CN102944558B - A kind of tryptic detection method - Google Patents
A kind of tryptic detection method Download PDFInfo
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- CN102944558B CN102944558B CN201210506087.0A CN201210506087A CN102944558B CN 102944558 B CN102944558 B CN 102944558B CN 201210506087 A CN201210506087 A CN 201210506087A CN 102944558 B CN102944558 B CN 102944558B
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Abstract
The invention discloses a kind of trypsase detection method, step is as follows: trypsase and core-shell type nano particle are reacted, obtain reaction product; The light absorption value of detection reaction product center-core-shell type nanometer particle; The light absorption value of metal nanoparticle in detection reaction product; Calculate the ratio of the light absorption value of metal nanoparticle and the light absorption value of core-shell type nano particle, obtain tryptic content according to typical curve; Wherein, described core-shell type nano particle is that protein or polypeptide bag are formed by metal.Present invention also offers the preparation method of described core-shell type nano particle.Trypsase detection method provided by the invention is simple to operate, and without the need to modifying probe or marking.
Description
Technical field
The present invention relates to protein detection field, particularly relate to a kind of tryptic detection method.
Background technology
Proteinase can specificity peptide bond in catalyzing hydrolysis target protein, relates to physiology, pathologic process that regulation and control are important.Trypsase is a kind of hydrolytic enzyme by pancreatic secretion, the c-terminus of lysine in peptide chain and arginine residues can be cut off, trypsase all has application in medicine, food and commercial production, energy dissolving deformation protein, to unmodified protein without effect, fester, sputum, blood clot etc. can be made to decompose, thinning, accelerate surface of a wound purification, also have the effect of anti-inflammatory; Clinically for local edema, hemotoncus and abscess etc. that pyothorax, hemothorax, surgery inflammation, ulcer, traumatic damage, fistula etc. produce; Trypsase, in the activation of other pancreas proenzymes of promotion, controls to have vital role in exocrine pancreatic function; Tryptic rising is Alcoholic Acute Pancreatitis indicator the most accurately; Tryptic shortage or variation will cause the pancreatic diseases such as meconium intestinal obstruction, hereditary pancreatitis.Therefore tryptic detection has important effect in healthy, medical and food quality control.
In prior art, tryptic detection method mainly contains fluorescence method, electrochemical process, colourimetry.Fluorescence method generally needs to select suitable fluorescent marker, and will carry out fluorescence labeling to probe, and electrochemical method relates to complicated electrode modification, complex steps.Metal and protein bound become metal nanometre cluster, when metal nanometre cluster mixes with trypsase, protein in metal nanometre cluster by after trypsin hydrolysis with metal separation, metallics after separation can be reunited, the color of solution can change, tryptic detection is realized by colorimetric, as (Biomaterials such as Lin, 2010,31,6087-6095) trypsin substrate and 1-sulfydryl hexanol are adsorbed on gold nano grain surface, when trypsin hydrolysis substrate, the stable environment of gold nano grain is destroyed, and produces and assembles; Zhang etc. (Analyst, 2011,136,3136-3141) can induce gold nano grain to assemble based on short poly arginine (Arg6), and poly arginine is gold nano grain can not be induced to assemble after little fragment by trypsin hydrolysis.Colorimetric method, without the need to modifying and mark, relatively simply, is easy to operation.
Summary of the invention
The present invention is intended to solve above-mentioned problems of the prior art, proposes a kind of method utilizing colorimetric to carry out trypsase detection, and step is simple, is easy to operation.
The invention provides a kind of trypsase detection method, comprise the following steps:
Trypsase and core-shell type nano particle are reacted, obtains reaction product;
The light absorption value of detection reaction product center-core-shell type nanometer particle;
The light absorption value of metal nanoparticle in detection reaction product;
Calculate the ratio of the light absorption value of metal nanoparticle and the light absorption value of core-shell type nano particle, obtain tryptic content according to typical curve;
Wherein, described core-shell type nano particle is that protein or polypeptide bag are formed by metal nanoparticle.
Preferably, described core-shell type nano particle is selected from poly-D-lysine-gold nano grain, poly-D-lysine-silver nano-grain, poly arginine-gold nano grain or poly arginine-silver nano-grain.
Preferably, described temperature of reaction is 30-40 DEG C.
Preferably, the described reaction time is 1-2h.
Preferably, described temperature of reaction is 37 DEG C, and the reaction time is 2h.
Preferably, in described core-shell type nano particle, the amount of substance of metal and the mass ratio of protein are (5-50) μm ol: (5-50) mg.
Preferably, in described core-shell type nano particle, the amount of substance of metal and the mass ratio of protein are 1 μm ol: 1mg.
Preferably, described core-shell type nano particle is prepared in accordance with the following methods:
By metallic ion and protein or polypeptide mixing, obtain mixed solution;
0.5-3h is hatched at mixed solution being positioned over 50-70 DEG C.
Preferably, described mixed solution hatches 3h at being positioned over 65 DEG C.
Trypsase detection method of the present invention utilizes protein or polypeptide bag to be formed core-shell type nano particle by metal, both as the substrate of trypsin acting, again as the indicator of color change, when not adding trypsase, solution display be the color of core-shell type nano particle, after mixing with trypsase, the protein of core-shell type nano grain surface is hydrolyzed, core-shell type nanoparticle structure is destroyed, metallic core exposes and assembles, form metal nanoparticle, the color of solution changes, the lighter of core-shell type nano particle, the color of metal nanoparticle is manifested, the ratio of these two kinds of color light absorption values is utilized to characterize the reduction of core-shell type nano particle, the i.e. amount that is hydrolyzed of protein, namely tryptic content is also characterized.
Beneficial effect of the present invention is, trypsase detection method provided by the invention is simple to operate, and without the need to modifying probe or marking.
Accompanying drawing explanation
Fig. 1 is the uv-visible absorption spectra of the core-shell type nano particle of preparation in embodiment 1.
Fig. 2 is the grain size distribution of the core-shell type nano particle of preparation in embodiment 1.
Fig. 3 is the canonical plotting of trypsinase concentration in embodiment 1 and absorbance ratio.
Fig. 4 is the canonical plotting of trypsinase concentration in embodiment 2 and absorbance ratio.
Fig. 5 is the canonical plotting of trypsinase concentration in embodiment 3 and absorbance ratio.
Fig. 6 is the specificity comparison diagram that in embodiment 5, trypsase detects.
Embodiment
In order to the technical scheme making those skilled in the art better understand the application, below in conjunction with the accompanying drawing in the embodiment of the present application, clear, complete description is carried out to the technical scheme in the embodiment of the present application.
The invention provides a kind of tryptic detection method, comprise the following steps:
Trypsase and core-shell type nano particle are reacted, obtains reaction product;
The light absorption value of detection reaction product center-core-shell type nanometer particle;
The light absorption value of metal nanoparticle in detection reaction product;
Calculate the ratio of the light absorption value of metal nanoparticle and the light absorption value of core-shell type nano particle, obtain tryptic content according to typical curve;
Wherein, described core-shell type nano particle is that protein or polypeptide bag are formed by metal.
Trypsase is the one of proteolytic enzyme, can selective hydrolysis protein, only acts on the c-terminus of arginine and lysine, all has a wide range of applications in medicine, food, commercial production.The indicator that the present invention changes using core-shell type nano particle as color, after utilizing the protein in trypsin hydrolysis core-shell type nano particle, metal occurs to assemble and forms metal nanoparticle, produces color change, utilizes colourimetry to detect trypsase.
Core-shell type nano particle described in detection method of the present invention is preferably prepared in accordance with the following methods:
By metallic ion and protein mixing, obtain mixed solution;
0.5-3h is hatched at mixed solution being positioned over 50-70 DEG C.
Preferably, described mixed solution hatches 3h at being positioned over 65 DEG C.
Core-shell type nano particle be a kind of by metal as core; the core-shell type nano particle that protein or peptide molecule assemble as blocking group; for water-soluble, can emission spectrum within the scope of visible ray to near-infrared region, also there is the advantages such as good biocompatibility, light stability be strong.
In the present invention, metal is preferably gold ion or silver ion, is more preferably gold ion, most preferably is the gold ion in gold chloride; Described protein or polypeptide are preferably poly arginine or poly-D-lysine, are more preferably poly-D-lysine; The mass ratio of the amount of substance of described metal and protein or polypeptide is preferably (5-50) μm ol: (5-50) mg, is more preferably (25-50) μm ol: (25-50) mg, most preferably is 1 μm ol: 1mg.
After obtaining mixed solution, at mixed solution being placed in 50-70 DEG C, hatch 0.5-3h, at being more preferably 65-70 DEG C, hatch 0.5-3h, at most preferably being 65 DEG C, hatch 3h, after reaction, obtain core-shell type nano particle of the present invention.
After obtaining core-shell type nano particle, trypsase is mixed with core-shell type nano particle, core-shell type nano particle is preferably poly-D-lysine-gold nano grain, poly-D-lysine-silver nano-grain, poly arginine-gold nano grain or poly arginine-silver nano-grain, be more preferably poly-D-lysine-gold nano grain, poly-D-lysine-silver nano-grain, most preferably be poly-D-lysine-gold nano grain; Trypsase and core-shell type nano particle hybrid reaction temperature adopt trypsase optimum temperature well known to those skilled in the art, are preferably 30-40 DEG C, are more preferably 37 DEG C; Trypsase and core-shell type nano particle hybrid reaction time are preferably 1-2h, are more preferably 2h.
After completion of the reaction, obtain reaction product, detect the light absorption value of core-shell type nano particle and metal nanoparticle, described core-shell type nano particle measures wavelength and is preferably wavelength corresponding when light absorption value in core-shell type nano particle uv-visible absorption spectroscopy obtains maximal value, and metal nanoparticle measures wavelength and is preferably wavelength corresponding when light absorption value in metal nanoparticle uv-visible absorption spectroscopy obtains maximal value.
Obtain the light absorption value of core-shell type nano particle and metal nanoparticle according to technique scheme after, calculate the ratio of described metal nanoparticle and core-shell type nano particle light absorption value, contrast according to described ratio and typical curve, obtain tryptic content.
Wherein, in described typical curve, in the detection of metal nanoparticle and core-shell type nano particle light absorption value and described technical scheme, the detection method of metal nanoparticle and core-shell type nano particle light absorption value is identical, according to certain concentration gradient configuration trypsin solution, be trypsase standard solution, by the trypsase standard solution of concentration known and core-shell type nano particle hybrid reaction, calculate the metal nanoparticle of the trypsase standard solution reaction product of each concentration and the ratio of core-shell type nano particle light absorption value, mapping according to metal nanoparticle and the ratio of core-shell type nano particle light absorption value and the logarithm of trypsinase concentration, (logarithm of trypsinase concentration is horizontal ordinate, the ratio of metal nanoparticle and core-shell type nano particle light absorption value is ordinate), carry out linear regression analysis, obtain the linear equation of trypsinase concentration and described ratio.The configuration of described trypsase standard solution adopts the allocation plan of trypsase standard solution well known in the art.Described trypsase concentration of standard solution is preferably (10
-9-10
-3) g/mL.
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment
reagent configures
Poly-L-Lysine Solution: (molecular weight: 30000 ~ 70000) 5mg is dissolved in 1mL ultrapure water, packing is frozen, for subsequent use to take poly-D-lysine.Poly-L-Lysine Solution concentration is 5mg/mL.
Poly arginine solution: (molecular weight: 30000 ~ 70000) 5mg is dissolved in 1mL ultrapure water, packing is frozen, for subsequent use to take poly arginine.Poly arginine solution concentration 5mg/mL.
Chlorauric acid solution: 1g tri-hydration gold chloride is dissolved in 50mL ultrapure water, is contained in brown reagent bottle, 4 DEG C of preservations, for subsequent use.Chlorauric acid solution concentration is 0.05mol/L.
Trypsin solution: take trypsase 1mg, is dissolved in 1mL PBS (10mM, pH=7.4), packing, frozen, for subsequent use.Trypsin solution concentration is 1mg/mL.
Bovine serum albumin(BSA) BSA solution: take BSA 1mg, is dissolved in 1mL PBS (10mmol/L, pH=7.4), 4 DEG C of preservations, for subsequent use.The concentration of BSA solution is 1mg/mL.
embodiment 1
Get 200 μ L, 5mg/mL Poly-L-Lysine Solution is dissolved in 780 μ L ultrapure waters, gets 20 μ L, 0.05mol/L chlorauric acid solution and Poly-L-Lysine Solution miscible are closed, and the concussion several seconds, at 65 DEG C, cultivate 3h, namely obtain the core-shell type nano particle of poly-D-lysine bag quilt.
Fig. 1 is the uv-visible absorption spectra of core-shell type nano particle, and the maximum absorption wavelength of core-shell type nano particle is 530nm; Figure 2 shows that the domain size distribution of core-shell type nano particle, the particle diameter of prepared core-shell type nano particle is about 20.49nm.
By the dilution of 1mg/mL trypsin solution for trypsase final concentration is respectively 250 μ g/mL, 10 μ g/mL, 1 μ g/mL, 0.1 μ g/mL, the standard solution of 0.01 μ g/mL.
Get the obtained core-shell type nanoparticulate dispersed of 50 μ L in 300 μ L PBS solution, and add 50 μ L trypsase standard solution, the final volume of reaction system is 400 μ L, and at 37 DEG C, reaction 2h, namely can be observed the change of solution colour before and after reacting.
Experiment is learnt, the maximum absorption wavelength of metal nanoparticle is 600nm.The present embodiment center-core-shell type nanometer particle pinkiness, because poly-D-lysine is hydrolyzed, the protein coat of core-shell type nano particle is destroyed, and metal occurs to reunite and forms metal nanoparticle, and after reaction, solution is purple.
Reacted solution is detected light absorption value respectively under 600nm and 530nm, and the ratio both calculating.With the logarithm of trypsase mass concentration for horizontal ordinate, with the ratio of reacted solution light absorption value under 600nm and 530nm for ordinate drawing standard curve, result as shown in Figure 3, positive correlation between the logarithm of trypsase mass concentration and the ratio of reacted solution light absorption value under 600nm and 530nm in the present embodiment, linear regression curves equation is Y=0.1207lgC+1.5478, R
2be 0.9737, wherein C is tryptic mass concentration, and Y is the ratio of reacted solution light absorption value under 600nm and 530nm, and the range of linearity is 10
-4-10
-8g/mL.Trypsase detection method of the present invention has higher sensitivity, and detection line can reach 10
-8g/mL.
embodiment 2
Get 40 μ L, 5mg/mL Poly-L-Lysine Solution is dissolved in 920 μ L ultrapure waters, gets 40 μ L, 0.05mol/L chlorauric acid solution and Poly-L-Lysine Solution miscible are closed, and the concussion several seconds, at 50 DEG C, cultivate 0.5h, namely obtain the core-shell type nano particle of poly-D-lysine bag quilt.
By the dilution of 1mg/mL trypsin solution for trypsase final concentration is respectively 250 μ g/mL, 10 μ g/mL, 1 μ g/mL, 0.1 μ g/mL, the standard solution of 0.01 μ g/mL.
Get the obtained core-shell type nanoparticulate dispersed of 50 μ L in 300 μ L PBS solution, and add 50 μ L trypsase standard solution, the final volume of reaction system is 400 μ L, and at 30 DEG C, reaction 2h, namely can be observed the change of solution colour before and after reacting.
Reacted solution is detected light absorption value respectively under 600nm and 530nm, and the ratio both calculating.With the logarithm of trypsase mass concentration for horizontal ordinate, with the ratio of reacted solution light absorption value under 600nm and 530nm for ordinate drawing standard curve, result as shown in Figure 4, positive correlation between the logarithm of trypsase mass concentration and the ratio of reacted solution light absorption value under 600nm and 530nm in the present embodiment, linear regression curves equation is Y=0.1258lgC+1.5517, R
2be 0.8506, wherein X is tryptic mass concentration, and Y is the ratio of reacted solution light absorption value under 600nm and 530nm.
embodiment 3
Get 400 μ L, 5mg/mL Poly-L-Lysine Solution is dissolved in 596 μ L ultrapure waters, gets 4 μ L, 0.05mol/L chlorauric acid solution and Poly-L-Lysine Solution miscible are closed, and the concussion several seconds, at 70 DEG C, cultivate 3h, namely obtain the core-shell type nano particle of poly-D-lysine bag quilt.
By the dilution of 1mg/mL trypsin solution for trypsase final concentration is respectively 250 μ g/mL, 10 μ g/mL, 1 μ g/mL, 0.1 μ g/mL, the standard solution of 0.01 μ g/mL.
Get the obtained core-shell type nanoparticulate dispersed of 50 μ L in 300 μ L PBS solution, and add 50 μ L trypsase standard solution, the final volume of reaction system is 400 μ L, and at 40 DEG C, reaction 1h, namely can be observed the change of solution colour before and after reacting.
Reacted solution is detected light absorption value respectively under 600nm and 530nm, and the ratio both calculating.With the logarithm of trypsase mass concentration for horizontal ordinate, with the ratio of reacted solution light absorption value under 600nm and 530nm for ordinate drawing standard curve, result as shown in Figure 5, positive correlation between the logarithm of trypsase mass concentration and the ratio of reacted solution light absorption value under 600nm and 530nm in the present embodiment, linear regression curves equation is Y=0.1574lgC+1.7239, R
2be 0.8982, wherein X is tryptic mass concentration, and Y is the ratio of reacted solution light absorption value under 600nm and 530nm.
embodiment 4
Get the core-shell type nanoparticulate dispersed of preparation in 50 μ L embodiments 1 in 300 μ L PBS solution, and add 50 μ L trypsin sample, the final volume of reaction system is 400 μ L, and at 37 DEG C, reaction 2h, namely can be observed the change of solution colour before and after reacting.
Reacted solution is detected light absorption value respectively under 600nm and 530nm, and the ratio both calculating is 1.0647.Be updated to by ratio in the typical curve of Fig. 3, the concentration calculating trypsin sample is 100 μ g/mL.
embodiment 5
Get the core-shell type nanoparticulate dispersed of preparation in 50 μ L embodiments 1 in 250 μ L PBS solution, and adding the trypsin solution of 100 μ L 1mg/mL, the final volume of reaction system is 400 μ L, at 37 DEG C, reaction 2h, namely can be observed the change of solution colour before and after reacting.
Reacted solution is detected light absorption value.
comparative example 1
Get the core-shell type nanoparticulate dispersed of preparation in 50 μ L embodiments 1 in 250 μ L PBS solution, and add 100 μ L BSA solution, the final volume of reaction system is 400 μ L, and at 37 DEG C, reaction 2h, namely can be observed the change of solution colour before and after reacting.
Reacted solution is detected light absorption value.
comparative example 2
Get the core-shell type nanoparticulate dispersed of preparation in 50 μ L embodiments 1 in 250 μ L PBS solution, and add 100 μ L ultrapure waters, the final volume of reaction system is 400 μ L, and at 37 DEG C, reaction 2h, namely can be observed the change of solution colour before and after reacting.
Reacted solution is detected light absorption value.
Fig. 6 is embodiment 5, comparative example 1, the change of the reacted solution of comparative example 2 light absorption value at different wavelengths, comparative example 1 and comparative example 2 maximum at 530nm place light absorption value, metal agglomeration does not occur, the poly-D-lysine of core-shell type nano grain surface is not hydrolyzed; And embodiment 5 is maximum at 600nm place light absorption value, embodiment 5 there occurs metal agglomeration, and lysine is by trypsin hydrolysis, and in comparative example 1, poly-D-lysine can not be hydrolyzed by BSA, illustrates that detection method of the present invention has specificity to trypsase.
Above-described embodiment of the present invention, does not form limiting the scope of the present invention.Any amendment done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within claims of the present invention.
Claims (4)
1. a trypsase detection method, comprises the following steps:
Trypsase and core-shell type nano particle are reacted 1-2h in 30-40 DEG C, obtains reaction product;
The light absorption value of detection reaction product center-core-shell type nanometer particle;
The light absorption value of metal nanoparticle in detection reaction product;
Calculate the ratio of the light absorption value of metal nanoparticle and the light absorption value of core-shell type nano particle, obtain tryptic content according to typical curve;
Wherein, described core-shell type nano particle is that polypeptide bag is formed by metal; and be selected from poly-D-lysine-gold nano grain, poly-D-lysine-silver nano-grain, poly arginine-gold nano grain or poly arginine-silver nano-grain; wherein the amount of substance of metal and the mass ratio of polypeptide are (5-50) μm ol:(5-50) mg
Wherein, described core-shell type nano particle is prepared as follows: by metallic ion and polypeptide mixing, obtain mixed solution, then mixed solution is placed in 50-70 DEG C and hatches 0.5-3h.
2. trypsase detection method according to claim 1, is characterized in that, described temperature of reaction is 37 DEG C, and the reaction time is 2h.
3. trypsase detection method according to claim 1, is characterized in that, in described core-shell type nano particle, the amount of substance of metal and the mass ratio of polypeptide are 1 μm of ol:1mg.
4. trypsase detection method according to claim 1, is characterized in that, described mixed solution hatches 3h at being positioned over 65 DEG C.
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| CN102680442A (en) * | 2012-04-19 | 2012-09-19 | 太原理工大学 | Method for detecting trypsin using unmarked fluorescence |
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| CN102680442A (en) * | 2012-04-19 | 2012-09-19 | 太原理工大学 | Method for detecting trypsin using unmarked fluorescence |
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