CN103196883A - Method for detecting activity of chymotrypsin by adopting gold nanoclusters - Google Patents
Method for detecting activity of chymotrypsin by adopting gold nanoclusters Download PDFInfo
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- CN103196883A CN103196883A CN2013101368726A CN201310136872A CN103196883A CN 103196883 A CN103196883 A CN 103196883A CN 2013101368726 A CN2013101368726 A CN 2013101368726A CN 201310136872 A CN201310136872 A CN 201310136872A CN 103196883 A CN103196883 A CN 103196883A
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Abstract
The invention discloses a method for detecting activity of chymotrypsin by adopting gold nanoclusters. The method disclosed by the invention mainly embodies detection of chymotrypsin content in a sample to be detected by adopting gold nanoclusters and specifically comprises the following steps: (a1) reacting chymotrypsin standard substance and the sample to be detected with the gold nanoclusters and detecting fluorescence intensity of a reaction system before and after reacting to acquire a change value of the fluorescence intensity before and after reacting; and (a2) drawing a standard curve according to the change value of the fluorescence intensity before and after reacting of the reaction system of the chymotrypsin standard substance and substituting the change value of the fluorescence intensity before and after reacting of the reaction system of the sample to be detected to acquire the content of the chymotrypsin in the sample to be detected. The experiments prove that the method for detecting activity of the chymotrypsin by adopting gold nanoclusters disclosed by the invention can be used for detecting the process of hydrolyzing substrate protein via the chymotrypsin in real time without the requirement for carrying out fluorescence labeling on the chymotrypsin action substrate and also can be used for detecting the content of the chymotrypsin in the sample to be detected with high sensitivity.
Description
Technical field
The present invention relates to a kind of method of measuring the chymotrypsin activity, particularly a kind of method of utilizing gold nano cluster to measure the chymotrypsin activity.
Background technology
Proteinase is the general name of the class of enzymes of aminosal, peptide bond, has participated in a series of physiology and pathologic process.Numerous disease comprises that cancer, apoplexy, infection, rheumatic arthritis and inflammation etc. all can have influence on the normal activity of proteinase.Therefore, the detection by proteinase activity has very important significance to the diagnosis of disease.In the method that the proteinase activity that adopts is at present measured, fluorescent method has accurately, quick, simple advantage.The detection of these fluorescent methods mainly is to adopt fluorescence resonance energy transmission (FRET) technology, and the fluorogenic substrate of selecting for use generally is the polypeptide that is marked with fluorescent dye, therefore needs synthetic polypeptide and expensive fluorescence labeling, and it is higher to detect cost.
Gold nano cluster is formed core by several to dozens of gold atoms, the molecular level aggregation that organic supramolecular or protein etc. combine as blocking group.Gold nano cluster has unique optical characteristics as a class novel nano-material, has widely at catalysis, bio-sensing and bio-imaging and uses.Gold nano cluster is obviously different with the character of gold nano grain, Fermi's wavelength of gold nano cluster size and electronics (＜1nm) suitable, be not enough to support its generation as the surface plasma body resonant vibration of gold nano grain owing to its size is too little.Gold nano cluster has some special optical characteristics, as has and rely on the photoluminescent property of particle diameter size etc.At present, the preparation method of gold nano cluster reduces golden presoma by some macromolecule, peptide chain, protein and DNA that have a sulfydryl.Wherein, adopt the synthetic gold nano cluster of protein template to have good biocompatibility and water-soluble.Adopt that the synthetic gold nano cluster of bovine serum albumin(BSA) (BSA) has simply, the advantage of green, " treating different things alike ".
Summary of the invention
The purpose of this invention is to provide a kind of new purposes of utilizing gold nano cluster, be specially gold nano cluster and in measuring the chymotrypsin activity, use.
Also protective money nanocluster application in the chymotrypsin content in measuring testing sample of the present invention.
A further object of the present invention provides a kind of method of utilizing gold nano cluster to measure chymotrypsin content in the testing sample.
The method of utilizing gold nano cluster to measure chymotrypsin content in the testing sample provided by the present invention specifically can comprise the steps:
(a1) chymotrypsin standard items or testing sample are mixed with gold nano cluster, form the experiment reaction system; Arrange simultaneously with described experiment reaction system and compare the blank reaction system that lacks described chymotrypsin standard items or described testing sample; Described experiment reaction system and described blank reaction system are reacted under identical conditions, measure the fluorescence intensity of described experiment reaction system and described blank reaction system after reaction finishes respectively, calculate the fluorescence intensity difference of described experiment reaction system and described blank reaction system;
(a2) the described fluorescence intensity difference drawing standard curve of the described chymotrypsin standard items that obtain according to step (a1), with the described typical curve of described fluorescence intensity difference substitution of described testing sample, thereby obtain the content of chymotrypsin in the described testing sample.
In said method, in the step (a2), the fluorescence intensity difference drawing standard curve of the described described chymotrypsin standard items that obtain according to step (a1), be that described fluorescence intensity difference with described chymotrypsin standard items is ordinate, be horizontal ordinate with the final concentration of described chymotrypsin standard items in containing the described experiment reaction system of described chymotrypsin standard items, the typical curve of drafting.
In said method, the fluorescence intensity difference of described experiment reaction system and described blank reaction system deducts the fluorescence intensity of the described experiment reaction system in reaction back for the fluorescence intensity of the described blank reaction system in reaction back.
In said method, described chymotrypsin standard items can be the chymotrypsin standard solution of series concentration, as concentration be followed successively by 50ng/ml, 250ng/ml, 500ng/ml, 2500ng/ml, 5000ng/ml, 25000ng/ml, 50000ng/ml, 250000ng/ml(are equivalent to 0.05U/mL, 0.25U/mL, 0.5U/mL, 2.5U/mL, 5U/mL, 25U/mL, 50U/mL, 250U/mL) the chymotrypsin standard solution; The solvent of described chymotrypsin standard solution can be the buffer system of pH7～8.5, and in one embodiment of the invention, the solvent of described chymotrypsin standard solution is specially 50mM NH
4HCO
3Aqueous solution.
In said method, the temperature of described reaction can be 35-40 ℃; The pH of described reaction can be 7-8.5; The time of described reaction can be 50min-70min.In one embodiment of the invention, the temperature of described reaction is specially 37 ℃, and the pH of described reaction is specially 7.8, and the time of described reaction is specially 1h.
In one embodiment of the invention, the liquid environment of described reaction can be 50mM NH
4HCO
3Aqueous solution.
In said method, contain chymotrypsin in the described testing sample, and do not contain other proteinase of the described gold nano cluster parcel albumen of degradable (as BSA) simultaneously.In one embodiment of the invention, described testing sample is the solution with the preparation of chymotrypsin standard items, is specially the chymotrypsin standard items are dissolved in 50mM NH
4HCO
3The solution that obtains in the aqueous solution; The concentration of chymotrypsin is that 750ng/ml(is 0.75U/mL in this solution).
In said method, in each described reaction system, the reaction ratio of described chymotrypsin and described gold nano cluster is (0.003U-15U): 24nmol, and the content of wherein said gold nano cluster is with the cubage of its blocking group BSA.
Concrete, in the present invention, the composition of described experiment reaction system that contains described chymotrypsin standard items is specific as follows: final concentration is the described chymotrypsin standard items of (0.01-50) U/mL, and final concentration is the described gold nano cluster of 80 μ M; Described chymotrypsin standard items and described gold nano cluster all are dissolved in 50mM NH
4HCO
3In the aqueous solution (pH7.8), the content of wherein said gold nano cluster is with the cubage of its blocking group BSA.
The composition of described experiment reaction system that contains described testing sample is specific as follows: containing final concentration is the described testing sample of 150ng/mL total protein, and final concentration is the described gold nano cluster of 80 μ M; Described testing sample and described gold nano cluster all are dissolved in 50mM NH
4HCO
3In the aqueous solution (pH7.8), the content of wherein said gold nano cluster is with the cubage of its blocking group BSA.
The composition of described blank system is specific as follows: final concentration is the described gold nano cluster of 80 μ M; Described gold nano cluster all is dissolved in 50mM NH
4HCO
3In the aqueous solution (pH7.8), the content of wherein said gold nano cluster is with the cubage of its blocking group BSA.
In above-mentioned application or method, the blocking group of described gold nano cluster is protein (as BSA).
The also application of protective money nanocluster in monitoring chymotrypsin hydrolysis substrate albumen of the present invention.
Another purpose of the present invention provides a kind of method of utilizing gold nano cluster to detect the reaction rate speed that chymotrypsin in the differential responses system is hydrolyzed to substrate protein.
The method of utilizing gold nano cluster to detect the reaction rate speed that chymotrypsin in the differential responses system is hydrolyzed to substrate protein provided by the present invention specifically can comprise the steps:
(b1) will react with gold nano cluster and the chymotrypsin of substrate protein as blocking group, form different reaction systems, monitor the fluorescence intensity of each reaction system in the course of reaction in real time;
(b2) according to the fluorescence intensity of each reaction system that real-time monitors in the step (b1), determine the reaction rate speed that chymotrypsin is hydrolyzed to substrate protein in the described differential responses system as follows: chymotrypsin is more fast to the reaction rate that described substrate protein is hydrolyzed in the more fast then respective reaction system of fluorescence intensity reduction of described reaction system; Chymotrypsin is more slow to the reaction rate that described substrate protein is hydrolyzed in the more slow then respective reaction system of fluorescence intensity reduction of described reaction system.
In said method, the temperature of described reaction can be 35-40 ℃; The pH of described reaction can be 7-8.5.In one embodiment of the invention, the temperature of described reaction is specially 37 ℃, and the pH of described reaction is specially 7.8.
In one embodiment of the invention, the liquid environment of described reaction can be 50mM NH
4HCO
3Aqueous solution.
In said method, in each described reaction system, the reaction ratio of described chymotrypsin and described gold nano cluster is (0.6U-6U): 24nmol, and the content of wherein said gold nano cluster is with the cubage of its blocking group BSA.
Concrete, in the present invention, the composition of different reaction systems is specific as follows described in the said method: final concentration is 2,10 or the described chymotrypsin standard items of 20U/mL, and final concentration is the described gold nano cluster of 80 μ M; Described chymotrypsin standard items and described gold nano cluster all are dissolved in 50mM NH
4HCO
3In the aqueous solution (pH7.8), the content of wherein said gold nano cluster is with the cubage of its blocking group BSA.
Also purpose of the present invention provides the method whether a kind of reaction that utilizes gold nano cluster monitoring chymotrypsin that substrate protein is hydrolyzed finishes.
The method whether reaction that utilizes gold nano cluster detection chymotrypsin that substrate protein is hydrolyzed provided by the present invention finishes specifically can comprise the steps:
(c1) will react the fluorescence intensity of real-time monitoring reaction system in the course of reaction with gold nano cluster and the chymotrypsin of substrate protein as blocking group;
(c2) according to the fluorescence intensity of the described reaction system that real-time monitors in the step (c1), determine as follows whether described chymotrypsin finishes the reaction that described substrate protein is hydrolyzed: if the fluorescence intensity of described reaction system is along with the carrying out that reacts no longer reduces, then described chymotrypsin finishes the reaction that described substrate protein is hydrolyzed; If the fluorescence intensity of described reaction system is along with the carrying out of reaction reducing always, then described chymotrypsin does not finish as yet to the reaction that described substrate protein is hydrolyzed.
In said method, the temperature of described reaction can be 35-40 ℃; The pH of described reaction can be 7-8.5.In one embodiment of the invention, the temperature of described reaction is specially 37 ℃, and the pH of described reaction is specially 7.8.
In one embodiment of the invention, the liquid environment of described reaction can be 50mM NH
4HCO
3Aqueous solution.
In said method, in each described reaction system, the reaction ratio of described chymotrypsin and described gold nano cluster is (0.6U-6U): 24nmol, and the content of wherein said gold nano cluster is with the cubage of its blocking group BSA.
Concrete, in the present invention, the composition of reaction system described in the said method is specific as follows: final concentration is 2,10 or the described chymotrypsin standard items of 20U/mL, and final concentration is the described gold nano cluster of 80 μ M; Described chymotrypsin standard items and described gold nano cluster all are dissolved in 50mM NH
4HCO
3In the aqueous solution (pH7.8), the content of wherein said gold nano cluster is with the cubage of its blocking group BSA.
In above each application and each method, described protein all can be bovine serum albumin(BSA) (BSA); Described substrate protein all can be bovine serum albumin(BSA) (BSA).
Concrete, the proportioning of gold atom and bovine serum albumin(BSA) is 10mmol:30g in the described gold nano cluster.Each described gold nano cluster comprises 25 gold atoms.
More concrete, in one embodiment of the invention, described gold nano cluster is to prepare according to the method that comprises the steps: with the 10mM HAuCl of equal-volume (as 5mL)
4Aqueous solution and 30mg/mL Bovine Serum Albumin in Aqueous Solution are in 37 ℃ of reaction 5min; Add the 1M NaOH aqueous solution of 1/20 volume (as 0.5mL) again in the reaction system in 37 ℃ of reaction 12h, from reaction product, obtain described gold nano cluster.
In above-mentioned each method, described fluorescence intensity all is at excitation wavelength 365nm, detects wavelength 640nm, entrance slit 10 nanometers, and detection obtains under the condition of exit slit 10 nanometers.
The present invention need not chymotrypsin effect substrate is carried out fluorescence labeling, can detect chymotrypsin in real time to the hydrolytic process of substrate protein, simultaneously can high-sensitive mensuration testing sample in the content of chymotrypsin.
Description of drawings
Fig. 1 measures the process of chymotrypsin activity for gold nano cluster.
Fig. 2 is fluorescent absorption spectrum and the emission spectrum of the gold nano cluster of preparation among the embodiment 1.
Fig. 3 be the gold nano cluster of 37 ℃ of following BSA parcels by the hydrolysis of variable concentrations chymotrypsin after fluorescence intensity change curve in time.
Fig. 4 utilizes the gold nano cluster of BSA parcel to detect the concentration of chymotrypsin and the relation (typical curve) of fluorescence intensity changing value.
Embodiment
Among the present invention, utilize the process of gold nano cluster mensuration chymotrypsin activity as shown in Figure 1, its principle is: in the building-up process of gold nano cluster, the phenolic hydroxyl group of the tyrosine on the bovine serum albumin(BSA) (BSA) and the sulfydryl of halfcystine tool reductibility under alkali condition are with Au
3+Be reduced into Au
0, the parcel of the gold nano cluster of BSA and stable then be the Au-S key realization that forms by cysteine residues and Au, therefore, when under the chymotrypsin effect, BSA is hydrolyzed, its structure is damaged, and then can influence the fluorescence intensity of gold nano cluster.
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Quantitative experiment among the following embodiment all arranges repeated experiments three times, results averaged.
Bovine serum albumin(BSA) (BSA): available from Amresco company, its catalog number is 0332.
The chymotrypsin standard items: available from Amresco company, its catalog number is 0164.The enzyme of these chymotrypsin standard items is lived and is 1000U/mg.Wherein, the enzyme work of chymotrypsin is defined as: (25 ℃ of condition determinations; PH value 7.0) under, transformed the needed enzyme amount of 1 μ mol substrate, and be a unit of activity (being 1U) in one minute.
The preparation of embodiment 1, gold nano cluster and sign
One, the preparation of the nano golden cluster of BSA parcel
Toward 5mL 10mM HAuCl
4Added 5mL 30mg/mL bovine serum albumin(BSA) (BSA) reactant aqueous solution 5 minutes on the following magnetic agitation of 37 ℃ of conditions limit in the aqueous solution, the back adds 0.5mL 1M NaOH aqueous solution, and reaction is 12 hours under 37 ℃, the dark-brown solution that obtains.With redistilled water dialysis 48 hours, remove impurity.The gold nano cluster of the BSA parcel that finally obtains, it is standby to be kept at 4 degree.
Two, the sign of the nano golden cluster of BSA parcel
Excitation spectrum and the emission spectrum of the gold nano cluster that the BSA for preparing with fluorescence spectrophotometer (PerkinElmer Model LS-55) determination step one wraps up.
Measurement result such as Fig. 2.As seen from the figure, the emission peak of the gold nano cluster of the BSA parcel that step 1 prepares is at 640nm, and fluorescent emission is stronger.What this description of step one prepared is the big very weak gold grain of fluorescence of golden cluster rather than size; In addition, emission peak positions is relevant with the gold atom quantity of forming gold nano cluster, emission peak in this position of 640nm illustrates that each gold nano cluster approximately comprises 25 gold atoms (list of references: Jianping Xie, Yuangang Zheng, Jackie Y.Ying.Protein-Directed Synthesis of Highly Fluorescent Gold Nanoclusters.J.AM.CHEM.SOC., 2009,131,888-889.).
Present embodiment will describe the hydrolytic process that the gold nano cluster that how to utilize the BSA parcel that embodiment 1 prepares detects chymotrypsin in detail.
The gold nano cluster of the BSA parcel that 1, embodiment 1 is prepared is dissolved in 50mM NH
4HCO
3In the aqueous solution (pH=7.8), obtain solution I.In the solution I, the concentration of the gold nano cluster of the BSA parcel that embodiment 1 prepares is that 0.1mM(is in BSA content).
2, get 60 μ l series concentration (0.1mg/mL, 0.05mg/mL, 0.01mg/mL; Be the enzyme unit that lives with quality conversion, be followed successively by 100U/mL, 50U/mL, 10U/mL) the chymotrypsin standard items (be dissolved in 50mM NH
4HCO
3Aqueous solution, pH=7.8) join respectively in the previously prepared 240 μ l solution I, mixing adds in the cuvette immediately, with fluorescence spectrophotometer (PerkinElmer Model LS-55) 37 ℃ down real-time monitoring reaction potpourris fluorescence intensity over time, excitation wavelength is 365nm, the detection wavelength is 640nm, entrance slit 10 nanometers, exit slit 10 nanometers.Experiment repeats 3 times, results averaged.
The result shows that on the one hand the gold nano cluster fluorescence intensity of the BSA parcel that embodiment 1 prepares weakens along with the prolongation of hydrolysis time, and the fluorescence signal of the reaction system that the chymotrypsin initial concentration is more high decline simultaneously is more obvious, sees Fig. 3 for details.And in theory in the reaction system initial concentration of chymotrypsin more high, hydrolysis reaction should be more fast.So according to above result, can draw as drawing a conclusion: fluorescence signal descends more obvious (fluorescence intensity reduces more fast), and the chymotrypsin hydrolysis reaction in the respective reaction system is more fast.The result shows on the other hand, the fluorescence intensity of the gold nano cluster of the BSA parcel that embodiment 1 prepares weakens along with the prolongation of hydrolysis time, and along with the passing fluorescence intensity in reaction time tends towards stability substantially, no longer reduce, this illustrates that corresponding time point has not had the peptide section to continue to leave the gold nano cluster surface, and namely hydrolysis reaction finishes.Based on the above results, visible fluorescence intensity has directly been reacted the hydrolysis reaction process of chymotrypsin to substrate protein BSA to a certain extent with the variation in reaction time.
One, the drafting of typical curve
The gold nano cluster of the BSA parcel that 1, embodiment 1 is prepared is dissolved in 50mM NH
4HCO
3In the aqueous solution (pH=7.8), obtain solution I.In the solution I, the concentration of the gold nano cluster of the BSA parcel that embodiment 1 prepares is that 0.1mM(is in BSA content).
2, with 60 μ l variable concentrations (50ng/ml, 250ng/ml, 500ng/ml, 2500ng/ml, 5000ng/ml, 25000ng/ml, 50000ng/ml, 250000ng/ml; Be the enzyme unit that lives with quality conversion, be followed successively by 0.05U/mL, 0.25U/mL, 0.5U/mL, 2.5U/mL, 5U/mL, 25U/mL, 50U/mL, 250U/mL) the chymotrypsin standard items (be dissolved in 50mM NH
4HCO
3Aqueous solution, pH=7.8) join respectively in the previously prepared 240 μ l solution I, 37 ℃ of reactions are after 1 hour, with fluorescence spectrophotometer (PerkinElmer Model LS-55) assaying reaction potpourri fluorescence intensity, measure blank reaction system (the 60 μ l 50mMNH that do not add the chymotrypsin standard items simultaneously
4HCO
3Aqueous solution (pH=7.8) joins previously prepared 240 μ l solution I) 37 ℃ of fluorescence intensities after hatching 1 hour, excitation wavelength is 365nm, the detection wavelength is 640nm, entrance slit 10 nanometers, exit slit 10 nanometers.Experiment repeats 3 times, results averaged.
3, the changing value (fluorescence intensity of blank reaction system deducts reaction afterreaction potpourri fluorescence intensity) with the reaction mixture fluorescence intensity is ordinate, logarithm with the final concentration (be followed successively by 10ng/mL, 50ng/ml, 100ng/ml, 500ng/ml, 1000ng/ml, 5000ng/ml, 10000ng/ml, 50000ng/ml) of chymotrypsin standard items in 300 μ l reaction systems is horizontal ordinate, the drawing standard curve.
The result is shown in Figure 4, the gold nano cluster fluorescence intensity is after reacting 1 hour with the variable concentrations chymotrypsin, the changing value of fluorescence signal is directly proportional with the logarithm of chymotrypsin concentration, and both have good linear relationship, and its linear equation is: y=-2.33877x+21.40956(R
2=0.99136).
Two, chymotrypsin Determination on content in the testing sample
Testing sample: the chymotrypsin standard items are dissolved in 50mM NH
4HCO
3In the aqueous solution (pH=7.8), the concentration that makes chymotrypsin is that 750ng/ml(is 0.75U/mL).
The gold nano cluster of the BSA parcel that 1, embodiment 1 is prepared is dissolved in 50mM NH
4HCO
3In the aqueous solution (pH=7.8), obtain solution I.In the solution I, the concentration of the gold nano cluster of the BSA parcel that embodiment 1 prepares is that 0.1mM(is in BSA content).
2,60 μ l testing samples (are dissolved in 50mM NH
4HCO
3Aqueous solution, pH=7.8) join in the previously prepared 240 μ l solution I, 37 ℃ of reactions are after 1 hour, with fluorescence spectrophotometer (PerkinElmer Model LS-55) assaying reaction potpourri fluorescence intensity, measure the fluorescence intensity of the blank reaction system that does not add testing sample simultaneously, excitation wavelength is 365nm, and the detection wavelength is 640nm, entrance slit 10 nanometers, exit slit 10 nanometers.Experiment repeats 3 times, results averaged.
3, in the typical curve equation that the fluorescence intensity changing value of step 2 gained reaction mixture (fluorescence intensity of blank reaction system deducts reaction afterreaction potpourri fluorescence intensity) substitution step 1 is obtained, be 753.04ng/ml by the content that calculates chymotrypsin in the testing sample.Because the enzyme of known chymotrypsin standard items is lived for 1000U/mg, so the content of the chymotrypsin in the testing sample is 0.75304U/mL.
Three, the mensuration of detectability
Determining of noise: measure blank sample under experiment condition, the standard deviation of the data that obtain is noise.Noise numerical value multiply by 3, brings typical curve into and obtains detectability, and noise numerical value multiply by 10, brings typical curve into and obtains quantitative limit.Specific as follows:
The gold nano cluster of the BSA parcel that 1, embodiment 1 is prepared is dissolved in 50mM NH
4HCO
3In the aqueous solution (pH=7.8), obtain solution I.In the solution I, the concentration of the gold nano cluster of the BSA parcel that embodiment 1 prepares is that 0.1mM(is in BSA content).
2, be the NH of 50mM with 60 μ l concentration
4HCO
3Aqueous solution (pH=7.8) joins in the previously prepared 240 μ l solution I, 37 ℃ hatch 1 hour after, measure the fluorescence intensity of mixed solution with fluorescence spectrophotometer (PerkinElmer Model LS-55), excitation wavelength is 365nm, the detection wavelength is 640nm, entrance slit 10 nanometers, exit slit 10 nanometers.Experiment repeats 3 times.
3,3 repeated experiments of statistic procedure 2 gained, the standard deviation of the fluorescence intensity level of the mixed solution that records, this standard deviation is noise.Noise numerical value multiply by 3, brings the typical curve that step 1 obtains into and obtains detectability, and noise numerical value multiply by 10, brings the typical curve that step 1 obtains into and obtains quantitative limit.
The result shows, is under 3 the situation in signal to noise ratio (S/N ratio), detects to be limited to 1.4ng/ml(and to be equivalent to 1.4 * 10
-3U/ml), be under 10 the situation, quantitatively to be limited to 5.7ng/ml(and to be equivalent to 5.7 * 10 in signal to noise ratio (S/N ratio)
-3U/ml).As seen, the content that utilizes the gold nano cluster of the BSA parcel that embodiment 1 prepares to detect chymotrypsin has higher sensitivity.
Claims (10)
1. gold nano cluster is used in measuring the chymotrypsin activity.
2. gold nano cluster application in the chymotrypsin content in measuring testing sample.
3. a method of utilizing gold nano cluster to measure chymotrypsin content in the testing sample comprises the steps:
(a1) chymotrypsin standard items or testing sample are mixed with gold nano cluster, form the experiment reaction system; Arrange simultaneously with described experiment reaction system and compare the blank reaction system that lacks described chymotrypsin standard items or described testing sample; Described experiment reaction system and described blank reaction system are reacted under identical conditions, measure the fluorescence intensity of described experiment reaction system and described blank reaction system after reaction finishes respectively, calculate the fluorescence intensity difference of described experiment reaction system and described blank reaction system;
(a2) the described fluorescence intensity difference drawing standard curve of the described chymotrypsin standard items that obtain according to step (a1), with the described typical curve of described fluorescence intensity difference substitution of described testing sample, thereby obtain the content of chymotrypsin in the described testing sample.
4. method according to claim 3, it is characterized in that: the temperature of described reaction is 35 ℃-40 ℃; The pH of described reaction is 7-8.5; The time of described reaction is 50min-70min.
5. the application of gold nano cluster in monitoring chymotrypsin hydrolysis substrate albumen.
6. a method of utilizing gold nano cluster to detect the reaction rate speed that chymotrypsin in the differential responses system is hydrolyzed to substrate protein comprises the steps:
(b1) will react with gold nano cluster and the chymotrypsin of substrate protein as blocking group, form different reaction systems, monitor the fluorescence intensity of each reaction system in the course of reaction in real time;
(b2) according to the fluorescence intensity of each reaction system that real-time monitors in the step (b1), determine the reaction rate speed that chymotrypsin is hydrolyzed to substrate protein in the described differential responses system as follows: chymotrypsin is more fast to the reaction rate that described substrate protein is hydrolyzed in the more fast then respective reaction system of fluorescence intensity reduction of described reaction system; Chymotrypsin is more slow to the reaction rate that described substrate protein is hydrolyzed in the more slow then respective reaction system of fluorescence intensity reduction of described reaction system.
7. the method whether reaction that utilizes gold nano cluster detection chymotrypsin that substrate protein is hydrolyzed finishes comprises the steps:
(c1) will react the fluorescence intensity of real-time monitoring reaction system in the course of reaction with gold nano cluster and the chymotrypsin of substrate protein as blocking group;
(c2) according to the fluorescence intensity of the described reaction system that real-time monitors in the step (c1), determine as follows whether described chymotrypsin finishes the reaction that described substrate protein is hydrolyzed: if the fluorescence intensity of described reaction system is along with the carrying out that reacts no longer reduces, then described chymotrypsin finishes the reaction that described substrate protein is hydrolyzed; If the fluorescence intensity of described reaction system is along with the carrying out of reaction reducing always, then described chymotrypsin does not finish as yet to the reaction that described substrate protein is hydrolyzed.
8. according to claim 6 or 7 described methods, it is characterized in that: the temperature of described reaction is 35 ℃-40 ℃; The pH of described reaction is 7-8.5.
9. according to arbitrary described application or method among the claim 1-8, it is characterized in that: the blocking group of described gold nano cluster is protein; Described protein is specially bovine serum albumin(BSA); Described substrate protein is bovine serum albumin(BSA).
10. according to arbitrary described application or method among the claim 4-9, it is characterized in that: the proportioning of gold atom and bovine serum albumin(BSA) is 10mmol:30g in the described gold nano cluster.
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| CN104237185A (en) * | 2014-09-13 | 2014-12-24 | 福建医科大学 | PH value measurement method based on N-acetyl-L-cysteine-gold nanocluster |
| CN105628636A (en) * | 2015-12-24 | 2016-06-01 | 合肥安德生制药有限公司 | Chymotrypsin activity detection method |
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| CN104237185B (en) * | 2014-09-13 | 2017-02-22 | 福建医科大学 | PH value measurement method based on N-acetyl-L-cysteine-gold nanocluster |
| CN105628636A (en) * | 2015-12-24 | 2016-06-01 | 合肥安德生制药有限公司 | Chymotrypsin activity detection method |
| CN105628636B (en) * | 2015-12-24 | 2018-08-03 | 安徽瑞达健康产业有限公司 | A kind of chymotrypsin vigor testing methods |
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