CN105536895A - Openable micro-fluidic chip and preparation method thereof - Google Patents

Abstract

The invention discloses an openable microfluidic chip, which comprises a first substrate and a second substrate, the first substrate has a concave part, the second substrate has a raised part, and the second The convex part of the substrate covers the surface of the concave part of the first substrate, and is combined with the first substrate by a polymer around it, and the polymer is used for reversibly connecting the first substrate and the second substrate. sheet to form an openable microfluidic structure. The invention also discloses a preparation method of the microfluidic chip. Through the present invention, the microfluidic chip can be used to capture cells or microparticles, and the captured cells or microparticles can be collected for further analysis.

Description

An openable microfluidic chip and its preparation method

technical field

The invention belongs to the field of microfluidic chips, and more specifically relates to an openable microfluidic chip and a preparation method thereof.

Background technique

The microfluidic chip is the core of the micro-total analysis system. The invention of the microfluidic chip promotes the development of analytical instruments used in chemistry, food, environment, medicine and life sciences in the direction of miniaturization, automation, integration and portability. Its advantages The method lies in detecting various chemical reactions through the microfluidic chip, significantly reducing reagent consumption, greatly improving analysis efficiency, and reducing costs. The design and production of microfluidic chips composed of microfluidic channel networks can be used in different analytical fields, including chemical analysis, food hygiene, environmental monitoring, medical testing, life sciences, criminal sciences, and national defense.

At present, in microfluidic chips, photoelectric technology or mechanical manipulation is mostly used to manipulate cells and microparticles. The optical method involves complex instruments, single manipulation, and many limitations. Electrical methods often require the integration of delicate electrodes on a chip, requiring complex power control, and voltages that adversely affect cells. However, the mechanical operation lacks the flexibility and reversibility of the operation; for example, the commonly used cell mechanical positioning elements such as microdams and microwells are fixed in height, and once the cells are captured, it is difficult to completely remove them. There are also some difficulties in further research. At the same time, the microfluidic chip in the prior art is usually composed of two substrates bonded together, the substrates are very tightly bonded, even if the cells and particles are captured, it is difficult to open to obtain the required cells and particles, or it is barely opened However, accurate capture cannot be guaranteed, and it is difficult to obtain the desired cells or particles in the microfluidic chip in a fixed-point or directional manner.

Contents of the invention

Aiming at the above defects or improvement needs of the prior art, the present invention provides an openable microfluidic chip, the purpose of which is to reversibly connect the first substrate and the second substrate through a polymer, thereby forming an openable microfluidic chip. Fluidic structure, so as to obtain the desired cells or particles in a directional manner.

In order to achieve the above object, according to one aspect of the present invention, an openable microfluidic chip is provided, including a first substrate and a second substrate, the first substrate has a concave portion, and the second substrate The sheet has a protruding part, the protruding part of the second substrate covers the surface of the concave part of the first substrate, and is bonded to the first substrate by a polymer around it, and the polymer is used for reversible The first substrate and the second substrate are connected to form an openable microfluidic structure.

Preferably, the width of the junction of the raised portion of the second substrate and the raised portion of the first substrate is 1mm˜2mm.

Preferably, the height of the raised portion of the second substrate is greater than 20 μm.

Preferably, the depression of the first substrate is rectangular, and the depth of the depression gradually decreases along the length direction of the rectangle, and the microfluidic chip is used for capturing cells or particles.

As a further preference, the depth of the depressed portion of the first substrate is 1 μm˜200 μm.

As a further preference, the length of the recessed portion of the first substrate is 50 mm to 70 mm, and the width is 10 mm to 22 mm.

As a further preference, the microfluidic chip is provided with an inlet and an outlet, which communicate with the deepest part and the shallowest part of the recessed part of the first substrate respectively.

As a further preference, the inlet and outlet are arranged on the first substrate.

As a further preference, the surface of the raised portion of the second substrate is covered with a gel layer of 1 μm˜5 μm.

As still further preferably, the gel layer is agarose, chitosan or polyvinyl alcohol.

Preferably, the first substrate and the second substrate are glass.

Preferably, the polymer is polydimethylsiloxane or epoxy.

According to another aspect of the present invention, a method for preparing the above-mentioned microfluidic chip is provided, comprising the following steps:

(1) A depression is obtained by etching on the surface of the first substrate, and a protrusion is obtained by etching on the surface of the second substrate, so that the protrusion of the second substrate and the depression of the first substrate are The shapes are the same, and the contour of the raised portion of the second substrate is 1 mm to 2 mm larger than the contour of the depressed portion of the first substrate;

(2) Aligning and bonding the protrusions of the second substrate with the depressions of the first substrate, so that the protrusions of the second substrate completely cover the depressions of the first substrate, and adhere to the first substrate;

(3) Filling prefabricated polymers around the protrusions of the second substrate, so that the protrusions of the second substrate are combined with the first substrate, and the polymer is cured to obtain The microfluidic chip.

Preferably, between the step (1) and the step (2), it further includes, covering the surface of the raised portion of the second substrate with a gel layer of 1 μm˜5 μm.

Generally speaking, compared with the prior art, the above technical solution conceived by the present invention can achieve the following beneficial effects due to the reversible connection of the first substrate and the second substrate through the polymer:

1. The second substrate is designed as a convex structure, covering the surface of the concave part of the first substrate, and at the same time, the polymer is used to fill the circumferential direction of the convex part of the second substrate, so that the first substrate and the second substrate The chip can be reversibly combined, so that it is convenient to open the microfluidic chip to obtain the cells or particles in it;

2. It is preferable to set the recessed part as a rectangle with uneven depth, so that cells or particles of different sizes are captured in different parts of the recessed part;

3. It is preferable to use glass as the material of the first substrate and the second substrate. Since glass is a transparent material, the capture process of cells and particles is more controllable;

4. The preparation method of the microfluidic chip is simple and efficient, and is convenient for directional acquisition of cells and particles required in the microfluidic chip.

Description of drawings

Fig. 1 is the first substrate structure schematic diagram in embodiment 1;

Fig. 2 is the second substrate structure schematic diagram in embodiment 1;

FIG. 3 is a schematic structural diagram of the microfluidic chip in Example 1;

4 is a schematic diagram of capturing cells using the microfluidic chip prepared in Example 1;

5 is a schematic diagram of paraffin-encapsulated captured cells in the microfluidic chip prepared in Example 1;

Figure 6 is a schematic diagram of transferring cells after the microfluidic chip prepared in Example 1 is turned on;

In all the drawings, the same reference numerals are used to denote the same elements or structures, wherein: 1-first substrate, 2-second substrate, 3-chip inlet, 4-chip outlet, 5-first Substrate depression, 6- second substrate protrusion, 7-PDMS, 8- deepest channel, 9- shallowest channel, 11-marker, 12-cell.

detailed description

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not constitute a conflict with each other.

The present invention provides an openable microfluidic chip, comprising a first substrate and a second substrate, the first substrate has a concave portion, the second substrate has a raised portion, the second The raised portion of the substrate covers the surface of the recessed portion of the first substrate, and is bonded to the first substrate through a polymer, and the width of the joint is 1 mm to 2 mm; the polymer is poly Methyl siloxane or epoxy resin is used for reversibly connecting the first substrate and the second substrate to form an openable microfluidic structure. The first substrate and the second substrate are preferably glass, so that the capture process of cells and particles is more controllable. The height of the raised part of the second substrate is greater than 20 μm, so that there is a certain gap at the joint part of the second substrate and the first substrate, so that the first substrate and the second substrate can be separated by a tool when opening.

Microfluidic chips for trapping both cells and microparticles could take advantage of this design. After the chip captures the cells and microparticles, the chip is turned on to obtain the desired cells or microparticles.

For example, the concave portion of the first substrate can be designed as a rectangular structure with a length of 50 mm to 70 mm and a width of 10 mm to 22 mm. The depth of the rectangular structure is 1 μm to 200 μm, and its depth gradually decreases in the length direction, so that different Cells and microparticles with diameters can be fixed at different positions of the rectangle, and the microfluidic chip is provided with an inlet and an outlet, which communicate with the deepest part and the shallowest part of the depression respectively. Its specific depth can be designed according to the size of the cells or particles that need to be captured. For example, when it is necessary to capture circulating tumor cells mixed with normal cells, the shallowest depth can be designed to be 5 μm, and the deepest depth can be designed to be 60 μm. Cells of different sizes from 15 μm to 30 μm were captured at different positions in the direction. The surface of the raised part can be covered with a gel layer with a thickness of 1 μm to 5 μm formed by materials such as agarose, chitosan, polyvinyl alcohol, etc., so that cells or particles can better adhere to the raised part during the use of the chip One side is convenient for subsequent transfer and separation when the microfluidic chip is turned on.

After using the chip for capture experiments, cells or particles usually adhere to one side of the raised part of the second substrate. At this time, the specific opening method of the microfluidic chip is as follows:

(1) Pass alcohol through the inlet to empty the gas (bubble) in the microfluidic chip;

(2) Use phosphate buffer solution PBS from the entrance (recipe: weigh NaCl8g, KCl0.2g, Na2HPO4 _{· 12H2O3.63g} , _KH2PO40.24g , dissolve in 900ml double distilled water, adjust the pH value with hydrochloric acid to 7.4, add water to 1L, store at room temperature for later use) empty the alcohol in the microfluidic chip;

(3) Pass paraffin through the entrance, so that the paraffin is filled with the microfluidic chip;

(4) After the paraffin is solidified, use tools such as blades to remove the polymer layer around the raised portion of the second substrate, separate the first substrate and the area around the raised portion of the second substrate, and the chip can be uncovered , to obtain cells or microparticles encapsulated in paraffin, then separate the paraffin containing the desired cells or microparticles, heat to dissolve the paraffin, and then take out the desired cells or microparticles for further research.

The preparation method of the microfluidic chip comprises the following steps:

(1) A depression is obtained by etching on the surface of the first substrate, and a protrusion is obtained by etching on the surface of the second substrate, so that the protrusion of the second substrate and the depression of the first substrate are The shapes are the same, and the contour of the raised portion of the second substrate is 1 mm to 2 mm larger than the contour of the depressed portion of the first substrate;

(2) Aligning and bonding the protrusions of the second substrate with the depressions of the first substrate, so that the protrusions of the second substrate completely cover the depressions of the first substrate, and adhere to the first substrate;

(3) Filling prefabricated polymers (usually a mixture of polymer monomers and crosslinking agents) around the protrusions of the second substrate, so that the protrusions of the second substrate are in contact with the The first substrate is combined, and the microfluidic chip is obtained after the polymer is cured.

Embodiment A highly precise and controllable single-sided cell transfer microfluidic chip with an openable lumen

Step 1 Prepare the first substrate

A glass slide with a length of 75mm and a width of 25mm is selected as the material of the first substrate 1, and the channel 5 is designed to be 60mm in length and 15mm in width, and its depth gradually decreases from left to right. The shallowest part is 5 μm, and the wet etching method is used to etch at a speed of 1 mm/min to obtain the corresponding morphology. And the inlet 3 and the outlet 4 are carved in the center of the deepest and shallowest respectively, and the side view and top view of the obtained first substrate are shown in Fig. 1a and Fig. 1b respectively.

Step 2. Prepare the second substrate

The second substrate 2 is designed as a boss 6 with a height of 61 mm×16 mm and a height of about 80 μm, and the second substrate is prepared by a method similar to that of the first substrate, and its side view and top view are shown in Figure 2a and Figure 2b respectively Show.

Step 3. Configure 2% mass fraction of agarose solution

Under the action of a magnetic stirrer, mix the solution evenly at a temperature of 60 degrees, and then use a 1000 μl pipette to take 500 μl of the solution and drop it on the boss, and put it into the homogenizer when the agarose has not cooled down into a gel. Shake well with parameters of 800rpm20s, 1400rpm40s, and finally dry on a hot stage at 50°C to obtain an agarose film of about 2 μm.

Step 4. Die Attach

Align the prepared first substrate with the second substrate with film, so that the boss completely covers the groove on the first substrate, and stack them together so that the first substrate and the second substrate are closely attached together .

Step 5. Polymer curing

Select prefabricated PDMS (the mass of polymer monomer and cross-linking agent is 10:1), after the PDMS is embedded around the boss, it is heated to 85°C for 10 minutes to cross-link and solidify the PDMS, and the chip is completed. The obtained chip The side view and top view are shown in Figure 3a and Figure 3b respectively, the deepest channel 8 is 60 μm, and the shallowest channel 9 is 5 μm.

The microfluidic chip can be used to test the controllable cell single-sided transfer microfluidic chip. The specific experimental process is as follows:

(1) Pass alcohol into the chip at a speed of 100 μm/min to empty the gas (bubble) in the microfluidic chip;

(2) Pass phosphate buffer solution PBS into the chip at a speed of 100 μm/min (recipe: weigh NaCl8g, KCl0.2g, Na2HPO4 _{· 12H2O3.63g} , _KH2PO40.24g , dissolve in 900ml double distilled In water, use hydrochloric acid to adjust the pH value to 7.4, add water to make up to 1L, store at room temperature for later use) and empty the alcohol in the channel;

(3) Pass the target cells with a concentration of 10 ³ /ml and a diameter of 10 μm to 20 μm and different sizes into the chip at a speed of 100 μm/min, and keep for 2 minutes;

(4) passing phosphate buffer solution PBS into the chip at a speed of 100 μm/min, so that the cells stay at different depths of the microfluidic chip according to their own size;

(5) Observe the position where the cell 11 stays under a microscope and make a mark 12, as shown in Figure 4;

(6) Pass paraffin into the chip at a speed of 100 μm/min;

(7) After the paraffin is solidified, observe the marked positions with a microscope. At this time, it can be seen that the position and quantity of the cells have not changed, as shown in Figure 5;

(8) At this time, soak the chip in water at 47 degrees Celsius to soften the gel material at the bottom. After ten minutes, use a special tool to remove the PDMS around the boss, and uncover the chip to get the sticky surface of the boss. The paraffin section, and then observe the marked positions under the microscope, at this time, it can be seen that the position and number of cells remain unchanged;

(9) After the paraffin is uncovered, the cells or particles encapsulated in the paraffin layer can be obtained, and then the paraffin of the part where the desired cells are located is taken out, and transferred to a petri dish, adding PBS and heating until the paraffin dissolves, and then use a microscope as an aid The desired cells can be taken out with a micro pipette for further research, as shown in Figure 6.

It is easy for those skilled in the art to understand that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, All should be included within the protection scope of the present invention.

Claims (10)

1. an openable micro-fluidic chip, it is characterized in that, comprise the first substrate and the second substrate, described first substrate has depressed part, described second substrate has lug boss, the lug boss of described second substrate is covered in the depressed part surface of described first substrate, and surrounding is combined with described first substrate by polymer.

2. micro-fluidic chip as claimed in claim 1, it is characterized in that, the lug boss of described second substrate and the width of described first substrate junction are 1mm ~ 2mm.

3. micro-fluidic chip as claimed in claim 1, it is characterized in that, the height of the lug boss of described second substrate is greater than 20 μm.

4. micro-fluidic chip as claimed in claim 1, it is characterized in that, the depressed part of described first substrate is rectangle, and described rectangle reduces gradually in the length direction degree of depth.

5. micro-fluidic chip as claimed in claim 3, it is characterized in that, the degree of depth of the depressed part of described first substrate is 1 μm ~ 200 μm.

6. micro-fluidic chip as claimed in claim 3, it is characterized in that, the length of the depressed part of described first substrate is 50mm ~ 70mm, and width is 10mm ~ 22mm.

7. micro-fluidic chip as claimed in claim 3, is characterized in that, described micro-fluidic chip is provided with entrance and exit, and it is connected with the bosom of the depressed part of described first substrate and the most shallow place respectively.

8. micro-fluidic chip as claimed in claim 1, it is characterized in that, described first substrate and the second substrate are glass.

9. micro-fluidic chip as claimed in claim 1, it is characterized in that, described polymer is dimethyl silicone polymer or epoxy resin.

10., as the preparation method of micro-fluidic chip as described in any one in claim 1-9, it is characterized in that, comprise the following steps:

(1) depressed part is obtained in the first substrate surface etching, lug boss is obtained in the second substrate surface etching, make the lug boss of described second substrate identical with the shape of the depressed part of described first substrate, the profile 1mm ~ 2mm larger than the profile of the depressed part of described first substrate of the lug boss of described second substrate;

(2) lug boss of described second substrate aimed at the depressed part of the first substrate fit, make the lug boss of described second substrate cover the depressed part of described first substrate completely, and with the fitting of described first substrate;

(3) surrounding to the lug boss of described second substrate fills prefabricated polymer, the lug boss of described second substrate is combined with described first substrate, namely obtains described micro-fluidic chip after described polymer cure.