CN104928178A - Three inlet concentration gradient generator and power function concentration gradient generation method - Google Patents
Three inlet concentration gradient generator and power function concentration gradient generation method Download PDFInfo
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Abstract
The invention discloses a three inlet concentration gradient generator and a power function concentration gradient generation method. The concentration gradient generator comprises three inlets, an outlet, a main passage and a series of flow distribution plates parallel to the main channel, and is prepared through a soft lithography method. The area where the flow distribution plates are located is a mixing area with fluid in different concentrations, and comprises three stages, wherein the first stage of the area comprises two flow distribution plates, the second stage of the area comprises four flow distribution plates, and the third stage of the area comprises eight flow distribution plates; the part, at the downstream of the flow distribution plates, of the main passage is the formation area of concentration gradient. Through control of flow and concentration of the three inlets, establishment of concentration gradient of different target power functions is realized. The concentration gradient generator is compact in structure, adjustable in concentration gradient and simple and feasible in adjustment method, and can be widely applied to the fields of cell directed migration, cell culture and the like in biology, pharmacy, medical engineering and the like.
Description
Technical field
The present invention relates to the Application Areas such as micro-fluid chip and biology, pharmacy, engineering in medicine, particularly relate to a kind of production method based on micro-fluidic concentration gradient generator and power function concentration gradient.
Background technology
The chemotactic response etc. of the migration of cell, the oriented growth of neuron axon and microorganism all affects by the concentration gradient of certain chemical substance.Therefore, realize controlled concentration gradient, in RESEARCH ON CELL-BIOLOGY and drug screening and engineering in medicine, there is important practical value.Microfluidic device because of its size small, accurate Laminar Flow can be produced, and effectively can control the mutual mixing of fluid, and be heavily used in the generation of concentration gradient.
Current microfluidic concentration gradient producer mainly comprises dispersion pattern and convection two class.Dispersion pattern utilizes the free diffusing of molecule and produces concentration gradient, and it generally for generation of linear concentration gradient, and is difficult to the concentration gradient realizing controlled other types.Convection be based on fluid separating for several times with mix, the complicated case such as non-linear concentration gradient and multiple concentration gradient superpositions can be realized.Comparatively speaking, the structure of convection concentration gradient generator is more complicated, needs longer straight channel or micro mixer.In addition, generally single concentration gradient generator only can for generation of a kind of concentration gradient, and namely the versatility of equipment is poor.In current concentration gradient device structure design, to realize multiple concentration gradient on a single device, then need to arrange a lot of fluid intakes, make device structure very complicated, and the requirement of external instrument is improved greatly.
Summary of the invention
In view of above technical problem, the invention provides a kind of three entrance concentration gradient generators simple and compact for structure and gradient production method.Its objective is and realize multiple different power function concentration gradient on single microfluidic device, providing novel method for solving the biomedical technical problem such as cell chemotaxis, drug screening.
The object of the invention is to be achieved through the following technical solutions: a kind of three entrance concentration gradient generators, one end is three access roades, and three entrances are connected with main channel; Location arrangements near entrance in main channel has a series of spreader plate; Described spreader plate refers to and is parallel to length direction, for the string configuration of fluid pill; The spreader plate of same position will be in the longitudinal direction as one-level, altogether needs 3 level shunt plate; 1st grade contains 2 pieces of spreader plates, and the 2nd grade contains 4 pieces of spreader plates, and 3rd level contains 8 pieces of spreader plates; Wherein, main channel is isolated into 3 passage aisles by 2 pieces of spreader plates of the first step, and these three passage aisles are connected with three access roades respectively; 8 pieces of spreader plates of 3rd level are perpendicular to evenly distributed on the direction of main channel; Perpendicular on direction, main channel, i-th piece of spreader plate of the 2nd grade is in the 2i-1 block of 3rd level and the centre of 2i block spreader plate; The jth block spreader plate of the 1st grade is in the 2j-1 block of the 2nd grade and the centre of 2j block spreader plate; The width of main channel is w, is highly less than or equal to w/10; The length of the 1st level shunt plate is
the length of the 2nd level shunt plate is
the length of 3rd level spreader plate is
the width of spreader plate is less than or equal to w/20; Be one section of empty main channel after 3rd level spreader plate, its opposite side is connected with exit passageway.
The making method of described concentration gradient generator is: first made the substrate containing described structure of PDMS by soft lithographic, then make itself and cover plate bonding by plasma treatment, finally passes through punching and other equipment connections at exit and entrance place.
A kind ofly apply the method that above-mentioned concentration gradient generator sets up power function concentration gradient, specific as follows: three entrances are defined as entrance one, entrance two and entrance three from top to bottom successively, then the concentration of entrance one is determined as required, the concentration of entrance three is 0, the concentration of entrance two is determined according to target power function, and its value is the concentration value of target power function in the midpoint perpendicular to flow direction; Wherein, the mean flow rate of entrance two is 20 μm/s, and the flow velocity sum of entrance one, three always equals the twice of entrance two flow velocity; When the power a of target power function is more than or equal to 1, the velocity ratio r=v of entrance one, two
1/ v
2as follows with the relation of variable b=ln (a) depending on power a: r=0.95 ?0.41b, thus obtain the flow velocity of entrance one and entrance three; As the power a=1/2 of target power function, the mean flow rate of entrance one is 13 μm/s, and the mean flow rate of entrance three is 8 μm/s; As the power a=1/3 of target power function, the mean flow rate of entrance one is 12 μm/s, and the mean flow rate of entrance three is 4 μm/s; When needing a certain power function concentration gradient, can obtain as follows:
(1) determine the concentration of each entrance solution according to the power a of target power function, and configure the solution of respective concentration;
(2) when a >=1, according to the relation of r and b, suitable entrance mean flow rate is obtained by power a; As a=1/2 or a=1/3, by above-mentioned given value determination entrance mean flow rate; According to the flow of each entrance of Size calculation of concentration gradient generator;
(2) three entrances of concentration gradient generator connect syringe pump respectively, and outlet connects liquid waste tank;
(3) first with distilled water, passage is filled up, then in entrance one, two, pass into the solution configured accordingly by the flux values calculating gained in step (2), in entrance three, pass into distilled water;
(4), after flow field is stablized, the foundation of required power function concentration gradient can be completed.
Beneficial effect of the present invention is:
(1) can only realize a kind of state of the art of concentration gradient compared to most of concentration gradient generator, the present invention on described single micro-fluidic chip, can realize different concentration gradients by regulating the concentration of each inlet flow rate and entrance two fluid.
(2) can form the device of multiple concentration gradient compared to other, the access road of present device is few.The hybrid channel direct neighbor being arranged so that same one-level of spreader plate, and the hybrid channel of two inter-stages is directly connected, the structure of mixing zone simplifies greatly, thus makes one-piece construction simply compact.
(3) for different power function concentration gradients (power is more than or equal to 0), the correlation of flow parameter and objective function parameters is linear relationship, simple and clear.After determining required aimed concn gradient, required operating parameters can be calculated easily.
(4) concentration gradient production method of the present invention can, while realizing different power function concentration gradient, keep the total flux of fluid constant.Namely, in the tests such as cell cultures, the shearing force that can control suffered by cell is constant.The engineering in medicine such as this vitro culture for cell have important practical value.
Accompanying drawing explanation
The orthographic plan of Fig. 1 concentration gradient generator structure of the present invention;
The experimental result of the different power function concentration gradients produced in concentration gradient generator described in Fig. 2;
The relation of Fig. 3 inlet velocity and power.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
As shown in Figure 1, a kind of three entrance concentration gradient generators of the present invention, one end is three access roades, and three entrances are connected with main channel; Location arrangements near entrance in main channel has a series of spreader plate; Described spreader plate refers to and is parallel to length direction, for the string configuration of fluid pill; The spreader plate of same position will be in the longitudinal direction as one-level, altogether needs 3 level shunt plate; 1st grade contains 2 pieces of spreader plates, and the 2nd grade contains 4 pieces of spreader plates, and 3rd level contains 8 pieces of spreader plates; Wherein, main channel is isolated into 3 passage aisles by 2 pieces of spreader plates of the first step, and these three passage aisles are connected with three access roades respectively; 8 pieces of spreader plates of 3rd level are perpendicular to evenly distributed on the direction of main channel; Perpendicular on direction, main channel, i-th piece of spreader plate of the 2nd grade is in the 2i-1 block of 3rd level and the centre of 2i block spreader plate; The jth block spreader plate of the 1st grade is in the 2j-1 block of the 2nd grade and the centre of 2j block spreader plate; The width of main channel is w, is highly less than or equal to w/10; The length of the 1st level shunt plate is
the length of the 2nd level shunt plate is
the length of 3rd level spreader plate is
the width of spreader plate is less than or equal to w/20; Be one section of empty main channel after 3rd level spreader plate, its opposite side is connected with exit passageway.
A kind ofly apply the method that above-mentioned concentration gradient generator sets up power function concentration gradient, specific as follows: as shown in Figure 1, three entrances are defined as entrance one, entrance two and entrance three from top to bottom successively, then the concentration of entrance one is determined as required, the concentration of entrance three is 0, the concentration of entrance two is determined according to target power function, and its value is the concentration value of target power function in the midpoint perpendicular to flow direction; Wherein, the mean flow rate of entrance two is 20 μm/s, and the flow velocity sum of entrance one, three always equals the twice of entrance two flow velocity, and its value is in table 1; When the power a of target power function is more than or equal to 1, the velocity ratio r=v of entrance one, two
1/ v
2as follows with the relation of variable b=ln (a) depending on power a: r=0.95 ?0.41b, thus obtain the flow velocity of entrance one and entrance three, calculation result is as shown in table 2, and by table 2 drafting pattern 3, visible r and b presents obvious linear relationship.As the power a=1/2 of target power function, the mean flow rate of entrance one is 13 μm/s, and the mean flow rate of entrance three is 8 μm/s; As the power a=1/3 of target power function, the mean flow rate of entrance one is 12 μm/s, and the mean flow rate of entrance three is 4 μm/s.
The entrance mean flow rate of the common power function of table 1
| Entrance one mean flow rate (μm/s) | Entrance three mean flow rate (μm/s) | |
| y＝x | 20 | 20 |
| y＝x 2 | 12 | 28 |
| y＝x 3 | 10 | 30 |
| y＝x 4 | 8 | 32 |
| y＝x 5 | 6 | 34 |
| y＝x 6 | 5 | 35 |
| y＝x 1/2 | 13 | 8 |
| y＝x 1/3 | 12 | 4 |
The numerical value of r and b of the different power function of table 2 (a >=1)
| r | b | |
| y＝x | 1.0 | 0 |
| y＝x 2 | 0.6 | 0.30 |
| y＝x 3 | 0.5 | 0.48 |
| y＝x 4 | 0.4 | 0.60 |
| y＝x 5 | 0.3 | 0.70 |
| y＝x 6 | 0.25 | 0.77 |
When needing a certain power function concentration gradient, can obtain as follows:
(1) determine the concentration of each entrance solution according to the power a of target power function, and configure the solution of respective concentration;
(2) when a >=1, according to the relation of r and b, suitable entrance mean flow rate is obtained by power a; As a=1/2 or a=1/3, by above-mentioned given value determination entrance mean flow rate; According to the size of concentration gradient generator, calculate the flow of each entrance;
(2) three entrances of concentration gradient generator connect syringe pump respectively, and outlet connects liquid waste tank;
(3) first with distilled water, passage is filled up, then in entrance one, two, pass into the solution configured accordingly by the flux values calculating gained in step (2), in entrance three, pass into distilled water;
(4), after flow field is stablized, the foundation of required power function concentration gradient can be completed.
The preparation method of described concentration gradient generator is as follows:
(1) photoetching: microchannel pattern is drawn in design, then outputs on film by pattern, adopts standard photolithography techniques, microchannel pattern is transferred to SU-8 and bear in the silicon substrate template of optical cement, namely formed by the formpiston of microchannel pattern after firmly treatment;
(2) Graphic transitions: by PDMS prepolymer and linking agent according to mass ratio 10:1 Homogeneous phase mixing, casting on flat is on the SU-8 mould of photoetching gained, put into vacuum chamber 15 minutes removing bubbles, then put into 70 DEG C of baking oven heating 2 hours, until take out after solidification.Cutting and stripping PDMS, then punch at each entrance and exit passage place with punch tool;
(3) bonding: by with the PDMS substrate of microstructure and the cover glass of clean also drying treatment, carry out permanent bonding with plasma machine, thus form closed microchannel, complete the making of concentration gradient device chip.
In following examples 1,2, the width of spreader plate is 10 μm; The length of the 1st level shunt plate is 209 μm, and the length of the 2nd level shunt plate is 420 μm, and the length of 3rd level spreader plate is 260 μm; The width of main channel is 400 μm, and length can adjust as required.The height of three entrance concentration gradient generators is 30 μm.
Embodiment 1: the generation of linear concentration gradient
(1) preparation work: fluorescein isothiocyanate (FITC) aqueous solution preparing 5mg/L and 2.5mg/L respectively.Be connected with three syringe pumps respectively by three entrances, exit passageway is connected with waste liquid pool.
(2) operating parameters: entrance one injects the FITC solution of 5mg/L, entrance two injects the FITC solution of 2.5mg/L, and entrance three injects deionized water.Entrance two flow velocity is 20 μm/s, and be 400 μm for width, be highly the concentration gradient generator of 30 μm, the width of entrance two is 168 μm, and can calculate entrance two flow is 6.05nL/min.According to the relation of aforementioned r=0.95-0.41lna, herein a=1, can obtain r=0.95, get and be approximately 1, namely the flow velocity of three access roades is equal.The width of entrance one and entrance three is 106 μm, and the flow that can be calculated entrance one and entrance three is 3.82nL/min.
(3) result obtains: under concentration gradient generator is placed in fluorescent microscope, gets 100 μm, 3rd level spreader plate downstream place and take pictures, according to the concentration gradient that Fluorescence Intensity Assays produces.The experimental result obtained after normalized is as shown in the linear result in Figure of description 2.
Embodiment 2:y=x
2the generation of concentration gradient
(1) preparation work: fluorescein isothiocyanate (FITC) aqueous solution preparing 5mg/L and 1.25mg/L respectively.Be connected with three syringe pumps respectively by three entrances, exit passageway is connected with waste liquid pool.
(2) operating parameters: entrance one injects the FITC solution of 5mg/L, entrance two injects the FITC solution of 1.25mg/L, and entrance three injects deionized water.Entrance two flow is still 6.05nL/min.According to the relation of aforementioned r=0.95-0.41lna, herein a=2, can obtain r=0.67, namely the speed of entrance three is 13.2 μm/s.The flow velocity that can obtain entrance one is thus 26.8 μm/s.The width of entrance one and entrance three is 106 μm, and the flow that can be calculated entrance one is 5.11nL/min, and the flow of entrance three is 2.52nL/min.
(3) result obtains: under concentration gradient generator is placed in fluorescent microscope, gets 100 μm, 3rd level spreader plate downstream place and take pictures, according to the concentration gradient that Fluorescence Intensity Assays produces.The experimental result obtained after normalized is as shown in the quafric curve in Figure of description 2.
Embodiment 3: main channel width is the method for design of the concentration gradient generator of 800 μm
Sometimes because the experiment condition such as flow accuracy and accurate to dimension is restricted, need the size of concentration gradient generator to carry out convergent-divergent.This sentences passage width and is amplified to 800 μm for example.In order to make fluid chemical field in each passage even, under the prerequisite that rate of flow of fluid remains unchanged, when passage width is increased to original twice, length should be increased to original four times.That is, the 1st level shunt plate length is 836 μm, and the 2nd level shunt plate length is 1680 μm, and 3rd level spreader plate length is 1040 μm.
Claims (2)
1. three entrance concentration gradient generators, is characterized in that, one end is three access roades, and three entrances are connected with main channel; Location arrangements near entrance in main channel has a series of spreader plate; Described spreader plate refers to and is parallel to length direction, for the string configuration of fluid pill; The spreader plate of same position will be in the longitudinal direction as one-level, altogether needs 3 level shunt plate; 1st grade contains 2 pieces of spreader plates, and the 2nd grade contains 4 pieces of spreader plates, and 3rd level contains 8 pieces of spreader plates; Wherein, main channel is isolated into 3 passage aisles by 2 pieces of spreader plates of the first step, and these three passage aisles are connected with three access roades respectively; 8 pieces of spreader plates of 3rd level are perpendicular to evenly distributed on the direction of main channel; Perpendicular on direction, main channel, the spreader plate of the 2nd grade is in the piece of 3rd level and the centre of block spreader plate; The spreader plate of the 1st grade is in the piece of the 2nd grade and the centre of block spreader plate; The width of main channel is highly be less than or equal to; The length of the 1st level shunt plate is, the length of the 2nd level shunt plate is, the length of 3rd level spreader plate is that the width of spreader plate is less than or equal to; Be one section of empty main channel after 3rd level spreader plate, its opposite side is connected with exit passageway.
2. an application rights requires that described in 1, concentration gradient generator sets up the method for power function concentration gradient, it is characterized in that, specific as follows: three entrances are defined as entrance one, entrance two and entrance three from top to bottom successively, then the concentration of entrance one is determined as required, the concentration of entrance three is 0, the concentration of entrance two is determined according to target power function, and its value is the concentration value of target power function in the midpoint perpendicular to flow direction; Wherein, the mean flow rate of entrance two is 20 μm/s, and the flow velocity sum of entrance one, three always equals the twice of entrance two flow velocity; When the power of target power function
awhen being more than or equal to 1, the velocity ratio of entrance one, two
r=
v 1 /
v 2 with depend on power
avariable
b=ln (
a) relation as follows:
r=0.95-0.41
b, thus obtain the flow velocity of entrance one and entrance three; When the power of target power function
awhen=1/2, the mean flow rate of entrance one is 13 μm/s, and the mean flow rate of entrance three is 8 μm/s; When the power of target power function
awhen=1/3, the mean flow rate of entrance one is 12 μm/s, and the mean flow rate of entrance three is 4 μm/s; When needing a certain power function concentration gradient, can obtain as follows:
(1) according to the power of target power function
adetermine the concentration of each entrance solution, and configure the solution of respective concentration;
(2) when
awhen>=1, according to
rwith
brelation, by power
aobtain suitable entrance mean flow rate; As a=1/2 or a=1/3, by above-mentioned given value determination entrance mean flow rate; According to the flow of each entrance of Size calculation of concentration gradient generator;
(2) three entrances of concentration gradient generator connect syringe pump respectively, and outlet connects liquid waste tank;
(3) first with distilled water, passage is filled up, then in entrance one, two, pass into the solution configured accordingly by the flux values calculating gained in step (2), in entrance three, pass into distilled water;
(4), after flow field is stablized, the foundation of required power function concentration gradient can be completed.
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