CN107238588B - A kind of super-molecule assembling body and preparation method of unmarked fluorescence detection trypsase - Google Patents
A kind of super-molecule assembling body and preparation method of unmarked fluorescence detection trypsase Download PDFInfo
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Abstract
A kind of super-molecule assembling body and preparation method of unmarked fluorescence detection trypsase, belong to the technical field of material, biology and analytical chemistry cross discipline, it is characterised in that the super-molecule assembling body is the compound water solution formed by quaternary ammonium cation Gemini surface active agent and heparin sodium by electrostatic interaction.Preparation method is to measure the critical micelle concentration of Gemini surface active agent first, and Gemini surface active agent and heparin sodium form super-molecule assembling body under the concentration, and hydrophobic dye Nile red enters assembling intracoelomic cavity, fluorescent emission enhancing.Then the nucleoprotamine de-assembly for having specificity to interact with heparin sodium is added, fluorescent emission intensity is reduced with the increase of protamine concentration.Adding trypsase hydrolyzes nucleoprotamine, and assembly restores, and fluorescent emission intensity increases with enzyme concentration and increased, and realizes the detection of enzymatic activity.Assembly of the present invention prepare it is simple, at low cost, have good salt-resistance and broad application prospect.
Description
Technical field
The present invention a kind of super-molecule assembling body and preparation method of unmarked fluorescence detection trypsase, belong to material, life
The technical field of object and analytical chemistry cross discipline.
Background technique
Trypsase is one of most important digestive ferment in breaks down proteins, the secretion of trypsinogen, activation, inhibition with
And the imbalance of circulation will lead to acute or chronic pancreatic disease, serious such as cancer of pancreas.About there are 35000 pancreases every year
Gland cancer is due to caused by the imbalance of serine protease especially trypsase.Furthermore trypsase not only plays digestive ferment
Effect, and the precursor for decomposing other enzymes such as chymotrypsinogen, procarboxypeptidase, phosphatide proenzyme can also be limited, play activation.
The method of the measurement trypsase to grow up at present mainly has ultraviolet spectrophotometry, colorimetric method, fluorescence method, exempts from
Epidemic disease method, mass spectrography (MS), liquid chromatography (HPLC), electrophoresis etc..Ultraviolet spectrophotometry is easy to operate with colorimetric method, but spirit
Sensitivity is low, poor reproducibility.Immunization high sensitivity, but using so that cost is also high, and needs certain due to monoclonal antibody
Operating technology.Mass spectrometer is expensive, high to the purity requirement of sample, chromatography and electrophoresis need more complicated operation and
Longer detection time.
Compared to these methods, fluorescence method is easy to operate, high sensitivity, real-time monitoring can be achieved, latent with clinical application
Possible.The method of general fluorescence detection is the substrate based on fluorescent marker, as patent 201410429549.2 uses fluorescence
The peptide chain of group's label, but this method higher cost and fluorescent marker may cause certain shadow to the selectivity of substrate
It rings.Thus there is researcher to develop the unmarked fluorescence detection method based on the i.e. Supramolecular Assembling of noncovalent interaction, assembling unit
Mainly apply water soluble fluorescent conjugated polymer and novel aggregation-induced emission molecule or fluorescence quantum, such as patent
201510719927.5 use fluorescent carbon point as probe, but the synthesis of these fluorescence probes with separate it is still more complicated,
Improve cost.Thus foundation prepares fluorescence detection method simple, at low cost and has great importance.
We developed a kind of unmarked trypsase detection method in the past, as described in patent 201210116073.8,
It is assembled using the polyelectrolyte of surfactant and oppositely charged, the de-assembly under being acted on by enzymatic hydrolysis is come real
The detection of existing trypsase.The system is that detection is realized by fluorescence attenuating, and relative to the raised method of fluorescence, fluorescence is reduced
Disturbing factor it is more, furthermore the salt-resistance of the system is poor, limits application of the system in physiological environment.Thus this hair
It is bright it is middle construct super-molecule assembling body using the preferable Gemini surface active agent of salt-resistance, and by the raised mode of fluorescence come real
Now detect.By retrieval, patent and report of the Gemini surface active agent for fluorescence detection are had no.
Summary of the invention
A kind of super-molecule assembling body of unmarked fluorescence detection trypsase of the present invention and the purpose of preparation method are to overcome
The deficiencies in the prior art, provide it is a kind of prepare it is simple, at low cost and with good salt-resistance unmarked fluorescence detection tryptose
Super-molecule assembling body of enzyme and preparation method thereof.
A kind of super-molecule assembling body of unmarked fluorescence detection trypsase of the present invention, it is characterised in that the Supramolecular Assembling
Body is the compound water solution formed by quaternary ammonium cation Gemini surface active agent and heparin sodium by electrostatic interaction, described
Quaternary ammonium cation Gemini surface active agent chemical structural formula are as follows:
Wherein n=12,14,16.
A kind of preparation method of the super-molecule assembling body of above-mentioned unmarked fluorescence detection trypsase, it is characterised in that including
Following steps:
The measurement of the critical micelle concentration CMC of I quaternary ammonium cation Gemini surface active agent；
It is 10mM, the phosphate buffer solution of pH 8.0 that quaternary ammonium cation Gemini surface active agent, which is dissolved in concentration,
In, compound concentration range is 10-2mol L-1~10-7mol L-1Gradient solution；10~50 μ L Buddhist nun sieve are pipetted with microsyringe
Red acetone soln, is added in gradient solution, makes final concentration of 10 of Nile red in gradient solution-6mol L-1, with 40~
100kHz opening ultrasonication 10~60 minutes carries out fluorescence emission spectrum detection after standing 0.5~12 hour；With emission peak
Intensity is ordinate, and the log concentration of surfactant is abscissa mapping, obtains sigmoid curve, first turning point on curve
Corresponding concentration is the critical micelle concentration CMC of Gemini surface active agent；
The measurement of II concentration of heparin:
It is 10mM that heparin sodium, which is dissolved in concentration, in the phosphate buffer solution of pH 8.0, compound concentration range is 10~
10-5mg mL-1Gradient solution；According to the critical micelle concentration of the obtained quaternary ammonium cation Gemini surface active agent of step I
CMC determines that the concentration of cation Gemini surfactant is 0.3~1.6 times of CMC, is added to heparin sodium according to volume ratio 1:1
Gradient solution in；The acetone soln that 10~50 μ L Nile reds are pipetted with microsyringe, is added in mixed solution, makes to mix
Final concentration of the 10 of Nile red in solution-6mol L-1, with 40~100kHz opening ultrasonication 10~60 minutes, stand 0.5
Fluorescence emission spectrum detection is carried out after~12 hours；Using emission peak intensity as ordinate, the log concentration of heparin sodium is abscissa
Mapping, obtains sigmoid curve, and the heparin sodium that second turning point corresponding concentration as forms stable super-molecule assembling body on curve is dense
Degree；
The preparation of III Supramolecular Assembling liquid solution:
According to the proportion of the quaternary ammonium cation Gemini surface active agent and heparin sodium that are determined in step II, by two kinds of solution
It is mixed according to volume ratio 1:1；The acetone soln that 10~50 μ L Nile reds are pipetted with microsyringe, is added in mixed solution,
Make final concentration of 10 of Nile red in mixed solution-6mol L-1, with 40~100kHz opening ultrasonication 10~60 minutes,
Supramolecular Assembling liquid solution is obtained after standing 0.5~12 hour.
A kind of preparation method of the super-molecule assembling body of unmarked fluorescence detection trypsase of the present invention, with prior art phase
Than having the advantages that
(1) novel Gemini surface active agent component composition body is used, super-molecule assembling body de-assembly and recovery group are passed through
The mode of dress is detected, and any organic synthesis process is not needed, and is prepared simple, at low cost；
(2) Gemini surface active agent has good salt-resistance, and it is good in physiological environment to assign super-molecule assembling body
Stability extends application of the system in physiological environment.
Detailed description of the invention
Fig. 1 is that addition is different dense in Supramolecular Assembling system (ethylene group didodecyldimethylammbromide bromide+heparin sodium)
Spend the fluorescence emission spectrogram of compound of nucleoprotamine.
Fig. 2 is that milt egg is added in Supramolecular Assembling system (ethylene group didodecyldimethylammbromide bromide+heparin sodium)
White (0.1mg mL-1), add the fluorescence emission spectrogram of compound of various concentration trypsase.
Specific embodiment
Super-molecule assembling body is to be formed by quaternary ammonium cation Gemini surface active agent and heparin sodium by electrostatic interaction
Compound water solution.The critical micelle concentration for measuring quaternary ammonium cation surfactant first, in the following Shuangzi table of the concentration
Face activating agent and heparin sodium form super-molecule assembling body, and hydrophobic dye Nile red enters assembling intracoelomic cavity, fluorescent emission enhancing.So
The nucleoprotamine for having specificity to interact with heparin sodium is added afterwards, nucleoprotamine leads to the assembly system of solutions in conjunction with heparin sodium
Dress, fluorescent emission intensity are reduced with the increase of protamine concentration.The protamine concentration for selecting fluorescence intensity basicly stable,
Trypsase, which is added, hydrolyzes nucleoprotamine, and assembly restores, and fluorescent emission intensity is increased with the increase of enzyme concentration, with this reality
The detection of existing enzymatic activity.Following embodiment is not intended to limit the scope of the invention to further explanation of the invention.
Embodiment 1:
(1) preparation of Supramolecular Assembling liquid solution
The measurement of the critical micelle concentration CMC of I quaternary ammonium cation Gemini surface active agent；
Quaternary ammonium cation surfactant used is ethylene group didodecyldimethylammbromide bromide, the structural formula such as following figure
It is shown:
It is 10mM, the phosphate buffer solution of pH 8.0 that ethylene group didodecyldimethylammbromide bromide, which is dissolved in concentration,
In, preparing 2mL concentration range is 10-2~10-7mol L-1Gradient solution；The third of 10 μ L Nile reds is pipetted with microsyringe
Ketone solution, is added in gradient solution, makes final concentration of 10 of Nile red in gradient solution-6mol L-1, super with 100kHz opening
Sonicated 10 minutes, fluorescence emission spectrum detection was carried out after standing 0.5 hour；Using emission peak intensity at 635nm as ordinate,
The log concentration of surfactant is abscissa mapping, obtains sigmoid curve, and first turning point corresponding concentration is double on curve
The critical micelle concentration CMC of sub- surfactant.The critical micelle concentration that fluorescence spectrometry obtains Gemini surface active agent is
1.75×10-5mol L-1。
The measurement of II concentration of heparin:
It is 10mM that heparin sodium, which is dissolved in concentration, in the phosphate buffer solution of pH 8.0, compound concentration range is 10~
10-5mg mL-1Gradient solution；According to the critical micelle concentration of the obtained quaternary ammonium cation Gemini surface active agent of step I
CMC determines that the concentration of Gemini surface active agent is 1.6CMC (2.8 × 10-5mol L-1), heparin is added to according to volume ratio 1:1
In the gradient solution of sodium；The acetone soln that 10 μ L Nile reds are pipetted with microsyringe, is added in mixed solution, makes to mix molten
Final concentration of the 10 of Nile red in liquid-6mol L-1, with 100kHz opening ultrasonication 10 minutes, carried out after standing 0.5 hour
Fluorescence emission spectrum detection；Using emission peak intensity at 635nm as ordinate, the log concentration of heparin sodium is abscissa mapping, is obtained
To sigmoid curve, second turning point corresponding concentration is the concentration of heparin for forming stable super-molecule assembling body on curve.Fluorescence
It is 0.05mg mL that spectroscopic assay, which obtains concentration of heparin,-1。
The preparation of III Supramolecular Assembling liquid solution:
According to the proportion of the quaternary ammonium cation Gemini surface active agent and heparin sodium that are determined in step II, by two kinds of solution
It is mixed according to volume ratio 1:1；The acetone soln that 10 μ L Nile reds are pipetted with microsyringe, is added in mixed solution, makes to mix
Close final concentration of 10 of Nile red in solution-6mol L-1, with 100kHz opening ultrasonication 10 minutes, after standing 0.5 hour
Obtain Supramolecular Assembling liquid solution.
(2) detection of trypsase
The determination of I protamine concentration:
Final concentration of 10 are separately added into Supramolecular Assembling liquid solution-7~1mg mL-1Nucleoprotamine phosphate it is slow
Solution (10mM, pH=8) is rushed, is placed at 37 DEG C after ten minutes, fluorescence intensity, with fluorescent emission peak intensity at 635nm
The ratio of decline obtains examination criteria curve to protamine concentration mapping is added.
The ratio of fluorescence intensity decline is (I0-I)/I0× 100%, I0Represent the blank control 635nm for only adding buffer solution
Locate fluorescent emission peak intensity, fluorescent emission peak intensity at 635nm after I representative addition various concentration nucleoprotamine.Fluorescence spectrum inspection
Survey the result shows that: fluorescent emission peak intensity decreasing ratio and protamine concentration have good linear relationship, protamine concentration
Reach 0.1mg mL-1Fluorescence intensity no longer declines substantially.
The detection of II trypsase:
0.1mg mL is added in Supramolecular Assembling liquid solution-1Nucleoprotamine, be then respectively adding final concentration of 0.01
~100 μ g mL-1Trypsase phosphate buffer solution (10mM, pH=8), after being placed 30 minutes at 37 DEG C, detect it is glimmering
Luminous intensity obtains examination criteria song with the raised ratio of fluorescent emission peak intensity at 635nm to trypsinase concentration mapping is added
Line.
The raised ratio of fluorescence intensity is (I-I0)/I0× 100%, I0Represent the blank control 635nm for only adding buffer solution
Locate fluorescent emission peak intensity, fluorescent emission peak intensity at 635nm after I representative addition various concentration trypsase.Fluorescence spectrum inspection
Survey the result shows that: fluorescent emission peak intensity lifting ratio and trypsinase concentration have good linear relationship, super-molecule assembling body
It can detecte trypsase in buffer solution.
The experiment of III salt-resistance:
0.1mg mL is added in Supramolecular Assembling liquid solution-1Nucleoprotamine, be separately added into final concentration of 0~
500mmol L-1Sodium chloride solution, fluorescence intensity.Finally it is separately added into final concentration of 100 μ g mL-1Trypsase
Solution, after being placed 30 minutes at 37 DEG C, fluorescence intensity.
Fluorescence spectrum testing result shows: under different salinity, fluorescence intensity has good recovery, Supramolecular Assembling
System has good salt-resistance.
Embodiment 2
(1) preparation of Supramolecular Assembling liquid solution
The measurement of the critical micelle concentration CMC of I quaternary ammonium cation Gemini surface active agent；
Quaternary ammonium cation surfactant used is the double dodecyldimethylamine base ammonium bromides of ethylene group, the structural formula such as following figure
It is shown:
It is 10mM, the phosphate buffer solution of pH 8.0 that the double dodecyldimethylamine base ammonium bromides of ethylene group, which are dissolved in concentration,
In, preparing 2mL concentration range is 10-2~10-7mol L-1Gradient solution；The third of 30 μ L Nile reds is pipetted with microsyringe
Ketone solution, is added in gradient solution, makes final concentration of 10 of Nile red in gradient solution-6mol L-1, super with 70kHz opening
Sonicated 30 minutes, fluorescence emission spectrum detection was carried out after standing 2 hours；Using emission peak intensity at 635nm as ordinate, table
The log concentration of face activating agent is abscissa mapping, obtains sigmoid curve, first turning point corresponding concentration is Shuangzi on curve
The critical micelle concentration CMC of surfactant.The critical micelle concentration that fluorescence spectrometry obtains Gemini surface active agent is 9.2
×10-6mol L-1。
The measurement of II concentration of heparin:
It is 10mM that heparin sodium, which is dissolved in concentration, in the phosphate buffer solution of pH 8.0, compound concentration range is 10~
10-5mg mL-1Gradient solution；According to the critical micelle concentration of the obtained quaternary ammonium cation Gemini surface active agent of step I
CMC determines that the concentration of Gemini surface active agent is CMC (9.2 × 10-6mol L-1), heparin sodium is added to according to volume ratio 1:1
Gradient solution in；The acetone soln that 30 μ L Nile reds are pipetted with microsyringe, is added in mixed solution, makes mixed solution
Final concentration of the 10 of middle Nile red-6mol L-1, with 70kHz opening ultrasonication 30 minutes, fluorescence was carried out after standing 2 hours
Emission spectrum detection；Using emission peak intensity at 635nm as ordinate, the log concentration of heparin sodium is abscissa mapping, obtains S-shaped
Curve, second turning point corresponding concentration is the concentration of heparin for forming stable super-molecule assembling body on curve.Fluorescence spectrum
It is 0.03mg mL that measurement, which obtains concentration of heparin,-1。
The preparation of III Supramolecular Assembling liquid solution:
According to the proportion of the quaternary ammonium cation Gemini surface active agent and heparin sodium that are determined in step II, by two kinds of solution
It is mixed according to volume ratio 1:1；The acetone soln that 30 μ L Nile reds are pipetted with microsyringe, is added in mixed solution, makes to mix
Close final concentration of 10 of Nile red in solution-6mol L-1, with 70kHz opening ultrasonication 30 minutes, after standing 2 hours
To Supramolecular Assembling liquid solution.
(2) detection of trypsase
The determination of I protamine concentration: with embodiment 1.
Fluorescence spectrum testing result shows: fluorescent emission peak intensity decreasing ratio and protamine concentration have well linearly
Relationship, protamine concentration reach 0.05mg mL-1Fluorescence intensity no longer declines substantially.
The detection of II trypsase: in addition to 0.05mg mL is added in Supramolecular Assembling liquid solution-1Nucleoprotamine,
Remaining same embodiment 1.
Fluorescence spectrum testing result shows: fluorescent emission peak intensity decreasing ratio and trypsinase concentration have well linearly
Relationship, super-molecule assembling body can detecte trypsase in buffer solution.
The experiment of III salt-resistance: 0.05mg mL is added in Supramolecular Assembling liquid solution-1Nucleoprotamine, remaining with implement
Mode 1.
Fluorescence spectrum testing result shows: under different salinity, fluorescence intensity has good recovery, Supramolecular Assembling
System has good salt-resistance.
Embodiment 3
(1) preparation of Supramolecular Assembling liquid solution
The measurement of the critical micelle concentration CMC of I quaternary ammonium cation Gemini surface active agent；
Quaternary ammonium cation surfactant used is ethylene group dihexadecyldimethylammonium bromide, the structural formula such as following figure
It is shown:
It is 10mM, the phosphate buffer solution of pH 8.0 that ethylene group dihexadecyldimethylammonium bromide, which is dissolved in concentration,
In, preparing 2mL concentration range is 10-2~10-7mol L-1Gradient solution；The third of 50 μ L Nile reds is pipetted with microsyringe
Ketone solution, is added in gradient solution, makes final concentration of 10 of Nile red in gradient solution-6mol L-1, super with 40kHz opening
Sonicated 60 minutes, fluorescence emission spectrum detection was carried out after standing 12 hours；Using emission peak intensity at 635nm as ordinate,
The log concentration of surfactant is abscissa mapping, obtains sigmoid curve, and first turning point corresponding concentration is double on curve
The critical micelle concentration CMC of sub- surfactant.The critical micelle concentration that fluorescence spectrometry obtains Gemini surface active agent is
1.05×10-6mol L-1。
The measurement of II concentration of heparin:
It is 10mM that heparin sodium, which is dissolved in concentration, in the phosphate buffer solution of pH 8.0, compound concentration range is 10~
10-5mg mL-1Gradient solution；According to the critical micelle concentration of the obtained quaternary ammonium cation Gemini surface active agent of step I
CMC determines that the concentration of Gemini surface active agent is 0.3CMC (3.15 × 10-7mol L-1), liver is added to according to volume ratio 1:1
In the gradient solution of plain sodium；The acetone soln that 50 μ L Nile reds are pipetted with microsyringe, is added in mixed solution, makes to mix
Final concentration of the 10 of Nile red in solution-6mol L-1, with 40kHz opening ultrasonication 60 minutes, carried out after standing 12 hours
Fluorescence emission spectrum detection；Using emission peak intensity at 635nm as ordinate, the log concentration of heparin sodium is abscissa mapping, is obtained
To sigmoid curve, second turning point corresponding concentration is the concentration of heparin for forming stable super-molecule assembling body on curve.Fluorescence
It is 0.01mg mL that spectroscopic assay, which obtains concentration of heparin,-1。
The preparation of III Supramolecular Assembling liquid solution:
According to the proportion of the quaternary ammonium cation Gemini surface active agent and heparin sodium that are determined in step II, by two kinds of solution
It is mixed according to volume ratio 1:1；The acetone soln that 50 μ L Nile reds are pipetted with microsyringe, is added in mixed solution, makes to mix
Close final concentration of 10 of Nile red in solution-6mol L-1, with 40kHz opening ultrasonication 60 minutes, after standing 12 hours
To Supramolecular Assembling liquid solution.
(2) detection of trypsase:
The determination of I protamine concentration: with embodiment 1.
Fluorescence spectrum testing result shows: fluorescent emission peak intensity decreasing ratio and protamine concentration have well linearly
Relationship, protamine concentration reach 0.01mg mL-1Fluorescence intensity no longer declines substantially.
The detection of II trypsase: 0.01mg mL is added in Supramolecular Assembling liquid solution-1Nucleoprotamine, remaining is same
Embodiment 1.
Fluorescence spectrum testing result shows: fluorescent emission peak intensity decreasing ratio and trypsinase concentration have well linearly
Relationship, super-molecule assembling body can detecte trypsase in buffer solution.
The experiment of III salt-resistance: 0.01mg mL is added in Supramolecular Assembling liquid solution-1Nucleoprotamine, remaining with implement
Mode 1.
Fluorescence spectrum testing result shows: under different salinity, fluorescence intensity has good recovery, Supramolecular Assembling
System has good salt-resistance.
Embodiment 4
(1) preparation of Supramolecular Assembling liquid solution
The measurement of the critical micelle concentration CMC of I quaternary ammonium cation Gemini surface active agent:
With embodiment 1.
Fluorescence spectrometry obtains the CMC of cation Gemini surfactant.
The measurement of II concentration of heparin:
It is 10mM that heparin sodium, which is dissolved in concentration, in the phosphate buffer solution of pH 8.0, compound concentration range is 10~
10-5mg mL-1Gradient solution；According to the critical micelle concentration of the obtained quaternary ammonium cation Gemini surface active agent of step I
CMC determines that the concentration of Gemini surface active agent is 0.8CMC, is added in the gradient solution of heparin sodium according to volume ratio 1:1；With
Microsyringe pipettes the acetone soln of 20 μ L Nile reds, is added in mixed solution, keeps the end of Nile red in mixed solution dense
Degree is 10-6mol L-1, with 80kHz opening ultrasonication 20 minutes, fluorescence emission spectrum detection is carried out after standing 1 hour, with
Emission peak intensity is ordinate at 635nm, and the log concentration of surfactant is that abscissa is mapped, and obtains sigmoid curve, on curve
Second turning point corresponding concentration is the concentration of heparin for forming stable super-molecule assembling body.
The preparation of III Supramolecular Assembling liquid solution:
According to the proportion of the quaternary ammonium cation Gemini surface active agent and heparin sodium that are determined in step II, by two kinds of solution
It is mixed according to volume ratio 1:1；The acetone soln that 20 μ L Nile reds are pipetted with microsyringe, is added in mixed solution, makes to mix
Close final concentration of 10 of Nile red in solution-6mol L-1, with 80kHz opening ultrasonication 20 minutes, after standing 1 hour
To Supramolecular Assembling liquid solution.
(2) detection of trypsase
The determination of I protamine concentration: with embodiment 1.
Fluorescence spectrum testing result shows: fluorescent emission peak intensity decreasing ratio and protamine concentration have well linearly
Relationship.
The detection of II trypsase: with embodiment 1.
Fluorescence spectrum testing result shows: fluorescent emission peak intensity decreasing ratio and trypsinase concentration have well linearly
Relationship, super-molecule assembling body can detecte trypsase in buffer solution.
III salt-resistance experiment: with embodiment 1.
Fluorescence spectrum testing result shows: under different salinity, fluorescence intensity has good recovery, Supramolecular Assembling
System has good salt-resistance.
Embodiment 5
(1) preparation of Supramolecular Assembling liquid solution
The measurement of the critical micelle concentration CMC of I quaternary ammonium cation Gemini surface active agent:
Except quaternary ammonium cation Gemini surface active agent structural formula used is as shown below, remaining is the same as embodiment 1.
Fluorescence spectrometry obtains the CMC of cation Gemini surfactant.
The measurement of II concentration of heparin:
Except quaternary ammonium cation Gemini surface active agent structural formula used is as shown above, cation Gemini surfactant
Concentration is other than 1.2CMC, other are the same as embodiment 1.
Fluorescence spectrometry obtains being formed the concentration of heparin of stable super-molecule assembling body.
The preparation of III Supramolecular Assembling liquid solution: with embodiment 1.
(2) detection of trypsase
The determination of I protamine concentration: with embodiment 1.
Fluorescence spectrum testing result shows: fluorescent emission peak intensity decreasing ratio and protamine concentration have well linearly
Relationship.
The detection of II trypsase: with embodiment 1.
Fluorescence spectrum testing result shows: fluorescent emission peak intensity decreasing ratio and trypsinase concentration have well linearly
Relationship, super-molecule assembling body can detecte trypsase in buffer solution.
III salt-resistance experiment: with embodiment 1.
Fluorescence spectrum testing result shows: under different salinity, fluorescence intensity has good recovery, Supramolecular Assembling
System has good salt-resistance.
Embodiment 6
(1) preparation of Supramolecular Assembling liquid solution
The measurement of the critical micelle concentration CMC of I quaternary ammonium cation Gemini surface active agent:
Except quaternary ammonium cation Gemini surface active agent structural formula used is as shown below, remaining is the same as embodiment 2.
Fluorescence spectrometry obtains the CMC of cation Gemini surfactant.
The measurement of II concentration of heparin:
Except quaternary ammonium cation Gemini surface active agent structural formula used is as shown above, cation Gemini surfactant
Concentration is other than 0.8CMC, other are the same as embodiment 2.
Fluorescence spectrometry obtains being formed the concentration of heparin of stable super-molecule assembling body.
The preparation of III Supramolecular Assembling liquid solution: with embodiment 2.
(2) detection of trypsase
The determination of I protamine concentration: with embodiment 2.
Fluorescence spectrum testing result shows: fluorescent emission peak intensity decreasing ratio and protamine concentration have well linearly
Relationship.
The detection of II trypsase: with embodiment 2.
Fluorescence spectrum testing result shows: fluorescent emission peak intensity decreasing ratio and trypsinase concentration have well linearly
Relationship, super-molecule assembling body can detecte trypsase in buffer solution.
III salt-resistance experiment: with embodiment 2.
Fluorescence spectrum testing result shows: under different salinity, fluorescence intensity has good recovery, Supramolecular Assembling
System has good salt-resistance.
Embodiment 7
(1) preparation of Supramolecular Assembling liquid solution
The measurement of the critical micelle concentration CMC of I quaternary ammonium cation Gemini surface active agent:
Except quaternary ammonium cation Gemini surface active agent structural formula used is as shown below, remaining is the same as embodiment 3.
Fluorescence spectrometry obtains the CMC of cation Gemini surfactant.
The measurement of II concentration of heparin:
Except quaternary ammonium cation Gemini surface active agent structural formula used is as shown above, cation Gemini surfactant
Concentration is other than 0.4CMC, other are the same as embodiment 3.
Fluorescence spectrometry obtains being formed the concentration of heparin of stable super-molecule assembling body.
The preparation of III Supramolecular Assembling liquid solution: with embodiment 3.
(2) detection of trypsase
The determination of I protamine concentration: with embodiment 3.
Fluorescence spectrum testing result shows: fluorescent emission peak intensity decreasing ratio and protamine concentration have well linearly
Relationship.
The detection of II trypsase: with embodiment 3.
Fluorescence spectrum testing result shows: fluorescent emission peak intensity decreasing ratio and trypsinase concentration have well linearly
Relationship, super-molecule assembling body can detecte trypsase in buffer solution.
III salt-resistance experiment: with embodiment 3.
Fluorescence spectrum testing result shows: under different salinity, fluorescence intensity has good recovery, Supramolecular Assembling
System has good salt-resistance.
Embodiment 8
(1) preparation of Supramolecular Assembling liquid solution
The measurement of the critical micelle concentration CMC of I quaternary ammonium cation Gemini surface active agent:
With embodiment 5.
Fluorescence spectrometry obtains the CMC of cation Gemini surfactant.
The measurement of II concentration of heparin:
Except quaternary ammonium cation Gemini surface active agent structural formula used is the same as embodiment 5, cationic gemini surface-active
Agent concentration is other than 0.6CMC, remaining is the same as embodiment 4.
Fluorescence spectrometry obtains being formed the concentration of heparin of stable super-molecule assembling body.
The preparation of III Supramolecular Assembling liquid solution: with embodiment 4.
(2) detection of trypsase
The determination of I protamine concentration: with embodiment 4.
Fluorescence spectrum testing result shows: fluorescent emission peak intensity decreasing ratio and protamine concentration have well linearly
Relationship.
The detection of II trypsase: with embodiment 4.
Fluorescence spectrum testing result shows: fluorescent emission peak intensity decreasing ratio and trypsinase concentration have well linearly
Relationship, super-molecule assembling body can detecte trypsase in buffer solution.
III salt-resistance experiment: with embodiment 4.
Fluorescence spectrum testing result shows: under different salinity, fluorescence intensity has good recovery, Supramolecular Assembling
System has good salt-resistance.
Claims (2)
1. a kind of super-molecule assembling body of unmarked fluorescence detection trypsase, it is characterised in that the super-molecule assembling body is by season
The compound water solution that ammonium type cation Gemini surfactant and heparin sodium are formed by electrostatic interaction, the quaternary ammonium type sun
Ion Gemini surface active agent chemical structural formula are as follows:
Or,
Wherein n=12,14,16.
2. a kind of preparation method of the super-molecule assembling body of unmarked fluorescence detection trypsase described in claim 1, special
Sign is to include the following steps:
The measurement of the critical micelle concentration CMC of I quaternary ammonium cation Gemini surface active agent；
Quaternary ammonium cation Gemini surface active agent is dissolved in concentration as 10 mM, in the phosphate buffer solution of pH 8.0, is matched
Concentration range processed is 10-2 mol L-1~10-7 mol L-1Gradient solution；10 ~ 50 μ L Nile reds are pipetted with microsyringe
Acetone soln is added in gradient solution, makes final concentration of 10 of Nile red in gradient solution-6 mol L-1, with 40 ~ 100kHz
Open ultrasonication 10 ~ 60 minutes carries out fluorescence emission spectrum detection after standing 0.5 ~ 12 hour；It is vertical with emission peak intensity
The log concentration of coordinate, surfactant is abscissa mapping, obtains sigmoid curve, first turning point corresponding concentration on curve
The as critical micelle concentration CMC of Gemini surface active agent；
The measurement of II concentration of heparin:
Heparin sodium is dissolved in concentration as 10 mM, in the phosphate buffer solution of pH 8.0, compound concentration range is 10 ~ 10-5
mg mL-1Gradient solution；According to the critical micelle concentration CMC of the obtained quaternary ammonium cation Gemini surface active agent of step I,
The concentration for determining cation Gemini surfactant is 0.3 ~ 1.6 times of CMC, and the ladder of heparin sodium is added to according to volume ratio 1:1
It spends in solution；The acetone soln that 10 ~ 50 μ L Nile reds are pipetted with microsyringe, is added in mixed solution, makes mixed solution
Final concentration of the 10 of middle Nile red-6 mol L-1, with 40 ~ 100kHz opening ultrasonication 10 ~ 60 minutes, it is small to stand 0.5 ~ 12
When after carry out fluorescence emission spectrum detection；Using emission peak intensity as ordinate, the log concentration of heparin sodium is abscissa mapping, is obtained
To sigmoid curve, second turning point corresponding concentration is the concentration of heparin for forming stable super-molecule assembling body on curve；
The preparation of III Supramolecular Assembling liquid solution:
According in step II determine quaternary ammonium cation Gemini surface active agent and heparin sodium proportion, by two kinds of solution according to
Volume ratio 1:1 mixing；The acetone soln that 10 ~ 50 μ L Nile reds are pipetted with microsyringe, is added in mixed solution, makes to mix
Close final concentration of 10 of Nile red in solution-6 mol L-1, with 40 ~ 100 kHz opening ultrasonication 10 ~ 60 minutes, stand
Supramolecular Assembling liquid solution is obtained after 0.5 ~ 12 hour.
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