阅读说明：本技术 基于半胱胺-n-乙酰-l-半胱氨酸-金纳米团簇荧光材料的酸性磷酸酶检测方法 (Acid phosphatase detection method based on cysteamine-N-acetyl-L-cysteine-gold nanocluster fluorescent material ) 是由 邓豪华 黄开源 陈伟 查代君 孙伟明 于 2019-09-25 设计创作，主要内容包括：本发明公开一种基于半胱胺-N-乙酰-L-半胱氨酸-金纳米团簇荧光材料的酸性磷酸酶检测方法,利用半胱胺-N-乙酰-L-半胱氨酸-金纳米团簇与磷酸吡哆醛的特异性相互作用后,金纳米团簇的荧光受抑制,酸性磷酸酶水解磷酸吡哆醛释放出吡哆醛,解除磷酸吡哆醛对半胱胺-N-乙酰-L-半胱氨酸-金纳米团簇荧光的抑制作用,从而表现出荧光发射光谱强度特征的变化,从而用于酸性磷酸酶的含量检测。酸性磷酸酶检测的线性范围为0.1~5 U/L,检测限为0.05 U/L。本检测方法具有操作简便、耗时短、灵敏度高、特异性强,绿色环保等优点,易于推广使用。(The invention discloses an acid phosphatase detection method based on a cysteamine-N-acetyl-L-cysteine-gold nanocluster fluorescent material, which is characterized in that after specific interaction of cysteamine-N-acetyl-L-cysteine-gold nanoclusters and pyridoxal phosphate is utilized, fluorescence of the gold nanoclusters is inhibited, the pyridoxal phosphate is hydrolyzed by the acid phosphatase to release the pyridoxal phosphate, inhibition of the pyridoxal phosphate on fluorescence of the cysteamine-N-acetyl-L-cysteine-gold nanoclusters is relieved, and change of fluorescence emission spectrum intensity characteristics is shown, so that the detection method is used for content detection of the acid phosphatase, the linear range of detection of the acid phosphatase is 0.1 ~ 5U/L, and the detection limit is 0.05U/L.)

1. A detection method of acid phosphatase based on gold nanoclusters of cysteamine-N-acetyl-L-cysteine is characterized in that the method is characterized in that the specific action of gold nanoclusters and pyridoxal phosphate is achieved, wherein the method comprises the steps of inhibiting fluorescence of the gold nanoclusters by utilizing the strong photoinduced electron transfer effect of the pyridoxal phosphate and the gold nanoclusters of cysteamine-N-acetyl-L-cysteine, catalyzing the pyridoxal phosphate by utilizing acid phosphatase to release the pyridoxal, and recovering the fluorescence of the gold nanoclusters to determine the activity of the acid phosphatase; the gold nanoclusters are prepared by the following steps: 0.75 mL of cysteamine with the concentration of 30mmol/L and 0.25 mL of N-acetyl-L-cysteine with the concentration of 30mmol/L are mixed in advance, 8 mL of ultrapure water is added for even mixing, 1 mL of chloroauric acid with the concentration of 20mmol/L is added, the mixture is placed in a water bath with the temperature of 90 ℃ for incubation for 1.5 hours, large-particle nano particles are removed by centrifugation, a dialysis bag with the molecular weight cutoff of 3500 is used for dialysis for 24 hours in double distilled water, and the gold nanocluster solution jointly protected by cysteamine and N-acetyl-L-cysteine is obtained; all glassware used in the preparation process is soaked in aqua regia, thoroughly cleaned with double distilled water and dried.

2. The method for detecting acid phosphatase based on gold cysteamine-N-acetyl-L-cysteine nanoclusters according to claim 1, wherein the activity of acid phosphatase can be judged according to the change of fluorescence intensity at 590nm of the maximum emission wavelength of fluorescence spectrum.

3. The method for detecting acid phosphatase based on gold cysteamine-N-acetyl-L-cysteine nanoclusters according to claim 1 or 2, wherein 0.2mL of pyridoxal phosphate solution with a concentration of 25 μmol/L and 0.2mL of acid phosphatase solution with different concentrations are added into 1.45 mL of acetate buffer solution with pH of 5.0 and 50 mmol/L, mixed uniformly and placed in a 37 ℃ water bath for reaction for 30 min; after the completion of the reaction, 0.15mL of the prepared gold nanocluster solution was mixed with the above reaction solution, and then the fluorescence emission intensity value (F) of the solution at an excitation wavelength of 425 nm and a maximum emission wavelength of 590nm was measured₅₉₀) Each group was assayed in triplicate; setting a control group without acid phosphatase, and calculating the fluorescence emission intensity F between the experimental group and the control group₅₉₀Within the range of 0.1 ~ 5U/L, the acid phosphatase and the activity fluorescence intensity difference DeltaF are in a linear relation, and the detection limit is 0.05U/L.

4. A method for detecting the content of acid phosphatase in serum based on cysteamine-N-acetyl-L-cysteine-gold nanoclusters is characterized by comprising the following steps: diluting human serum by 20 times by using a pH5.0, 50 mmol/L acetate buffer solution to obtain a sample solution, adding 0.2mL of a pyridoxal phosphate solution with the concentration of 25 mu mol/L and 0.2mL of the sample solution into 1.45 mL of a pH5.0, 50 mmol/L acetate buffer solution, uniformly mixing, and placing in a 37 ℃ water bath kettle for reaction for 30 min; after completion, 0.15mL of the gold nanocluster solution was mixed with the above reaction solution, and then the fluorescence emission intensity value (F) of the solution at an excitation wavelength of 425 nm and a maximum emission wavelength of 590nm was measured₅₉₀) (ii) a Setting a control group, and calculating the fluorescence emission intensity F between the experimental group and the control group₅₉₀The difference (. DELTA.F) to obtain the content of acid phosphatase in the human serum sample; the gold nanoclusters are prepared by the following steps: 0.75 mL of 30mmol/L cysteamine and 0.25 mL of 30mmol/L N-acetyl-L-cysteine are mixed in advance, 8 mL of ultrapure water is added and mixed evenly, 1 mL of 20mmol/L chloroauric acid is added, the mixture is placed in a water bath at 90 ℃ for incubation for 1.5 h, large-particle nano particles are removed by centrifugation, and a dialysis bag with the molecular weight cutoff of 3500 is used for dialysis in the double bagDialyzing in distilled water for 24 h to obtain a gold nanocluster solution jointly protected by cysteamine and N-acetyl-L-cysteine; all glassware used in the preparation process is soaked in aqua regia, thoroughly cleaned with double distilled water and dried.

5. A method for determining an acid phosphatase inhibitor based on cysteamine-N-acetyl-L-cysteine-gold nanoclusters is characterized by comprising the following steps: adding 0.2mL of pyridoxal phosphate solution with the concentration of 25 mu mol/L and 0.2mL of acid phosphatase solution with the final concentration of 4U/L into 1.45 mL of acetate buffer solution with different concentrations of sodium vanadate and with the pH value of 5.0 and the concentration of 50 mmol/L, uniformly mixing, and placing in a water bath kettle at 37 ℃ for reaction for 30 min; after completion, 0.15mL of the gold nanocluster solution was mixed with the above reaction solution, and then the fluorescence emission intensity value (F) of the solution at an excitation wavelength of 425 nm and a maximum emission wavelength of 590nm was measured₅₉₀) Each group was run in parallel three times; obtaining the semi-inhibitory concentration IC of sodium vanadate by software fitting₅₀The value was 0.18. mu. moL/L; the gold nanoclusters are prepared by the following steps: 0.75 mL of cysteamine with the concentration of 30mmol/L and 0.25 mL of 30mmol/L N-acetyl-L-cysteine are mixed in advance, 8 mL of ultrapure water is added for mixing, 1 mL of chloroauric acid with the concentration of 20mmol/L is added, the mixture is placed in a water bath with the temperature of 90 ℃ for incubation for 1.5 hours, large-particle nano particles are removed by centrifugation, dialysis bags with the molecular weight cutoff of 3500 are used for dialysis for 24 hours in double distilled water, and the gold nanocluster solution jointly protected by cysteamine and N-acetyl-L-cysteine is obtained; all glassware used in the preparation process is soaked in aqua regia, thoroughly cleaned with double distilled water and dried.

Technical Field

The invention provides a novel acid phosphatase activity detection method based on cysteamine-N-acetyl-L-cysteine-gold nanocluster fluorescent materials, and belongs to the technical fields of analytical chemistry and nanometer.

Background

It is well known that acid phosphatase is an enzyme capable of catalyzing the hydrolysis of monophosphate under weakly acidic conditions. The content change of the acid phosphatase is an important index for diagnosing diseases such as prostatic cancer, thrombophlebitis, gaucher disease, Paget disease, hepatitis, hyperparathyroidism, multiple myeloma and the like. Therefore, the development of a rapid and simple method for detecting acid phosphatase is of great clinical significance.

The fluorescence analysis method is widely used for measuring the acid phosphatase because of the advantages of high sensitivity, good selectivity, low cost, quick signal response time, simple operation and the like. However, the fluorescence analysis method for acid phosphatase developed at present has various disadvantages, such as complicated operation steps, use of toxic metal ions, and susceptibility to interference by other enzymes. Therefore, it is still very important to develop an accurate, reliable, simple and convenient fluorescence analysis method for the activity of the acid phosphatase.

The gold nanocluster is a novel fluorescent nanomaterial and has the advantages of large Stokes shift, strong bleaching resistance, easiness in modification and the like, so that the gold nanocluster is widely concerned by researchers. The invention establishes a novel method for detecting the activity of acid phosphatase by taking pyridoxal phosphate as an enzyme substrate and taking gold nanoclusters jointly protected by cysteamine and N-acetyl-L-cysteine as a fluorescent probe.

Disclosure of Invention

In view of the above-mentioned deficiencies in the prior art, the present invention aims to provide a method for fluorescence detection of acid phosphatase based on cysteamine-N-acetyl-L-cysteine-gold nanoclusters. The method comprises the steps of quenching fluorescence of gold nanoclusters by utilizing the strong photoinduced electron transfer effect of cysteamine-N-acetyl-L-cysteine-gold nanoclusters and pyridoxal phosphate, carrying out catalytic hydrolysis on the pyridoxal phosphate by acid phosphatase to release the pyridoxal phosphate, and recovering the fluorescence of the gold nanoclusters, so that rapid and sensitive detection on the acid phosphatase is realized, and the method is used for detecting actual samples.

In order to realize the purpose of the detection method, the invention adopts the following technical scheme:

a method for detecting acid phosphatase based on cysteamine-N-acetyl-L-cysteine gold nanoclusters. The method is characterized by the specific action of gold nanoclusters and pyridoxal phosphate, wherein the strong photoinduced electron transfer action of the pyridoxal phosphate and cysteamine-N-acetyl-L-cysteine-gold nanoclusters is utilized to inhibit fluorescence of the gold nanoclusters, the pyridoxal phosphate is catalyzed by acid phosphatase to release the pyridoxal phosphate, and the fluorescence of the gold nanoclusters is recovered to determine the activity of acid phosphatase; the gold nanoclusters are prepared by the following steps: 0.75 mL of cysteamine with the concentration of 30mmol/L and 0.25 mL of N-acetyl-L-cysteine with the concentration of 30mmol/L are mixed in advance, 8 mL of ultrapure water is added for even mixing, 1 mL of chloroauric acid with the concentration of 20mmol/L is added, the mixture is placed in a water bath with the temperature of 90 ℃ for incubation for 1.5 h, large-particle nano particles are removed by centrifugation, dialysis bags with the molecular weight cutoff of 3500 are used for dialysis for 24 h in double distilled water, and the gold nanocluster solution jointly protected by cysteamine and N-acetyl-L-cysteine is obtained. All glassware used in the preparation process is soaked in aqua regia, thoroughly cleaned with double distilled water and dried.

The method for detecting the acid phosphatase based on the cysteamine-N-acetyl-L-cysteine gold nanocluster is characterized in that the activity of the acid phosphatase can be judged according to the change of fluorescence intensity at the position of 590nm of the maximum emission wavelength of a fluorescence spectrum.

The detection method of the acid phosphatase based on the cysteamine-N-acetyl-L-cysteine gold nanocluster is characterized in that 0.2mL of pyridoxal phosphate solution with the concentration of 25 mu mol/L and 0.2mL of acid phosphatase solution with different concentrations are added into 1.45 mL of acetate buffer solution with the pH value of 5.0 and the concentration of 50 mmol/L, and the mixture is uniformly mixed and then placed into a water bath kettle with the temperature of 37 ℃ for reaction for 30 min; after the completion of the reaction, 0.15mL of the prepared gold nanocluster solution was mixed with the above reaction solution, and then the fluorescence emission intensity value (F) of the solution at an excitation wavelength of 425 nm and a maximum emission wavelength of 590nm was measured₅₉₀) Each group was assayed in triplicate; setting a control group without acid phosphatase, and calculating the fluorescence emission intensity F between the experimental group and the control group₅₉₀Difference (. DELTA.F) between the fluorescence intensity of acid phosphatase and the fluorescence intensity of the activity in the range of 0.1 ~ 5U/LThe Delta F is in a linear relation, and the detection limit is 0.05U/L.

The invention relates to a method for detecting the content of acid phosphatase in serum based on cysteamine-N-acetyl-L-cysteine-gold nanoclusters, which is characterized by comprising the following steps: diluting human serum by 20 times by using a pH5.0, 50 mmol/L acetate buffer solution to obtain a sample solution, adding 0.2mL of a pyridoxal phosphate solution with the concentration of 25 mu mol/L and 0.2mL of the sample solution into 1.45 mL of a pH5.0, 50 mmol/L acetate buffer solution, uniformly mixing, and placing in a 37 ℃ water bath kettle for reaction for 30 min; after completion, 0.15mL of the gold nanocluster solution was mixed with the above reaction solution, and then the fluorescence emission intensity value (F) of the solution at an excitation wavelength of 425 nm and a maximum emission wavelength of 590nm was measured₅₉₀) (ii) a Setting a control group, and calculating the fluorescence emission intensity F between the experimental group and the control group₅₉₀The difference (. DELTA.F) to obtain the content of acid phosphatase in the human serum sample; the gold nanoclusters are prepared by the following steps: 0.75 mL of cysteamine with the concentration of 30mmol/L and 0.25 mL of N-acetyl-L-cysteine with the concentration of 30mmol/L are mixed in advance, 8 mL of ultrapure water is added for even mixing, 1 mL of chloroauric acid with the concentration of 20mmol/L is added, the mixture is placed in a water bath with the temperature of 90 ℃ for incubation for 1.5 hours, large-particle nano particles are removed by centrifugation, a dialysis bag with the molecular weight cutoff of 3500 is used for dialysis for 24 hours in double distilled water, and the gold nanocluster solution jointly protected by cysteamine and N-acetyl-L-cysteine is obtained; all glassware used in the preparation process is soaked in aqua regia, thoroughly cleaned with double distilled water and dried.

The invention discloses a method for determining an acid phosphatase inhibitor based on cysteamine-N-acetyl-L-cysteine-gold nanoclusters, which is characterized by comprising the following steps of: 0.2mL of a pyridoxal phosphate solution with a concentration of 25. mu. mol/L and 0.2mL of an acid phosphatase solution with a final concentration of 4U/L were added to 1.45 mL of acetate buffers with different concentrations of sodium vanadate, pH5.0, 50 mmol/L, and the mixture was mixed and reacted in a 37 ℃ water bath for 30 min. After completion, 0.15mL of the gold nanocluster solution was mixed with the above reaction solution, and then the fluorescence emission intensity value (F) of the solution at an excitation wavelength of 425 nm and a maximum emission wavelength of 590nm was measured₅₉₀) Each group is parallel to threeSecondly; obtaining the semi-inhibitory concentration IC of sodium vanadate by software fitting₅₀The value was 0.18. mu. moL/L. The gold nanoclusters are prepared by the following steps: 0.75 mL of cysteamine with the concentration of 30mmol/L and 0.25 mL of 30mmol/L N-acetyl-L-cysteine are mixed in advance, 8 mL of ultrapure water is added for mixing, 1 mL of chloroauric acid with the concentration of 20mmol/L is added, the mixture is placed in a water bath with the temperature of 90 ℃ for incubation for 1.5 hours, large-particle nano particles are removed by centrifugation, dialysis bags with the molecular weight cutoff of 3500 are used for dialysis for 24 hours in double distilled water, and the gold nanocluster solution jointly protected by cysteamine and N-acetyl-L-cysteine is obtained; all glassware used in the preparation process is soaked in aqua regia, thoroughly cleaned with double distilled water and dried.

Specifically, the invention adopts the following technical scheme:

preparation of gold nanoclusters

0.75 mL of cysteamine (30 mmol/L) and 0.25 mL of N-acetyl-L-cysteine (30 mmol/L) are mixed in advance, 8 mL of ultrapure water is added for even mixing, 1 mL of chloroauric acid (20 mmol/L) is added, the mixture is placed in a water bath at 90 ℃ for incubation for 1.5 h, large-particle nano particles are removed by centrifugation, dialysis bags with the molecular weight cutoff of 3500 are used for dialysis for 24 h in double distilled water, and the gold nanocluster solution jointly protected by cysteamine and N-acetyl-L-cysteine is obtained. All glassware used in the preparation process is soaked in aqua regia, thoroughly cleaned with double distilled water and dried.

Method for detecting acid phosphatase

0.2mL of a pyridoxal phosphate solution having a concentration of 25. mu. mol/L and 0.2mL of an acid phosphatase solution having different enzyme activities were added to 1.45 mL of an acetate buffer solution (pH 5.0, 50 mmol/L), mixed well, and then placed in a 37 ℃ water bath to react for 30 min. After the reaction, 0.15mL of the gold nanocluster solution prepared in the step (I) was mixed with the reaction solution, and then the fluorescence emission intensity value (F) of the solution at 590nm was measured₅₉₀) (excitation wavelength 425 nm), each group was assayed in triplicate. Setting a control group without acid phosphatase, and calculating the fluorescence emission intensity F between the experimental group and the control group₅₉₀Difference (Δ F). Finally using acid phosphoric acidThe enzyme activity is the abscissa, and the Delta F is the ordinate to establish a working curve for detecting the acid phosphatase.

Compared with the defects of the prior art, the invention has the advantages that:

(1) according to the method, after the specific interaction between the cysteamine-N-acetyl-L-cysteine-gold nanocluster and pyridoxal phosphate is utilized, the fluorescence of the gold nanocluster is inhibited, the pyridoxal phosphate is hydrolyzed by acid phosphatase to release the pyridoxal, and the inhibition effect of the pyridoxal phosphate on the fluorescence of the cysteamine-N-acetyl-L-cysteine-gold nanocluster is relieved, so that the change of the fluorescence emission spectrum intensity characteristic is shown, and the method is used for detecting the content of the acid phosphatase.

(2) The acid phosphatase substrate adopted by the invention has the characteristics of environmental protection, good biocompatibility, low cost and the like.

(3) The cysteamine-N-acetyl-L-cysteine-gold nanocluster used by the invention has simple and quick preparation process.

(4) The invention has the advantages of high detection sensitivity, strong specificity, quick, simple and convenient operation, easy popularization and use and the like.

Drawings

FIG. 1 shows fluorescence emission spectra. (A) A gold nanocluster; (B) gold nanoclusters + pyridoxal phosphate; (C) gold nanoclusters + pyridoxal phosphate + acid phosphatase.

FIG. 2 shows the pH optimization of the reaction in the acid phosphatase assay system.

FIG. 3 shows the reaction temperature optimization of the acid phosphatase measurement system.

FIG. 4 shows the reaction time optimization of the acid phosphatase assay system.

FIG. 5 is a fluorescence emission spectrum of a reaction system under the action of acid phosphatases with different enzyme activities.

FIG. 6 is a standard curve for acid phosphatase determination.

FIG. 7 shows an interference assay for acid phosphatase detection.

Fig. 8 is a graph of sodium vanadate inhibition.

Detailed Description