CN102435660A - Microfluidic chip-based probiotics electrophoresis detection method - Google Patents
Microfluidic chip-based probiotics electrophoresis detection method Download PDFInfo
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- CN102435660A CN102435660A CN2011103843551A CN201110384355A CN102435660A CN 102435660 A CN102435660 A CN 102435660A CN 2011103843551 A CN2011103843551 A CN 2011103843551A CN 201110384355 A CN201110384355 A CN 201110384355A CN 102435660 A CN102435660 A CN 102435660A
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Abstract
The invention discloses a microfluidic chip-based probiotics electrophoresis detection method which comprises the steps of: firstly preparing tetrabromoethane-poly ethylene oxide (TBE-PEO) running buffer, preparing bacteria suspending liquid by using the TBE-PEO running buffer, carrying out the step of derivatization, and using a pretreated microfluidic chip to carry out electrophoresis combination laser-induced fluorescence detection. The detection method has the advantages of being simple and easy in operation steps, short in analysis time, less in sample dosage, high in sensitivity, accurate in analysis results and the like, and is applicable to the detection for probiotics in a probiotics product.
Description
Technical field
The invention belongs to the electrophoretic analysis technical field of micro-fluidic chip, relate to a kind of kind of utilizing micro-fluid control chip electrophoretic-LIF coupling technique separation detection probio particularly.
Background technology
Probio is meant human body and animal bacteria beneficial, can bring into play beneficial effect through improving host's gut flora ecologic equilibrium, to improve the host health level.Probiotic composition has been played the part of important role in daily life.Therefore, the kind of probio is particularly important to guarantee its Products Quality in the detection probiotic composition.The detection technique of bacterium has serum method, flow cytometry, protein analysis, composition analysis, signature analysis etc., and above analytical technology has increased workload virtually because of analysis time is long.In addition, because having, bacterium forms the reliability that the characteristics complicated, that similarity is high have reduced analysis result.
The present invention has overcome defectives such as the Bacteria Detection analysis of technology time is long in the prior art, workload is big, the analysis result reliability is limited; Proposed a kind of probio electrophoretic detection, utilized the kind of micro-fluid control chip electrophoretic-LIF coupling technique separation detection probio based on micro-fluidic chip.Advantages such as it is simple that detection method of the present invention has operation steps, and analysis time is short, and amount of samples is few, and is highly sensitive, and analysis result is accurate are applicable to that the probio that carries out in the probiotic composition detects.
Summary of the invention
The present invention proposes a kind of probio electrophoretic detection, may further comprise the steps based on micro-fluidic chip:
The first step prepares running buffer
Tris, boric acid, EDTA be dissolved in obtain the TBE storing solution in the deionized water, add polyethylene oxide and obtain the TBE-PEO storing solution, obtain running buffer, under 4 ℃ of temperature, store for future use through said TBE storing solution dilution and after regulating pH;
Second step preparation bacterial suspension
Probio is suspended in the said running buffer that the first step prepares,, is suspended in again in the said running buffer and obtains bacterial suspension through centrifugal removal supernatant;
The 3rd goes on foot the step of deriving
The said bacterial suspension that second step was prepared mixes with fluorescent dye, and derive in the place in dark, through centrifugal removal supernatant;
The pre-service of the 4th step micro-fluidic chip and microchannel thereof
Adopt sodium hydroxide solution, deionized water, running buffer that micro-fluidic chip is washed successively;
The 5th step electrophoresis detection step
The micro-fluidic chip that the 4th step of employing warp handles is through electrophoresis sample introduction, separation, laser-Induced Fluorescence Detection, and the kind that warp the 3rd is gone on foot probio in the bacterial suspension of handling detects.
Wherein, in the TBE storing solution in the said first step, Tris concentration is 4.0mmol/L, and boric acid concentration is that 4.0mmol/L, EDTA concentration are 0.09mmol/L; The massfraction of polyethylene oxide is 0.5% in the said TBE-PEO storing solution.
Wherein, in the said first step, regulating pH is 8.5.
Wherein, the fluorescent dye in said the 3rd step is SYTO 62.
Wherein, the concentration of the sodium hydroxide solution in said the 4th step is 1mol/L.
Wherein, in said the 5th step, the voltage during the electrophoresis sample introduction is set to the first liquid bath 1300V, the second liquid bath 650V, and the 3rd liquid bath 0V, the 4th liquid bath 1000V continues 15s; Voltage during electrophoretic separation is set to the first liquid bath 1300V, the second liquid bath 2150V, and the 3rd liquid bath 1300V, the 4th liquid bath 0V continues 200s.
Problem to prior art exists the objective of the invention is to release a kind of method based on micro-fluid control chip electrophoretic-LIF coupling separation detection probio kind.This method has advantages such as quick and precisely reliable.
To achieve these goals, the present invention adopts following technical scheme:
The first step prepares running buffer
A certain amount of Tris, boric acid, EDTA are dissolved in the deionized water, obtain concentration and be respectively Tris4.0 mmol/L, the TBE storing solution of boric acid 4.0 mmol/L and EDTA0.09 mmol/L.Take by weighing 0.2 g polyethylene oxide (PEO) and be dissolved in the TBE-PEO storing solution that obtains containing PEO 0.5% among the 40 ml TBE.The TBE-PEO storing solution is obtained running buffer with the dilution of TBE storing solution and after transferring its pH value, be stored in 4 ℃ of refrigerators.With 0.2 μ m filter paper running buffer is filtered before the formal experiment.
Second step preparation bacterial suspension
Get Bifidobacterium, lactobacillus acidophilus, Lactobacillus casei and the enterococcus faecalis of cultivating 22 h respectively; Centrifugal 3 min of 3400rpm; Be suspended in the running buffer after removing nutrient culture media, remove supernatant through centrifugal 3 min of 3400 rpm again, being suspended at last and being made into concentration in the running buffer is 1.0 * 10
7The bacterial suspension of CUF.With the whirlpool appearance whirlpool several seconds, make in the suspending liquid bacterium resuspended before using.
The 3rd goes on foot the step of deriving
Selecting excitation wavelength for use is 635 fluorescent dye, and this dyestuff can permeate through cell membranes labeling nucleic acid (DNA and RNA).The concentration of bacterial suspension is 1.0 * 10
7CUF gets this suspending liquid of 1.5 mL and mixes the back with the fluorescent dye of 3.0 μ l, 5 mmol/L behind 30 min that derive under the dark condition, through centrifugal 3 min of 3400 rpm, removes supernatant.
The pre-service of the 4th step micro-fluidic chip and microchannel thereof
Getting a new cross chip, to use concentration be that the sodium hydroxide solution of 1 mol/L washes 20 min, and deionized water rinsing 10 min wash 10 min with tbe buffer liquid again.Before each experiment, the sodium hydroxide solution with 1mol/L cleans 2 min earlier, uses deionized water rinsing 1 min again, washes 2 min with running buffer at last.After each experiment finishes, wash 3 min with absolute ethyl alcohol.
The 5th step electrophoresis detection step
The micro-fluidic chip that the 4th step of employing warp handles is through electrophoresis sample introduction, separation, laser-Induced Fluorescence Detection, and the kind that warp the 3rd is gone on foot probio in the bacterial suspension of handling detects.Wherein, the voltage during the electrophoresis sample introduction is set to the first liquid bath 1300V, the second liquid bath 650V, and the 3rd liquid bath 0V, the 4th liquid bath 1000V continues 15s; Voltage during electrophoretic separation is set to the first liquid bath 1300V, the second liquid bath 2150V, and the 3rd liquid bath 1300V, the 4th liquid bath 0V continues 200s.
Technical scheme of the present invention is further characterized in that, the pH=8.5 of running buffer, and the concentration of used polyethylene oxide PEO is 0.025 %.
Technical scheme of the present invention is further characterized in that the whirlpool time is 60-90 second.
Technical scheme of the present invention is further characterized in that fluorescent reagent is SYTO 62.
The present invention adopts SYTO 62 dyestuffs that Bifidobacterium, lactobacillus acidophilus, Lactobacillus casei and enterococcus faecalis are derived, and in the running buffer of TBE-PEO preparation, carries out separation detection.This method has the following advantages: operation steps is simple, and analysis time is short, and amount of samples is few, and is highly sensitive, and analysis result is accurate.The inventive method can be used for the analyzing and testing to actual product (as: sour milk, candy, medicine etc.) probio.
Description of drawings
Shown in Figure 1 is the microfluidic chip structure synoptic diagram.The 1st, sample cell, the 2nd, buffer pool, the 3rd, sample waste liquid pool, the 4th, damping fluid waste liquid pool, the 5th, sample channel, the 6th, sample intake passage, the 7th, microchannel, the 8th, microchannel.
Testing result synoptic diagram for utilizing the inventive method that four kinds of probios are detected shown in Figure 2.
Embodiment
In conjunction with following specific embodiment and accompanying drawing, the present invention is done further detailed description, protection content of the present invention is not limited to following examples.Under spirit that does not deviate from inventive concept and scope, variation and advantage that those skilled in the art can expect all are included among the present invention, and are protection domain with the appending claims.
The present invention is based on the probio electrophoretic detection of micro-fluidic chip, may further comprise the steps:
The first step prepares running buffer
A certain amount of Tris, boric acid, EDTA are dissolved in the deionized water, obtain concentration and be respectively Tris4.0 mmol/L, the TBE storing solution of boric acid 4.0 mmol/L and EDTA0.09 mmol/L.Take by weighing 0.2 g polyethylene oxide (PEO) and be dissolved in the TBE-PEO storing solution that obtains containing PEO 0.5% among the 40 ml TBE.The TBE-PEO storing solution is obtained running buffer with the dilution of TBE storing solution and after transferring its pH value, be stored in 4 ℃ of refrigerators.With 0.2 μ m filter paper running buffer is filtered before the formal experiment.
Second step preparation bacterial suspension
Get Bifidobacterium, lactobacillus acidophilus, Lactobacillus casei and the enterococcus faecalis of cultivating 22 h respectively; Centrifugal 3 min of 3400rpm; Be suspended in the running buffer after removing nutrient culture media, remove supernatant through centrifugal 3 min of 3400 rpm again, being suspended at last and being made into concentration in the running buffer is 1.0 * 10
7The bacterial suspension of CUF.With the whirlpool appearance whirlpool several seconds, make in the suspending liquid bacterium resuspended before using.
The 3rd goes on foot the step of deriving
Selecting excitation wavelength for use is 635 fluorescent dye, and this dyestuff can permeate through cell membranes labeling nucleic acid (DNA and RNA).The concentration of bacterial suspension is 1.0 * 10
7CUF gets this suspending liquid of 1.5 mL and mixes the back with the fluorescent dye of 3.0 μ l, 5 mmol/L behind 30 min that derive under the dark condition, through centrifugal 3 min of 3400 rpm, removes supernatant.
The pre-service of the 4th step micro-fluidic chip and microchannel thereof
Getting a new cross chip, to use concentration be that the sodium hydroxide solution of 1 mol/L washes 20 min, and deionized water rinsing 10 min wash 10 min with tbe buffer liquid again.Before each experiment, the sodium hydroxide solution with 1mol/L cleans 2 min earlier, uses deionized water rinsing 1 min again, washes 2 min with running buffer at last.After each experiment finishes, wash 3 min with absolute ethyl alcohol.
The 5th step electrophoresis detection step
The micro-fluidic chip that the 4th step of employing warp handles is through electrophoresis sample introduction, separation, laser-Induced Fluorescence Detection, and the kind that warp the 3rd is gone on foot probio in the bacterial suspension of handling detects.Wherein, the voltage during the electrophoresis sample introduction is set to the first liquid bath 1300V, the second liquid bath 650V, and the 3rd liquid bath 0V, the 4th liquid bath 1000V continues 15s; Voltage during electrophoretic separation is set to the first liquid bath 1300V, the second liquid bath 2150V, and the 3rd liquid bath 1300V, the 4th liquid bath 0V continues 200s.
Embodiment
Present embodiment concrete operations step is fully according to the method described above operated, and same steps as repeats no more.
In the first step, regulate the pH=8.5 of running buffer, the concentration of PEO is 0.025 %; In second step, the whirlpool time is 60-90 second; In the 3rd step, fluorescent dye is SYTO 62.
The 5th step of present embodiment is following to the detection step of four kinds of probio electrophoretic analysiss:
The present invention is applicable to the bacterial species check and analysis of probio.In the present embodiment, the concentration of choosing Bifidobacterium (Bifidobacterium), lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus casei (Lactobacillus casei) and enterococcus faecalis (Enterococccus faecalis) mixed liquor is 1.0 * 10
7CUF.
At first, in four liquid baths of micro-fluidic chip, add running buffer respectively, treat to change the damping fluid in the first liquid bath sample cell 1 into sample after baseline steadily; Adopt compression sample introduction voltage to be set to sample introduction voltage, as shown in Figure 1, sample gets into the 3rd liquid bath sample waste liquid pool 3 by liquid bath 1 through sample channel 5, sample intake passage 6; Convert separation voltage to after 15 seconds; Sample in microchannel 7 separated after, 8 places are to be detected in the microchannel, the sample after the separation gets into the 4th liquid bath damping fluid waste liquid pool 4.Concrete electrophoretic separation condition sees the following form:
Laser-induced fluorescence detection system: it is 635nm that light source adopts emission wavelength, the semiconductor laser of power 4 mW.Laser beam is perpendicular to the plane of DNA micro-fluidic chip, and (635 ± 10 nm, rich optics incorporated company converges in Shenyang) filter through preposition bandpass filter, are converged at 8 places, microchannel of DNA micro-fluidic chip by condenser lens.Laser instrument wherein, preposition bandpass filter, the condenser lens three constitutes integrated module.Fluorescence detecting system is by collecting lens, aperture, and bandpass filter (670 ± 10 nm, rich optics incorporated company converges in Shenyang), photomultiplier (R928 type, Binsong Photon Technology Co., Ltd. Beijing) four parts are formed.On the inclined to one side 45 ° of directions of DNA micro-fluidic chip quadrature, detect the fluorescence of outgoing.
High voltage power supply: four contact high pressure outputs can be provided, and every contact voltage output (scope 0-6000 V) can be controlled separately.Control system comprises hardware and software two parts.The fluorescence signal that photomultiplier is collected is handled through signals collecting and amplification module, is input to external computing machine, carries out data processing and demonstration by computing machine through Chip Microcomputer A/D conversion.All parameter settings, control and the signals collecting of instrument are all through RS232 Serial Port Line and computer realization communication.
The present embodiment experimental result is as shown in Figure 2; The fluorescence peak that on fluorescence spectrum figure, has presented Bifidobacterium, lactobacillus acidophilus, Lactobacillus casei and enterococcus faecalis respectively; This shows that it is that the running buffer that 8.5 TBE-PEO storing solution is processed has carried out good separation and check and analysis with four kinds of probios that detection method of the present invention is utilized pH.
Method of the present invention is particularly suitable for microorganisms such as separation detection bacterium, and is simple to operate and quick and precisely.
Claims (6)
1. the probio electrophoretic detection based on micro-fluidic chip is characterized in that, said method comprising the steps of:
The first step prepares running buffer
Tris, boric acid, EDTA be dissolved in obtain the TBE storing solution in the deionized water, add polyethylene oxide and obtain the TBE-PEO storing solution, obtain running buffer, under 4 ℃ of temperature, store for future use through said TBE storing solution dilution and after regulating pH;
Second step preparation bacterial suspension
Probio is suspended in the said running buffer that the first step prepares,, is suspended in again in the said running buffer and obtains bacterial suspension through centrifugal removal supernatant;
The 3rd goes on foot the step of deriving
The said bacterial suspension that second step was prepared mixes with fluorescent dye, and derive in the place in dark, through centrifugal removal supernatant;
The pre-service of the 4th step micro-fluidic chip and microchannel thereof
Adopt sodium hydroxide solution, deionized water, running buffer that micro-fluidic chip is washed successively;
The 5th step electrophoresis detection step
The micro-fluidic chip that the 4th step of employing warp handles is through electrophoresis sample introduction, separation, laser-Induced Fluorescence Detection, and the kind that warp the 3rd is gone on foot probio in the bacterial suspension of handling detects.
2. the probio electrophoretic detection based on micro-fluidic chip according to claim 1 is characterized in that, in the TBE storing solution in the said first step, Tris concentration is 4.0mmol/L, and boric acid concentration is that 4.0mmol/L, EDTA concentration are 0.09mmol/L; The massfraction of polyethylene oxide is 0.5% in the said TBE-PEO storing solution.
3. the probio electrophoretic detection based on micro-fluidic chip according to claim 1 is characterized in that, in the said first step, regulating pH is 8.5.
4. the probio electrophoretic detection based on micro-fluidic chip according to claim 1 is characterized in that, the fluorescent dye in said the 3rd step is SYTO 62.
5. the probio electrophoretic detection based on micro-fluidic chip according to claim 1 is characterized in that, the concentration of the sodium hydroxide solution in said the 4th step is 1mol/L.
6. the probio electrophoretic detection based on micro-fluidic chip according to claim 1 is characterized in that, in said the 5th step; Voltage during the electrophoresis sample introduction is set to the first liquid bath 1300V, the second liquid bath 650V, the 3rd liquid bath 0V; The 4th liquid bath 1000V continues 15s; Voltage during electrophoretic separation is set to the first liquid bath 1300V, the second liquid bath 2150V, and the 3rd liquid bath 1300V, the 4th liquid bath 0V continues 200s.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102961966A (en) * | 2012-11-29 | 2013-03-13 | 西安建筑科技大学 | Method for specific continuous separation of micro-scale particles |
| CN103234949A (en) * | 2013-01-30 | 2013-08-07 | 大连海事大学 | Microalgae activity detection method and device in ship ballast water |
| CN106940305A (en) * | 2016-01-05 | 2017-07-11 | 中国科学院苏州纳米技术与纳米仿生研究所 | Antibiotic detection means and detection method based on micro-fluidic chip |
-
2011
- 2011-11-28 CN CN2011103843551A patent/CN102435660A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| ORATHAI LIM等: "Capillary zone electrophoresis for enumeration of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in yogurt", 《JOURNAL OF CHROMATOGRAPHY B》 * |
| SHUANG CHENG等: "Rapid separation of four probiotic bacteria in mixed samples using microchip electrophoresis with laser-induced fluorescence detection", 《MICROCHIM ACTA》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102961966A (en) * | 2012-11-29 | 2013-03-13 | 西安建筑科技大学 | Method for specific continuous separation of micro-scale particles |
| CN103234949A (en) * | 2013-01-30 | 2013-08-07 | 大连海事大学 | Microalgae activity detection method and device in ship ballast water |
| CN106940305A (en) * | 2016-01-05 | 2017-07-11 | 中国科学院苏州纳米技术与纳米仿生研究所 | Antibiotic detection means and detection method based on micro-fluidic chip |
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Application publication date: 20120502 |