CN104531504A - Micro-fluidic chip device for separating bacteria by using laminar flow and chemotaxis effects - Google Patents
Micro-fluidic chip device for separating bacteria by using laminar flow and chemotaxis effects Download PDFInfo
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- CN104531504A CN104531504A CN201410783551.XA CN201410783551A CN104531504A CN 104531504 A CN104531504 A CN 104531504A CN 201410783551 A CN201410783551 A CN 201410783551A CN 104531504 A CN104531504 A CN 104531504A
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Abstract
The invention belongs to the field of micro-fluidic chips, and discloses a micro-fluidic chip device for separating bacteria by using laminar flow and chemotaxis effects. The device comprises two parts namely a micro-fluidic chip for separating the bacteria and an inducing reagent concentration control system, wherein the micro-fluidic chip is an approximately 'X'-shaped PDMS-glass chip which is integrated with an inducing reagent inlet, a bacterium inlet, a bacterium separation channel, a bacterium collection outlet and a waste liquid outlet; and the inducing reagent concentration control system consists of a plurality of groups of injection pumps and injectors, parameters such as flow rates and the like can be controlled by virtue of the injection pumps, and thus an inducing reagent of which the concentration dynamically changes with time can be obtained. According to the micro-fluidic chip device disclosed by the invention, operations of separating interested bacteria from a bacterial mixed liquid can be achieved by mainly using the characteristics that the bacteria swim with tendency to high attractant concentration and low repellent concentration directions under the action of laminar flows and chemical inducing reagents. The micro-fluidic chip device disclosed by the invention can be applied to research and analysis of the fields of food safety, water quality monitoring, pharmaceutical engineering, clinical medicines and the like.
Description
Technical field
The invention belongs to biological micro-fluidic chip field, be specifically related to a kind of micro flow control chip device utilizing laminar flow and chemotaxis separation of bacterial.
Background technology
Bacteria distribution refers to the process isolating bacterium interested from the mixture or various bacteria mixture of bacterium and cell, and it is all of great significance on foodstuffs industry, water quality detection, biotechnology, pharmaceutical industry and clinical medicine.
Traditional bacteria distribution carries out in nutrient agar, and culture condition is harsh, time-consuming, and separation and Culture process is subject to the pollution of miscellaneous bacteria, therefore cultivates Chang Buneng acquisition desired result based on the bacteria distribution of this method.In recent years, the development of microflow control technique makes the sharp separation of bacterium become possibility.Utilize the swimming of bacterium in laminar flow and rolling characteristic, people establish the micro-fluidic chip of multiple separation of bacterial, but the separation efficiency of these bacteria distribution chips is still on the low side.Therefore, set up one fast and efficiently bacterial isolation method seem particularly important.
There is the bacterium of motor capacity, its film surface exists and variously has narrow spectrum chemoreceptor, bacterium can experience the change of surrounding chemical inductor concentration by them, and by the signal transduction system in born of the same parents, the chemical information sensed is transformed into intracellular signal, and then by the direction of motion of this signal control bacterial flagellum, produce corresponding Sperm chemotactic response, go after profits and advoid disadvantages.When running into chemical attractant, bacterium is to the direction straight line swimming of high density attractant.Otherwise run into and drive when scolding agent, bacterium produces tumbling motion and immediately along driving the direction swimming of scolding agent concentration gradient to successively decrease.When attractant or drive scold the concentration signal of agent to change in time time, the motion of bacterium also presents the characteristic with strength of signal and frequency dependent.This chemotaxis behavior of bacterium makes application microflow control technique carry out bacteria distribution fast and efficiently becomes possibility.
Based on this, the present invention proposes a kind of micro flow control chip device based on fluid mechanics principle accuracy controlling inductor concentration gradient environment, utilizes the chemotaxis behavior of bacterium in laminar flow to be separated fast and efficiently by bacterium.
Summary of the invention
Purpose of design of the present invention be to provide a kind of fast, the micro flow control chip device of high efficiency separation bacterium, microflow control technique is utilized to carry out fixing quantity to the time gradient of chemical induction reagent (attractant and drive scold agent) concentration and spatial gradient in design, in conjunction with the swimming of bacterium in laminar flow, rolling and chemotactic characteristic, realize the bacteria distribution operation under micro-dimension.
In the present invention, this device comprises micro-fluidic chip and induction agent concentration control system two portions (Fig. 1) of separation of bacterial.Wherein, micro-fluidic chip be a kind ofly be integrated with induction agent entrance, PDMS-glass-chip (Fig. 2) that bacterium entrance, bacteria distribution passage, bacterium collect approximate " X " type of outlet and waste liquid outlet.
Technical scheme of the present invention is as follows:
A kind of micro flow control chip device utilizing laminar flow and chemotaxis separation of bacterial, this micro flow control chip device comprises: induction agent A entrance, bacterium entrance, induction agent B entrance, induction agent A injection channel, bacterium injection channel, induction agent B injection channel, bacteria distribution passage, bacterium output channel, waste liquid output channel, bacterium collects outlet and waste liquid outlet.Wherein, bacteria distribution passage is a sliver transvers section is flat rectangular passage, its height H much smaller than width W and length L and size in micron or millimeter magnitude, entrance communicates with induction agent A injection channel, bacterium injection channel and induction agent B injection channel, and outlet communicates with bacterium output channel and waste liquid output channel.
Induction agent entrance, bacterium entrance, induction agent injection channel, bacterium injection channel, bacteria distribution passage, bacterium output channel, waste liquid output channel, bacterium collection outlet and waste liquid outlet composition are similar to the PDMS-glass-chip (Fig. 2) of " X " type.
In the present invention (Fig. 2), the width design of induction agent A injection channel and induction agent B injection channel is consistent; In addition, bacterium injection channel width is less than induction agent injection channel width, and bacterium can be contacted fully with induction agent, thus improves the accuracy rate be separated.Geometrical dimension liquid motion speed in the bacteria distribution passage of micron and millimeter magnitude is slow, presents laminar flow characteristics, and ignore the motion of fluid in y-axis direction and change, can obtain the flow velocity of solution in X-type microchannel according to Poiseuille's law is:
Wherein p is pressure, and μ is soltion viscosity coefficient.To above formula integration along the z-axis direction, the flow Q that can obtain unit width meets:
In the approximately equalised situation of viscous modulus of hypothesis induction agent A, induction agent B and bacterium mixed solution, induction agent A, induction agent B are equal with the flow of bacterium mixed solution on unit width, that is:
Wherein Q
a, Q
band Q
0represent the flow of induction agent A, induction agent B and bacterium mixed solution respectively, W
1, W
2and W
3represent the width that induction agent A, bacterium mixed solution and induction agent B are shared in bacteria distribution passage respectively.
From deriving above, the line of delimitation, flow field (Fig. 3 chain lines) of induction agent A, induction agent B and bacterium mixed solution and the flow Q of induction agent A, induction agent B and bacterium mixed solution in bacteria distribution passage
a, Q
band Q
0ratio is relevant, controls this throughput ratio and can change marginal position, flow field.
Based on the laminar flow characteristics of above-mentioned fluid, after induction agent A, induction agent B and bacterium mixed solution enter bacteria distribution passage, demixing phenomenon will be produced.If when not having chemical inducer, dynamic bacterium is by first straight line swimming one segment distance reposefully, and then rolling suddenly once changes direction of motion, then swimming forward, then rolls.According to the rolling characteristic of different bacterium kind macroscopically by the swimming of generation two kinds of different directions: a bacterioid moves about in the left side be tending towards to fluid flowing, embodies left swimming characteristics; Another kind is then have right swimming characteristics.This two type games makes bacterium near mixed solution and induction agent laminar flow line of delimitation, produce the diffusional effect of macroscopic view.Once run into chemical induction reagent, this free swimming rolling characteristic of bacterium will be destroyed.When running into attractant, by the direction straight line swimming to high density attractant, there is not rollover phenomenon, show as forward chemotaxis in bacterium; Otherwise run into and drive when scolding agent, bacterium can produce tumbling motion immediately and along driving the direction swimming of scolding agent concentration gradient to successively decrease, embody negative sense chemotaxis.Obviously, induction agent A and induction agent B is chosen as reasonably to drive respectively and scolds agent or attractant, the transverse movement of bacterium can be strengthened, and then improve the separation efficiency of bacterium.Particularly, when bacterium shows as left swimming characteristics, induction agent A elects attractant as, and induction agent B scolds agent for driving; Otherwise when it has right swimming characteristics, induction agent A then selects to drive to scold agent, and induction agent B is attractant.Like this, the bacterium of the separable different sorts different qualities of this micro-fluidic chip is utilized.
Because bacterium easily adapts to the environment that induction agent concentration remains unchanged for a long period of time, also according to actual needs, induction agent concentration control system can be utilized dynamically to change attractant and drive and to scold agent concentration over time.Induction agent concentration control system comprises: many group syringe pumps and syringe, and syringe comprises solute syringe, solvent injection device, and syringe is connected with syringe pump, by arranging the parameter such as flow, speed in syringe pump, and then controls the inject state of syringe.Solute syringe and solvent injection device are connected to three-way interface by bilateral interface and silicone tube, form the device that can generate dynamic concentration, then it is connected to corresponding micro-fluidic chip ingress by bilateral interface, silicone tube.By arranging different solutes, the flow of solvent, speed parameter, obtain the induction agent solution of different concns, namely concentration can the induction agent solution of dynamic change in time.
The principle of the induction agent solution generating concentration dynamic change is set forth below for induction agent solution A.As shown in Figure 4, Q
a1(t), Q
a2(t) and Q
at () represents the flow of the solute of induction agent A, solvent and solution respectively, φ
a1and φ
at () represents effective chemical inductive substance concentration in the solute of induction agent A and solution respectively, φ
a1be constant, not containing effective chemical inducer matter in solvent, obtain according to mass conservation law and fluid continuity:
Obtained by formula (4):
Therefore at the flow Q of given required induction agent solution A
a(t), concentration φ
athe concentration φ of (t) and solute
a1, the flow Q of solute and solvent can be obtained
a1(t) and Q
a2(t).Like this, controlled the flow of induction agent A solute syringe and induction agent A solvent injection device by syringe pump, make them according to Q
a1(t) and Q
a2t () changes, can generate required concentration by φ
at induction agent solution A that () changes.
Micro flow control chip device for separating of bacterium provided by the invention, simple to operate, the chemotactic characteristic of application-level flow and bacterium, successfully can isolate interested bacterium, for the bacterial studies analysis of the related fields such as food safety, pharmaceutical engineering, clinical medicine from bacterium mixed solution (bacterium and cell mixture or various bacteria mixed solution).
Accompanying drawing explanation
Fig. 1 is the micro flow control chip device structure iron of separation of bacterial.
Fig. 2 is micro-fluidic chip microchannel schematic diagram.
Fig. 3 is approximate " X " type access diagram.
Fig. 4 is that induction agent solution A produces schematic diagram.
Fig. 5 is experimental system schematic diagram.
In figure: I induction agent concentration control system, II bacteria distribution micro-fluidic chip; III micro-fluidic chip microchannel vertical view, IV micro-fluidic chip microchannel front view;
1 induction agent A solute syringe, 2 induction agent A solvent injection devices, 3 bacterium mixed solution syringes, 4 induction agent B solute syringes, 5 induction agent B solvent injection devices, 6 induction agent solution A, 7 bacterium mixed solutions, 8 induction agent B solution, 9 micro-fluidic chip microchannels, 10 PDMS-glass-chips, 11 bacteriums export, and 12 waste liquids export; 13 induction agent concentration control systems, 14 calculator display organizations, 15 laser co-focusings or fluorescent microscope, 16 bacteria distribution micro-fluidic chips, 17 devil liquor recovery process;
9-1 induction agent A entrance, 9-3 bacterium entrance, 9-2 induction agent B entrance, 9-4 induction agent A injection channel, 9-5 bacterium injection channel, 9-6 induction agent B injection channel, 9-7 bacteria distribution passage, 9-8 bacterium output channel, 9-9 waste liquid output channel, 9-10 bacterium collects outlet, 9-11 waste liquid outlet.
Embodiment
Below in conjunction with accompanying drawing and technical scheme, further illustrate the specific embodiment of the present invention.
Figure 1 shows that the micro flow control chip device structure iron of separation of bacterial, comprise induction agent concentration control system I and bacteria distribution micro-fluidic chip II two portions.Wherein, induction agent concentration control system I is combined by five groups of syringes and syringe pump; Bacteria distribution micro-fluidic chip II, comprising: induction agent A entrance 9-1, bacterium entrance 9-3, induction agent B entrance 9-2, induction agent A injection channel 9-4, bacterium injection channel 9-5, induction agent B injection channel 9-6, bacteria distribution passage 9-7, bacterium output channel 9-8, waste liquid output channel 9-9, bacterium collect outlet 9-10 and waste liquid outlet 9-11.Wherein, bacteria distribution passage 9-7 entrance communicates with induction agent A injection channel 9-4, bacterium injection channel 9-5 and induction agent B injection channel 9-6, and outlet communicates with bacterium output channel 9-8 and waste liquid output channel 9-9.The all channel architectures of chip adopt standardized micro-processing method, complete with PDMS, and with clean glass sheet bonded seal, form common glass-PDMS chip.
In the present embodiment, this micro-fluidic chip and programmable infusion pump, laser co-focusing (or fluorescence) microscope, computer constitute complete cellular segregation system (Fig. 5).The bacterium entrance 9-3 of a syringe 3 and chip that helicobacter pylori sample mixed solution is housed is connected, the injection of other two kinds of induction agent is connected realization by two groups of programmable infusion pump with syringe respectively, two are equipped with respectively syringe 1 and the syringe 2 of 1% blood plasma (attractant A) and solvent, are connected to induction agent A entrance 9-1 by bilateral interface, silicone tube and three-way interface; Syringe 4 and the syringe 5 of 2% bile (drive and scold agent B) and solvent will be housed again, be connected to induction agent B entrance 9-2 by bilateral interface, silicone tube and three-way interface, and all syringes are connected with programmable infusion pump.Induction agent A and B of concentration changes with time can be obtained by the fluctuations in discharge controlling syringe pump, and be injected in chip, realize the separation to helicobacter pylori.
In the present embodiment, all channel heights of micro-fluidic chip inside are 30 μm, and induction agent A injection channel 9-4 and induction agent B injection channel 9-6 width are consistent, and are 210 μm; Bacterium injection channel width is 80 μm; Bacteria distribution passage is long is 16mm, and wide is the rectangular channel of 500 μm; Two output channels, bacterium output channel 9-8 is identical with the width of waste liquid output channel 9-9, equals 200 μm.
Further, utilize other detection techniques to detect the bacterium of collecting after separation, analyze the purity, activity etc. that obtain collected bacterium, for follow-up study analysis.The present invention can according to the chemotaxis difference of different bacterium, select different attractants and drive to scold agent, and make the occurrence dynamics change in time of its concentration according to needs, success is quick, efficient, highly purified from bacterium mixed solution (comprising bacterium and cell mixture, various bacteria mixed solution) isolates interested bacterium, for researching and analysing of the fields such as food safety, water quality monitoring (WQM), pharmaceutical engineering, clinical medicine.
Claims (3)
1. utilize a micro flow control chip device for laminar flow and chemotaxis separation of bacterial, it is characterized in that, this micro flow control chip device comprises:
Three sample inlets: induction agent A entrance 9-1, bacterium entrance 9-3 and induction agent B entrance
9-2; Article three, input channel: induction agent A injection channel 9-4, bacterium injection channel 9-5 and induction
Reagent B injection channel 9-6;
, a main channel: bacteria distribution passage 9-7;
Article two, output channel: bacterium output channel 9-8 and waste liquid output channel 9-9;
Two outlets: bacterium collects outlet 9-10 and waste liquid outlet 9-11;
Induction agent entrance, bacterium entrance, induction agent injection channel, bacterium injection channel, bacteria distribution passage, bacterium output channel, waste liquid output channel, bacterium collection outlet and waste liquid outlet composition are similar to the PDMS-glass-chip of " X " type;
Bacteria distribution passage is a sliver transvers section is flat rectangular passage, its height H be less than width W and length L and size in micron or millimeter magnitude, sample inlet communicates with induction agent A injection channel, bacterium injection channel and induction agent B injection channel, and outlet communicates with bacterium output channel and waste liquid output channel;
The width of induction agent A injection channel and induction agent B injection channel is consistent, and bacterium injection channel width is less than induction agent injection channel width, and bacterium can be contacted fully with induction agent.
2. according to micro flow control chip device according to claim 1, it is characterized in that, the travelling trend different according to different bacterium selects different reagent, when the bacterium of required separation has left swimming characteristics, induction agent A is bacterium attractant, and induction agent B scolds agent for driving; Otherwise when the bacterium of required separation has right swimming characteristics, induction agent A scolds agent for driving, induction agent B is bacterium attractant.
3. according to the micro flow control chip device described in claim 1 or 2, it is characterized in that, controlled the fluctuations in discharge of induction agent solute and solvent by syringe pump, obtain the induction agent of required concentration occurrence dynamics change in time.
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Cited By (6)
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CN104965082A (en) * | 2015-05-21 | 2015-10-07 | 大连理工大学 | Micro-fluidic chip used for simultaneous detection of bladder cancer cells, calculi, blood cells, and bacteria in urine |
CN108660053A (en) * | 2018-03-13 | 2018-10-16 | 大连大学 | A kind of body eutrophication indicating means based on micro-fluidic chemotactic chip |
CN110935492A (en) * | 2018-09-21 | 2020-03-31 | 浙江麦大基因科技有限公司 | Micro-fluidic detection system and method for real-time flow velocity of fluid |
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CN104965082A (en) * | 2015-05-21 | 2015-10-07 | 大连理工大学 | Micro-fluidic chip used for simultaneous detection of bladder cancer cells, calculi, blood cells, and bacteria in urine |
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CN112964684A (en) * | 2021-02-08 | 2021-06-15 | 大连理工大学 | Method for determining average flow velocity of variable cross-section flat microchannel in height direction under microscale based on space-time gradient distribution of substance concentration |
CN114917754A (en) * | 2022-05-06 | 2022-08-19 | 东南大学 | Microfluidic colloidal particle separation device and separation method |
CN114917754B (en) * | 2022-05-06 | 2023-02-17 | 东南大学 | Microfluidic colloidal particle separation device and separation method |
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