CN104383919B - Trypsin colorimetric detection method based on nanocluster mimic enzyme with visible-light activity - Google Patents
Trypsin colorimetric detection method based on nanocluster mimic enzyme with visible-light activity Download PDFInfo
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- CN104383919B CN104383919B CN201410525739.4A CN201410525739A CN104383919B CN 104383919 B CN104383919 B CN 104383919B CN 201410525739 A CN201410525739 A CN 201410525739A CN 104383919 B CN104383919 B CN 104383919B
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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, more particularly, to new visible light induced nano
The preparation of cluster analogue enztme and its application in terms of detection trypsase.
Background technology：
Native enzyme can be reacted with catalytic chemistry, contrasts chemical catalysis, and native enzyme has higher catalysis activity.Thus, sky
So enzyme has a wide range of applications [James in every field such as pesticide producing, pharmacy procedure and food industry
B.Chem.Soc.Rev.2009,38,185-196].But, native enzyme is inactivated and typically to acid, alkali, heat by ectocine easily
Unstable, and expensive, and these factors all limit their extensive application.Therefore, the research of analogue enztme causes extensively
General interest.
Recent studies have shown that, the fast development of nanometer technology provides more wide space for the research of analogue enztme.Arrive
So far it has been found that multiple nano material such 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.；Zhou W.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, that is, in peroxide
The oxidation reaction of catalytic characteristics substrate in the presence of change hydrogen.Contrast native enzyme, nano material analogue enztme has many advantages as become
This is cheap, synthesis is controlled, high catalysis activity and more preferable stability.But using natural peroxide enzyme or nanometer material
Corrosive H in a large number need to be added during material Mimetic Peroxidase2O2As oxidant to make it have preferable catalysis activity.High
Concentration H2O2Using make using peroxidase or nano material Mimetic Peroxidase carry out living things system mensure become
Obtain more difficult [Cook C.J.Nat.Biotechnol.1997,15,467-471].
The discovery of metal nano clustered materials, causes everybody and widely pays close attention to.Due to quantum confinement effect raising so that
These extra small metal nanometre clusters have uncommon optics, electrology characteristic [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；Zhu M.；Aikens C.M.；
Hollander F.J.；Schatz G.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 are associated with disease, and such as trypsase is controlling
Play key player in exocrine pancreatic function, be 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 one kind has the photoactive nanometer of visible ray
The preparation method of cluster analogue enztme is simultaneously applied to tryptic colorimetric determination.Gold/silver nanoclusters do not need any strong oxygen
Agent, presents higher quasi-enzyme catalytic activity under the induction of visible ray.As far as we know, this be find first gold/
One new spectrochemical property of silver nanoclusters.The nano-cluster with quasi-enzyme catalytic activity being found prepares simple, catalysis work
Property high, good stability, the more environmentally-friendly green of catalytic condition.Decompose the protein template on nano-cluster surface by trypsase, lead
Cause nano-cluster surface state to change and cause gathering thus causing the reduction of catalysis activity.Based on its efficient quasi-enzyme catalytic performance,
Achieve tryptic efficient, convenient detection.Detect that tryptic detection is limited to 0.6 μ g/mL, far below the urine of patient
With the trypsin amount in blood.
Content of the invention：
It is an object of the invention to provide a kind of new non-photoactive nanoparticles cluster analogue enztme, utilize its photolytic activity analogue enztme simultaneously
Property, can easily and quickly detect trypsase.
The purpose of the present invention can be achieved by the following technical measures：
After a, a certain amount of bovine serum albumin(BSA), dimercaptosuccinic acid are mixed with chlorine gold (III) acid or silver nitrate (I) solution,
Add appropriate reducing agent and adjust pH to alkalescence using NaOH solution；After stirring 24h under the conditions of 37 DEG C, obtain gold/silver nanoparticle
Clustered materials；
B, 48h that the gold of gained/silver nanoclusters material is dialysed are to remove reaction impurities；Take the gold/silver nanoclusters material of 100 μ L
Material, adds the trypsase of variable concentrations, and after placing 2h under the conditions of 37 DEG C, being subsequently adding feature substrate and 2mL pH is 4.0
0.2mol/L hac buffer, constant volume to 5mL, at room temperature, be placed under xenon lamp use aobvious after radiation of visible light 10min
Color.
The purpose of the present invention also can be achieved by the following technical measures：
Described bovine serum albumin(BSA), the summation of the amount of the material of dimercaptosuccinic acid are chlorine gold (III) acid or silver nitrate (I)
The amount of material 1/30-1/10, the ratio of bovine serum albumin(BSA) and the amount of the material of dimercaptosuccinic acid is 1: 1-1: 10；Described
Reducing agent, selected from hydrogen peroxide, ascorbic acid, gallic acid, formaldehyde, glucose, the amount of reducing agent is chlorine gold (III) acid or nitre
0.1-3 times of the amount of material of acid silver (I)；Described feature substrate has DOPAC, and 3,3 ', 5,5 '-tetramethyl
Benzidine, double (3- ethyl benzo thiazole phenanthroline -6- sulfonic acid) di-ammonium salts of 2,2 '-connection nitrogen base, 3,3 '-diaminobenzidine, feature bottom
Thing concentration is 0.1mM-10mM.
Brief description：
Fig. 1 be do not exist nanocluster material under illumination condition (a), exist under nanocluster material non-illuminated conditions (b) with
And there is nanocluster material (c) and 3 × 10 under illumination condition-4The suction of the TMB reaction of mol/L
Receive spectrogram.
Impact that Fig. 2 is different reactive intermediate scavengers to the simulation enzyme performance of gold nanoclusters (substrate is 3,3 ', 5,
5 '-tetramethyl benzidine).
Fig. 3 is that gold nanoclusters photolytic activity simulation enzyme system makees substrate detection pancreas egg using TMB
The selectivity of white enzyme.
Fig. 4 is that gold nanoclusters photolytic activity simulation enzyme system makees substrate detection pancreas egg using TMB
The linear relationship chart of white enzyme.
Embodiment 1：
The bovine serum albumin(BSA) of a, 5mL 50mg/mL, 1mL 1.25 × 10-3The dimercaptosuccinic acid of mol/L and 5ml
After chlorine gold (III) the acid mixing of 0.01mol/L, add 1mL 5.0 × 10-2The hydrogenperoxide steam generator of mol/L is simultaneously adjusted using NaOH
Section pH to pH=9；After stirring 24h under the conditions of 37 DEG C, obtain gold nanoclusters material；
B, 48h (changing a water every four hours) that the gold nanoclusters material of gained is dialysed are to remove reaction impurities；Take 100
The gold nanoclusters material of μ L, adds the trypsase of variable concentrations, after placing 2h, is subsequently adding 0.3mL under the conditions of 37 DEG C
5.0×10-3The acetic acid of the 0.2mol/L of the feature substrate TMB of mol/L and 2mL pH=4.0 delays
Rush liquid, constant volume to 5mL, place and develop the color after illumination 10min under visible light.Produce in the oxidation of TMB
Characteristic absorption (the λ of thingmax=652nm) place measure system absorbance.
Embodiment 2：
The 50mg/mL bovine serum albumin(BSA) of a, 5mL, 1mL 1.75 × 10-3The dimercaptosuccinic acid of mol/L and 5ml
After chlorine gold (III) the acid mixing of 0.01mol/L, it is added dropwise over 500 μ L 1.0 × 10-3The ascorbic acid of mol/L, and use NaOH
Adjust pH to 11；After stirring 12h under the conditions of 37 DEG C, obtain gold nanoclusters material；
B, 48h (changing a water every four hours) that the gold nanoclusters material of gained is dialysed are to remove reaction impurities；Take 100
The gold nanoclusters material of μ L, adds the trypsase of variable concentrations, after placing 2h, is subsequently adding 0.5mL under the conditions of 37 DEG C
5.0×10-3Double (3- ethyl benzo thiazole phenanthroline -6- sulfonic acid) di-ammonium salts of feature substrate 2,2 '-connection nitrogen base of mol/L and 2mL pH=
The acetate buffer solution of 4.0 0.2mol/L, constant volume to 5mL, place and develop the color after illumination 10min under visible light.In 2,2 '-connection nitrogen
Characteristic absorption (the λ of the oxidation product of double (3- ethyl benzo thiazole phenanthroline -6- sulfonic acid) di-ammonium salts of basemax=417nm) place's mensure system
Absorbance.
Embodiment 3：
The bovine serum albumin(BSA) of a, 5mL 50mg/mL, 1mL 4.25 × 10-3The dimercaptosuccinic acid of mol/L and 5ml
After the silver nitrate mixing of 0.01mol/L, it is added dropwise over 1mL 5.0 × 10-2The formaldehyde of mol/L, and adjust pH to 11 using NaOH；
After stirring 12h under the conditions of 37 DEG C, obtain silver nanoclusters material；
B, 48h (changing a water every four hours) that the silver nanoclusters material of gained is dialysed are to remove reaction impurities；Take 100
The silver nanoclusters material of μ L, adds the trypsase of variable concentrations, after placing 2h under the conditions of 37 DEG C, add 0.5mL 5.0 ×
10-3The acetate buffer solution of the 0.2mol/L of the feature substrate TMB 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.Oxidation product in TMB
Characteristic absorption (λmax=652nm) place measure system absorbance.
Claims (3)
1. the trypsase colorimetric detection method based on having visible ray photoactive nano-cluster analogue enztme, its feature exists
In：
A, bovine serum albumin(BSA) and the amount being equivalent to its material are that mixture made by 1-10 times of dimercaptosuccinic acid, this mixture
Chlorine gold (III) acid of 10-30 times of the amount of the material total with being equivalent to it or after silver nitrate (I) solution mixes, addition is equivalent to
0.1-3 times of the reducing agent of amount of substance of chlorine gold (III) acid or silver nitrate (I) simultaneously adjusts pH to alkalescence using NaOH solution；?
After stirring reaction 24h under the conditions of 37 DEG C, obtain gold/silver nanoclusters material；
B, 48h that the gold of gained/silver nanoclusters material is dialysed are to remove reaction impurities；Take the gold/silver nanoclusters material of 100 μ L,
Add the trypsase of variable concentrations, after placing 2h under the conditions of 37 DEG C, being subsequently adding feature substrate and 2mL pH is 4.0
The hac buffer of 0.2mol/L, constant volume to 5mL；Under room temperature, it is placed under xenon lamp and develops the color after using radiation of visible light 10min.
2. the trypsase colorimetric based on there is visible ray photoactive nano-cluster analogue enztme according to claim 1
Detection method it is characterised in that described reducing agent, selected from hydrogen peroxide, ascorbic acid, gallic acid, formaldehyde, glucose.
3. the trypsase colorimetric based on there is visible ray photoactive nano-cluster analogue enztme according to claim 1
Detection method it is characterised in that described feature substrate has a DOPAC, TMB, 2,
Double (3- ethyl benzo thiazole phenanthroline -6- sulfonic acid) di-ammonium salts of 2 '-connection nitrogen base, 3,3 '-diaminobenzidine, feature concentration of substrate is
0.1mM-10mM.
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| CN102590166A (en) * | 2012-02-10 | 2012-07-18 | 中国科学院长春应用化学研究所 | Test method for trypsin |
| CN103175800A (en) * | 2013-03-11 | 2013-06-26 | 中国科学院苏州生物医学工程技术研究所 | Colorimetric analysis method for quickly measuring trypsin |
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| CN102590166A (en) * | 2012-02-10 | 2012-07-18 | 中国科学院长春应用化学研究所 | Test method for trypsin |
| CN103175800A (en) * | 2013-03-11 | 2013-06-26 | 中国科学院苏州生物医学工程技术研究所 | Colorimetric analysis method for quickly measuring trypsin |
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