CN101556260B - Method for detecting microorganism isoelectric point by adopting immobilized pH gradient capillary isoelectric focusing (CIEF) - Google Patents
Method for detecting microorganism isoelectric point by adopting immobilized pH gradient capillary isoelectric focusing (CIEF) Download PDFInfo
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- CN101556260B CN101556260B CN200910084833XA CN200910084833A CN101556260B CN 101556260 B CN101556260 B CN 101556260B CN 200910084833X A CN200910084833X A CN 200910084833XA CN 200910084833 A CN200910084833 A CN 200910084833A CN 101556260 B CN101556260 B CN 101556260B
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Abstract
The invention relates to a method for detecting microorganism isoelectric point by adopting immobilized pH gradient capillary isoelectric focusing (CIEF), belonging to the field of bioanalysis. The method comprises the steps of: firstly carrying out isoelectric focusing on carrier ampholyte solution by adopting constant voltage in a capillary to lead ampholyte with continuous pH gradient to be formed on the inner wall of the capillary; secondly injecting sample solution to the capillary and carrying out isoelectric focusing by adopting the continuous voltage; then adopting immobilized atmospheric pressure to push the sample from focusing section inside the capillary to pass a detecting window at equal speed so as to determine focus point position of the sample; and finally figuring out pH value of the focus point according to linear relation between the pH gradient and the length of the capillary. The method can be applied to the simultaneous online detection of various microorganisms and has universality for microorganism isoelectric point detection.
Description
Technical field
The present invention belongs to the bioanalysis field for the method for immobilized pH gradient capillary isoelectric focusing mensuration microorganism isoelectric point.
Background technology
Microorganism has surface charge and both sexes characteristic, has isoelectric point (pI).Microorganism surface charge and isoelectric point characteristic are to be determined by microbial cell film or the surperficial equimolecular overall charged character of amino acid residue, glycoprotein and lipid of cell membrane.Therefore, all available its surface charge characteristic of microorganism and the isoelectric point characteristic when different microorganisms and different growth metabolism state characterizes.The charged characteristic of research microorganism surface charge can characterize the physio-biochemical characteristics of microorganism self, and the biochemical reaction of studying its growth metabolism state, detection and sign microorganism surface etc. is had important theory and using value.Still do not confirm the assay method report of microorganism isoelectric point in the biological study at present.
Capillary isoelectric focusing can carry out the separation and the evaluation of protein based on the difference of target molecule isoelectric points such as protein.There is report that the capillary isoelectric focusing electrophoresis is applied to the microorganism correlative study in recent years.The report method is that microbiological specimens and isoelectric point label such as are carried out jointly at point focusing, with the different microorganism of initial gross separation isoelectric point, and according to its isoelectric point scope of relative migration time estimation of microbiological specimens and isoelectric point label.Because of used ampholyte has very strong ultraviolet background absorption; And ampholyte is skewness in kapillary; Ultraviolet detection jitter and the high background noise of microorganism be will cause if use the migration in pipe of EOF driving microorganism to wait, the stability and the sensitivity of microorganism detection therefore reduced greatly.The assay method and the report that biologically still do not have at present general definite microorganism isoelectric point.
Summary of the invention
The objective of the invention is in order to solve microorganism problem, and provide a kind of capillary isoelectric focusing of immobilization pH gradient to measure the method for microorganism isoelectric point.
The objective of the invention is to realize through following technical scheme:
The capillary isoelectric focusing of a kind of immobilization pH gradient of the present invention is measured the method for microorganism isoelectric point, and concrete measuring process is:
1) at first kapillary is carried out pre-service; Above with 0.1M HCl flushing 10min successively, water flushing 5min is above, 1M NaOH flushing 20min is above, 0.1M NaOH flushing 20min is above, water flushing 20min is above and more than the washed with methanol 20min, it is subsequent use that nitrogen dries up the back; Isocyanates added inject kapillary behind the anhydrous pyridine solution, more than 12 hours, make isocyanates be bonded to capillary tube inner wall 65～75 ℃ of reactions; Use toluene more successively, methyl alcohol, tri-distilled water, the phosphate buffer flushing capillary tube inner wall of 20mM pH7.0, each washes more than the 10min:
Wherein kapillary is quartzy fusion kapillary, internal diameter 50～200 μ m, and length 20～100cm, the one of which end is fired detection window, and through the UV-detector test sample, isocyanates and anhydrous pyridine solution proportion scope are 0.3: 5～10.
2) carrier ampholyte solution A mpholine is dissolved in the 20mM phosphate buffer with 1%～5% volume ratio, mixed solution is injected kapillary; To insert the negative electrode damping fluid near an end of detection window; The other end inserts anode buffer liquid; The constant voltage that adds 10～25KV is then carried out isoelectric focusing, treats to focus on completion after the current stabilization, and the kapillary two ends are inserted in the deionized water; Standing and reacting made the ampholyte that forms continuous pH gradient on the capillary tube inner wall more than 12 hours:
The NaOH solution of negative electrode damping fluid 15～20mM wherein, anode buffer liquid is the H of 15～20mM
3PO
4Solution.
3) will go up the kapillary that makes of step washes more than the 1h with the phosphate buffer of pH 7.0; Being prepared into concentration in the phosphate buffer with sample microorganism adding pH7.0 is 104～105cfu/ml sample solution, and sample solution is injected kapillary, adopts the constant voltage isoelectric focusing of 10～25KV; After treating that electric current steadily, focus on and accomplish, stop making alive; Use fixedly air pressure, sample in the kapillary is begun to be pressurized to the time through detection window from focusing at the uniform velocity promotion process of a section inspection, writing down to sample.
4) re-inject sample; Obtaining the fixedly air pressure that adopts in the step 3) makes sample flow through time of detection window from the kapillary injection side; Because capillary pipe length is known, depresses the at the uniform velocity migration velocity of sample solution in kapillary thereby obtain this fixed air, the sample that obtains according to step 3) very much again begins to be pressurized to the time through detection window; Obtain this fixed air depress sample from focus point to through the distance of detection window, it is poor to the distance L of entrance point to try to achieve focus point;
5), obtain the representation formula of the pH value of focus point according to the linear relationship of pH gradient and long capillary tube:
PH asks=pH beginning+pH poor (L poor/L is total)
Wherein the pH minimum value of carrier ampholyte solution is selected in " pH beginning " expression for use, and the pH range differences of carrier ampholyte solution is selected in " pH is poor " expression for use; " L is poor " expression focus point is to the distance of entrance point; " L is total " representes total length capillaceous;
" L is poor " substitution linear equation of 6) step 4) being tried to achieve can be obtained the pH value that sample is pointed out in focusing.
The invention has the beneficial effects as follows
1) adopts immobilization pH gradient, can form the pH gradient of steady and continuous, avoid in solution, using ampholyte to form the dynamic ph gradient, avoid the use of the pI label and manage interior isoelectric point demarcation at capillary tube inner wall.The interference of the common migration of ampholyte to microorganism detection avoided in the migration of pressure-driven sample.
2) use air pressure to drive sample and move, need not use electric field and EOF to drive, the sample migration velocity is accelerated, shorten detection time, improve analysis efficiency.
3) this method adopts chemical bonding to connect ampholyte, and ampholyte is fixed on capillary tube inner wall, does not drive with air pressure and moves, and it is steady therefore to detect baseline.PH isoelectric focusing after the immobilization does not need in kapillary, to add ampholyte, has therefore improved the ultraviolet detection sensitivity to microorganism.
4) under particular analysis instrument and condition (fixed capillary length, effective length, focus voltage, air pressure etc.), only needing to measure an analytical parameters is the time that microbiological specimens migrates to detection window, just can calculate its isoelectric point.Assay method is simply quick, accurately the test sample isoelectric point.Can be used for the online detection simultaneously of multiple microorganism, detection has universality for the microorganism isoelectric point.
Description of drawings
Fig. 1: the schematic diagram that detects microorganism migration velocity in kapillary;
Fig. 2: the schematic diagram that detects the microorganism isoelectric point;
1-detection window wherein.
Embodiment
With fused quartz kapillary (100 μ m i.d. kapillary column lengths 38.5; Effective length 30cm; Detection window is apart from endpiece 8.5cm) successively with 0.1M HCl flushing 10min, water flushing 5min, 1M NaOH flushing 20min, 0.1M NaOH flushing 20min, water flushing 20min and washed with methanol 20min, it is subsequent use that nitrogen dries up the back.The 0.3ml isocyanates is added the 5ml anhydrous pyridine, inject kapillary with manual pump behind the mixing, 70 ℃ are reacted 12h down, make isocyanates be bonded to capillary tube inner wall.After the reaction, use toluene successively, methyl alcohol, tri-distilled water, phosphate buffer (PBS, 20mM pH 7.0) respectively washes 10min towards kapillary.Inject carrier ampholyte solution (Ampholine pH 3.5-10.0 is dissolved in the 20mM phosphate-buffered liquor with 4% volume ratio) with manual pump to capillary tube inner wall.The negative electrode damping fluid is a 20mM NaOH solution, and anode buffer liquid is 20mM H
3PO
4Solution, 15kV focuses on down.It is steady to focus on the 10min after-current, focuses on to accomplish, and the kapillary two ends are sealed with tri-distilled water, leaves standstill 12h and reacts, and makes the pH gradient be fixed on capillary tube inner wall.
E.coli JM83 bacterial classification is received in the common LB nutrient culture media, and 37 ℃ of shaking tables are cultivated 12h.(3400r/min, 5min), abandoning supernatant adds deionized water wash to cultured sample through the hydro-extractor separation.Centrifugal, the washing 3 times, to remove residual nutrient culture media.The microbiological specimens of deposition is processed bacteria suspension with the PBS damping fluid, i.e. the viable bacteria microbiological specimens.
Add small amount of sample at immobilized pH gradient capillary post entrance point, add 50mbar air pressure, the start-of-record pressurization promotion time, the record sample arrives the time t=3.1min of detection window.The distance L of known kapillary entrance point distance detecting window '=30cm, can know migration velocity the V=L '/t=30cm/3.1min=9.68cm/min of sample uniform motion under the constant air pressure conditions.(Fig. 1)
Under constant air pressure conditions, microbiological specimens is filled with kapillary from the capillary sample inlet end, the isoelectric focusing of power 15kV voltage, it is stable to focus on the 10min after-current, focuses on to accomplish, and stops voltage.Use the 50mbar constant pressure, promote sample process detection window in the kapillary.Is T=1.2min from beginning to be pressurized to microbiological specimens through time of detection window.But the distance of calculation sample focus point distance detecting window is L "=V * T=9.6 * 1.2=11.6cm.(Fig. 2)
The pH gradient that has the linearity of stable continuous in the kapillary of isoelectric focusing, promptly there are linear relationship in capillary pipe length and pH gradient.Kapillary column length in the experiment after the immobilization of pH gradient is 38.5cm, pH scope 3.5～10, and obtaining linear relationship is y=0.1688x+3.5.With X=L-L "=30-11.6=18.4cm brings above-mentioned linear equation into, and trying to achieve the corresponding pH value of focus point Y is 6.6, and promptly the sample isoelectric point is 6.6.
Claims (2)
1. the capillary isoelectric focusing of immobilization pH gradient is measured the method for microorganism isoelectric point, it is characterized in that concrete measuring process is following:
1) at first kapillary is carried out pre-service; Above with 0.1M HCl flushing 10min successively, water flushing 5min is above, 1M NaOH flushing 20min is above, 0.1M NaOH flushing 20min is above, water flushing 20min is above and more than the washed with methanol 20min, it is subsequent use that nitrogen dries up the back; Isocyanates added inject kapillary behind the anhydrous pyridine solution, more than 12 hours, make isocyanates be bonded to capillary tube inner wall 65～75 ℃ of reactions; Use toluene more successively, methyl alcohol, tri-distilled water, the phosphate buffer flushing capillary tube inner wall of 20mM pH7.0, each washes more than the 10min;
2) carrier ampholyte solution A mpholine is dissolved in the 20mM phosphate buffer with 1%～5% volume ratio, mixed solution is injected kapillary; To insert the negative electrode damping fluid near an end of detection window; The other end inserts anode buffer liquid; The constant voltage that adds 10～25KV is then carried out isoelectric focusing, treats to focus on completion after the current stabilization, and the kapillary two ends are inserted in the deionized water; Standing and reacting made the ampholyte that forms continuous pH gradient on the capillary tube inner wall more than 12 hours;
The NaOH solution of negative electrode damping fluid 15～20mM wherein, anode buffer liquid is the H of 15～20mM
3PO
4Solution;
3) will go up the kapillary that makes of step washes more than the 1h with the phosphate buffer of pH 7.0; Being prepared into concentration in the phosphate buffer with sample microorganism adding pH7.0 is 10
4～10
5The cfu/ml sample solution injects kapillary with sample solution, adopts the constant voltage isoelectric focusing of 10～25KV; After treating that electric current steadily, focus on and accomplish, stop making alive; Use fixedly air pressure, sample in the kapillary is begun to be pressurized to the time through detection window from focusing at the uniform velocity promotion process of a section inspection, writing down to sample;
4) re-inject sample; Obtaining the fixedly air pressure that adopts in the step 3) makes sample flow through time of detection window from the kapillary injection side; Because capillary pipe length is known, depresses the at the uniform velocity migration velocity of sample solution in kapillary thereby obtain this fixed air, the sample that obtains according to step 3) very much again begins to be pressurized to the time through detection window; Obtain this fixed air depress sample from focus point to through the distance of detection window, it is poor to the distance L of entrance point to try to achieve focus point;
5), obtain the representation formula of the pH value of focus point according to the linear relationship of pH gradient and long capillary tube:
PH asks=pH beginning+pH poor (L poor/L is total)
Wherein the pH minimum value of carrier ampholyte solution is selected in " pH beginning " expression for use, and the pH range differences of carrier ampholyte solution is selected in " pH is poor " expression for use: " L is poor " expression focus point is to the distance of entrance point; " L is total " representes total length capillaceous;
" L is poor " substitution linear equation of 6) step 4) being tried to achieve can be obtained the pH value that sample is pointed out in focusing.
2. the capillary isoelectric focusing of immobilization pH gradient as claimed in claim 1 is measured the method for microorganism isoelectric point; It is characterized in that: wherein kapillary is quartzy fusion kapillary; Internal diameter 50～200 μ m; Length 20～100cm, the one of which end is fired detection window, through the UV-detector test sample; Isocyanates and anhydrous pyridine solution proportion scope are 0.3: 5～10.
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| CN104034789A (en) * | 2013-04-02 | 2014-09-10 | 上海交通大学 | Protein isoelectric focusing electrophoresis device |
| CN104788536A (en) * | 2015-04-28 | 2015-07-22 | 扬州大学 | Testing device for isoelectric focusing separation of amphoteric electrolyte and application method of testing device |
| WO2018057885A1 (en) * | 2016-09-23 | 2018-03-29 | University Of Notre Dame Du Lac | One-step capillary isoelectric focusing and mobilization of analytes |
| CN109675344A (en) * | 2018-12-26 | 2019-04-26 | 上海交通大学 | Solidify the capillary isoelectric focusing hydrophily integral post and preparation method of pH gradient |
| CN109718576A (en) * | 2018-12-26 | 2019-05-07 | 上海交通大学 | Solidify the capillary isoelectric focusing open tubular column and preparation method thereof of pH gradient |
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| CN1032661A (en) * | 1987-10-23 | 1989-05-03 | 先灵公司 | Method of purifying protein |
| EP0617048A1 (en) * | 1993-03-26 | 1994-09-28 | Waters Investments Limited | Method of capillary isoelectric focusing of proteins and peptides with fraction collection for post-run analysis |
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Patent Citations (7)
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| CN1032661A (en) * | 1987-10-23 | 1989-05-03 | 先灵公司 | Method of purifying protein |
| EP0617048A1 (en) * | 1993-03-26 | 1994-09-28 | Waters Investments Limited | Method of capillary isoelectric focusing of proteins and peptides with fraction collection for post-run analysis |
| JP2828426B2 (en) * | 1995-03-31 | 1998-11-25 | 株式会社分子バイオホトニクス研究所 | Isoelectric focusing marker for fluorescence detection |
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