Micro-fluidic chip based on electric cracking and protein purification and application of micro-fluidic chip in biological sample analysis

Abstract

The invention relates to a micro-fluidic chip based on electric cracking and protein purification. A metal electrode layer is combined on a glass substrate; the bacterial lysis and protein purification detection module is connected with the glass substrate; the bacterial lysis and protein purification detection module is internally provided with a lysis cavity, an enzyme substrate cavity and a chromogenic reaction cavity which are communicated through a micro-channel at the bottom, the metal electrode layer comprises a silver paste interdigital electrode, and the silver paste interdigital electrode is aligned with the lysis cavity, is positioned below the lysis cavity and is in an exposed state in the lysis cavity; the silver paste interdigital electrode generates a strong electric field to realize efficient splitting decomposition of bacteria. The invention also relates to application of the micro-fluidic chip in biological sample analysis. According to the present invention, the electric splitting method and the microfluidic technology are integrated, the splitting and the accurate quantitative detection of the bacteria are achieved, and compared with the prior art, the method has the great advantages in the detection sensitivity, the process automation, the integration level and the like, and has the wide application prospect in the molecular diagnosis aspect.

Description

TECHNICAL FIELD

[0001] The present application relates to microfluidics, and more particularly to a microfluidic chip based on electrical lysis and protein purification and its application in biological sample analysis. BACKGROUND

[0002] Rapid detection of foodborne pathogens is crucial for food safety. Escherichia coli O157:H7 is a major foodborne pathogen with extremely strong pathogenicity, which can cause serious illness even at low concentrations. Studies have shown that only 50-100 bacteria (equivalent to one thousandth of the dose of ordinary pathogenic bacteria) can cause infection. This bacterium can produce various pathogenic substances such as Shiga-like toxin, plasmid O157, etc., and infected individuals may exhibit clinical manifestations ranging from no symptoms to fatal complications such as hemolytic-uremic syndrome, so Escherichia coli O157:H7 is a foodborne pathogen that requires special vigilance.

[0003] However, traditional detection methods such as bacterial culture, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) have many limitations. These methods not only have complex operations and long time consumption, but also are heavily dependent on professional laboratory equipment and personnel. This makes it difficult for them to meet the urgent needs of food safety rapid detection, environmental real-time monitoring, and clinical emergency testing scenarios. SUMMARY

[0004] In order to solve the problems of the traditional detection methods existing in the prior art, the present application aims to provide a microfluidic chip based on electrical lysis and protein purification and its application in biological sample analysis.

[0005] The microfluidic chip based on electrical lysis and protein purification according to the present application comprises a glass substrate, a metal electrode layer combined on the glass substrate, and a bacterial lysis and protein purification detection module connected together with the glass substrate; wherein the bacterial lysis and protein purification detection module is provided with a lysis chamber, an enzyme substrate chamber and a color development reaction chamber communicated through a microchannel at the bottom, the metal electrode layer comprises a silver paste interdigital electrode, the silver paste interdigital electrode is located below the lysis chamber in alignment with the lysis chamber and presents a bare state in the lysis chamber, and when the power is turned on, the silver paste interdigital electrode generates a strong electric field to achieve efficient lysis of bacteria.

[0006] In a preferred embodiment, the metal electrode layer comprises a plurality of silver paste interdigital electrodes arranged side by side in sequence on the glass substrate, the bacterial lysis and protein purification detection module comprises a plurality of independent lysis chambers, and the silver paste interdigital electrodes correspond one-to-one to the lysis chambers.

[0007] In the preferred embodiment, the bacterial lysis and protein purification detection module is further provided with a first washing cavity and at least one second washing cavity in sequence communicated through the microfluidic channel at the bottom, the first washing cavity is located between the lysis cavity and the enzyme substrate cavity, and the at least one second washing cavity is located between the enzyme substrate cavity and the color development reaction cavity.

[0008] In the preferred embodiment, the lysis cavity is added with magnetic beads, the surface of the magnetic beads is modified with specific antibodies, and the specific antibodies specifically capture target antigens.

[0009] In the preferred embodiment, the target antigen is beta-glucuronidase (GUS) of Escherichia coli, and the specific antibody is a GUS antibody.

[0010] In the preferred embodiment, each interdigital width of the silver paste interdigital electrode is 80-100 μm, and the interdigital spacing between adjacent interdigital electrodes is 80-100 μm.

[0011] In the preferred embodiment, the bacterial lysis and protein purification detection module is a PDMS layer.

[0012] In the preferred embodiment, the metal electrode layer is printed on the glass substrate by a screen printing technique, and the bacterial lysis and protein purification detection module and the glass substrate are connected together by an ionic bonding method.

[0013] According to the application of the microfluidic chip based on electrical lysis and protein purification in biological sample analysis, the application comprises injecting lysis liquid, enzyme substrate and reaction color development liquid into the lysis cavity, the enzyme substrate cavity and the color development reaction cavity respectively, and each cavity is separated by an oil phase to prevent reaction reagents from interfering with each other; the sample to be detected is injected into the lysis cavity, the silver paste interdigital electrode is connected to a power supply, lysis is realized by adjusting the voltage, the lysis product enzyme is dragged into the enzyme substrate cavity for reaction, and the reaction product is dragged into the color development reaction cavity to complete the color development reaction; the light signal generated by the color development reaction is proportional to the concentration of the lysis product enzyme, so that detection is realized.

[0014] In the preferred embodiment, the power supply connected to the silver paste interdigital electrode is an alternating current with a voltage of 10-30 V and a frequency of 50-200 kHz, and a pulse waveform is adopted.

[0015] The application integrates the electrical lysis method and the microfluidic technology together, realizes lysis of bacteria and accurate quantitative detection, has great advantages in detection sensitivity, process automation and integration compared with the prior art, and has a wide application prospect in molecular diagnosis. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1is the overall structure schematic diagram of the microfluidic chip based on electrical lysis and protein purification according to the application.

[0017] Figure 2 is the microfluidic chip of Figure 1 .

[0018] Figure 3 is the structure schematic diagram of the silver paste interdigital electrode module of Figure 2 . DETAILED DESCRIPTION

[0019] The application will be further described below in connection with specific embodiments. It should be understood that the following embodiments are only used to illustrate but not limit the scope of the application. Unless otherwise specified, the technical means used in the embodiments are the conventional operations in the art, or according to the experimental methods suggested by the instrument and equipment manufacturers. The reagents and materials used in the embodiments are commercially available unless otherwise specified.

[0020] As shown in Figures 1-2 , the microfluidic chip based on electrical lysis and protein purification according to the application includes a glass substrate 101, a metal electrode layer 102 and a bacterial lysis and protein purification detection module 103. Among them, the metal electrode layer 102 is printed on the glass substrate 101 by screen printing technology, and the bacterial lysis and protein purification detection module 103 and the glass substrate 101 are connected together by ionic bonding. Of course, the application is not limited to the above specific manufacturing method, other suitable manufacturing methods, such as photolithography technology for electrode patterning, or chemical bonding method for interlayer connection, are also within the protection scope of the application.

[0021] In the preferred embodiment, the bacterial lysis and protein purification detection module 103 is a PDMS layer. The bacterial lysis and protein purification detection module 103 is provided with a lysis chamber 104, a first washing chamber 105, an enzyme substrate chamber 106, a second washing chamber 107, a third washing chamber 108 and a color development reaction chamber 109 connected in sequence from left to right. The chambers are connected by microfluidic channels at the bottom. By filling the oil phase into these microfluidic channels, the reaction reagents in each chamber can be effectively prevented from interfering with each other. Of course, the application is not limited to the above specific number and arrangement order of the chambers. For example, the number of the second washing chamber and the third washing chamber can be adjusted according to actual needs, and even one or more of the washing chambers can be omitted to adapt to different detection requirements and application scenarios.

[0022] The microfluidic chip includes a plurality of lysis chambers 104, and the example in the figure is five, but this number is only an example and not a limitation. Each lysis chamber 104 is respectively connected with a first washing chamber 105, an enzyme substrate chamber 106, a second washing chamber 107, a third washing chamber 108 and a reaction color development chamber 109, so as to realize the detection of multiple samples at the same time.

[0023] In a preferred embodiment, magnetic beads are added to each lysis chamber 104, their surfaces modified with specific antibodies. These antibodies are capable of specifically capturing the target antigen. Through manipulation of an external magnetic field, the magnetic beads are sequentially drawn into the first wash chamber 105, the enzyme substrate chamber 106, the second wash chamber 107, the third wash chamber 108, and the reaction and color development chamber 109. It should be noted that the movement of the magnetic beads is achieved through the external magnetic field, which does not disrupt the isolation layer formed by the oil phase, thereby ensuring that the liquid reagents in each chamber remain independent and do not interfere with each other. In a preferred embodiment, the target antigen is Escherichia coli β-glucuronidase (GUS), and the specific antibody is an antibody against GUS.

[0024] In a preferred embodiment, the metal electrode layer 102 includes a plurality of silver paste interdigitated electrodes, which are arranged side by side in sequence on the glass substrate 101. Five are shown in the embodiment in the figure, but this number is only an example and not a limitation. Each silver paste interdigitated electrode is aligned with the corresponding lysis chamber 104, is located below each lysis chamber 104 and is exposed in the lysis chamber 104. When the power is turned on, the silver paste interdigitated electrodes generate a strong electric field, which in turn generates a transmembrane potential, thereby achieving efficient lysis of bacteria. This design allows for the simultaneous detection of multiple samples, thereby improving detection efficiency and throughput.

[0025] like Figure 3 As shown, the width 201 of each interdigit of the silver paste interdigital electrode is 80-100 μm, and the interdigital spacing 202 between adjacent interdigits is 80-100 μm. In a most preferred embodiment, the interdigital width 201 is 100 μm, and the interdigital spacing 202 is 100 μm.

[0026] Particularly, the application integrates the electric lysis technology and the magnetic control technology in the microfluidic chip. Specifically, on the basis of the original chip, the interdigital electrode is further printed on the glass substrate 101, the interdigital electrode uses silver paste material with excellent conductivity in material, which avoids the generation of heavy metal ions in the electrolysis process, and solves the problem that the heavy metal ions generated by the copper alloy electrode in the electric lysis process affect the protein purification and further affect the detection result in the prior art. In fact, silver paste material is rarely used in interdigital electrodes, and is more commonly used in the photovoltaic industry, and the conductivity of the silver paste electrode is better than that of the copper alloy. The inventor found through many experiments that the copper alloy is oxidized to form copper oxide and copper ions under the condition of electrification, which affects the experimental results. On the contrary, silver in the silver paste electrode is more stable. The use of the optimized silver paste interdigital electrode of the application has better lysis effect, the two ends of the electrode are connected with the power supply oscilloscope to generate a strong electric field, a transmembrane potential is generated to realize the lysis of bacteria, the magnetic beads modified with antibodies are added to combine with the antigen protein, and the antigen protein can be washed and purified by using the magnet for transportation. The application can shorten the time of bacterial lysis, simplify the premixing step, and realize the integration of bacterial lysis, protein extraction and purification, and magnetic control technology with high sensitivity and simple operation.

[0027] The following will be described in detail by taking the detection of E. coli O157:H7 as an example.

[0028] The application of the microfluidic chip according to the application in biological sample analysis includes a preparation stage. Specifically, the lysis solution, the first washing solution, the enzyme substrate, the second washing solution, the third washing solution and the reaction color developing solution are respectively injected into the lysis cavity 104, the first washing cavity 105, the enzyme substrate cavity 106, the second washing cavity 107, the third washing cavity 108 and the reaction color developing cavity 109, and the cavities are separated by oil to prevent the reaction reagents from interfering with each other.

[0029] The application of the microfluidic chip according to the application in biological sample analysis also includes the lysis of E. coli and the purification of protein antigens. Specifically, the sample containing E. coli to be detected is injected into the lysis cavity 104, the silver paste interdigital electrode is connected to the power supply, the lysis of E. coli is realized by adjusting the voltage, the intracellular GUS released by the broken bacteria is captured by the GUS antibody modified on the magnetic beads, and the magnetic beads are dragged into the first washing cavity 105, the enzyme substrate cavity 106, the second washing cavity 107 and the third washing cavity 108 in sequence by the hand-held magnet for sufficient washing, and the washed protein molecules are guided into the reaction color developing cavity 109 by the magnet. It should be understood that the purpose of multiple washing is to remove impurities and other non-specifically combined proteins in the lysis solution, to improve the purity of GUS, and to ensure that the GUS entering the reaction color developing cavity finally has high purity.

[0030] In a preferred embodiment, the power source for the silver paste interdigital electrode is an alternating current with a voltage between 10V and 30V to ensure efficient lysis. In a preferred embodiment, the voltage is 20V. In a preferred embodiment, the frequency of the alternating current is between 50,000Hz and 200,000Hz to ensure efficiency and consistency of the lysis process. In a preferred embodiment, the frequency is 1,000,000Hz. In a preferred embodiment, the alternating current uses a pulsed waveform to optimize the distribution of the electric field and reduce the thermal effects of the electrode.

[0031] The application of the microfluidic chip according to the present application in biological sample analysis also includes color reaction detection. Specifically, under the action of the heating module, the antigen protein molecules complete the color reaction in the reaction color development cavity 109, and different wavelengths emitted by the fluorescent group are detected by a fluorescence microscope, so that precise detection of bacteria can be achieved. It should be understood that the intensity of the fluorescent signal is proportional to the concentration of GUS, thereby realizing quantitative detection of E. coli.

[0032] The microfluidic chip according to the present application can directly detect E. coli O157:H7 in a complex sample without bacterial proliferation culture and expensive equipment, thereby providing an efficient solution for foodborne pathogen detection. Specifically, the present application uses screen printing technology to print a layer of silver paste interdigital electrode on a glass substrate based on the original chip, and the upper PDMS is ionically bonded and packaged with the glass substrate. The silver paste interdigital electrode is aligned below the lysis cavity, and by connecting with a power source, the interdigital silver paste electrode generates a strong electric field, thereby generating a transmembrane potential, and irreversible electroporation can be formed on the surface of the ruptured bacterial membrane within a few seconds, thereby realizing lysis of E. coli. Moreover, the present application realizes the integration of antigen protein extraction, purification and color development reaction on the microfluidic chip by adding modified antibody magnetic beads to combine with the antigen protein and using a magnet for transportation. The present application can realize rapid detection of E. coli on the same chip. In addition, the electric lysis microfluidic chip according to the present application has potential applications in the field of molecular diagnosis. In summary, the present application provides a microfluidic chip integrating interdigital electrode lysis, bacterial protein extraction, purification and detection, and applications thereof, which has broad application prospects in the field of molecular diagnosis.

[0033] The above description is only a preferred embodiment of the present application, and is not intended to limit the scope of the present application. The above-described embodiments of the present application can be variously changed. Any simple, equivalent changes and modifications made in accordance with the content of the claims and the specification of the present application fall within the scope of the claims of the present application. The present application does not describe all conventional technical contents.

Claims

1. A microfluidic chip based on electrolysis and protein purification, characterized in that: The microfluidic chip includes: Glass substrate (101); a metal electrode layer (102) bonded to the glass substrate (101); and a bacterial lysis and protein purification detection module (103), which is connected to the glass substrate (101); The bacterial lysis and protein purification detection module (103) is provided with a lysis chamber (104), an enzyme substrate chamber (106) and a color reaction chamber (109) connected through a microchannel at the bottom. The metal electrode layer (102) includes a silver paste interdigitated electrode. The silver paste interdigitated electrode is aligned with the lysis chamber (104) and is located below the lysis chamber (104) and is exposed in the lysis chamber (104). When the power is turned on, the silver paste interdigitated electrode generates a strong electric field to achieve efficient bacterial lysis.

2. The microfluidic chip according to claim 1, characterized in that The metal electrode layer (102) includes a plurality of silver paste interdigitated electrodes arranged in sequence side by side on the glass substrate (101); the bacterial lysis and protein purification detection module (103) includes a plurality of independent lysis chambers (104); and the silver paste interdigitated electrodes correspond one to one to the lysis chambers (104).

3. The microfluidic chip according to claim 1, characterized in that The bacterial lysis and protein purification detection module (103) is further provided with a first washing chamber (105) and at least one second washing chamber (107) which are connected in sequence through a microchannel at the bottom. The first washing chamber (105) is located between the lysis chamber (104) and the enzyme substrate chamber (106), and the at least one second washing chamber (107) is located between the enzyme substrate chamber (106) and the color development reaction chamber (109).

4. The microfluidic chip according to claim 1, characterized in that Magnetic beads are added to the lysis chamber (104), and the surface of the magnetic beads is modified with specific antibodies, and the specific antibodies specifically capture the target antigen.

5. The microfluidic chip according to claim 4, characterized in that: The target antigen is β-glucuronidase (GUS) of Escherichia coli, and the specific antibody is a GUS antibody.

6. The microfluidic chip according to claim 1, characterized in that The width of each interdigital finger of the silver paste interdigital electrode is 80-100 μm, and the interdigital spacing between adjacent interdigital fingers is 80-100 μm.

7. The microfluidic chip according to claim 1, characterized in that The bacterial lysis and protein purification detection module (103) is a PDMS layer.

8. The microfluidic chip according to claim 1, characterized in that The metal electrode layer (102) is printed on the glass substrate (101) by screen printing technology, and the bacterial lysis and protein purification detection module (103) and the glass substrate (101) are connected together by ion bonding.

9. Use of the microfluidic chip according to any one of claims 1 to 8 in biological sample analysis, characterized in that: The application includes injecting a lysis solution, an enzyme substrate and a reaction color development solution into a lysis chamber (104), an enzyme substrate chamber (106) and a color development reaction chamber (109) respectively, wherein each chamber is separated by an oil phase so that the reaction reagents do not interfere with each other; injecting a sample to be detected into the lysis chamber (104), connecting the silver paste interdigitated electrode to a power source, achieving lysis by adjusting the voltage, and dragging the lysis product enzyme into the enzyme substrate chamber (106) for reaction, and dragging the reaction product into the color development reaction chamber (109) to complete the color development reaction; the light signal generated by the color development reaction is proportional to the concentration of the lysis product enzyme, thereby achieving detection.

10. The use according to claim 9, characterized in that The power source connected to the silver paste interdigital electrodes is alternating current, with a voltage between 10V and 30V, a frequency between 50,000 Hz and 200,000 Hz, and a pulse waveform.

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