A kind of method detecting enzymatic activity and chemiluminescence reaction substrate performanceTechnical field
The present invention relates to bioanalytical chemistry technical field, be specifically related to a kind of method detecting enzymatic activity and chemiluminescence reaction substrate performance.
Background technology
Chemiluminescence is that material is carrying out a kind of optical radiation phenomenon adjoint in chemical reaction process, its luminescence mechanism be the Cucumber in reaction system absorb reaction release energy and by ground state transition to excited state, when excited state returns ground state, energy is discharged with the form of optical radiation, produce luminescence phenomenon.Chemiluminometry is then one trace and the trace analysis method of respective components content in determining to react according to a certain moment chemiluminescence intensity or chemiluminescence total amount, there is high sensitivity, the range of linearity be wide, equipment is simple, easy and simple to handle, easily be automated and analyze the feature such as fast.In recent years, the coupling with methods such as flow injection, galvanochemistry, high performance liquid chromatography and Capillary Electrophoresis, makes chemiluminometry be widely used in the fields such as Pharmaceutical Analysis, clinical analysis, environmental analysis and material analysis.Chemical luminous substrate is researched and developed, and selects, in the work such as stability test, all needs effective performance evaluation means.General enterprises is all adopt mode on probation in ELISA or self-control kit to evaluate.It is too many all to there is influence factor in these two kinds of methods, wastes time and energy, high in cost of production problem.
Concentration and active (or claiming enzyme activity) of enzyme are by the problem of showing great attention in field of biology, and how fast, the concentration and the activity that measure enzyme are exactly the bases realizing proper use of enzyme and enzyme preparation.The assay method that current most of producer adopts is the methods such as electrophoresis, immunization, bioanalysis.These method ubiquity testing costs are high, the shortcomings such as the cycle is long, not accurate enough.
Summary of the invention
Based on this, be necessary to provide fast a kind of, easy, the method for direct-detection enzymatic activity and chemiluminescence reaction substrate performance is with at least one in solving the problems of the technologies described above.
A kind of method detecting enzymatic activity and chemiluminescence reaction substrate performance, comprise: glow cup, chemiluminescence reaction substrate A, chemical reaction luminous substrate B, match with described chemiluminescence reaction substrate A, chemical reaction luminous substrate B the enzyme solutions, the lighting apparatus that detect, with the foundation of described chemiluminescence reaction substrate A, chemical reaction luminous substrate B and described enzyme solutions luminescence system, detect the performance of described enzyme solutions activity or enzyme concentration or described chemiluminescence reaction substrate according to the luminescence system of described foundation;
The foundation of described luminescence system comprises the following steps:
1) described chemiluminescence reaction substrate A and chemical reaction luminous substrate B are modulated by a certain percentage mix, and be placed in described glow cup, for subsequent use;
2) described enzyme solutions is diluted to a series of different concentration;
3) enzyme solutions diluted described in step 2 is fully mixed with the mixed liquor of described chemiluminescence reaction substrate A and chemical reaction luminous substrate B as in described glow cup, and insert the mensuration of carrying out light signal strength in described lighting apparatus, draw light signal strength;
4) light signal strength described in during a series of variable concentrations obtained according to step 3 establish described enzyme solutions concentration or and chemiluminescence signal intensity between linear relationship.
Further, described detection enzyme solutions activity comprises the following steps:
1) described chemiluminescence reaction substrate A and chemical reaction luminous substrate B are modulated by a certain percentage mix, and be placed in described glow cup, for subsequent use;
2) described enzyme solutions is diluted to certain concentration;
3) enzyme solutions diluted described in step 2 is fully mixed with the mixed liquor of described chemiluminescence reaction substrate A and chemical reaction luminous substrate B as in described glow cup, and insert the mensuration of carrying out light signal strength in described lighting apparatus, draw light signal strength X;
4) light signal strength X step 3 obtained and the luminescence system of described foundation compare, and calculate the activity of described enzyme solutions or the performance of concentration or described chemiluminescence reaction substrate.
Further, described chemiluminescence reaction substrate A and chemical reaction luminous substrate B can produce any one chemiluminescence reaction substrate chemiluminescent with described enzyme solutions, comprise and are not limited to the reaction substrate of luminol-hydrogen peroxide reaction substrate, phosphoric acid or phosphoric acid ester, the chemical reaction substrate containing ruthenium.
Further, described enzyme solutions is to carry out chemiluminescence reaction with described chemiluminescence reaction substrate A and chemical reaction luminous substrate B and to produce the enzyme of light signal, comprises and is not limited to hydrogen peroxidase, horseradish peroxidase or alkaline phosphatase.
Further, described chemiluminescence reaction substrate A can be luminol luminescent solution A liquid, and described chemical reaction luminous substrate B can be luminol luminescent solution B liquid.
Further, described luminol luminescent solution A liquid comprises luminol, Tris-damping fluid, and described luminol luminescent solution B liquid comprises peroxide ingredient.
Further, described luminol luminescent solution A liquid and described luminol luminescent solution B liquid mix according to the ratio modulation of 1:1.
Further, the material of described glow cup is without the plastics absorbed to visible ray.
Further, the inner chamber of described glow cup is cuboid, and the uniform wall thickness of described glow cup.
Further, described lighting apparatus is the equipment that can measure light signal strength, comprises and is not limited to camera, photoelectric cell, photomultiplier, chemical luminescent detecting equipment.
Further, described lighting apparatus is CR-0302 type light signal reading apparatus.
Beneficial effect of the present invention:
(1) provided by the invention detect the method for enzymatic activity and chemiluminescence reaction substrate performance based on chemoluminescence method can fast and effeciently multiple activity and the concentration that can cause the enzyme of chemiluminescence phenomenon of Accurate Determining.This method detects the time only needing 2-3 minute at every turn, and cost is low.Very auitable enzyme manufacturing enterprise or use enterprise carry out Fast Evaluation to the performance of enzyme preparation.
(2) as long as the enzyme solutions that the method detecting enzymatic activity and chemiluminescence reaction substrate performance based on chemoluminescence method provided by the invention adopts concentration known just can the performance of evaluating chemical luminous substrate rapidly and accurately.Particularly be applicable to two kinds of chemical luminous substrates compare.
Accompanying drawing explanation
Luminous photo legend when Fig. 1 is different horseradish peroxidase concentration.
Fig. 2 is the linear relationship curve between chemiluminescence signal intensity and the concentration of enzyme.
Embodiment
A kind of method detecting enzymatic activity and chemiluminescence reaction substrate performance provided by the invention, comprise glow cup, chemiluminescence reaction substrate A, chemical reaction luminous substrate B, match with described chemiluminescence reaction substrate A, chemical reaction luminous substrate B the enzyme solutions, the lighting apparatus that detect, with the foundation of described chemiluminescence reaction substrate A, chemical reaction luminous substrate B and described enzyme solutions luminescence system, detect the performance of described enzyme solutions activity or described chemiluminescence reaction substrate according to the luminescence system of described foundation;
The foundation of described luminescence system comprises the following steps:
1) described chemiluminescence reaction substrate A and chemical reaction luminous substrate B are modulated by a certain percentage mix, and be placed in described glow cup, for subsequent use;
2) described enzyme solutions is diluted to a series of different concentration; In detail, using the enzyme solutions of concentration known as reference enzyme solution, enzyme solutions to be measured is measured and evaluated.Enzyme solutions to be measured is made into the enzyme solutions of a series of variable concentrations, concentration range is 1pg/ml-1ug/ml, is preferably 1ng/ml-100ng/ml, and the chemiluminescence light signal strength that the enzyme solutions of variable concentrations produces is different, and linear with the concentration of enzyme solutions.
3) enzyme solutions diluted described in step 2 is placed in described glow cup fully to mix with the mixed liquor of described chemiluminescence reaction substrate A and chemical reaction luminous substrate B, and insert the mensuration of carrying out light signal strength in described lighting apparatus, draw light signal strength;
4) light signal strength described in during a series of variable concentrations obtained according to step 3 establish described enzyme solutions concentration or and chemiluminescence signal intensity between linear relationship.
Further, described detection enzyme solutions activity comprises the following steps:
1) described chemiluminescence reaction substrate A and chemical reaction luminous substrate B are modulated by a certain percentage mix, and be placed in described glow cup, for subsequent use;
2) described enzyme solutions is diluted to certain concentration;
3) enzyme solutions diluted described in step 2 is fully mixed with the mixed liquor of described chemiluminescence reaction substrate A and chemical reaction luminous substrate B as in described glow cup, and insert the mensuration of carrying out light signal strength in described lighting apparatus, draw light signal strength X;
4) light signal strength X step 3 obtained and the luminescence system of described foundation compare, and calculate the activity of described enzyme solutions or the performance of concentration or described chemiluminescence reaction substrate.
Preferably, described chemiluminescence reaction substrate A and chemical reaction luminous substrate B can produce any one chemiluminescence reaction substrate chemiluminescent with described enzyme solutions, comprise and are not limited to the reaction substrate of luminol-hydrogen peroxide reaction substrate, phosphoric acid or phosphoric acid ester, the chemical reaction substrate containing ruthenium.
Further, described enzyme solutions is to carry out chemiluminescence reaction with described chemiluminescence reaction substrate A and chemical reaction luminous substrate B and to produce the enzyme of light signal, comprises and is not limited to hydrogen peroxidase, horseradish peroxidase or alkaline phosphatase.
Further, described chemiluminescence reaction substrate A can be luminol luminescent solution A liquid, and described chemical reaction luminous substrate B can be luminol luminescent solution B liquid.The chemiluminescence reaction substrate A that the present invention adopts also can be different luminol or its derivative class thing.Described luminol luminescent solution A liquid comprises luminol and Tris-damping fluid, and described luminol luminescent solution B liquid comprises peroxide ingredient.During allotment mixing, luminol luminescent solution A liquid and described luminol luminescent solution B liquid mix according to the ratio modulation of 1:1.
Preferably, described glow cup adopts visible ray substantially without the plastic production absorbed.The inner chamber of glow cup is cuboid, and the uniform wall thickness of described glow cup.The inner chamber capacity of glow cup is about 300ul.Whole inner chamber is cuboid, makes the thickness of each position solution without significant difference.Use this glow cup to have evenly luminous, easy and simple to handle, measure the advantages such as accurate.The inner chamber of glow cup is specifically of a size of the thick 3mm of the wide 5mm of long 20mm.Natch, the inner cavity size of glow cup can adjust as required, but preferably thickness uniformly to reduce error at measurment.
Preferably, described lighting apparatus is the equipment that can measure light signal strength, comprises and is not limited to camera, photoelectric cell, photomultiplier, chemical luminescent detecting equipment.Natch, other can all can apply equipment such as the high sensitivity camera system of sensed light signal intensity.The lighting apparatus adopted in an embodiment of the present invention is CR-0302 type light signal reading apparatus.
The present invention can be applied to and detect enzyme solutions activity,
Particularly, with the activity of reference enzyme reagent for 100, the activity of enzyme reagent to be measured is measured and evaluated.The solution of same concentrations is made into reference to enzyme reagent and enzyme reagent to be measured, concentration range is 1pg/ml-1ug/ml, be preferably 1ng/ml-100ng/ml, different enzyme reagent has the suitableeest concentration, and preferably concentration is about the chemiluminescence light signal produced is about the half of the optical signal detecting equipment Inspection upper limit.
A certain amount of chemical luminous substrate is prepared in glow cup in advance according to its request for utilization.Subsequently to wherein adding a certain amount of enzyme solutions.Chemiluminescence intensity is measured with lighting apparatus after abundant mixing.Record chemiluminescence signal intensity.Calculate enzymatic activity according to the following formula:
The signal intensity of the signal intensity/reference enzyme of enzymatic activity=100 × enzyme to be measured
The present invention can also be applied to and detect enzyme solutions concentration.
Particularly, when measuring the effective concentration of enzyme, the enzyme solutions of concentration known is first used to prepare the enzyme solutions of a series of variable concentrations.The concentration of enzyme solutions is formulated as 0ng/ml, 1ng/ml, 2ng/ml, 3ng/ml, 5ng/ml, 10ng/ml, 18ng/ml, 20ng/ml, 25ng/ml.Then measure the light signal strength sent when the enzyme solutions of variable concentrations and the chemiluminescence reaction substrate A of same dose and chemiluminescence reaction substrate B carry out chemiluminescence reaction with said method, draw the relation curve between chemiluminescence signal intensity and enzyme solutions concentration.As shown in Figure 1, be luminous photo legend during different horseradish peroxidase concentration.Wherein 1 is the luminous photo of the horseradish peroxidase solution of 0ng/ml for concentration, and 2 is the luminous photo of the horseradish peroxidase solution of 1ng/ml for concentration, and 3 is the luminous photo of the horseradish peroxidase solution of 10ng/ml for concentration.The concentration of chemiluminescence signal intensity and enzyme presents good linear as shown in Figure 2.Then equally chemiluminescent mensuration is carried out to the enzyme solutions of concentration to be measured, contrast according to the relation curve between the luminous signal strength chemical luminous signal intensity recorded and enzyme solutions concentration, extrapolate the effective concentration of enzyme.
The present invention can also evaluate the performance of chemical luminous substrate.
Particularly, can be evaluated accurately the performance of chemical luminous substrate by this method, and the interference of other factors such as kit performance can not be subject to.Under the effect of the enzyme preparation of same quantity, with the luminescent properties with reference to chemical luminous substrate for 100, the signal produced by chemical luminous substrate to be measured compares with the signal with reference to substrate, can evaluate the performance of chemical luminous substrate to be measured.Such as match silent fly " the SuperSignalELISAFemto maximum sensitivity substrate " of Company for reference substrate with the U.S..The light signal strength that the chemical luminous substrate adopted in the present invention produces is match to write from memory to fly 95% of Products.Then the performance scores of this chemical luminous substrate is 95.
Embodiment one: the detection of enzymatic activity.
Use chemical luminous substrate: SF-2 type luminol ultra-sensitive chemical luminous substrate,
Use sense light device: CR-0302 type light signal reading apparatus
Get glow cup 1, in glow cup, add 100ul luminol luminescent solution A liquid and 100ul luminol luminescent solution B liquid respectively.Add that 10ul diluted subsequently horseradish peroxidase solution to be measured, enzyme concentration 10ng/ml, takes pictures after mixing immediately, 30 seconds timing ups.Note avoiding bubble.Obtaining signal value is 1068.Same method measures with reference to horseradish peroxidase solution (U.S.'s match is silent flies product), and acquisition signal value is the activity value of 1100. these tested horseradish peroxidases is 97.
Embodiment two: the detection of enzyme solutions concentration.
Use chemical luminous substrate: SF-2 type luminol ultra-sensitive chemical luminous substrate
Use sense light device: CR-0302 type light signal reading apparatus
100ul luminol luminescent solution A liquid and 100ul luminol luminescent solution B liquid is added respectively in glow cup.Add 10ul variable concentrations horseradish peroxidase solution (each enzyme concentration is 100,80,60,40,20,10,5,2ng/ml) subsequently, take pictures immediately after each sample mixing, 30 seconds timing ups.Relation curve is drawn according to each signal value result.
To enzyme solutions to be measured, operate equally, after obtaining signal value, from working curve, read the concentration of this enzyme solutions.
Embodiment three: chemical luminous substrate performance evaluation.
Use enzyme: horseradish peroxidase, concentration 10ng/ml.
With reference to chemical luminous substrate: SuperSignalELISAFemto maximum sensitivity substrate,
Tested object: SF-2 type luminol ultra-sensitive chemical luminous substrate,
Use sense light device: CR-0302 type light signal reading apparatus
Get glow cup 1, in glow cup, add homemade 100ul luminol luminescent solution A liquid and 100ul luminol luminescent solution B liquid respectively.Add the horseradish peroxidase solution that 10ul has diluted subsequently, enzyme concentration 10ng/ml, takes pictures after mixing immediately, 30 seconds timing ups.Note avoiding bubble.Acquisition signal value is that the chemical luminous substrate of 1072. use references carries out same mensuration, and obtaining signal value is 1128.The performance number then making chemical luminous substrate by oneself is 95.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.