Micro-fluidic printing chip with buffer liquid storage tank, system and liquid drop printing method

Abstract

The invention relates to a microfluidic printing chip with a buffer liquid storage pool, a system and a droplet printing method, the microfluidic printing chip comprises a microfluidic channel, a sample liquid storage pool and the buffer liquid storage pool, the microfluidic channel comprises a sample inlet, a flow limiting section and a printing outlet, the sample inlet is connected with the flow limiting section, the flow limiting section is connected with the printing outlet, and the sample liquid storage pool is connected with the buffer liquid storage pool. The sample liquid storage pool is connected with the sample inlet, the buffer liquid storage pool is connected with the connecting point of the flow limiting section and the printing outlet, and a liquid supplementing opening is formed in the lower portion of the side wall of the buffer liquid storage pool so that the liquid level of the buffer liquid storage pool can be higher than the liquid supplementing opening. The generation of liquid drops is accurately regulated and controlled through the gas pressure of the flow limiting section, and mixing and pollution of multi-component liquid are avoided from the source. The liquid supplementing opening is formed in the lower portion of the side wall of the buffer liquid storage pool so that the liquid level of the buffer liquid storage pool can be higher than the liquid supplementing opening, the limitation of the microliter level liquid storage amount of a chip can be solved, uninterrupted printing is achieved, and new bubbles are not introduced in the liquid supplementing process.

Description

Technical Field

[0001] This invention relates to the field of microfluidics, and in particular to a microfluidic printing chip, system, and droplet printing method with a buffer reservoir. Background Technology

[0002] Microfluidic printing chips combine "microfluidics" and "printing" into one. First, picoliter-nanoliter droplets / microfibers are "pre-fabricated" into monodisperse units within the chip using microvalve, micropump, or electrowetting array. Then, they are sprayed point by point onto an open substrate according to CAD coordinates to achieve digital assembly of 2D / 3D biological structures.

[0003] Droplet fabrication technologies include passive microfluidics and active printing. Passive microfluidics utilizes fluid shear forces within microchannels to generate droplets. While achieving high throughput, it suffers from susceptibility to cross-contamination, and the droplet formation location and component ratios are difficult to control flexibly, failing to meet the fabrication requirements of programmable arrays. For example, traditional T-channel droplet generation systems rely on complex flow field adjustments, are prone to crosstalk during multi-component mixing, and cannot deposit individual droplets at specific points. Furthermore, the liquid storage capacity of existing microfluidic printed chips is only microliters, further limiting long-term continuous printing. Among active printing technologies, traditional pneumatic printing is the most widely used. However, traditional pneumatic printing methods suffer from hydrostatic pressure drift: as printing progresses, the liquid level in the reservoir decreases, and the total pressure (air pressure + hydraulic pressure) acting on the nozzle continuously decreases slightly; at the microfluidic scale, the droplet volume drifts accordingly, failing to meet the stringent sample consistency requirements of single-cell sequencing. Summary of the Invention

[0004] This invention provides a microfluidic printing chip, system, and droplet printing method with a buffer reservoir, aiming to solve at least one of the technical problems existing in the prior art.

[0005] The technical solution of this invention is a microfluidic printing chip with a buffer reservoir, comprising: A microfluidic channel, comprising a sample inlet, a flow-limiting section, and a printing outlet, wherein the sample inlet is connected to the flow-limiting section, and the flow-limiting section is connected to the printing outlet; A sample storage tank, which is connected to the sample inlet; A buffer reservoir is connected to the connection point of the flow limiting section and the printing outlet. A replenishment port is provided on the lower part of the side wall of the buffer reservoir so that the liquid level of the buffer reservoir is higher than the replenishment port.

[0006] According to some embodiments of the present invention, the buffer storage tank is provided with a vent.

[0007] According to some embodiments of the present invention, a cross channel is provided at the connection point of the buffer reservoir, the flow limiting section and the printing outlet, and the microfluidic printing chip with the buffer reservoir includes a waste liquid outlet, which is connected to the cross channel.

[0008] According to some embodiments of the present invention, the multiple microfluidic channels are arranged independently of each other.

[0009] According to some embodiments of the present invention, a nanoliter jetting terminal is included, the nanoliter jetting terminal being connected to the printing outlet.

[0010] The technical solution of the present invention also relates to a microfluidic printing system, comprising: The microfluidic printed chip with a buffer reservoir as described in any one of claims 1-5; An injection pump is connected to the replenishment port of a buffer reservoir; A main control computer, which is connected to the injection pump.

[0011] According to some embodiments of the present invention, it includes: an active microvalve connected to the vent of a buffer reservoir; A pressure pump, which is connected to the active micro-valve.

[0012] According to some embodiments of the present invention, a liquid level sensor is included, the liquid level sensor being connected to a buffer storage tank.

[0013] According to some embodiments of the present invention, a temperature-controlled box is included, wherein the microfluidic printing chip is disposed inside the temperature-controlled box.

[0014] The technical solution of the present invention also relates to a droplet printing method, comprising the following steps: S100. The temperature control box is activated according to the printing material and the temperature of the temperature control box is set so as to reduce the fluid resistance of the microfluidic channel through temperature control. The temperature control box encloses the microfluidic printing chip. S200. Set the initial liquid level of the buffer storage tank to a preset level, and read the current liquid level of the buffer storage tank through a liquid level sensor connected to the buffer storage tank. S300, set the droplet volume, active microvalve parameters, displacement platform path and multi-channel triggering sequence, wherein the active microvalve parameters include active microvalve pressure and active microvalve opening time; S400: Based on the set droplet volume, active microvalve parameters, displacement platform path, and multi-channel trigger timing, the main control computer sends instructions to control the displacement platform to move to the first target coordinate and position it; the active microvalve connected to the microfluidic printing chip in the corresponding channel is controlled to open according to the active microvalve parameters, so that the gas drives the liquid to be ejected through the nanoliter injection terminal to form droplets, thereby completing droplet printing; S500. If the current liquid level of the buffer reservoir is lower than the preset liquid level, liquid replenishment is triggered. Liquid is replenished to the buffer reservoir by an injection pump until the current liquid level of the buffer reservoir reaches the preset liquid level, so as to keep the droplet printing process stable and thus keep the size of the printed droplets consistent.

[0015] The present invention also relates to a computer device, including a memory and a processor, wherein the processor executes the method described above when executing a computer program stored in the memory.

[0016] The present invention also relates to a computer-readable storage medium having program instructions stored thereon, which, when executed by a processor, implement the method described above.

[0017] The beneficial effects of the present invention include: the microfluidic printing chip includes: a microfluidic channel, a sample reservoir, and a buffer reservoir. The microfluidic channel includes a sample inlet, a flow-limiting section, and a printing outlet. The sample inlet is connected to the flow-limiting section, the flow-limiting section is connected to the printing outlet, the sample reservoir is connected to the sample inlet, and the buffer reservoir is connected to the connection point of the flow-limiting section and the printing outlet. A replenishment port is provided at the lower part of the side wall of the buffer reservoir so that the liquid level in the buffer reservoir is higher than the replenishment port.

[0018] The microfluidic printing chip incorporates a microfluidic channel, including a sample inlet, a flow-limiting section, and a printing outlet. Droplet generation is precisely controlled by the gas pressure in the flow-limiting section, preventing the mixing and contamination of multiple liquid components from the outset. A replenishment port is located at the lower part of the side wall of the buffer reservoir, ensuring the liquid level in the buffer reservoir is higher than the replenishment port. This overcomes the limitation of liquid volume in micro-scale chip printing, enabling uninterrupted printing without introducing new air bubbles during replenishment. Timely replenishment of liquid into the buffer reservoir through the replenishment port corrects pressure shifts and maintains stability during extended printing processes.

[0019] Furthermore, additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of a microfluidic printing chip with a buffer reservoir in an embodiment of the present invention.

[0021] Figure 2 This is a schematic diagram of a microfluidic printed chip without a sample reservoir in an embodiment of the present invention.

[0022] Figure 3 This is a schematic diagram of the buffer storage tank in an embodiment of the present invention.

[0023] Figure 4 This is a schematic diagram of the microfluidic printing system in an embodiment of the present invention.

[0024] Figure 5 This is an optional flowchart of the droplet printing method in the embodiments of the present invention.

[0025] Figure 6 This is an optional flowchart of the droplet printing method in the embodiments of the present invention.

[0026] Figure 7 This is a schematic diagram of the printing process when there is sufficient liquid for pneumatic printing in an embodiment of the present invention.

[0027] Figure 8 This is a schematic diagram of the buffer storage tank structure in this embodiment of the invention for mechanism research.

[0028] Figure 9 This is a comparison chart of the linearity and spacing of droplets continuously printed for 1000ms with and without nanoliter jetting terminals in this embodiment of the invention.

[0029] Figure 10 This is a schematic diagram of the droplet printing and mixing results of a nano-scale multi-path collaborative printing jet nozzle in an embodiment of the present invention.

[0030] Figure 11 This is a schematic diagram illustrating the applicable scope of the single-cell printed chip with an integrated buffer reservoir in this embodiment of the invention.

[0031] Explanation of reference numerals in the attached figures: 100. Microfluidic channel; 110. Sample inlet; 120. Flow restriction section; 130. Printing outlet; 140. Cross channel; 200. Sample reservoir; 300. Buffer reservoir; 310. Liquid replenishment port; 320. Vent port; 400. Waste liquid outlet. Detailed Implementation

[0032] The following will provide a clear and complete description of the concept, specific structure, and technical effects of the present invention in conjunction with the embodiments and accompanying drawings, so as to fully understand the purpose, solution, and effects of the present invention. It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0033] It should be noted that, unless otherwise specified, when a feature is referred to as "fixed" or "connected" to another feature, it can be directly fixed or connected to the other feature, or indirectly fixed or connected to the other feature. Furthermore, the descriptions of "upper," "lower," "left," "right," "top," and "bottom" used in this invention are only relative to the relative positional relationships of the various components of the invention in the accompanying drawings.

[0034] Furthermore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in this specification is for the purpose of describing particular embodiments only and not for limiting the invention. The term "and / or" as used herein includes any combination of one or more of the associated listed items.

[0035] It should be understood that although the terms first, second, third, etc., may be used to describe various elements in this invention, these elements should not be limited to these terms. These terms are only used to distinguish elements of the same type from each other. For example, a first element may also be referred to as a second element without departing from the scope of this invention, and similarly, a second element may also be referred to as a first element.

[0036] Reference Figures 1 to 11 In some embodiments, the microfluidic printing chip with a buffer reservoir of the present invention includes: a microfluidic channel 100, a sample reservoir 200, and a buffer reservoir 300. The microfluidic channel 100 includes a sample inlet 110, a flow-limiting section 120, and a printing outlet 130. The sample inlet 110 is connected to the flow-limiting section 120, and the flow-limiting section 120 is connected to the printing outlet 130. The sample reservoir 200 is connected to the sample inlet 110. The buffer reservoir 300 is connected to the connection point of the flow-limiting section 120 and the printing outlet 130. A replenishment port 310 is provided on the lower part of the side wall of the buffer reservoir 300 so that the liquid level of the buffer reservoir 300 is higher than that of the replenishment port 310.

[0037] As can be seen, a microfluidic printing chip internally constructs a microfluidic channel 100, which includes a sample inlet 110, a flow-limiting section 120, and a printing outlet 130. The gas pressure in the flow-limiting section 120 precisely controls droplet generation, preventing the mixing and contamination of multiple liquid components from the source. By providing a replenishment port 310 on the lower part of the side wall of the buffer reservoir 300, the liquid level in the buffer reservoir 300 is higher than that at the replenishment port 310. This overcomes the limitation of liquid storage capacity in chip micro-upgrades, enabling "uninterrupted printing" without introducing new air bubbles during replenishment. Timely delivery of liquid into the buffer reservoir 300 through the replenishment port 310 corrects pressure deviations and maintains stability during long-term printing processes.

[0038] It should be noted that the printing objects (droplets) of the microfluidic printing chip include: pure droplets (such as pure water, biological culture medium, matrix gel, etc.), droplets encapsulating cells (such as metacercariae, microalgae, sperm), and droplets encapsulating microparticles (such as plastic microspheres). The sample reservoir 200 can hold cell suspensions or chemical reagents, and the buffer reservoir 300 is used for printing gases or liquids. The buffer reservoir 300 is used to spray the liquid flowing through the cross channel 140 in the sample reservoir 200 into a container outside the chip via the printing outlet 130. The lower channel of the cross channel 140 connects to the printing outlet 130 for printing droplets; the recognition area of ​​the cross channel 140 is located at the intersection and upstream of the cross channel, and this recognition area has image recognition capabilities for cell sorting.

[0039] Specifically, the function of the flow-limiting section 120 is to stabilize the liquid flow rate. Tiny pressure fluctuations introduced into the microfluidic channel 100 are amplified internally, and the flow-limiting section is needed to reduce this impact. The microfluidic channels are of the same size and dimensions, and multiple microfluidic channels 100 are arranged parallel to each other. The flow-limiting section 120 consists of multiple curved channels, which achieve the effect of stabilizing the liquid flow rate. These curved channels are U-shaped.

[0040] In some embodiments, the buffer reservoir 300 is provided with a vent 320, and the bottom of the buffer reservoir 300 is inverted conical.

[0041] In a specific embodiment, the buffer storage tank 300 is provided with a vent 320 at the top, and the buffer storage tank 300 is arranged with a large cross-section cavity.

[0042] See Figure 3 To address the challenges of bubble interference and flow field fluctuations, this invention designs the buffer reservoir 300 as a hexagonal conical-bottom reservoir, serving as the core hub connecting the pressure pump (drive end) and the injection pump (replenishment end). The top vent 320 connects to a pressure pump, and the air pressure directly acts on the liquid surface to provide driving force. The liquid in the buffer reservoir 300 effectively absorbs the mechanical pulsation during the injection pump's replenishment and the impact generated by the active micro-valve switching, ensuring stable air pressure and a consistently stable printing flow field. The printing process is as follows: Figure 7 As shown, a replenishment port 310 is located below the liquid surface on the side wall of the buffer reservoir 300, connecting to the injection pump to achieve "simultaneous printing and replenishment." The inverted conical design at the bottom of the buffer reservoir 300 collects the liquid, eliminating dead volume. The buffer reservoir 300 utilizes bypass injection to prevent liquid dripping and air entrainment. Based on a large cross-section cavity design, the buffer reservoir 300 ensures that the buoyancy of microbubbles exceeds the fluid drag force, causing them to float and be discharged naturally, acting as a "bubble filter" to block bubbles from entering the microchannels at the source. This design achieves hydrodynamic decoupling between the driving air path and the replenishment liquid path, ensuring that the pressure transmitted to the nozzle remains stable during continuous replenishment.

[0043] in, Figure 7 (a) represents the initial state; Figure 7 (b) indicates the start of printing; Figure 7 (c) indicates the end of printing; Figure 7 (d) indicates that the flow field has returned to stability. Figure 3 (a) represents a plan view (blue represents the sample). Figure 3 (b) represents the 3D rendering of the chip.

[0044] Understandably, the buffer reservoir 300 connects both the pressure pump (drive end) and the injection pump (replenishment end). This unique dual-interface design not only enables long-term continuous operation with "printing and replenishing simultaneously," but also effectively buffers pressure pulsations during replenishment and drive processes using the volume effect of the buffer reservoir 300, maintaining a stable flow field. Simultaneously, the structural design achieves gas-liquid separation, preventing microbubbles from entering the microchannels at the source and ensuring bubble-free generation of nanoliter droplets. Based on this, the system integrates inlet liquid level monitoring and compensation, outlet window droplet counting detection, and local temperature control, constructing a closed-loop system for the entire process of "stabilized flow-drive-verification." This chip enables: efficient, stable, and bubble-free printing of single multi-type biological microparticles and multi-component droplets; effective control of droplet deviation and real-time counting and verification of jet output, significantly improving printing accuracy and data reliability; precise mixing of multi-color liquids and programmable micro-pattern printing. This technology combines advanced pneumatic drive technology with multi-channel collaborative control, solving the problems of pressure drift and bubble interference caused by liquid level changes in traditional pneumatic printing. It enables continuous manufacturing of high-viscosity matrices and special single cells (sperm, parasites, microalgae) with long duration, high activity, high precision and zero missing prints, expanding its application in the fields of precision drug delivery and tissue engineering.

[0045] In some embodiments, a cross channel 140 is provided at the connection point of the buffer reservoir 300, the flow limiting section 120 and the printing outlet 130. The microfluidic printing chip with the buffer reservoir includes a waste liquid outlet 400, which is connected to the cross channel 140.

[0046] See Figure 2 In some embodiments, multiple microfluidic channels 100 are arranged independently of each other.

[0047] Specifically, the microfluidic printing chip can internally construct multiple independent microfluidic channels 100, each corresponding to an independent liquid source interface and a nano-scale printing jet terminal, enabling the printing of multi-component droplet arrays. The buffer reservoir can be equipped with a non-contact liquid level detection interface (capacitive or photoelectric) for real-time sensing of liquid volume changes.

[0048] Microfluidic printed chips with integrated buffer reservoir structures can be applied to the following scenarios: i. Droplet printing chip. Capable of printing nanoscale arrays of multicolor droplets.

[0049] ii. Cell-printed chip. Capable of stably printing various single cells such as algae and parasitic metacercariae, and can successfully print and recover rare sperm at ultra-low concentrations (5 cells / mL).

[0050] iii. Matrix adhesive printing chip. It not only enables the printing of matrix adhesive but also allows for stable replenishment of the matrix adhesive.

[0051] It should be understood that the microfluidic printing chip based on the integrated buffer reservoir 300 proposed in this invention utilizes its unique flow field buffering and gas-liquid separation characteristics to construct a universal printing environment for biological particles of different sizes and morphologies. This technology breaks through the limitation of traditional microfluidic printing on the single physical characteristics of samples, achieving cross-scale compatibility from extremely small reproductive cells (such as sperm) to complex morphological phytoplankton (such as microalgae), and even large-sized pathogens (such as parasitic metacercariae). Through the smooth control of fluid pressure by the buffer reservoir 300, stable transport and highly active encapsulation of various irregular and cross-scale biological particles are ensured in the flow channel, realizing the wide application capability of "one chip structure adapting to multiple biological objects".

[0052] Specifically, a multi-channel timing control algorithm was developed. By setting the coordinates of the displacement platform and the triggering sequence of the active microvalve through programming, different liquids can be continuously printed at the same substrate location, forming composite droplets in situ (such as the reaction system of reagent A + reagent B). Breaking through the limitations of traditional single printhead and single material, and relying on the independent control characteristics of multi-channel, it enables the digital and programmable array preparation of various liquids, supporting customized printing of linear arrays, rectangular arrays, and complex patterns.

[0053] In a specific embodiment, the bottom of the sample storage tank 200 is inverted cone shape, and the sample storage tank 200 is provided with an air inlet.

[0054] In some embodiments, the microfluidic printing chip with a buffer reservoir includes: a nanoliter jetting terminal ( Figure 1 (Not shown in the image), the nanoliter jetting terminal is connected to the printing outlet 130.

[0055] In a specific embodiment, the buffer storage tank 300 is arranged with a large cross-section cavity.

[0056] In a specific embodiment, the channel width of the current limiting section 120 is 0.1mm-0.3mm. Specifically, the channel width of the current limiting section 120 is 0.2mm, and the channel width of the current limiting section 120 can fluctuate within the range of 0.1mm.

[0057] In a specific embodiment, the surface roughness of the nano-injection terminal is set to ≤0.1μm to reduce droplet residue and displacement.

[0058] Specifically, the microfluidic printed chip adopts a PDMS photolithography integrated molding process to ensure the sealing and independence of the microfluidic channel 100. Each microfluidic channel 100 includes a liquid inlet section, a flow-limiting section 120, and a nano-elevation terminal interface. The flow-limiting section 120 has a channel width of 0.2 mm, which forms impedance matching with the nano-elevation terminal (inner diameter 0.1 mm) to control the liquid flow rate to be stable at 5-10 μL / min. The nano-elevation terminal is coaxially fixed to the chip outlet with UV adhesive, and the end surface roughness is ≤0.1 μm to reduce droplet residue and displacement.

[0059] See Figure 9 , Figure 9 The image above shows the droplet linearity and spacing results of continuous printing for 1000ms without a nanoliter jetting terminal. Figure 9 The image below shows the linearity and spacing of droplets printed continuously for 1000ms using nanoliter jetting terminals.

[0060] Specifically, UV adhesive, also known as photosensitive adhesive or ultraviolet curing adhesive, is an adhesive that must be cured by ultraviolet light.

[0061] In a specific embodiment, the inner diameter of the nano-ejection terminal is 0.06-0.16 mm, and the outer diameter of the nano-ejection terminal is 0.23-0.31 mm. Specifically, the inner diameter of the nano-ejection terminal is 0.1 mm, and the outer diameter is 0.23 mm. The inner and outer diameters of the nano-ejection terminal can fluctuate within a range of 0.1 mm.

[0062] It should be understood that the design of an integrated multi-channel independent chip structure enables the supply of multi-component liquids without cross-contamination, optimizes the parameters of the channels and nanoliter injection terminals, and adapts to the pneumatic drive characteristics of the active microvalve to ensure the stability of droplet injection.

[0063] See Figure 4 In some embodiments, the microfluidic printing system of the present invention includes: The aforementioned microfluidic printed chip with a buffer reservoir; An injection pump is connected to the replenishment port 310 of the buffer reservoir 300. The main control computer is connected to the injection pump.

[0064] Specifically, the syringe pump can automatically replenish fluid. The main control computer integrates fluid level control and replenishment algorithms.

[0065] In a specific embodiment, the main control computer is connected to the pressure pump and the main control computer is connected to the liquid level sensor.

[0066] In some embodiments, the microfluidic printing system includes: An active micro-valve is connected to the vent 320 of the buffer storage tank 300. Pressure pump, which is connected to an active micro-valve.

[0067] Specifically, the active micro-valve can be multi-channel.

[0068] In a specific embodiment, the pressure pump is connected to the vent 320. The pressure pump is also connected to the air inlet; the active micro-valve is connected to the air inlet.

[0069] In some embodiments, the microfluidic printing system includes a liquid level sensor connected to a buffer reservoir 300.

[0070] Specifically, the buffer reservoir 300 may integrate a level sensor. The level sensor is used to monitor the amount of liquid stored in the buffer reservoir 300.

[0071] Specifically, to address the hydrostatic pressure drift problem, this system establishes a closed-loop control model. A capacitive sensor (i.e., the aforementioned liquid level sensor) is integrated into the side wall of the buffer storage tank. By detecting the liquid level in the buffer storage tank 300 in real time, liquid is replenished in a timely manner when the liquid level in the buffer storage tank 300 is lower than the preset liquid level, thereby ensuring that the droplet volume variation coefficient (CV) remains at an extremely low level during long-term printing.

[0072] In some embodiments, the microfluidic printing system includes a temperature control box, wherein the microfluidic printing chip is disposed inside the temperature control box.

[0073] Specifically, the temperature-controlled box (semiconductor cooling) encloses the entire microfluidic printed chip.

[0074] Specifically, for high-viscosity bio-inks (such as Matrigel), Matrigel's viscosity is approximately 20-50 mPa·s (liquid) at 4°C, rapidly increasing to >1000 mPa·s (colloidal) above 25°C. The temperature-controlled chamber maintains the flow channel at 4°C, significantly reducing fluid resistance. According to the flow resistance formula... viscosity A reduction of several tens of times means that, at the same flow rate, the required driving air pressure can be greatly reduced.

[0075] A temperature-controlled box encapsulating microfluidic channels and nanoliter jet terminals was constructed, enabling precise control of a fixed temperature range (≤10°C) through semiconductor cooling elements: for high-viscosity matrix adhesives (such as Matrigel), low temperature (4°C) was used to maintain their low-viscosity liquid state, significantly reducing the required driving gas pressure and minimizing shear damage to cells.

[0076] In a specific embodiment, the microfluidic printing system includes: The displacement platform is used to locate the target coordinates of the droplet ejection. Droplet counting sensor, used to count the number of droplets; High-speed camera used to monitor droplet morphology.

[0077] Specifically, the droplet counting sensor (i.e. the aforementioned droplet counting sensor) is installed directly below the nanoliter injection terminal to monitor the droplet falling.

[0078] It should be understood that, to address the "dry printing" problem caused by nozzle clogging or insufficient driving force in open-loop printing, a window-type photoelectric drop sensor (i.e., the aforementioned droplet counting sensor) is installed below the nanoliter injection terminal. When a nanoliter droplet passes through the detection window (infrared beam area) of the droplet counting sensor, it causes a momentary change in light intensity. The droplet counting sensor converts this into an electrical pulse signal and transmits it to the host computer. The host computer uses this signal as a "printing complete" confirmation handshake signal, and only instructs the displacement platform to move to the next position after receiving the signal, thereby achieving strict closed-loop stepping control.

[0079] Understandably, this invention uses a buffer reservoir structure for mechanism research to address the pressure offset problem in traditional pneumatic printing. In traditional microfluidic chip reservoirs, the volume of generated droplets is positively correlated with printing pressure and the opening time of the active microvalve (see...). Figure 8 (a) and Figure 8 (b) However, the drop in liquid level due to sample loss (increase in the air-to-volume ratio in the buffer reservoir) causes a significant change in the volume of the printed droplets (see [reference]). Figure 8 (c) Generally speaking, the higher the air volume ratio in the buffer reservoir, the smaller the printed droplet volume.

[0080] in, Figure 8 (a) represents impact pressure. Figure 8 (b) represents the opening time of the active microvalve. Figure 8 (c) represents the effect of the air volume ratio in the buffer reservoir on the size of the generated droplets.

[0081] To achieve uninterrupted printing and prevent the introduction of new air bubbles during replenishment, the replenishment device features a bypass connected to the buffer reservoir, which links to a software-controlled syringe pump. During replenishment, the liquid in the syringe connected to the pump is promptly delivered to the buffer reservoir, correcting pressure shifts and maintaining stability during extended printing processes.

[0082] Reference Figure 5 In some embodiments, the droplet printing method of the present invention includes at least the following steps: S100: The temperature control box is activated according to the printing material and the temperature of the temperature control box is set so as to reduce the fluid resistance of the microfluidic channel 100 through temperature control. The temperature control box encloses the microfluidic printing chip. S200: Set the initial liquid level of the buffer storage tank 300 to a preset level, and read the current liquid level of the buffer storage tank 300 through a liquid level sensor connected to the buffer storage tank 300. S300, set droplet volume, active microvalve parameters, displacement platform path and multi-channel trigger timing, wherein the active microvalve parameters include active microvalve pressure and active microvalve opening time; S400, based on the set droplet volume, active microvalve parameters, displacement platform path and multi-channel trigger timing, sends instructions through the main control computer to control the displacement platform to move to the first target coordinate and position it; controls the active microvalve connected to the microfluidic printing chip in the corresponding channel to open according to the active microvalve parameters, so that the gas drives the liquid to be ejected through the nanoliter injection terminal to form droplets, thereby completing droplet printing; S500 If the current liquid level of the buffer reservoir 300 is lower than the preset liquid level, liquid replenishment is triggered. Liquid is replenished to the buffer reservoir 300 through the injection pump until the current liquid level of the buffer reservoir 300 reaches the preset liquid level, so as to keep the droplet printing process stable and thus keep the size of the printed droplets consistent.

[0083] The droplets can be dyed droplets, typically pure water; they can also be droplets encapsulating cells, microalgae, parasitic metacercariae, or sperm. A preset liquid level is manually set, ensuring it is higher than the replenishment port. Replenishment maintains a stable liquid level in the buffer reservoir 300 at the preset level, stabilizing the droplet printing process and ensuring consistent droplet volume printed through the printing outlet 130. The replenishment is controlled by an intelligent algorithm on the main control computer, allowing for either printing first followed by replenishment, or printing and replenishment simultaneously.

[0084] Furthermore, step S400 includes: Based on the set droplet volume, active microvalve parameters, displacement platform path, and multi-channel trigger timing, the main control computer sends instructions to control the displacement platform to move to the first target coordinate and position it; the active microvalve connected to the microfluidic printing chip in the corresponding channel is controlled to open according to the active microvalve parameters, so that the gas drives the liquid to be ejected through the nanoliter injection terminal to form droplets, and the droplets are deposited on the substrate; the positioning and injection process is repeated to obtain a single-component droplet array, thus completing the printing of the single-component droplet array.

[0085] See Figure 6 In a specific embodiment, the droplet printing method includes: The temperature control box is activated and its temperature is set according to the printing material, so as to reduce the fluid resistance of the microfluidic channel 100 through temperature control. The temperature control box encloses the microfluidic printing chip. The system controls the injection of multi-component liquids into the sample inlet 110 corresponding to the microfluidic printed chip, starts the pressure pump and main control computer, adjusts the initial pressure to fill the microfluidic channel 100 with liquid, opens the waste liquid outlet 400 to discharge the residual liquid, until the flow field of the microfluidic channel 100 is stable. The initial liquid level of the buffer storage tank 300 is set to reach the preset liquid level, and the current liquid level of the buffer storage tank 300 is read through the liquid level sensor; Set the droplet volume, active microvalve parameters, displacement platform path, and multi-channel triggering sequence. The active microvalve parameters include the active microvalve pressure and the active microvalve opening time. Based on the set droplet volume, active microvalve parameters, displacement platform path, and multi-channel triggering sequence, the main control computer sends instructions to control the displacement platform to move to the first target coordinate and position it; the active microvalve of the corresponding channel is controlled to open according to the active microvalve parameters, so that the gas drives the liquid to be sprayed through the nanoliter injection terminal to form droplets, and the droplets are deposited on the substrate; the positioning and spraying process is repeated to obtain a single-component droplet array, thus completing the printing of the single-component droplet array. Based on the set droplet volume, active microvalve parameters, displacement platform path, and multi-channel triggering sequence, the main control computer sends instructions to control the displacement platform to move to the target coordinates of the composite droplet and position it. The active microvalve of the corresponding channel is controlled to open according to the active microvalve parameters, so that the gas drives the liquid to form droplets through the nanoliter injection terminal. Thus, the multi-component liquids are superimposed at the same position to form a composite droplet, thereby obtaining a multi-component droplet array and completing the printing of the multi-component droplet array. The droplet status is monitored in real time by a high-speed camera. If the droplet shifts or has an abnormal volume, the parameters are automatically fine-tuned. After printing a single-component droplet array or a multi-component droplet array, the active microvalve is closed, and cleaning fluid is introduced to flush the microfluidic channel 100 and discharged through the waste liquid outlet 400. Specifically, when a droplet passes the droplet counting sensor below the nanoliter injection terminal, the droplet counting sensor is controlled to send a droplet detection signal to the main control computer; if the main control computer does not receive the droplet detection signal within a preset time window, it determines that the printing has failed and automatically executes retry or error reporting logic; if the main control computer receives the droplet detection signal within the preset time window, it controls the displacement platform to move to the next target coordinate. If the current liquid level in the buffer reservoir 300 is lower than the preset liquid level, a replenishment operation is triggered. The buffer reservoir 300 is replenished by an injection pump until the current liquid level in the buffer reservoir 300 reaches the preset liquid level, at which point the replenishment operation ends. This ensures that the droplet printing process remains stable, resulting in consistent droplet sizes.

[0086] Specifically, when the current liquid level in the buffer reservoir 300 is lower than the preset liquid level, the system maintains a constant printing air pressure while using a software-controlled flow rate mechanism with an integrated syringe pump to replenish the liquid in the buffer reservoir 300, achieving "printing while replenishing liquid." This overcomes the liquid capacity limitation of microfluidic chips, supporting continuous printing tasks lasting several hours. This method eliminates flow rate decay during long-term printing, and combined with the millisecond-level response (minimum 0.5ms) of the active microvalve, it significantly improves volume uniformity over long periods while ensuring that the energy consumption for generating nanoliter droplets (tens to hundreds of nL) remains consistent with the original solution.

[0087] Specifically, the printing verification logic is established using the pulse signal fed back by the droplet counting sensor in the printing window. When the host computer sends the spray command, the system opens the "detection window period." If the droplet counting sensor in the printing window does not detect the droplet passing through, it is determined as "missed print" or "blocked," and the system automatically triggers the reprint program or alarms and pauses. Combined with the shape monitoring of a high-speed camera, comprehensive quality control is achieved from "whether it exists" to "how well it is."

[0088] Specifically, system initialization and liquid loading: The temperature control box is activated and the temperature is set according to the printing material. For example, if printing Matrigel, a high-viscosity bio-ink containing cells, the temperature is set to 4°C; ordinary cells, sperm, microalgae, etc., do not require the use of a temperature control box; the multi-component liquid is injected into the sample inlet 110 corresponding to the microfluidic printing chip, the pressure pump and the main control computer are activated, the initial pressure is adjusted to fill each microfluidic channel 100 with liquid (without air bubbles), the waste liquid outlet 400 is opened to discharge the residual liquid until the flow field is stable.

[0089] Liquid level self-check and printing parameter programming settings: The liquid level sensor reads the initial height of the buffer storage tank 300. Printing parameters are input via the host computer software: droplet volume (nL), active microvalve parameters (pressure mbar, opening time ms), displacement platform path (coordinates, moving speed), and multi-channel trigger sequence (e.g., printing order of composite droplets). Then, the system is adjusted to the appropriate air pump pressure and active microvalve opening time to ensure successful droplet printing.

[0090] Single-component / multi-component collaborative printing and closed-loop detection: The main control computer sends a synchronization command, and the displacement platform moves to the first target coordinate and positions itself; the corresponding channel active microvalve opens according to the preset active microvalve parameters, and gas drives the liquid to form droplets through the nano-lift injection terminal, which are precisely deposited on the substrate; the positioning and injection process is repeated to complete the single-component array printing. The displacement platform moves to the target coordinate of the composite droplet, and the main control computer triggers the active microvalve of different channels in sequence according to the timing, so that the multi-color / multi-component liquids are superimposed at the same position to form a composite droplet. The droplet passes through the window drop counting sensor below the nano-lift injection terminal; the window drop counting sensor sends a "droplet detected" signal to the main control computer (count +1); if the main control computer does not receive the "droplet detected" signal within the preset time window, the software judges the printing failure and automatically executes retry or error reporting logic; after confirmation of success, the platform moves to the next coordinate.

[0091] Automatic liquid replenishment during printing: Before each print job, the system reads the current liquid level in the buffer reservoir 300. If the current liquid level in the buffer reservoir 300 is lower than the preset level, liquid replenishment is triggered. The replenishment system is integrated into the software-controlled syringe pump system, and then replenishment begins until the liquid level in the buffer reservoir 300 reaches the preset level. This process does not interfere with the ongoing print job.

[0092] Process monitoring and system cleaning: The droplet status is monitored in real time by a high-speed camera. If there is any deviation or abnormal volume, the software will automatically fine-tune the parameters. After printing, the active micro valve is closed, the cleaning fluid is introduced into the flushing channel, and discharged through the waste liquid outlet at 400°.

[0093] In a specific embodiment, the pressure of the active microvalve is adjusted to 150-400 mBar; the width of the 120 channel of the flow-limiting section is arranged to be 0.2 mm, forming an impedance match with the nanoliter injection terminal with an inner diameter of 0.1 mm and an outer diameter of 0.23 mm, so as to stabilize the control liquid flow rate at 5-10 μL / min; by adjusting the opening time of the active microvalve to 1 ms, the droplet volume is obtained to be 7.7-28.2 nL; by adjusting the opening time of the active microvalve to 2 ms, the droplet volume is obtained to be 25.8-47.2 nL.

[0094] Specifically, see Figure 7On-demand dripping control: Based on fluid dynamics and the response characteristics of the active microvalve, the droplet volume is adjusted by controlling the opening time of the active microvalve. A 1ms opening time corresponds to 7.7-28.2 nL, and a 2ms opening time corresponds to 25.8-47.2 nL. At a pressure of 200 mBar, the active microvalve can achieve stable, bubble-free spraying, avoiding liquid dispersion. Spatiotemporal synchronization mechanism: Employing a real-time communication protocol based on Ethernet, the signal delay between the main control computer, the displacement platform, and the active microvalve drive module is ≤1ms. A coordinate-time mapping table is established, using the position signal of the displacement platform as the synchronization signal for triggering the active microvalve, ensuring precise droplet deposition at preset coordinates.

[0095] Understandably, see Figure 10 Composite droplet preparation: Taking the "red + yellow = orange" composite droplet as an example, the program steps are as follows: the displacement platform moves to coordinates (X1, Y1) → triggers the active microvalve of the red channel (opens in 1ms) → delays for 50ms → triggers the active microvalve of the yellow channel (opens in 1ms) → the displacement platform moves to the next coordinate, completing in-situ composite. The 50ms delay is the time for the displacement platform to move; after the red channel is printed, the displacement platform moves the previously printed position of the red channel below the yellow channel.

[0096] Complex pattern printing: The target pattern is pixelated to generate a pixel coordinate matrix and corresponding color / component information; the software automatically converts it into a displacement platform path command and an active micro-valve trigger sequence to drive the system to complete modular printing with a minimum pixel spacing of 250μm.

[0097] It should be noted that the integrated multi-channel independent structure avoids liquid mixing contamination at the source, supports simultaneous printing of more than three different liquids, and is compatible with various components such as biological reagents and chemical raw materials to meet the needs of reaction system construction. Active pneumatic control enables droplet volume regulation from 7.7 to 47.2 nL, and the nanoliter jetting terminal achieves linearity R² ≥ 0.87 and spacing coefficient of variation ≤ 5.0%, far superior to traditional technologies (R² < 0.3, coefficient of variation > 3.2%). Timing control enables in-situ compounding of multiple components at the same location without the need for external mixing equipment; it supports customizable patterns, from simple arrays to complex graphics, which can be quickly realized to meet personalized experimental needs. The printing success rate reaches 97.83%, the channels are modularly expandable, and with automated processes, ≥1000 droplet arrays can be prepared within 1 hour; only manual liquid loading is required, and the rest of the process is fully automated, reducing the operational threshold. While ensuring high throughput (>1000 cells / hour), the number of droplets in each microwell is precisely recorded by a droplet counting sensor, providing irrefutable sample preparation data support for subsequent drug screening or single-cell analysis. The microfluidic printing chip is mass-produced using photolithography; the modular system design allows for expansion of the number of channels and printing area as needed, adapting to large-scale applications in different scenarios. The automatic liquid replenishment system overcomes the limitation of microliter (μL) level liquid storage, supporting milliliter (mL) level continuous throughput, enabling hourly droplet and single-cell printing, suitable for large-scale drug screening and tissue engineering scaffold construction. Temperature control reduces the viscosity of the thermosensitive material, enabling low-pressure printing of high-viscosity matrix gels and improving cell viability. The single-cell printing chip with an integrated 300-cell buffer reservoir successfully and stably prints single cells / microalgae, sperm, parasites, etc. (e.g., ...) in actual testing. Figure 11 As shown in the figure, it demonstrates excellent applicability.

[0098] in, Figure 11 (a) indicates sperm printing. Figure 11 (b) indicates the printing of a parasite (metacercariae). Figure 11 (c) indicates algae printing. Figure 11 (d) indicates substrate adhesive printing.

[0099] Specifically, the microfluidic printing chip contains multiple independent microfluidic channels 100, supporting simultaneous printing of multiple different liquids. Specifically, the nanoliter droplet array is an ordered structure formed by discrete droplets with volumes in the nanoliter range arranged according to a preset pattern, used in scenarios such as biological detection and reaction synthesis. Active microvalves are microvalves that operate based on external energy input or external control signals, including solenoid valves, pneumatic valves, and piezoelectric valves.

[0100] Understandably, the proposed "on-demand dripping" active control strategy precisely controls the timing and volume (tens to hundreds of nanoliters) of a single nanoliter droplet by programming the opening time (minimum 0.5ms), pressure (tens to thousands of mBar), and frequency of the active microvalve, replacing the traditional continuous pressure-driven mode. A collaborative system is constructed between a microfluidic printing chip and a high-precision displacement platform (minimum displacement 10μm). The displacement platform achieves precise two-dimensional planar positioning, the active microvalve is responsible for timed spraying, and synchronous control via host computer software enables droplet printing at any position.

[0101] By leveraging the high-resistance guiding effect of the nanoliter jetting terminals (linearity R² ≥ 0.87, spacing variation coefficient ≤ 5.0%) and the rapid response characteristics of the active microvalve, large-scale droplet arrays can be rapidly generated while maintaining nanoliter printing accuracy. Employing a modular design, the number of channels can be expanded from 3 to over 10. Combined with an automated control process, a printing success rate of 97.83% is achieved, resolving the inherent trade-off between accuracy and efficiency in traditional technologies.

[0102] This invention also provides a computer device including a memory and a processor, wherein the processor performs the above-described method when executing a computer program stored in the memory.

[0103] This invention also provides a computer-readable storage medium storing program instructions thereon, which, when executed by a processor, implement the method described above.

[0104] It should be understood that the method steps in the embodiments of the present invention can be implemented or carried out by computer hardware, a combination of hardware and software, or by computer instructions stored in a non-transitory computer-readable storage medium. The method can use standard programming techniques. Each program can be implemented in a high-level procedural or object-oriented programming language to communicate with the computer system. However, if necessary, the program can be implemented in assembly or machine language. In any case, the language can be a compiled or interpreted language. Furthermore, for this purpose, the program can run on a programmed application-specific integrated circuit (ASIC).

[0105] Furthermore, the procedures described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The procedures described herein (or variations and / or combinations thereof) may be executed under the control of one or more computer systems configured with executable instructions, and may be implemented by hardware or a combination thereof as code (e.g., executable instructions, one or more computer programs, or one or more applications) that commonly executes on one or more processors. The computer program comprises a plurality of instructions executable by one or more processors.

[0106] Furthermore, the method can be implemented in any suitable type of computing platform, including but not limited to personal computers, minicomputers, mainframes, workstations, networked or distributed computing environments, standalone or integrated computer platforms, or in communication with charged particle tools or other imaging devices, etc. Aspects of the invention can be implemented as machine-readable code stored on a non-transitory storage medium or device, whether removable or integrated into a computing platform, such as a hard disk, optical read and / or write storage medium, RAM, ROM, etc., such that it is readable by a programmable computer, and when the storage medium or device is read by the computer, it can be used to configure and operate the computer to perform the processes described herein. Furthermore, the machine-readable code, or portions thereof, can be transmitted via wired or wireless networks. The invention described herein includes these and other different types of non-transitory computer-readable storage media when such media comprises instructions or programs that implement the steps described above in conjunction with a microprocessor or other data processor. When programmed according to the methods and techniques described in the invention, the invention may also include the computer itself.

[0107] A computer program can be applied to input data to perform the functions described herein, thereby transforming the input data to generate output data stored in non-volatile memory. The output information can also be applied to one or more output devices, such as a display. In a preferred embodiment of the invention, the transformed data represents physical and tangible objects, including specific visual depictions of physical and tangible objects generated on the display.

[0108] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention, as long as they achieve the technical effects of the present invention by the same means, should be included within the scope of protection of the present invention. Within the scope of protection of the present invention, the technical solutions and / or implementation methods can have various modifications and variations.

Claims

1. A microfluidic printing chip with a buffer reservoir, characterized in that, include: A microfluidic channel (100) includes a sample inlet (110), a flow-limiting section (120), and a printing outlet (130). The sample inlet (110) is connected to the flow-limiting section (120), and the flow-limiting section (120) is connected to the printing outlet (130). A sample storage tank (200) is connected to the sample inlet (110); A buffer reservoir (300) is connected to the flow limiting section (120) and the printing outlet (130) at the connection point. A replenishment port (310) is provided on the lower part of the side wall of the buffer reservoir (300) so that the liquid level of the buffer reservoir (300) is higher than the replenishment port (310).

2. The microfluidic printing chip with a buffer reservoir according to claim 1, characterized in that, The buffer storage tank (300) is equipped with a vent (320).

3. The microfluidic printing chip with a buffer reservoir according to claim 1, characterized in that, A cross channel (140) is provided at the connection point of the buffer storage tank (300), the flow limiting section (120) and the printing outlet (130). The microfluidic printing chip with a buffer reservoir includes a waste liquid outlet (400), which is connected to the cross channel (140).

4. The microfluidic printing chip with a buffer reservoir according to claim 1, characterized in that, The multiple microfluidic channels (100) are arranged independently of each other.

5. The microfluidic printing chip with a buffer reservoir according to claim 1, characterized in that, include: A nano-ejection terminal is connected to the printing outlet (130).

6. A microfluidic printing system, characterized in that, include: The microfluidic printed chip with a buffer reservoir as described in any one of claims 1-5; An injection pump is connected to the replenishment port (310) of a buffer reservoir (300); A main control computer, which is connected to the injection pump.

7. The microfluidic printing system according to claim 6, characterized in that, include: An active micro-valve is connected to the vent (320) of the buffer reservoir (300); A pressure pump, which is connected to the active micro-valve.

8. The microfluidic printing system according to claim 6, characterized in that, include: A liquid level sensor is connected to a buffer storage tank (300).

9. The microfluidic printing system according to claim 6, characterized in that, include: A temperature-controlled box, wherein the microfluidic printing chip is disposed inside the temperature-controlled box.

10. A droplet printing method, applied to the microfluidic printing system according to any one of claims 6 to 9, characterized in that, Includes the following steps: S100: The temperature control box is activated according to the printing material and the temperature of the temperature control box is set so as to reduce the fluid resistance of the microfluidic channel (100) through temperature control. The temperature control box encloses the microfluidic printing chip. S200, Set the initial liquid level of the buffer storage tank (300) to a preset liquid level, and read the current liquid level of the buffer storage tank (300) through a liquid level sensor, wherein the liquid level sensor is connected to the buffer storage tank (300); S300, set the droplet volume, active microvalve parameters, displacement platform path and multi-channel triggering sequence, wherein the active microvalve parameters include active microvalve pressure and active microvalve opening time; S400: Based on the set droplet volume, active microvalve parameters, displacement platform path, and multi-channel trigger timing, the main control computer sends instructions to control the displacement platform to move to the first target coordinate and position it; the active microvalve connected to the microfluidic printing chip in the corresponding channel is controlled to open according to the active microvalve parameters, so that the gas drives the liquid to be ejected through the nanoliter injection terminal to form droplets, thereby completing droplet printing; S500. If the current liquid level of the buffer reservoir (300) is lower than the preset liquid level, liquid replenishment is triggered. Liquid is replenished to the buffer reservoir (300) by injection pump until the current liquid level of the buffer reservoir (300) reaches the preset liquid level, so as to keep the droplet printing process stable and the size of the printed droplets is consistent.

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