Micro-fluidic chip based on photocuring liquid drops and escherichia coli detection method

Abstract

The invention relates to a micro-fluidic chip based on light-cured liquid drops, which comprises an oil phase liquid inlet used for introducing an oil phase containing a photopolymerization initiator and a light-cured monomer; the water phase liquid inlet is used for introducing a water phase containing a test sample, a bacterial lysis reagent and an enzymatic reaction substrate; the liquid drop generation intersection is used for forming water-in-oil liquid drops; the tree-shaped dispersion structure comprises a multi-stage bifurcated micro-channel and an inlet dispersion assembly and is used for realizing preliminary dispersion of liquid drops; the incubation chamber is a flat plate cavity, an internal dispersion assembly is arranged in the incubation chamber, liquid drops are solidified and fixed through an oil phase, bacteria are cracked through a water phase, and a substrate is catalyzed to generate a fluorescent product. The invention also relates to a method for detecting escherichia coli by using the micro-fluidic chip. The water-in-oil liquid drops are cured in situ, the problem that the liquid drops are easy to crack and fuse in incubation is thoroughly solved, the spatial fixity of the cured liquid drops facilitates the positioning collection of fluorescence signals, the signal drift interference in the traditional flowing liquid drop detection is reduced, and the method is suitable for ddPCR technology.

Description

Technical Field

[0001] This invention relates to microfluidic chips, and more specifically to a microfluidic chip based on photocurable droplets and a method for detecting Escherichia coli. Background Technology

[0002] Escherichia coli (E. coli) primarily resides in the large intestine, accounting for approximately 1% of the intestinal flora. It is a short, motile, non-spore-forming Gram-negative bacillus with blunt ends. E. coli can synthesize vitamins B and K and is not pathogenic under normal living conditions; however, it can cause inflammation if it enters the gallbladder, bladder, or other organs. Its detection in water and food is considered an indicator of fecal contamination. Therefore, as an important indicator bacterium for water safety and food hygiene, rapid, trace detection of E. coli is of great significance for preventing foodborne illnesses and environmental pollution.

[0003] Currently, E. coli detection typically involves multiple-tube fermentation and membrane filtration. However, while these methods are low-cost and simple to operate, they are time-consuming and susceptible to interference from other microorganisms. With the increasing frequency of water pollution incidents, they are insufficient to meet the demand for rapid detection.

[0004] Microfluidic chip technology has become a research hotspot for E. coli detection due to its advantages of integration, high throughput, and low consumption. Each droplet can serve as an independent reaction unit, enabling high-throughput detection, and each droplet can contain one or more E. coli cells. However, after droplet generation, they are often transported through a single channel or a simple branching structure, which can easily lead to droplet aggregation and stacking due to flow rate fluctuations. After entering the incubation area, the droplets are unevenly distributed, and some droplets overlap and block each other, seriously affecting the accuracy of fluorescence detection. Droplets may also break or fuse during transport and incubation, leading to bacterial leakage and reaction system disorder. Summary of the Invention

[0005] To address the problems of droplet fusion and breakage in the existing technologies, this invention aims to provide a microfluidic chip based on photocurable droplets and a method for detecting Escherichia coli.

[0006] The microfluidic chip based on photocurable droplets according to the present invention comprises: an oil phase inlet for introducing an oil phase containing a photocurable reagent, the photocurable reagent comprising a photopolymerization initiator and a photocurable monomer; an aqueous phase inlet for introducing an aqueous phase comprising a test sample, a bacterial lysis reagent, and an enzyme-catalyzed reaction substrate; a droplet generation cross-section located downstream of the channels of the oil phase inlet and the aqueous phase inlet, for encapsulating the aqueous phase with the oil phase to form water-in-oil droplets; a dendritic dispersion structure connected downstream of the droplet generation cross-section, comprising multi-level branched microchannels and an inlet dispersion component disposed at the end of the channels, for initially dispersing the droplets; and an incubation chamber, which is a cavity structure formed on a flat plate surface, the inlet of which is connected to the end of the dendritic dispersion structure, and an internal dispersion component for carrying the droplet array. The droplets are fixed by the photocurable reagent in the oil phase, and the bacterial lysis reagent in the aqueous phase lyses the bacteria in the test sample, releases enzymes, and catalyzes the enzyme-catalyzed reaction substrate to generate fluorescent products, thereby realizing the solidification and reaction of the droplets.

[0007] In a preferred embodiment, the multi-level branched microchannels of the tree-like dispersion structure are initially a single main channel, which is then branched into 2-8 parallel branch channels through 1-3 levels of branching. Each branch channel achieves the initial dispersion of droplets through diversion.

[0008] In a preferred embodiment, the inlet dispersion component of the tree-like dispersion structure is an inlet dispersion column, with gaps between adjacent inlet dispersion columns, through which droplets are transported to avoid aggregation.

[0009] In a preferred embodiment, the internal dispersion component of the incubation chamber is an internal dispersion column, which is distributed in a dot matrix on the surface of the flat cavity to prevent the incubation chamber from collapsing while separating the droplets into a single-layer spread array.

[0010] In a preferred embodiment, the droplet generation intersection has a cross-shaped or T-shaped structure, and the oil phase wraps the water phase with shear force to form an oil-in-water droplet.

[0011] The method for detecting *E. coli* using the microfluidic chip according to the present invention includes the following steps: preparing an oil phase and an aqueous phase respectively, introducing them through corresponding inlets to form water-in-oil droplets at the droplet generation intersection; transporting the droplets through the branched channels of the dendritic dispersion structure, combing them through the inlet dispersion component, and entering the incubation chamber, where they form a droplet array under the action of the internal dispersion component; irradiating the droplets with ultraviolet light to solidify the oil phase and fix the aqueous phase droplets in situ; incubating at a constant temperature to cause *E. coli* in the test sample to lyse and release enzymes, catalyzing the enzyme-catalyzed reaction substrate to generate fluorescent products; detecting the fluorescence signal and calculating the *E. coli* concentration using statistical methods.

[0012] In a preferred embodiment, the bacterial lysis reagent is a PELB composite lysis reagent, and 1-2 μL of bacterial lysis reagent and 0.5-2 μL of enzymatic reaction substrate are added to every 100 μL of test sample in the aqueous phase.

[0013] In a preferred embodiment, the enzyme-catalyzed reaction substrate is 4-methylumbelliferone-β-D-glucuronide, and the fluorescent product is 4-methylumbelliferone.

[0014] In a preferred embodiment, the concentration of the enzyme-catalyzed reaction substrate is 0.05-2 mmol / L.

[0015] In a preferred embodiment, the photocurable monomer is a polymer containing (meth)acrylate functional groups and / or a monomer containing (meth)acrylate functional groups.

[0016] According to the microfluidic chip and E. coli detection method based on photocurable droplets of the present invention, water-in-oil droplets are solidified in situ, completely solving the problem of droplet breakage and fusion during incubation. The spatial fixation of the solidified droplets facilitates the localization and acquisition of fluorescence signals, reducing signal drift interference in traditional flowing droplet detection, and is suitable for digital droplet PCR (ddPCR) technology. In particular, the present invention combines a three-stage dispersion (multi-branched microchannels, an inlet dispersion column, and an internal dispersion column) single-bacterial isolation design with the enzymatic reaction enhancement effect of the solidification system, enabling trace E. coli detection without a pre-enrichment step. This method offers a 1000-fold increase in sensitivity compared to existing microfluidic technologies, providing a highly efficient, stable, and universal new solution for trace microbial detection. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the structure of a microfluidic chip according to a preferred embodiment of the present invention.

[0018] Figure 2 yes Figure 1 The droplet formation and dispersion diagram.

[0019] Figure 3 yes Figure 1 The droplet array diagram.

[0020] Figure 4 yes Figure 1 Image of droplet array cured by ultraviolet light. Detailed Implementation

[0021] The preferred embodiments of the present invention are given below with reference to the accompanying drawings and described in detail.

[0022] like Figure 1As shown, a microfluidic chip according to a preferred embodiment of the present invention includes an oil phase inlet 1, an aqueous phase inlet 2, a droplet generation cross-section 3, a tree-like dispersion structure 4, an incubation chamber 5, an inlet dispersion column 6, an internal dispersion column 7, and a fluorescence detection alignment part 8. The structures are connected in an orderly manner according to functional logic to jointly realize the droplet generation and detection process.

[0023] Oil phase inlet 1 is the fluid input interface on the left side of the chip, used to introduce an oil phase mixture containing photocuring reagents (including photopolymerization initiators and photocurable monomers), diluents, and surfactants. Under ultraviolet light irradiation, the photopolymerization initiator generates active free radicals, initiating the polymerization reaction of the photocurable monomers to form a cross-linked polymer network, thereby achieving oil phase curing. The photocurable monomers are polymers containing (meth)acrylate functional groups and / or monomers containing (meth)acrylate functional groups. The diluent adjusts the viscosity to ensure flow properties; the surfactant reduces the oil-water interfacial tension and stabilizes the droplet morphology. Its output end, after passing through a "branching and then merging" diversion region, leads to one side of the droplet generation intersection 3.

[0024] The aqueous phase inlet 2 is located within the split-flow area of ​​the oil phase inlet 1 and is connected to a constant-pressure pump to deliver an aqueous phase mixture. This aqueous phase mixture consists of the test sample, PELB composite lysis reagent, and enzymatic reaction substrate (4-MUG). In a preferred embodiment, 100 μL of sample is mixed with 1.5 μL of PELB composite lysis reagent and 1 μL of 4-MUG to form the aqueous phase mixture. The test sample is a sample containing the target *E. coli*, which is the target source for detection, such as the liquid enriched from the water quality sample to be tested. The PELB composite lysis reagent, acting as a cell wall disruptor, under incubation conditions (e.g., incubation at 40°C for 3 hours), breaks down the cell membrane and cell wall of *E. coli* through osmosis, releasing intracellular β-glucuronidase (a marker enzyme specific to *E. coli*), providing catalytic conditions for subsequent enzymatic reactions. The enzyme-catalyzed substrate 4-MUG (4-methylumbelliferone-β-D-glucuronide) is a specific substrate of β-glucuronidase. Under enzyme catalysis, it undergoes a hydrolysis reaction to generate 4-MU (4-methylumbelliferone), which has strong fluorescent properties. Its concentration is usually controlled at 0.1-1 mmol / L (e.g., 0.4 mg / mL). This concentration range ensures sufficient reaction between the substrate and the enzyme while avoiding interference from excessive substrate on the fluorescence signal. The output end of the aqueous phase inlet 2 meanders to the other side of the droplet generation intersection 3, where it intersects with the oil phase. In this embodiment, the output end of the aqueous phase inlet 2 is connected to a curved microchannel to balance the pressure and facilitate the adjustment of the flow rates of the oil and water phases, allowing both to reach the droplet generation intersection 3 simultaneously, thus facilitating droplet generation.

[0025] The droplet generation intersection 3 is located at the downstream junction of the oil phase and water phase channels, and has a cross-shaped or T-shaped structure. The oil phase wraps the water phase with shear force to form W / O (Water-in-Oil) single droplets (bacterial liquid droplets).

[0026] The initial segment of the tree-like dispersion structure 4 consists of multi-level branched microchannels extending from the droplet generation intersection 3. Initially, it is a single main channel, which then branches symmetrically into 2-4 parallel branch channels through 1-2 levels. Each branch channel extends at equal intervals, achieving initial dispersion of droplets through channel diversion. The ends of these branched channels are connected to a flat rectangular plate (i.e., the carrier base plate of the incubation chamber 5). The plate and the branched channels are integrally formed, with the channel ends seamlessly connected to the left edge of the plate, ensuring that droplets flow smoothly into the plate surface. In the left inlet region of the plate (i.e., the junction of the branched channels and the plate), a row of inlet dispersion columns 6 protruding vertically from the plate surface are distributed. The inlet dispersion columns 6 are elongated and arranged side-by-side along the left edge of the plate. A gap matching the droplet diameter is left between adjacent inlet dispersion columns 6 (e.g., when the droplet diameter is 50-100 μm, the gap width is 60-110 μm, and the gap width is 10-20 μm larger than the droplet diameter). This allows the droplet flow dispersed through the branching channels to be further blocked and organized by the inlet dispersion columns 6, preventing droplet aggregation when entering the open plate area. At this point, the inlet dispersion columns 6 act as the end component of the tree-like dispersion structure 4, continuing the dispersion function by relying on the plate carrier. Figure 2 As shown.

[0027] Downstream of the inlet dispersion column 6, inside the incubation chamber 5, multiple internal dispersion columns 7 protruding from the surface of the plate are distributed to prevent the incubation chamber 5 from collapsing. The internal dispersion columns 7 are cylindrical and uniformly arranged in a lattice pattern, forming a continuous dispersion logic of "inlet sorting → internal separation". After the droplets enter the plate through the inlet dispersion column 6, they further spread under the physical barrier of the internal dispersion columns 7, ultimately forming a uniformly distributed array of droplets on the surface of the incubation chamber 5, such as... Figure 3 As shown.

[0028] The fluorescence detection alignment part 8 is located on the right edge of the incubation chamber 5, is integrally formed with the carrier plate of the incubation chamber 5, and is coplanar with the droplet array in the incubation chamber 5, thereby providing a precise alignment reference for the probe of the external fluorescence detector.

[0029] After entering the incubation chamber 5 through the dendritic dispersion structure 4, the inlet dispersion column 6, and the internal dispersion column 7, the droplets spread out in a monolayer on the flat substrate of the incubation chamber 5. An ultraviolet lamp above the chip irradiates the droplets, causing the photopolymerization initiator in the oil phase of the droplets to absorb light energy and decompose into free radicals, initiating the polymerization of photocurable monomers to form a cross-linked polymer network. Within seconds, the previously flowing droplets rapidly solidify into a solid film, preventing droplet breakage or fusion and "locking" the aqueous droplets on the flat surface. In this way, the droplets are solidified in situ, preventing them from flowing and colliding with each other, reducing the possibility of breakage and fusion. The incubation chamber 5 maintains a constant temperature for incubation via a temperature control module. The PELB complex lysis reagent in the aqueous phase penetrates the E. coli cell wall, releasing intracellular β-glucuronidase; this enzyme specifically binds to 4-MUG in the aqueous phase, catalyzing its hydrolysis into 4-MU (fluorescent product). During the 3-hour incubation period, the solid oil phase film prevents the diffusion of aqueous components, allowing the enzyme and substrate to react fully in a closed microenvironment, resulting in the continuous accumulation of fluorescent products. After the reaction was completed, the fluorescence intensity of each droplet was collected. The signal strength was positively correlated with the number of E. coli. Based on the statistical method of Poisson distribution, the concentration of E. coli in the original sample was finally calculated.

[0030] In existing technologies, E. coli detection often faces sensitivity bottlenecks due to "extremely low levels of β-glucuronidase in bacteria" and "signal interference caused by droplet flow." It is generally believed in the industry that complex pre-concentration or sacrificing reaction efficiency for stability is necessary. This invention employs a three-stage dispersion design—a dendritic dispersion structure 4, an inlet dispersion column 6, and an internal dispersion column 7—to ensure that droplets spread out in a single layer without overlap in the incubation chamber, avoiding interference from "multi-bacterial mixing" caused by the fusion of multiple droplets. After the droplets enter the incubation chamber 5, ultraviolet light triggers photocuring of the oil phase, forming a solid film within seconds that "locks in" the aqueous phase. Compared to traditional flow systems, this invention, through the synergy of "single-bacterial isolation + solidification locking," improves detection sensitivity by 1000 times, enabling the detection of trace amounts of E. coli as low as 0.01 CFU / mL without pre-concentration.

[0031] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. That is, all simple and equivalent changes and modifications made based on the claims and description of this invention fall within the protection scope of the claims. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A microfluidic chip based on photocurable droplets, characterized in that, The microfluidic chip includes: An oil phase inlet is used to introduce an oil phase containing a photocurable reagent, which includes a photopolymerization initiator and a photocurable monomer; Aqueous phase inlet, used to introduce an aqueous phase, which includes test sample, bacterial lysis reagent and enzyme reaction substrate; The droplet generation intersection is located at the downstream junction of the channels of the oil phase inlet and the water phase inlet, and is used to encapsulate the water phase with the oil phase to form water-in-oil droplets. A tree-like dispersion structure, connected downstream of the droplet generation intersection, includes multi-level branched microchannels and an inlet dispersion component located at the end of the channel, for the initial dispersion of droplets; as well as The incubation chamber is a cavity structure formed on a flat plate surface. The inlet is connected to the end of the dendritic dispersion structure. Inside, there is an internal dispersion component to hold the droplet array. The droplets are fixed by a photocuring reagent in the oil phase, and a bacterial lysis reagent in the aqueous phase lyses the bacteria in the test sample, releases enzymes, and catalyzes the enzyme-catalyzed reaction substrate to generate fluorescent products, thus realizing the solidification and reaction of the droplets.

2. The microfluidic chip according to claim 1, characterized in that, The multi-level branched microchannels of the tree-like dispersion structure are initially a single main channel, which branches into 2-8 parallel branch channels through 1-3 levels of branching. Each branch channel achieves the initial dispersion of droplets through diversion.

3. The microfluidic chip according to claim 1, characterized in that, The inlet dispersion component of the tree-like dispersion structure is an inlet dispersion column, with gaps between adjacent inlet dispersion columns, through which droplets are transported to avoid aggregation.

4. The microfluidic chip according to claim 1, characterized in that, The internal dispersion component of the incubation chamber is an internal dispersion column, which is distributed in a dot matrix on the surface of the flat cavity to prevent the incubation chamber from collapsing while separating the droplets into a single-layer spread array.

5. The microfluidic chip according to claim 1, characterized in that, The droplet generation intersection has a cross-shaped or T-shaped structure, and the oil phase wraps the water phase with shear force to form an oil-in-water droplet.

6. A method for detecting Escherichia coli using the microfluidic chip according to any one of claims 1-5, characterized in that, Includes the following steps: Oil and aqueous phases are prepared separately and introduced into each phase through corresponding inlets to form water-in-oil droplets at the droplet generation intersection. The droplets are transported through the branched channels of the tree-like dispersion structure, and after being combed by the inlet dispersion component, they enter the incubation chamber, where they form a droplet array under the action of the internal dispersion component. Ultraviolet light irradiation solidifies the oil phase of the droplets, while fixing the aqueous phase droplets in situ. Constant temperature incubation causes the E. coli in the test sample to lyse and release enzymes, which catalyze the enzyme-catalyzed reaction to generate fluorescent products from the substrate. The concentration of Escherichia coli was calculated by detecting fluorescence signals and combining them with statistical methods.

7. The method according to claim 6, characterized in that, The bacterial lysis reagent is a PELB composite lysis reagent. In the aqueous phase, 1-2 μL of bacterial lysis reagent and 0.5-2 μL of enzymatic reaction substrate are added to every 100 μL of test sample.

8. The method according to claim 6, characterized in that, The substrate for the enzymatic reaction is 4-methylumbelliferone-β-D-glucuronide, and the fluorescent product is 4-methylumbelliferone.

9. The method according to claim 6, characterized in that, The concentration of the enzyme-catalyzed reaction substrate is 0.05-2 mmol / L.

10. The method according to claim 6, characterized in that, The photocurable monomer is a polymer containing (meth)acrylate functional groups and / or a monomer containing (meth)acrylate functional groups.

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