Summary of the invention
The purpose of the tryptic method of a kind of unmarked fluoroscopic examination of the present invention is the deficiency that overcomes prior art, and a kind of simple to operate, quick, sensitive, unmarked fluoroscopic examination trypsase method that need not organic synthesis is provided.
The tryptic method of a kind of unmarked fluoroscopic examination of the present invention is characterized in that it being that a kind of under the enzymatic hydrolysis effect, separating based on super-molecule assembling body assembled the method that realizes that trypsase detects, and its concrete steps are:
The organic solution of I preparation fluorescence probe:
With the hydrophobic dye is fluorescence probe, hydrophobic dye is dissolved in the organic solvent of commercially available spectroscopic pure acetone, chloroform or methyl alcohol, and compound concentration is 10
-3~ 10
-6Mol L
-1The organic solution of fluorescence probe;
II is measured the critical micelle concentration CMC of surfactant:
Is 10 mM with surfactant dissolves in concentration, and in the PBS of pH 8.0, the compound concentration scope is 1mol L
-1~ 10
-7Mol L
-1Gradient solution, the fluorescence probe organic solution with microsyringe removing step I preparation joins in the gradient solution, the final concentration that makes the gradient solution middle probe is 10
-8~ 10
-6Mol L
-1, with the uncovered ultrasonic Treatment of 40 ~ 100kHz 10 ~ 60 minutes, leave standstill and carry out fluorescence spectrum after 0.5 ~ 12 hour and detect, measure the critical micelle concentration CMC of surfactant;
III preparation super-molecule assembling body solution:
The critical micelle concentration CMC of the surfactant that obtains according to the step II confirms that surfactant concentration is 0.1 ~ 1.6CMC, and it is 10 mM that the polyelectrolyte of oppositely charged is dissolved in concentration, processes polyelectrolyte solution in the PBS of pH 8.0; According to polyelectrolyte and surfactant charge ratio is 1:1; In the solution of surfactant, add isopyknic polyelectrolyte solution; The polyelectrolyte of surfactant and oppositely charged forms super-molecule assembling body through hydrophobic with electrostatic interaction, mixes, and leaves standstill 10 ~ 15 minutes; Obtain super-molecule assembling body solution, the concentration expressed in percentage by weight of this super-molecule assembling body solution is 0.01 ~ 0.2%;
The IV preparation detects liquid:
With the fluorescence probe organic solution of microsyringe removing step I preparation, join respectively in the resulting super-molecule assembling body solution of III once more, the final concentration that makes this super-molecule assembling body solution middle probe is 10
-8~ 10
-6Mol L
-1,, leave standstill after 0.5 ~ 12 hour and obtain detecting liquid with the uncovered ultrasonic Treatment of 40 ~ 100kHz 10 ~ 60 minutes;
V is measured the catalyzing hydrolysis time:
The detection liquid that the step IV obtains was cultivated 10 ~ 15 minutes down at 35 ~ 37 ℃; It is 10 mM that trypsase is dissolved in concentration; In the PBS of pH 8.0; In detecting liquid, add the trypsase buffer solution of equal volume respectively, only to add buffer solution as blank, trypsase final concentration scope is 0.01 ~ 100 μ g mL
-1, under 35 ~ 37 ℃, carry out fluorescence spectrum and follow the tracks of detection, with the ratio of fluorescence emission peak strength degradation the time is mapped, obtain the linear time range that descends of fluorescence emission peak intensity under the variable concentrations trypsase catalyzing hydrolysis condition;
VI is measured tryptic activity:
According to the time range that the step V obtains, get the maximal value of collinearity time range under each concentration conditions, map to adding trypsinase concentration with the ratio of fluorescence emission peak strength degradation, obtain the examination criteria curve.
The tryptic method of above-mentioned a kind of unmarked fluoroscopic examination; It is characterized in that described hydrophobic dye is that commercially available purity is higher than 98% the glimmering dyestuff BODIPY of pyrene, Nile red, cumarin 481, tonyred or fluorine boron, the glimmering dyestuff BODIPY of fluorine boron is meant BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY 581/591, BODIPY TR or BODIPY 630/650.
The tryptic method of above-mentioned a kind of unmarked fluoroscopic examination; It is characterized in that described surfactant is electronegative alkyl sulfate or alkyl sulfonate, alkyl sulfate is the pure lauryl sodium sulfate of commercially available analysis, sodium tetradecyl sulfate or sodium hexadecyl sulfate; Alkyl sulfonate is the pure sodium dodecylsulphonate of commercially available analysis, myristyl sodium sulfonate or sodium cetanesulfonate.
The tryptic method of above-mentioned a kind of unmarked fluoroscopic examination is characterized in that described polyelectrolyte is can be by the polypeptide of trypsase catalyzing hydrolysis, and polypeptide is that commercially available purity is higher than 99%, and length is 10 ~ 16 positively charged arginine sequence A rg
10 ~ Arg
16
The beneficial effect that the method for a kind of unmarked fluoroscopic examination tryptic activity of the present invention and existing compared with techniques have:
The mode of 1) separating assembling through super-molecule assembling body detects, and does not need complicated organic synthesis and fixation procedure, and preparation is simple, cost is low, pollution-free;
2) at the inner embedding fluorescent dye of super-molecule assembling body, detect through fluorescent method, highly sensitive, detection time is short, realizes easily detecting in real time.
Embodiment
With hydrophobic dye as fluorescence probe; At first measured the critical micelle concentration of surfactant, below the surfactant critical micelle concentration, the polyelectrolyte of surfactant and oppositely charged forms super-molecule assembling body through hydrophobic with electrostatic interaction then; Hydrophobic dye is embedded in the assembly inner chamber; Adding the enzyme that polyelectrolyte is had hydrolytic action at last, is low-molecular-weight polyelectrolyte with the polyelectrolyte catalyzing hydrolysis of HMW, induces assembly to separate assembling; The fluorescence probe of assembly inner chamber embedding is discharged, realize the detection of enzymatic activity through the reduction of fluorescence intensity.Following embodiment is to further specify of the present invention, rather than limits scope of the present invention.
Embodiment 1:
The organic solution of I preparation fluorescence probe:
With the pyrene is fluorescence probe, and pyrene is dissolved in the acetone, and compound concentration is 10 in 10 mL volumetric flasks
-2Mol L
-1The acetone soln of pyrene is further with acetone diluted to 10
-5Mol L
-1
II is measured the critical micelle concentration CMC of surfactant:
It is 10 mM that lauryl sodium sulfate SDS is dissolved in concentration, in the PBS of pH 8.0, in 10 mL volumetric flasks, prepares 10
-2Mol L
-1Solution, further do doubling dilution with damping fluid, obtaining concentration range is 1mol L
-1~ 10
-7Mol L
-1Gradient solution;
The organic solution of the fluorescence probe that obtains with microsyringe removing step I preparation joins respectively in the gradient solution of preparation, and the final concentration that makes fluorescence probe in the gradient solution is 5 * 10
-7Mol L
-1With the uncovered ultrasonic Treatment of 100kHz 10 minutes, leave standstill after 0.5 hour and detect fluorescence excitation spectrum.
With fluorescence excitation spectrum I
338 / I
333 Ratio to the mapping of the logarithm value of surfactant concentration, match obtains s type curve, first catastrophe point corresponding concentration of curve is the critical micelle concentration of surfactant.Fluorescence spectrometry obtains the critical micelle concentration of surfactant.
III preparation super-molecule assembling body solution:
Confirm that surfactant concentration is 0.6 CMC, with Arg
10Being dissolved in concentration is 10 mM, in the PBS of pH 8.0, is that 1:1 adds isopyknic Arg according to polyelectrolyte and surfactant charge ratio
10Buffer solution, the polyelectrolyte of surfactant and oppositely charged makes up super-molecule assembling body through hydrophobic and electrostatic interaction, mixes, and leaves standstill 10 minutes, obtains assembly solution, the concentration expressed in percentage by weight of assembly solution is 0.075%;
The IV preparation detects liquid:
With the fluorescence probe organic solution of microsyringe removing step I preparation, join respectively in the resulting assembly solution of III once more, the final concentration that makes fluorescence probe in the assembly solution is 5 * 10
-7Mol L
-1,, leave standstill after 0.5 hour and obtain detecting liquid with the uncovered ultrasonic Treatment of 100kHz 10 minutes;
V is measured the catalyzing hydrolysis time:
Cultivated 10 minutes down at 35 ℃ in the detection liquid that the step IV obtains; It is 10 mM that trypsase is dissolved in concentration; In the PBS of pH 8.0; The trypsase that in detecting liquid, adds the equal volume variable concentrations respectively is to add buffer solution as blank, concentration range from 0.01 to 100 μ g mL
-1, under 35 ℃, carry out fluorescence spectrum and follow the tracks of detection, with the ratio of fluorescence emission peak strength degradation the time is mapped, obtain the linear time range that descends of fluorescence emission peak intensity under the variable concentrations trypsase catalyzing hydrolysis condition;
VI is measured tryptic activity:
According to the time range that the step V obtains, get the maximal value of collinearity time range under each concentration conditions, map to adding trypsinase concentration with the ratio of fluorescence emission peak strength degradation, obtain the examination criteria curve.
The ratio that fluorescence intensity descends is (I-I
0)/I
0* 100 %, I
0Representative only adds the blank fluorescence emission peak place fluorescence intensity of buffer solution, emission peak place fluorescence intensity behind the I representative adding variable concentrations trypsase.
The fluorescence spectrum testing result shows: emission peak positions fluorescence intensity decreasing ratio and trypsinase concentration have good linear relationship.
Specificity experiment: under the same test condition, in assembly solution, add alkaline phosphatase, lysozyme, glucose oxidase, the trypsase of same concentrations respectively, cultivate down for 37 ℃ and carry out fluorescence spectrum after half an hour and detect.
The fluorescence spectrum testing result shows: assembly system detects trypsase has excellent specificity, and other albumen can not influence detection.
Embodiment 2
The organic solution of I preparation fluorescence probe:
With the Nile red is fluorescence probe, and Nile red is dissolved in the methyl alcohol, and compound concentration is 10 in 10 mL volumetric flasks
-1Mol L
-1The methanol solution of Nile red further is diluted to 10 with methyl alcohol
-3Mol L
-1
II is measured the critical micelle concentration CMC of surfactant:
It is 10 mM that sodium tetradecyl sulfate is dissolved in concentration, in the PBS of pH 8.0, in 10 mL volumetric flasks, prepares 10
-2Mol L
-1Solution, further do doubling dilution with damping fluid, obtaining concentration range is 1mol L
-1~ 10
-7Mol L
-1Gradient solution;
The organic solution of the fluorescence probe that obtains with microsyringe removing step I preparation joins respectively in the gradient solution of preparation, and the final concentration that makes fluorescence probe in the gradient solution is 10
-6Mol L
-1With the uncovered ultrasonic Treatment of 70 kHz 30 minutes, leave standstill after 1 hour and detect fluorescence excitation spectrum.
To the mapping of surfactant concentration logarithm value, match obtains s type curve with fluorescence emission spectrum emission peak intensity, and first catastrophe point corresponding concentration of curve is the critical micelle concentration of surfactant.Fluorescence spectrometry obtains the critical micelle concentration of surfactant.
III preparation super-molecule assembling body solution:
The fixed surface surfactant concentration is 0.1CMC, with Arg
16Being dissolved in concentration is 10 mM, in the PBS of pH8.0, is that 1:1 adds isopyknic Arg according to polyelectrolyte and surfactant charge ratio
16Buffer solution, the polyelectrolyte of surfactant and oppositely charged makes up super-molecule assembling body through hydrophobic and electrostatic interaction, mixes, and leaves standstill 15 minutes, obtains assembly solution, the concentration expressed in percentage by weight of assembly solution is 0.01%;
The IV preparation detects liquid:
With the fluorescence probe organic solution of microsyringe removing step I preparation, join respectively in the resulting assembly solution of III once more, the final concentration that makes assembly solution middle probe is 10
-6Mol L
-1,, leave standstill after 1 hour and obtain detecting liquid with the uncovered ultrasonic Treatment of 70kHz 30 minutes;
V is measured the catalyzing hydrolysis time:
Cultivated 15 minutes down at 37 ℃ in the detection liquid that the step IV obtains; It is 10 mM that trypsase is dissolved in concentration; In the PBS of pH 8.0; The trypsase that in detecting liquid, adds the equal volume variable concentrations respectively is to add buffer solution as blank, concentration range from 0.01 to 100 μ g mL
-1, under 37 ℃, carry out fluorescence spectrum and follow the tracks of detection, with the ratio of fluorescence emission peak strength degradation the time is mapped, obtain the linear time range that descends of fluorescence emission peak intensity under the variable concentrations trypsase catalyzing hydrolysis condition;
VI is measured tryptic activity:
According to the time range that the step V obtains, get the maximal value of collinearity time range under each concentration conditions, map to adding trypsinase concentration with the ratio of fluorescence emission peak strength degradation, obtain the examination criteria curve.
The ratio that fluorescence intensity descends is (I-I
0)/I
0* 100 %, I
0Representative only adds the blank fluorescence emission peak place fluorescence intensity of buffer solution, emission peak place fluorescence intensity behind the I representative adding variable concentrations trypsase.
The fluorescence spectrum testing result shows: emission peak positions fluorescence intensity decreasing ratio and trypsinase concentration have good linear relationship.
Specificity experiment: under the same test condition, in assembly solution, add alkaline phosphatase, lysozyme, glucose oxidase, the trypsase of same concentrations respectively, cultivate down for 37 ℃ and carry out fluorescence spectrum after half an hour and detect.
The fluorescence spectrum testing result shows: assembly system detects trypsase has excellent specificity, and other albumen can not influence detection.
Embodiment 3
The organic solution of I preparation fluorescence probe:
With the tonyred is fluorescence probe, and tonyred is dissolved in the chloroform, and compound concentration is 10 in 10 mL volumetric flasks
-1Mol L
-1The chloroformic solution of tonyred further is diluted to 10 with chloroform
-3Mol L
-1
II is measured the critical micelle concentration CMC of surfactant:
It is 10 mM that sodium hexadecyl sulfate is dissolved in concentration, in the PBS of pH 8.0, in 10 mL volumetric flasks, prepares 10
-2Mol L
-1Solution, further do doubling dilution with damping fluid, obtaining concentration range is 1mol L
-1~ 10
-7Mol L
-1Gradient solution;
The organic solution of the fluorescence probe that obtains with microsyringe removing step I preparation joins respectively in the gradient solution of preparation, and the final concentration that makes fluorescence probe in the gradient solution is 10
-6Mol L
-1With the uncovered ultrasonic Treatment of 40 kHz 60 minutes, leave standstill after 12 hours and detect fluorescence excitation spectrum.
To the mapping of surfactant concentration logarithm value, match obtains s type curve with fluorescence emission spectrum emission peak intensity, and first catastrophe point corresponding concentration of curve is the critical micelle concentration of surfactant.Fluorescence spectrometry obtains the critical micelle concentration of surfactant.
III preparation super-molecule assembling body solution:
The fixed surface surfactant concentration is 0.8 CMC, with Arg
12Being dissolved in concentration is 10 mM, in the PBS of pH 8.0, is that 1:1 adds isopyknic Arg according to polyelectrolyte and surfactant charge ratio
16Buffer solution, the polyelectrolyte of surfactant and oppositely charged makes up super-molecule assembling body through hydrophobic and electrostatic interaction, mixes, and leaves standstill 10 minutes, obtains assembly solution, the concentration expressed in percentage by weight of assembly solution is 0.12%;
The IV preparation detects liquid:
With the fluorescence probe organic solution of microsyringe removing step I preparation, join respectively in the resulting mixed solution of III once more, the final concentration that makes the mixed solution middle probe is 10
-6Mol L
-1,, leave standstill after 12 hours and obtain detecting liquid with the uncovered ultrasonic Treatment of 40kHz 60 minutes;
V is measured the catalyzing hydrolysis time:
Cultivated 10 minutes down at 37 ℃ in the detection liquid that the step IV obtains; It is 10 mM that trypsase is dissolved in concentration; In the PBS of pH 8.0; The trypsase that in detecting liquid, adds the equal volume variable concentrations respectively is to add buffer solution as blank, concentration range from 0.01 to 100 μ g mL
-1, under 37 ℃, carry out fluorescence spectrum and follow the tracks of detection, with the ratio of fluorescence emission peak strength degradation the time is mapped, obtain the linear time range that descends of fluorescence emission peak intensity under the variable concentrations trypsase catalyzing hydrolysis condition;
VI is measured tryptic activity:
According to the time range that the step V obtains, get the maximal value of collinearity time range under each concentration conditions, map to adding trypsinase concentration with the ratio of fluorescence emission peak strength degradation, obtain the examination criteria curve.
The ratio that fluorescence intensity descends is (I-I
0)/I
0* 100 %, I
0Representative only adds the blank fluorescence emission peak place fluorescence intensity of buffer solution, emission peak place fluorescence intensity behind the I representative adding variable concentrations trypsase.The fluorescence spectrum testing result shows: emission peak positions fluorescence intensity decreasing ratio and trypsinase concentration have good linear relationship.
Specificity experiment: under the same test condition, in assembly solution, add alkaline phosphatase, lysozyme, glucose oxidase, the trypsase of same concentrations respectively, cultivate down for 37 ℃ and carry out fluorescence spectrum after half an hour and detect.
The fluorescence spectrum testing result shows: assembly system detects trypsase has excellent specificity, and other albumen can not influence detection.
Embodiment 4
The organic solution of I preparation fluorescence probe:
Removing used fluorescence probe is cumarin 481, and other are with embodiment 2;
II is measured the critical micelle concentration CMC of surfactant:
Removing used surfactant is sodium dodecylsulphonate, and other are with embodiment 2;
Fluorescence spectrometry obtains the critical micelle concentration of surfactant.
III preparation super-molecule assembling body solution:
Removing surfactant concentration is 1.6CMC, and used polyelectrolyte is Arg
14 , the concentration expressed in percentage by weight of assembly solution is 2%, other are with embodiment 1;
The IV preparation detects liquid: with embodiment 2;
V is measured the catalyzing hydrolysis time: with embodiment 2;
VI is measured tryptic activity: with embodiment 1;
The fluorescence spectrum testing result shows: emission peak positions fluorescence intensity decreasing ratio and trypsinase concentration have good linear relationship.
The specificity experiment:
The fluorescence spectrum testing result shows: assembly system detects trypsase has excellent specificity, and other albumen can not influence detection.
Embodiment 5
The organic solution of I preparation fluorescence probe:
Removing used fluorescence probe is BODIPY FL, and concentration is 10
-5Mol L
-1, other are with embodiment 2;
II is measured the critical micelle concentration CMC of surfactant:
Removing used surfactant is the myristyl sodium sulfonate, and the final concentration of fluorescence probe is 10
-8Mol L
-1, other are with embodiment 2;
Fluorescence spectrometry obtains the critical micelle concentration of surfactant.
III preparation super-molecule assembling body solution:
Removing surfactant concentration is 0.6CMC, and used polyelectrolyte is Arg
13 , the concentration expressed in percentage by weight of assembly solution is 0.08%, other are with embodiment 1;
The IV preparation detects liquid: with embodiment 2;
V is measured the catalyzing hydrolysis time: with embodiment 2;
VI is measured tryptic activity: with embodiment 1;
The fluorescence spectrum testing result shows: emission peak positions fluorescence intensity decreasing ratio and trypsinase concentration have good linear relationship.
The specificity experiment:
The fluorescence spectrum testing result shows: assembly system detects trypsase has excellent specificity, and other albumen can not influence detection.
Embodiment 6
The organic solution of I preparation fluorescence probe:
Removing used fluorescence probe is BODIPY R6G, and other are with embodiment 5;
II is measured the critical micelle concentration CMC of surfactant:
Removing used surfactant is sodium cetanesulfonate, and other are with embodiment 5;
Fluorescence spectrometry obtains the critical micelle concentration of surfactant.
III preparation super-molecule assembling body solution:
Removing surfactant concentration is 0.5CMC, and used polyelectrolyte is Arg
15 , the concentration expressed in percentage by weight of assembly solution is 0.07%, other are with embodiment 1;
The IV preparation detects liquid: with embodiment 2;
V is measured the catalyzing hydrolysis time: with embodiment 2;
VI is measured tryptic activity: with embodiment 1;
The fluorescence spectrum testing result shows: emission peak positions fluorescence intensity decreasing ratio and trypsinase concentration have good linear relationship.
The specificity experiment:
The fluorescence spectrum testing result shows: assembly system detects trypsase has excellent specificity, and other albumen can not influence detection.
Embodiment 7
The organic solution of I preparation fluorescence probe:
Removing used fluorescence probe is BODIPY TMR, and other are with embodiment 5;
II is measured the critical micelle concentration CMC of surfactant:
The final concentration that removes fluorescence probe is 10
-7Mol L
-1, other are with embodiment 2
Fluorescence spectrometry obtains the critical micelle concentration of surfactant.
III preparation super-molecule assembling body solution:
Removing surfactant concentration is 0.9CMC, and used polyelectrolyte is Arg
11 , the concentration expressed in percentage by weight of assembly solution is 0.12 %, other are with embodiment 1;
The IV preparation detects liquid: with embodiment 2;
V is measured the catalyzing hydrolysis time: with embodiment 2;
VI is measured tryptic activity: with embodiment 1;
The fluorescence spectrum testing result shows: emission peak positions fluorescence intensity decreasing ratio and trypsinase concentration have good linear relationship.
The specificity experiment:
The fluorescence spectrum testing result shows: assembly system detects trypsase has excellent specificity, and other albumen can not influence detection.
Embodiment 8
The organic solution of I preparation fluorescence probe:
Removing used fluorescence probe is BODIPY 581/591, and other are with embodiment 5;
II is measured the critical micelle concentration CMC of surfactant:
The final concentration that removes fluorescence probe is 10
-7Mol L
-1, other are with embodiment 3;
Fluorescence spectrometry obtains the critical micelle concentration of surfactant.
III preparation super-molecule assembling body solution:
Removing surfactant concentration is CMC, and used polyelectrolyte is Arg
16 , other are with embodiment 1, and the concentration expressed in percentage by weight of assembly solution is 0.15%;
The IV preparation detects liquid: with embodiment 2;
V is measured the catalyzing hydrolysis time: with embodiment 2;
VI is measured tryptic activity: with embodiment 1;
The fluorescence spectrum testing result shows: emission peak positions fluorescence intensity decreasing ratio and trypsinase concentration have good linear relationship.
The specificity experiment:
The fluorescence spectrum testing result shows: assembly system detects trypsase has excellent specificity, and other albumen can not influence detection.
Embodiment 9
The organic solution of I preparation fluorescence probe:
Removing used fluorescence probe is BODIPY TR, and other are with embodiment 5;
II is measured the critical micelle concentration CMC of surfactant:
The final concentration that removes fluorescence probe is 10
-7Mol L
-1, other are with embodiment 4;
Fluorescence spectrometry obtains the critical micelle concentration of surfactant.
III preparation super-molecule assembling body solution:
Removing surfactant concentration is 1.2CMC, and used polyelectrolyte is Arg
14 , other are with embodiment 1, and the concentration expressed in percentage by weight of assembly solution is 0.15%;
The IV preparation detects liquid: with embodiment 2;
V is measured the catalyzing hydrolysis time: with embodiment 2;
VI is measured tryptic activity: with embodiment 1;
The fluorescence spectrum testing result shows: emission peak positions fluorescence intensity decreasing ratio and trypsinase concentration have good linear relationship.
The specificity experiment:
The fluorescence spectrum testing result shows: assembly system detects trypsase has excellent specificity, and other albumen can not influence detection.
Embodiment 10
The organic solution of I preparation fluorescence probe:
Removing used fluorescence probe is BODIPY 630/650, and other are with embodiment 5;
II is measured the critical micelle concentration CMC of surfactant: with embodiment 5;
Fluorescence spectrometry obtains the critical micelle concentration of surfactant.
III preparation super-molecule assembling body solution:
Removing surfactant concentration is 0.4CMC, and used polyelectrolyte is Arg
12 , the concentration expressed in percentage by weight of assembly solution is 0.05%, other are with embodiment 1;
The IV preparation detects liquid: with embodiment 2;
V is measured the catalyzing hydrolysis time: with embodiment 2;
VI is measured tryptic activity: with embodiment 1;
The fluorescence spectrum testing result shows: emission peak positions fluorescence intensity decreasing ratio and trypsinase concentration have good linear relationship.
The specificity experiment:
The fluorescence spectrum testing result shows: assembly system detects trypsase has excellent specificity, and other albumen can not influence detection.