A method for detecting water quality by embedding coliform bacteria in alginate hydrogel

Abstract

The application discloses a microfluidic chip and a method for detecting water quality by embedding coliform group in alginate hydrogel, and the microfluidic chip comprises a sample inlet, an oil phase inlet, an alginate aqueous solution inlet, a droplet mixing flow channel, an incubation chamber and an outlet; wherein the oil phase inlet is connected with the sample flow channel through two oil phase flow channels which are divided into two parts and meet at a cross-shaped port, the alginate aqueous solution inlet is connected with the flow channel from the cross-shaped port through a water phase flow channel and meets at a T-shaped port; a contraction section flow channel and a first serpentine flow channel are sequentially arranged between the T-shaped port and the droplet mixing flow channel, and are respectively used for extruding and fusing the bacterial liquid droplet and the alginate droplet. The application solves the problem that the detection effect is poor due to the spontaneous fusion of droplets caused by the increase of incubation temperature in the prior art by combining the alginate with the calcium salt solution to form the hydrogel through ion cross-linking, and has a good application prospect in the detection of coliform group in water samples.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of water quality detection, and more particularly to a method for detecting water quality by embedding coliform bacteria in alginate hydrogel. BACKGROUND

[0002] Escherichia coli (E. coli for short) is a kind of bacteria commonly found in the intestines of humans and many other warm-blooded animals, usually existing as part of the normal flora of the intestines, generally harmless to the host, and even beneficial in some cases. However, certain mutant strains or specific serotypes can cause diseases such as food poisoning, urinary tract infection, sepsis, etc. These pathogenic E. coli can produce toxins or have the ability to invade host tissues. Therefore, detecting E. coli in food and water sources has important public health significance. Because E. coli is ubiquitous in feces, its presence and quantity are often used as an indicator microorganism for water quality detection. If E. coli is detected in a water sample, it usually means that the water quality may be contaminated. In the field of environmental protection, detection of E. coli can evaluate the environmental quality of water bodies and soil, and guide wastewater treatment and environmental protection work.

[0003] Traditional culture detection techniques (plate culture, MPN method, etc.) are simple to operate, can perform qualitative analysis, and have high accuracy for E. coli identification. However, they require a long time, usually several days to obtain results, and the process is cumbersome, which is not conducive to rapid detection. Molecular detection techniques have fast analysis speed, high sensitivity, and strong specificity. For example, real-time fluorescent quantitative PCR technology can achieve the transition from qualitative detection to quantitative detection, simplifying the experimental process and reducing pollution and errors. However, it has high technical requirements and expensive equipment, and may require professional operators.

[0004] In view of the above phenomenon, some studies have combined single droplet microfluidic technology with enzyme reaction and fluorescence counting method for embedding E. coli. However, a large number of E. coli-embedded droplets are arranged in a single layer in the reaction chamber, and E. coli is cultured and lysed in the droplets for detection, which requires several hours of incubation, and the temperature is maintained at 50℃ during incubation. However, when two droplets come into contact, the surface between them is replaced by a common interface, reducing the total surface area of the system. Due to the effect of surface tension, the system tends to reduce surface energy, so two droplets will spontaneously fuse to reduce the total energy of the system. In addition, an increase in temperature usually reduces the viscosity and surface tension of the liquid, making the droplets more likely to fuse. Therefore, it is crucial to reduce the probability of droplet fusion by changing experimental conditions. SUMMARY

[0005] The application aims to provide a microfluidic chip and a method for detecting water quality by embedding coliform bacteria in alginate hydrogel, so as to solve the problem of poor detection effect caused by spontaneous fusion of droplets due to the increase of incubation temperature in the existing detection technology of coliform bacteria in water quality samples.

[0006] In order to solve the above problems, the application adopts the following technical solutions:

[0007] According to a first aspect of the application, a microfluidic chip is provided, comprising: a sample inlet, an oil phase inlet, the sample inlet being connected with a sample flow channel, the oil phase inlet being connected with the sample flow channel through two oil phase flow channels which are divided into two, and the two oil phase flow channels and the sample flow channel meet at a "cross" port; an alginate aqueous solution inlet, the alginate aqueous solution inlet being connected with the flow channel from the "cross" port through a water phase flow channel at a "T" port; a droplet mixing flow channel connected with the "T" port, the droplet mixing flow channel being branched in a tree structure; an incubation chamber connected with the end of the droplet mixing flow channel, the incubation chamber being used for single-layer arrangement of droplets; and an outlet; wherein the flow channel between the "T" port and the droplet mixing flow channel is provided with a contraction section flow channel and a first serpentine flow channel in sequence, respectively used for extrusion and fusion of bacterial liquid droplets and alginate droplets therein, and the first serpentine flow channel has a widened flow channel size relative to the contraction section flow channel.

[0008] Preferably, the ratio of the pipe diameter of the first serpentine flow channel to the contraction section flow channel is (1.5-2):1.

[0009] Preferably, the sample inlet and the "cross" port are connected through a second serpentine flow channel.

[0010] Preferably, the incubation chamber comprises: fences close to the droplet mixing flow channel, and a plurality of pillars dispersed in the incubation chamber.

[0011] Preferably, the size of the channel between adjacent fences is 150-200 μm.

[0012] According to a second aspect of the application, a method for detecting water quality by embedding coliform bacteria in alginate hydrogel is provided, comprising the following steps: S1: providing a microfluidic chip as described above;

[0013] S2: adding Mg 2+S1: the sample is prepared by adding divalent metal salt other than Mg2+, PELB complex lysis reagent and enzymatic reaction substrate 4-CMUG, the sample is input from the sample inlet while the oil phase is input through the oil phase inlet, the sample is sheared by the oil phase at the "cross" port to generate W / O type bacterial liquid droplets; S2: the aqueous alginate solution is input from the aqueous alginate solution inlet and is sheared into W / O type alginate droplets at the "T" port; S3: the bacterial liquid droplets and the alginate droplets are made to flow downstream alternately by controlling the flow rates of the phases, then the bacterial liquid droplets and the alginate droplets are extruded into long strip-shaped droplets in the contraction section flow channel, and then the adjacent bacterial liquid droplet and alginate droplet become round and fuse into a large droplet in the widened first serpentine flow channel, and ion cross-linking occurs to form a hydrogel droplet; S4: the hydrogel droplet is mixed uniformly in the droplet mixing flow channel, enters the incubation chamber and is arranged in a single layer therein; S5: after incubation at 40℃ for 3-4h, the β-glucuronidase secreted by the E. coli catalyzes the hydrolysis of the substrate 4-MUG into specific fluorescent substance 4-MU, which is observed and imaged under a fluorescence microscope, and the number of fluorescent droplets is calculated by using Poisson distribution, so that the detection of E. coli in the water quality sample to be detected is realized.

[0014] Preferably, the size of the generated alginate droplets is larger than that of the bacterial liquid droplets by adjusting the flow rates of the sample, the oil phase and the aqueous alginate solution respectively.

[0015] Preferably, the diameter ratio of the generated alginate droplets to the bacterial liquid droplets is (1-1.5):1 by adjusting the flow rates of the sample, the oil phase and the aqueous alginate solution respectively.

[0016] Preferably, the divalent metal salt other than Mg2+ is CaCl2, and the aqueous alginate solution is an aqueous sodium alginate solution. However, it should be understood that the present application is not limited to sodium alginate. In addition, the salt added in the sample preparation of the present application is not limited to calcium salt, but can also be other divalent metal salt other than Mg2+. 2+ Preferably, the divalent metal salt other than Mg2+ is CaCl2, and the aqueous alginate solution is an aqueous sodium alginate solution. However, it should be understood that the present application is not limited to sodium alginate. In addition, the salt added in the sample preparation of the present application is not limited to calcium salt, but can also be other divalent metal salt other than Mg2+. 2+

[0017] Preferably, the concentration of CaCl2 in the sample is 0.1%-0.5% w / w, and the concentration of the aqueous sodium alginate solution is 0.1-2% w / w.

[0018] ​According to the application, a method for generating and stabilizing single droplets in a microfluidic chip is provided. By combining alginate with a calcium salt solution, ion cross-linking occurs to form a hydrogel. In principle, ion cross-linking reaction occurs between alginate droplets and bacterial liquid droplets as soon as they come into contact, forming a hydrogel droplet, preventing further fusion of the droplets, and the strength of the hydrogel droplet can be further controlled by adjusting the concentration of alginate. The system concentration of local fluorescent substances can be increased to exceed the detection limit of the detection instrument, thereby reducing the detection time. The application discloses a technical solution for the first time, which utilizes the ion cross-linking performance of alginate and calcium salt, so that the two droplets do not fuse again after the first fusion, thereby maintaining the stability of the droplets. Prior art has never reported similar reports before.

[0019] One of the key points of the application is that by adjusting the flow rates of the sample, oil phase and alginate aqueous solution respectively, the bacterial liquid droplets and alginate droplets are generated at intervals, and then pass through the contraction section flow channel to the widened second serpentine flow channel, so that the same droplets cannot come into contact, thereby ensuring that one adjacent bacterial liquid droplet and one alginate droplet become round and then fuse into a large droplet, and ion cross-linking occurs to form a hydrogel droplet. On this basis, the generation speed of the single droplet can also be appropriately reduced to ensure that the droplets are not disturbed by the following droplets before fusion.

[0020] In summary, according to the microfluidic chip and the method for detecting water quality by embedding coliform bacteria in alginate hydrogel provided by the application, by combining alginate with a calcium salt solution, ion cross-linking occurs to form a hydrogel, which solves the problem that the detection effect of the existing detection technology for coliform bacteria in water samples is poor due to spontaneous fusion of droplets caused by increased incubation temperature. Even during the high-temperature incubation process of 3-4 hours, the droplets do not fuse again, realizing accurate detection of coliform bacteria in water samples, reducing pollution and errors, and the operation is simple. The method has a good application prospect in the detection of coliform bacteria in water samples. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 is a structural schematic diagram of a microfluidic chip according to a preferred embodiment of the application;

[0022] Figure 2 is a schematic diagram of the generation of two droplets alternately;

[0023] Figure 3 is a process schematic diagram of the fusion of two droplets into a hydrogel droplet in a flow channel with changing pipe diameter;

[0024] Figure 4 is a dispersion schematic diagram of the fused hydrogel droplet;

[0025] Figure 5 This is a schematic diagram of hydrogel droplets incubating in a chamber.

[0026] Figure 6 Sodium alginate and Ca 2+ Crosslinking principle diagram. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and materials used in the embodiments are commercially available.

[0028] Example 1: Construction of a microfluidic chip

[0029] like Figure 1 The image shows a microfluidic chip provided according to the present invention, comprising: an oil phase inlet 1, a sample inlet 2, an alginate aqueous solution inlet 3, a contraction section channel 4, a first serpentine channel 5, a second serpentine channel 6, a droplet mixing channel 7, an incubation chamber 8, a fence 9, a support column 10, and an outlet 11.

[0030] The sample inlet 2 is connected to a sample flow channel. The oil phase inlet 1 is connected to the sample flow channel via two bisected oil phase flow channels at a cross-shaped junction 12. The alginate aqueous solution inlet 3 is connected to the flow channel from the cross-shaped junction at a T-shaped junction 13 via an aqueous phase flow channel. The T-shaped junction 13 and the droplet mixing flow channel 7 are connected sequentially by a constricted flow channel 4 and a widened first serpentine flow channel 5, which respectively achieve droplet compression and fusion. The droplet mixing flow channel 7 has a tree-like branching structure to achieve the dispersion of fused droplets. The sample inlet 2 is connected to the cross-shaped junction via a second serpentine flow channel 6. The incubation chamber 8 is connected to the end of the droplet mixing flow channel 7 for the single-layer arrangement of fused droplets. The incubation chamber 8 is equipped with a fence 9 near the droplet mixing flow channel 7 and several support pillars 10 dispersed within it.

[0031] Combination Figure 2 , Figure 3 As shown, by controlling the flow rate of each phase, bacterial liquid droplet A and alginate droplet B flow downstream alternately one after the other. Then, bacterial liquid droplet A and alginate droplet B are squeezed into long strip droplets in the contraction section of the flow channel 4. Subsequently, in the widened first serpentine flow channel 5, an adjacent bacterial liquid droplet and an alginate droplet become round and gradually merge into a large droplet, while ionic cross-linking occurs to form hydrogel droplet C.

[0032] Combination Figure 1 , Figure 3As shown, the first serpentine flow channel 5 has a widened flow channel size relative to the converging section flow channel 4. According to the preferred embodiment of the present application, the ratio of the tube diameter of the first serpentine flow channel 5 to the converging section flow channel 4 is (1.5-2): 1. Most preferably, it is 1.5: 1.

[0033] Example 2 A method for detecting water quality by embedding coliform bacteria in alginate hydrogel

[0034] According to the present embodiment, a method for detecting water quality by embedding coliform bacteria in alginate hydrogel is provided, comprising the following steps:

[0035] 1) Provide the microfluidic chip shown in Example 1.

[0036] 2) Add CaCl2, PELB compound lysis reagent and enzymatic reaction substrate 4-CMUG to the water quality sample to be detected (preferably the liquid after enrichment of the water quality sample to be detected) to prepare a sample, wherein the concentration of CaCl2 is 0.2% w / w (within the range of 0.1%-0.5% w / w), the concentration of enzymatic reaction substrate 4-CMUG is 0.4 mg / mL (within the range of 0.2-0.7 mg / mL), which is delivered from the sample inlet 2 by a constant pressure pump, while the oil phase is input through the oil phase inlet 1, so that the sample is sheared by the oil phase entering from the oil phase inlet 1 at the "cross" port 12 to generate W / O bacterial liquid droplets.

[0037] 3) The aqueous alginate solution enters from the aqueous alginate solution inlet 3 and is sheared into W / O type alginate droplets at the "T" port 13.

[0038] 4) By adjusting the flow rates of the sample, oil phase and aqueous alginate solution respectively, the size of the generated alginate droplets is larger than that of the bacterial liquid droplets, as shown in Figure 3 Preferably, the diameter ratio of the generated alginate droplets B to the bacterial liquid droplets A is (1-1.5): 1. At the same time, by controlling the flow rates of the phases, the bacterial liquid droplets A and the alginate droplets B are alternately flowed downstream, followed by the bacterial liquid droplets A and the alginate droplets B being extruded into long strip-shaped droplets at the converging section flow channel 4, respectively, and then one adjacent bacterial liquid droplet A and one adjacent alginate droplet B become circular and fuse into a large droplet in the widened first serpentine flow channel 5. It should be understood that the alginate droplets B and the bacterial liquid droplets A will undergo ionic crosslinking reaction as soon as they come into contact, thereby forming a hydrogel droplet C, and the reaction mechanism is as shown in Figure 6 .

[0039] 5) As shown in Figure 4 , Figure 5 , the hydrogel droplets C are mixed uniformly in the droplet mixing flow channel 7, and then enter the incubation chamber 8 and arrange in a single layer therein.

[0040] 6) After 3-4 hours of incubation at 40℃, the β-glucuronidase secreted by the E. coli catalyzes the hydrolysis of the substrate 4-MUG into the specific fluorescent substance 4-MU, which is observed and imaged under a fluorescence microscope, and the number of fluorescent substances is calculated using the Poisson distribution, so that the detection of E. coli in the water quality sample to be detected can be realized.

[0041] The above is only a preferred embodiment of the present application, and is not intended to limit the scope of the present application. The above embodiment of the present application can be variously changed. Any simple, equivalent change and modification made according to the content of the claims and the specification of the present application falls within the scope of protection of the present patent. The present application is not described in detail, and is a routine technical content.

Claims

1. A method for water quality detection using alginate hydrogel-embedded coliform bacteria, characterized in that, Includes the following steps: S1: A microfluidic chip is provided; the microfluidic chip includes: a sample inlet, an oil phase inlet, the sample inlet being connected to a sample channel, the oil phase inlet being connected to the sample channel at a "+" joint via two oil phase channels that are split into two; an alginate aqueous solution inlet, the alginate aqueous solution inlet being connected to a channel from the "+" joint at a "T" joint via an aqueous phase channel; a droplet mixing channel connected to the "T" joint, the droplet mixing channel having a tree-like branching structure; an incubation chamber connected to the end of the droplet mixing channel for single-layer arrangement of droplets; and an outlet; wherein, the channel between the "T" joint and the droplet mixing channel is provided with a constriction section channel and a first serpentine channel in sequence, for the compression and fusion of bacterial droplets and alginate droplets therein, respectively, and the diameter ratio of the first serpentine channel to the constriction section channel is (1.5~2):1; S2: Add Mg removal agent to the water sample to be tested. 2+ The sample was prepared by using divalent metal salts other than those in the sample inlet, PELB complex lysis reagent and enzyme-catalyzed reaction substrate 4-CMUG. The sample was then introduced into the sample inlet and simultaneously into the oil phase inlet, so that the sample was sheared by the oil phase at the "+" shaped inlet to generate W / O type bacterial droplets. S3: Alginate aqueous solution enters from the alginate aqueous solution inlet and is sheared into W / O type alginate droplets at the "T" shaped opening; S4: By controlling the flow rate of each phase, the bacterial liquid droplets and alginate droplets flow downstream alternately one after the other. By adjusting the flow rates of the sample, oil phase, and alginate aqueous solution respectively, the diameter ratio of the generated alginate droplets to the bacterial liquid droplets is (1~1.5):

1. Then, the bacterial liquid droplets and alginate droplets are squeezed into long strip droplets in the contraction section of the flow channel. Subsequently, in the widened first serpentine flow channel, an adjacent bacterial liquid droplet and an alginate droplet become round and then merge into a large droplet. At the same time, ionic cross-linking occurs to form a hydrogel droplet. S5: The hydrogel droplets are mixed evenly in the droplet mixing channel, enter the incubation chamber and are arranged in a single layer therein; S6: After incubation at 40℃ for 3-4 hours, β-glucuronidase secreted by Escherichia coli catalyzes the hydrolysis of substrate 4-MUG into specific fluorescent substance 4-MU. The substance is observed and imaged under a fluorescence microscope, and the number of fluorescent molecules is calculated using the Poisson distribution, thus enabling the detection of Escherichia coli in the water sample to be tested.

2. The method according to claim 1, characterized in that, In the microfluidic chip, the sample inlet and the cross-shaped opening are connected by a second serpentine flow channel.

3. The method according to claim 1, characterized in that, In the microfluidic chip, the incubation chamber includes: a fence near the droplet mixing channel, and a number of pillars dispersed within the incubation chamber.

4. The method according to claim 3, characterized in that, In the microfluidic chip, the channel size between adjacent fences is 150~200μm.

5. The method according to claim 1, characterized in that, The removal of Mg 2+ The divalent metal salt other than CaCl2 is used, and the alginate aqueous solution is a sodium alginate aqueous solution.

6. The method according to claim 5, characterized in that, The concentration of CaCl2 in the sample was 0.1%~0.5% w / w, and the concentration of sodium alginate aqueous solution was 0.1%~2% w / w.

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