio of 1:4, and the carboxyl group of the glutathione ligand is activated by magnetic stirring at 25°C for 30 min to obtain a quantum dot dispersion with water-soluble ligands with active properties.

[0149] (3) Labeling of protein molecules to quantum dots

[0150] According to the method provided in step (4) of Example 1, 5 mL of the quantum dot dispersion with water-soluble ligands with active properties and 1 mL of a glucose oxidase (GOx) solution with a concentration of 20 mg / mL are mixed, and then vacuum dried to prepare quantum dots with surface-bound glutathione ligands and labeled with protein molecules (PbS CQDs-GSH-GOx).

[0151] (4) Preparation of the electrochemical biosensor

[0152] According to the method provided in step (5) of Example 1, the PbS CQDs-GSH-GOx and PBS above are mixed uniformly to prepare a solution with a concentration of 10 mg / mL, and then 10 μL of the solution is uniformly spin-coated on the working electrode of the screen-printed gold three-electrode, and after drying at room temperature, the PbS CQDs-GSH-GOx modified electrochemical biosensor is obtained.

[0153] Then, the above electrochemical biosensor is used to detect glucose test solutions with 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) by using the method provided in Example 1.

[0154] It is found in the experiment that when the PbS CQDs-GSH-GOx modified electrochemical biosensor detects glucose solutions with different concentrations, the current peak value at about 0 mV increases obviously with the increase of the detected glucose concentration (0.1 μmol / L ~ 100 mmol / L), and the current peak value reaches 18 μA. The current peak value at about 0 mV of the above DPV test curve is linearly fitted, and it is calculated that the response sensitivity of the electrochemical biosensor for detecting glucose is 1.62 μA / dec, which can realize rapid response and high sensitivity detection of glucose and improve the detection accuracy.

[0155] It is easy for those skilled in the art to understand that the above description is only the preferred embodiment of the present application, and is not used to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for constructing a high performance electrochemical sensor using protein molecular tags, characterized by, Comprising the following steps: S1, mixing the surface-bound organic ligand sulfide colloidal quantum dots and weakly polar organic solvent or non-polar organic solvent, then mixing with a solution containing water-soluble ligand, carrying out liquid phase ligand replacement, then centrifuging, washing, drying to obtain surface-bound water-soluble ligand sulfide colloidal quantum dots; The water-soluble ligand is a water-soluble ligand containing thiol and carboxyl; S2, mixing the surface-bound water-soluble ligand sulfide colloidal quantum dots and carboxyl activator, protein molecules, activating the carboxyl of the water-soluble ligand to label the protein molecules, obtaining sulfide colloidal quantum dots surface-bound water-soluble ligand and labeled protein molecules.

2. The protein molecular 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 mercaptoacetic acid.

3. The method of claim 1 or 2, wherein the protein molecule is a protein molecule that is expressed in a cell. 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 molecular labeling method according to claim 1, characterized in that, In step S2, the carboxyl activator includes EDC and / or NHS; and / or, The protein molecules are one or more of glucose oxidase, catalase, cholesterol oxidase, triglyceride oxidase, urease, alkaline phosphatase, lactate dehydrogenase, antigen and antibody.

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

6. The method of claim 1 or 4, wherein In step S2, the mass ratio of the surface-bound water-soluble ligand sulfide colloidal quantum dots to the protein molecules is 1:(1-5).

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

8. A method of constructing a high-performance electrochemical biosensor using the sulfide colloid quantum dots according to claim 7, characterized by, Comprising the following steps: The dispersion liquid of the sulfide colloidal quantum dots is coated on the working electrode of a planar three-electrode, and after drying into a film, an electrochemical biosensor is prepared.

9. The method of claim 8, wherein, The coating method is one or more of spin coating, drop coating, spray coating and electro-spray printing; and / or, The working electrode is a gold electrode.

10. A high performance electrochemical biosensor, characterized by, Prepared by using the method according to claim 8 or 9.

Images