CN110044988B - Method for detecting multienzyme by bent capillary electrophoresis - Google Patents
Method for detecting multienzyme by bent capillary electrophoresis Download PDFInfo
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- CN110044988B CN110044988B CN201910342854.0A CN201910342854A CN110044988B CN 110044988 B CN110044988 B CN 110044988B CN 201910342854 A CN201910342854 A CN 201910342854A CN 110044988 B CN110044988 B CN 110044988B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3271—Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
- G01N27/3272—Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
- G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The invention belongs to the field of biological analysis, and particularly relates to a method for detecting multienzyme by bent capillary electrophoresis. Firstly, combining quantum dots and polypeptide to form a double-enzyme probe, then respectively bending the capillary tube used for capillary electrophoresis into semi-circles with different numbers, fixing, firstly sampling the double-enzyme probe, sampling hydrolase after an interval of 10s, after a certain time, the hydrolase catches up with the double-enzyme probe, fully mixing in the bent capillary tube, and detecting the fluorescence intensity of the double-enzyme probe by a fluorescence spectrometer. The technical scheme is simple to operate and high in repeatability, and a novel high-sensitivity multi-enzyme detection technology is established.
Description
Technical Field
The invention belongs to the technical field of nano biology, and particularly relates to a method for detecting multienzyme by bent capillary electrophoresis.
Background
The enzyme sensors which are rapidly developed and widely applied at present tightly combine two steps of reaction and detection, have the characteristics of rapidness, convenience, sensitivity and accuracy, play a great role in enzyme detection, and further promote the development of enzyme detection methods. In general, a single enzyme substrate is used to detect a particular enzyme, which has the disadvantage of a single detection selectivity and is complicated to operate.
On the other hand, capillary electrophoresis has wide application prospect in the field of biological analysis as a high-resolution, high-sensitivity, high-flux and low-sample small-size micro-separation technology. Meanwhile, fluorescence detection and capillary electrophoresis are combined, so that the detection line is greatly improved, and the application of the capillary is expanded. Analysis and determination of samples is usually performed using straight capillaries.
Disclosure of Invention
For solving the problem that the prior art has no rapidnessThe invention aims to provide a method for detecting multiple enzymes by bent capillary electrophoresis. The condition that the hydrolase hydrolyzes the double-enzyme probe in the capillary is researched by utilizing the characteristic that the bending of the capillary is beneficial to the full reaction of the sample in the capillary. Detection was carried out by capillary electrophoresis analysis, and the peak area (S) of the fluorescent dye Cy5 was calculated665nm) Peak area with QDs (S)605nm) The FRET change condition is judged according to the ratio, so that the detection of the hydrolase is realized.
The technical scheme adopted by the invention is as follows: combining quantum dots and polypeptide to form a double-enzyme probe, respectively bending a capillary tube used for electrophoresis into semi-circles with different numbers, fixing, firstly sampling the double-enzyme probe, sampling hydrolase after an interval of 10s, and detecting the fluorescence intensity of the double-enzyme probe by a fluorescence spectrometer; the bent capillary tube is a quartz capillary tube.
The hydrolase is PreScission protease and thrombin.
The polypeptide contains (1) double enzyme cutting sites; (2) a fluorescent dye Cy5 for FRET with the quantum dots; (3) and quantum dot-bound 6 × histidine.
The polypeptide contains LEVLFQGP and LVPRGS as double enzyme cutting sites.
The polypeptide sequence is Cy5-D3LEVLFQGPGLVPRGSGP9G2H6(Cy5-LEVLVP-QD)。
The quantum dots are Zn-containing quantum dots, such as CdSe/ZnS, CdTe/ZnS, CdSe/ZnSe or CdTe/ZnSe.
The double-enzyme probe is obtained by mixing and incubating 1 mu M quantum dot and 8 mu M polypeptide in equal volume for 10 min.
The inner diameter of the bent capillary tube is 75 micrometers, the outer diameter of the bent capillary tube is 365 micrometers, the number of the bent semicircles is 1-4, and the diameter of the bent semicircles is 3 cm.
The bent capillary tube is a 180-degree equal-width circular arc bent capillary tube.
The sample injection time of the double-enzyme probe is 15s, the sample injection time of the hydrolase is 15s, and the sample injection interval of the hydrolase is 10 s.
After the technical scheme is adopted, the invention has the beneficial effects that: since CE-FL can detect multiple enzymes simultaneously using one substrate, this design allows rapid screening for unknown enzymes from microorganisms or other environments. The method is simple to operate and high in repeatability, and further expands the application of the fluorescent probe in the field of biological analysis.
Drawings
FIG. 1: (A) PSP and (B) thrombin in-tube hydrolysis of the complex at different degrees of capillary flexion. Wherein (a) only Cy 5-levvlvp-QD, (b) straight capillary, (c) bend 1 semicircle, (d) bend 2 semicircles, (e) bend 4 semicircles. [ His-PSP ]]＝10U/ml，[Thrombin]10U/ml. (excitation light source lambda)ex420nm, black line, 605nm, QDs donor detection channel, grey, 665nm, Cy5 acceptor detection channel)
FIG. 2: detecting the activity of (A) His-PSP and (B) thrombin at different concentrations. (a)0U/ml, (b)5U/ml, (c)10U/ml, (d)20U/ml, (e) 40U/ml.
FIG. 3: s665nm/S605nmA trend plot of the value of (D) as a function of enzyme concentration. (a) His-PSP, (b) thrombin.
FIG. 4: the Cy5-LEV-QD complex was hydrolyzed inside the PSP tube at different degrees of bending of the capillary. Wherein (a) only Cy5-LEV-QD, (b) straight capillary, (c) bending 1 semicircle, (d) bending 2 semicircles, (e) bending 4 semicircles. [ His-PSP ] ═ 10U/ml.
Detailed Description
The invention will be further described in the following examples, but it is to be understood that these examples are for illustrative purposes only and are not to be construed as limiting the practice of the invention.
Example 1
The method adopts a conventional solid phase Fmoc method, namely, a monomer amino acid protected by Fmoc on solid phase resin is deprotected to expose an amino group, and a peptide bond is formed with a carboxyl group of the amino acid in a solution through a condensation reaction, so that the amino acid is connected to the resin, and the peptide chain is extended from a C end to an N end.
1. Basic materials:
(1) resin and linker molecule: the resin selected by the solid phase Fmoc method is Rink Amide-
And (3) resin. The resin has very good swelling property, can better perform condensation reaction between peptide chains, and has enough network space to meet the growing peptide chains. Polypeptide molecules are immobilized on the resin using HBTU and HOBt as linker molecules.
(2) Monomer (b): the monomers used for the synthesis are chemically modified alpha-amino acids.
2. The reaction steps are as follows:
in the first step, the first amino acid is covalently attached to the resin
Adding appropriate condensing agent such as HBTU and HOBt to make the carboxyl terminal of the protected amino acid form co-lipid with resin to complete the fixation of the amino acid;
second, deprotection
The Fmoc on the amino group was removed with 20% piperidine in basic solvent to expose the amino group.
Third step, activation and crosslinking
Activating carboxyl on the next amino group by adopting activating agents HBTU and HOBt, and crosslinking with amino on the resin to form peptide bonds.
And fourthly, repeating the second step and the third step, repeatedly and circularly adding the monomer amino acid, and synthesizing from right to left according to the sequence of the Cy5-LEVLVP until the synthesis is finished.
Fifth, dye labeling
The Fmoc on the amino group was removed with 20% piperidine in basic solvent to expose the amino group. EDC/Cy5/DIEA/HOBT was dissolved in DMF and added to the resin and the mixture was left to react overnight in the dark.
3. And (3) post-synthesis treatment:
(1) elution and deprotection: the peptide chain is eluted from the resin with the deprotection agent trifluoroacetic acid (TFA) and the protecting groups are removed.
(2) HPLC analysis and purification, freeze-drying and storage.
The polypeptide sequence Cy5-LEVLVP was synthesized by the above method.
4. Conversion of lipid-soluble QDs to water-soluble QDs via GSH
mu.L of GSH solution (250. mu.L of methanol solution containing 18mg of GSH and 5mg of KOH) was added to 600. mu.L of oil-soluble QDs (20. mu.M) and shaken vigorously. Then 600. mu.L of aqueous NaOH (1mM) was added and the QDs transferred to the top aqueous layer. The QDs thus obtained were centrifuged and dispersed in 600. mu.L of borate buffer (pH7.4,10mM) for use.
5. Synthesis of the double enzyme probe:
mu.M QDs were incubated for 10min with equal volumes of 8. mu.M Cy5-LEVLVP mixed to form a stable complex Cy 5-LEVLVP-QD.
6. The interaction between hydrolase and double-enzyme probe in capillary is studied:
the capillary was bent into 0, 1, 2 or 4 half circles (diameter 3cm), and in each of these 4 cases, Cy5-LEVLVP-QD 15s was first injected, 10U/ml PSP 15s was injected at intervals of 10s, and the hydrolysis of Cy5-LEVLVP-QD was detected by capillary electrophoresis. When the number of semicircles is 0, S is caused by hydrolysis of the complex in the PSP tube665nm/S605nmThe value of (A) is 1.44 (FIG. 1A, curve b), the PSP cleavage effect is most pronounced when the number of semi-circles is 4, S665nm/S605nmThe value of (d) was reduced to 0.89 (fig. 1A, curve e).
And (3) testing conditions are as follows: the interaction process of the hydrolase and the double-enzyme probe is detected by fluorescence capillary electrophoresis, and the electrophoresis conditions are as follows: 25mM boric acid buffer (pH 9.3) at a voltage of 20 kV.
Example 2
According to the method of example 1, 10U/ml thrombin was hydrolyzed in the capillary with the double enzyme probe.
Similarly, the thrombin cutting effect on the compound becomes more and more obvious with the increase of the number of semicircles, S665nm/S605nmFrom 1.65 (FIG. 1B, curve B) to 1.10 (FIG. 1B, curve e).
Example 3
Bending the capillary into 4 semicircles with a diameter of 3cm, injecting Cy5-LEVLVP-QD 15s, introducing PSP (5U/ml, 10U/ml, 20U/ml or 40U/ml) with different concentrations for 15s after 10s interval, passing through the hairThe tube electrophoresis was used to detect the hydrolysis of Cy 5-LEVLVP-QD. Over a time frame, the increase in enzyme concentration decreases the FRET signal intensity of the dual enzyme probe. Calculating the ratio of the QDs donor detection channel peak area to the Cy5 receptor channel peak area (S)665nm/S605nm) The fitting was performed to yield the curve equation y of 1.378 x exp (-x/11.307) + 0.407. In the equation, y is S665nm/S605nmX is the PSP concentration.
The other steps are the same as in example 1.
Example 4
Thrombin was also detected according to the method of example 3.
Calculating the ratio of the QDs donor detection channel peak area to the Cy5 receptor channel peak area (S)665nm/S605nm) The fitting was performed to obtain the curve equation y of 1.467 x exp (-x/19.063) +0.355, where y is S665nm/S605nmAnd x is the thrombin concentration.
Comparative example 1
The polypeptide Cy5-LEV (Cy 5-D) containing only PSP cleavage sites was synthesized according to the method of example 13LEVLFQGPGP9G2H6) And the bent capillary PSP cleavage Cy5-LEV-QD complex study was performed as in example 1. As a result, it was found that the hydrolysis of the complex formed by the polypeptide of the single cleavage site and QDs in the capillary was also affected by the degree of bending of the capillary (FIG. 4).
Claims (9)
1. A method for detecting multienzyme by bent capillary electrophoresis is characterized in that: combining quantum dots and polypeptide to form a double-enzyme probe, respectively bending a capillary tube used for electrophoresis into semi-circles with different numbers, fixing, firstly sampling the double-enzyme probe, sampling hydrolase after an interval of 10s, and detecting the fluorescence intensity of the double-enzyme probe by a fluorescence spectrometer; the bent capillary tube is a quartz capillary tube;
the polypeptide contains (1) double enzyme cutting sites; (2) a fluorescent dye Cy5 for FRET with the quantum dots; (3) and quantum dot-bound 6 × histidine.
2. The method for detecting multienzyme by bent capillary electrophoresis according to claim 1, wherein: the hydrolase is PreScission protease and thrombin.
3. The method for detecting multienzyme by bent capillary electrophoresis according to claim 1, wherein: the polypeptide contains LEVLFQGP and LVPRGS as double enzyme cutting sites.
4. The method for detecting multienzyme by bent capillary electrophoresis according to claim 1, wherein: the polypeptide sequence is Cy5-D3LEVLFQGPGLVPRGSGP9G2H6。
5. The method for detecting multienzyme by bent capillary electrophoresis according to claim 1, wherein: the quantum dots are quantum dots containing Zn, CdSe/ZnS, CdTe/ZnS, CdSe/ZnSe or CdTe/ZnSe.
6. The method for detecting multienzyme by bent capillary electrophoresis according to claim 1, wherein: the double-enzyme probe is obtained by mixing and incubating 1 mu M quantum dot and 8 mu M polypeptide in equal volume for 10 min.
7. The method for detecting multienzyme by bent capillary electrophoresis according to claim 1, wherein: the inner diameter of the bent capillary tube is 75 micrometers, the outer diameter of the bent capillary tube is 365 micrometers, the number of the bent semicircles is 1-4, and the diameter of the bent semicircles is 3 cm.
8. The method for detecting multiple enzymes by bent capillary electrophoresis as claimed in claim 1, wherein the bent capillary is a 180 ° constant-width circular arc bent capillary.
9. The method for detecting multienzyme by bent capillary electrophoresis as claimed in claim 1, wherein the sample injection time of the double-enzyme probe is 15s, the sample injection time of the hydrolase is 15s, and the sample injection interval of the hydrolase is 10 s.
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