阅读说明：本技术 具有高时间分辨率的单液滴更替捕获微芯片系统及其应用 (Single drop replacement capture microchip system with high time resolution and applications thereof ) 是由 孙英男 田晴晴 宋宇涵 吕祥萍 张书圣 于 2021-10-08 设计创作，主要内容包括：本发明公开了具有高时间分辨率的单液滴更替捕获微芯片系统及其应用,属于微芯片及细胞分析技术领域,芯片系统内设置有微通道,通道高度远小于单液滴直径；通道内设置有凹陷的势阱,势阱内径小于单液滴直径,势阱用于捕获单液滴。本发明结构简单、操作方便,液体更替捕获过程具有高时间分辨率,因此通过对单液滴的更替捕获控制,可原位完成化学反应及细胞的高时间分辨率分析。(The invention discloses a microchip system for replacing and capturing single liquid drops with high time resolution and application thereof, belonging to the technical field of microchips and cell analysis, wherein a microchannel is arranged in a chip system, and the height of the microchannel is far smaller than the diameter of a single liquid drop; a sunken potential well is arranged in the channel, the inner diameter of the potential well is smaller than the diameter of the single liquid drop, and the potential well is used for capturing the single liquid drop. The invention has simple structure and convenient operation, and the liquid replacement and capture process has high time resolution, so that the chemical reaction and the high time resolution analysis of cells can be completed in situ by the replacement and capture control of the single liquid drop.)

1. Single drop replacement capture microchip system with high temporal resolution, characterized in that,

a channel is arranged in the chip system, and the height of the channel is smaller than the diameter of the single liquid drop;

and a sunken potential well is arranged in the channel, the inner diameter of the potential well is smaller than the diameter of the single liquid drop, and the potential well is used for capturing the single liquid drop instead.

2. The single-droplet replacement capture microchip system with high temporal resolution of claim 1,

the potential well is at the top or bottom of the channel.

3. Use of a single drop replacement capture system with high temporal resolution as claimed in claim 1 or 2 for capturing a single drop.

4. Method for single droplet replacement capture control using a single droplet replacement capture system with high temporal resolution as claimed in claim 1 or 2,

under the action of oil phase, single liquid drop is at speed Q_oTo the potential well, there are three cases:

(1) no capture mode: when single drop movement speed Q_oCritical speed Q for capturing more than or equal to single drop_cWhen the liquid drops are in use, the single liquid drops directly pass through the potential well and are not captured;

(2) an alternate mode: when the single drop movement speed is larger than the critical speed Q of single drop replacement₁I.e. Q₁＜Q_o＜Q_cWhen the single liquid drop is captured at the potential well, the next single liquid drop completely replaces the previous single liquid drop and is captured at the potential well, and the replacement of the single liquid drop is realized;

(3) parking mode: when the single drop movement speed is lower than the critical speed Q of single drop replacement₁When is, Q_o≤Q₁When the single liquid drop is captured and stops in the potential well, the subsequent single liquid drop can not replace the captured single liquid drop and then bypasses;

by varying Q_oSingle droplet capture control is achieved.

5. Use of a single drop replacement capture system with high temporal resolution according to claim 1 or 2 or of a method according to claim 4 in a high temporal resolution analysis system.

6. Use of a single drop replacement capture system with high temporal resolution according to claim 1 or 2 or a method according to claim 4 in high temporal resolution cell analysis.

7. Use according to claim 6, characterized in that it comprises the following steps:

(1) preparing a solution containing cells into single liquid drops, and capturing the single liquid drops at a potential well;

culturing the cells captured at the potential well, and allowing the cells to settle and grow adherent;

(3) preparing an exciting reagent into a single liquid drop, introducing the single liquid drop into the channel, replacing the original culture liquid drop, and exciting adherent cells;

(4) preparing a reagent for stopping excitation into a single liquid drop, introducing the single liquid drop into the microchannel, and replacing the original liquid drop in a replacement mode so as to stop exciting the cell;

(5) cells under or after excitation are observed and detected.

8. The use according to claim 7,

by adjusting the cell density in the solution containing the cells, single cell level analysis is achieved.

9. The use according to claim 7,

when the cells are settled, the chip system adopts a structure that the potential well is positioned at the top of the channel.

Technical Field

The invention relates to the technical field of microchips and cell analysis, in particular to a single-droplet replacement capture microchip system with high time resolution and application thereof.

Background

The methods which can be used for researching single cell level signal transduction at present mainly comprise mass spectrometry, flow analysis, FACS analysis technology and the like; although the method has the advantages of single cell analysis, high sensitivity and multi-parameter analysis, two limitations still exist:

one is the need for large amounts of dispersed cell suspension; for adherent cells or tissues, the dispersed cell suspension destroys the inherent cell-cell and cell-tissue communication link to a certain extent, thereby causing uncertain disturbance of cell signal paths;

secondly, the steps of cell culture, sample treatment and the like in the existing method mainly depend on manual operation, so that the time resolution is generally in the order of several minutes and cannot meet the time resolution required by rapid signal transduction analysis; for example, the process by which cells are stimulated to generate phosphorylation responses is typically in the range of seconds to fractions of a second, and thus the transient resolution required to study the rapid kinetics of protein phosphorylation reactions should be on the order of seconds.

The key to achieving high temporal resolution is the rapid replacement of the excitation reagents, and current research methods for analyzing cell signaling with high temporal resolution mainly include continuous fluid-based microfluidic technologies and individual droplet-based digital microfluidic technologies. Among them, continuous fluid-based microfluidic technology requires a high liquid flow rate in order to achieve fast switching of chemical excitation (for obtaining high time resolution), thereby generating a large shear force in a microfluidic channel, which may result in generating an additional interference signal related to the continuous fluid; furthermore, continuous fluid based microfluidic systems often require integrated in-channel microvalve structures to avoid cell-to-cell crosstalk; but the related structure is not only complicated to prepare, but also has higher requirements on the operation. For the digital microfluidic technology based on independent liquid drops, researchers have proposed a method for realizing generation and transportation of independent liquid drops by means of electrowetting phenomenon to complete detection of cell signal paths; the rapid signal transduction analysis device can complete rapid replacement of an exciting reagent under the action of low shear force, and achieves rapid signal transduction analysis. However, the method needs to operate liquid drops by means of the action of an electric field, the chip relates to circuit design and integration, and the preparation and operation of the chip are complex; and the method has limited analysis flux and is difficult to expand.

Therefore, it is desired to provide a microchip system which has a low shearing action and can realize a high time-resolved analysis.

Disclosure of Invention

In view of the above, the present invention provides a single droplet capture microchip system based on surface energy effect, which can realize reaction process analysis with high time resolution by capturing and controlling a single droplet.

In order to achieve the purpose, the invention adopts the following technical scheme:

the microchip system is captured by replacing single liquid drops with high time resolution, a channel is arranged in the chip system, and the height of the channel is smaller than the diameter of the single liquid drops; a sunken potential well is arranged in the channel, the inner diameter of the potential well is smaller than the diameter of the single liquid drop, and the potential well is used for capturing the single liquid drop.

Because the height of the channel is smaller than the diameter of the single liquid drop in the natural state, the single liquid drop is squeezed in the channel to be flat. When the single liquid drop passes through the potential well, the surface energy of the single liquid drop is reduced due to the concave structure in the channel, and a small part of the single liquid drop enters the potential well so that the whole single liquid drop is captured; and then other single liquid drops flow through the potential well at a proper flow rate, so that the replacement of the single liquid drops can be realized.

The single droplet capture microchip system described above is applied to capture a single droplet.

Further, potential wells may be disposed at the top or bottom of the channel.

One of the control methods for performing single droplet replacement capture using the single droplet replacement capture microchip system with high time resolution described above:

under the action of oil phase, single liquid drop is at speed Q_oTo the potential well, there are three cases:

(1) no capture mode: when single drop movement speed Q_oCritical speed Q for capturing more than or equal to single drop_cWhen the liquid drops are in use, the single liquid drops directly pass through the potential well and are not captured;

(2) an alternate mode: when single drop movesThe speed is greater than the critical speed Q of single-drop replacement₁I.e. Q₁＜Q_o＜Q_cWhen the single liquid drop is captured at the potential well, the next single liquid drop completely replaces the previous single liquid drop and is captured at the potential well, and the replacement of the single liquid drop is realized;

(3) parking mode: when the single drop movement speed is lower than the critical speed Q of single drop replacement₁When is, Q_o≤Q₁When the single liquid drop is captured and stops in the potential well, the subsequent single liquid drop can not replace the captured single liquid drop and then bypasses;

by varying Q_oSingle droplet capture control is achieved.

The single droplet replacement capture microchip system or the single droplet replacement capture control method with high time resolution can be applied to a high time resolution analysis system, which can be either a cell-based analysis system or a non-cell reaction analysis system.

Preferably, the single droplet replacement capture microchip system with high time resolution or the single droplet replacement capture control method is used for cell analysis under high time resolution, and comprises the following operation steps:

(1) preparing a solution containing cells into single liquid drops, and capturing the single liquid drops at a potential well;

(2) culturing the cells captured at the potential well, and allowing the cells to settle and grow adherent;

(3) preparing an exciting reagent into a single liquid drop, introducing the single liquid drop into a channel, and replacing the original liquid drop in a replacement mode so as to excite the cells growing adherent to the wall in situ;

(4) preparing a reagent for stopping excitation into a single liquid drop, introducing the single liquid drop into the microchannel, and replacing the original liquid drop in a replacement mode so as to stop exciting the cell;

(5) and carrying out in-situ real-time observation and detection on the cells during or after excitation.

The chip system based on the surface energy difference effect can realize the quick and complete replacement of the exciting reagent under high time resolution, and further can be used for the research of quick cell signal transduction.

Further preferably, the single cell level analysis is achieved by adjusting the cell density in the solution containing the cells.

Further preferably, the chip system is configured such that the potential well is located at the top of the channel when the cells are sedimented.

Preferably, with high time resolution single droplet replacement capture microchip system, including a micro channel, the first inlet, the second inlet, the third inlet and the outlet;

the micro-channel comprises a single liquid drop generation area, a conveying area and a capture area which are sequentially communicated;

the single-droplet generation area is respectively communicated with a first inlet externally connected with a water phase and a second inlet externally connected with an oil phase through a channel; the channel communicated with the second inlet is intersected with the channel communicated with the first inlet at two sides of the channel, and a water phase at the intersection is stable to form liquid drops; after the liquid drops occur, the liquid drops are conveyed to a conveying area through a downstream channel; the conveying area is communicated with a third inlet externally connected with an oil phase; the liquid drops are conveyed to the capturing region by the oil phase in the conveying region, the capturing region is provided with a sunken potential well, and different modes of the liquid drops in the capturing region can be adjusted by controlling the injection speed of the oil phase at the third inlet; the capture zone is in communication with the outlet.

Furthermore, the structure that produces a single droplet (single droplet generation zone) and the single droplet flow control structure (delivery zone) can be replaced with other structures that can perform the same function.

The single droplet with high time resolution described above replaces the use of the capture microchip system in capturing a single droplet.

The method for single-droplet capture control using the single-droplet replacement capture microchip system with high time resolution as described above:

injecting oil phase from the third inlet, and allowing the single drop to act at a speed Q_oTo the potential well, there are three cases:

(1) no capture mode: q_oCritical speed Q for capturing more than or equal to single drop_cWhen the liquid drops are in use, the single liquid drops directly pass through the potential well and are not captured;

(2) an alternate mode: critical speed Q for single drop replacement₁＜Q_o＜Q_cWhen the single liquid drop is captured in the potential well,the latter single liquid drop completely replaces the former single liquid drop and is captured in the potential well, so that the replacement of the single liquid drop is realized;

(3) parking mode: q_o≤Q₁When the single liquid drop is captured and stopped in the potential well, the subsequent single liquid drop can not replace the captured single liquid drop and bypasses;

by varying Q_oSingle droplet capture control is achieved.

The microchip system for single-droplet replacement and capture with high time resolution is simple to operate, can realize the capture and replacement of single droplets by changing the flow rate of the oil phase injected from the third inlet, and has outstanding advantages and application potentials in the aspects of precise regulation and control of the single-droplet replacement speed and the replacement efficiency.

Further preferably, the method for analyzing cells at high time resolution using the single droplet replacement capture microchip system or the single droplet replacement capture control method with high time resolution described above comprises the steps of:

injecting a cell culture solution containing cells from a first inlet to form a single liquid drop, and capturing the single liquid drop at a potential well;

culturing the cells captured at the potential well, and settling and adhering the cells to the wall;

injecting an exciting reagent from the first inlet, so that the exciting reagent forms a single liquid drop to replace the cell culture solution at the potential well;

injecting an excitation termination reagent from the first inlet, so that the termination reagent forms a single droplet to replace the excitation droplet at the potential well;

and observing and detecting the excited cells.

Furthermore, the chip can be used for preparing a template based on a photoetching process, then turning the pattern by using PDMS (polydimethylsiloxane), and finally obtaining the microchip system through a plasma bonding process.

In conclusion, the microchip system for single-droplet replacement capture with high time resolution has simple structure and convenient operation; can realize single-droplet capture and single-droplet replacement control, is suitable for analyzing response mechanism and kinetic process of cell rapid signal transduction under high time resolution, and can also be used for high-resolution analysis of other non-cell reactions.

The method has the following characteristics:

(1) the analysis of the rapid reaction process under the sub-second time resolution can be realized;

(2) the complete replacement of single liquid drop can be realized, and the replacement efficiency can reach 100% theoretically;

(3) a rapid and complete replacement process between droplets without producing high levels of shear forces;

(4) the research of quantitative in-situ analysis of the single cell signal transduction path under high time resolution can be realized; and cell detection and analysis can be completed in situ on adherent cells;

(5) the microchip system has simple structure and simple and convenient operation.

Drawings

FIG. 1 is a schematic diagram of a single drop replacement capture microchip system with high temporal resolution;

in the figure, 1 is a first inlet, 2 is a second inlet, 3 is a third inlet, and 4 is an outlet;

a. channel width 100 μm, b channel width 200 μm, c channel width 400 μm, e channel length 1mm, f channel length 2 mm.

FIG. 2 is an enlarged view of a portion of the trapping region of FIG. 1;

FIG. 3 is a schematic view of a single droplet capture, alternate mode;

FIG. 4 is a flowchart illustrating a photolithography process for preparing a template;

FIG. 5 is a video image taken with a high speed microscope of a single droplet replacement process;

FIG. 6 is a schematic view showing the manner in which cells adhere to the wall;

FIG. 7 is an imaging view showing adherent growth of cells in a potential well;

FIG. 8 shows the staining pattern of the viability assay of cells at the potential well.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

As shown in figure 1, with high time resolution single droplet replacement capture microchip system, including a micro channel, the first inlet 1, the second inlet 2, the third inlet 3 and the outlet 4.

The micro-channel comprises a single liquid drop generation area, a conveying area and a capture area which are communicated in sequence.

The single-droplet generation area is communicated with a first inlet 1 (externally connected with a water phase) and a second inlet 2 (externally connected with an oil phase) through a channel; the channel communicating with the second inlet is in T shape with the channel communicating with the first inlet on two sides.

The conveying area is communicated with the T-shaped structure of the single liquid drop generating area through a channel; and the delivery zone communicates with the third inlet 3 through channels provided on both sides.

The capture zone is communicated with the conveying zone; potential trap type micropores are arranged in the capture region; and the potential well micropores are positioned on the same extension line with the T-shaped structure of the single droplet generation region and the communication channel of the capture region delivery region; the direction of flow of the fluid over the capture zone (the side remote from the delivery zone) is communicated by a passage to the outlet 4.

Example 2

Example 1 preparation of single drop replacement capture microchip system with high time resolution:

1. template design

Height of microchannel (h)₁)50 μm; the diameter (d) of the micropores (potential wells) of the trapping region was 75 μm and the depth (h)₂)50 μm (FIG. 2), the remaining dimensions are shown in FIG. 1.

2. Preparation of the template

Cleaning a silicon wafer in a standard step, and treating for about 3min by using oxygen plasma;

(II) multilayer spin coating (negative coating, optionally Etertec HQ-6100) on clean silicon wafers (FIGS. 4-a and 4-b) at 100 ℃ to the desired thickness;

(III) use of a chrome mask plate, 7mW/cm²Exposure to ultraviolet light for 40s (FIG. 4-c);

(IV) soaking and developing in 1% potassium carbonate aqueous solution (figure 4-d);

(V) spin-coating again (FIG. 4-e), and then spin-coating again at 7mW/cm²Exposing for 40s under ultraviolet to perform secondary curing; the secondary exposure step is adopted, so that the post-baking step is avoided, and the processing time can be greatly reduced;

and (VI) developing (figure 4-f), post-baking and hardening, etching and removing photoresist (4-g) after exposure to obtain the multilayer microstructure.

PDMS mold turnover

Adopting Dow Corning PDMS (Dow Corning SYLGARD 184), mixing the basic components and the curing agent according to the weight ratio of 10: 1 weight ratio and completely mixing; pouring into a vacuum drying oven to remove bubbles;

(II) pouring the mixture into a template gently to avoid mixing bubbles, and standing the mixture until the mixture is spread evenly;

(III) curing at 85 ℃ for 20min in a drying oven;

(IV) uncovering the film, cutting and punching;

(V) selecting plasma method bonding or dipping liquid PDMS for curing bonding according to experiment requirements;

(VI) finally, the first inlet, the second inlet, the third inlet and the outlet are cannulated and connected to an external syringe or syringe pump to obtain the complete microchip system.

Example 3

Single drop capture was performed using the single drop replacement capture microchip system with high time resolution prepared in example 2:

the first inlet is communicated with the water phase injector, the second inlet is communicated with the oil phase injector, water phase droplets are formed in the single droplet generation area at a specific speed, and the water phase droplets further flow downstream in the micro-channel.

Injecting an oil phase from the third inlet, conveying the single liquid drop A to the direction of the potential well under the action of the oil phase, wherein the height of the capture area is far smaller than the spherical diameter of the single liquid drop A in a natural state, and the single liquid drop A is in an extrusion state and stores surface energy; making the single drop A less than the critical speed Q at which the single drop is captured_cThe single liquid drop A is delivered to the potential well, the surface energy is released and captured by the potential well, and the delivery is continuedWhen a single liquid drop B is sent and arrives at a potential well, the following three conditions exist (figure 3):

(1) no capture mode: velocity of single droplet B (i.e. flow rate of oil phase in catch zone) Q_oCritical speed Q for capturing more than or equal to single drop_cWhen the single liquid drop A is separated from the potential well, the single liquid drop B and the subsequent single liquid drops directly pass through the potential well and are not captured;

(2) an alternate mode: critical speed Q for single drop replacement₁＜Q_o＜Q_cBased on a Hele-Shaw cell theory model, the single liquid drop B completely replaces the single liquid drop A and is captured in a potential well;

(3) parking mode: q_o≤Q₁When the single liquid drop A is captured and stopped in the potential well, the single liquid drop B can not replace the single liquid drop A and bypasses the potential well.

It can be seen that by changing Q_oSingle droplet capture control can be achieved.

Further, reagent replacement efficiency is evaluated by using different fluorescent liquid drops, a programmable high-precision injection pump is used for injecting fluorescent liquid into the first inlet, and images in the microchannel are shot by a high-speed camera installed on an inverted microscope; as shown in fig. 5, in the replacement mode, the red and blue single droplets are sequentially brought into the capture region, the blue droplet pushes the red droplet out of the potential well, and in the process, there is no color change, and the red micro droplet is completely pushed out of the potential well, so that the replacement efficiency can be considered to be 100% theoretically.

Furthermore, the number of the camera frames for completing the liquid drop replacement at one time is counted, and the time required for completing the liquid drop replacement at one time can be calculated. The experimental results show that the average time of single replacement is 0.4s, and the second-order time resolution required by the analysis of rapid signal transduction dynamics can be completely met.

Example 4

Single droplet capture was performed according to the method of example 3 using the single droplet replacement capture microchip system with high time resolution prepared in example 2:

injecting cell suspension (cells are suspended in a culture medium) into a first inlet, generating single cell suspension liquid drops in a single liquid drop generation region, wherein the volume of the single liquid drop is about 10nL, conveying the single liquid drops to a middle potential well region, capturing the single cell suspension liquid drops by the potential well due to reduction of surface energy, stopping flowing of an oil phase, and putting the microchip system into a cell culture box to enable encapsulated cells to sink to the bottom; experiments show that cell sedimentation and wall adhesion can be realized after 2-3h of standing; as shown in fig. 6, the wells are positioned such that they are positioned at the top of the channel when attached to the wall. Further, in situ detection of cells at the potential well can be performed:

placing the microchip system at a temperature, CO₂And a humidity controller, and imaging the bright field of the cells at the potential well in the chip, as shown in FIG. 7.

Furthermore, when the cells in the potential well region are cultured, a culture medium can be injected into the first inlet, the replacement mode is achieved by adjusting the speed of the oil phase communicated with the third inlet, substances required by the cells are further conveyed to the potential well region, and the original liquid drops at the potential well are replaced, so that the purpose of culturing the cells in the potential well is achieved. Cell viability can be assessed using Hoechst and FDA staining reagents, as shown in fig. 8, with normal cell viability for MCF7 growing adherently at the potential well in the chip system. Indicating that the system and the method have no damage to the cell activity.

Furthermore, when the potential well region cells are cultured, an exciting reagent or a staining reagent can be injected into the first inlet, the potential well region cells pass through the potential well region in the replacement mode, and the change of fluorescence in the cells is monitored through a fluorescence microscope system.

The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.