CN104383919B - Trypsin colorimetric detection method based on nanocluster mimic enzyme with visible-light activity - Google Patents

Abstract

The invention provides the preparation of the nano-cluster mimic enzyme with visible light activity and the application of the colorimetric method to detect trypsin. Gold/silver nanoclusters with bovine serum albumin and mercaptosuccinic acid as surface modifiers have catalytic properties similar to peroxidase under visible light irradiation, and can catalyze the oxidation of chromogen substrates. Compared with peroxidase, this photoactive nanomaterial mimic enzyme does not need to use a high concentration of oxidant, has high catalytic activity, good stability, and the catalytic conditions are more environmentally friendly and green. Trypsin decomposes the protein template on the surface of the nanoclusters, resulting in changes in the surface state of the nanoclusters leading to aggregation and a decrease in its catalytic activity. The linear range of the detection of trypsin by the present invention is 9.0× ^10-7-1.0 × ^10-3 g/mL, and the detection limit is 0.6 μg/mL, which is far lower than the trypsin content in the patient's urine and blood.

Description

Colorimetry of trypsin based on nanocluster-mimicking enzymes with visible-light photoactivity Detection method

Technical field:

The invention relates to the field of nanotechnology and the field of biological analysis and detection, in particular to the preparation of a novel visible light-induced nano-cluster imitation enzyme and its application in the detection of trypsin.

Background technique:

Natural enzymes can catalyze chemical reactions, and compared with chemical catalysis, natural enzymes have higher catalytic activity. Therefore, natural enzymes are widely used in various fields such as pesticide production, pharmaceutical process and food industry [James B. Chem. Soc. Rev. 2009, 38, 185-196]. However, natural enzymes are easily inactivated by external influences and are generally unstable to acids, alkalis, and heat, and are expensive, which limit their wide application. Therefore, the study of mimetic enzymes has aroused widespread interest.

Recent studies have shown that the rapid development of nanotechnology provides a broader space for the study of mimic enzymes. So far, a variety of nanomaterials such as metal and bimetallic nanomaterials have been discovered [Jv Y.; Li BX,; Cao R. Chem. Commun. 2010, 46, 8017-8019; He WW; Wu XC; Liu JB; Hu XN; Zhang K.; HouS.; Zhou WY; Xie, SSChem.Mater.2010, 22, 2988-2994], metal oxide nanomaterials [GaoL.Z.; Zhuang J.; Y.; Gu N.; Wang TH; Feng J.; Yang DL; Perrett S.; Yan X. Nat. Nanotechnol. 2007, 2, 577-583; .Commun.2012,48,2540-2542], carbon nanomaterials [Song YJ; Qu KG; Zhao C.; RenJ.S.; Qu XGAdv.Mater.2010, 22, 2206-2210], etc. The activity of a biocatalyst, which catalyzes the oxidation of a characteristic substrate in the presence of hydrogen peroxide. Compared with natural enzymes, nanomaterial mimic enzymes have many advantages such as low cost, controllable synthesis, high catalytic activity and better stability. However, when using natural peroxidase or nanomaterial peroxidase mimetic enzyme, a large amount of corrosive H ₂ O ₂ should be added as an oxidant to make it have ideal catalytic activity. The use of high concentrations of H ₂ O ₂ makes it difficult to measure biological systems using peroxidase or nanomaterial peroxide-mimicking enzymes [Cook CJ Nat. Biotechnol. 1997, 15, 467-471].

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 [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.; Goodson T.J.Am.Chem.Soc.2008, 130, 5032-5033; R. J. Am. Chem. Soc. 2008, 130, 5883-5885]. Proteases are involved in a variety of important physiological and pathological control processes and the activity of proteases is associated with diseases. For example, trypsin plays an important role in controlling exocrine function of the pancreas and is a biomarker of pancreatitis [Byrne M.F.; Mitchell R.M.; Stiffler H.; Jowell P.S.; Branch M.S.; Pappas T.N.; Tyler D.; The invention provides a preparation method of nano-cluster mimic enzyme with visible light activity and its application in colorimetric detection of trypsin. Au/Ag nanoclusters do not require any strong oxidant, and exhibit high enzyme-like catalytic activity under the induction of visible light. To the best of our knowledge, this is the first discovery of a new photochemical property of gold/silver nanoclusters. The discovered nanoclusters with enzyme-like catalytic activity are simple to prepare, have high catalytic activity, good stability, and the catalytic conditions are more environmentally friendly and green. The protein template on the surface of the nanoclusters was decomposed by trypsin, resulting in changes in the surface state of the nanoclusters leading to aggregation and a decrease in catalytic activity. Based on its high-efficiency enzyme-like catalytic performance, the efficient and convenient detection of trypsin is realized. The detection limit of trypsin is 0.6μg/mL, which is much lower than the trypsin content in the patient's urine and blood.

Invention content:

The purpose of the present invention is to provide a novel photoactive nano-cluster mimic enzyme, and simultaneously use its photoactive mimic enzyme property to detect trypsin conveniently and rapidly.

The purpose of the present invention can be achieved through the following technical measures:

a. After mixing a certain amount of bovine serum albumin, mercaptosuccinic acid and chloroauric (III) acid or silver (I) nitrate solution, add an appropriate amount of reducing agent and use NaOH solution to adjust the pH to alkaline; at 37 ° C After stirring for 24h, the gold/silver nano-cluster material is obtained;

b. Dialyze the obtained gold/silver nanocluster material for 48 hours to remove reaction impurities; take 100 μL of gold/silver nanocluster material, add different concentrations of trypsin, place it at 37°C for 2 hours, then add the characteristic substrate and 2 mL of 0.2 mol/L acetic acid buffer solution with a pH of 4.0, dilute to 5 mL, place under a xenon lamp at room temperature and irradiate with visible light for 10 min to develop color.

The purpose of the present invention can also be achieved through the following technical measures:

The sum of the amount of substances of described bovine serum albumin and mercaptosuccinic acid is 1/30-1/10 of the amount of substances of chloroauric (III) acid or silver nitrate (I), bovine serum albumin and sulfhydryl The ratio of the amount of substances of succinic acid is 1:1-1:10; the reducing agent is selected from hydrogen peroxide, ascorbic acid, gallic acid, formaldehyde, glucose, and the amount of the reducing agent is chloroauric (III) acid Or 0.1-3 times the amount of substance of silver nitrate (I); the characteristic substrate has 3,4-dihydroxyphenylacetic acid, 3,3',5,5'-tetramethylbenzidine, 2, 2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 3,3'-diaminobenzidine, the characteristic substrate concentration is 0.1mM-10mM.

Description of drawings:

Figure 1 shows the 3×10 ^-4 mol/L 3 without nanocluster material under light condition (a), the presence of nanocluster material without light condition (b) and the presence of nanocluster material under light condition (c) , 3',5,5'-Tetramethylbenzidine reaction absorption spectrum.

Figure 2 is the effect of different active intermediate scavengers on the simulated enzyme performance of gold nanoclusters (the substrate is 3,3',5,5'-tetramethylbenzidine).

Figure 3 shows the selectivity of the gold nanocluster photoactive mimic enzyme system using 3,3',5,5'-tetramethylbenzidine as a substrate to detect trypsin.

Figure 4 is a linear relationship diagram for the detection of trypsin by the photoactive analog enzyme system of gold nanoclusters using 3,3',5,5'-tetramethylbenzidine as a substrate.

Implementation example 1:

a. After mixing 5mL of 50mg/mL bovine serum albumin, 1mL of 1.25× ^10-3 mol/L mercaptosuccinic acid and 5ml of 0.01mol/L chloroauric acid (III), add 1mL of 5.0× ^10-2 mol /L of hydrogen peroxide solution and use NaOH to adjust the pH to pH=9; after stirring at 37°C for 24 hours, a gold nanocluster material is obtained;

b. Dialyze the obtained gold nanocluster material for 48 hours (change the water every four hours) to remove reaction impurities; take 100 μL of gold nanocluster material, add different concentrations of trypsin, and place it at 37° C. for 2 hours, then Add 0.3mL of 5.0×10 ^-3 mol/L characteristic substrate 3,3',5,5'-tetramethylbenzidine and 2mL of 0.2mol/L acetate buffer solution with pH=4.0, and dilute to 5mL , placed under visible light for 10 min to develop color. The absorbance of the system was measured at the characteristic absorption (λ _max =652nm) of the oxidation product of 3,3',5,5'-tetramethylbenzidine.

Implementation example 2:

a. After mixing 5mL of 50mg/mL bovine serum albumin, 1mL of 1.75×10 ^-3 mol/L mercaptosuccinic acid and 5ml of 0.01mol/L chloroauric acid (III), add 500μL of 1.0×10 ^{- 3} mol/L ascorbic acid, and use NaOH to adjust the pH to 11; after stirring at 37°C for 12 hours, the gold nanocluster material was obtained;

b. Dialyze the obtained gold nanocluster material for 48 hours (change the water every four hours) to remove reaction impurities; take 100 μL of gold nanocluster material, add different concentrations of trypsin, and place it at 37° C. for 2 hours, then Add 0.5 mL of 5.0×10 ^-3 mol/L characteristic substrate 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and 2 mL of 0.2 mol/L acetate buffer solution, dilute to 5mL, and place under visible light for 10min to develop color. The absorbance of the system was measured at the characteristic absorption (λ _max =417nm) of the oxidation product of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt.

Implementation example 3:

a. After mixing 5mL 50mg/mL bovine serum albumin, 1mL 4.25× ^10-3 mol/L mercaptosuccinic acid and 5ml 0.01mol/L silver nitrate, add 1mL 5.0× ^10-2 mol/L dropwise formaldehyde, and use NaOH to adjust the pH to 11; after stirring at 37°C for 12 hours, silver nanocluster materials were obtained;

b. Dialyze the obtained silver nanocluster material for 48 hours (change the water every four hours) to remove reaction impurities; take 100 μL of silver nanocluster material, add different concentrations of trypsin, place it at 37°C for 2 hours, add 0.5mL 5.0×10 ^-3 mol/L characteristic substrate 3,3',5,5'-tetramethylbenzidine and 2mL 0.2mol/L acetate buffer solution with pH=4.0, dilute to 5mL, place The color develops after 10 min of exposure to visible light. The absorbance of the system was measured at the characteristic absorption (λ _max =652nm) of the oxidation product of 3,3',5,5'-tetramethylbenzidine.

Claims (3)

1. The trypsin colorimetric detection method based on the nano-cluster mimic enzyme with visible light photoactivity, is characterized in that: a, bovine serum albumin and mercaptosuccinic acid equivalent to 1-10 times the amount of its substance are made into a mixture, and the mixture is mixed with gold chloride (III) equivalent to 10-30 times the amount of its total substance After mixing the acid or silver (I) nitrate solution, add a reducing agent equivalent to 0.1-3 times the amount of chloroauric (III) acid or silver (I) nitrate and use NaOH solution to adjust the pH to alkaline; at 37 ° C After stirring and reacting for 24 hours under the same conditions, a gold/silver nano-cluster material is obtained; b. Dialyze the obtained gold/silver nanocluster material for 48 hours to remove reaction impurities; take 100 μL of gold/silver nanocluster material, add different concentrations of trypsin, place it at 37°C for 2 hours, then add the characteristic substrate and 2mL of 0.2mol/L acetic acid buffer solution with a pH of 4.0, dilute to 5mL; place under a xenon lamp at room temperature and irradiate with visible light for 10min to develop color. 2. the trypsin colorimetric detection method based on the nanocluster mimic enzyme with visible light photoactivity according to claim 1, is characterized in that described reducing agent is selected from hydrogen peroxide, ascorbic acid, gallic acid, formaldehyde ,glucose. 3. the trypsin colorimetric detection method based on the nanocluster mimetic enzyme with visible light light activity according to claim 1, is characterized in that described characteristic substrate has 3,4-dihydroxyphenylacetic acid, 3, 3',5,5'-tetramethylbenzidine, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, 3,3'-diaminobenzidine Aniline, the characteristic substrate concentration is 0.1mM-10mM.