An apparatus and method for generating microdroplets

Abstract

This application discloses a device for generating microdroplets, comprising at least a separable first part and a second part. The first part has at least an inlet and a fluid conduit, and the second part has at least an array of micropits. The relative physical positions of the first and second parts allow the device to have at least three states: a solution injection position where solution is injected through the inlet and flows within the fluid conduit; a liquid diffusion position where the inlet contacts a high-concentration solution, allowing the high-concentration solution to diffuse within the fluid conduit; and a concentration gradient microdroplet formation position where the fluid conduit partially or completely overlaps with the micropits, causing the solution in the fluid conduit to be segmented and enter the micropits, forming microdroplets containing target substances of different concentrations. This application utilizes a small microfluidic system to generate droplets with continuous concentration gradients; it achieves a large dynamic range for screening droplet arrays with continuous concentration gradients and allows for parallel multi-step operations on the droplets, enabling multiple studies within the droplets.

Description

Technical Field

[0001] This application relates to the field of microfluidic chips, and in particular to an apparatus and method for generating microdroplets. Background Technology

[0002] In scientific research, industrial development, and clinical diagnostics, there is a strong need for user-friendly and low-cost screening systems that cover a wide dynamic range. Traditional screening tests rely primarily on manual pipetting for continuous dilutions in microliter or milliliter-sized test tubes or microplates, which is both time-consuming and labor-intensive. Automated liquid handling robotic systems can simplify the screening process and significantly increase test throughput; however, such systems still consume large amounts of reagents and typically have high equipment and maintenance costs.

[0003] Microfluidics has proven to be an increasingly promising screening tool due to its advantages in small reaction volumes (typically in the picoliter and nanoliter range), high throughput, and ensemble potential. Furthermore, many microfluidic methods can be partitioned to randomly restrict or selectively capture objects of interest in a single event, performing “digital screening” of single molecules or single cells. This strategy opens up new opportunities for studying molecular or cellular heterogeneity. Moreover, operating in small volumes, such as nanoliters and below, not only allows for better control of the screening microenvironment, but its confinement effect can also potentially improve screening speed and analytical sensitivity. Therefore, microfluidics has been used in numerous screening applications in research and diagnostics, including protein crystallization, drug development, reaction kinetic studies, and antimicrobial susceptibility testing.

[0004] To enable screening tests over a wide dynamic range, researchers have investigated microfluidic gradient generation devices with different mechanisms. Based on the principle of laminar flow, microfluidic concentration gradient generators containing microchannel networks achieve diffusion mixing of input substances or different input substances, thereby generating droplets with gradient concentrations. Furthermore, droplets with gradient concentrations can be generated by controlling droplet formation conditions or manipulating in-situ droplets. Multilayer centrifugal microfluidic devices can also generate droplets with concentration gradients. Microfluidic methods based on wettability modes or surface energy levels can also generate droplets with gradient concentrations for screening tests. Additionally, pneumatically valved microfluidic devices can generate concentration gradients through precise control of microvalve. However, most of these methods require precise control of the fluid to generate a stable gradient distribution or involve cumbersome device fabrication and operation processes.

[0005] Microfluidic sliding chip devices achieve fluid manipulation through the relative movement or "sliding operation" of two closely contacting microfluidic glass chips (two layers with microstructures). Sliding chips can set screening conditions for nanoliter protein crystallization via free interface diffusion, and can also achieve multiple dilutions of nanoscale droplet arrays through multi-step sliding. While traditional sliding chips provide a simple method for generating concentration gradients, they have several drawbacks, such as: the need for precise micro-alignment to establish a continuous fluid path; the requirement for manual establishment of concentration gradients in some devices, which can lead to discontinuous gradients and errors; reliance on precise control of auxiliary devices such as pumps, making the experimental setup less simple and portable; and the need for a clean environment and expertise. Furthermore, traditional sliding chips struggle to perform additional operations on gradient droplets, such as merging them with another droplet array. Summary of the Invention

[0006] The purpose of this application is to provide an apparatus and method for generating microdroplets, which uses a small microfluidic system to generate droplets with continuous gradient concentrations and to achieve parallel multi-step diffusion operations on microdroplets in the same aqueous solution.

[0007] One aspect of this application discloses a device for generating microdroplets, comprising at least a separable first part and a second part, the first part having at least an inlet and a fluid conduit, and the second part having at least an array of micropits. The relative physical positions of the first part and the second part allow the device to have at least three states:

[0008] Solution injection location: The first part is configured to inject solution from the inlet and allow it to flow within the fluid conduit;

[0009] Liquid diffusion location: The inlet of the first part comes into contact with the high-concentration solution, allowing the high-concentration solution to diffuse in the fluid pipe;

[0010] Location of concentration gradient microdroplets: The fluid conduit partially or completely overlaps with the micropit, the solution in the fluid conduit is divided and enters the micropit, forming microdroplets containing target substances of different concentrations.

[0011] In a preferred embodiment, when the device is in the liquid diffusion position, the contact between the inlet of the first part and the high-concentration solution is achieved by changing the relative positions of the first part and the second part.

[0012] In a preferred embodiment, the fluid conduit of the first portion may be one or more; when there are multiple fluid conduits, the multiple fluid conduits are configured such that, at the solution injection position, different types of solutions are injected from the inlet.

[0013] In a preferred embodiment, the surface of the fluid conduit is partially or entirely modified, the modification being selected from either hydrophobic or hydrophilic modification.

[0014] In a preferred embodiment, the dimensions of the fluid conduit range from 1 μm to 10 cm in width, 100 μm to 100 cm in length, and 1 μm to 1 cm in depth.

[0015] In a preferred embodiment, when there are multiple fluid conduits, the multiple fluid conduits have different geometries and surface modification treatments, so that different positions of the multiple fluid conduits can generate different surface tensions on the liquid.

[0016] In a preferred embodiment, the second portion further includes multiple non-connected flow channels, wherein the distance between two non-connected flow channels located on the same straight line is equivalent to the length of the fluid conduit on the first portion.

[0017] In a preferred embodiment, the multiple non-connected flow channels are arranged in two straight lines, wherein the first straight line is located at the upper part of the second part and the second straight line is located at the lower part of the second part.

[0018] In a preferred embodiment, when the device is in the solution injection position, the fluid conduit coincides with the flow channel located in the first straight line, and connects the previously disconnected flow channels.

[0019] In a preferred embodiment, the second part further includes a liquid storage tank, which is configured such that when the device is in a liquid diffusion position, the fluid conduit partially overlaps with and communicates with the liquid storage tank.

[0020] In a preferred embodiment, the state of the device further includes: a second connection position: the liquid inlet, the fluid conduit, and two non-communicating flow channels located at the lower part of the second section are interconnected.

[0021] Another aspect of this application discloses a method for generating microdroplets, comprising the following steps:

[0022] (S is in a preferred embodiment and assembled to the injection solution location;)

[0023] (S2) Inject the solution into the fluid pipeline;

[0024] (S3) At the inlet, the high-concentration solution comes into contact with the solution in the fluid pipeline;

[0025] (S4) Place the device at the liquid diffusion location and leave it for a period of time to form a diffusion gradient;

[0026] (S5) The relative positions of the first and second parts of the moving device are adjusted to reach the positions where the concentration gradient microdroplets are formed, thereby generating microdroplets with a concentration gradient.

[0027] In a preferred embodiment, (S3) further includes:

[0028] (S31) Inject the high-concentration solution into the storage tank;

[0029] (S32) and place the device in the first connection position so that the fluid pipe partially overlaps with the liquid storage tank.

[0030] In a preferred embodiment, the following steps are also included:

[0031] (S6) Place the device in the second connection position to re-establish the connection path;

[0032] (S7) The second aqueous solution is injected into the connected pathway formed in (S6);

[0033] (S8) Place the device back at the location where the concentration gradient microdroplets are formed, so that the second solution is mixed with droplets with different concentration gradients.

[0034] In a preferred embodiment, the following steps are also included:

[0035] (S_cal) calculates the concentration of the target substance in the high-concentration solution after diffusion in all microdroplets by taking the concentration integral of the mixed solution in each micro-pit.

[0036] Another aspect of this application discloses the use of generating microdroplets with a concentration gradient to determine the minimum inhibitory concentration of Escherichia coli within 3 hours.

[0037] Another aspect of this application discloses the use of generating microdroplets with concentration gradients to detect the antibiotic sensitivity of Escherichia coli samples from patients with urinary tract infections to nitrofurantoin.

[0038] This application has at least the following technical effects:

[0039] A small microfluidic system was used to generate droplets with continuous gradient concentrations.

[0040] Droplet arrays with continuous concentration gradients enable screening over a large dynamic range;

[0041] It requires no precise calculations or operations, is easy to use, and can be performed by non-professionals.

[0042] The system is stable, reliable, and has small errors;

[0043] Small in size, low in price, and easy to operate;

[0044] It allows for parallel, multi-step operations on droplets and enables multiple studies to be conducted within the droplet.

[0045] The specification of this application contains numerous technical features distributed across various technical solutions. Listing all possible combinations of these technical features (i.e., technical solutions) would make the specification excessively lengthy. To avoid this problem, the various technical features disclosed in the above-described invention, the various technical features disclosed in the following embodiments and examples, and the various technical features disclosed in the accompanying drawings can be freely combined to form various new technical solutions (all of which should be considered as described in this specification), unless such a combination of technical features is technically infeasible. For example, one example discloses feature A+B+C, and another example discloses feature A+B+D+E. Features C and D are equivalent technical means that serve the same function, and technically only one needs to be used; they cannot be used simultaneously. Feature E can technically be combined with feature C. Therefore, the solution A+B+C+D should not be considered as described because it is technically infeasible, while the solution A+B+C+E should be considered as described. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of a method for forming microdroplets with a concentration gradient between upper and lower chips according to one embodiment of this application;

[0047] Figure 2 This is a schematic diagram of an upper chip according to one embodiment of this application;

[0048] Figure 3 This is a top view of the upper chip according to one embodiment of this application;

[0049] Figure 4 This is a top view of the chip according to one embodiment of this application;

[0050] Figure 5 This is a schematic diagram of the lower chip according to one embodiment of this application;

[0051] Figure 6 This is a top view of the chip according to one embodiment of this application;

[0052] Figure 7a This is a schematic diagram of the upper and lower chip assembly according to one embodiment of this application;

[0053] Figure 7b This is a top view of the upper and lower chip assembly according to one embodiment of this application;

[0054] Figure 8a This is a schematic diagram of the formation of gradient concentration microdroplet positions according to one embodiment of this application;

[0055] Figure 8b This is a top view of the location of the microdroplets forming a gradient concentration according to one embodiment of this application;

[0056] Figures 9 to 13 This is a schematic diagram of a method for generating concentration gradient microdroplets according to one embodiment of this application;

[0057] Figure 14 This is a schematic diagram of the upper chip according to another embodiment of this application;

[0058] Figure 15 This is a schematic diagram of the lower chip according to another embodiment of this application;

[0059] Figure 16 This is a schematic diagram of the injection solution position according to another embodiment of this application;

[0060] Figure 17 This is a schematic diagram of the liquid diffusion location according to another embodiment of this application;

[0061] Figure 18 This is a schematic diagram of the location of microdroplets forming a gradient concentration according to another embodiment of this application;

[0062] Figure 19 This is a schematic diagram of the location of microdroplets forming a gradient concentration according to another embodiment of this application;

[0063] Figures 20 to 27 This is a schematic diagram of a method for generating concentration gradient microdroplets according to another embodiment of this application;

[0064] Figure 28 This is a schematic diagram of an upper chip with a "pearl chain" structure according to another embodiment of this application;

[0065] Figure 29 This is a schematic diagram of the process for generating concentration gradient microdroplets according to another embodiment of this application;

[0066] Figure 30 This is a schematic diagram of the microdroplet concentration diffusion trend simulated by a computer according to another embodiment of this application;

[0067] Figure 31 This is a computer-simulated schematic diagram of a "pearl necklace" structure according to another embodiment of this application;

[0068] Figure 32This describes the characterization of HCl diffusion in water on a gd-SlipChip using simulation and a two-color pH indicator experiment. A) A graph shows the simulated HCl concentration distribution in the pearl chain channel after 0.5 h, 1 h, 2 h, and 3 h. B) A 3D bar chart shows the simulated HCl concentration in the gradient droplets after 0.5 h, 1 h, 2 h, and 3 h. C) The pH value of each well is calculated based on the concentration values ​​from the 3D bar chart. D) A brightfield image shows the HCl concentration in the gradient droplets after 0.5 h, 1 h, 2 h, and 3 h using a two-color pH indicator.

[0069] Figure 33 This represents the AST results of wild-type Escherichia coli (ATCC 25922) on gd-SlipChip for nitrofurantoin; where A) bright-field images of four droplets at 0 h, 1 h, 2 h, and 3 h under a 20x objective lens; B) nitrofurantoin concentration in 20 droplets after 24 hours of diffusion; C) bacterial count in 20 droplets at 0 h, 1 h, 2 h, and 3 h. The Y-axis is logarithmic, base 2, n=3, and the error bars represent the standard deviation.

[0070] Figure 34 This indicates the results of nitrofurantoin resistance testing on clinical Escherichia coli isolates on a gd-SlipChip; the heatmap represents the changes in bacterial count within the gd-SlipChip droplets; A) Clinical Escherichia coli b08712 is sensitive to nitrofurantoin; B) Clinical Escherichia coli b08920 is sensitive to nitrofurantoin; C) Clinical Escherichia coli b09314 is sensitive to nitrofurantoin; D) Clinical Escherichia coli b10046 is resistant to nitrofurantoin. Detailed Implementation

[0071] In the following description, numerous technical details are presented to facilitate the reader's better understanding of this application. However, those skilled in the art will understand that the technical solutions claimed in this application can be implemented even without these technical details and various variations and modifications based on the following embodiments.

[0072] The following is a brief summary of some of the innovative aspects of the embodiments of this application:

[0073] This invention includes a diffusion-generated concentration gradient-based gd-SlipChip, and designs a numerical simulation tool to simulate the fluid trends within the chip, and uses the diffusion coefficient to calculate the concentration of substances at different locations within the chip.

[0074] The sliding chip consists of an upper chip and a lower chip. The upper chip includes a portion of the fluid conduit. The assembly of the upper and lower chips forms a complete fluid conduit. The fluid conduit does not overlap with the aforementioned micropits. An aqueous solution containing microorganisms can be injected into the chip through this fluid conduit. Specifically, the microorganisms can be bacteria or fungi.

[0075] By changing the relative physical positions of the two chips, the liquid in the original fluid channel is divided into independent volumes and mixed with the material in the micro-pits.

[0076] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0077] In one embodiment, the upper chip is as follows Figures 2 to 4 As shown. The upper chip includes a connected fluid channel C and has an inlet for injecting an aqueous solution. The lower chip is as follows. Figures 5 to 6 As shown, the lower chip has one or more micropits. The micropits are arranged linearly. Biological or chemical substances can be pre-embedded in the micropits of the lower chip. The upper and lower chips are assembled together, as shown... Figure 7a As shown in 7b, an aqueous solution S is injected into the fluid channel C of the upper chip, and this position is the injection solution position.

[0078] By changing the relative positions of the two chips, contact is achieved between the inlet of fluid pipe C and a solution H containing a high concentration of the target substance T. According to the diffusion principle, the target substance T can diffuse through the fluid pipe into which the aqueous solution S is injected. The diffusion rate can be calculated and simulated using Fick's law, and this rate is related to parameters such as the properties of the target substance T, the viscosity of the aqueous solution S, temperature, and time. In this embodiment, the injection point is also the liquid diffusion point.

[0079] like Figure 8a , 8b As shown, by changing the relative positions of the upper and lower chips, the fluid channel C partially or completely overlaps with the micro-pit. The aqueous solution S in the fluid channel is divided and enters the micro-pit W, forming microdroplets D containing different concentrations of the target substance T. This location is where gradient concentration microdroplets are formed.

[0080] In other embodiments of this application, the upper chip may also have multiple interconnected fluid channels L1, L2, ... that can be used to inject aqueous solutions N1, N2, ... By changing the relative positions of the upper and lower chips, the aqueous solutions N1, N2, ... can be sent into the micro-pit W and fused with the micro-droplets D.

[0081] The surfaces of fluid conduits C, L1, L2… and micro-pits W have undergone surface modification (e.g., partial or complete hydrophobic treatment).

[0082] The fluid conduits C, L1, L2… have different geometric structures or surface modifications, allowing different locations within the conduits to generate varying surface tensions for the liquid. When they partially or completely overlap with the micro-pits W, it facilitates the segmentation of the aqueous solution S within the fluid conduits, allowing it to enter the micro-pits W.

[0083] In this embodiment, the method for forming microdroplets with a concentration gradient using the device is as follows:

[0084] Assemble the upper and lower chips into the solution injection position (e.g., Figure 9 (as shown)

[0085] Inject the aqueous solution into the fluid pipeline (e.g.) Figure 10 (as shown)

[0086] Place a high-concentration liquid (such as...) at one end of the fluid pipeline. Figure 11 (as shown)

[0087] After being left for a period of time, a diffusion gradient is formed (e.g.) Figure 12 (as shown)

[0088] The relative positions of the upper and lower chips change, generating droplets with a concentration gradient (such as...). Figure 13 (As shown).

[0089] In another implementation, the upper chip, such as Figure 14 As shown. The upper chip includes a fluid conduit C and has at least two inlets: inlet 1 and inlet 2. The lower chip is as follows. Figure 15 As shown, it includes a micro-pit array. The lower chip also includes multiple non-connected flow channels, which are arranged in at least two straight lines, one at the top of the lower chip and the other at the bottom; and at least two non-connected flow channels are located on the same straight line and are spaced apart by a distance equivalent to the length of the fluid channel C of the upper chip. The lower chip also includes a high-concentration reservoir. This reservoir is located between the two straight lines forming the flow channels. The upper and lower chips are assembled together, as shown... Figure 16 As shown, this position is the solution injection position. At this time, the fluid channel C of the upper chip and the two flow channels on the upper part of the lower chip are on a straight line, and the fluid channel C is connected to the two flow channels of the lower chip.

[0090] like Figure 17 As shown, by changing the relative positions of the two chips, the liquid reservoir of the lower chip is connected to the fluid pipe C of the upper chip, and this position is the liquid diffusion position of the chip.

[0091] like Figure 18As shown, by changing the relative positions of the upper and lower chips, the fluid channel C partially or completely overlaps with the micro-pit. The aqueous solution S in the fluid channel is divided and enters the micro-pit W, forming microdroplets D containing different concentrations of the target substance T. This location is where gradient concentration microdroplets are formed.

[0092] like Figure 19 As shown, by changing the relative positions of the upper and lower chips, the fluid channel C of the upper chip moves away from the micro-pit of the lower chip, and the fluid channel C of the upper chip and the two flow channels at the bottom of the lower chip are aligned in a straight line, with the fluid channel C connected to the two flow channels of the lower chip. At this point, the chip is in the second liquid inlet position.

[0093] In this embodiment, the method for forming microdroplets with a concentration gradient using the device is as follows:

[0094] 1. Assemble the upper and lower chips into the solution injection position (e.g., Figure 20 (as shown)

[0095] 2. Inject the aqueous solution into the fluid pipeline, or inject the high-concentration solution into the storage tank (e.g., Figure 21 (as shown)

[0096] 3. The chip moves to the first connection position, so that the fluid pipe partially overlaps with the liquid storage tank (e.g., Figure 22 (as shown)

[0097] 4. After a period of time, the solution diffuses from the high-concentration storage tank into the flow channel, forming a concentration gradient (e.g., Figure 23 (as shown)

[0098] 5. Move the upper and lower chips to the segmentation position (droplet generation position) to form droplets with different concentration gradients (e.g., Figure 24 (as shown)

[0099] 6. Move the upper and lower chips to the second connection position to re-establish a connected pathway. The original gradient concentration droplets remain in the micro-pits (e.g., Figure 25 (as shown)

[0100] 7. Pour the second aqueous solution into the connected pathway (e.g., Figure 26 (as shown)

[0101] 8. Move the upper and lower chips to the split position (droplet generation position), and mix the second solution with droplets having different concentration gradients (e.g., Figure 27 (As shown).

[0102] In yet another embodiment, the gd-SlipChip utilizes a slip-induced self-separating mechanism to generate droplets; the device consists of two closely contacting glass chips. For example... Figure 28As shown, the upper chip includes a "chain of pearls" fluid conduit and an inlet; the "chain of pearls" structure includes the following parameters:

[0103] Table 1

[0104]

[0105] The lower chip comprises a reservoir (or tank), droplet expansion holes (micropits), and flow channels. The surface of the glass chip is silanized to become hydrophobic, and a thin layer of tetradecane is placed between the two chips to reduce sliding friction and form water-in-oil droplets. At the first location, a buffer solution is introduced into the "pearl chain" channel, and a high-concentration stock solution is introduced into the reservoir. Then, the upper chip slides downward relative to the lower chip, connecting the "pearl chain" channel and the reservoir. The target molecule begins to diffuse from the reservoir into the "pearl chain" channel and continues to diffuse distally along the channel. After establishing a concentration gradient in the "pearl chain" channel, the upper chip slides downward relative to the lower chip, aligning the "pearl chain" channel with the circular expansion hole, and the liquid self-dives into droplets with gradient concentrations. Then, the upper chip slides downward again relative to the base plate, aligning with the "pearl chain" channel that can be connected to the inlet, introducing a second aqueous reagent into the "pearl chain" channel. Finally, the upper chip slides upward, merging the second aqueous solution with the pre-formed gradient droplets.

[0106] Figure 29 A schematic diagram of the gd-SlipChip is provided, illustrating the sample loading steps and the mechanism of the gd-SlipChip: A) Diffusion buffer (slanted lines) is introduced into the pearl chain microfluidic channel using a pipette, and high-concentration stock solution (dots) is introduced into the storage solution well. B) The upper chip moves relative to the lower chip, bringing the pearl chain channel into contact with the storage solution well and initiating diffusion. C) The upper chip moves relative to the lower chip, bringing the pearl chain channel into contact with the micropits of the lower chip, forming a gradient droplet. D) The upper chip moves relative to the lower chip, allowing a second aqueous reagent to be introduced into the pearl chain channel. E) The upper chip moves relative to the substrate to deliver the second reagent into the gradient droplets, forming a second series of droplets (slanted lines).

[0107] To better understand the technical solution of this application, specific examples are provided below. The details listed in these examples are for ease of understanding and are not intended to limit the scope of protection of this application.

[0108] Example 1

[0109] To calculate the concentration of each droplet in the gradient concentration droplet, we developed a numerical simulation tool in COMSOL Multiphysics 5.6 to simulate the diffusion trend in the gd-slip chip. In the software, we modeled the pearl chain diffusion channels and storage solution pores based on the actual geometry and relative positions, with simulation parameters based on Fick's diffusion law. Figure 30 The simulation interface of COMSOL Multiphysics 5.6 is shown, along with some details of the model.

[0110] To demonstrate the accuracy of the numerical simulation tool and the concentration calculation based on the diffusion coefficient, we characterized the diffusion trend of hydrogen chloride (HCl) in water over a three-hour diffusion period. Using a 0.1 M HCl solution as a stock solution, the simulation tool was applied to simulate the diffusion profile of HCl in the pearl chain channel at 0.5 h, 1 h, 2 h, and 3 h. Figure 32 A). The concentration of HCl in droplets with different diffusion time gradients can be calculated by dividing the channels and taking the concentration integral. Figure 32 B).

[0111] The simulation results were compared with experimental results using a dual-color pH indicator (methyl orange and aniline blue) combined with the diffusion of hydrogen chloride (HCl). The dual-color pH indicator was purple at pH < 4.2 and green at pH > 4.2. However, due to the nanoliter volume of the droplets in the gd-slipship, the droplets were yellow at pH > 4.2. Water was added to the pearl chain channel as a diffusion buffer, and 0.1 M HCl solution was added to the reservoir. By sliding, one end of the pearl chain channel overlapped with the reservoir, initiating the diffusion of HCl. After diffusion, the upper chip slid relative to the lower chip, causing the pearl channel chain to overlap with the expansion pore, forming droplets of different HCl concentrations. Then, a solution containing the dual-color pH indicator was introduced into the pearl chain channel using a pipette. After sliding, the dual-color pH indicator could be delivered into the HCl gradient droplets. The final color change of the droplets at different diffusion times was very consistent with the simulation results generated by COMSOL software. Figure 32 D).

[0112] The following figures illustrate the characterization of HCl diffusion in water on a gd-SlipChip using simulation and a two-color pH indicator experiment. 32A) The graph shows the simulated HCl concentration distribution in the pearl chain channel after 0.5 h, 1 h, 2 h, and 3 h. 32B) The 3D histogram shows the simulated HCl concentration in the gradient droplets after 0.5 h, 1 h, 2 h, and 3 h. 32C) The pH value for each well was calculated based on the concentration values ​​from the 3D histogram. 32D) The brightfield image shows the HCl concentration in the gradient droplets after 0.5 h, 1 h, 2 h, and 3 h using a two-color pH indicator.

[0113] To validate this diffusion system (comprising a microfluidic sliding chip based on diffusion-generated gradient concentration droplets and numerical simulation software), it was applied to bacterial antibiotic resistance testing. We performed phenotypic antimicrobial susceptibility testing (AST) on *E. coli* on the gd-SlipChip and measured its minimum inhibitory concentration (MIC). MIC represents the sensitivity of a specific strain to a specific antibiotic and plays a crucial role in the selection of treatment strategies. Dilution methods, such as broth dilution, which use culture media to dilute antibiotics into gradient concentration solutions to culture bacteria, are commonly used to determine MIC. However, these methods typically involve many manual preparation steps, limiting throughput and requiring technicians to perform them in a laboratory setting. Gradient methods, such as the E-test, with predefined antibiotic concentration gradients, can simplify the AST workflow, but they may lead to inaccurate MIC measurements for some antibiotics. MIC results can also be obtained by automated systems such as VITEK and Phoenix. However, these systems are too expensive for routine MIC testing. Furthermore, most existing methods still require an incubation time of 6 hours or longer, which does not meet the desire for rapid MIC testing.

[0114] This application provides a promising method for determining the MIC of *E. coli* in less than 3 hours using the gd-SlipChip. First, droplets containing gradient concentrations of nitrofurantoin are generated using the gd-SlipChip. Nitrofurantoin, a representative nitrofuran derivative, is a synthetic broad-spectrum antibiotic. Nitrofurantoin's antibacterial mechanism is multifaceted. It is absorbed by nitroreductases in bacteria and converted to its active form via nitroreduction. Intermediate metabolites in this process inhibit enzymes involved in the synthesis of DNA, RNA, and cell wall glycoproteins within the bacteria. Nitrofurantoin is pre-embedded in a reservoir in the lower chip, and the concentration of nitrofurantoin in the droplets can be calculated using simulation tools. Then, a solution containing bacteria can be introduced into the upper chip, which can be mixed with the pre-formed droplets containing gradient concentrations of nitrofurantoin. The device is incubated at 37 °C, and bright-field images of each droplet are captured every hour from the start of droplet mixing to determine the number of bacteria in each droplet. First, standard wild-type *E. coli* (ATCC 25922) was tested, with a previously characterized MIC of 32 μg / mL. In droplets 1–5, the nitrofurantoin concentration ranged from 29–69 μg / mL, and no significant change in *E. coli* count was observed (Figures 33A–B). In droplets 9–20, the nitrofurantoin concentration was below 6 μg / mL, and even after 2 hours, significant *E. coli* growth was observed (Figures 33A–B). In droplets 6–8, the nitrofurantoin concentration ranged from 9–21 μg / mL, with minimal bacterial growth and noticeable bacterial elongation. The measured MIC of *E. coli* for nitrofurantoin was 29 μg / mL (Figures 33B–C), which is very consistent with the previously characterized value.

[0115] Example 2

[0116] Phenotypic antibiotic susceptibility testing for nitrofurantoin was performed on clinical isolates of *E. coli* from patients with urinary tract infections (UTIs) using the gd-SlipChip. The inventors obtained four clinical *E. coli* isolates, identified as *E. coli* (b08712), *E. coli* (b08920), *E. coli* (b09314), and *E. coli* (b10046). Using standard AST, we determined that *E. coli* (b08712), *E. coli* (b08920), and *E. coli* (b09314) were nitrofurantoin-sensitive, while *E. coli* (b10046) was nitrofurantoin-resistant. We generated gradient concentration droplets of nitrofurantoin on the gd-SlipChip, then introduced a solution containing clinical *E. coli* into the device and mixed it with the nitrofurantoin gradient droplets using a simple loading and sliding operation. Bright-field images of each droplet were captured and analyzed to determine the bacterial count, and growth curves of *E. coli* were plotted. During a 3-hour incubation period, no significant growth was observed in droplets containing nitrofurantoin at concentrations above 32 μg / ml for *E. coli* (b08712), *E. coli* (b08920), and *E. coli* (b09314), indicating their sensitivity to nitrofurantoin (Figures 34A-C). However, significant growth was observed in *E. coli* (b10046) even in droplets containing high concentrations of nitrofurantoin, suggesting that this strain is resistant to nitrofurantoin. Figure 34 D). The drug resistance of all four clinical E. coli isolates was highly consistent with the clinical test results provided by the hospital laboratory.

[0117] It should be noted that in this patent application, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this patent application, if it refers to performing an action according to an element, it means performing the action at least according to that element, including two cases: performing the action only according to that element, and performing the action according to that element and other elements. Expressions such as "multiple," "repeatedly," and "various" include two, two times, two kinds, and more than two, more than two times, and more than two kinds.

[0118] This specification includes combinations of various embodiments described herein. Individual references to “one embodiment” or a particular embodiment, etc., do not necessarily refer to the same embodiment; however, these embodiments are not mutually exclusive unless indicated to be mutually exclusive or are readily apparent to those skilled in the art. It should be noted that the word “or” is used in a non-exclusive sense throughout this specification unless the context explicitly indicates or requires it.

[0119] All documents mentioned in this application are considered to be incorporated in their entirety into the disclosure of this application so that they can serve as a basis for modifications if necessary. Furthermore, it should be understood that after reading the foregoing disclosure of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. An apparatus for generating microdroplets, characterized by, The device comprises at least a first part and a second part, the first part has at least a liquid inlet and a fluid channel, the second part has at least a micro-pit array, a liquid storage tank and a plurality of disconnected flow channels, the flow channels are arranged in two straight lines, one line is located in the upper part of the lower chip, and the other line is located in the lower part of the lower chip; the distance between the two flow channels in the same line is equivalent to the length of the fluid channel in the first part; the liquid storage tank is located between the two straight lines formed by the flow channels and contains a high-concentration solution; the first part and the second part can be in at least the following states by changing the relative position of the two parts: solution injection position: the first part is configured to inject solution from the liquid inlet and flow in the fluid channel, at this time, the fluid channel and the liquid inlet of the first part are located in the same line with the two flow channels in the upper part of the second part, and the fluid channel is connected with the two flow channels in the second part; liquid diffusion position: the liquid storage tank of the second part is connected with the fluid channel of the first part, so that the high-concentration solution diffuses in the fluid channel; concentration gradient micro-droplet formation position: the fluid channel overlaps with the micro-pit part, the solution in the fluid channel is divided and enters the micro-pit to form micro-droplets containing different concentrations of target substances; the state of the device also includes: second liquid injection position: the liquid inlet, the fluid channel and the two disconnected flow channels in the lower part of the second part are connected with each other.

2. The apparatus of claim 1, wherein, The fluid channel of the first part can be one or more; when there are multiple fluid channels, the multiple fluid channels are configured to inject different kinds of solution from the liquid inlet at the solution injection position.

3. The apparatus of claim 1, wherein, The surface of the fluid channel is partially or entirely modified, and the modification is selected from one of hydrophobic or hydrophilic modification.

4. The apparatus of claim 1, wherein, The size of the fluid channel ranges from 1 μm to 10 cm in width, 100 μm to 100 cm in length and 1 μm to 1 cm in depth.

5. The apparatus of claim 1, wherein, When there are multiple fluid channels, the multiple fluid channels have different geometric structures and surface modification treatments, so that different positions of the multiple fluid channels can generate different surface tension to the liquid.

6. A method for generating microdroplets, characterized by, The method comprises the following steps: (S1) using the device as claimed in any one of claims 1-5 and assembling it to the solution injection position; (S2) injecting solution into the fluid channel; (S3) injecting high-concentration solution into the liquid storage tank; (S4) placing the device in the liquid diffusion position to partially overlap the fluid channel with the liquid storage tank, so that the high-concentration solution contacts with the solution in the fluid channel, and the high-concentration solution diffuses from the liquid storage tank to the fluid channel to form a diffusion gradient after a period of time; (S5) moving the relative position of the first part and the second part to the concentration gradient micro-droplet formation position to generate micro-droplets with concentration gradient.

7. The method of claim 6, wherein, The method further comprises the following steps: (S6) placing the device in the second connection position to form a connected path again. (S7) injecting a second aqueous solution into the interconnected channels formed in (S6); (S8) placing the device again in the position of the microdroplets with concentration gradient, so that the second solution mixes with the droplets having a different concentration gradient.

8. The method of claim 7, wherein, Further comprising the steps of: Integrating the concentration of the mixed solution in each micro-pit, and calculating the concentration of the target substance in the high-concentration solution after diffusion in all microdroplets.

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