Intermittent vibration micro-droplet generation method and device, storage medium and electronic equipment

Abstract

The embodiment of the invention provides an intermittent vibration micro-droplet generation method, which comprises the following steps: immersing a liquid outlet end of a sample adding needle into a second liquid by using a mobile device, the liquid outlet end of the sample adding needle is filled with a first liquid, the first liquid and the second liquid are not dissolved mutually, and the second liquid is filled with a second liquid; and the density of the first liquid is greater than that of the second liquid. And driving the first liquid in the sampling needle to be continuously discharged by using liquid driving equipment. The fixed end of the sample adding needle is driven by vibration equipment, so that the sample adding needle performs intermittent reciprocating vibration, and the first liquid discharged from the liquid outlet end of the sample adding needle forms micro-droplets in the second liquid. The embodiment of the invention further provides an intermittent vibration micro-droplet generation device.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microdroplet preparation, in particular to a method for preparing microdroplets for digital nucleic acid amplification quantitative analysis (ddPCR) by microdroplets, and more particularly to an intermittent vibration microdroplet generation method and device, a storage medium and an electronic device. BACKGROUND

[0002] Droplet Digital PCR (ddPCR) is a method for absolute quantification of nucleic acid molecules. Compared with quantitative PCR (qPCR), it has significant advantages in precision, accuracy and sensitivity. At the same time, ddPCR eliminates the dependence on quantitative standard curve and improves the tolerance to amplification inhibitors. Therefore, ddPCR is called the third generation PCR technology. Based on the similar principle of ddPCR, a series of digital isothermal nucleic acid amplification (dNAA) technologies have been developed, including digital RPA (Recombinase Polymerase Amplification, RPA), digital LAMP (digital loop-mediated amplification, dLAMP) and the like. The above technologies are collectively referred to as digital nucleic acid amplification technology.

[0003] The working principle of ddPCR is to perform microdroplet processing on the reaction solution before PCR amplification, that is, to disperse the reaction solution containing nucleic acid templates into tens of thousands of nanoliter microdroplets. Each microdroplet contains no or one to several nucleic acid target molecules to be detected, and each microdroplet serves as an independent PCR reaction unit. After PCR amplification, the microdroplets containing the nucleic acid target molecules to be detected produce fluorescence signals, and the microdroplets not containing the nucleic acid target molecules to be detected do not produce fluorescence signals. Finally, according to the Poisson distribution principle and the proportion of positive microdroplets, the initial concentration or copy number of the nucleic acid target molecules to be detected is calculated.

[0004] Since the size and uniformity of microdroplets greatly affect the digital nucleic acid amplification detection results, the preparation technology of microdroplets is particularly important. The technical solutions for generating microdroplets by vibration in the related art mainly include symmetric reciprocating vibration for generating microdroplets and asymmetric reciprocating vibration for generating microdroplets.

[0005] In the process of implementing the present inventive concept, the inventors have found that at least the following problems exist in the related art: the production and processing technology requirements of the sample adding needle are high when generating microdroplets by reciprocating vibration, and if the thickness of the liquid outlet of the sample adding needle is uneven or has burrs, the size of the microdroplets generated by the vibration or reciprocating movement of the sample adding needle is uneven, which affects the digital nucleic acid amplification detection result; although the production and processing technology requirements of the sample adding needle are low when generating microdroplets by asymmetric reciprocating vibration, even if the thickness of the liquid outlet of the sample adding needle is uneven or has burrs, the size of the microdroplets generated by the vibration or reciprocating movement of the sample adding needle is very uniform, but the microdroplets generated by asymmetric reciprocating vibration are easily disturbed by the sample adding needle and are difficult to settle, and even the generated microdroplets are disturbed by the sample adding needle, which also affects the digital nucleic acid amplification detection result. SUMMARY

[0006] Therefore, the present application aims to solve the technical problem of providing an intermittent vibration microdroplet generation method and device, storage medium and electronic equipment, which solves the problem of uneven size of microdroplets generated by reciprocating vibration in the related art, and even the problem of fusion or stirring of microdroplets under the disturbance of the sample adding needle.

[0007] To solve the above technical problems, the specific embodiment of the present application provides an intermittent vibration microdroplet generation method, comprising: immersing the liquid outlet end of a sample adding needle into a second liquid by using a moving device, wherein the liquid outlet end of the sample adding needle is filled with a first liquid, the first liquid and the second liquid are immiscible, and the density of the first liquid is greater than the density of the second liquid; continuously discharging the first liquid in the sample adding needle by using a liquid driving device; driving the fixed end of the sample adding needle by using a vibration device to make the sample adding needle do intermittent reciprocating vibration, so that the first liquid discharged from the liquid outlet end of the sample adding needle forms microdroplets in the second liquid.

[0008] The specific embodiment of the present application also provides an intermittent vibration microdroplet generation device, comprising: a base; a moving device arranged on the base to immerse the liquid outlet end of a sample adding needle into a second liquid, wherein the liquid outlet end of the sample adding needle is filled with a first liquid, the first liquid and the second liquid are immiscible, and the density of the first liquid is greater than the density of the second liquid; a liquid driving device arranged on the base to continuously discharge the first liquid in the sample adding needle; and a vibration device arranged on the base to drive the fixed end of the sample adding needle, so that the first liquid discharged from the liquid outlet end of the sample adding needle forms microdroplets in the second liquid.

[0009] Another aspect of the embodiments of the present application provides an electronic device comprising one or more processors and a storage device, wherein the storage device is configured to store executable instructions, and the executable instructions, when executed by the processor, implement the method of the embodiments of the present application.

[0010] Another aspect of the embodiments of the present application provides a computer readable storage medium storing computer executable instructions, and the instructions, when executed by a processor, implement the method of the embodiments of the present application.

[0011] Another aspect of the embodiments of the present application provides a computer program comprising computer executable instructions, and the instructions, when executed, implement the method of the embodiments of the present application.

[0012] According to the above embodiments of the present application, the intermittent reciprocating vibration sample adding needle generates microdroplets, which can at least partially solve the problem of uneven size of microdroplets generated by the reciprocating vibration sample adding needle in the related art, and even the problem of microdroplet fusion or stirring under the disturbance of the sample adding needle, and thus can generate microdroplets of uniform size, and the technical effect of microdroplet fusion or stirring does not occur.

[0013] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0014] The following drawings accompanying the specification are part of the specification and serve to illustrate exemplary embodiments of the application and together with the description of the specification to explain the principle of the application.

[0015] Figure 1 A schematic diagram of a sample adding needle with uneven thickness of the outlet end wall and burrs in the related art.

[0016] Figure 2 A schematic diagram of uneven size of microdroplets generated when the burr of the flash exists in the outlet end of the sample adding needle in the related art.

[0017] Figure 3 A picture of the microdroplets being stirred when generating the microdroplets by the asymmetric reciprocating vibration method in the related art.

[0018] Figure 4 A flowchart of an intermittent vibration microdroplet generation method provided by an embodiment of the present application.

[0019] Figure 5 A flowchart of another intermittent vibration microdroplet generation method provided by an embodiment of the present application.

[0020] Figure 6Flow chart of another intermittent vibration microdroplet generation method provided for the embodiment of the present application.

[0021] Figure 7 Flow chart of using adsorption material to adsorb the third liquid remaining outside the liquid outlet end of the sample adding needle provided for the embodiment of the present application.

[0022] Figure 8A Velocity-time relationship diagram in a periodic intermittent reciprocating vibration provided for the embodiment of the present application.

[0023] Figure 8B Another velocity-time relationship diagram in a periodic intermittent reciprocating vibration provided for the embodiment of the present application.

[0024] Figure 9A Another velocity-time relationship diagram in a periodic intermittent reciprocating vibration provided for the embodiment of the present application.

[0025] Figure 9B Comparison diagram of single-period two-droplet generation test results provided for the embodiment of the present application.

[0026] Figure 10 Structural schematic diagram of an intermittent vibration microdroplet generation device provided for the embodiment of the present application.

[0027] Figure 11 Structural schematic diagram of adsorption material of an intermittent vibration microdroplet generation device provided for the embodiment of the present application.

[0028] Figure 12 Structural schematic diagram of a sample adding needle provided for the embodiment of the present application.

[0029] Figure 13 Structural schematic diagram of a sample adding needle adapter provided for the embodiment of the present application.

[0030] Figure 14 Structural schematic diagram of a metal sample adding needle provided for the embodiment of the present application.

[0031] Figure 15A Single-side vibration schematic diagram of a periodic generation of one droplet, the position of the liquid outlet end of the sample adding needle being in a sine wave distribution provided for the embodiment of the present application.

[0032] Figure 15B Single-side vibration schematic diagram of a periodic generation of one droplet, the position of the liquid outlet end of the sample adding needle being in a sawtooth wave distribution provided for the embodiment of the present application.

[0033] Figure 15CA single-side vibration schematic diagram that generates one droplet in one period, and the end position of the sample injection needle liquid outlet is in square wave distribution is provided for a specific embodiment of the present application.

[0034] Figure 15D A single-side vibration schematic diagram that generates one droplet in one period, and the end position of the sample injection needle liquid outlet is in triangular wave distribution is provided for a specific embodiment of the present application.

[0035] Figure 16A A double-side vibration schematic diagram that generates one droplet in one period, and the end position of the sample injection needle liquid outlet is in sine wave distribution is provided for a specific embodiment of the present application.

[0036] Figure 16B A double-side vibration schematic diagram that generates one droplet in one period, and the end position of the sample injection needle liquid outlet is in square wave distribution is provided for a specific embodiment of the present application.

[0037] Figure 16C A double-side vibration schematic diagram that generates one droplet in one period, and the end position of the sample injection needle liquid outlet is in sawtooth-sine combined wave distribution is provided for a specific embodiment of the present application.

[0038] Figure 16D A double-side vibration schematic diagram that generates one droplet in one period, and the end position of the sample injection needle liquid outlet is in sawtooth wave distribution is provided for a specific embodiment of the present application.

[0039] Figure 16E A double-side vibration schematic diagram that generates one droplet in one period, and the end position of the sample injection needle liquid outlet is in triangular wave distribution is provided for a specific embodiment of the present application.

[0040] Figure 17A A double-side vibration schematic diagram that generates two droplets in one period, and the end position of the sample injection needle liquid outlet is in sawtooth-sine combined wave distribution by adjusting the length of the static period is provided for a specific embodiment of the present application.

[0041] Figure 17B A double-side vibration schematic diagram that generates two droplets in one period, and the end position of the sample injection needle liquid outlet is uniformly distributed over time by adjusting the length of the static period is provided for a specific embodiment of the present application.

[0042] Figure 17C A double-side vibration schematic diagram that generates two droplets in one period, and the end position of the sample injection needle liquid outlet is in sine wave distribution by adjusting the length of the static period is provided for a specific embodiment of the present application.

[0043] Figure 17D A double-side vibration schematic diagram that generates two droplets in one period, and the end position of the sample injection needle liquid outlet is in sawtooth wave distribution by adjusting the length of the static period is provided for a specific embodiment of the present application.

[0044] Figure 17EA periodic generation of two liquid drops, through mediation of the length of the static period, the end position of the liquid outlet is triangular wave distribution of the double-sided vibration schematic diagram provided for the specific embodiment of the application.

[0045] Figure 18 A periodic generation of two liquid drops, through mediation of the length of the static period, the end position of the liquid outlet is a sine-triangle combined wave distribution of the asymmetric vibration schematic diagram provided for the specific embodiment of the application.

[0046] Figure 19 A periodic generation of one or two liquid drops, through mediation of the length of the static period, the end position of the liquid outlet is a sine wave distribution of the symmetric vibration schematic diagram provided for the specific embodiment of the application.

[0047] Figure 20A A periodic generation of one or two liquid drops, through mediation of the length of the static period, the end position of the liquid outlet is a sine-triangle combined wave distribution of the symmetric vibration schematic diagram provided for the specific embodiment of the application.

[0048] Figure 20B A periodic generation of one or two liquid drops, through mediation of the length of the static period, the end position of the liquid outlet is a sine-triangle combined wave distribution of the symmetric vibration schematic diagram provided for the specific embodiment of the application.

[0049] Figure 20C A periodic generation of one or two liquid drops, through mediation of the length of the static period, the end position of the liquid outlet is a sine-triangle combined wave distribution of the symmetric vibration schematic diagram provided for the specific embodiment of the application.

[0050] BRIEF DESCRIPTION OF DRAWINGS

[0051] 1 base 2 mobile device

[0052] 3 liquid driving device 4 vibration device

[0053] 5 sample needle adapter 6 adsorbing material

[0054] L1 first liquid L2 second liquid

[0055] L3 third liquid P sample needle

[0056] D microdroplet 61 vertical adsorbing material

[0057] 62 horizontal adsorbing material P1 collimation guide section

[0058] P2 liquid storage section P3 opening section

[0059] P4 liquid outlet hole 51 height positioning stop surface

[0060] 52 coaxial positioning structure 53 sealing ring DETAILED DESCRIPTION

[0061] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the following will clearly explain the spirit of the present application with the attached drawings and detailed description. Any person skilled in the art can make changes and modifications to the technology taught by the present application without departing from the spirit and scope of the present application.

[0062] The schematic embodiments of the present application and the descriptions thereof are used to explain the present application, but are not as limitations of the present application. In addition, the same or similar reference signs of elements / components used in the drawings and embodiments are used to represent the same or similar parts.

[0063] As to the "first", "second", and the like used herein, they do not particularly refer to the order or sequence, nor are used to limit the present application, but are merely used to distinguish elements or operations described by the same technical terms.

[0064] As to the directional terms used herein, such as "upper", "lower", "left", "right", "front", "back", and the like, they are merely with reference to the directions of the drawings. Therefore, the directional terms used are used to explain, not to limit the present application.

[0065] As to the "comprise", "include", "have", "contain", and the like used herein, they are all open terms, that is, they mean comprising but not limited to.

[0066] As to the "and / or" used herein, it includes any or all combinations of the described things.

[0067] As to "a plurality of" herein, it includes "two" and "more than two"; as to "a plurality of groups" herein, it includes "two groups" and "more than two groups".

[0068] As to the phrases "substantially", "about", and the like used herein, they are used to modify any quantity or error that can be slightly changed, but the slight changes or errors do not change the nature. Generally, the slight changes or errors modified by such phrases can be 20% in some embodiments, 10% in some embodiments, 5% or other values in some embodiments. Those skilled in the art should understand that the aforementioned values can be adjusted according to actual needs, and are not limited thereto.

[0069] All terms used herein (including technical and scientific terms) have meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein should be interpreted to have meanings consistent with the context of the present specification, and should not be interpreted in an idealized or overly formal manner.

[0070] In the case of using expressions such as "at least one of A, B, and C", it should generally be understood that such expressions are used to enable those skilled in the art to interpret the expression to include at least one of A or B or C, for example. In the case of using expressions such as "at least one of A, B, or C", it should generally be understood that such expressions are used to enable those skilled in the art to interpret the expression to include at least one of A, or B, or C, for example. It will be further understood by those skilled in the art that substantially any disjunctive word or phrase presenting two or more alternative items, whether in the description, claim, or drawings, can be understood to contemplate the possibility of including at least one of the items, even when the items are not listed together with the disjunctive word or phrase. For example, the phrase "A or B" can be understood to include "A" or "B" or "A and B."

[0071] An intermittent vibration microdroplet generation method according to an optional embodiment of the present application can include the following operations: immersing a liquid outlet end of a sample adding needle into a second liquid by using a moving device, wherein the liquid outlet end of the sample adding needle is filled with a first liquid, the first liquid and the second liquid are immiscible, and the density of the first liquid is greater than the density of the second liquid; continuously discharging the first liquid in the sample adding needle by using a liquid driving device; and driving a fixed end of the sample adding needle by using a vibration device to make the sample adding needle do intermittent reciprocating vibration, so that the first liquid discharged from the liquid outlet end of the sample adding needle forms microdroplets in the second liquid.

[0072] Figure 1 A schematic diagram of a sample adding needle with uneven wall thickness and burrs at the liquid outlet end in the related art.

[0073] In an optional embodiment of the present application, as shown in Figure 1 , Figure 1 the left drawing in FIG. 1 shows that the wall thickness of the liquid outlet end of the sample adding needle is uneven, Figure 1 the right drawing in FIG. 1 shows that the liquid outlet end of the sample adding needle has burrs. Under the existing processing conditions, it is difficult to avoid the uneven wall thickness and burrs of the liquid outlet end of the sample adding needle (plastic sample adding needle) produced by one-time injection molding. The uneven wall thickness or burrs caused by the processing defects are usually in the order of tens to hundreds of microns, and the preparation defects of the sample adding needle have an adverse effect on the uniformity of droplet preparation.

[0074] Figure 2A schematic diagram of the size of the microdroplets generated when the fly edge burr exists at the outlet end of the sample adding needle in the related art.

[0075] In an optional embodiment of the present application, compared with the metal sample adding needle, the disposable injection molding sample adding needle can greatly reduce the cost, but due to the large volume of the outlet end of the disposable injection molding sample adding needle, when the fly edge burr exists at the outlet end of the disposable injection molding sample adding needle, the size of the microdroplets generated by the two-side symmetrical wave will be affected by the asymmetric wall thickness of the outlet end and the fly edge burr, resulting in the inconsistency of the volume of the microdroplets on both sides. As shown in Figure 2 , the volume of the larger microdroplet is about 1.8 times that of the smaller microdroplet. The uneven size of the microdroplets will affect the digital nucleic acid amplification detection result. The quantification of ordinary digital PCR is based on the Poisson distribution model, which assumes that nucleic acids are randomly distributed in the droplets. If the sizes of two droplets are not equal, it is not a random distribution, and the probability of large droplets is larger. Therefore, it is not possible to calculate the number of molecules using the simple Poisson distribution formula. It is necessary to simultaneously count the fluorescence signal and the size of all droplets, and establish a new mathematical model. If the fluorescence signal and the size of all droplets are not counted simultaneously, and a new mathematical model is established, the number of molecules is calculated by the Poisson distribution formula, which will affect the accuracy of the digital nucleic acid amplification detection result.

[0076] Figure 3 A picture of the microdroplets being disturbed when the microdroplets are generated by the asymmetric reciprocating vibration method in the related art.

[0077] In an optional embodiment of the present application, as shown in Figure 3 , even if the wall thickness of the outlet end of the disposable injection molding sample adding needle is asymmetric or there is a burr, the size of the microdroplets generated by the asymmetric reciprocating vibration method is still relatively uniform. The production and processing technology requirements of the disposable injection molding sample adding needle are relatively low. Compared with the metal sample adding needle, the volume of the outlet end of the disposable injection molding sample adding needle is larger, and the microdroplets generated by the asymmetric reciprocating vibration method are easily disturbed by the disposable injection molding sample adding needle. The microdroplets are difficult to settle, and sometimes even the generated microdroplets are disturbed by the sample adding needle. If the generated microdroplets are disturbed by the sample adding needle, the digital nucleic acid amplification detection result will also be affected.

[0078] Figure 4 A flowchart of an intermittent vibration microdroplet generation method provided by an embodiment of the present application.

[0079] In an optional embodiment of the present application, as shown in Figure 4 , the intermittent vibration microdroplet generation method can include the following operations S101-S103:

[0080] In operation S101, the mobile device is used to immerse the liquid outlet end of the sample needle into a second liquid, wherein the liquid outlet end of the sample needle is filled with a first liquid, the first liquid is immiscible with the second liquid, and the density of the first liquid is greater than that of the second liquid.

[0081] In an optional embodiment of the present application, the mobile device can move the sample needle up and down, left and right, and forward and backward. The sample needle has a liquid outlet end and a fixed end, and the liquid outlet end is also a free end. The material of the sample needle can include metal, non-metal, alloy, and a combination of metal and non-metal. The first liquid can be a reaction liquid containing a nucleic acid template. The second liquid can be placed in an open container. Since the first liquid is immiscible with the second liquid, and the density of the first liquid is greater than that of the second liquid, the microdroplets of the first liquid formed in the second liquid slowly sink and lay on the bottom of the open container.

[0082] Then, in operation S102, the liquid driving device is used to drive the first liquid in the sample needle to continuously discharge.

[0083] In an optional embodiment of the present application, the liquid driving device can include a hydraulic pump, a hydraulic valve, and other precise flow control devices.

[0084] Next, in operation S103, the vibration device is used to drive the fixed end of the sample needle to make the sample needle perform intermittent reciprocating vibration, so that the first liquid discharged from the liquid outlet end of the sample needle forms microdroplets in the second liquid.

[0085] In an optional embodiment of the present application, the vibration device can include a vibration motor, a dot chronograph, etc. One cycle of intermittent reciprocating vibration can include a stationary phase and a rapid swing phase. The intermittent reciprocating vibration can include symmetric intermittent reciprocating vibration and asymmetric intermittent reciprocating vibration.

[0086] In an optional embodiment of the present application, the intermittent reciprocating vibration includes a stationary phase and a motion phase. In the motion phase, the sample needle moves in the second liquid, and the microdroplets of the first liquid formed in the second liquid slowly sink without disturbance. In the stationary phase, the sample needle is stationary, the sample needle does not disturb the second liquid, and the microdroplets of the first liquid formed in the second liquid can slowly sink without disturbance, so that the microdroplets are not easily fused or broken, and the accuracy of the digital nucleic acid amplification detection result is improved.

[0087] In an optional embodiment of the present application, the following will be described in combination with Figures 5-9B The intermittent vibration microdroplet generation method will be described in detail.

[0088] Figure 5 Another flowchart of the intermittent vibration microdroplet generation method provided by the embodiment of the present application.

[0089] In an optional embodiment of the present application, as shown in FIG. 1, before the liquid outlet end of the sample needle is immersed into the second liquid in operation S101, the intermittent vibration micro-droplet generation method can further include the following operations S201-S204: Figure 5

[0090] In operation S201, the fixed end of the sample needle is connected to the liquid channel by using a sample needle adapter, and the liquid channel is filled with a third liquid.

[0091] In an optional embodiment of the present application, one end of the sample needle adapter is connected to the liquid channel, and the other end of the sample needle adapter is connected to the sample needle. The third liquid can be the same as the second liquid or different from the second liquid. The density of the third liquid is less than the density of the first liquid. The third liquid and the first liquid are immiscible. The material of the liquid channel can include plastic, metal, alloy, etc. The liquid channel can be transparent or non-transparent.

[0092] Next, in operation S202, the third liquid in the liquid channel is driven by using a liquid driving device to fill the sample needle through the fixed end of the sample needle.

[0093] In an optional embodiment of the present application, the liquid driving device drives the third liquid in the liquid channel to fill the sample needle, so that the sample needle is filled with the third liquid.

[0094] Then, in operation S203, the liquid outlet end of the sample needle is immersed into the first liquid.

[0095] In an optional embodiment of the present application, the first liquid can be contained in an open container or a test tube.

[0096] Secondly, in operation S204, a negative pressure is formed in the sample needle by using a liquid driving device, so that the first liquid is sucked into the liquid outlet end of the sample needle, and the density of the first liquid is greater than the density of the third liquid.

[0097] In an optional embodiment of the present application, under the action of the negative pressure, the first liquid is sucked into the sample needle. The sample needle is usually arranged vertically, and the liquid outlet end of the sample needle is located at the lower side. Since the density of the first liquid is greater than the density of the third liquid, the first liquid is gathered at the liquid outlet end of the sample needle and does not enter the liquid channel.

[0098] ​In an optional embodiment of the present application, the third liquid can be referred to as a carrier oil. Before the first liquid is sucked into the sample needle, the liquid path pipeline and the sample needle are filled with the third liquid. After the first liquid is sucked into the sample needle, the upper part of the liquid path pipeline and the sample needle is filled with the third liquid, and the lower part of the sample needle is filled with the first liquid. The third liquid and the first liquid are in close contact and immiscible with each other, so that the first liquid does not contact air at all, avoiding the contamination of the first liquid. Moreover, the flow rate of the first liquid continuously discharged from the sample needle by the third liquid is more stable than the flow rate of the first liquid continuously discharged from the sample needle by the gas. The size of the microdroplets formed in the second liquid is more balanced, further improving the accuracy and reliability of the digital nucleic acid amplification detection result.

[0099] Figure 6 A flow chart of another intermittent vibration microdroplet generation method provided by an embodiment of the present application.

[0100] In an optional embodiment of the present application, as shown in FIG. 2, before the operation S203 of immersing the liquid outlet end of the sample needle into the first liquid, the intermittent vibration microdroplet generation method can further include the operation S301 of: Figure 6

[0101] In the operation S301, the third liquid remaining outside the liquid outlet end of the sample needle is adsorbed by using an adsorption material.

[0102] In an optional embodiment of the present application, the adsorption material can include sponge, cloth, silk, cotton products, etc. Before the other end of the sample needle is immersed into the first liquid, the third liquid remaining outside the liquid outlet end of the sample needle is adsorbed by using the adsorption material, which can avoid the contamination of the first liquid in the container by the third liquid remaining outside the liquid outlet end of the sample needle, and ensure the separation of the third liquid and the first liquid in the sample needle, further improving the accuracy and reliability of the digital nucleic acid amplification detection result.

[0103] In an optional embodiment of the present application, before the operation S101 of immersing the liquid outlet end of the sample needle into the second liquid by using the mobile device, the first liquid remaining outside the liquid outlet end of the sample needle can also be adsorbed by using the adsorption material, which can prevent the problem that the first liquid remaining outside the other end of the sample needle is contaminated by air, and then microdroplets are formed in the second liquid, and the size of the generated microdroplets is not uniform, reducing the accuracy and reliability of the digital nucleic acid amplification detection result.

[0104] Figure 7 A flow chart of adsorbing the third liquid remaining outside the liquid outlet end of the sample needle by using an adsorption material provided by an embodiment of the present application.

[0105] In an optional embodiment of the present application, as shown in FIG. 2, before the operation S203 of immersing the liquid outlet end of the sample needle into the first liquid, the intermittent vibration microdroplet generation method can further include the operation S301 of: Figure 7 ​As shown in FIG. 1, operation S301 can include operations S3011 and S3012.

[0106] In operation S3011, the third liquid remaining around the liquid outlet end of the sample adding needle is adsorbed by the adsorbing material.

[0107] In an optional embodiment of the present application, in order to completely discharge the gas in the sample adding needle, under the driving of the liquid driving device, the third liquid in the liquid path pipeline fills the sample adding needle through the fixed end of the sample adding needle and discharges a small amount of third liquid from the liquid outlet end of the sample adding needle. At this time, the third liquid can remain around the liquid outlet end of the sample adding needle, and the third liquid can also hang below the liquid outlet end of the sample adding needle.

[0108] In operation S3012, the third liquid hanging below the liquid outlet end of the sample adding needle is adsorbed by the adsorbing material.

[0109] In an optional embodiment of the present application, the sample adding needle adapter can be moved left and right and forward and backward by the moving device, thereby driving the sample adding needle to move left and right and forward and backward. The liquid outlet end of the sample adding needle touches the adsorbing material left and right and forward and backward, thereby adsorbing the third liquid remaining around the liquid outlet end of the sample adding needle. Then, the sample adding needle adapter can be moved up and down by the moving device, thereby driving the sample adding needle to move up and down. The liquid outlet end of the sample adding needle touches the adsorbing material on the downside, thereby adsorbing the third liquid hanging below the liquid outlet end of the sample adding needle.

[0110] In an optional embodiment of the present application, adsorbing the third liquid remaining around the liquid outlet end of the sample adding needle and the third liquid hanging below the liquid outlet end of the sample adding needle by the adsorbing material can avoid the third liquid remaining around the liquid outlet end of the sample adding needle from polluting the first liquid in the container. The third liquid hanging below the liquid outlet end of the sample adding needle has contacted air and can have been contaminated. Before the liquid outlet end of the sample adding needle sucks in the first liquid, the third liquid hanging below the liquid outlet end of the sample adding needle is sucked back into the sample adding needle, and the sucked-back third liquid further pollutes the first liquid sucked in through the liquid outlet end of the sample adding needle. At the same time, the third liquid and the first liquid in the sample adding needle can be separated from each other, thereby further improving the accuracy and reliability of the digital nucleic acid amplification detection result.

[0111] Figure 8A An intermittent reciprocating vibration cycle speed-time relationship diagram is provided for a specific embodiment of the present application. Figure 8B Another intermittent reciprocating vibration cycle speed-time relationship diagram is provided for a specific embodiment of the present application. In an optional embodiment of the present application, as shown in FIG. 1, operation S103 can include operations S1031 and S1032. Figure 8A 、 Figure 8B As shown in FIG. 1, operation S301 can include operations S3011 and S3012.

[0112] One end of the sample adding needle is driven by a vibrating device to make the sample adding needle do periodic intermittent reciprocating vibration, wherein one intermittent reciprocating vibration period comprises a static period and a rapid swing period.

[0113] In an optional embodiment of the present application, referring to Figure 8A , the abscissa is time T, and the ordinate is speed V. The sample adding needle moves at acceleration in a first direction, and after the speed reaches a first peak value, the sample adding needle moves at deceleration. After the speed decreases to 0, the sample adding needle is static for a first time period. The sample adding needle moves at acceleration in a second direction, and after the speed reaches a second peak value, the sample adding needle moves at deceleration. After the speed decreases to 0, the sample adding needle is static for a second time period. If the first direction and the second direction are opposite, the first peak value and the second peak value are equal, and the first time period and the second time period are equal, then the sample adding needle moves like a pendulum. If the first peak value and the second peak value are not equal, then the sample adding needle moves in asymmetric vibration. Referring to Figure 8B , the abscissa is time T, and the ordinate is speed V. The sample adding needle moves at deceleration in a first direction, and after the speed decreases to 0, the sample adding needle moves at acceleration in a second direction, and after the speed reaches a first peak value, the sample adding needle is suddenly static for a first time period. Then, the sample adding needle moves at deceleration in the second direction, and after the speed decreases to 0, the sample adding needle moves at acceleration in the first direction, and after the speed reaches a second peak value, the sample adding needle is suddenly static for a second time period. The sample adding needle adapter can suddenly move in the first direction under the action of a magnetic field. Because the sample adding needle adapter is hindered by the elastic member when moving, the speed becomes slower and slower, and after the speed decreases to 0, the elastic member releases the elastic force, the sample adding needle adapter moves at acceleration in the second direction, and after the speed reaches a first peak value, the sample adding needle encounters an obstacle and is suddenly static for a first time period. The sample adding needle adapter suddenly moves in the second direction under the action of an opposite magnetic field. Because the sample adding needle adapter is hindered by the elastic member when moving, the speed becomes slower and slower, and after the speed decreases to 0, the elastic member releases the elastic force, the sample adding needle adapter moves at acceleration in the first direction, and after the speed reaches a second peak value, the sample adding needle encounters an obstacle and is suddenly static for a second time period. If the first peak value and the second peak value are not equal, then the sample adding needle moves in asymmetric vibration.

[0114] In an optional embodiment of the present application, the sample adding needle does periodic intermittent reciprocating vibration in an interval defined by a first extreme point and a second extreme point. The first extreme point can be the farthest distance reached by the sample adding needle in one direction, and the second extreme point can be the farthest distance reached by the sample adding needle in another direction.

[0115] In an optional embodiment of the present application, during the static period, the sample adding needle is located between the first extreme point and the second extreme point, for example, Figure 8BDuring the first and second time periods, the sampling needle is positioned between the first and second poles. Alternatively, during the resting period, the position of the sampling needle coincides with the position of the first pole, or the position of the sampling needle coincides with the position of the second pole. For example, Figure 8A In the first time interval, the needle is located at the first pole; in the second time interval, the needle is located at the second pole.

[0116] In an optional embodiment of the present invention, the sampling needle generates one, two, or more microdroplets within one intermittent reciprocating vibration cycle. For example... Figure 8A , Figure 8B As shown, there are two static time periods within one intermittent reciprocating vibration cycle. Therefore, one or two microdroplets are generated within one intermittent reciprocating vibration cycle, which can achieve good precipitation. The generated microdroplets will not be disturbed by the sampling needle. Figure 8A , Figure 8B The intermittent reciprocating vibration shown can generate a microdroplet when the speed of the sampling needle reaches its maximum value.

[0117] Figure 9A Another graph showing the relationship between velocity and time within a cycle of intermittent reciprocating vibration, provided as a specific embodiment of the present invention. Figure 9B A comparison chart of single-cycle two-droplet generation test results provided for a specific embodiment of the present invention.

[0118] In optional embodiments of the present invention, such as Figure 9A As shown, during the quiescent period, the position of the sampling needle coincides with the position of the first pole, or the position of the sampling needle coincides with the position of the second pole. When two microdroplets are generated within one intermittent reciprocating vibration cycle, one intermittent reciprocating vibration cycle includes a first rapid oscillation period M1, a first quiescent period S1, a second rapid oscillation period M2, and a second quiescent period S2. The size of the two microdroplets can be adjusted by regulating the duration of the first quiescent period S1 and the second quiescent period S2.

[0119] In optional embodiments of the present invention, such as Figure 9B As shown, experiments indicate that due to manufacturing errors and defects in the sampling needle leading to asymmetry in the needle outlet structure, non-perpendicular mounting of the sampling needle on the adapter, non-perpendicularity of the needle tip, and symmetry of the mechanical mechanism, when the sampling needle is driven by a symmetrical waveform where the first static period S1 equals the second static period S2, the two generated droplets may have errors in droplet volume. Figure 9B As shown, when S1 = S2 = 2.76 ms, the two droplets generated within one vibration cycle are of different sizes.

[0120] The time length of the first static period S1 and the second static period S2 is adjusted, and when S1=3.61 ms and S2=1.91 ms are adjusted, it is found that the volumes of the two microdroplets generated in a vibration period are basically the same.

[0121] In actual experiments, due to the randomness of the installation direction of the needle and the processing flaws, the size relationship of S1 and S2 does not have a certain rule, and needs to be observed and adjusted according to the specific situation each time to finally generate microdroplets in the first fast swing period M1 and microdroplets in the second fast swing period M2, realize the uniform size of the microdroplets generated by intermittent vibration, and further improve the precision and reliability of the digital nucleic acid amplification detection result.

[0122] Figure 10 A structure diagram of an intermittent vibration microdroplet generation device provided by an embodiment of the present application.

[0123] In an optional embodiment of the present application, as shown in Figure 10 The intermittent vibration microdroplet generation device can include a base 1, a moving device 2, a liquid driving device 3 and a vibration device 4.

[0124] Specifically, the moving device 2 is arranged on the base 1, and the moving device 2 immerses the liquid outlet end of the sample needle P into the second liquid L2. The liquid outlet end of the sample needle P is filled with the first liquid L1, the first liquid L1 and the second liquid L2 are mutually insoluble, and the density of the first liquid L1 is greater than the density of the second liquid L2. The liquid driving device 3 is arranged on the base 1, and the liquid driving device 3 is used to drive the first liquid L1 in the sample needle P to continuously discharge. The vibration device 4 is arranged on the base 1, and the vibration device 4 is used to drive the fixed end of the sample needle P, so that the first liquid L1 discharged from the liquid outlet end of the sample needle P forms a microdroplet D in the second liquid L2.

[0125] In an optional embodiment of the present application, the mobile device 2 can control the up-down, left-right and forward-backward movement of the sample needle P with the aid of a guide rail or a gear, so that the liquid outlet end of the sample needle P is immersed in the second liquid L2. Under the control of the liquid driving device 3, the first liquid L1 in the sample needle P can be continuously discharged at a constant flow rate, ensuring the uniform size of the generated microdroplets. The vibration device 4 can be controlled by an intermittent sawtooth wave, a sine wave, a cosine wave or the like, to drive the sample needle P to make intermittent vibration. Under the action of inertia and the resistance generated by the second liquid L2, the first liquid L1 discharged from the liquid outlet end of the sample needle P can form uniform microdroplets D in the second liquid L2. In addition, the intermittent vibration allows a certain interval to be maintained between the generated microdroplets, avoiding the disturbance of the vibration of the sample needle P to the generated microdroplets, thereby causing the microdroplets to fuse or be stirred. The sample needle P only moves after the microdroplets sink out of the disturbance range. Therefore, the microdroplet generating device can use a larger diameter of the sample needle P, and the processing difficulty is reduced, and the cost of the sample needle P is greatly reduced.

[0126] In an optional embodiment of the present application, the intermittent reciprocating vibration includes a stationary phase and a motion phase. In the motion phase, the sample needle moves in the second liquid, and the microdroplets of the first liquid formed in the second liquid. In the stationary phase, the sample needle is stationary, and the sample needle no longer disturbs the second liquid. The microdroplets of the first liquid formed in the second liquid can slowly sink without disturbance, and the microdroplets are not easy to fuse or be stirred, thereby improving the accuracy of the digital nucleic acid amplification detection result.

[0127] In an optional embodiment of the present application, the intermittent vibration microdroplet generating device can further include a sample needle adapter 5.

[0128] Specifically, the sample needle adapter 5 is arranged on the base 1, and the sample needle adapter 5 is used to connect the fixed end of the sample needle P and a liquid path pipeline T, wherein the liquid path pipeline T is filled with a third liquid L3. The liquid driving device 3 is further used to drive the third liquid L3 in the liquid path pipeline T to fill the sample needle P in sequence through the sample needle adapter 5 and the fixed end of the sample needle P. The mobile device 2 is further used to immerse the liquid outlet end of the sample needle P in the first liquid L1. The liquid driving device 3 is further used to form a negative pressure in the sample needle P, so as to suck the first liquid L1 from the liquid outlet end of the sample needle P, wherein the density of the first liquid L1 is greater than the density of the third liquid L3.

[0129] In an optional embodiment of the present application, the sample adding needle adapter 5 can be connected with a group of sample adding needles at the same time, for example, by setting different sample adding needle adapters 5, a group of 4, 6, 8, 16, 24 sample adding needles can be connected at the same time, so as to improve the generation efficiency of micro-droplets and increase the practicability of the intermittent vibration micro-droplet generation device.

[0130] Figure 11 A structure diagram of an adsorbing material of an intermittent vibration micro-droplet generation device is provided for an embodiment of the present application.

[0131] In an optional embodiment of the present application, as shown in Figure 11 , the intermittent vibration micro-droplet generation device can further include an adsorbing material 6.

[0132] Specifically, the adsorbing material 6 is arranged on the base 1, and the adsorbing material 6 is used to adsorb the third liquid L3 remaining outside the liquid outlet end of the sample adding needle P.

[0133] In an optional embodiment of the present application, the adsorbing material 6 can include vertical adsorbing material 61 and horizontal adsorbing material 62. The vertical adsorbing material 61 is used to adsorb the third liquid L3 remaining around the liquid outlet end of the sample adding needle P. The horizontal adsorbing material 62 is used to adsorb the third liquid L3 hanging below the liquid outlet end of the sample adding needle P.

[0134] In an optional embodiment of the present application, as shown in Figure 11 , the shape of the adsorbing material 6 is similar to a container, which can include vertical adsorbing material 61 and horizontal adsorbing material 62. The mobile device 2 moves the sample adding needle adapter 5 forward and backward, left and right, and the sample adding needle adapter 5 drives the sample adding needle P to move forward and backward, left and right. The liquid outlet end of the sample adding needle P touches the vertical adsorbing material 61, and the third liquid L3 remaining around the liquid outlet end of the sample adding needle P is adsorbed. The mobile device 2 moves the sample adding needle adapter 5 up and down, and the sample adding needle adapter 5 drives the sample adding needle P to move up and down. The liquid outlet end of the sample adding needle P touches the horizontal adsorbing material 62, and the third liquid L3 hanging below the liquid outlet end of the sample adding needle P is adsorbed. In order to prevent the adsorbing material 6 from damaging the sample adding needle P when adsorbing the remaining third liquid L3, the adsorbing material 6 is usually made of soft material, for example, the adsorbing material can be made of sponge, cloth, silk, cotton products, etc.

[0135] Figure 12 A structure diagram of a sample adding needle is provided for an embodiment of the present application. Figure 13 A structure diagram of a sample adding needle adapter is provided for an embodiment of the present application.

[0136] In an optional embodiment of the present application, as shown in Figure 12 , Figure 13As shown, the material of the sample adding needle P can be non-metal. The sample adding needle P can sequentially include a collimating guide section P1, a liquid storage section P2 and an opening section P3 from top to bottom. The sample adding needle adapter 5 can sequentially include a height positioning stop surface 51, a coaxial positioning structure 52 and a sealing ring 53 from top to bottom. The height positioning stop surface 51 and the coaxial positioning structure 52 cooperate with the collimating guide section P1 to determine the installation position of the sample adding needle P on the sample adding needle adapter 5. The sealing ring 53 is in contact with the upper end of the liquid storage section P2 to achieve the sealed connection of the sample adding needle P and the sample adding needle adapter 5.

[0137] In an optional embodiment of the present application, the height positioning stop surface 51 and the coaxial positioning structure 52 cooperate with the collimating guide section P1 to ensure that the sample adding needle P is clamped at a predetermined position on the sample adding needle adapter 5. The sealing ring 53 is in contact with the upper end of the liquid storage section P2 to achieve the sealed connection of the sample adding needle P and the sample adding needle adapter 5. At the same time, the sample adding needle P can be quickly installed on the sample adding needle adapter 5, and the sample adding needle P can be particularly easily detached from the sample adding needle adapter 5.

[0138] In an optional embodiment of the present application, the outer taper of the opening section P3 is greater than the inner taper of the opening section P3. The outer taper of the opening section P3 can be 10-20 degrees (for example, 13 degrees, 15 degrees, 17 degrees, 19 degrees, etc.), and the inner taper of the opening section P3 can be 5-10 degrees (for example, 5 degrees, 6 degrees, 7 degrees, 9 degrees, etc.). The outer taper of the opening section P3 is greater than the inner taper of the opening section P3, which can make the liquid outlet end wall of the sample adding needle P thinner and finer. When the sample adding needle P reciprocates intermittently, the microdroplets of the first liquid formed in the second liquid are not easily disturbed, the microdroplets of the first liquid formed in the second liquid can slowly sink without being disturbed, and the microdroplets are not easily fused or broken, thereby improving the accuracy of the digital nucleic acid amplification detection result.

[0139] Figure 14 A structure diagram of a metal sample adding needle is provided for a specific embodiment of the present application.

[0140] In an optional embodiment of the present application, as shown in the figure, Figure 14 the sample adding needle P is a metal sample adding needle, and the outer surface of the metal sample adding needle has a perfluorinated coating film.

[0141] In an optional embodiment of the present application, the metal sample adding needle can be recycled, thereby reducing the cost of digital nucleic acid amplification detection and reducing the generation of medical waste. The outer surface of the metal sample adding needle has a perfluorinated coating film, so that the third liquid or the first liquid is not easily left on the outer surface of the metal sample adding needle, and the liquid left on the outer surface of the metal sample adding needle can be easily removed by adsorption or flushing.

[0142] In an optional embodiment of the present application, a liquid outlet hole P4 is formed on the front side of the metal sample adding needle. The side hole of the metal sample adding needle can generate micro-droplets by horizontal vibration of the metal sample adding needle, and can also generate micro-droplets by vertical vibration, thereby expanding the use range of the metal sample adding needle.

[0143] In an optional embodiment of the present application, the metal sample adding needle adopts a reduced diameter structure. Figure 14 The cross-sectional diameter of the front end portion F of the metal sample adding needle is smaller than the cross-sectional diameter of the rear end portion B of the metal sample adding needle. That is, the metal sample adding needle can adopt a variable diameter metal tube shape, and the liquid outlet end is tapered, thereby further reducing the disturbance of the liquid outlet end of the sample adding needle to the second liquid when generating micro-droplets by intermittent vibration, so that the micro-droplets formed by the first liquid in the second liquid can slowly sink without disturbance, and the micro-droplets are not easily fused or broken, thereby improving the accuracy and reliability of the digital nucleic acid amplification detection result.

[0144] In an optional embodiment of the present application, the depth of the liquid outlet end of the sample adding needle P inserted into the second liquid L2 can be-0.3mm-0.9mm. For example, the depth of the liquid outlet of the sample adding needle P inserted into the second liquid L2 can be-0.3mm, -0.2mm, -0.1mm, 0mm, 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, etc. The depth is negative, indicating that the liquid outlet of the sample adding needle P is above the liquid surface. Due to the specific adhesion of the liquid, when the liquid outlet of the sample adding needle P is located at a certain distance above the liquid surface, the liquid outlet of the sample adding needle P will not be separated from the second liquid. The depth is positive, indicating that the liquid outlet of the sample adding needle P is below the liquid surface.

[0145] In an optional embodiment of the present application, since the intermittent vibration method is used to generate micro-droplets, the generated micro-droplets have sufficient sinking time, and the generated micro-droplets sink out of the disturbance range of the sample adding needle P, and then the sample adding needle P moves again. The re-movement of the sample adding needle P will not disturb or break the micro-droplets generated in the early stage. Therefore, the liquid outlet of the sample adding needle P is inserted into the oil surface at a depth range of-0.3mm-0.9mm, which is higher than the existing-0.3mm-0.3mm, and the positioning accuracy requirement of the intermittent vibration micro-droplet generating device is greatly reduced, thereby further reducing the production cost of the intermittent vibration micro-droplet generating device.

[0146] Figure 15A A single-sided vibration schematic diagram is provided for a specific embodiment of the present application, in which one droplet is generated per cycle, and the position of the liquid outlet end of the sample adding needle is in a sine wave distribution. Figure 15B A single-sided vibration schematic diagram is provided for a specific embodiment of the present application, in which one droplet is generated per cycle, and the position of the liquid outlet end of the sample adding needle is in a sawtooth wave distribution. Figure 15CA single-side vibration schematic diagram that a period generates one droplet and the position of the sample needle liquid outlet end is in square wave distribution is provided for a specific embodiment of the present application. Figure 15D A single-side vibration schematic diagram that a period generates one droplet and the position of the sample needle liquid outlet end is in triangular wave distribution is provided for a specific embodiment of the present application.

[0147] As shown in Figure 15A , Figure 15B , Figure 15C , Figure 15D , one intermittent asymmetric reciprocating vibration (single-side vibration of the sample needle liquid outlet end) period forms one micro-droplet, the micro-droplet falls as a parabola, and the micro-droplet formed by the first liquid in the second liquid can slowly sink without disturbance and is not prone to fusion or stirring.

[0148] Figure 16A A double-side vibration schematic diagram that a period generates one droplet and the position of the sample needle liquid outlet end is in sine wave distribution is provided for a specific embodiment of the present application. Figure 16B A double-side vibration schematic diagram that a period generates one droplet and the position of the sample needle liquid outlet end is in square wave distribution is provided for a specific embodiment of the present application. Figure 16C A double-side vibration schematic diagram that a period generates one droplet and the position of the sample needle liquid outlet end is in sawtooth-sine combined wave distribution is provided for a specific embodiment of the present application. Figure 16D A double-side vibration schematic diagram that a period generates one droplet and the position of the sample needle liquid outlet end is in sawtooth wave distribution is provided for a specific embodiment of the present application. Figure 16E A double-side vibration schematic diagram that a period generates one droplet and the position of the sample needle liquid outlet end is in triangular wave distribution is provided for a specific embodiment of the present application.

[0149] As shown in Figure 16A , Figure 16B , Figure 16C , Figure 16D , Figure 16E , one intermittent symmetric reciprocating vibration (double-side vibration of the sample needle liquid outlet end) period forms one micro-droplet, the micro-droplet falls vertically, the disturbance of the left and right vibration of the sample needle liquid outlet end to the micro-droplet is offset (the disturbance of the left and right vibration to the oil phase is offset), and the micro-droplet formed by the first liquid in the second liquid can slowly sink without disturbance and is not prone to fusion or stirring.

[0150] Figure 17A A double-side vibration schematic diagram that a period generates two droplets, the length of the static period is adjusted, and the position of the liquid outlet end is in sawtooth-sine combined wave distribution is provided for a specific embodiment of the present application. Figure 17B A double-side vibration schematic diagram that a period generates two droplets, the length of the static period is adjusted, and the position of the liquid outlet end is uniformly distributed with time is provided for a specific embodiment of the present application.Figure 17C A periodic generation of two droplets, a double-sided vibration schematic diagram of a sine wave distribution of the length of the static period and the position of the outlet end is provided for a specific embodiment of the application. Figure 17D A periodic generation of two droplets, a double-sided vibration schematic diagram of a sawtooth wave distribution of the length of the static period and the position of the outlet end is provided for a specific embodiment of the application. Figure 17E A periodic generation of two droplets, a double-sided vibration schematic diagram of a triangular wave distribution of the length of the static period and the position of the outlet end is provided for a specific embodiment of the application.

[0151] As shown in Figure 17A , Figure 17B , Figure 17C , Figure 17D , Figure 17E , a 2-microdroplet is formed by a periodic intermittent symmetrical reciprocating vibration, the microdroplet falls vertically, the disturbance of the left and right vibration of the outlet end of the sample needle to the microdroplet is offset (the left and right vibration disturbs the oil phase and is offset), and by adjusting the size of the first static period S1 and the second static period S2, the size of the two microdroplets is consistent, the microdroplet formed by the first liquid in the second liquid can slowly sink without disturbance, and the microdroplet fusion or crushing is not easy to occur.

[0152] Figure 18 An asymmetric vibration schematic diagram of a sine-triangle combined wave distribution of the length of the static period and the position of the outlet end is provided for a specific embodiment of the application, which lacks a gap period and generates two droplets in one cycle.

[0153] As shown in Figure 18 , a non-intermittent asymmetric vibration cycle, the microdroplet is cut off at the fast movement period of the outlet end of the sample needle (i.e. the microdroplet is separated from the outlet end of the sample needle), and due to the lack of an intermittent period (i.e. no static stage), the disturbance of the outlet end of the sample needle to the second liquid oil phase is strong, in order to reduce the disturbance of the outlet end of the sample needle to the second liquid oil phase, the outlet end of the sample needle cannot be inserted too deep into the second liquid, resulting in a shallow depth of the outlet end of the sample needle inserted into the second liquid, which requires a higher positioning accuracy of the vibration microdroplet generation device, and increases the production cost of the vibration microdroplet generation device.

[0154] Figure 19 A symmetrical vibration schematic diagram of a sine wave distribution of the length of the static period and the position of the outlet end is provided for a specific embodiment of the application, which lacks a gap period and generates one or two droplets in one cycle.

[0155] As shown in Figure 19As shown, the lack of an interval (i.e., no static phase) and the symmetrical vibration of the sample dispensing needle can result in the generation of one or two microdroplets per vibration cycle. The generated microdroplets are unstable in volume, requiring an increase in the vibration frequency of the sample dispensing needle to generate smaller microdroplets. This contradicts the time required for the microdroplets to descend, causing the generated microdroplets to be broken up, thus reducing the accuracy and reliability of digital nucleic acid amplification detection results.

[0156] Figure 20A This is a schematic diagram of a symmetrical vibration in a specific embodiment of the present invention, in which two static periods are equal within an intermittent reciprocating vibration cycle and the outlet end position exhibits a sinusoidal-triangular combined wave distribution. Figure 20B This is a schematic diagram of a symmetrical vibration in a specific embodiment of the present invention, in which two static periods are equal within an intermittent reciprocating vibration cycle and the outlet end position is distributed in a triangular wave pattern. Figure 20C This is a schematic diagram of a symmetrical vibration in a specific embodiment of the present invention, in which two static periods are equal within an intermittent reciprocating vibration cycle and the position of the liquid outlet end is distributed in a sinusoidal wave.

[0157] like Figure 20A , Figure 20B and Figure 20C As shown, the sample needle outlet vibrates intermittently and symmetrically, forming two microdroplets in one cycle. The first stationary period S1 and the second stationary period S2 are equal. Due to the asymmetry in needle manufacturing, the asymmetry in instrument assembly, and the deviation of the actual vibration waveform from the ideal value, the droplets on both sides are of different sizes, which reduces the accuracy and reliability of digital nucleic acid amplification detection results.

[0158] The above experiments show that when one or two microdroplets are generated within one intermittent reciprocating vibration cycle, the microdroplets formed by the first liquid and the second liquid can sink slowly and undisturbed, with little chance of merging or breaking up. If only one microdroplet is generated within one intermittent reciprocating vibration cycle, regardless of whether the sampling needle outlet vibrates on one or both sides, the microdroplet formed by the first liquid and the second liquid can sink slowly and undisturbed, with little chance of merging or breaking up. However, if two microdroplets are generated within one intermittent reciprocating vibration cycle, the duration of the first and second static periods needs to be adjusted to ensure that the two microdroplets are of equal size within one cycle.

[0159] According to embodiments of the present invention, the method flow according to embodiments of the present invention can be implemented as a computer software program. For example, embodiments of the present invention include a computer program product comprising a computer program carried on a computer-readable storage medium, the computer program containing program code for performing the method shown in the flowchart. According to embodiments of the present invention, the electronic devices, devices, apparatuses, modules, units, etc., described above can be implemented by computer program modules.

[0160] The application further provides a computer readable storage medium, which can be included in the device / apparatus / system described in the above embodiments, or can exist independently without being assembled into the device / apparatus / system. The computer readable storage medium carries one or more programs, which, when executed, implement the method according to the embodiments of the application.

[0161] According to the embodiments of the application, the computer readable storage medium can be a non-volatile computer readable storage medium, which can include, but is not limited to, a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), a portable compact disc read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any appropriate combination thereof. In this application, a computer readable storage medium can be any tangible medium that contains or stores a program for use by or in connection with an instruction execution system, apparatus, or device.

[0162] The flow diagrams and the block diagrams in the drawings are illustrations of possible architectures, functions, and operations of systems, methods, and computer program products according to various embodiments of the present application. In this regard, each block in the flow diagrams or block diagrams can represent a module, a segment, or a portion of code, which comprises one or more executable instructions for implementing the specified logical functions. It should also be noted that in some alternative implementations, the functions noted in the blocks can occur out of the order noted in the figures. For example, two blocks shown in succession may, in fact, be executed substantially concurrently or the blocks may sometimes be executed in the reverse order, depending upon the functionality involved. It will also be noted that each block of the block diagrams and / or flow diagrams, and combinations thereof, can be implemented by special purpose hardware-based systems that perform the specified functions or operations, or combinations of special purpose hardware and computer instructions.

[0163] In the description of the specification, the description of the terms "embodiment", "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the application. In the specification, the illustrative description of the above terms does not necessarily mean the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0164] Those skilled in the art will appreciate that features recited in the various embodiments and / or claims of the present application can be combined or / and integrated in various combinations, even if such combinations are not expressly recited in the present application. In particular, the features recited in the various embodiments and / or claims of the present application can be combined and / or integrated in various combinations, without departing from the spirit and teachings of the present application. All such combinations and / or integrations are within the scope of the present application.

[0165] The above describes embodiments of the present application. However, these embodiments are merely for illustrative purposes, and are not intended to limit the scope of the present application. Although each embodiment is described above separately, this does not mean that the measures in each embodiment cannot be used advantageously in combination. The scope of the present application is defined by the appended claims and their equivalents. Those skilled in the art can make various substitutions and modifications without departing from the scope of the present application, and all such substitutions and modifications shall fall within the scope of the present application.

Claims

1. An intermittent oscillating microdroplet generation method, characterized by, The method comprises: immersing a liquid outlet end of a sample needle into a second liquid, wherein the liquid outlet end of the sample needle is filled with a first liquid, the first liquid is immiscible with the second liquid, and the first liquid has a density greater than that of the second liquid; driving the first liquid in the sample needle to continuously discharge by using a liquid driving device; and driving a fixed end of the sample needle by using a vibration device to make the sample needle do intermittent reciprocating vibration, so that the first liquid discharged from the liquid outlet end of the sample needle forms microdroplets in the second liquid. Before the step of immersing the liquid outlet end of the sample needle into the second liquid, the method further comprises:

2. The method of claim 1, wherein the method is a batch method. communicating the fixed end of the sample needle and a liquid channel pipeline by using a sample needle adapter, wherein the liquid channel pipeline is filled with a third liquid; filling the sample needle through the fixed end of the sample needle by driving the third liquid in the liquid channel pipeline by using a liquid driving device; immersing the liquid outlet end of the sample needle into the first liquid; and forming a negative pressure in the sample needle by using a liquid driving device to suck the first liquid into the liquid outlet end of the sample needle, wherein the first liquid has a density greater than that of the third liquid. Before the step of immersing the liquid outlet end of the sample needle into the first liquid, the method further comprises:

3. The method of claim 2, wherein the method is a batch method. absorbing the third liquid remaining outside the liquid outlet end of the sample needle by using an adsorption material. The step of absorbing the third liquid remaining outside the liquid outlet end of the sample needle by using an adsorption material comprises:

4. The method of claim 3, wherein the method is a batch method. absorbing the third liquid remaining around the liquid outlet end of the sample needle by using an adsorption material; and absorbing the third liquid hanging below the liquid outlet end of the sample needle by using an adsorption material. The step of driving the fixed end of the sample needle by using a vibration device to make the sample needle do intermittent reciprocating vibration comprises:

5. The method of claim 1, wherein the method is a batch method. driving the fixed end of the sample needle by using a vibration device to make the sample needle do periodic intermittent reciprocating vibration, wherein one intermittent reciprocating vibration period comprises a stationary period and a rapid swing period. The sample needle does periodic intermittent reciprocating vibration within an interval defined by a first extreme point and a second extreme point, 6. The method of claim 5, wherein the method is a batch method. In the stationary period, the sample needle is located between the first extreme point and the second extreme point; or in the stationary period, the position of the sample needle coincides with the position of the first extreme point, or the position of the sample needle coincides with the position of the second extreme point. The sample needle generates one or two microdroplets in one intermittent reciprocating vibration period.

7. The method of claim 6, wherein the method is a batch method. In the stationary period, the position of the sample needle coincides with the position of the first extreme point, or the position of the sample needle coincides with the position of the second extreme point, and when two microdroplets are generated in one intermittent reciprocating vibration period, one intermittent reciprocating vibration period comprises a first rapid swing period, a first stationary period, a second rapid swing period, and a second stationary period, 8. The method of claim 7, wherein the method is a batch method. The size of the two microdroplets is adjusted by adjusting the length of the first stationary period and the second stationary period. The method comprises:

9. An intermittent vibration microdroplet generation device, characterized by, a base (1); ​ a moving device (2) arranged on the base (1) to immerse the liquid outlet end of the sample needle (P) into the second liquid (L2), wherein the liquid outlet end of the sample needle (P) is filled with the first liquid (L1), the first liquid (L1) is immiscible with the second liquid (L2), and the density of the first liquid (L1) is greater than the density of the second liquid (L2); a liquid driving device (3) arranged on the base (1) to drive the first liquid (L1) in the sample needle (P) to continuously discharge; a vibrating device (4) arranged on the base (1) to drive the fixed end of the sample needle (P) so that the first liquid (L1) discharged from the liquid outlet end of the sample needle (P) forms a microdroplet (D) in the second liquid (L2).

10. The intermittent vibration microdroplet generation device of claim 9, wherein, Further comprising: a sample needle adapter (5) arranged on the base (1) to communicate the fixed end of the sample needle (P) and a liquid path pipeline (T), wherein the liquid path pipeline (T) is filled with a third liquid (L3), wherein the liquid driving device (3) is further used to drive the third liquid (L3) in the liquid path pipeline (T) to fill the sample needle (P) in sequence via the sample needle adapter (5) and the fixed end of the sample needle (P); the moving device (2) is further used to immerse the liquid outlet end of the sample needle (P) into the first liquid (L1); and the liquid driving device (3) is further used to form a negative pressure in the sample needle (P) so as to suck the first liquid (L1) from the liquid outlet end of the sample needle (P), wherein the density of the first liquid (L1) is greater than the density of the third liquid (L3).

11. The intermittent vibration microdroplet generation device of claim 10, wherein, Further comprising: an adsorbing material (6) arranged on the base (1) to adsorb the third liquid (L3) remaining outside the liquid outlet end of the sample needle (P).

12. The intermittent oscillating microdroplet generation device of claim 11, wherein, The adsorbing material (6) comprises: a vertical adsorbing material (61) used to adsorb the third liquid (L3) remaining around the liquid outlet end of the sample needle (P); and a horizontal adsorbing material (62) used to adsorb the third liquid (L3) hanging below the liquid outlet end of the sample needle (P).

13. The intermittent vibration microdroplet generator of claim 10, wherein, The material of the sample needle (P) is non-metal, and the sample needle (P) comprises, from top to bottom, a collimating guide section (P1), a liquid storage section (P2), and an opening section (P3).

14. The intermittent vibration microdroplet generator of claim 13, wherein, The sample needle adapter (5) comprises, from top to bottom, a height positioning stop surface (51), a coaxial positioning structure (52), and a sealing ring (53) in contact with the sample needle (P), the height positioning stop surface (51), the coaxial positioning structure (52), and the collimating guide section (P1) cooperate to determine the installation position of the sample needle (P) on the sample needle adapter (5); the sealing ring (53) is in contact with the upper end of the liquid storage section (P2) to realize the sealed connection between the sample needle (P) and the sample needle adapter (5).

15. The intermittent vibration microdroplet generator of claim 13, wherein, The outer taper of the opening section (P3) is greater than the inner taper of the opening section (P3).

16. The intermittent vibration microdroplet generator of claim 9, wherein, The sample adding needle (P) is a metal sample adding needle, and an outer surface of the metal sample adding needle is provided with a full fluorine plating film.

17. The intermittent vibration microdroplet generator of claim 16, wherein, A front end side surface of the metal sample adding needle is provided with a liquid outlet hole (P4).

18. The intermittent vibration microdroplet generator of claim 16, wherein, The metal sample adding needle adopts a reduced diameter structure.

19. The intermittent vibration microdroplet generator of claim 9, wherein, A depth of a liquid outlet end of the sample adding needle (P) inserted into the second liquid (L2) is-0.3mm-0.9mm.

20. A computer-readable storage medium, characterized in that, The computer readable storage medium has stored computer executable instructions, and the instructions are used for implementing the method in any one of claims 1-8 when executed by a processor.

21. An electronic device, comprising: The computer readable storage medium has stored computer executable instructions, and the instructions are used for implementing the method in any one of claims 1-8 when executed by a processor.

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