WO2023236760A1

WO2023236760A1 - Printing apparatus matched with microfluidic chip and printing method

Info

Publication number: WO2023236760A1
Application number: PCT/CN2023/095303
Authority: WO
Inventors: 陈华英; 余恩; 赵文涛
Original assignee: 珠海大略科技有限公司
Priority date: 2022-06-10
Filing date: 2023-05-19
Publication date: 2023-12-14
Also published as: CN114958566A

Abstract

Disclosed herein are a printing apparatus matched with a microfluidic chip and a printing method. A printing head comprises: a printing channel configured for outputting particles and a specific liquid, and an auxiliary channel located on one or two sides of the printing channel or surrounding printing channel. When an image recognition system judges that a certain number of particles or a certain volume of liquid is output through a printing port, an auxiliary printing liquid is rapidly output through the auxiliary channel and blended with the droplets around the printing port, thus facilitating the natural drop of the droplets under the action of gravity. The present invention helps the natural drop of the liquid droplets containing the printing liquid or particles by increasing the volume of the droplets, thereby effectively avoiding the influence of conventional rapid spraying-based printing methods on the particles inside the liquid, especially on cell activity.

Description

A printing device and printing method matching microfluidic chips

Technical field

The invention relates to the field of particle printing, in particular to a printing device and a printing method that are matched with a microfluidic chip.

Background technique

Single-cell analysis has become a powerful tool for regenerative medicine, clinical diagnostics, and cell therapy by obtaining data that is often overlooked in traditional population-based studies, and seeding single-cell prints into separate culture chambers is a key requirement for single-cell analysis. one.

Successful isolation of single cells from the cell population and subsequent transfer to designated containers at extremely low volumes while maintaining cell viability is the first and critical step for subsequent cellular and molecular analyses, including single cell cloning, PCR, protein expression, gene Sequencing. Efficient single-cell printing methods have become the key technology for single-cell printing systems. At present, there are many studies at home and abroad that can perform single cell sorting. However, in the vast majority of studies, single cells still stay in the original microfluidic chip after separation, or they can only do some limited work downstream of the microfluidic chip. Single cell analysis cannot inoculate single cells into other containers with certainty without any damage, which limits the development of single cell analysis. Therefore, single cell printing and extraction technology is crucial.

Existing advanced single-cell printing is based on piezoelectric, pressure printing, tube drainage or contact methods. Although these methods make full use of the advantages of microfluidic technology, they either require high equipment requirements and are cumbersome, or the manual printing efficiency is low, and the manufacturing process is complicated.

Peter Koltay and others from the University of Freiburg in Germany introduced a device that uses a fully automatic prototype instrument to print single living cells with non-contact inkjet driven by piezoelectric ceramics. The technology is based on a transparent microfluidic chip and a camera-assisted automatic detection system. Cells inside the chip are confined in microdroplets and controlled by electrical signals in piezoelectric ceramics Single cells are printed by squeezing the liquid chamber that stores the cells so that the droplets fall out. The distribution nozzle is placed at the outlet of the single-cell printing chip designed by Feng, Lin and others of Nagoya University in Japan. Finally, the screened oocytes stop at the distribution nozzle hole and are sprayed by the upper airflow into the culture array to achieve single-cell printing. Chen Huaying of Harbin Institute of Technology and others used one of the simplest printing methods. After the chip screened individual cells through a pneumatic microvalve, the cells were directly drained and inoculated into the microwell plate through a pipe inserted by a needle above the chip. Chen Xiang and others from Shanghai Jiao Tong University punched holes under the chip, inserted capillary glass tubes, and printed droplets containing cells or particles onto the collection substrate. Lidong Qin and others from the Anderson Cancer Center in the United States introduced Hand-Held Single-Cell Pipettes (hSCP) that can be used for single cell isolation. This type of hSCP consists of a dual channel pipette and an hSCP tip. The chip at the hSCP tip is designed with a hook for single cell capture, and the dual-channel pipette connected to the tip can separate single cells through the four steps of preparation, capture, rinse, and release through positive and negative pressure operations. Similarly, Lidong Qin's team also designed a single-cell pipette consisting of three modules: SCP-Tip, Air-Displacement Pipette (ADP) and ADP-Tips for single-cell transcriptome sequencing. Some researchers achieve the purpose of printing by forming tiny droplets that contact the micropores below to achieve passive dripping of the droplets.

The above method has greatly promoted the development of single-cell printing, but it is still necessary to design new printing technology to achieve advanced single-cell biochemical analysis. Therefore, the present invention novelly proposes an easy-to-operate method based on the previous work. Microfluidic printing outlet capable of efficient printing of single cells/particles.

In summary, compared with the present invention, the background technology has obvious shortcomings. A brief summary is as follows:

The piezoelectric single-cell printing of Peter Koltay et al. has high requirements on equipment, is complex to manufacture, expensive, and requires other precision equipment to provide electrical signals. The method of Feng, Lin et al. and the pipette-type single-cell printing of Lidong Qin et al. cannot ensure gentle fluid shear force through pressurized printing, which is more harmful to cells. The most important thing is that pressurization will cause It greatly affects the hydrodynamic pressure distribution of the flow channel inside the chip, causing fluid backflow inside the chip, which is not suitable for high-throughput cell printing. List The upward punching and drainage method of Huaying et al. can easily cause cells to settle at the outlet due to gravity. Although the downward punching and drainage method of Chen Xiang et al. avoids cell precipitation, the operation is more complicated, and cells can easily get stuck in the gap between the capillary glass tube and the chip outlet, making it difficult to achieve single-cell certainty. Print.

The above methods and their shortcomings are briefly summarized as follows:

Table 1 Shortcomings of various background technologies

The purpose of the invention is to develop a printing outlet that assists a single cell printing system to realize printing inoculation. By dynamically adjusting the flow rate of the auxiliary printing channel to control droplet formation through image recognition of cells and particles, efficient and continuous printing of single or multiple cells or particles is achieved. The outlet includes one or more auxiliary printing channels parallel to the main printing channel, which can control the flow rate at will, in addition to the main working channel. When the printing channel prints out single cells or particles to form microdroplets that have not yet been dropped, the auxiliary printing channel will flow out liquid to form large droplets that can drip quickly and merge with the microdroplets at the printing outlet. Because the liquid flow in the auxiliary printing channel and the size of the droplets have no effect on the liquid flow in the printing channel, the flow rate of the auxiliary printing channel can be increased to accelerate the formation of droplets, which greatly improves the efficiency of printing cells or particles. This method does not require additional equipment, is low cost, simple to manufacture, and has flexible control methods. In addition, this method can also control the time interval of droplet formation through image recognition, so that a falling droplet contains a desired number of cells or particles. Ultimately, high-precision printing of single or multiple cells/particles can be achieved.

The purposes of the invention include:

1) Use the auxiliary printing channel to increase the flow rate to quickly form falling droplets, achieving single or multiple fine Rapid printing of cells or particles;

2) The printing outlet is formed in one step without secondary processing, so that no other factors block the printed cells or microspheres in the channel. The auxiliary printing channel is not directly connected to the printing outlet and has no impact on the hydrodynamic characteristics of the fluid in the chip;

3) Identify the number and flow rate of particles based on images, electrical signals, optical signals, etc., and then use the auxiliary printing channel liquid flow to control the droplet size and dripping time to achieve single cells/particles, double cells/particles, three cells/particles, etc. Printing of quantitative cells/particle droplets.

4) According to different control flow rates, rapid printing of liquid droplets wrapping different contents can be achieved.

Contents of the invention

In order to solve the problems existing in the prior art, the present invention provides a printing device and a printing method that are matched with the microfluidic chip; dynamically adjusting the flow rate of the auxiliary printing channel through image recognition of cells and particles to control droplet formation, achieving efficient and continuous The printing of single or multiple cells or particles can also be achieved with the printing of water-in-oil or oil-in-water droplets.

The specific technical solutions are as follows:

A printing device that matches the microfluidic chip, including a single-sided auxiliary printing channel, a double-sided auxiliary printing channel and a four-sided surrounding auxiliary channel, are all used to help particles print quickly.

Preferably, the single-sided auxiliary printing channel includes: a particle sample inlet; a printing channel is provided at the bottom of the particle sample inlet, an auxiliary printing channel is provided on one side of the printing channel, and an auxiliary liquid supply inlet is provided at the top of the auxiliary printing channel.

Preferably, the double-sided auxiliary printing channel includes a particle sample inlet, a printing channel is set at the bottom of the particle sample inlet, auxiliary printing channels are set on both sides of the printing channel, an auxiliary liquid supply inlet is set at the bottom of the auxiliary printing channel, and the printing outlet is set at the printing The size of the bottom end of the channel, the tip of the print outlet, is used to determine the size of the final droplet.

Preferably, the four-sided surrounding auxiliary channel includes an auxiliary liquid supply outlet, an auxiliary printing channel and an auxiliary printing channel annular liquid; an auxiliary liquid supply outlet is arranged at the top of the auxiliary printing channel, and an auxiliary printing channel annular liquid is arranged at the bottom of the auxiliary liquid supply outlet.

Preferably, the present invention can also form oil-in-water or water-in-oil droplets by controlling the flow rate.

Preferably, the printing method of a printing device matching the microfluidic chip includes the following steps:

Step 1: Before the system works, the printing system needs to be placed vertically;

Step 2: The printing channel is open, the auxiliary printing channel is closed, and the image recognition system is used to determine that single or multiple particles flow out with the liquid to the outside of the printing port to form tiny droplets;

Step 3: Open the auxiliary printing channel, inject a certain amount of auxiliary printing liquid into the outlet at a specific time according to the required droplet size, so that it merges with the tiny droplets in step 2 to form large droplets and drips immediately;

Step 4: Close the auxiliary printing channel;

Step 5: Repeat the above steps 2 to 4 to achieve loop printing.

Preferably, the printing channel in step 2 can be liquid water without any particles. At this time, if the auxiliary channel is oil, this method can be used to produce water-in-oil droplets. The amounts of water and oil in the droplets can be adjusted;

On the contrary, the printing channel is oil and the auxiliary channel is water, producing oil-in-water droplets. The size of the droplets is related to the tip size of the printing outlet. The smaller the tip of the chip, the smaller the droplets that can be dropped;

When the printing channel is a liquid with particles or cells, the auxiliary channel is oil. At this time, water-in-oil is printed, with single or multiple particles or cells in the water;

When there is liquid with particles or cells in the printing channel, the auxiliary channel can be the same water or cell culture medium as the liquid in the printing channel. At this time, large droplets will be printed and drip directly.

Preferably, this inventive principle is applied to the chip through the matching printing principle of the microfluidic chip, including the following forms: one-time molding and manufacturing, combined bonding of the print head and chip, and wraparound liquid printing.

The beneficial effects of the printing device and printing method matched with the microfluidic chip of the present invention are as follows:

1. Easy to operate, automated, efficient and fast

You only need to control the flow rate of the auxiliary printing outlet to generate large enough droplets to merge with the droplets at the printing outlet and drip, without any other unnecessary operations. Printing time no longer depends on the flow rate of the print outlet, achieving fast and efficient printing.

2. Simple design and processing, reducing costs

The newly added auxiliary printing outlet can be manufactured together with the chip printing outlet. The microfluidic chip can be exposed to form a channel at one time. The additional cost is basically negligible, and the one-time molded outlet will not create gaps to trap particles.

3. Non-invasive printing of internal channel flow fields

The auxiliary printing outlet is outside the printing outlet and does not affect the flow rate in the cell flow channel. There is no need to use a larger printing flow rate in order to improve printing efficiency. It has little impact on the fluid mechanics characteristics in the printing channel, thus not affecting cell activity. It is suitable for many applications. single-cell printing system.

4. Encapsulation of any number of cells or particles can be achieved.

The number of particles is identified based on the optical signal image, and then the flow rate of the auxiliary printing channel is controlled by electrical signals to control the size and dripping time of the droplets to achieve the printing of multiple droplets.

5. Droplet printing that can wrap different contents can be realized

Different wrapped droplets are formed according to the flow rate, enabling printing of water-in-oil or oil-in-water droplets.

Description of the drawings

Figure 1 is a schematic structural diagram of the method of the present invention; A) a schematic diagram of the auxiliary printing channel on one side; B) a schematic diagram of the auxiliary printing channel on both sides.

Figure 2 Schematic diagram of the principle of printing single particles in the printing structure; A) The sorted single particles form micro droplets at the exit of the printing channel, B) Open the auxiliary printing channel, and quickly form large droplets at the outlet, C) The liquid in the auxiliary printing channel continues The large flow rate output causes the droplets at the outlet to slowly expand and wrap with the exit of the printing channel. The micro-droplets containing a single particle merge into one droplet. D) The auxiliary printing channel continues to pass liquid to make the lower droplet reach a sufficient size, and the droplets fall to complete a round of single-particle printing.

Figure 3 Schematic diagram of the printing structure printing single particle process; A) Schematic diagram of image recognition dynamic control-assisted printing. After the particles arrive at the exit of the printing channel and are recognized by the camera image and receive a high-frequency pulse, the program controls to increase the flow rate of the auxiliary printing channel to accelerate the formation of large droplets at the exit end; B) Droplet fusion diagram. The liquid in the auxiliary printing channel continues to be output at a high flow rate, causing the droplets at the outlet to slowly expand and merge with the micro-droplets containing single particles at the outlet of the printing channel to form one droplet; C) Droplet falling diagram. The auxiliary printing channel continues to pass liquid to make the lower droplets reach a sufficient size, and the droplets fall to complete a round of single-particle printing.

Figure 4 Schematic flow chart of multi-particle printing process for printing structure. Double particle printing as an example; A) Image recognition dynamic control auxiliary printing schematic; after the particles arrive at the exit of the printing channel and are recognized by the camera's image and receive two high-frequency pulses, the program control begins to increase the assistance. The flow rate of the printing channel accelerates the formation of large droplets at the exit end; B) Droplet fusion diagram; the liquid in the auxiliary printing channel continues to be output at a high flow rate, causing the droplets at the outlet to slowly expand and merge with the microparticles containing two particles at the exit of the printing channel. The droplets merge into one droplet; C) Droplet dripping diagram; the auxiliary printing channel continues to pass liquid to make the lower droplets reach a sufficient size, and the droplets drop to complete a round of double-particle printing.

Figure 5 is a schematic diagram of photolithography of the printing channel, auxiliary printing channel and upstream sorting microfluidic chip designed by the present invention, including a printing channel and an auxiliary printing channel.

Figure 6 is a schematic diagram of the bonding between the print head and the upstream microfluidic chip. The print head includes an auxiliary printing channel and a printing channel, and the microfluidic chip includes an auxiliary printing liquid supply channel and a particle channel. A) Connection diagram of the print head and microfluidic chip, align the cell channel with the cell inlet on the print head, and align the auxiliary printing liquid supply channel with the auxiliary printing channel on the print head; B) Print head and microfluidic chip Bonding diagram.

Figure 7 is a schematic diagram of the bonding of the auxiliary printing channel chip and the upstream microfluidic chip. The print head contains an auxiliary printing channel, and the microfluidic chip contains a printing channel; A) Auxiliary printing channel chip and microfluidic chip Control chip connection diagram; B) Assist printing channel chip and microfluidic chip alignment bonding diagram.

Figure 8 is a schematic diagram of the bonding of the annular surrounding auxiliary printing channel chip and the upstream microfluidic chip. The print head contains an auxiliary liquid supply inlet, an auxiliary printing channel and an auxiliary printing channel annular liquid outlet. The microfluidic chip contains a particle channel; A ) Diagram of annular surrounding auxiliary printing channel chip; B) Connection diagram of auxiliary printing channel chip and microfluidic chip; C) Alignment and bonding diagram of auxiliary printing channel chip and microfluidic chip.

Figure 9 Schematic diagram of oil-in-water and water-in-oil droplet printing. The wrapped liquid flows out slowly, and the wrapping liquid flows out quickly, quickly forming wrapped droplets and falling; A) Schematic diagram of water-in-oil droplet printing; B) Water-in-water droplet printing Oil droplet printing schematic.

Detailed ways

The specific embodiments of the present invention are described below to facilitate those skilled in the art to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the technical field, as long as various changes These changes are obvious within the spirit and scope of the invention as defined and determined by the appended claims, and all inventions and creations utilizing the concept of the invention are protected.

The present invention is mainly a printing device that can be matched with a microfluidic chip and help particles to be printed quickly. The printing design is shown in Figure 1. Figure 1A is a single-sided auxiliary printing channel design, including a Particle sample inlet, an auxiliary liquid supply inlet, an auxiliary printing channel and a printing channel. Figure 1B shows the design of the double-sided auxiliary printing channel, which includes a particle sample inlet, an auxiliary liquid supply inlet, two auxiliary printing channels and a printing channel. aisle. When printing particles, according to Figure 2

Follow these steps to implement:

Step 4: Close the auxiliary printing channel;

Step 5: Repeat the above steps 2 to 4 to achieve loop printing.

Furthermore, this fast printing method can also be combined with an image recognition system to realize automated printing and save operating steps.

The printing channel can be liquid without any particles, such as water. If the auxiliary channel is oil, this method can be used to produce water-in-oil droplets. The amounts of water and oil are adjustable. Conversely, oil-in-water droplets can be produced.

When the printing channel is a liquid with particles or cells, the auxiliary channel can be oil. At this time, water-in-oil is printed, with single or multiple particles or cells in the water.

When there is liquid with particles or cells in the printing channel, the auxiliary channel can be the same as the liquid in the printing channel, such as water or cell culture medium. At this time, the printed liquid will be large droplets that drip directly.

The function of the auxiliary channel is: (1) Provide a different liquid from the printing channel to form water-in-oil or oil-in-water, and make it drip quickly by forming larger droplets (2) Provide the same liquid as the printing channel , mainly to increase the volume of liquid and make it drip quickly.

As shown in Figure 3, follow the following steps when printing particles:

Step 4: Close the auxiliary printing channel;

Step 5: Repeat the above steps 2 to 4 to achieve loop printing.

In particular, multi-particle printing can also be achieved, as shown in Figure 4 as an example of printing double particles. Since they are all automated printing, the implementation steps are the same as the single particle printing steps mentioned above, except that the image recognition system is set to two high-voltage pulses and then the syringe pump switch is turned on.

The above is the working principle of the present invention, and there are many forms of applying this method to popular microfluidic chips. This printing method is a structural design downstream of the particle sorting system. After the upstream sorting system completes the particle sorting, it is quickly printed through this printing structure. The method of the present invention can be divided into four types when applied to the upstream microfluidic system. Form: One is to directly lithograph the printing channel, auxiliary printing channel and upstream sorting microfluidic chip designed in the present invention together, as shown in Figure 5, including a printing channel and an auxiliary printing channel; the other The printing channel and auxiliary printing channel of this printing method can be manufactured on a separate printing head, and then the printing head and the upstream microfluidic chip are bonded together, and the auxiliary printing channel entrance is connected to the auxiliary printing liquid supply of the microfluidic chip channel, the printing channel entrance is connected to the particle channel of the upstream microfluidic particle screening chip,

As shown in Figure 6, the print head includes an auxiliary printing channel and a printing channel, and the microfluidic chip includes an auxiliary printing liquid supply channel and a particle channel; there is also a microfluidic chip that originally has an auxiliary printing channel. When creating a printing outlet, the auxiliary printing channel of this printing method is manufactured on a separate chip, and the chip is bonded to the upstream microfluidic chip.

As shown in Figure 7, the print head contains an auxiliary printing channel, and the microfluidic chip contains a printing channel. The liquid flowing out of the last auxiliary printing channel flows out in an annular shape. The annular channel surrounds the particle printing channel, so that the liquid in the auxiliary printing channel can better wrap the particle sample flowing out of the printing channel.

As shown in Figure 8, the print head contains an auxiliary liquid supply inlet, an auxiliary printing channel and an auxiliary printing channel annular liquid outlet, and the microfluidic chip contains a particle channel.

At a deeper level, the present invention can also be applied to droplet microfluidics to form water pockets by controlling the flow rate. Oil or water-in-oil droplets. As shown in Figure 9, water is introduced into the printing channel, and oil is introduced into the auxiliary printing channel. The steps to form water-in-oil droplets are as follows:

Step 1: Before the system works, the printing system needs to be placed vertically.

Step 2: Keep the auxiliary printing channel closed, open the printing channel, and allow a certain amount of printing liquid (water) to flow out of the printing port to form small water droplets;

Step 3: Close the printing channel and open the auxiliary printing channel, so that a certain amount of auxiliary liquid (oil) flows out, merges with the small water droplets in the printing port to form large droplets, and drips quickly.

Step 4: Close the auxiliary printing channel;

Step 5: Repeat steps 2, 3, and 4 above to print water-in-oil droplets in a cycle.

When printing oil-in-water droplets, follow the same steps as above, except that the oil is introduced through the printing channel and the water is introduced through the auxiliary printing channel.

Through the printing principle of microfluidic chip matching, this inventive principle is applied to the chip, including the following forms: one-time molding and manufacturing, combined bonding of the print head and chip, and wrap-around liquid printing.

One-time molding manufacturing refers to the integrated molding with the upstream chip on the same device.

Combination bonding of print head and chip means that the print head is made separately and then assembled with the chip.

Surrounding liquid printing means that the auxiliary printing channel wraps the printing channel in the three-dimensional direction.

Claims

A printing device matching the microfluidic chip is characterized by including a single-sided auxiliary printing channel, a double-sided auxiliary printing channel and a four-sided surrounding auxiliary channel, all of which are used to help particles be printed quickly.
The printing device matching the microfluidic chip according to claim 1, characterized in that the unilateral auxiliary printing channel includes: a particle sample inlet; a printing channel is provided at the bottom of the particle sample inlet, and the printing channel is An auxiliary printing channel is provided on one side, and an auxiliary liquid supply inlet is provided at the top of the auxiliary printing channel.
The printing device matched with the microfluidic chip according to claim 1, characterized in that the two-sided auxiliary printing channel includes a particle sample inlet, a printing channel is provided at the bottom of the particle sample inlet, and both sides of the printing channel An auxiliary printing channel is provided on each side. An auxiliary liquid supply inlet is provided at the bottom of the auxiliary printing channel. The printing outlet is provided at the bottom of the printing channel. The size of the tip of the printing outlet is used to determine the size of the final droplet.
The printing device matched with the microfluidic chip according to claim 1, characterized in that the four-sided surrounding auxiliary channel includes an auxiliary liquid supply outlet, an auxiliary printing channel and an auxiliary printing channel annular liquid; An auxiliary liquid supply outlet is provided at the top, and an auxiliary printing channel annular liquid is provided at the bottom of the auxiliary liquid supply outlet.
The printing device matched with the microfluidic chip according to claim 1, characterized in that the present invention can also form oil-in-water or water-in-oil droplets by controlling the flow rate.
A printing method for a printing device matching a microfluidic chip is characterized by including the following steps:

Step 1: Before the system works, the printing system needs to be placed vertically;

Step 2: The printing channel is open, the auxiliary printing channel is closed, and the image recognition system is used to determine that single or multiple particles flow out with the liquid to the outside of the printing port to form tiny droplets;

Step 3: Open the auxiliary printing channel, and add a certain amount of auxiliary printing liquid according to the required droplet size. Inject it into the outlet at a specific time, so that it merges with the tiny droplets in step 2 to form large droplets and drips immediately;

Step 4: Close the auxiliary printing channel;

Step 5: Repeat the above steps 2 to 4 to achieve loop printing.
The printing method of a printing device matching a microfluidic chip according to claim 4, characterized in that the printing channel in the second step can be liquid water without any particles. At this time, the auxiliary channel can be oil. This method produces water-in-oil droplets with adjustable amounts of water and oil;

On the contrary, the printing channel is oil and the auxiliary channel is water, producing oil-in-water droplets. The size of the droplets is related to the tip size of the printing outlet. The smaller the chip tip is, the smaller the droplets can be. ;

When the printing channel is a liquid containing particles or cells, and the auxiliary channel is oil, water-in-oil is printed, with single or multiple particles or cells in the water;

When the printing channel contains liquid containing particles or cells, the auxiliary channel can be the same water or cell culture medium as the liquid in the printing channel. At this time, large droplets are printed and dripped directly.
The printing method of a printing device matching a microfluidic chip according to claim 4 or claim 5, characterized in that the inventive principle is applied to the chip through the printing principle of the microfluidic chip matching, including the following forms : One-time molding manufacturing, print head and chip combination bonding, and wrap-around liquid printing.