A microfluidic chip integrating bacterial blotting and droplet detection and its application.

Abstract

This invention provides a microfluidic chip integrating bacterial imprinting and droplet detection, and its application. The chip comprises, from top to bottom, a microfluidic channel layer, a glassy carbon electrode layer, and a glass substrate layer. The microfluidic channel layer includes a sample inlet, a biochemical reaction solution inlet, an oil phase inlet, a waste liquid inlet, an E. coli capture chamber, and a droplet observation chamber. The surface of the glassy carbon electrode layer is treated with bacterial imprinting to form a bacterial imprint region corresponding to the E. coli capture chamber, for capturing and enriching VBNC E. coli in the sample. The glass substrate layer is aligned with the droplet observation chamber of the microfluidic channel layer for droplet spreading and observation. This invention achieves highly sensitive detection of VBNC E. coli in drinking water by integrating bacterial enrichment and droplet detection. This invention significantly reduces the requirements for operator skills and auxiliary equipment, while also making it potentially applicable in the field of molecular diagnostics.

Description

Technical Field

[0001] This invention relates to the field of microfluidics, and more specifically to a microfluidic chip that integrates bacterial imprinting and droplet detection, and its applications. Background Technology

[0002] Drinking water is an indispensable part of daily life, and its quality significantly impacts people's well-being, thus requiring professional testing and analysis. Microbial contamination is a major hazard in drinking water, with coliform bacteria being a primary pathogen causing pollution. Direct consumption of water with excessive coliform levels can pose serious health risks. Currently, drinking water is commonly disinfected using chlorination and ultraviolet light. Under prolonged exposure to chlorination and ultraviolet radiation, E. coli may enter a viable but non-culturable (VBNC) state to increase its survival rate. Studies have found that VBNC E. coli exhibit variations in size and shape; cells may become smaller or larger, and their shape may become irregular, but they still maintain metabolic activity and toxicity. Because VBNC E. coli is unculturable, the gold standard for E. coli detection—the plate assay—can miss it, underestimating the number of E. coli in drinking water. If people consume large amounts of drinking water containing VBNC E. coli, the probability of these bacteria reviving and exerting their virulence in the body increases significantly, potentially posing incalculable harm to human health. Therefore, it is necessary to develop a rapid and efficient method for detecting VBNC (Vitamin B NC) in drinking water.

[0003] Researchers have developed a series of microfluidic chips for Escherichia coli (VBNC) detection, such as immunomagnetic bead separation chips, droplet detection chips, and digital PCR chips, which can accurately and reliably quantify VBNC. However, because VBNC bacteria are a type of bacteria that survives environmental stress and are relatively few in number, effectively detecting VBNC E. coli in samples presents certain challenges. Bacterial blotting technology utilizes materials prepared using molecular imprinting techniques to specifically recognize and capture target bacteria. This involves synthesizing polymer materials with porous structures that match the specific structure of target molecules (such as bacteria), thereby achieving selective recognition and capture of target molecules. This provides a new approach for the efficient detection of E. coli in drinking water. Summary of the Invention

[0004] The purpose of this invention is to provide a microfluidic chip that integrates bacterial blotting and droplet detection and its application, thereby solving the problem that existing technologies cannot effectively detect VBNC Escherichia coli in drinking water.

[0005] To solve the above problems, the present invention adopts the following technical solution:

[0006] According to a first aspect of the present invention, a microfluidic chip integrating bacterial blotting and droplet detection is provided, comprising, from top to bottom, a microfluidic channel layer, including: a sample inlet, a biochemical reaction solution inlet, an oil phase inlet, a waste liquid outlet, an E. coli capture chamber, and a droplet observation chamber, wherein the sample inlet is connected to the E. coli capture chamber via a sample channel, the biochemical reaction solution inlet is connected to the E. coli capture chamber via a biochemical reaction solution channel, and the oil phase inlet is connected to the E. coli capture chamber via an oil phase channel. The detection liquid channels at the opening converge and communicate with a droplet channel, which is connected to the droplet observation cavity; a glass carbon electrode layer, the size of which is smaller than that of the microfluidic channel layer, has a surface treated with bacterial imprinting to form a bacterial imprint region corresponding to the E. coli capture cavity, for capturing and enriching VBNC E. coli in the sample; and a glass substrate layer, the size of which is equal to that of the microfluidic channel layer and aligned with the droplet observation cavity of the microfluidic channel layer for droplet spreading and observation.

[0007] Preferably, the oil phase inlet is connected to two oil phase channels, and the two oil phase channels converge with the detection liquid channel at the outlet of the E. coli capture chamber at a cross intersection and are connected to the droplet channel.

[0008] Preferably, the two oil phase flow channels extend from the oil phase inlet in opposite directions and converge at the cross joint to form a rectangular flow channel, which surrounds the sample inlet, the biochemical reaction solution inlet, and the E. coli capture chamber.

[0009] Preferably, the E. coli capture chamber and the droplet observation chamber are arranged on the left and right sides of the microfluidic channel layer, respectively.

[0010] Preferably, the waste liquid outlet is located at the end of the droplet observation cavity.

[0011] Preferably, the E. coli capture chamber is circular, and the droplet observation chamber is square.

[0012] Preferably, the microfluidic channel layer, glass carbon electrode layer, and glass substrate layer are installed and fixed by clamps and screws.

[0013] Preferably, the bacterial imprint treatment of the glassy carbon electrode layer includes the following steps: 1) Before use, the surface of the glassy carbon electrode layer is polished to a mirror finish with a 0.05-0.3 μm alumina slurry, and then ultrasonically cleaned with water and ethanol; 2) In a solution containing 0.05 M pyrrole and 10 5Electrochemical copolymerization was carried out in 2 mL of 0.1 M KCl solution containing CFU / mL VBNC E. coli template by cyclic voltammetry; 3) VBNC E. coli was imprinted on the glassy carbon electrode layer by applying a cyclic voltage of -0.4 to +0.7 V relative to the saturated calomel reference electrode at a scan rate of 50 mV / s under gentle stirring; 4) The modified glassy carbon electrode layer was treated with 5% w / v SDS / HAC for 4.0 hours under static conditions to remove the bacterial template, thereby obtaining a glassy carbon electrode layer with a characteristic bacterial imprinted region on its surface.

[0014] According to a second aspect of the present invention, a microfluidic chip integrating bacterial imprinting and droplet detection is provided for the detection of VBNC (bacterial blotting) Escherichia coli in drinking water, thereby realizing water quality monitoring.

[0015] The present invention provides a microfluidic chip integrating bacterial blotting and droplet detection, mainly comprising two components: bacterial blotting and droplet detection. First, the bacterial blotting component is responsible for specifically enriching VBNC (Vitamin B Count) Escherichia coli in drinking water; then, specific biochemical reactions can occur within the generated droplets to detect the presence of VBNC E. coli. The main inventive point of this invention lies in the first-time combination of bacterial blotting and droplet detection technologies, providing specific monitoring of VBNC E. coli, while the use of microfluidic technology makes experimental operations more precise and controllable.

[0016] The microfluidic chip integrating bacterial blotting and droplet detection, and its application, provided by the present invention, have the following advantages over the prior art:

[0017] 1) According to the structural design of the microfluidic channel layer of the present invention, it not only has the function of reagent separation during the isolation of VBNC Escherichia coli, but also only needs to continuously inject oil phase into the oil cavity to provide pressure during subsequent droplet biochemical detection, so that water and oil can enter the micro cavity in an orderly manner and fill it, without the need for complex equipment such as negative pressure pumps or centrifuges to drive liquid flow, thus realizing simple semi-automatic sample injection.

[0018] 2) Integrating bacterial enrichment and droplet detection together enables highly sensitive detection of VBNC Escherichia coli in drinking water.

[0019] 3) This invention greatly reduces the requirements for sample culture and the professional skills of operators and auxiliary equipment during the detection process, and at the same time makes this invention potentially applicable in the field of molecular diagnostics. Attached Figure Description

[0020] Figure 1 This is an exploded view of a microfluidic chip structure according to a preferred embodiment of the present invention;

[0021] Figure 2 This is a schematic diagram of the microfluidic channel layer.

[0022] Figure 3 A schematic diagram of the bacterial imprinting process for glassy carbon electrode layers;

[0023] Figure 4 This is a fixture structure for microfluidic chips. Detailed Implementation

[0024] 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.

[0025] like Figure 1 The image shows a microfluidic chip integrating bacterial blotting and droplet detection according to a preferred embodiment of the present invention. This microfluidic chip can not only specifically enrich VBNC Escherichia coli in drinking water, but also perform specific biochemical reactions to detect VBNC Escherichia coli. The microfluidic chip mainly includes a microfluidic channel layer 1, a glassy carbon electrode layer 2, and a glass substrate layer 3 arranged sequentially from top to bottom, and then assembled by clamping with a fixture 4.

[0026] Combination Figure 1 , Figure 2 As shown, the microfluidic channel layer 1 is rectangular, with a length of 50 mm, a width of 23 mm, and a thickness of 3 mm. This microfluidic channel layer 1 includes: a sample inlet 11, a biochemical reaction solution inlet 12, an oil phase inlet 13, a waste liquid outlet 14, an E. coli capture chamber 15, and a droplet observation chamber 16. The channels and chambers are all located at the bottom. The sample inlet 11 is connected to the E. coli capture chamber 15 via a sample channel. The biochemical reaction solution inlet 12 is connected to the E. coli capture chamber 15 via a biochemical reaction solution channel. The oil phase inlet 13, through two oil phase channels, converges with the detection liquid channel at the outlet of the E. coli capture chamber 15 at a cross 17 and connects to a droplet channel, which is further connected to the droplet observation chamber 16. The droplet observation chamber 16 contains several micropillars 18, which provide support for the entire droplet observation chamber 16. According to this preferred embodiment, two oil phase flow channels extend from the oil phase inlet in opposite directions and converge at the crossroads to form a rectangular flow channel. This rectangular flow channel surrounds the sample inlet 11, the biochemical reaction liquid inlet 12, and the E. coli capture chamber 15, achieving a better spatial arrangement.

[0027] The glassy carbon electrode layer 2 is smaller than the microfluidic channel layer 1. Its surface is treated with bacterial imprinting to form a bacterial imprint region 21 corresponding to the E. coli capture cavity 15, for capturing and enriching VBNC E. coli in the sample. In this embodiment, the bacterial imprint region 21 is circular and is formed by dropping a bacterial solution onto the glassy carbon electrode layer 2.

[0028] The glass substrate 3 is the same size as the microfluidic channel layer 1 and is aligned with the droplet observation cavity 16 of the microfluidic channel layer 1 so that the droplets can be spread out and observed in the droplet observation cavity 16.

[0029] like Figure 3 As shown, bacterial imprinting treatment is required on the glassy carbon electrode layer 2. The specific steps are as follows: 1) Before use, the surface of the glassy carbon electrode layer is polished to a mirror finish with 0.05-0.3 μm alumina slurry, and then ultrasonically cleaned with water and ethanol; 2) In 2 mL of a solution containing 0.05 M pyrrole and VBNC E. coli template (10 5 Electrochemical copolymerization was carried out in 0.1M KCl solution (CFU / mL) by cyclic voltammetry; 3) VBNC Escherichia coli was imprinted on a glassy carbon electrode layer by applying a cyclic voltage of -0.4 to +0.7V relative to a saturated calomel reference electrode at a scan rate of 50mV / s under gentle stirring; 4) The prepared modified glassy carbon electrode layer was treated with SDS / HAC (5% w / v) for 4.0 hours under static conditions to remove the bacterial template, thereby forming a bacterial imprint material.

[0030] The reaction mechanism of bacterial blotting is as follows: When an electric current and pressure are applied to the top and bottom of a carbon electrode, VBNC (virtually occurring non-virtualized coliform bacteria) from a bacterial solution dropped onto the electrode leave an imprint on the glassy carbon electrode under the influence of the electric field. After blotting, the surface of the glassy carbon electrode will have many structures of different VBNC E. coli morphologies. When a sample is introduced, those with similar structures will be captured, thus achieving the enrichment of VBNC E. coli in the sample. It should be understood that the content of E. coli in drinking water is relatively low. Therefore, when using a higher concentration of bacterial stock solution for blotting, the structural types on the electrode surface are far more numerous than the structural types of VBNC in the sample, facilitating the capture of VBNC E. coli in subsequent experiments.

[0031] like Figure 4 As shown, the fixture 4 consists of upper and lower layer structures 41 and 42. The three-layer chip—microfluidic channel layer 1, glass carbon electrode layer 2, and glass substrate layer 3—is clamped between the upper and lower layers in a top-to-bottom order, and then secured with screws around the perimeter. The lower layer of the fixture has an opening on the left side for easy placement and removal of the chip.

[0032] The microfluidic chip provided in the above preferred embodiment is used as follows:

[0033] First, the drinking water sample is injected into the chip through the sample inlet 11 using a syringe. The drinking water sample enters the E. coli capture chamber 15 along the sample flow channel and flows over the upper surface of the glassy carbon electrode layer 2. At this time, the VBNC E. coli contained in the drinking water sample is captured by the glassy carbon electrode layer 2, and the waste liquid flows out from the waste liquid outlet 14.

[0034] Subsequently, the biochemical reaction solution composed of PELB composite lysis reagent and enzyme-catalyzed reaction substrate 4-CMUG is delivered by a constant pressure pump from the biochemical reaction solution inlet 12 into the E. coli capture chamber 15. The VBNC E. coli captured on the glassy carbon electrode layer 2 is lysed under the action of PELB composite lysis reagent and enters the biochemical reaction solution. Then, it flows into the flow channel connecting the E. coli capture chamber 15 and the cross 17. At the cross 17, the biochemical reaction solution is sheared by the oil phase to generate W / O type (water-in-oil) single internal droplets. Then, the droplets flow along the droplet flow channel into the droplet observation chamber 16 and spread out in a single layer therein.

[0035] After incubation at 42℃ for 2 hours, the β-glucuronidase (GUS enzyme) secreted by Escherichia coli catalyzes the hydrolysis of the substrate 4-MUG (4-methylumbelliferyl-β-D-galactoside) into the specific fluorescent substance 4-MU (4-methylumbelliferyl ketone). Finally, the substance is detected under a fluorescence microscope, which can realize the detection of the number of VBNC Escherichia coli in drinking water samples.

[0036] 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. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A microfluidic chip integrating bacterial blotting and droplet detection, characterized in that, Including settings from top to bottom: A microfluidic channel layer includes: a sample inlet, a biochemical reaction solution inlet, an oil phase inlet, a waste liquid inlet, an E. coli capture chamber, and a droplet observation chamber. The sample inlet is connected to the E. coli capture chamber via a sample channel. The biochemical reaction solution inlet is connected to the E. coli capture chamber via a biochemical reaction solution channel. The oil phase inlet intersects with the detection liquid channel at the outlet of the E. coli capture chamber via the oil phase channel and communicates with a droplet channel. The droplet channel is connected to the droplet observation chamber. A glassy carbon electrode layer, the size of which is smaller than that of the microfluidic channel layer, has its surface treated with bacterial imprinting to form a bacterial imprint region corresponding to the E. coli capture cavity, for capturing and enriching VBNC E. coli in the sample; the bacterial imprinting process is as follows: 1) Before use, the surface of the glassy carbon electrode layer is polished to a mirror finish with 0.05~0.3μm alumina slurry, and then ultrasonically cleaned with water and ethanol; 2) In a solution containing 0.05 M pyrrole and 10 5 Electrochemical copolymerization was performed in 2 mL of 0.1 M KCl solution containing CFU / mL VBNC *E. coli* template by cyclic voltammetry; 3) Under gentle stirring, a cyclic voltage of -0.4 to +0.7 V relative to a saturated calomel reference electrode was applied at a scan rate of 50 mV / s to imprint VBNC *E. coli* onto the glassy carbon electrode layer; 4) The modified glassy carbon electrode layer was treated with 5% w / v SDS / HAC for 4.0 hours under static conditions to remove the bacterial template, thereby obtaining a glassy carbon electrode layer with a characteristic bacterial imprinted region on its surface; and A glass substrate layer, the size of which is equal to that of the microfluidic channel layer and aligned with each other, for droplet spreading and observation.

2. The microfluidic chip according to claim 1, characterized in that, The oil phase inlet is connected to two oil phase channels, which converge at a cross-shaped intersection with the detection liquid channel at the outlet of the E. coli capture chamber and are connected to the droplet channel.

3. The microfluidic chip according to claim 2, characterized in that, The two oil phase flow channels extend from the oil phase inlet in opposite directions and converge at the cross-shaped junction to form a rectangular flow channel that surrounds the sample inlet, the biochemical reaction solution inlet, and the E. coli capture chamber.

4. The microfluidic chip according to claim 1, characterized in that, The E. coli capture chamber and the droplet observation chamber are respectively arranged on the left and right sides of the microfluidic channel layer.

5. The microfluidic chip according to claim 1, characterized in that, The waste liquid outlet is located at the end of the droplet observation chamber.

6. The microfluidic chip according to claim 1, characterized in that, The E. coli capture chamber is circular, and the droplet observation chamber is square.

7. The microfluidic chip according to claim 1, characterized in that, The microfluidic channel layer, glass carbon electrode layer, and glass substrate layer are installed and fixed by clamps and screws.

8. The application of a microfluidic chip integrating bacterial blotting and droplet detection as described in any one of claims 1 to 7 in the detection of VBNC (Vitamin B 2, C. coli) in drinking water.

Images