Microfluidic chip and method for quantitatively detecting coliform bacteria

Abstract

The present application relates to a kind of microfluidic chip, second substrate is sealed and stacked above first substrate, recessed sample channel is formed on first substrate, sample channel includes connected capture zone, reaction zone and detection zone, first ceramic film is arranged on capture zone, second ceramic film covers first ceramic film and makes that immune magnetic bead is clamped and fixed between first ceramic film and second ceramic film, through sample port is formed on second substrate, sample port is arranged in alignment with second ceramic film to facilitate by sample port Sample or eluent is introduced into chip, electrode piece is arranged in detection zone to realize quantitative detection by the voltage change of electrode piece.The present application also relates to the method for quantitative detection of coliform group of the above-mentioned microfluidic chip.According to the microfluidic chip and the method for quantitative detection of coliform group of the present application, it has the advantages of simple operation, high sensitivity, high integration and fast detection.

Description

Technical Field

[0001] This invention relates to electrochemical detection, and more specifically to a microfluidic chip and a method for quantitative detection of coliform bacteria therein. Background Technology

[0002] A healthy aquatic environment is fundamental to people's daily lives, and microbiological indicators are crucial components of water quality testing. The plate culture method for detecting microbial numbers takes 48 hours, a lengthy process. Even for pathogenic Escherichia coli, which has become the leading cause of clinical infections in recent years, the gas production method for detecting specific microorganisms requires at least 24 hours to produce results. This time-consuming process also poses potential infection risks to testing personnel.

[0003] The quantity of *E. coli* and coliform bacteria are both important indicators for the detection of microorganisms in water. Currently, methods for simultaneously detecting *E. coli* and coliform bacteria include chromogenic plate assays and multiple tube fermentation (MPN) assays. In China, plate culture is currently considered the "gold standard" for microbial detection, but it is time-consuming and somewhat complex. Internationally, microbial detection mainly relies on immunoassays, molecular biology methods, and the increasingly developed mass spectrometry. Immunoassays utilize the specific recognition of antibodies on immunogenic regions of microbial surfaces for detection, including enzyme-linked immunosorbent assays (ELISA) and immunochromatography. However, these methods suffer from drawbacks such as large batch-to-batch variability in antibody preparation, poor stability, and low precision. Molecular biology methods primarily use polymerase chain reactions (PCR) for species identification. While highly specific, PCR reactions are susceptible to aerosol contamination and require specific environmental conditions. Mass spectrometry mainly employs matrix-assisted time-of-flight mass spectrometry (MATS), identifying species based on characteristic peaks in the protein expression profiles of microorganisms. However, the high cost of mass spectrometers limits its widespread application, and this method cannot meet the quantitative detection needs of microorganisms. Summary of the Invention

[0004] To address the problems of complex operation, susceptibility to contamination, high cost, and inability to quantitatively detect in the prior art, this invention provides a microfluidic chip and a method for quantitatively detecting coliform bacteria.

[0005] According to the microfluidic chip of the present invention, it includes a first substrate, a second substrate, a first ceramic membrane, a second ceramic membrane, immunomagnetic beads, and electrode sheets. The second substrate is sealed and stacked on top of the first substrate. A recessed sample channel is formed on the first substrate. The sample channel includes a connected capture area, a reaction area, and a detection area. The first ceramic membrane is disposed on the capture area. The second ceramic membrane covers the first ceramic membrane and clamps and fixes the immunomagnetic beads between the first and second ceramic membranes. A through sample port is formed on the second substrate. The sample port is aligned with the second ceramic membrane to facilitate the introduction of sample or eluent into the chip through the sample port. The electrode sheets are disposed in the detection area to achieve quantitative detection through voltage changes of the electrode sheets.

[0006] In a preferred embodiment, the surface of the immunomagnetic beads is coated with specific antibodies for adsorbing coliform bacteria, which are then eluted from the immunomagnetic beads using an elution buffer.

[0007] In a preferred embodiment, the electrode sheet is a gold electrode sheet.

[0008] In a preferred embodiment, the immunomagnetic beads are spherical droplets contained in a cavity composed of a first ceramic membrane and a second ceramic membrane.

[0009] In a preferred embodiment, the first substrate and the second substrate are PDMS sheets, respectively.

[0010] In a preferred embodiment, the first and second ceramic membranes used to fix the immunomagnetic beads are identical discs, which are arranged corresponding to the capture area.

[0011] In a preferred embodiment, the electrode sheet is a rectangular sheet, which is arranged corresponding to the detection area.

[0012] The method for quantitative detection of coliform bacteria using the microfluidic chip of the present invention includes: a sample entering a capture zone through a sample port; immunomagnetic beads between a first ceramic membrane and a second ceramic membrane in the capture zone adsorbing coliform bacteria from the sample; an eluent entering the capture zone through the sample port to elute the coliform bacteria from the immunomagnetic beads; the coliform bacteria entering a reaction zone; and the reaction product entering a detection zone to react with an electrode sheet and cause a voltage change.

[0013] In a preferred embodiment, the β-glucosidase carried by the coliform bacteria catalyzes the reaction of phenylcyanide to produce cyanide. The cyanide enters the detection area and reacts with the electrode plate, causing a voltage change. The rate of voltage change over time is directly proportional to the concentration of β-glucosidase.

[0014] In a preferred embodiment, the coliform bacteria are Escherichia coli.

[0015] The microfluidic chip and the method for quantitative detection of coliform bacteria according to the present invention have the advantages of simple operation, high sensitivity, high integration and rapid detection. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the structure of a microfluidic chip according to a preferred embodiment of the present invention.

[0017] Figure 2 Show Figure 1 The linear relationship between the current and the logarithmic concentration of coliform bacteria when the microfluidic chip is used for detection. Detailed Implementation

[0018] The preferred embodiments of the present invention are given below with reference to the accompanying drawings and described in detail.

[0019] like Figure 1 As shown, a microfluidic chip according to a preferred embodiment of the present invention includes a first substrate 1, a second substrate 2, a first ceramic membrane 3, a second ceramic membrane 4, immunomagnetic beads 5, and an electrode sheet 6. The second substrate 2 is sealed and stacked on top of the first substrate 1. A recessed sample channel 11 is formed on the first substrate 1. The sample channel 11 includes a connected capture area 111, a reaction area 112, and a detection area 113. The first ceramic membrane 3 is disposed on the capture area 111. The second ceramic membrane 4 covers the first ceramic membrane 3, and the immunomagnetic beads 5 are clamped and fixed between the first ceramic membrane 3 and the second ceramic membrane 4. A through sample port 21 is formed on the second substrate 2. The sample port 21 is aligned with the second ceramic membrane 4 to facilitate the introduction of a sample or eluent into the chip through the sample port 21. The electrode sheet 6 is disposed in the detection area 13 to achieve quantitative detection through voltage changes of the electrode sheet 6.

[0020] In this embodiment, the first substrate 1 and the second substrate 2 are polydimethylsiloxane (PDMS) sheets, and their edges are bonded and sealed by oxygen plasma treatment.

[0021] In this embodiment, the capture area 111, reaction area 112 and detection area 113 of the sample channel 11 of the first substrate 1 are artificially divided areas, and are not strictly distinguished in the actual product.

[0022] In this embodiment, the first ceramic membrane 3 and the second ceramic membrane 4 are identical discs used to immobilize the immunomagnetic beads 5, and their shape and size correspond to the capture area 111 of the sample channel 11 of the first substrate 1. It should be understood that the specific shape and size of the first ceramic membrane 3 and the second ceramic membrane 4 are merely examples and not limitations.

[0023] In this embodiment, the surface of the immunomagnetic beads 5 is coated with specific antibodies for adsorbing coliform bacteria. The adsorbed coliform bacteria can be eluted from the immunomagnetic beads 5 using an elution buffer. In this embodiment, the coliform bacteria are *Escherichia coli* (E. coli). In this embodiment, the immunomagnetic beads 5 are spherical droplets contained within a chamber formed by a first ceramic membrane 3 and a second ceramic membrane 4. It should be understood that the surfaces of the first ceramic membrane 3 and the second ceramic membrane 4 themselves have micron-sized pore structures, through which the immunomagnetic beads 5 are fixed between the first ceramic membrane 3 and the second ceramic membrane 4. It should be understood that the shape, size, and number of the immunomagnetic beads 5 are merely examples and not limitations.

[0024] In this embodiment, electrode 6 is a gold electrode. The β-glucosidase carried by coliform bacteria catalyzes the reaction of phenylcyanide to produce cyanide. The cyanide then enters the detection area 113 and reacts with the gold electrode. In this embodiment, electrode 6 is a rectangular sheet, and its shape and size correspond to the detection area 113 of the sample channel 11 of the first substrate 1. It should be understood that the specific shape and size of electrode 6 are merely examples and not limitations.

[0025] A method for quantitative detection of coliform bacteria using a microfluidic chip according to a preferred embodiment of the present invention includes: a sample entering a capture zone 111 through a sample port 21; immunomagnetic beads 5 between a first ceramic membrane 3 and a second ceramic membrane 4 in the capture zone 111 adsorbing the coliform bacteria in the sample; an eluent entering the capture zone 111 through the sample port 21 to elute the coliform bacteria from the immunomagnetic beads 5; the coliform bacteria entering a reaction zone 112; and the reaction product entering a detection zone 113 reacting with an electrode plate 6 and causing a voltage change. The rate of change of voltage in the reaction system over time is directly proportional to the concentration of β-glucosidase, such as... Figure 2 As shown, by monitoring the change of voltage over time, the activity of β-glucosidase in the sample can be quantitatively analyzed, the number of coliform bacteria can be determined, and thus the concentration of coliform bacteria in the sample can be quantified.

[0026] Thus, according to the microfluidic chip of the present invention, coliform bacteria are captured by immunomagnetic beads 5 and detected by electrode sheet 6, achieving capture and detection on a single chip. This enables rapid, sensitive, and quantitative detection of coliform bacteria in water, and has broad application prospects. It should be understood that the capture step can increase the concentration of coliform bacteria in subsequent detection, thereby raising the detection limit. Without the capture step, the low concentration of coliform bacteria in the sample might prevent detection. In other words, the microfluidic chip of the present invention innovatively embeds electrode sheet 6 into the microfluidic chip for detection, while simultaneously introducing the capture step of immunomagnetic beads 5 for enrichment, greatly improving detection sensitivity and making online detection of coliform bacteria possible.

[0027] 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 of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A microfluidic chip, characterized in that, The microfluidic chip includes a first substrate, a second substrate, a first ceramic membrane, a second ceramic membrane, immunomagnetic beads, and electrode plates. The surfaces of the first and second ceramic membranes themselves have micron-sized pore structures. The second substrate is sealed and stacked on top of the first substrate. A recessed sample channel is formed on the first substrate, including a connected capture area, a reaction area, and a detection area. The first ceramic membrane is disposed on the capture area. The second ceramic membrane covers the first ceramic membrane, such that immunomagnetic beads, as spherical droplets, are disposed between the first and second ceramic membranes within a cavity formed by the first and second ceramic membranes. The surface of the immunomagnetic beads is coated with specific antibodies for adsorbing coliform bacteria. A through-type sample channel is formed on the second substrate. The sample inlet and the second ceramic membrane are aligned to allow the sample to be introduced into the capture area of ​​the microfluidic chip first. Specific antibodies on the immunomagnetic beads adsorb and enrich the coliform bacteria in the sample. Then, the eluent is introduced into the capture area of ​​the microfluidic chip through the sample inlet. The coliform bacteria adsorbed on the immunomagnetic beads are eluted from the immunomagnetic beads by the eluent. The eluted coliform bacteria enter the reaction area, where the β-glucosidase carried by the coliform bacteria catalyzes the reaction of phenylcyanide to produce cyanide. An electrode is placed in the detection area to achieve quantitative detection through voltage changes on the electrode. The cyanide enters the detection area and reacts with the electrode, causing a voltage change. The rate of voltage change over time is proportional to the concentration of β-glucosidase.

2. The microfluidic chip according to claim 1, characterized in that, The electrode sheet is a gold electrode sheet.

3. The microfluidic chip according to claim 1, characterized in that, Immunomagnetic beads are spherical droplets contained in a chamber composed of a first ceramic membrane and a second ceramic membrane.

4. The microfluidic chip according to claim 1, characterized in that, The first and second substrates are PDMS substrates, respectively.

5. The microfluidic chip according to claim 1, characterized in that, The first and second ceramic membranes used to immobilize the immunomagnetic beads are identical discs, which are set corresponding to the capture area.

6. The microfluidic chip according to claim 1, characterized in that, The electrode sheet is rectangular and is positioned corresponding to the detection area.

7. A method for quantitative detection of coliform bacteria using a microfluidic chip according to any one of claims 1-6, characterized in that, The method includes: a sample entering a capture zone through a sample port; immunomagnetic beads between a first ceramic membrane and a second ceramic membrane in the capture zone adsorbing coliform bacteria from the sample; an eluent entering the capture zone through the sample port to elute the coliform bacteria from the immunomagnetic beads; the coliform bacteria entering a reaction zone; and the reaction product entering a detection zone to react with an electrode and cause a voltage change.

8. The method according to claim 7, characterized in that, The β-glucosidase carried by coliform bacteria catalyzes the reaction of phenylcyanide to produce cyanide. The cyanide enters the detection area and reacts with the electrode plate, causing a voltage change. The rate of voltage change over time is directly proportional to the concentration of β-glucosidase.

9. The method according to claim 7, characterized in that, The coliform bacteria are Escherichia coli.

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