CN108535240A - The method for detecting trypsase with bovine serum albumin-copper nano-cluster catalysis luminol chemiluminescence - Google Patents

Abstract

The invention discloses a method for detecting trypsin by using bovine serum albumin-copper nano-clusters to catalyze luminol chemiluminescence. After mixing, incubate at a constant temperature; S2: Add luminol-hydrogen peroxide detection solution to the mixed solution for chemiluminescent reaction, and calculate the concentration of trypsin to be tested according to the chemiluminescent intensity and the established standard curve. The detection method of the invention has the advantages of low price, fast detection speed, high precision, good reproducibility and strong anti-interference ability, and can realize real-time monitoring.

Description

Chemiluminescence detection of trypsin catalyzed by bovine serum albumin-copper nanoclusters Methods

technical field

The invention relates to the field of nanometer analysis and detection, in particular to an analysis method for quantitatively detecting trypsin by using bovine serum albumin-copper nano-clusters (BSA-CuNCs) as a catalyst to catalyze luminol chemiluminescence.

Background technique

Trypsin is one of the most important digestive enzymes in protein decomposition, it can selectively hydrolyze the peptide chain composed of lysine or arginine carboxyl in protein. Trypsin plays an important role in the control of pancreatic exocrine function, and the imbalance of trypsinogen secretion, activation, inhibition and circulation can lead to acute or chronic pancreatic diseases. Enzyme-linked immunosorbent assay is a common method for trypsin detection, but this method is expensive, time-consuming and usually requires a large number of samples, and the sample pre-analysis purification process will lead to sample loss, which will affect the accuracy of the test results and reproducibility. In the past many years, chemiluminescent detection methods based on bioanalysis have been widely used to detect proteolytic enzymes, proteins, nucleic acids, small molecules, etc., and this method also provides an effective and rapid method for detecting enzymes. Therefore, it is very necessary and meaningful to develop a trypsin detection method that is easy to prepare, can be monitored in real time and has low cost.

Chemiluminescence refers to the phenomenon of luminescence caused by chemical reactions. Chemiluminescence analysis is an analytical method that uses chemiluminescence reactions to measure the light signals emitted by chemiluminescent substances when they transition from an excited state to a ground state. Luminol (3-aminophthalohydrazide, 5-amino-2,3-dihydro-1,4-naphthalenedione) is the most commonly used chemiluminescence agent. In recent years, the introduction of emerging nanotechnology and novel materials has significantly improved the detection sensitivity and specificity of chemiluminescence, and is expected to achieve multiplex detection. The discovery of metal nanocluster materials has aroused widespread concern. Due to the improvement of the quantum confinement effect, these ultra-small metal nanoclusters have unusual optical and electrical properties, such as Wang G.; Huang T.; Murray R.W.; Menard L.; J.Am.Chem.Soc.2005,127,812 -813; Ramakrishna G.; Varnavski O.; Kim J.; Lee D.; R. J. Am. Chem. Soc. 2008, 130, 5883-5885 et al. As a new type of chemiluminescence response unit, noble metal nanoclusters can significantly improve the efficiency of luminol chemiluminescence reaction, which is of great significance for the expansion and development of new luminol chemiluminescence reaction systems. There have been many reports on luminol chemiluminescence involving gold, platinum, silver and other nanoparticles, such as He Y, He X, Liu X, et al.Dynamically tunablechemiluminescence of luminol functionalized silver nanoparticles and its application to protein sensing arrays[J ].Anal.Chem.,2014,86:12166-12171, etc., but the high price of noble metal nanoclusters limits its biological applications.

Contents of the invention

The technical problem to be solved by the present invention is to provide an analytical method for quantitatively detecting trypsin by using bovine serum albumin-copper nanoclusters as a catalyst to catalyze luminol chemiluminescence. The detection method is low in price, fast in detection speed, high in precision, Good reproducibility, strong anti-interference ability, real-time monitoring can be realized.

In order to solve the above-mentioned technical problems, the present invention provides a method for detecting trypsin by catalyzing luminol with bovine serum albumin-copper nanoclusters. - Chemiluminescence of the hydrogen peroxide system, trypsin can decompose the bovine serum protein template on the surface of the copper nanoclusters, resulting in a change in the surface state of the copper nanoclusters, resulting in aggregation and a decrease in catalytic activity. The experiment found that the addition of trypsin has an effect on luminol- The hydrogen peroxide-copper nano-cluster chemiluminescence system has an obvious inhibitory effect, and the reduction of the chemiluminescence intensity of the system is linearly related to the logarithm of the trypsin concentration, based on which the quantitative analysis and detection of trypsin is achieved.

The detection method comprises the following steps:

S1: Take the trypsin solution to be tested, bovine serum albumin-copper nanocluster catalyst, mix and incubate at constant temperature;

S2: Add luminol-hydrogen peroxide detection solution to the mixed solution for chemiluminescence reaction, and calculate the concentration of trypsin to be tested according to the chemiluminescence intensity and the established standard curve.

In the present invention, the preparation method of bovine serum albumin-copper nano-cluster catalyst is as follows: mix bovine serum albumin and copper sulfate pentahydrate solution, stir at 30-40°C for 5-10min, and then use sodium hydroxide solution to adjust pH to alkaline, and then stirred at 50-60° C. for 8-16 hours to obtain bovine serum albumin-copper nano-cluster catalyst.

Further, in step S1, the molar ratio of copper sulfate pentahydrate to bovine serum albumin is (0.5-1):1.

Further, in step S1, the pH is adjusted to 10-12 with sodium hydroxide solution.

Further, the obtained bovine serum albumin-copper nano-cluster catalyst needs to be dialyzed in ultrapure water to remove unreacted impurities, and after the dialyzing is completed, it is stored at 2-6° C. for use.

Further, during dialysis, the water was changed every four hours, and the dialysis time was 40-60 hours.

In the present invention, the establishment method of standard curve comprises:

Take n groups of equal amounts and different concentrations of newly configured trypsin solutions, add an appropriate amount of bovine serum albumin-copper nanocluster catalysts, mix and incubate at a constant temperature, then add luminol-hydrogen peroxide detection solution to perform chemiluminescence reaction, detecting its chemiluminescent intensity; and

According to the concentration of trypsin in each group and the corresponding decrease in luminous intensity, draw a relationship curve between the decrease in luminous intensity and the logarithm of the trypsin concentration, which is the standard curve.

When establishing a standard curve, the value of n should be a positive integer greater than 5, for example, n can be 8, 10, 12, etc. The amounts of the trypsin solution and the bovine serum albumin-copper nanocluster catalyst are determined according to the situation, for example, the amount of the trypsin solution is 500 μL, and the amount of the bovine serum albumin-copper nanocluster catalyst is 50 μL.

Further, the concentration range of the newly prepared trypsin solution is 0.2 μg/mL˜0.6 mg/mL.

Further, when detecting the sample to be tested and drawing a standard curve, the temperature of the mixed solution is incubated at a constant temperature of 30-45°C, and the incubation time is 0.5h-2h; the luminol-hydrogen peroxide detection system is luminol and peroxide Hydrogen aqueous solution, wherein the concentration of luminol is 1.0×10 ^-6 ~5×10 ^-5 M, the concentration of hydrogen peroxide is 0.02 ~ 0.2M, and the pH of the system is 9.0.

According to the detection method of the present invention, a kit for quantitative detection of trypsin can be prepared, thereby simplifying the detection operation. Further, according to the detection method of the present invention, biochips and biosensors can also be prepared to realize automatic and real-time monitoring of detection, such as biosensors for detecting trypsin content in human blood or urine.

The beneficial effects of the present invention are:

1. The detection method of the present invention adopts bovine serum albumin-copper nano-clusters as a catalyst to catalyze luminol chemiluminescence to improve luminous efficiency. Compared with the traditional method using noble metal nanoclusters as catalysts, the preparation method of the catalyst bovine serum albumin-copper nanoclusters in the present invention is simple, the raw materials are cheap and easy to obtain, the catalytic activity is high, the stability is good, and the cost of detection is reduced.

2. The detection method of the present invention has strong anti-impurity interference ability, for 10μg/L glucose, glucose oxidase, lysozyme, urea and 5.0×10-4mol/L Na ⁺ , K ⁺ , Ca ²⁺ , Mg ^{2 +} and other interfering substances have good anti-interference effect, thus effectively ensuring the accuracy of detection.

3. With the detection method of the present invention, the detection limit of trypsin can be as low as 0.12 μg/mL, which is far lower than the trypsin content in the patient's urine and blood, and is suitable for detecting the trypsin content in the patient's urine and blood .

4. The detection method of the present invention has no pollution in the detection process, high speed, convenient operation, good reproducibility, and real-time monitoring can be realized.

Description of drawings

Fig. 1 is the kinetic curve of the luminescence intensity of luminol-hydrogen peroxide mixed solution in the presence of (b)/no (a) bovine serum albumin-copper nanocluster;

Fig. 2 is the inhibitory effect diagram of different concentrations of trypsin on luminol-hydrogen peroxide-copper nanocluster chemiluminescent system;

Fig. 3 is the standard curve figure of the trypsin that embodiment 1 establishes;

Fig. 4 is a result diagram of the selectivity experiment of the detection method of the present invention to trypsin.

Wherein: in Fig. 3, C is the trypsin concentration, I ₀ is the luminous intensity value without adding trypsin, and I is the luminous intensity value after adding trypsin;

In Fig. 4, I ₀ is the chemiluminescence intensity of the luminol-hydrogen peroxide-copper nanocluster system, and I is the chemiluminescence intensity of the luminol-hydrogen peroxide-copper nanocluster system in the presence of various interferents.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the examples given are not intended to limit the present invention.

In the following embodiments, sodium hydroxide, copper sulfate pentahydrate, luminol, and hydrogen peroxide are all analytically pure and purchased from Sinopharm Chemical Reagent Co., Ltd.; bovine serum albumin and trypsin are purchased from Sigma-Aldrich.

Example 1

A method for detecting trypsin by catalyzing luminol chemiluminescence with bovine serum albumin-copper nano clusters as a catalyst, comprising the following steps:

(1) Mix bovine serum albumin and copper sulfate pentahydrate solution at a molar ratio of 1:2, stir the mixture at 30°C for 5 minutes, adjust the pH to 10 with sodium hydroxide solution at the end of the stirring, Down stirring 8h, obtain bovine serum albumin-copper nano-cluster material;

(2) Mix the bovine serum albumin-copper nanocluster material prepared in step (1) into ultrapure water for dialysis for 40 hours, change the water every four hours to remove unreacted various impurities, and after the dialysis finishes, the cow The serum albumin-copper nanocluster catalyst is stored at 2-6°C until use;

(3) Take ten 3mL sample tubes and add 500 μL of freshly prepared trypsin solution to the final concentrations of 0.4, 2.0, 10, 20, 30, 46, 60, 160, 400, 520, and 1200 μg/mL, respectively. Add 50 μL of the bovine serum albumin-copper nanocluster catalyst prepared in step (2), mix well, and incubate the mixed solution at a constant temperature, the incubation temperature is 30°C, and the incubation time is 0.5h;

(4) Add 450uL of a mixture of luminol (its final concentration is 1.10×10 ^-4 M) and hydrogen peroxide (its final concentration is 0.45M) to the 10 sample tubes in step (3), and quickly dissolve the Place the cuvette in the chemiluminescent light path, and quickly detect the chemiluminescent intensity of the luminescent agent at 425nm.

Figure 1 shows the kinetic curve of the luminescence intensity of the luminol-hydrogen peroxide mixture with/without the presence of bovine serum albumin-copper nanoclusters. It can be seen from the figure that hydrogen peroxide oxidizes luminol to produce very weak chemiluminescence (a), and with the addition of bovine serum albumin-copper nanoclusters, the luminescence intensity of the mixture is significantly enhanced (b), and the two The maximum emission wavelength of the luminescent system remains consistent, both at 425nm, which shows that under the catalysis of bovine serum albumin-copper nanoclusters, the chemiluminescent body of the luminol-hydrogen peroxide mixed solution is still the oxidation product of luminol, That is, the excited state of 3-aminophthalate anion (3-APA*), bovine serum albumin-copper nanoclusters only played a catalytic role, and did not produce new luminophores.

Fig. 2 shows the luminous intensity change diagram of the luminol-hydrogen peroxide-copper nanocluster chemiluminescence system after adding different concentrations of trypsin. It can be seen from the figure that trypsin has an obvious inhibitory effect on the luminol-hydrogen peroxide-copper nanocluster chemiluminescence system.

Take logC (C is trypsin concentration) as the abscissa, and I ₀ -I (I ₀ is the luminous intensity value without trypsin, I is the luminous intensity value after adding trypsin) as the ordinate, according to logC and I The corresponding relationship of ₀ -I draws a standard curve, and the results are shown in Figure 3. It can be seen that within the concentration range of 0.2 μg/mL to 0.6 mg/mL, the change of luminescence intensity is linearly related to the logarithm of the trypsin concentration, based on which the quantitative detection and analysis of trypsin has been realized.

Example 2

A method for detecting trypsin by catalyzing luminol chemiluminescence with bovine serum albumin-copper nano clusters as a catalyst, comprising the following steps:

(1) Mix bovine serum albumin and copper sulfate pentahydrate solution according to the molar ratio of 3:4, stir the mixture at 35°C for 7.5min, adjust the pH to 11 with sodium hydroxide solution after stirring, and adjust the pH to 11 at 55°C Down stirring 12h, obtain bovine serum albumin-copper nano-cluster material;

(2) Mix the bovine serum albumin-copper nanocluster material prepared in step (1) into ultrapure water and dialyze for 50h, change the water every four hours to remove unreacted various impurities, after the dialysis, the bovine The serum albumin-copper nanocluster catalyst is stored at 2-6°C until use;

(3) Take 10 3mL sample tubes, add 500 μL of freshly prepared trypsin solution, and the final concentrations are 0.4, 2.0, 10, 20, 30, 46, 60, 160, 400, 520, 1200 μg/mL, and then Add an equivalent amount of 50 μL of the bovine serum albumin-copper nanocluster catalyst prepared in step (2), mix well, and incubate the mixed solution at a constant temperature, the incubation temperature is 37.5°C, and the incubation time is 1.25h;

(4) Add 450uL of a mixture of luminol (its final concentration is 1.10×10 ^-4 M) and hydrogen peroxide (its final concentration is 0.45M) to the 10 sample tubes in step (3), and quickly dissolve the Place the cuvette in the chemiluminescent light path, and quickly detect the chemiluminescent intensity of the luminescent agent at 425nm.

Example 3

A method for detecting trypsin by catalyzing luminol chemiluminescence with bovine serum albumin-copper nano clusters as a catalyst, comprising the following steps:

(1) Mix bovine serum albumin and copper sulfate pentahydrate solution according to a certain amount of substances, stir the mixed solution at 40°C for 10 minutes, adjust the pH to 12 with sodium hydroxide solution after stirring, and adjust the pH to 12 at 50- Stirring at 60°C for 16 hours to obtain a bovine serum albumin-copper nanocluster material;

(2) Mix the bovine serum albumin-copper nanocluster material prepared in step (1) into ultrapure water and dialyze for 60h, change the water every four hours to remove unreacted various impurities, after the dialysis ends, the cow The serum albumin-copper nanocluster catalyst is stored at 2-6°C until use;

(3) Take ten 3mL sample tubes and add 500 μL of freshly prepared trypsin solution to the final concentrations of 0.4, 2.0, 10, 20, 30, 46, 60, 160, 400, 520, and 1200 μg/mL, respectively. Add an equal amount of 50 μL of the bovine serum albumin-copper nanocluster catalyst prepared in step (2), mix well, and incubate the mixed solution at a constant temperature, the incubation temperature is 45°C, and the incubation time is 2h.

(4) Add 450uL of a mixture of luminol (its final concentration is 1.10×10 ^-4 M) and hydrogen peroxide (its final concentration is 0.45M) to the 10 sample tubes in step (3), and quickly dissolve the Place the cuvette in the chemiluminescent light path, and quickly detect the chemiluminescent intensity of the luminescent agent at 425nm.

Example 4

The selectivity experiment for trypsin is as follows:

Take freshly prepared 10 μg/L trypsin glucose, glucose oxidase, lysozyme, urea, and 5.0×10 ^-4 mol/L Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ solutions respectively, and add appropriate amount of cattle Serum protein-copper nanocluster catalyst, after mixing and constant temperature incubation, was added to luminol-hydrogen peroxide detection solution for chemiluminescence reaction, and the chemiluminescence intensity was detected. The obtained results are shown in Figure 4.

From Figure 4, it can be concluded that 10μg/L of glucose, glucose oxidase, lysozyme, urea and 5.0×10 ^{- 4} mol/L of Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ all contribute to the chemical stability of the system. The luminescence intensity has no great influence, but the addition of 10 μg/L trypsin can significantly reduce the chemiluminescence intensity of the system, indicating that the detection system has good selectivity for trypsin. When the detection system is used for the detection of trypsin, it has Strong anti-interference ability, thus ensuring the accuracy of detection.

The above-mentioned embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or transformations made by those skilled in the art on the basis of the present invention are all within the protection scope of the present invention. The protection scope of the present invention shall be determined by the claims.

Claims (10)

1. A method for detecting trypsin using bovine serum albumin-copper nanoclusters to catalyze luminol chemiluminescence. 2. the method for detecting trypsin with bovine serum albumin-copper nanocluster catalysis luminol chemiluminescence as claimed in claim 1, is characterized in that, comprises: S1: Take the trypsin solution to be tested, bovine serum albumin-copper nanocluster catalyst, mix and incubate at constant temperature; S2: Add luminol-hydrogen peroxide detection solution to the mixed solution for chemiluminescence reaction, and calculate the concentration of trypsin to be tested according to the chemiluminescence intensity and the established standard curve. 3. the method for detecting trypsin with bovine serum albumin-copper nanocluster catalysis luminol chemiluminescence as claimed in claim 1, it is characterized in that, in step S1, the preparation method of bovine serum albumin-copper nanocluster catalyst is : Mix bovine serum albumin and copper sulfate pentahydrate solution, stir at 30-40°C for 5-10 minutes, then adjust the pH to alkaline with sodium hydroxide solution, then stir at 50-60°C for 8-10 minutes After 16 hours, the bovine serum albumin-copper nanocluster catalyst was obtained. 4. the method for detecting trypsin with bovine serum albumin-copper nanocluster catalysis luminol chemiluminescence as claimed in claim 3, is characterized in that, in step S1, the mol ratio of copper sulfate pentahydrate and bovine serum albumin It is (0.5～1):1. 5 . The method for detecting trypsin with bovine serum albumin-copper nanoclusters catalyzed by luminol chemiluminescence as claimed in claim 3 , wherein in step S1 , the pH is adjusted to 10-12 with sodium hydroxide solution. 6. the method for detecting trypsin with bovine serum albumin-copper nano-cluster catalysis luminol chemiluminescence as claimed in claim 3, it is characterized in that, the bovine serum albumin-copper nano-cluster catalyst obtained needs to be placed in ultrapure water Dialyze to remove unreacted impurities. After dialysis, store at 2-6°C for later use. 7. the method for detecting trypsin with bovine serum albumin-copper nanocluster catalysis luminol chemiluminescence as claimed in claim 1, it is characterized in that, the establishment method of standard curve comprises: Take n groups of equal amounts and different concentrations of newly configured trypsin solutions, add an appropriate amount of bovine serum albumin-copper nanocluster catalysts, mix and incubate at a constant temperature, then add luminol-hydrogen peroxide detection solution to perform chemiluminescence reaction, detecting its chemiluminescent intensity; and According to the concentration of trypsin in each group and the corresponding decrease in luminous intensity, draw a relationship curve between the decrease in luminous intensity and the logarithm of the trypsin concentration, which is the standard curve. 8. the method for detecting trypsin with bovine serum albumin-copper nanocluster catalysis luminol chemiluminescence as claimed in claim 7, it is characterized in that, the concentration range of the trypsin solution of described new configuration is 0.2 μ g/mL～ 0.6mg/mL. 9. the method for detecting trypsin with bovine serum albumin-copper nanocluster catalysis luminol chemiluminescence as claimed in claim 1 or 7, it is characterized in that, the temperature of constant temperature incubation is 30-45 ℃, and the time is 0.5h～ 2h. 10. the method for detecting trypsin with bovine serum albumin-copper nanocluster catalysis luminol chemiluminescence as claimed in claim 1 or 7, it is characterized in that, luminol-hydrogen peroxide detection liquid is luminol and peroxide The aqueous solution of hydrogen oxide has a pH of 9.0, wherein the concentration of luminol is 1.0×10 ^-6 to 5×10 ^-5 M, and the concentration of hydrogen peroxide is 0.02 to 0.2M.