CN116673079A - A three-dimensional focusing high-throughput microfluidic chip and its application and manufacturing method - Google Patents

Abstract

The invention discloses a three-dimensional focusing high-flux microfluidic chip and an application and a manufacturing method thereof, wherein a microfluidic structure is arranged in a chip main body, the microfluidic structure comprises a sample channel, a sheath liquid channel, a detection channel and a waste liquid channel, and the sample channel is arranged between the two sheath liquid channels; the sample channel comprises a sample inlet channel and a sample converging channel which are communicated; the sheath liquid channel comprises a sheath liquid inlet channel and a sheath liquid converging channel which are communicated; the sample inlet channel and the sheath liquid inlet channel are both arranged on one side of the chip main body, and the waste liquid channel is arranged on the other side of the chip main body. The invention abandons the traditional inlet design vertical to the plane of the chip, adopts the side surface to set the sample inlet and the sample outlet, avoids the interference of the chip interface and the microscope lens, reduces the size of the chip, greatly reduces the flow resistance of the channel and realizes the high-speed flow of cells.

Description

A three-dimensional focusing high-throughput microfluidic chip and its application and manufacturing method

technical field

The invention relates to the technical field of microfluidic chips, in particular to a three-dimensional focused high-throughput microfluidic chip and its application and manufacturing method.

Background technique

Time-domain stretched single-cell imaging technology is a new type of cell imaging detection method, using broadband pulsed laser as the light source, stretching the broadband pulsed laser in the time domain and performing dispersion in the space and irradiating the cells. The signal is decoded to recover a clear cell image. This detection method has a very high image acquisition speed (up to tens of millions to billions of frames per second), and is very suitable for the detection of orders of magnitude huge cell samples.

However, in the practical application of time-domain stretching single-cell imaging technology, its performance is restricted by microfluidic chips. As the carrier of most cell detection, cells are detected or captured during the flow process, and the flow rate of cells is difficult to match the imaging detection speed of time-domain stretched single-cell imaging technology. At present, the fastest microfluidic chip for cell imaging detection can make cells move at a speed of 25m/s, but it is still lower than the upper limit of 60m/s allowed by the current time-domain stretching single-cell imaging system, and this The first microfluidic chip is an all-glass chip. Although it has the advantages of good optical performance and high material strength, its processing equipment is expensive, the process is complicated, and the cost of a single microfluidic chip is extremely high.

Contents of the invention

In order to solve the problems existing in the prior art, the present invention provides a three-dimensional focused high-throughput microfluidic chip, including a chip cover plate, a chip body and a chip bottom plate,

The chip body is provided with a microfluidic structure, the microfluidic structure includes a sample channel, a sheath fluid channel, a detection channel and a waste fluid channel, and the sample channel is arranged between two sheath fluid channels;

The sample channel includes a connected sample inlet channel and a sample confluence channel;

The sheath fluid channel includes a sheath fluid inlet channel and a sheath fluid confluence channel;

The sample confluence channel merges with the sheath fluid confluence channel at the confluence section, and communicates with the detection channel at the confluence section;

The detection channel communicates with the waste liquid channel;

Both the sample inlet channel and the sheath fluid inlet channel are arranged on one side of the chip body, and the waste liquid channel is arranged on the other side of the chip body.

Further, the initial height of the sheath confluence channel is greater than twice the initial height of the sample confluence channel;

And the initial height of the sheath fluid confluence channel is greater than the diameter of the detection cells.

Further, both the sheath fluid confluence channel and the sample confluence channel are structures with a wide front and a narrow rear.

Further, a sample reservoir is provided at the communication place between the sample inlet channel and the sample confluence channel;

A sheath fluid reservoir is provided at the place where the sheath fluid inlet channel communicates with the sheath fluid confluence channel;

A waste liquid storage pool is provided at the communication point between the detection channel and the waste liquid channel.

Further, the detection channel has a rectangular cross-section.

Further, if there is a gap between the chip body, the chip cover plate and the chip bottom plate, the gap is filled with resin.

Further, the sample inlet channel, the sheath fluid inlet channel and the waste liquid channel are made of capillary steel needles.

The present invention also provides the application of the above-mentioned three-dimensional focused high-throughput microfluidic chip in single-cell imaging detection, including,

Use a syringe pump to inject the sample into the main body of the chip, and the sample flows through the sample inlet channel, the sample liquid reservoir and the sample confluence channel, and at the same time use the syringe pump to inject the sheath liquid into the chip main body, and the sheath liquid flows through the sheath liquid inlet channel, the sheath liquid reservoir Confluence channel with sheath fluid;

Subsequently, the sample and the sheath fluid converge at the confluent section, and the cells in the sample are detected at the detection channel.

The present invention also provides a method for manufacturing the above-mentioned three-dimensional focused high-throughput microfluidic chip, including:

Perform photolithography to form a mold;

preparing a chip body using the mold;

A three-dimensional focused high-throughput microfluidic chip is obtained by assembling the chip cover plate, the chip body and the chip bottom plate.

Compared with the prior art, the present invention has the following beneficial effects:

1. The present invention abandons the traditional entrance design perpendicular to the plane of the chip, and instead adopts the side setting of the sample inlet and outlet ports, which avoids the interference between the chip interface and the microscope lens, thereby reducing the size of the chip and greatly reducing the channel flow resistance. The microfluidic chip of the present invention can withstand very high flow and pressure, realize high-speed flow of cells, and meet the requirements of time-domain stretching optical microscope as far as possible.

2. The present invention aims at the focusing of cells. By setting the sample channel and the sheath fluid channel at different heights, all the cells are driven to flow to one side of the rectangular channel; the flow rate of the liquid and cells is increased, and the velocity gradient on the channel section is increased, so that The effect of the inertial lift force on the cells is more obvious, and they quickly stabilize at a fixed vertical height under the action of high inertial forces, thereby realizing the three-dimensional focusing of the cells.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description These are some embodiments of the present invention. Those skilled in the art can also obtain other drawings based on these drawings without creative work.

Figure 1 shows a schematic structural view of a three-dimensional focused high-throughput microfluidic chip in an embodiment of the present invention;

Fig. 2 shows a schematic flow chart of preparing a three-dimensional focused high-throughput microfluidic chip in an embodiment of the present invention;

Explanation of reference signs:

1. Chip cover plate; 2. Chip main body; 21. Sample channel; 211. Sample inlet channel; 212. Sample reservoir; 213. Sample confluence channel; 22. Sheath fluid channel; 221. Sheath fluid inlet channel; 222. Sheath liquid storage tank; 223, sheath liquid confluence channel; 23, confluence section; 24, detection channel; 25, waste liquid storage tank; 26, waste liquid channel; 3, chip bottom plate; 4, microstructure mold; 5, Single throw silicon wafer; 6. Microstructure; 7. Quartz glass slide; 8. Capillary steel tube; 9. Resin glue.

Detailed ways

Neither the endpoints of the ranges nor any values disclosed herein are limited to such precise ranges or values, and these ranges or values are understood to include values approaching these ranges or values. For numerical ranges, between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, these values Ranges should be considered as specifically disclosed in the present invention.

The following will clearly and completely describe the technical solutions in the embodiments of the present invention in combination with the specific embodiments of the present invention and the accompanying drawings. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Referring to Fig. 1, an embodiment of the present invention provides a three-dimensional focused high-throughput microfluidic chip, which can be applied to single-cell imaging detection, including a chip cover 1, a chip body 2 and a chip bottom plate 3, and the chip cover Both the plate 1 and the chip base plate 3 are made of quartz, the chip body 2 is PDMS with a microfluidic structure, the chip body 2 is fixed in the middle by the chip cover plate 1 and the chip base plate 3, the chip body 2, the chip cover plate 1 and the The gaps in the chip base plate 3 are filled with resin, and the resin also adheres them firmly.

The microfluidic structure of the chip main body 2 includes a sample channel 21, a sheath fluid channel 22, a confluence section 23, a detection channel 24 and a waste fluid channel 26, and the sample channel 21 is arranged between two sheath fluid channels 22; The channel 21 includes a sample inlet channel 211, a sample liquid reservoir 212, and a sample confluence channel 213 that are connected in sequence; The sample confluence channel 213 and the sheath fluid confluence channel 223 merge at the confluence section 23, and communicate with the detection channel 24 at the confluence section 23; the detection channel 24 is sequentially connected with the waste liquid storage tank 25 and The waste liquid channel 26 communicates, and the cross-section of the detection channel 24 is rectangular; the sample inlet channel 211 and the sheath liquid inlet channel 221 are both arranged on one side of the chip main body 2, and the waste liquid channel 26 is arranged on The other side of the chip body 2 .

The initial height of the sheath fluid confluence channel 223 is greater than twice the initial height of the sample confluence channel 213; and the initial height of the sheath fluid confluence channel 223 is greater than the diameter of the detection cells. It should be noted that the starting height mentioned in the present invention refers to the height away from the end of the converging section 23 . Both the sheath fluid confluence channel 223 and the sample confluence channel 213 have a structure with a wide front and a narrow rear.

An embodiment of the present invention specifically provides a three-dimensional focused high-throughput microfluidic chip applied to single-cell imaging detection with a diameter of 5-25 μm. The chip main body 2 has a length of 10 mm, a width of 10 mm, and a height of 1 mm; the sample inlet The material of the channel 211 and the sheath liquid inlet channel 221 is a 21G capillary steel needle; the shape of the sample liquid storage tank 212 and the sheath liquid storage tank 222 are the same as a cylinder with a diameter of 1.5 mm and a height of 1 mm; the sample confluence channel 213 The initial height is 30 μm, the initial height of the sheath fluid confluence channel 223 is 70 μm, the height of the confluence section 23 is 70 μm; the width of the detection channel 24 is 100 μm, and the height is 70 μm; the waste liquid storage tank 25 is 2 mm and the height is 1 mm. Cylinder; the material of the waste liquid channel 26 is a 19G capillary steel needle.

An embodiment of the present invention provides the application of a three-dimensional focused high-throughput microfluidic chip in the imaging detection of single cells (5-25 μm in diameter),

Set the flow rate of the syringe pump to 3.36mL/min, inject the sample into the chip main body 2, the sample flows through the sample inlet channel 211, the sample liquid reservoir 212 and the sample confluence channel 213, and set the flow rate of the syringe pump to 6.72mL/min at the same time, use The syringe pump injects the sheath fluid into the chip main body 2, and the sheath fluid flows through the sheath fluid inlet channel 221, the sheath fluid reservoir 222 and the sheath fluid confluence channel 223;

Subsequently, the sample and the sheath fluid converge at the confluence section 23 . At this time, the flow rate in the detection channel 24 is 16.8 mL/min, and the average flow rate is 40 m/s. Cells in the sample are detected in the detection channel 24 .

The working principle of the three-dimensional focused high-throughput microfluidic chip is as follows:

Firstly, the cell sample is driven by the pump, through the pipeline from the sample inlet channel 211, the sample liquid reservoir 212 and the sample confluence channel 213 of the microfluidic chip; at the same time, the sheath fluid is also driven by the pump, through the pipeline From the sheath fluid inlet channel 221 of the microfluidic chip, the sheath fluid reservoir 222 and the sheath fluid confluence channel 223; the cell sample flows from the sample confluence channel 213 into the confluence section 23 to merge with the sheath fluid, and then enters the narrow and long detection channel 24 and In the waste liquid storage tank 25, the cells will accelerate when they enter the detection channel 24 from the lower part of the confluence section 23, and stabilize at a point on the cross section in the optical imaging detection channel 24, and flow to the waste liquid storage after passing the detection The pool 25 flows out from the waste liquid channel 26.

In summary, the three-dimensional focusing high-throughput microfluidic chip of the present invention abandons the traditional inlet design perpendicular to the chip plane, and instead uses the side to set the sample inlet and outlet ports, avoiding the gap between the chip interface and the microscope lens. Interference, thereby reducing the size of the chip, greatly reducing the flow resistance of the channel. The microfluidic chip of the present invention can withstand very high flow and pressure, realize high-speed flow of cells, and meet the requirements of time-domain stretching optical microscope as far as possible. In addition, for the focus of the cells, the sample channel and the sheath channel are set at different heights to drive all the cells to flow to one side of the rectangular channel; the flow rate of the liquid and cells is increased, and the velocity gradient on the channel section is increased, so that the cells are subjected to The role of inertial lift is more obvious, and it quickly stabilizes at a fixed vertical height under the action of high inertial force, thereby achieving three-dimensional focusing of cells.

An embodiment of the present invention also provides a method for manufacturing a three-dimensional focused high-throughput microfluidic chip comprising the following steps:

Step 1: As shown in FIG. 2( a ), make a microstructure mold 4 on a single-polished silicon wafer 5 with SU-8 negative photoresist by conventional photolithography means.

Step 2: paste the SU8 male mold obtained in step 1 into a flat-bottomed box that can accommodate single-throw silicon wafers 5, as a casting mold;

Step 3: vacuumize the mixture of PDMS and curing agent and pour it into the casting mold obtained in step 2, and bake it at 80°C for 2 hours to cure. After the PDMS is cured, a microstructure 6 is obtained, as shown in Figure 2(b).

Step 3: peeling off the microstructure 6 from the single-polished silicon wafer 5;

Step 4: Use a puncher to punch holes at the inlet and outlet of the microchannel on the microstructure 6 as the sample reservoir 212, the sheath fluid reservoir 222 and the waste fluid reservoir 25, as shown in Figure 2 (c) Show.

Step 5: Use a puncher to punch holes from the side of the microstructure 6 toward the sample reservoir 212, the sheath fluid reservoir 222 and the waste fluid reservoir 25, and these holes will be used as catheter insertion holes, as shown in Figure 2 ( d) as shown

Step 6: Blow the microstructure 6 and the quartz glass slide 7 that have been punched clean, use plasma to perform surface treatment on the microstructure 6 and the quartz glass slide 7 for channel sealing, and stick the treated surfaces together;

Step 7: seal the microstructure 6 and another liquid reservoir and blow it off with a quartz glass slide 7, use plasma again to perform surface treatment on the upper surface of the microstructure 6 and the quartz glass slide 7, and bond the treated surfaces together together. At this moment, the holes of the sample liquid storage tank 212, the sheath liquid storage tank 222 and the waste liquid storage tank 25 are sealed into cavities by two quartz glass slides 7, as shown in Figure 2(e)

Step 8: As shown in Figure 2(f), the microstructure 6 has the catheter insertion holes made in step 7, and inserts capillary steel pipes 8 at these interfaces respectively, and the capillary steel pipes 8 cannot be inserted into the sample liquid reservoir 212, the sheath liquid reservoir In the liquid pool 222 and the waste liquid storage pool 25, the chip main body 2 is obtained.

Step 9: Pour the mixed resin glue 9 into the gap between the two quartz glass slides 7 and the chip main body 2, and seal and fix the chip main body 2 and the capillary steel pipe 8.

The manufacture of a three-dimensional focused high-throughput microfluidic chip can be completed by the above nine steps.

Finally, it should be noted that: the above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it still It is possible to modify the technical solutions recorded in the foregoing embodiments, or to perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. within the spirit and principles of the present invention shall be included in the within the protection scope of the present invention.

Claims (9)

1. A three-dimensional focused high-throughput microfluidic chip, comprising a chip cover plate, a chip body and a chip base plate, characterized in that, The chip body is provided with a microfluidic structure, the microfluidic structure includes a sample channel, a sheath fluid channel, a detection channel and a waste fluid channel, and the sample channel is arranged between two sheath fluid channels; The sample channel includes a connected sample inlet channel and a sample confluence channel; The sheath fluid channel includes a sheath fluid inlet channel and a sheath fluid confluence channel; The sample confluence channel merges with the sheath fluid confluence channel at the confluence section, and communicates with the detection channel at the confluence section; The detection channel communicates with the waste liquid channel; Both the sample inlet channel and the sheath fluid inlet channel are arranged on one side of the chip body, and the waste liquid channel is arranged on the other side of the chip body. 2. The high-throughput microfluidic chip according to claim 1, wherein the initial height of the sheath confluence channel is greater than twice the initial height of the sample confluence channel; And the initial height of the sheath fluid confluence channel is greater than the diameter of the detection cells. 3 . The high-throughput microfluidic chip according to claim 1 , wherein both the sheath fluid confluence channel and the sample confluence channel have structures that are wide at the front and narrow at the rear. 4. The high-throughput microfluidic chip according to claim 1, characterized in that, a sample reservoir is provided at a place where the sample inlet channel communicates with the sample confluence channel; A sheath fluid reservoir is provided at the place where the sheath fluid inlet channel communicates with the sheath fluid confluence channel; A waste liquid storage pool is provided at the communication point between the detection channel and the waste liquid channel. 5 . The high-throughput microfluidic chip according to claim 1 , wherein the detection channel has a rectangular cross-section. 6 . The high-throughput microfluidic chip according to claim 1 , wherein if there is a gap between the chip body, the chip cover plate and the chip bottom plate, the gap is filled with resin. 7. The high-throughput microfluidic chip according to claim 1, wherein the material of the sample inlet channel, the sheath fluid inlet channel and the waste liquid channel is capillary steel needles. 8. The application of the three-dimensional focused high-throughput microfluidic chip of claim 5 in single-cell imaging detection, characterized in that, comprising: Use a syringe pump to inject the sample into the main body of the chip, and the sample flows through the sample inlet channel, the sample liquid reservoir and the sample confluence channel, and at the same time use the syringe pump to inject the sheath liquid into the chip main body, and the sheath liquid flows through the sheath liquid inlet channel, the sheath liquid reservoir Confluence channel with sheath fluid; Subsequently, the sample and the sheath fluid converge at the confluent section, and the cells in the sample are detected at the detection channel. 9. A method for manufacturing a three-dimensional focused high-throughput microfluidic chip according to any one of claims 1 to 7, characterized in that, comprising: Perform photolithography to form a mold; preparing a chip body using the mold; A three-dimensional focused high-throughput microfluidic chip is obtained by assembling the chip cover plate, the chip body and the chip bottom plate.