Multiplex PCR (Polymerase Chain Reaction) micro-fluidic chip integrating electric cracking and nucleic acid purification detection and application of multiplex PCR micro-fluidic chip

Abstract

The invention provides a multiplex PCR (Polymerase Chain Reaction) micro-fluidic chip integrating electric cracking and nucleic acid purification detection and application thereof. The multiplex PCR micro-fluidic chip comprises a PDMS (Polydimethylsiloxane) layer and a glass substrate which are connected together in an ionic bonding manner, wherein the PDMS layer is internally provided with a cracking cavity, a first washing cavity, a second washing cavity and an amplification cavity which are sequentially communicated through a micro-channel at the bottom; a silver paste interdigital electrode is printed on the glass substrate through a screen printing technology, the silver paste interdigital electrode is aligned with the cracking cavity below the cracking cavity, and after a power supply is switched on, a strong electric field can be generated through current to realize electric cracking of bacteria. The chip is simple in structure, suitable for batch process manufacturing, low in cost and suitable for popularization and application. According to the present invention, the electric splitting method and the microfluidic technology are integrated, such that the splitting and the accurate quantitative detection of the bacteria are achieved, the great advantages in the detection sensitivity, the process automation, the integration level and the like are provided, and the wide application prospect in the molecular diagnosis aspect is provided.

Description

Technical Field

[0001] The present invention relates to the field of microfluidics, and more specifically to a multiplex PCR microfluidics chip integrating electrolysis and nucleic acid purification detection and applications thereof. Background Art

[0002] Nucleic acid detection technology is the most widely used molecular diagnostic technology in recent years. In the diagnosis of infectious diseases, this technology can quickly and efficiently amplify the characteristic nucleotide sequences of pathogens, and has been widely used in clinical diagnosis, disease screening and other fields. Traditional nucleic acid detection steps usually include: nucleic acid extraction and purification, nucleic acid amplification and detection. Among them, nucleic acid extraction and purification are the key in molecular biology experiments. It is related to whether high-quality nucleic acids can be extracted, which can directly affect the success or failure of subsequent experiments. For nucleic acid extraction and purification, the reagents provided by the current kits often have cumbersome steps, take a long time, and are prone to cross-contamination due to the operation method or experimental environment, so there are high requirements for the experimental ability and detection environment of medical personnel.

[0003] Nucleic acid extraction is a basic step in pathogen research, and bacterial lysis is the first step. Bacteria have thick cell walls and polysaccharide capsules on their surfaces. Ordinary chemical reagents are not efficient in lysing bacteria, and other physical methods are needed to assist in lysis, such as heating, sound waves, grinding, electric current, etc. Bacterial lysis can be divided into mechanical, physical, chemical or biological methods. The mechanical method is to physically decompose the cell membrane through mechanical shear force. Common mechanical equipment includes high-pressure homogenizers and bead mills. The physical method is a non-contact method that uses external forces to destroy the cell membrane. These forces include heat energy, high pressure, electrical energy and sound energy. The chemical method is to destroy the cell membrane by changing the pH of the lysis buffer or adding surfactants to the lysis buffer to dissolve membrane proteins and rupture the membrane to release its contents. The biological method is to destroy the bacterial / cell structure through enzymatic hydrolysis. Commonly used enzymatic enzymes include lysozyme, cellulase, etc. However, traditional lysis methods have limitations such as complex equipment, chemical reagent pollution, high cost, and large sample consumption.

[0004] Nucleic acid amplification and detection is to efficiently and quickly amplify the characteristic nucleotide sequences of the extracted and purified pathogens and perform fluorescent or visual detection to obtain immediate diagnostic results. The duration, sensitivity and specificity of the nucleic acid amplification method will also directly affect the interpretation of subsequent results. At present, the commonly used method for nucleic acid amplification detection is real-time fluorescent quantitative PCR, which can perform qualitative and quantitative analysis of nucleic acids in samples and is the current gold standard for nucleic acid amplification detection. However, its disadvantage is that the quantitative analysis of this method must rely on the existence of a standard curve, and it is impossible to achieve absolute quantification of nucleic acid molecules. In addition, when the nucleic acid content is low, it is impossible to detect low-abundance target genes, resulting in false negative results.

[0005] However, there are still some problems to be solved in the application of integrated detection operating devices in the field of molecular diagnosis. Either the entire process of nucleic acid extraction, washing, purification, and detection cannot be realized, or the effect of electrolysis is poor. Summary of the invention

[0006] The purpose of the present invention is to provide a multiplex PCR microfluidic chip integrating electrolysis and nucleic acid purification detection and its application, so as to solve the problems of low bacterial lysis efficiency and long lysis time in the prior art, as well as the problems of difficult integrated detection and complicated operation steps in nucleic acid amplification and detection technology.

[0007] In order to solve the above problems, the present invention adopts the following technical solutions:

[0008] According to a first aspect of the present invention, there is provided a multiplex PCR microfluidic chip integrating electrolysis and nucleic acid purification detection, comprising a PDMS layer and a glass substrate connected together by ionic bonding, wherein: the PDMS layer is provided with a lysis chamber, a first washing chamber, a second washing chamber and an amplification chamber which are sequentially connected through a microchannel at the bottom; silver paste interdigitated electrodes are printed on the glass substrate by screen printing technology, the silver paste interdigitated electrodes are aligned with the lysis chamber below the lysis chamber, and can generate a strong electric field by current when the power is turned on to achieve electrolysis of bacteria.

[0009] Preferably, the multiplex PCR microfluidic chip comprises a plurality of lysis chambers, each of which is connected to a first washing chamber, a first and a second washing chamber, and an amplification chamber, respectively, so as to realize the detection of multiple samples simultaneously.

[0010] Preferably, the multiplex PCR microfluidic chip comprises a plurality of silver paste interdigital electrodes, each of which is aligned with a lysis chamber.

[0011] Preferably, the plurality of silver paste interdigital electrodes are arranged side by side in sequence on the glass substrate.

[0012] Preferably, the width of each interdigital finger of the silver paste interdigital electrode is 100 μm, and the gap distance between adjacent interdigital fingers is 100 μm.

[0013] According to a second aspect of the present invention, there is provided a use of the multiplex PCR microfluidic chip as described above in detecting pathogens.

[0014] The application comprises the following steps: S1, preparation stage: injecting nucleic acid lysis solution, first washing solution, second washing solution and reaction solution into the lysis chamber, first washing chamber, second washing chamber and amplification chamber respectively, and each chamber is separated by oil phase so that liquid reagents do not interfere with each other; S2, extraction and purification of nucleic acid: injecting a sample containing bacteria into the lysis chamber, connecting the silver paste cross-finger electrode to the power supply, achieving bacterial lysis by adjusting the voltage, and nucleic acid molecules released by bacterial rupture are adsorbed by magnetic beads, and the magnetic beads are dragged into the first washing chamber and the second washing chamber in turn by a handheld magnet to complete washing, and the washed nucleic acid molecules are manipulated by the magnet to enter the amplification chamber; S3, PCR amplification detection: under the action of the heating module, the nucleic acid molecules complete the PCR reaction in the amplification chamber, and the different wavelengths emitted by the fluorescent group are detected by a fluorescence microscope to achieve accurate detection of bacteria.

[0015] Preferably, the silver paste interdigital electrode is connected to a power supply, the voltage of the alternating current is 20V, the frequency is 100,000 Hz, and the waveform is a pulse.

[0016] The inventor further consulted the literature on the basis of existing research, summarized the deficiencies in past experiments, and creatively integrated the electrolysis technology and magnetic control technology into the microfluidic chip. On the basis of the original chip, the present invention further prints the interdigital electrodes on the glass substrate, uses the silver paste material with excellent conductivity on the material, 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 electrolysis process in the prior art affect the purification of nucleic acids and thus affect the detection results. According to literature research, silver paste materials are rarely used for interdigital electrodes, and are more used in the photovoltaic industry. Secondly, the conductivity of the silver paste electrode is due to copper alloy. After many experiments, the inventor found that the copper alloy is oxidized to form copper oxide and copper ions when powered on, which affects the experimental structure. On the contrary, the silver in the silver paste electrode is more stable. Using the optimized silver paste interdigital electrode of the present invention, the lysis effect is better, the two ends of the electrode are connected to the power oscilloscope to generate a strong electric field, and the transmembrane potential is generated to achieve the lysis of Escherichia coli. By adding magnetic beads to bind to nucleic acids, the extraction and purification of nucleic acids can be achieved by using magnet transportation. The present invention shortens the time of nucleic acid extraction, simplifies the premixing steps, realizes high sensitivity and simple operation integrating nucleic acid lysis, extraction and purification with magnetic control technology, can realize multiple PCR reactions, and detect multiple pathogens at the same time.

[0017] A multiplex PCR microfluidic chip integrating bacterial lysis nucleic acid extraction and purification with microfluidic chip detection and its application provided by the present invention has the following beneficial effects compared with the prior art:

[0018] 1) The present invention uses screen printing technology to print a layer of silver paste interdigital electrodes on a glass substrate based on the original chip, and ionically bonds and packages the upper PDMS with the glass substrate. The silver paste interdigital electrodes are aligned with the lysis chamber below the lysis chamber, and are connected to a power source. The interdigital silver paste electrodes generate a strong electric field, thereby generating a transmembrane potential, and irreversible electroporation can be formed on the ruptured surface of the bacterial membrane within a few seconds, thereby achieving bacterial lysis;

[0019] 2) By adding magnetic beads to bind to nucleic acids and using magnets for transport, nucleic acid extraction and purification can be achieved. The present invention realizes an integrated process of nucleic acid extraction, purification and amplification on a microfluidic chip, and can realize nucleic acid extraction and purification and PCR detection of bacteria as well as precise quantitative detection on the same chip;

[0020] 3) The chip has a simple structure, is suitable for batch manufacturing, is low-cost, and is suitable for popularization and use;

[0021] 4) The multiplex PCR microfluidic chip provided by the present invention has potential applicability in the field of molecular diagnosis.

[0022] In summary, the present invention provides a multiplex PCR microfluidic chip integrating interdigital electrode lysis, bacterial nucleic acid extraction, purification and detection and its application, which has broad application prospects in molecular diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A schematic diagram of the structure of a microfluidic chip integrating interdigital electrode lysis of bacteria, extraction, purification and detection of nucleic acid according to a preferred embodiment of the present invention;

[0024] Figure 2 For Figure 1 An exploded schematic diagram of the microfluidic chip shown;

[0025] Figure 3 For Figure 1 A three-dimensional image of the microfluidic chip shown;

[0026] Figure 4 An interdigital electrode provided according to a preferred embodiment of the present invention;

[0027] Figure 5 Electroporation of E. coli after electrolysis by interdigitated electrodes in a microfluidic chip;

[0028] Figure 6 is the gel electrophoresis diagram of nucleic acid extracted after E. coli lysis in the microfluidic chip;

[0029] The meanings of the reference numerals are as follows:

[0030] 101: PDMS layer; 102: interdigitated silver paste electrode; 103: glass substrate; 201: lysis chamber; 202: first washing chamber; 203: second washing chamber; 204: amplification chamber; 301: interdigitated width; 302: interdigitated spacing; 401: electroporation. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the instrument and equipment manufacturers. The reagents and materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0032] Example 1

[0033] Combination Figure 1-Figure 3 As shown, a microfluidic chip integrating bacterial lysis by interdigitated electrodes and nucleic acid extraction, purification and detection is provided according to this embodiment. The chip includes a PDMS layer 101 and a glass substrate 103 connected together by ionic bonding, wherein silver paste interdigitated electrodes 102 are printed on the glass substrate 103 by screen printing technology.

[0034] The PDMS layer 101 is provided with a lysis chamber 201, a first washing chamber 202, and a second washing chamber 203 which are connected from left to right in sequence, wherein the lysis chamber 201, the first washing chamber 202, and the second washing chamber 203 are connected via microchannels at the bottom, and by filling these channels with oil phase, the reaction reagents in each chamber can be prevented from cross-interference.

[0035] The microfluidic chip includes five lysis chambers 201. Accordingly, a silver paste interdigital electrode 102 is provided at the bottom of each lysis chamber 201. Figure 4 As shown, the width 301 of each interdigital finger of the silver paste interdigital electrode is 80-100 μm, preferably 100 μm; the interdigital distance 302 between adjacent interdigital fingers is 80-100 μm, preferably 100 μm.

[0036] Example 2

[0037] According to this embodiment, an application of a multiplex PCR microfluidic chip in detecting pathogens is provided. The application includes the following steps:

[0038] 1) Preparation stage: Provide a Figure 1 In the multiplex PCR microfluidic chip shown, first, the nucleic acid lysing solution, the first washing solution, the second washing solution, and the reaction solution are respectively injected into the lysing chamber 201, the first washing chamber 202, the second washing chamber 203, and the amplification chamber 204, and each chamber is separated by an oil phase so that the liquid reagents do not interfere with each other;

[0039] 2) Extraction and purification of nucleic acid: A sample containing bacteria is injected into the lysis chamber 201, and the silver paste interdigitated electrode 102 is connected to a power supply, 20V AC, and a frequency of 100,000 Hz, so that a strong electric field is generated in the lysis chamber 201, generating a transmembrane potential. The lysis solution is conductive. When the lysis solution is added and the power supply is connected, the positive and negative electrodes form a loop and generate a strong electric field to act on the Escherichia coli, causing irreversible electroporation 401 of the bacterial membrane, such as Figure 5 As shown, the nucleic acid is released, and the released nucleic acid molecules are adsorbed by the magnetic beads. The handheld magnet drags the magnetic beads into the first washing chamber 202 and the second washing chamber 203 in sequence to complete the washing. The washed nucleic acid molecules are manipulated by the magnet to enter the amplification chamber 204;

[0040] 3) Amplification detection: Under the action of the heating module, the nucleic acid molecules complete PCR amplification in the amplification chamber 204, and finally the instrument detects the different wavelengths emitted by the fluorescent group to achieve accurate quantitative detection of the pathogen;

[0041] 4) Gel electrophoresis test: The amplified product was verified by gel electrophoresis. Figure 6 As shown, the PCR reaction amplified the target gene fragment.

[0042] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A multiplex PCR microfluidic chip integrating electrolysis and nucleic acid purification detection, characterized in that: The invention comprises a PDMS layer and a glass substrate connected together by ion bonding, wherein: The PDMS layer is provided with a lysis chamber, a first washing chamber, a second washing chamber and an amplification chamber which are sequentially connected through a microchannel at the bottom; Silver paste interdigital electrodes are printed on the glass substrate by screen printing technology. The silver paste interdigital electrodes are aligned with the lysis chamber below the lysis chamber. After the power is turned on, a strong electric field can be generated by the current to realize the electric lysis of bacteria.

2. The multiplex PCR microfluidic chip according to claim 1, characterized in that: The multiplex PCR microfluidic chip comprises a plurality of lysis chambers, each of which is respectively connected to a first washing chamber, a first and a second washing chamber, and an amplification chamber, thereby realizing the detection of a plurality of samples simultaneously.

3. The multiplex PCR microfluidic chip according to claim 2, characterized in that: The multiplex PCR microfluidic chip comprises a plurality of silver paste interdigital electrodes, each of which is aligned with a lysis chamber.

4. The multiplex PCR microfluidic chip according to claim 3, characterized in that: The plurality of silver paste interdigital electrodes are sequentially arranged side by side on the glass substrate.

5. The multiplex PCR microfluidic chip according to claim 1, characterized in that: The width of each interdigital finger of the silver paste interdigital electrode is 100 μm, and the gap distance between adjacent interdigital fingers is 100 μm.

6. Use of the multiplex PCR microfluidic chip according to any one of claims 1 to 5 in detecting pathogens.

7. The use according to claim 6, characterized in that: The application comprises the following steps: S1, preparation stage: inject nucleic acid lysis solution, first washing solution, second washing solution and reaction solution into the lysis chamber, first washing chamber, second washing chamber and amplification chamber respectively, and separate the chambers by oil phase so that the liquid reagents do not interfere with each other; S2, extraction and purification of nucleic acids: inject the sample containing bacteria into the lysis chamber, connect the silver paste interdigitated electrode to the power supply, and achieve bacterial lysis by adjusting the voltage. The nucleic acid molecules released by the rupture of the bacteria are adsorbed by the magnetic beads. The magnetic beads are dragged into the first washing chamber and the second washing chamber in turn by the handheld magnet to complete the washing. The washed nucleic acid molecules are manipulated by the magnet to enter the amplification chamber; S3, PCR amplification detection: Under the action of the heating module, the nucleic acid molecules complete the PCR reaction in the amplification chamber. The different wavelengths emitted by the fluorescent group are detected by a fluorescence microscope to achieve accurate detection of bacteria.

8. The use according to claim 7, characterized in that: The silver paste interdigital electrode is connected to a power supply with an alternating current voltage of 20 V, a frequency of 100,000 Hz, and a pulse waveform.

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