CN116359532A - Method for dispensing droplets and application - Google Patents

Abstract

The liquid drop distribution method and the application thereof realize the evaluation of the validity of the volume parameters of the liquid printed by the chip, and provide a method for the accuracy and the stability of the volume printed by the liquid of the instrument and the equipment.

Description

Method for dispensing droplets and application

Technical Field

The application relates to the field of cell clone printing, in particular to a liquid drop distribution method and application.

Background

In order to generate monoclonal cell lines in conventional techniques, the cells need to be transferred individually to the containers of the microtiter plate, and droplets of the encapsulated individual cells are typically ejected into the microtiter plate by dispensing means. After the cells are deposited in the corresponding containers of the microtiter plate, the cells may be grown and then transferred to a bioreactor.

In conventional cell clone printing, the dispensing apparatus typically has an optical detection device for determining whether the dispensed liquid sample contains cells or particles of a predetermined condition, and a dispenser for spraying the liquid sample on demand. It is generally necessary to capture the droplets ejected by the dispenser by an additionally configured high speed camera and then evaluate the dispensed droplet volume size. The traditional dispensing device is complex, other devices are added to realize liquid drop volume calculation, the number of instruments and devices is large, the whole liquid drop dispensing process is complex, the efficiency is low, and accurate and efficient liquid drop dispensing cannot be realized.

Disclosure of Invention

Based on the above, the application provides a liquid drop distribution method and application. The liquid drop distribution method can conveniently and rapidly realize liquid drop distribution, so that the technical problems of complex traditional distribution devices, complex process and low efficiency are solved.

In one aspect, the present disclosure provides a droplet dispensing method, including the steps of:

the method comprises the steps of adding sample solution into a dispenser, opening an optical detection device and an analysis device, setting working parameters of an actuator, triggering the actuator to squeeze the dispenser, enabling the sample solution to be sprayed out of the dispenser in a liquid drop mode, detecting particulate matter information of the sample solution in a target area of the dispenser by the optical detection device, analyzing the particulate matter information by the analysis device, and judging whether the working parameters set by the actuator meet preset requirements or not according to analysis results.

And if the preset requirement is met, controlling the actuator to work according to the set working parameter, and if the preset requirement is not met, adjusting the working parameter of the actuator until the preset requirement is met.

In one embodiment, the dispenser has a loading chamber and a nozzle in communication with the loading chamber.

In one embodiment, the actuator is disposed in correspondence with the dispenser.

In one embodiment, the operating parameters set by the actuator upon actuation include at least one of drive signal pulse width time, frequency, and amplitude.

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

In one embodiment, after the actuator is triggered, when there is only one particle to be dispensed in the target area, the particle is taken as a target particle, and the optical detection device detects the position information of the target particle in the target area.

In one embodiment, the optical detection device records a first relative position of the target particulate matter within the target area after the actuator is first triggered.

Triggering the actuator again, wherein the optical detection device records a second relative position of the target particulate matter in the target area of the dispenser when the target particulate matter is still in the target area of the dispenser; when the target particle leaves the target area or other particles appear in the target area, the actuator is triggered again until the optical detection device selects a new unique particle as the target particle in the target area, and the first relative position and the second relative position of the target particle can be determined.

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

In one embodiment, when the relative displacement is effective, calculating a droplet volume of the sample solution ejected by the dispenser each time, and if the droplet volume meets a preset requirement, indicating that the working parameter of the actuator meets the preset requirement; and if the liquid drop volume does not meet the preset requirement, adjusting the working parameters of the actuator, and continuously selecting target particles for detection and analysis until the liquid drop volume meets the preset requirement.

And when the relative displacement is invalid, re-triggering the actuator, and re-selecting the target particulate matters for detection and analysis until the relative displacement is valid.

A droplet dispensing apparatus comprising the droplet dispensing method described above, wherein the droplet dispensing apparatus comprises a dispenser, an actuator, an optical detection device, and an analysis device.

The liquid drop distribution device provided by the application realizes the validity assessment of the volume parameters of the liquid printed by the chip, and provides a method for the accuracy and stability of the liquid printed volume of the instrument and equipment.

Drawings

FIG. 1 is a schematic illustration of a droplet dispensing apparatus;

fig. 2 is a schematic view of a dispenser in a droplet dispensing apparatus.

Detailed Description

The present application will be described in further detail with reference to embodiments and examples. It should be understood that these embodiments and examples are provided solely for the purpose of illustrating the application and are not intended to limit the scope of the application in order to provide a more thorough understanding of the present disclosure. It is also to be understood that this application may be embodied in many different forms and is not limited to the embodiments and examples described herein, but is capable of numerous changes or modifications without departing from the spirit of the application, as equivalent forms are intended to be within the scope of this application. Furthermore, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present application, it being understood that the present application may be practiced without one or more of these details.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

Terminology

As used herein, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.

In the present application, "suitable" is described in "suitable combination mode", "suitable mode", "any suitable mode", etc., so as to implement the technical scheme of the present application, solve the technical problem of the present application, and achieve the technical effect expected in the present application.

In this application, "preferred," "better," "preferred," and "preferred" are merely examples of better performing implementations or examples, and it should be understood that they are not limiting the scope of the application.

In this application, "further," "still further," "particularly," and the like are used for descriptive purposes and are not to be construed as limiting the scope of the present application.

In this application, reference is made to a numerical interval (i.e., a numerical range), where the optional numerical distribution is considered continuous, and includes two numerical endpoints (i.e., a minimum value and a maximum value) of the numerical range, and each numerical value between the two numerical endpoints, unless otherwise indicated. Where a numerical range merely refers to integers within the numerical range, including both end integers of the numerical range, and each integer between the two ends, unless otherwise indicated, each integer is recited herein as directly, such as where t is an integer selected from 1-10, and where t is an integer selected from any one of the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Further, when a plurality of range description features or characteristics are provided, these ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

In the present application, the technical features described in an open manner include a closed technical scheme composed of the listed features, and also include an open technical scheme including the listed features.

The technical scheme of the application is to provide a liquid drop distribution method, which comprises the following steps:

the sample solution is added into the dispenser 111, the optical detection device 113 and the analysis device 114 are opened, the working parameters of the actuator 112 are set, the actuator 112 is triggered to squeeze the dispenser 111, the sample solution is ejected from the dispenser 111 in the form of liquid drops, the optical detection device 113 detects the particulate matter information of the sample solution in the target area 117 of the dispenser, and whether the working parameters set by the actuator meet the preset requirements is judged according to the analysis result. For example, detecting the particulate matter information includes one or more of particulate matter size, quantity, morphology, roundness, and color.

If the preset requirement is met, the actuator 112 is controlled to work according to the set working parameters, and if the preset requirement is not met, the working parameters of the actuator 112 are adjusted until the preset requirement is met.

Wherein the dispenser 111 has a loading chamber 115 and a nozzle 116 in communication with the loading chamber.

In a specific example, the actuator 112 is disposed corresponding to the dispenser 111. It is understood that the corresponding arrangement is that if the actuator 112 and the dispenser 111 are not in contact or only in contact without pressing force when they are not in operation, the actuator 112 is in pressing contact with the dispenser 111 when the actuator and the dispenser are in operation, and the actuator 112 presses the dispenser 111 to spray the sample solution from the dispenser 111.

In a specific example, the dispenser 111 further comprises an output member with an output conduit, which output member communicates at one end with the loading chamber and at the other end with the nozzle 116. In particular, the output channel comprises a microchannel having a very small flow cross section, so that the sample solution itself cannot flow out of the microchannel. Wherein, the output element may be a microfluidic chip, and the actuator 112 is disposed corresponding to the output element.

In one particular example, the actuator may be a piezoelectric actuator, an electromagnetic actuator, or a pneumatic actuator.

In one specific example, the operating parameters set by the actuator 112 upon actuation include at least one of a drive signal pulse width time, frequency, and amplitude.

Further alternatively, the particulate matter is a microparticle or a cell. The sample solution may be a cell suspension or a particle suspension. The particles are insoluble objects and may be living cells, gel particles, oil droplets, liquid-filled particles or solid particles. For example, the liquid may be a buffered suspension containing living cells.

In a specific example, when there is only one particle to be dispensed in the target area 117 after the actuator 112 is triggered, the optical detection device 113 detects the position information of the target particle in the target area by using the particle as the target particle.

In one specific example, the optical detection device 113 records a first relative position of the target particulate matter within the target area 117 of the dispenser after the actuator 112 is first triggered.

Triggering the actuator 112 again, the optical detection means 113 registering a second relative position of the target particles within the target area 117 of the dispenser when the target particles are still within the target area 117 of the dispenser; when the target particle leaves the target area 117 of the dispenser or other particles are present in the target area 117 of the dispenser, the actuator 112 is re-activated until the optical detection device 113 selects a new unique particle as the target particle in the target area 117 of the dispenser and is able to determine a first relative position and a second relative position of the target particle.

In one specific example, the analysis device 114 calculates a deviation angle and a 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 based on the deviation angle.

When the relative displacement is effective, calculating a droplet volume of the sample solution ejected by the dispenser 111 each time, and if the droplet volume meets a preset requirement, indicating that the working parameter of the actuator 112 meets the preset requirement; if the drop volume does not meet the preset requirement, the working parameters of the actuator 112 are adjusted to continue to select the target particulate matter for detection and analysis until the drop volume meets the preset requirement.

When the relative displacement is not effective, the actuator 112 is then re-triggered and the target particulate matter is re-selected for detection and analysis until the relative displacement is effective.

In one particular example, the number of times the actuator is triggered n is greater than or equal to 2, and n is an integer.

In a specific example, the analysis device 114 records the first relative position and the second relative position in terms of coordinates, and marks (x 1, y 1) and (x 2, y 2), where the origin of coordinates is the upper left corner of the rectangular frame of the target area 117, the x-axis and the y-axis are the lateral and vertical deviation angles of the target area, θ, the relative displacement is L, the depth of the loading cavity 115 is H, and the width of the loading cavity 115 is W.

The liquid drop volume calculation formula of the sample solution is as follows: Δx= |x2-x1|, Δy= |y2-y1|, θ=arctan (Δx/Δy), when θ is less than or equal to 5 °, l=Δy; v ejected droplets = L x S = H x W x L.

In one specific example, the relative displacement is effective when the deviation angle θ=arctan (Δx/Δy). Ltoreq.5°, otherwise ineffective.

As shown in fig. 1, the present application further provides a droplet dispensing apparatus 10, where the droplet dispensing apparatus 10 includes the droplet dispensing method described above for dispensing droplets, and the droplet dispensing apparatus 10 includes a dispenser 111, an actuator 112, an optical detection device 113, and an analysis device 114.

Wherein, the optical detection device 113 is used for detecting the sample solution condition of the target area in the sample adding cavity 115 of the dispenser 111.

The analysis device 114 is configured to process the detection information detected by the optical detection device 113, and determine whether the working parameter set by the actuator 112 meets a preset requirement according to a processing result.

Embodiments of the present application will be described in detail below with reference to examples. It should be understood that these examples are illustrative only of the present application and are not intended to limit the scope of the present application. The experimental methods, in which specific conditions are not noted in the following examples, are preferably referred to in the guidelines given in the present application, may be according to the experimental manual or conventional conditions in the art, may be according to the conditions suggested by the manufacturer, or may be referred to experimental methods known in the art.

In the specific examples described below, the measurement parameters relating to the raw material components, unless otherwise specified, may have fine deviations within the accuracy of weighing. Temperature and time parameters are involved, allowing acceptable deviations from instrument testing accuracy or operational accuracy.

Example 1

Operation procedure

Step S1: adding the sample solution to the dispenser 111 and opening the optical detection means 113 and the analysis means 114;

step S2, the actuator 112 is triggered after the working parameters of the actuator 112 are set;

step S3 of processing, by the analysis device 114, detection information detected by the optical detection device 113 in the target area 117;

step S4, determining whether the working parameters set by the actuator 112 meet the preset requirements according to the processing result.

This step can be refined as:

1. first a target area 117 is provided in the area of the dispenser 111 that is observed and the actuator 112 is triggered, causing a change in the volume of liquid in the dispenser 111.

2. When the target area 117 has only one cell, the optical detection device 113 records the cell position coordinate position information (x 1, y 1).

3. The actuator 112 triggers and causes the displacement of the original cells within the target area 117. If at this point a new cell is present in the target area 117 or an old cell leaves the target area 117, the actuator 112 is triggered again and the process returns to step 2, otherwise step 4 is entered.

4. The optical detection device 113 records new displacement coordinate information (x 2, y 2) of the cells in the target area 117.

5. The analysis device 114 determines from the two coordinate information, and if the deviation angle θ=arc tan (Δx/Δy) is less than or equal to 5 °, Δx= |x2-x1|, Δy= |y2-y1|, calculates the relative displacement distance L of the cell by l=Δy; if the deviation angle θ=arc tan (Δx/Δy) > 5 °, the detected relative displacement is deemed to be L invalid, and a new target recognition calculation is performed. The analyzer 114 calculates the droplet volume from the droplet=l×s=h×w×l ejected by V by the target cell displacement distance L calculated in step S4.

If the liquid drop volume meets the required printing volume range, the parameters of the whole system are effective, and modification is not needed. If the actually calculated droplet size does not accord with the printing volume range, the step S2 can be returned to be debugged, and the real-time adjustment is carried out until the printing volume accords with the printing volume range.

If the debugging is unsuccessful (for example, the sizes of the droplets printed each time are inconsistent, so that the debugging test is too many or the droplets cannot be printed), the system rectifying direction can be provided according to the actual phenomenon, whether the direct or indirect part in the printing result in the system accords with the design is analyzed, and the printing droplet size result is synchronously verified according to the rectifying part.

For example, 80. Mu.L of the cells with a density of 1X 10 will be loaded ⁶ Placement of cells/ml solution into dispenser 111, appropriate dispenser 111 operating parameters (actuator trigger frequency 2Hz, trigger amplitude 40V, pulse width 500 μs) were set. At this time, the solution in the dispenser 111 is dripped 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 region, and records the coordinate information as (10, 12). The trigger is triggered next time, so that the original cells in the target area are displaced and changed. The optical detection device records new displacement coordinate information of the cell (10.2,7). The analysis device judges according to the two coordinates, and calculates the deviation angle theta to be 2.3 degrees. The deviation angle satisfies the effective calculation condition of relative displacement (the deviation angle theta is less than or equal to 5 degrees), so that the relative displacement of cellsThe distance L is equal to 5 μm (Deltay= |7-12|), and the final analysis device calculated the volume of each dispensed droplet to be 200pL (the width in the dispenser was 1mm, the depth was 40 μm, and the cross-sectional area was 0.04 mm) ² )。

If the user changes the operating parameters (actuator trigger frequency 2Hz, trigger amplitude 10V, pulse width 500 mus). At this time, the optical detection device 113 detects that only one cell is in the target area 117, and records the coordinate information as (10, 12.1). The trigger is triggered next time, and the optical detection device records new displacement coordinate information (10.1, 12.0) of the cells. At this time, the deviation angle calculated by the analysis device is 45 degrees, the deviation angle does not satisfy the effective calculation condition that the relative displacement is Leffective, and the analysis device calculates that the liquid drop volume result is abnormal. Therefore, the operation parameters set by the user are considered to be insufficient for the normal use of the single cell dispensing device, and need to be adjusted. The specific parameter adjustments are shown in table 1.

TABLE 1

Trigger frequency (Hz)	Trigger amplitude (V)	Pulse width (mu s)	Distance of displacement (μm)	Drop 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

The above examples merely represent a few embodiments of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but are not to be construed as limiting the scope of the claims. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Further, it will be understood that various changes or modifications may be made to the present application by those skilled in the art after reading the foregoing teachings, and equivalents thereof will be within the scope of the present application. It should also be understood that those skilled in the art, based on the technical solutions provided in the present application, can obtain technical solutions through logical analysis, reasoning or limited experiments, all fall within the protection scope of the claims attached to the present application. The scope of the patent application is therefore intended to be limited by the content of the appended claims, the description and drawings being presented to the extent that the claims are defined.

Claims (10)

1. A method of dispensing droplets, comprising the steps of:

adding a sample solution into a dispenser, opening an optical detection device and an analysis device, setting working parameters of an actuator, triggering the actuator to squeeze the dispenser, enabling the sample solution to be sprayed out of the dispenser in a liquid drop form, detecting particulate matter information of the sample solution in a target area of the dispenser by the optical detection device, analyzing the particulate matter information by the analysis device, and judging whether the working parameters set by the actuator meet preset requirements according to analysis results;

and if the preset requirement is met, controlling the actuator to work according to the set working parameter, and if the preset requirement is not met, adjusting the working parameter of the actuator until the preset requirement is met.

2. The method of dispensing droplets of claim 1, wherein the dispenser has a loading chamber and a nozzle in communication with the loading chamber.

3. The method of claim 1, wherein the actuator is disposed in correspondence with the dispenser.

4. The method of claim 1, wherein the operating parameters set by the actuator upon actuation include at least one of a drive signal pulse width time, frequency, and amplitude.

5. The method of claim 1, wherein the particulate matter is particulates or cells.

6. The method according to any one of claims 1 to 5, wherein when only one particle to be dispensed is present in the target area after the actuator is activated, the particle is used as a target particle, and the optical detection device detects positional information of the target particle in the target area.

7. The droplet dispensing method according to claim 6, wherein the optical detection means detecting positional information of the target particulate matter within the target area includes:

after the actuator is triggered for the first time, the optical detection device records a first relative position of the target particles in the target area;

triggering the actuator again, and recording a second relative position of the target particles in the target area by the optical detection device when the target particles are still in the target area; when the target particle leaves the target area or other particles appear in the target area, the actuator is triggered again until the optical detection device selects a new unique particle as the target particle in the target area, and the first relative position and the second relative position of the new target particle can be determined.

8. The liquid droplet dispensing method according to claim 7, wherein the analyzing means calculates a deviation angle and a relative displacement of the target particulate matter based on the first relative position and the second relative position, and judges whether the relative displacement of the target particulate matter is effective based on the deviation angle.

9. The method of claim 8, wherein when the relative displacement is effective, calculating a drop volume of the sample solution each time the dispenser ejects, and if the drop volume meets a preset requirement, indicating that an operating parameter of the actuator meets the preset requirement; if the liquid drop volume does not meet the preset requirement, adjusting the working parameters of the actuator, and continuously selecting target particles for detection and analysis until the liquid drop volume meets the preset requirement;

and when the relative displacement is invalid, re-triggering the actuator, and re-selecting the target particulate matters for detection and analysis until the relative displacement is valid.

10. A droplet dispensing apparatus comprising the droplet dispensing method of any one of claims 1 to 9, wherein the droplet dispensing apparatus comprises a dispenser, an actuator, an optical detection device, and an analysis device.

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