Method and Application of Droplet Dispensing

Abstract

The droplet dispensing method and application provided by this application achieve the effective evaluation of the liquid volume parameter for chip printing, and provide a method for the accuracy and stability of the liquid printing volume of instrument equipment. The droplet dispensing device and method of this application can achieve real-time measurement and precise control, and have the characteristics of rapid startup, convenient adjustment, high efficiency, and accurate volume manipulation.

Description

Technical Field

[0001] This application relates to the field of cell cloning printing, and particularly to a method and application for droplet dispensing. Background Art

[0002] In traditional techniques, in order to generate monoclonal cell lines, cells need to be individually transferred to the containers of a microtiter plate. Generally, a dispensing device sprays droplets containing single cells into the microtiter plate. After the cells are deposited in the corresponding containers of the microtiter plate, the cells can grow and then can be transferred to a bioreactor.

[0003] In traditional cell cloning printing, the dispensing device usually has an optical detection device for determining whether the dispensed liquid sample contains cells or particles meeting preset conditions, and a dispenser for spraying the liquid sample as required. Generally, a separately configured high-speed camera is needed to capture the droplets ejected by the dispenser, and then the volume of the dispensed droplets is evaluated. Traditional dispensing devices are relatively complex, and additional equipment is required to calculate the droplet volume. There are many instruments and devices, and the whole process of droplet dispensing is rather cumbersome, resulting in low efficiency and being unable to achieve accurate and efficient droplet dispensing. Summary of the Invention

[0004] Based on this, this application provides a method and application for droplet dispensing. The droplet dispensing method of this application can conveniently and quickly achieve droplet dispensing to solve the technical problems of relatively complex traditional dispensing devices, cumbersome processes, and low efficiency.

[0005] One aspect of the technical solution of this application provides a method for droplet dispensing, including the following steps:

[0006] Add a sample solution to the dispenser, turn on the optical detection device and the analysis device, set the working parameters of the actuator, trigger the actuator to squeeze the dispenser, so that the sample solution is ejected from the dispenser in the form of droplets. The optical detection device detects the particle information of the sample solution in the target area of the dispenser, and the analysis device analyzes the particle information and determines whether the working parameters set by the actuator meet the preset requirements according to the analysis results.

[0007] If the preset requirements are met, control the actuator to work according to the set working parameters. If the preset requirements are not met, adjust the working parameters of the actuator until the preset requirements are met.

[0008] In one embodiment, the dispenser has a sample loading cavity and a nozzle communicating with the sample loading cavity.

[0009] In one embodiment, the actuator is correspondingly arranged with the dispenser.

[0010] In one embodiment, the operating parameters set by the actuator during actuation include at least one of the driving signal pulse width time, frequency, and amplitude.

[0011] In one embodiment, the particulate matter is a microparticle or a cell.

[0012] In one embodiment, after triggering the actuator, when there is only one particulate matter to be dispensed in the target area, the particulate matter is used as the target particulate matter, and the optical detection device detects the position information of the target particulate matter in the target area.

[0013] In one embodiment, after the actuator is triggered for the first time, the optical detection device records the first relative position of the target particulate matter in the target area.

[0014] When the actuator is triggered again, when the target particulate matter is still in the target area of the dispenser, the optical detection device records the second relative position of the target particulate matter in the target area of the dispenser; when the target particulate matter leaves the target area or other particulate matters appear in the target area, the actuator is triggered again until the optical detection device selects a new unique particulate matter as the target particulate matter in the target area and can determine the first relative position and the second relative position of the target particulate matter.

[0015] In one embodiment, the analysis device calculates the deviation angle and relative displacement of the target particulate matter according to the first relative position and the second relative position, and determines whether the relative displacement of the target particulate matter is effective according to the deviation angle.

[0016] In one embodiment, when the relative displacement is effective, the droplet volume of the sample solution ejected by the dispenser each time is calculated. If the droplet volume meets the preset requirements, it means that the operating parameters of the actuator meet the preset requirements; if the droplet volume does not meet the preset requirements, the operating parameters of the actuator are adjusted to continue to select the target particulate matter for detection and analysis until the droplet volume meets the preset requirements.

[0017] When the relative displacement is invalid, the actuator is triggered again to reselect the target particulate matter for detection and analysis until the relative displacement is effective.

[0018] A droplet dispensing device, the droplet dispensing device performs droplet dispensing by the above-mentioned droplet dispensing method, wherein the droplet dispensing device includes a dispenser, an actuator, an optical detection device, and an analysis device.

[0019] The droplet dispensing device provided by this application realizes the effective evaluation of the volume parameters of the liquid printed on the chip, and provides a method for the accuracy and stability of the liquid printing volume of the instrument and equipment. The droplet dispensing device and method of this application can achieve real-time measurement and precise control, and have the characteristics of rapid startup, convenient adjustment, high efficiency, and accurate volume control. Brief Description of the Drawings

[0020] Figure 1 It is a schematic diagram of the droplet dispensing device;

[0021] Figure 2 It is a schematic diagram of the dispenser in the droplet dispensing device. Detailed Description of the Embodiments

[0022] The following further describes this application in detail in combination with the embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate this application and not to limit the scope of this application. The purpose of providing these embodiments and examples is to make the understanding of the disclosed content of this application more thorough and comprehensive. It should also be understood that this application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of this application, and the equivalent forms obtained also fall within the protection scope of this application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of this application. It should be understood that this application can be implemented without one or more of these details.

[0023] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the technical field to which this application belongs.

[0024] Terms

[0025] As used in this application, "its combination", "any combination thereof", "any combination mode thereof", etc. include all suitable combination modes of any two or more of the listed items.

[0026] In this application, "suitable combination mode", "suitable mode", "any suitable mode", etc., the "suitable" described therein is subject to being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0027] In this application, "preferred", "better", "more preferable", "preferably" are only used to describe the embodiments or examples with better effects. It should be understood that they do not constitute a limitation on the protection scope of this application.

[0028] In this application, "further", "even further", "especially", etc. are used for descriptive purposes and represent differences in content, but should not be understood as a limitation on the protection scope of this application.

[0029] In this application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the selectable numerical values are considered continuous within the above numerical intervals, and include the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, when t is an integer selected from 1 to 10, it means that t is an integer selected from any one of the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this article should be understood to include any and all sub-ranges subsumed therein.

[0030] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, as well as an open technical solution including the listed features.

[0031] The technical solution of this application is to provide a droplet dispensing method, including the following steps:

[0032] Add the sample solution to the dispenser 111, turn on the optical detection device 113 and the analysis device 114, set the working parameters of the actuator 112, trigger the actuator 112 to squeeze the dispenser 111, so that the sample solution is ejected from the dispenser 111 in the form of droplets. The optical detection device 113 detects the particulate matter information of the sample solution in the target area 117 of the dispenser, and judges whether the working parameters set by the actuator meet the preset requirements according to the analysis results. For example, detecting the particulate matter information includes one or more of particulate matter size, quantity, morphology, roundness, and color.

[0033] If the preset requirements are met, control the actuator 112 to work according to the set working parameters. If the preset requirements are not met, adjust the working parameters of the actuator 112 until the preset requirements are met.

[0034] Among them, the dispenser 111 has a sample addition cavity 115 and a nozzle 116 communicating with the sample addition cavity.

[0035] In a specific example, the actuator 112 is arranged corresponding to the dispenser 111. Herein, the corresponding arrangement can be understood as that when the actuator 112 and the dispenser 111 are not in the working state, they do not contact or only contact without extrusion force. When the actuator and the dispenser are in the working state, the actuator 112 makes extrusion contact with the dispenser 111, and the actuator 112 extrudes the dispenser 111 to eject the sample solution from the dispenser 111.

[0036] In a specific example, the dispenser 111 further includes an output element with an output pipeline. One end of the output element communicates with the sample adding cavity, and the other end communicates with the nozzle 116. Particularly, the output pipeline includes a micro pipeline which has a very small flow cross-section so that the sample solution itself cannot flow out of the micro pipeline. Herein, the output element can be a microfluidic chip, and the actuator 112 is arranged corresponding to the output element.

[0037] In a specific example, the actuator can be a piezoelectric actuator, an electromagnetic actuator or a pneumatic actuator.

[0038] In a specific example, the working parameters set when the actuator 112 is actuated include at least one of the driving signal pulse width time, frequency and amplitude.

[0039] Further optionally, the particulate matter is a microparticle or a cell. The sample solution can be a cell suspension or a particulate suspension. The particle is an insoluble object, which can be a live cell, a gel particle, an oil droplet, a particle filled with liquid or a solid particle. For example, the liquid can be a buffer suspension containing live cells.

[0040] In a specific example, after the actuator 112 is triggered, when there is only one particulate matter to be dispensed in the target area 117, taking the particulate matter as the target particulate matter, the optical detection device 113 detects the position information of the target particulate matter in the target area.

[0041] In a specific example, after the actuator 112 is triggered for the first time, the optical detection device 113 records the first relative position of the target particulate matter in the target area 117 of the dispenser.

[0042] Trigger the actuator 112 again. When the target particulate matter is still within the target area 117 of the dispenser, the optical detection device 113 records the second relative position of the target particulate matter within the target area 117 of the dispenser; when the target particulate matter leaves the target area 117 of the dispenser or other particulate matters appear within the target area 117 of the dispenser, the actuator 112 is triggered again until the optical detection device 113 selects a new unique particulate matter within the target area 117 of the dispenser as the target particulate matter and can determine the first relative position and the second relative position of the target particulate matter.

[0043] In a specific example, the analysis device 114 calculates the deviation angle and relative displacement of the target particulate matter based on the first relative position and the second relative position, and determines whether the relative displacement of the target particulate matter is valid according to the deviation angle.

[0044] Among them, when the relative displacement is valid, calculate the droplet volume of the sample solution ejected by the dispenser 111 each time. If the droplet volume meets the preset requirements, it indicates that the working parameters of the actuator 112 meet the preset requirements; if the droplet volume does not meet the preset requirements, adjust the working parameters of the actuator 112 and continue to select the target particulate matter for detection and analysis until the droplet volume meets the preset requirements.

[0045] When the relative displacement is invalid, trigger the actuator 112 again and reselect the target particulate matter for detection and analysis until the relative displacement is valid.

[0046] In a specific example, the number of times n of triggering the actuator satisfies n≥2 and n is an integer.

[0047] In a specific example, the analysis device 114 records the first relative position and the second relative position in the form of coordinates respectively, and records them as (x1, y1) and (x2, y2). The origin of the coordinate is the upper left corner of the rectangular frame of the target area 117, the x-axis and the y-axis are the horizontal and vertical directions of the target area respectively. The deviation angle is θ, the relative displacement is L, the depth of the sample addition cavity 115 is H, and the width of the sample addition cavity 115 is W.

[0048] The calculation formula for the droplet volume of the sample solution is: Δx = |x2 - x1|, Δy = |y2 - y1|, θ = arctan(Δx / Δy). When θ≤5°, L = Δy; V of the ejected droplet = L * S = H * W * L.

[0049] In a specific example, when the deviation angle θ = arctan(Δx / Δy)≤5°, the relative displacement is valid, otherwise it is invalid.

[0050] As Figure 1 shown, the present application also provides a droplet dispensing device 10, which includes the above-mentioned droplet dispensing method for droplet dispensing. Among them, the droplet dispensing device 10 includes a dispenser 111, an actuator 112, an optical detection device 113, and an analysis device 114.

[0051] Among them, the optical detection device 113 is used to detect the sample solution situation in the target area within the sample addition cavity 115 of the dispenser 111.

[0052] The analysis device 114 is used to process the detection information detected by the optical detection device 113, and judge whether the working parameters set by the actuator 112 meet the preset requirements according to the processing results.

[0053] Next, the implementation scheme of the present application will be described in detail in combination with embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions in the following embodiments, the guidance given in the present application is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or refer to the experimental methods known in the art.

[0054] In the following specific embodiments, regarding the measurement parameters of the raw material components, if there is no special description, there may be slight deviations within the weighing accuracy range. Regarding the temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0055] Example 1

[0056] Operation process

[0057] Step S1: Add the sample solution to the dispenser 111 and turn on the optical detection device 113 and the analysis device 114;

[0058] Step S2, set the working parameters of the actuator 112 and then trigger the actuator 112;

[0059] Step S3, process the detection information detected by the optical detection device 113 in the target area 117 through the analysis device 114;

[0060] Step S4, judge whether the working parameters set by the actuator 112 meet the preset requirements according to the processing results.

[0061] This step can be refined as:

[0062] 1. First, set the target area 117 within the observed area of the dispenser 111 and trigger the actuator 112, causing a volume change of the liquid in the dispenser 111.

[0063] 2. When there is only one cell in the target area 117, the optical detection device 113 records the position coordinate information (x1, y1) of this cell.

[0064] 3. The actuator 112 is triggered, causing a displacement change of the original cells in the target area 117. If a new cell appears in the target area 117 or the original cells leave the target area 117 at this time, the actuator 112 is triggered again and the process returns to step 2; otherwise, it proceeds to step 4.

[0065] 4. The optical detection device 113 records the new displacement coordinate information (x2, y2) of the cells in the target area 117.

[0066] 5. The analysis device 114 makes a judgment based on the two coordinate information. If the deviation angle θ = arc tan(Δx / Δy) ≤ 5°, where Δx = |x2 - x1| and Δy = |y2 - y1|, then the relative displacement distance L of the cell is calculated by L = Δy; if the deviation angle θ = arc tan(Δx / Δy) > 5°, it is considered that the detected relative displacement L is invalid, and a new target recognition calculation is performed. The analysis device 114 calculates the displacement distance L of the target cell through step S4, and calculates the droplet volume according to Vsprayed droplet = L * S = H * W * L.

[0067] If the droplet volume meets the required printing volume range, it means that the parameters of the whole machine system are valid and do not need to be modified. If the actually calculated droplet size does not meet the printing volume range, the process can return to step S2 for debugging and real-time adjustment until the printed volume meets the printing volume range.

[0068] If the debugging is not successful (for example: the droplet sizes printed each time are inconsistent, resulting in too many debugging tests or unable to print droplets), then according to the actual situation, the direction for system rectification can be provided, and it is analyzed whether the direct or indirect parts in the system meet the design requirements for the printing results. The printing droplet size results are verified synchronously according to the rectified parts.

[0069] For example, a container filled with 80 μL and a cell density of 1×10 6The solution of cells / ml is placed into the dispenser 111, and appropriate operating parameters of the dispenser 111 are set (actuator trigger frequency 2 Hz, trigger amplitude 40 V, pulse width 500 μs). At this time, the solution in the dispenser 111 is dispensed drop by drop as needed, and the volume of the liquid in the dispenser 111 starts to change. The optical detection device 113 detects that there is only one cell in the area and records the coordinate information as (10, 12). The actuator makes the next trigger, causing a displacement change of the original cells in the target area. The optical detection device records the new displacement coordinate information of the cell as (10.2, 7). The analysis device makes a judgment based on the two coordinates and calculates that the deviation angle θ is 2.3°. This deviation angle meets the effective calculation condition for relative displacement (deviation angle θ ≤ 5°), so the relative displacement distance L of the cell is equal to 5 μm (Δy = |7 - 12|). Finally, the analysis device calculates that the volume of each dispensed droplet is 200 pL (the width in the dispenser is 1 mm, the depth is 40 μm, and the cross-sectional area is 0.04 mm 2 ).

[0070] If the user changes the operating parameters (actuator trigger frequency 2 Hz, trigger amplitude 10 V, pulse width 500 μs). At this time, the optical detection device 113 detects that there is only one cell in the target area 117 and records the coordinate information as (10, 12.1). The actuator makes the next trigger, and the optical detection device records the new displacement coordinate information of the cell as (10.1, 12.0). At this time, the deviation angle calculated by the analysis device is 45°, and this deviation angle does not meet the effective calculation condition for the relative displacement of L. The result of the analysis device calculating the droplet volume is abnormal. Therefore, it is considered that the operating parameters set by the user do not meet the normal use of the single-cell dispensing device and need to be adjusted. The specific parameter adjustment is shown in Table 1.

[0071] Table 1

[0072] Trigger frequency (Hz) Trigger amplitude (V) Pulse width (μs) Displacement distance (μm) Droplet volume (pL) 2 10 500 0 0 2 20 500 0 0 2 35 500 3.75 150 2 40 500 5 200 2 45 500 8.75 350 2 50 500 10.5 420

[0073] The above-described embodiments merely represent several implementation manners of the present application, facilitating a specific and detailed understanding of the technical solution of the present application. However, it should not be construed as a limitation on the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can be made, and these all fall within the protection scope of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning, or limited experiments based on the technical solution provided by the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A droplet dispensing method, characterized in that, It includes the following steps: Add the sample solution to the dispenser, turn on the optical detection device and the analysis device, set the working parameters of the actuator, trigger the actuator to squeeze the dispenser, so that the sample solution is ejected from the dispenser in the form of droplets. The optical detection device detects the particulate matter information of the sample solution in the target area of the dispenser, and the analysis device analyzes the particulate matter information and determines whether the working parameters set by the actuator meet the preset requirements according to the analysis results; If the preset requirements are met, control the actuator to work according to the set working parameters. If the preset requirements are not met, adjust the working parameters of the actuator until the preset requirements are met; After triggering the actuator, when there is only one particulate matter to be dispensed in the target area, use the particulate matter as the target particulate matter, and the optical detection device detects the position information of the target particulate matter in the target area; The optical detection device detecting the position information of the target particulate matter in the target area includes: After the actuator is triggered for the first time, the optical detection device records the first relative position of the target particulate matter in the target area; Trigger the actuator again. When the target particulate matter is still in the target area, the optical detection device records the second relative position of the target particulate matter in the target area; when the target particulate matter leaves the target area or other particulate matters appear in the target area, trigger the actuator again until the optical detection device selects a new unique particulate matter as the target particulate matter in the target area and can determine the first relative position and the second relative position of the new target particulate matter; 2. The droplet dispensing method according to claim 1, wherein The dispenser has a sample addition cavity and a nozzle communicated with the sample addition cavity.

3. The droplet dispensing method according to claim 1, characterized in that, The actuator is correspondingly arranged with the dispenser.

4. The droplet dispensing method according to claim 1, wherein The working parameters set by the actuator during actuation include at least one of the driving signal pulse width time, frequency and amplitude.

5. The droplet dispensing method according to claim 1, characterized in that, The particulate matter is a microparticle or a cell.

6. The droplet dispensing method according to claim 1, wherein The analysis device calculates the deviation angle and relative displacement of the target particulate matter according to the first relative position and the second relative position, and determines whether the relative displacement of the target particulate matter is effective according to the deviation angle.

7. The droplet dispensing method according to claim 6, wherein When the relative displacement is effective, calculate the droplet volume of the sample solution ejected by the dispenser each time. If the droplet volume meets the preset requirements, it means that the working parameters of the actuator meet the preset requirements; if the droplet volume does not meet the preset requirements, adjust the working parameters of the actuator to continue to select the target particulate matter for detection and analysis until the droplet volume meets the preset requirements; When the relative displacement is invalid, trigger the actuator again and reselect the target particulate matter for detection and analysis until the relative displacement is effective.

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