Gold nanocluster electrochemiluminescence active oxygen rapid determination box and determination method and application

Abstract

The invention relates to the field of biosensing, in particular to a gold nanocluster electrochemiluminescence active oxygen rapid determination box, a determination method and application. The gold nanocluster electrochemical luminescence active oxygen rapid determination box and the determination method provided by the invention can react with a co-reactant under negative voltage to generate a strong electrochemical luminescence signal, and can rapidly and circularly react with the gold nanocluster and the co-reactant thereof under negative voltage after an active oxygen target is added, so that the electrochemical luminescence valence state of the gold nanocluster is rapidly regulated and controlled; the electrochemical luminescence signal of the gold nanocluster is rapidly quenched, one-step rapid and sensitive detection of the active oxygen is realized, the test period of a single sample is 11 seconds, and the electric signal can be repeatedly used for testing after being subjected to electroreduction to recover the electric signal and is applied to rapid detection of the concentration of the active oxygen in seminal plasma. The method solves the problem of rapid, high-sensitivity, accurate and quantitative determination of unstable and easily inactivated seminal plasma active oxygen, realizes rapid, high-sensitivity and accurate determination of active oxygen in seminal plasma, and effectively promotes the technical effects of disease diagnosis.

Description

Technical Field

[0001] The invention relates to the field of biosensing, and in particular to a gold nanocluster electrochemiluminescence active oxygen rapid determination box and a determination method and application. Background Art

[0002] Reactive oxygen species (ROS) are oxygen-containing chemicals produced during cellular metabolism, including hydrogen peroxide, superoxide anions, and hydroxyl radicals. It has been reported that in about 70% of men with idiopathic infertility, the level of ROS in seminal plasma is elevated, and excessive ROS in seminal plasma has been shown to negatively affect human sperm function. Therefore, the detection of ROS in seminal plasma plays a vital role in the evaluation of male idiopathic infertility.

[0003] Traditional methods for detecting reactive oxygen species include spectrophotometry, iodine reduction titration, high performance liquid chromatography, and fluorescence. Among these methods, spectrophotometry and iodine reduction titration are relatively simple to operate, but have limited sensitivity; high performance liquid chromatography and fluorescence are highly sensitive, but complex to operate and require a certain reaction time. In addition, the level of reactive oxygen species in seminal plasma is characterized by low concentration, instability, short half-life in the air, and immediate dissipation after leaving the human body. Therefore, there is an urgent need to establish a rapid and highly sensitive assay to accurately detect reactive oxygen species in seminal plasma, thereby promoting disease diagnosis.

[0004] At present, electrochemiluminescence (ECL) detection technology has been widely developed. Applying voltage can induce rapid luminescence response. With its inherent high sensitivity and rapid response advantages, electrochemiluminescence has shown great potential in developing feasible reactive oxygen detection strategies, providing a possible solution to this arduous challenge. As a new type of luminophore, gold nanoclusters (AuNCs) have attracted widespread attention in the field of electrochemiluminescence analysis due to their inherent advantages such as size-controlled luminescence, high quantum yield, low toxicity and good water solubility. For example, a method for constructing an electrochemiluminescence sensor based on cerium dioxide and nano-platinum dual-enhanced luminol luminescence disclosed in the patent document with publication number CN110927226A successively used CeO2-Au@Pt solution, heptapeptide HGC solution, primary antibody Ab1 solution, BSA solution, PCT solution, luminol-Au star@BSA-Ab2 solution to form 6 layers of modification layers on the electrode to construct an electrochemiluminescence sensor and achieve high-sensitivity detection of PCT.

[0005] However, the above-mentioned patents do not solve the problem of rapid, highly sensitive and accurate quantitative determination of unstable and easily inactivated reactive oxygen species in seminal plasma. How to achieve rapid, highly sensitive and accurate determination of reactive oxygen species in seminal plasma is a problem that is difficult for technicians in this field to solve. Summary of the invention

[0006] In order to solve the problem of how to achieve rapid, highly sensitive and accurate determination of reactive oxygen species in seminal plasma mentioned in the above background technology, an embodiment of the present invention provides a gold nanocluster electrochemiluminescence reactive oxygen species rapid determination kit, comprising: a substrate solution, the substrate solution comprising potassium persulfate and phosphate; The gold nanocluster film is formed by mixing HAuCl4, N-acetyl-L-cysteine ​​and NaOH, coating the mixture on the electrode surface and drying the mixture.

[0007] On the basis of the above embodiment, further, the working electrode is a glassy carbon electrode; The three-electrode system further includes an auxiliary electrode and a reference electrode. The auxiliary electrode is a platinum electrode, and the reference electrode is a saturated silver chloride electrode.

[0008] On the basis of the above embodiment, further, the preparation steps of the gold nanocluster solution are as follows: S100, mixing HAuCl4 solution, N-acetyl-L-cysteine ​​solution, and NaOH solution; S101, incubating the mixed solution described in S100 at 37-45°C for 3-10 h; S102. Purify the culture solution described in S101 using a dialysis bag and store it at 4°C.

[0009] On the basis of the above embodiment, further, the concentration of the HAuCl4 solution is 20-25 mg / mL; the concentration of the N-acetyl-L-cysteine ​​solution is 0.06-0.10 mol / L; the concentration of the NaOH solution is 0.5-0.6 mol / L; The HAuCl4 solution, N-acetyl-L-cysteine ​​solution, and NaOH solution are mixed in a volume ratio of 1.5-2.5:15-25:2-4.

[0010] On the basis of the above embodiment, further, the substrate solution is 70-100 mmol / L potassium persulfate 1× phosphate buffer, pH=7.

[0011] On the basis of the above embodiments, further, the gold nanocluster electrochemiluminescence reactive oxygen rapid determination kit can restore the electrochemiluminescence signal to the level before the test after electrical reduction, and continue to be used in a new test cycle.

[0012] The present invention also provides a method for rapid determination of active oxygen species by electrochemiluminescence of active gold nanoclusters, which uses the electrochemiluminescence active oxygen species rapid determination kit of the gold nanoclusters as described in any one of the above items, comprising: S200, applying a negative voltage to the electrode coated with the gold nanocluster film to pre-reduce the gold nanocluster; S201, inserting the three-electrode system into the substrate solution, applying an instantaneous negative voltage, generating a strong electrochemiluminescent signal, and recording it by an electrochemiluminescence instrument; S202, adding a reactive oxygen standard of known concentration to the substrate solution, applying an instantaneous negative voltage, the electrochemiluminescence signal is immediately quenched, the degree of light intensity quenching is positively correlated with the concentration of the reactive oxygen standard, and recorded by an electrochemiluminescence instrument; S203, replacing the substrate solution, applying a negative voltage to the electrode for electroreduction, and restoring the electrochemiluminescence signal; S204, repeating steps S202 and S203 using reactive oxygen standard substances of different concentrations, and drawing a standard curve by plotting the degree of change of the electrochemiluminescence signal and the concentration of the reactive oxygen standard substance; S205, adding active oxygen test solution to the substrate solution, applying instantaneous negative voltage, quenching the electrochemiluminescence signal, and recording by an electrochemiluminescence instrument; S206. Calculate the active oxygen solution concentration in the active oxygen test solution based on the standard curve prepared in S204.

[0013] On the basis of the above embodiment, further, the active oxygen includes at least one of hydrogen peroxide, superoxide anion and hydroxyl radical.

[0014] On the basis of the above-mentioned embodiment, further, the method for rapid determination of electrochemiluminescence reactive oxygen species by gold nanoclusters adopts a step pulse method for electrochemiluminescence detection, and the test cycle of a single sample is 11 s.

[0015] The present invention also provides an application of the gold nanocluster electrochemiluminescence active oxygen rapid determination method as described in any one of the above items in the rapid detection of active oxygen in seminal plasma.

[0016] The present invention has the following beneficial effects: The present invention provides a gold nanocluster electrochemiluminescent active oxygen rapid determination kit, which can react with a coreactant potassium persulfate under negative voltage conditions to generate a strong electrochemiluminescent signal, and the gold nanocluster electrochemiluminescent active oxygen rapid determination kit can be reused after electrical reduction, and the method of use is simple and easy to use, effectively saving cost and time.

[0017] The present invention also provides a method for rapid determination of electrochemiluminescence reactive oxygen species by gold nanoclusters, which can rapidly regulate the electrochemiluminescence valence state of gold nanoclusters through rapid cyclic reactions of target reactive oxygen species with gold nanoclusters and their co-reactants under negative voltage conditions, thereby rapidly quenching the electrochemiluminescence intensity of gold nanoclusters and achieving a one-step rapid and sensitive detection of reactive oxygen species.

[0018] The present invention also provides an application of a gold nanocluster electrochemiluminescence active oxygen rapid determination method in the rapid detection of active oxygen in seminal plasma. The instant and sensitive active oxygen rapid determination method effectively avoids the inaccuracy caused by the low concentration, instability and short half-life of active oxygen in seminal plasma in the air, thereby effectively promoting the accurate diagnosis of diseases.

[0019] Other features and beneficial effects of the present invention will be described in the following description, and some of the technical features and beneficial effects can be obviously derived from the description or understood by implementing the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, a brief introduction is given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, some of the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a schematic diagram of a gold nanocluster electrochemiluminescence reactive oxygen rapid determination kit and determination method provided in Examples 1 and 2 of the present invention; Figure 2 This is a data analysis diagram of the electroreduction and recovery repeatability test of the gold nanocluster electrochemiluminescence active oxygen rapid determination kit provided in Example 1 of the present invention; Figure 3 This is a capture graph of an ECL curve of a single reactive oxygen sample test cycle measured by a gold nanocluster electrochemiluminescence reactive oxygen rapid determination kit provided in Example 2 of the present invention; Figure 4 The standard curve of the active oxygen standard measured by the gold nanocluster electrochemiluminescence active oxygen rapid determination kit provided in Example 2 of the present invention; Figure 5 This is a schematic diagram of a new strategy for detecting male infertility using the gold nanocluster electrochemiluminescence reactive oxygen rapid determination method provided in Example 3 of the present invention; Figure 6 This is a data analysis diagram of the active oxygen concentration in clinical samples using the gold nanocluster electrochemiluminescence active oxygen rapid determination method provided in Example 3 of the present invention; Figure 7 The standard curve of the active oxygen standard product measured by the gold nanocluster electrochemiluminescence active oxygen rapid determination kit using the existing gold nanocluster provided in Comparative Example 1 of the present invention; Figure 8 This is a data analysis diagram of the light intensity difference of active oxygen standard measured by the gold nanocluster electrochemiluminescence active oxygen rapid determination kit provided in Example 2 of the present invention and Comparative Example 1. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; the technical features designed in different implementation modes of the present invention described below can be combined with each other as long as they do not conflict with each other; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0023] Example 1 Embodiment 1 of the present invention provides a gold nanocluster electrochemiluminescence reactive oxygen rapid determination kit, comprising: A three-electrode system, wherein the working electrode in the three-electrode system is pretreated and coated with a gold nanocluster film on the surface; a substrate solution, the substrate solution comprising potassium persulfate and phosphate; The gold nanocluster film is formed by mixing HAuCl4, N-acetyl-L-cysteine ​​and NaOH, coating the mixture on the electrode surface and drying the mixture.

[0024] Wherein, when used specifically, the working electrode is a glassy carbon electrode; The three-electrode system further includes an auxiliary electrode and a reference electrode. The auxiliary electrode is a platinum electrode, and the reference electrode is a saturated silver chloride electrode.

[0025] Wherein, when used specifically, the steps for preparing the gold nanocluster solution are as follows: S100, mixing HAuCl4 solution, N-acetyl-L-cysteine ​​solution, and NaOH solution; S101, incubating the mixed solution described in S100 at 37-45°C for 3-10 h; S102. Purify the culture solution described in S101 using a dialysis bag and store it at 4°C.

[0026] The concentration of the HAuCl4 solution is 20-25 mg / mL; the concentration of the N-acetyl-L-cysteine ​​solution is 0.06-0.10 mol / L; the concentration of the NaOH solution is 0.4-0.6 mol / L; The HAuCl4 solution, N-acetyl-L-cysteine ​​solution, and NaOH solution are mixed in a volume ratio of 1.5-2.5:15-25:2-4; When the test is carried out, the electrode coated with the gold nanocluster film needs to be pre-reduced under a negative voltage to make the positive monovalent gold nanoclusters (AuNCs) coated on the electrode +) are reduced to zero valence to form electroreduced gold nanoclusters (ER-AuNCs).

[0027] Wherein, when used specifically, the substrate solution is 70 mmol / L potassium persulfate 1× phosphate buffer, pH=7; When the test is carried out, the potassium persulfate in the 70 mmol / L potassium persulfate 1× phosphate buffer is a co-reactant. Under the action of instantaneous negative voltage, S2O8 2- Electroreduction reaction will occur to generate SO4 ·- , while the ER-AuNCs will be reduced to the gold nanocluster radical anion state (AuNCs ·- ), the SO4 ·- With AuNCs ·- Will react to produce SO4 2- and unstable excited-state gold nanoclusters (AuNCs * ), unstable AuNCs * It will release energy and emit photons, thus producing a strong electrochemical luminescence signal; the specific reaction mechanism logic reference Figure 1 (A) is sorted out.

[0028] The gold nanocluster electrochemiluminescent reactive oxygen rapid assay kit can restore the electrochemiluminescent signal to the level before the test after electrical reduction, and continue to be used in a new test cycle; When the test was carried out, the gold nanocluster electrochemiluminescence reactive oxygen rapid determination kit was tested with a reactive oxygen standard at a concentration of 10 mmol / L for post-test reproducibility verification. The reproducibility verification results were as follows: Figure 2 As shown, the high-order data are the electrochemiluminescence signal data after electrical reduction. In 5 repeated experiments, the standard deviation of the data group is 3.7%, and the data repeatability is good. It can be seen that after the gold nanocluster electrochemiluminescence reactive oxygen rapid determination kit is used for testing, the electrochemiluminescence signal can be restored by electrical reduction and continued to be used in subsequent tests.

[0029] In this embodiment, preferably, the glassy carbon electrode is a 3 mm glassy carbon electrode.

[0030] In this embodiment, preferably, the mixed solution in S100 is cultured at 37° C. for 3 h.

[0031] In this embodiment, preferably, the concentration of the HAuCl4 solution is 20 mg / mL; the concentration of the N-acetyl-L-cysteine ​​solution is 0.08 mol / L; and the concentration of the NaOH solution is 0.5 mol / L.

[0032] In this embodiment, preferably, the HAuCl4 solution, N-acetyl-L-cysteine ​​solution, and NaOH solution are mixed in a volume ratio of 2:20:3.

[0033] In this embodiment, preferably, the substrate solution is 70 mmol / L potassium persulfate 1× phosphate buffer, pH=7.

[0034] In this embodiment, preferably, the negative voltage condition for pre-reduction of the electrode coated with the gold nanocluster film is -2 V, which lasts for 60 s.

[0035] In this embodiment, preferably, the instantaneous negative voltage is -2 V, the power is on for 1 s, and the rest is 10 s.

[0036] The gold nanocluster electrochemiluminescence reactive oxygen rapid determination kit provided in this embodiment can produce a rapid, sensitive, strong and easy-to-capture electrochemiluminescence response to the reactive oxygen in the system through gold nanoclusters, and can be used in the rapid detection of reactive oxygen, especially in some application scenarios where reactive oxygen is easily deactivated and / or high reactive oxygen detection accuracy is required.

[0037] Example 2 This embodiment provides a method for rapid determination of active oxygen species by electrochemiluminescence of gold nanoclusters based on embodiment 1, comprising: S200, applying a negative voltage to the electrode coated with the gold nanocluster film to pre-reduce the gold nanocluster; S201, inserting the three-electrode system into the substrate solution, applying an instantaneous negative voltage, generating a strong electrochemiluminescent signal, and recording it by an electrochemiluminescence instrument; S202, adding a reactive oxygen standard of known concentration to the substrate solution, applying an instantaneous negative voltage, the electrochemiluminescence signal is immediately quenched, the degree of light intensity quenching is positively correlated with the concentration of the reactive oxygen standard, and recorded by an electrochemiluminescence instrument; S203, replacing the substrate solution, applying a negative voltage to the electrode for electroreduction, and restoring the electrochemiluminescence signal; S204, repeating steps S202 and S203 using reactive oxygen standard substances of different concentrations, and drawing a standard curve by plotting the degree of change of the electrochemiluminescence signal and the concentration of the reactive oxygen standard substance, referring to Figure 2 As shown; S205, adding active oxygen test solution to the substrate solution, applying instantaneous negative voltage, quenching the electrochemiluminescence signal, and recording by an electrochemiluminescence instrument; S206. Calculate the active oxygen solution concentration in the active oxygen test solution based on the standard curve prepared in S204.

[0038] Wherein, during specific operation, the active oxygen includes at least one of hydrogen peroxide, superoxide anion and hydroxyl free radical.

[0039] In this embodiment, specifically, the active oxygen species are hydrogen peroxide and hydroxyl radicals.

[0040] When testing, under instantaneous negative voltage, H2O2 can generate OH and OH - , among which OH - The SO4 produced by electroreduction in Example 1 ·- Reaction to generate OH and SO4 2- , and ·OH has a strong oxidizing ability and can preferentially oxidize the ER-AuNCs to positive monovalent gold nanoclusters AuNCs. + , which leads to the rapid quenching of the electrochemiluminescence signal; the specific reaction mechanism logic reference Figure 1 (B) is sorted out.

[0041] When the test is carried out, the test sample is directly added to the substrate solution and the test is immediately started. The step pulse method is used for electrochemiluminescence detection. The initial potential is 0 V, the pulse time is 10 s, the termination potential is -2 V, the pulse time is 1 s, and the single sample test cycle is 11 s. The test ECL curve is as follows: Figure 3 As shown in the figure, 0~10 s is the 0 V potential pulse segment, and in the 10~11 s segment, the high point peak is the electrochemiluminescence signal value when no test sample is added, and the low point value is the electrochemiluminescence signal value when the luminescence signal is instantly quenched after adding the test sample. It takes a total of 11 s to complete the single sample test.

[0042] On the basis of this embodiment, further, as Figure 4 As shown, the electrochemiluminescence intensity change value (ΔECL) of the gold nanocluster film in the gold nanocluster electrochemiluminescence reactive oxygen rapid determination kit is related to the reactive oxygen concentration ( ) at 1.0×10 -8 ~1.0×10 -3 mol / L and 1.0×10 -3 ~1.0×10 -1 mol / L, and the detection limit was 0.75×10 -10 mol / L, can be used for rapid detection of reactive oxygen species.

[0043] Example 3 Based on Examples 1 and 2, this example provides an application of a gold nanocluster electrochemiluminescence reactive oxygen rapid determination method in the rapid detection of reactive oxygen in seminal plasma. For a specific strategy diagram, refer to Figure 5 ,include: S300, obtaining seminal plasma samples: collect freshly collected semen samples, quickly take 1 mL of the supernatant by centrifugation at a speed of 900-1200 rpm / min for 5-10 min; S301, according to Examples 1 and 2, prepare a gold nanocluster electrochemiluminescence reactive oxygen rapid determination kit, reactive oxygen standard, and an electrochemiluminescence instrument, and calculate the electrochemiluminescence intensity change value (ΔECL) of the gold nanocluster film and the reactive oxygen concentration ( )Standard curve, please refer to Figure 4 ; S302, taking 10 μL of the seminal plasma sample and directly adding it into the substrate solution, shaking to mix, and recording the change of electrochemiluminescence intensity by an electrochemiluminescence instrument at an instantaneous voltage of -2 V; S303, according to the obtained standard curve Figure 4 , calculate the concentration of active oxygen in seminal plasma; Among them, during the specific measurement, the concentration of reactive oxygen in seminal plasma was inversely proportional to the electrochemiluminescence intensity of gold nanoclusters, and positively correlated with the degree of light intensity quenching.

[0044] Excessive reactive oxygen in seminal plasma can damage the integrity of sperm and ultimately lead to male infertility.

[0045] In this embodiment, preferably, the rotation speed in S300 is 1200 rpm / min, and the duration is 5 min.

[0046] On the basis of this example, 57 clinical semen samples were collected and divided into a normal group and an asthenospermia group. The reactive oxygen species in seminal plasma were detected according to the above steps. The data results are as follows: Figure 6 As shown. The data cluster on the left side of the data graph is the normal group. The level of reactive oxygen in the seminal plasma of the normal group is low, and the average level of reactive oxygen concentration is around 200 μmol / L; the data cluster on the right side of the data graph is the asthenospermia group. The level of reactive oxygen in the seminal plasma is generally high, and the average level of reactive oxygen concentration is around 600 μmol / L. However, those skilled in the art generally know that in clinical medicine, male asthenospermia is not entirely determined by the level of reactive oxygen in seminal plasma, so there may be a situation where the data discreteness is high. However, despite the above-mentioned influences, the level of reactive oxygen in the seminal plasma of the normal group and the asthenospermia group can still be significantly distinguished, and the normal group and the asthenospermia group can be effectively distinguished by this gold nanocluster electrochemiluminescence reactive oxygen rapid determination method.

[0047] In this embodiment, a gold nanocluster electrochemiluminescence reactive oxygen rapid determination method was used to perform a one-step rapid determination of reactive oxygen in seminal plasma, and 57 clinical semen samples were used to verify the application effect of the said rapid determination method of reactive oxygen in the rapid detection of reactive oxygen in seminal plasma. The verification results showed that the gold nanocluster electrochemiluminescence reactive oxygen rapid determination method was able to quickly and accurately distinguish between the normal group and the asthenospermia group in the 57 clinical semen samples, achieving the technical effect of rapid and highly sensitive and accurate determination of reactive oxygen in seminal plasma.

[0048] Comparative Example 1 The present invention also provides a gold nanocluster electrochemiluminescence active oxygen rapid determination kit using existing gold nanoclusters as a comparative example. The difference between the kit and Example 1 is that the working electrode is coated with a gold nanocluster solution prepared using histidine as a raw material for preparing the gold nanoclusters, and other conditions and test methods are consistent with Examples 1 and 2.

[0049] The preparation method using histidine as the raw material for preparing gold nanoclusters is based on the preparation method provided in the patent document with publication number CN116550987A, which is as follows: S400, take 100 mL of 0.86 mmol / L chloroauric acid aqueous solution and react with 100 mL of 2.3 mmol / L histidine aqueous solution in a 250 mL round-bottom flask at 97°C for 12 h; S401, slowly cooling the reaction solution in S400 to room temperature, centrifuging at 12500 rpm for 10 min, taking the supernatant to obtain a gold nanocluster solution, and storing it at 4°C; S402, pre-treating the glassy carbon electrode in the three-electrode system with aluminum oxide polishing powder and wiping it clean with dust-free paper; S403, taking 7 μL of the gold nanocluster solution and applying it on the surface of the glassy carbon electrode to form a gold nanocluster film after drying; S404, applying a voltage of -2 V to the electrode coated with the gold nanocluster film to pre-reduce the gold nanocluster to zero valence; S405, inserting the three-electrode system into the substrate solution, applying a -2 V instantaneous voltage, generating an electrochemiluminescent signal, and recording it by an electrochemiluminescence instrument; S406, adding a reactive oxygen standard of known concentration to the substrate solution, applying a -2 V instantaneous voltage, quenching the electrochemiluminescence signal, and the degree of light intensity quenching is positively correlated with the concentration of the reactive oxygen standard, and recording by an electrochemiluminescence instrument; S407, replacing the substrate solution, applying a negative voltage to the electrode for electroreduction, and restoring the electrochemiluminescence signal; S408, using the same step concentration of the active oxygen standard as in Example 2, repeating steps S406 and S407, and plotting the change degree of the electrochemiluminescence signal and the concentration of the active oxygen standard to draw a standard curve, referring to Figure 7 shown.

[0050] In this comparative example, the gold nanocluster electrochemiluminescence reactive oxygen rapid determination kit using the existing gold nanocluster is used to make a standard curve with the same step concentration in reference example 2 as shown in FIG. Figure 7 As shown, only when the active oxygen concentration ( ) is 1.0×10 -3 ~1.0×10 -1 mol / L range; further, the electrochemiluminescence signal intensity series captured at different active oxygen standard concentrations in Example 2 and this comparative example were differentially analyzed, and the analysis results were referenced to Figure 8 As shown, the light intensity difference results of Comparative Example 1 are lower than those of Example 2 at each step concentration of each reactive oxygen species. In particular, in the two stages where the reactive oxygen species concentration is relatively high and relatively low and approaches the detection limit, respectively, the difference in the light intensity difference results of the two is more significant. It can be seen that the gold nanocluster electrochemiluminescence reactive oxygen rapid determination kit provided in this comparative example is significantly inferior to the gold nanocluster electrochemiluminescence reactive oxygen rapid determination kit provided by the technical solution of the present invention in terms of sensitivity and detection range.

[0051] In addition, those skilled in the art should understand that, although there are many problems in the prior art, each embodiment or technical solution of the present invention can be improved in only one or several aspects, without having to solve all the technical problems listed in the prior art or background technology at the same time. Those skilled in the art should understand that the content not mentioned in a claim should not be used as a limitation on the claim.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gold nanocluster electrochemiluminescence active oxygen rapid determination kit, characterized in that: include: A three-electrode system, wherein the working electrode in the three-electrode system is pretreated and coated with a gold nanocluster film on the surface; a substrate solution, the substrate solution comprising potassium persulfate and phosphate; The gold nanocluster film is formed by mixing HAuCl4, N-acetyl-L-cysteine ​​and NaOH, coating the mixture on the electrode surface and drying the mixture.

2. The gold nanocluster electrochemiluminescence reactive oxygen rapid assay kit according to claim 1, characterized in that: The working electrode is a glassy carbon electrode; The three-electrode system further comprises an auxiliary electrode and a reference electrode, wherein the auxiliary electrode is a platinum electrode and the reference electrode is a saturated silver chloride electrode.

3. The gold nanocluster electrochemiluminescence reactive oxygen rapid assay kit according to claim 1, characterized in that: The steps for preparing the gold nanocluster solution are as follows: S100, mixing HAuCl4 solution, N-acetyl-L-cysteine ​​solution, and NaOH solution; S101, incubating the mixed solution described in S100 at 37-45°C for 3-10 h; S102. Purify the culture solution described in S101 using a dialysis bag and store it at 4°C.

4. The gold nanocluster electrochemiluminescence reactive oxygen rapid assay kit according to claim 3, characterized in that: The concentration of the HAuCl4 solution is 20-25 mg / mL; The concentration of the N-acetyl-L-cysteine ​​solution is 0.06-0.10 mol / L; The concentration of the NaOH solution is 0.4-0.6 mol / L; The HAuCl4 solution, N-acetyl-L-cysteine ​​solution and NaOH solution are mixed in a volume ratio of 1.5-2.5:15-25:2-4.

5. The gold nanocluster electrochemiluminescence reactive oxygen rapid assay kit according to claim 1, characterized in that: The substrate solution is 70-100 mmol / L potassium persulfate 1× phosphate buffer, pH=7.

6. The gold nanocluster electrochemiluminescence reactive oxygen rapid assay kit according to claim 1, characterized in that: The gold nanocluster electrochemiluminescent active oxygen rapid determination kit can restore the electrochemiluminescent signal to the level before the test after electrical reduction, and continue to be used in a new test cycle.

7. A method for rapid determination of active oxygen by electrochemiluminescence of gold nanoclusters, characterized in that: The gold nanocluster electrochemiluminescence reactive oxygen rapid determination kit as described in any one of claims 1 to 6 is used, comprising the following steps: S200, applying a negative voltage to the electrode coated with the gold nanocluster film to pre-reduce the gold nanocluster; S201, inserting the three-electrode system into the substrate solution, applying an instantaneous negative voltage, generating a strong electrochemiluminescent signal, and recording it by an electrochemiluminescence instrument; S202, adding a reactive oxygen standard of known concentration to the substrate solution, applying an instantaneous negative voltage, the electrochemiluminescence signal is immediately quenched, the degree of light intensity quenching is positively correlated with the concentration of the reactive oxygen standard, and recorded by an electrochemiluminescence instrument; S203, replacing the substrate solution, applying a negative voltage to the electrode for electroreduction, and restoring the electrochemiluminescence signal; S204, repeating steps S202 and S203 using reactive oxygen standard substances of different concentrations, and drawing a standard curve by plotting the degree of change of the electrochemiluminescence signal and the concentration of the reactive oxygen standard substance; S205, adding active oxygen test solution to the substrate solution, applying instantaneous negative voltage, quenching the electrochemiluminescence signal, and recording by an electrochemiluminescence instrument; S206. Calculate the active oxygen solution concentration in the active oxygen test solution based on the standard curve prepared in S204.

8. The method for rapid determination of active oxygen species by electrochemiluminescence of gold nanoclusters according to claim 7, characterized in that: The active oxygen includes at least one of hydrogen peroxide, superoxide anion and hydroxyl radical.

9. The method for rapid determination of active oxygen species by electrochemiluminescence of gold nanoclusters according to claim 7, characterized in that: The gold nanocluster electrochemiluminescence active oxygen rapid determination method adopts a step pulse method for electrochemiluminescence detection, and a single sample test cycle is 11 s.

10. Use of the gold nanocluster electrochemiluminescence active oxygen rapid determination method as claimed in any one of claims 7 to 9 in the rapid detection of active oxygen in seminal plasma.

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