CN104383919A - Preparation method of nanocluster mimic enzyme with visible-light activity and use of nanocluster mimic enzyme in colourimetry detection of trypsin - Google Patents
Preparation method of nanocluster mimic enzyme with visible-light activity and use of nanocluster mimic enzyme in colourimetry detection of trypsin Download PDFInfo
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Abstract
The invention provides a preparation method of nanocluster mimic enzyme with visible-light activity and a use of the nanocluster mimic enzyme in colourimetry detection of trypsin. Gold/silver nanoclusters modified by bovine serum albumin and mercaptosuccinic acid as surface modification agents has peroxidase-like catalytic characteristics under visible light irradiation and can catalyze chromogenic substrate oxidation. Compared with the peroxidase, the nanocluster mimic enzyme with visible-light activity is free of a high-concentration oxidizing agent and has high catalytic activity, good stability and eco-friendly catalytic conditions. Trypsin decomposes a protein template on the surface of the nanocluster so that the nanocluster surface state is changed and causes aggregation thereby reducing catalytic activity. The preparation method has a trypsin detection linear range of 9.0*10<-7> to 1.0*10<-3>g/mL and has a detection limit of 0.6 micrograms per milliliter far lower than trypsin content of urine and blood of patients.
Description
Technical field:
The present invention relates to nanosecond science and technology field and bioanalysis detection field, particularly relate to the preparation of novel visible light induced nano bunch analogue enztme and detecting the application in trypsase.
Background technology:
Native enzyme can catalyzed chemical reaction, and contrast chemical catalysis, native enzyme has higher catalytic activity.Thus, native enzyme has a wide range of applications [James B.Chem.Soc.Rev.2009,38,185-196] in every field such as pesticide producing, pharmacy procedure and food industry.But native enzyme is subject to ectocine inactivation and general to acid, alkali, thermally labile, and expensive, these factors all limit their extensive use.Therefore, the research of analogue enztme causes broad interest.
Recent studies have shown that, the fast development of nanometer technology is that the research of analogue enztme provides more wide space.Up to the present, it is found that multiple nano material is as metal and bimetal nano material [Jv Y.; Li B.X.; Cao R.Chem.Commun.2010,46,8017-8019; He W.W.; Wu X.C.; Liu J.B.; Hu X.N.; Zhang K.; Hou S.; ZhouW.Y.; Xie, S.S.Chem.Mater.2010,22,2988-2994], metal oxide nano-material [Gao L.Z.; Zhuang J.; Nie L.; Zhang J.B.; Zhang Y.; Gu N.; Wang T.H.; Feng J.; Yang D.L.; Perrett S.; Yan X.Nat.Nanotechnol.2007,2,577-583; Mu J.S.; Wang Y.; Zhao M.; Zhang L.Chem.Commun.2012,48,2540-2542], carbon nanomaterial [Song Y.J.; Qu K.G.; Zhao C.; Ren J.S.; Qu X.G.Adv.Mater.2010,22,2206-2210] etc. all there is the activity of similar peroxidase, i.e. the oxidation reaction of catalytic characteristics substrate in the presence of hydrogen peroxide.Contrast native enzyme, nano material analogue enztme have many advantages as with low cost, synthesize controlled, high catalytic activity and better stability.But the H of macro-corrosion need be added when using natural peroxide enzyme or nano material Mimetic Peroxidase
2o
2as oxidant to make it have desirable catalytic activity.High concentration H
2o
2use make the mensuration utilizing peroxidase or nano material Mimetic Peroxidase to carry out living things system become comparatively difficulty [Cook C.J.Nat.Biotechnol.1997,15,467-471].
The discovery of metal nano clustered materials, causes everybody and pays close attention to widely.Due to the raising of quantum confinement effect, these extra small metal nanometre clusters are made to have uncommon optics, electrology characteristic [WangG.; Huang T.; Murray R.W.; Menard L.; J.Am.Chem.Soc.2005,127,812-813; RamakrishnaG.; Varnavski O.; Kim J.; Lee D.; Goodson T.J.Am.Chem.Soc.2008,130,5032-5033; Zhu M.; Aikens C.M.; Hollander F.J.; SchatzG.C.; Jin R.J.Am.Chem.Soc.2008,130,5883-5885].Protease participates in multiple important physiology and the control flow of pathology and the activity of protease and disease association join, and such as trypsase, controlling to play key player in exocrine pancreatic function, is pancreatitic biomarker [Byrne M.F.; Mitchell R.M.; Stiffler H.; Jowell P.S.; Branch M.S.; Pappas T.N.; Tyler D.; Baillie J.Can.J.Gastroenterol.2002,16,849-854].The invention provides and a kind ofly there is the preparation method of visible ray photoactive nano-cluster analogue enztme and be applied to tryptic colorimetric determination.Gold/silver nanoclusters, without any need for strong oxidizer, presents higher quasi-enzyme catalytic active under the induction of visible ray.As far as we know, this is the new spectrochemical property of of the gold/silver nanoclusters of Late Cambrian.The nano-cluster preparation with quasi-enzyme catalytic activity found is simple, and catalytic activity is high, good stability, and catalytic condition is environment-friendly and green more.Decomposed the protein template on nano-cluster surface by trypsase, cause nano-cluster surface state to change and cause gathering thus the reduction causing catalytic activity.Based on its efficient quasi-enzyme catalytic performance, achieve tryptic efficient, convenient detection.Detect tryptic detection and be limited to 0.6 μ g/mL, far below the trypsin amount in the urine of patient and blood.
Summary of the invention:
The object of this invention is to provide a kind of novel non-photoactive nanoparticles bunch analogue enztme, utilize its photolytic activity analogue enztme character simultaneously, trypsase can be detected easily and quickly.
Object of the present invention realizes by following technical measures:
After a, a certain amount of bovine serum albumin(BSA), dimercaptosuccinic acid mix with chlorine gold (III) acid or silver nitrate (I) solution, add appropriate reducing agent and use NaOH solution to regulate pH to alkalescence; Stir 24h under 37 DEG C of conditions after, obtain gold/silver nanoclusters material;
B, by the gold of gained/silver nanoclusters material dialysis 48h to remove reaction impurities; Get the gold/silver nanoclusters material of 100 μ L, add the trypsase of variable concentrations, place 2h under 37 DEG C of conditions after, then add the hac buffer that feature substrate and 2mL pH are the 0.2mol/L of 4.0, constant volume is to 5mL.At room temperature, develop the color with after radiation of visible light 10min under being placed on xenon lamp.
Object of the present invention also realizes by following technical measures:
The summation of described bovine serum albumin(BSA), the amount of substance of dimercaptosuccinic acid is the 1/30-1/10 of the amount of substance of chlorine gold (III) acid or silver nitrate (I), and bovine serum albumin(BSA) is 1: 1-1: 10 with the ratio of the amount of substance of dimercaptosuccinic acid; Described reducing agent, is selected from hydrogen peroxide, ascorbic acid, gallic acid, formaldehyde, glucose, and the amount of reducing agent is 0.1-3 times of the amount of substance of chlorine gold (III) acid or silver nitrate (I); Described feature substrate has DOPAC, TMB, 2, two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts of 2 '-Lian nitrogen base, and 3,3 '-diaminobenzidine, feature concentration of substrate is 0.1mM-10mM.
Accompanying drawing illustrates:
Fig. 1 be do not exist nanocluster material under illumination condition (a), to there is under nanocluster material non-illuminated conditions (b) and there is nanocluster material (c) and 3 × 10 under illumination condition
-4the abosrption spectrogram of the TMB reaction of mol/L.
Fig. 2 is that different reactive intermediate scavenger is on the impact (substrate is TMB) of the analogue enztme performance of gold nanoclusters.
Fig. 3 be gold nanoclusters photolytic activity analogue enztme system use TMB do substrate detect tryptic selective.
Fig. 4 is that gold nanoclusters photolytic activity analogue enztme system uses TMB to detect tryptic linear relationship chart as substrate.
Embodiment 1:
The bovine serum albumin(BSA) of a, 5mL 50mg/mL, 1mL 1.25 × 10
-3the dimercaptosuccinic acid of mol/L adds 1mL 5.0 × 10 after mixing with chlorine gold (III) acid of 5ml 0.01mol/L
-2the hydrogenperoxide steam generator of mol/L also uses NaOH to regulate pH to pH=9; Stir 24h under 37 DEG C of conditions after, obtain gold nanoclusters material;
B, by the gold nanoclusters material of gained dialysis 48h (changing a water every four hours) to remove reaction impurities; Get the gold nanoclusters material of 100 μ L, add the trypsase of variable concentrations, place 2h under 37 DEG C of conditions after, then add 0.3mL 5.0 × 10
-3the acetate buffer solution of the feature substrate TMB of mol/L and the 0.2mol/L of 2mL pH=4.0, constant volume, to 5mL, is placed and is developed the color after illumination 10min under visible light.At the characteristic absorption (λ of the oxidation product of TMB
max=652nm) place measures the absorbance of system.
Embodiment 2:
The 50mg/mL bovine serum albumin(BSA) of a, 5mL, 1mL 1.75 × 10
-3the dimercaptosuccinic acid of mol/L dropwise adds 500 μ L 1.0 × 10 after mixing with chlorine gold (III) acid of 5ml 0.01mol/L
-3the ascorbic acid of mol/L, and use NaOH to regulate pH to 11; Stir 12h under 37 DEG C of conditions after, obtain gold nanoclusters material;
B, by the gold nanoclusters material of gained dialysis 48h (changing a water every four hours) to remove reaction impurities; Get the gold nanoclusters material of 100 μ L, add the trypsase of variable concentrations, place 2h under 37 DEG C of conditions after, then add 0.5mL 5.0 × 10
-3the acetate buffer solution of the 0.2mol/L of two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts of feature substrate 2, the 2 '-Lian nitrogen base of mol/L and 2mL pH=4.0, constant volume, to 5mL, is placed and is developed the color after illumination 10min under visible light.At the characteristic absorption (λ of the oxidation product of two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts of 2,2 '-Lian nitrogen bases
max=417nm) place measures the absorbance of system.
Embodiment 3:
The bovine serum albumin(BSA) of a, 5mL 50mg/mL, 1mL 4.25 × 10
-3after the dimercaptosuccinic acid of mol/L mixes with the silver nitrate of 5ml 0.01mol/L, dropwise add 1mL 5.0 × 10
-2the formaldehyde of mol/L, and use NaOH to regulate pH to 11; Stir 12h under 37 DEG C of conditions after, obtain silver nanoclusters material;
B, by the silver nanoclusters material of gained dialysis 48h (changing a water every four hours) to remove reaction impurities; Get the silver nanoclusters material of 100 μ L, add the trypsase of variable concentrations, place 2h under 37 DEG C of conditions after, add 0.5mL 5.0 × 10
-3the acetate buffer solution of the feature substrate TMB of mol/L and the 0.2mol/L of 2mL pH=4.0, constant volume, to 5mL, is placed and is developed the color after illumination 10min under visible light.At the characteristic absorption (λ of the oxidation product of TMB
max=652nm) place measures the absorbance of system.
Claims (4)
1. there is preparation and the application of colorimetric determination trypsase thereof of the photoactive nano-cluster analogue enztme of visible ray, it is characterized in that:
After a, a certain amount of bovine serum albumin(BSA), dimercaptosuccinic acid mix with chlorine gold (III) acid or silver nitrate (I) solution, add appropriate reducing agent and use NaOH solution to regulate pH to alkalescence; Under 37 DEG C of conditions after stirring reaction 24h, obtain gold/silver nanoclusters material;
B, by the gold of gained/silver nanoclusters material dialysis 48h to remove reaction impurities; Get the gold/silver nanoclusters material of 100 μ L, add the trypsase of variable concentrations, place 2h under 37 DEG C of conditions after, then add the hac buffer that feature substrate and 2mL pH are the 0.2mol/L of 4.0, constant volume is to 5mL.Under room temperature, develop the color with after radiation of visible light 10min under being placed on xenon lamp.
2. preparation and the application of colorimetric determination trypsase thereof with the photoactive nano-cluster analogue enztme of visible ray according to claim 1, it is characterized in that the summation of the amount of substance of described bovine serum albumin(BSA), dimercaptosuccinic acid is the 1/30-1/10 of the amount of substance of chlorine gold (III) acid or silver nitrate (I), bovine serum albumin(BSA) is 1: 1-1: 10 with the ratio of the amount of substance of dimercaptosuccinic acid.
3. preparation and the application of colorimetric determination trypsase thereof with the photoactive nano-cluster analogue enztme of visible ray according to claim 1, it is characterized in that described reducing agent, be selected from hydrogen peroxide, ascorbic acid, gallic acid, formaldehyde, glucose, the amount of reducing agent is 0.1-3 times of the amount of substance of chlorine gold (III) acid or silver nitrate (I).
4. preparation and the application of colorimetric determination trypsase thereof with the photoactive nano-cluster analogue enztme of visible ray according to claim 1, it is characterized in that described feature substrate has DOPAC, 3,3 ', 5,5 '-tetramethyl benzidine, 2, two (3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) di-ammonium salts of 2 '-Lian nitrogen base, 3,3 '-diaminobenzidine, feature concentration of substrate is 0.1mM-10mM.
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