WO2023115717A1 - 微流控芯片、基于微流控芯片的检测系统及细菌的检测方法 - Google Patents

微流控芯片、基于微流控芯片的检测系统及细菌的检测方法 Download PDF

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WO2023115717A1
WO2023115717A1 PCT/CN2022/080095 CN2022080095W WO2023115717A1 WO 2023115717 A1 WO2023115717 A1 WO 2023115717A1 CN 2022080095 W CN2022080095 W CN 2022080095W WO 2023115717 A1 WO2023115717 A1 WO 2023115717A1
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microfluidic chip
microchannel
pathogenic bacteria
hairpin oligonucleotide
reaction
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PCT/CN2022/080095
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English (en)
French (fr)
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蒋宇扬
高丹
孙冬丽
樊婷婷
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清华大学深圳国际研究生院
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Publication of WO2023115717A1 publication Critical patent/WO2023115717A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the technical field of microfluidic chips, in particular to a microfluidic chip, a detection system based on a microfluidic chip and a detection method for bacteria, in particular to a microfluidic chip for pathogenic bacteria, a microfluidic chip based Fluidic chip detection system and pathogenic bacteria detection method.
  • Foodborne pathogens are pathogenic bacteria that can cause food poisoning or use food as a medium for transmission. Pathogenic bacteria directly or indirectly contaminate food and water sources, which can lead to infectious diseases of livestock and poultry or human intestinal infectious diseases and food poisoning, which is one of the root causes of food safety problems. Common foodborne pathogens mainly include pathogenic Escherichia coli, Salmonella, Staphylococcus aureus, and Listeria monocytogenes.
  • Escherichia coli O157:H7 is one of the main foodborne pathogenic bacteria, it is a representative strain of enterohaemorrhagic Escherichia coli (EHEC), the infection dose is extremely low, The incubation period is 3-10 days, and the course of disease is 2-9 days, which can lead to hemorrhagic colitis (HC), usually with sudden severe abdominal pain and watery diarrhea, followed by hemorrhagic diarrhea after a few days, and some patients may develop hemolytic uremic syndrome Syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP), severe cases can lead to death.
  • HUS hemolytic uremic syndrome Syndrome
  • TTP thrombotic thrombocytopenic purpura
  • the methods for detecting foodborne pathogens can be divided into indirect methods and direct methods.
  • the indirect method is mainly to detect the secretions of food-borne pathogenic bacteria, such as endotoxin and nucleic acid, and indirectly obtains the presence or absence of pathogenic bacteria, which often requires complicated sample pretreatment.
  • the direct method is to directly quantitatively detect the quantity or concentration of foodborne pathogenic bacteria in the sample, including plate counting method and biosensor method.
  • the plate counting method is the gold standard for the detection of foodborne pathogens, which has high accuracy and reliability, but requires professional technicians to cultivate and pre-enrich the samples for a long time, and the detection time is as long as 2 -3 days, consume a lot of time and manpower and material resources.
  • Microfluidic chip technology is a kind of miniaturization and integration of biological or chemical experiments (such as sample reaction preparation, analysis and purification, detection and analysis, etc.) complex analysis process. Because microfluidic chip technology has the advantages of small sample and reagent consumption, parallel detection of multiple samples, high throughput, fast analysis speed, and easy integration and automation, it is widely used in the field of analysis and detection. A growing number of studies have developed methods for the detection of foodborne pathogens based on microfluidic cores, such as colorimetry, fluorescence, electrochemical, surface Raman scattering, surface plasmon resonance, and lateral flow Article and other methods.
  • the present invention proposes a microfluidic chip, a detection system based on the microfluidic chip and a detection method for bacteria based on catalytic hairpin self-assembly and chemiluminescence.
  • Chemiluminescence is a method of converting the concentration signal of the detection target into chemiluminescent intensity.
  • the main signal generation mechanism is based on the chemiluminescence reaction of the enzyme-catalyzed substrate and oxidant (such as luminol-hydrogen peroxide system).
  • Chemiluminescence relies on the chemical reaction of trace analytes to produce chemiluminescence, so it has high sensitivity.
  • no excitation light source is required, thereby avoiding the interference of light scattering, and the instrument and operation of chemiluminescence method are simple, with a wide calibration range, and its miniaturization applicability provides great potential for the detection of foodborne pathogens.
  • Catalysed hairpin assembly is a superior signal amplification strategy, the basic principle of which is composed of carefully designed two hairpin oligonucleotides and a chain oligonucleotide, using stem-loop
  • the "foothold" in the structure can be changed by the sequence-complementary naked nucleic acid through the principle of base complementary pairing and topological reaction kinetics, thereby completing the self-assembly and disassembly process between oligonucleotides.
  • the net result of this reaction is the generation of two hybridized strands of the hairpin oligonucleotides, which, by introducing specific groups in the hairpin oligonucleotides, immobilize the hybridized strands on the substrate and generate an assay signal. Since the chain oligonucleotide is repeatedly used, as long as the chain oligonucleotide is associated with the target analyte, the concentration signal of the target analyte can be amplified through the above reaction.
  • the first aspect of the present invention provides a microfluidic chip, the microfluidic chip is composed of a bonded substrate and a cover sheet, the cover sheet is provided with a mixing microchannel and a reaction microchannel, the mixing microchannel The channel and the reaction microchannel are connected to each other, and the reaction microchannel has a microcolumn array inside.
  • the mixing microchannel is a serpentine mixing microchannel
  • the reaction microchannel is a boat-shaped reaction microchannel
  • the boat-shaped reaction microchannel includes two bows and a hull between the two bows, one bow is used as an inlet, and the other is used as an outlet;
  • the mixing microchannel includes an inlet and an outlet
  • the mixing microchannel includes 2 inlets;
  • the outlet of the mixing microchannel is connected to the bow inlet of the boat-shaped reaction microchannel;
  • the length of the reaction microchannel is 16mm, the width is 3mm, and the streamlined design of gradual expansion at the entrance and tapering at the exit avoids the generation of dead ends, so that the mixed reagents can be distributed after entering the boat-shaped channel. more uniform;
  • the serpentine mixing microchannel has a width of 200 ⁇ m, a depth of 150 ⁇ m, and a length of 20 cm. Its narrow and elongated characteristics allow the chemiluminescent reagents to be fully mixed to a greater extent in terms of hydrodynamics, and can quickly produce higher chemical Light signals, and folding the mixing channel into a serpentine shape can achieve a space-saving effect.
  • the microcolumn has a diameter of 200 ⁇ m, a depth of 150 ⁇ m, and a microcolumn spacing of 200 ⁇ m, which increases the internal surface area of the boat-shaped reaction microchannel, provides more space for its internal surface modification, and can be larger in hydrodynamics To a certain extent, increase the collision probability between the initiator strand and the micropillar that catalyzes the hairpin self-assembly reaction, and increase the binding efficiency of the initiator strand and the hairpin oligonucleotide 1;
  • the material of the cover sheet is polydimethylsiloxane (PDMS), which has strong biocompatibility, is easy to modify the surface, has good light transmittance, and is suitable for chemiluminescent detection.
  • PDMS polydimethylsiloxane
  • the microcolumn is modified with hairpin oligonucleotide 1 (H1), and the hairpin oligonucleotide 1 is used to catalyze the hairpin self-assembly reaction; preferably, the H1 carries out fluorescence To verify the successful modification of H1, the formal experiment uses H1 without fluorescent label.
  • H1 hairpin oligonucleotide 1
  • the microcolumn is modified with streptavidin, and the hairpin oligonucleotide 1 is modified on the microcolumn by combining biotin and streptavidin.
  • the second aspect of the present invention provides a detection system based on a microfluidic chip, including the microfluidic chip, a sampling unit, and a detection and analysis unit, and the sampling unit is mixed with the inlet of the microchannel of the microfluidic chip connected;
  • the detection and analysis unit includes a PMT detector and an ultra-weak chemiluminescence analyzer, and the light inlet of the PMT detector matches the reaction microchannel;
  • the sampling unit includes a micro-injection pump, a catheter and a syringe
  • the microfluidic chip can be connected to the syringe through the catheter, and the flow rate and flow rate can be precisely controlled by the micro-injection pump, and the chemiluminescent reagent is automatically perfused after programming;
  • the detection system further includes a waste liquid pool communicated with the outlet of the microfluidic chip.
  • the third aspect of the present invention provides a detection kit based on a microfluidic chip, including the microfluidic chip, the hybridization chain formed by the specific nucleic acid aptamer (Apt) of pathogenic bacteria and the priming chain (I) (I/Apt), hairpin oligonucleotide 2 (H2) and horseradish peroxidase (HRP) modified gold nanoparticles (H2-AuNP-HRP), luminol and hydrogen peroxide;
  • the specific nucleic acid aptamer, trigger strand, hairpin oligonucleotide 1 and hairpin oligonucleotide 2 of the pathogenic bacteria meet the following conditions (1) or (2);
  • the specific nucleic acid aptamer of the pathogenic bacteria has m bases, and the 3' end of the specific nucleic acid aptamer of the pathogenic bacteria has n complementary paired bases with the priming strand, wherein n/m is 1/4 ⁇ 1/3;
  • the 5' end of the priming strand is paired with 6-10 exposed bases at the 3' end of the hairpin oligonucleotide 1;
  • the 5' end of the hairpin oligonucleotide 1 is complementary to 6 to 10 exposed bases at the 3' end of the hairpin oligonucleotide 2;
  • the 5' end of the hairpin oligonucleotide 1 is modified with biotin;
  • the 5' end of the hairpin oligonucleotide 2 is labeled with a sulfhydryl group, and there is an amino acid residue on the horseradish peroxidase sulfhydryl group, the hairpin oligonucleotide 2 and horseradish peroxidase are modified to gold nanoparticles through gold-sulfur bonds to form hairpin oligonucleotide 2 and horseradish peroxidase modified gold nanoparticles;
  • the specific nucleic acid aptamer of the pathogenic bacteria has m bases, and the 5' end of the specific nucleic acid aptamer of the pathogenic bacteria has n complementary paired bases with the priming strand, wherein n/m is 1/4 ⁇ 1/3;
  • the 3' end of the priming strand is paired with 6-10 exposed bases at the 5' end of the hairpin oligonucleotide 1;
  • the 3' end of the hairpin oligonucleotide 1 is complementary to 6 to 10 exposed bases at the 5' end of the hairpin oligonucleotide 2;
  • the 3' end of the hairpin oligonucleotide 1 is modified with biotin; the 3' end of the hairpin oligonucleotide 2 is labeled with a sulfhydryl group, and there is an amino acid residue on the horseradish peroxidase sulfhydryl group, the hairpin oligonucleotide 2 and horseradish peroxidase are modified to gold nanoparticles through gold-sulfur bonds to form hairpin oligonucleotide 2 and horseradish peroxidase modified gold nanoparticles.
  • the hairpin oligonucleotide 2 (H2) is fluorescently labeled to verify the successful modification of the hairpin oligonucleotide 1 (H1), and the formal experiment uses aptamer 2 without fluorescent labeling.
  • the horseradish peroxidase has a characteristic absorption peak at 420nm, and the horseradish peroxidase is verified by whether there is a characteristic absorption peak at 420nm before and after the gold nanoparticle modification.
  • the sequence of the specific nucleic acid aptamer of the pathogenic bacteria is shown in SEQ ID NO.1
  • the sequence of the trigger chain is shown in SEQ ID NO.1.
  • the sequence of the hairpin oligonucleotide 1 is shown in SEQ ID NO.3
  • the sequence of the hairpin oligonucleotide 2 is shown in SEQ ID NO.4.
  • the fourth aspect of the present invention provides the application of the microfluidic chip, the detection system based on the microfluidic chip or the detection kit based on the microfluidic chip in the detection of pathogenic bacteria;
  • the pathogenic bacteria are food-borne pathogenic bacteria.
  • pathogenic bacteria can be combined with the hybrid chain (I/Apt) on the specific nucleic acid aptamer (Apt) of pathogenic bacteria, thereby competing for the initiator strand (I) that catalyzes the hairpin self-assembly reaction.
  • the initiator strand (I) that catalyzes the hairpin self-assembly reaction can trigger the self-assembly of hairpin oligonucleotide 1 (H1) and hairpin oligonucleotide 2 (H2) to form a hybrid strand (H1/H2), specifically , the 5' end of the priming strand (I) is paired with the exposed base at the 3' end of the hairpin oligonucleotide 1 (H1), thereby opening the hairpin oligonucleotide 1 (H1), and the opened hairpin oligonucleotide 1 (H1) is opened.
  • the 5' end of clip oligonucleotide 1 (H1) complementarily pairs with the exposed base at the 3' end of hairpin oligonucleotide 2 (H2), thereby opening hairpin oligonucleotide 2 (H2) , as the hairpin oligonucleotide 2 (H2) binds to the hairpin oligonucleotide 1 (H1) to form a hybrid strand (H1/H2), the priming strand (I) is competed, and the The priming strand (I) continues to open hairpin oligonucleotide 1 (H1), and so on until all hairpin oligonucleotides (H2) are bound to hairpin oligonucleotide 1 (H1).
  • H2 hairpin oligonucleotide 2
  • H1 hairpin oligonucleotide 1
  • HRP modified horseradish peroxidase
  • the fifth aspect of the present invention provides a microfluidic chip-based bacterial detection method, characterized in that, comprising the following steps:
  • the sample to be tested, the specific nucleic acid aptamer of pathogenic bacteria and the hybridized chain formed by the priming chain, and the hairpin oligonucleotide 2 are evenly mixed, injected into the reaction microchannel of the microfluidic chip, and left to stand , the catalytic hairpin self-assembly reaction triggered by the competitive binding of pathogenic bacteria occurs;
  • chemiluminescence spectrum of the reaction microchannel Collect the chemiluminescence spectrum of the reaction microchannel, and use the chemiluminescence spectrum to determine the presence or absence and concentration of pathogenic bacteria in the sample to be tested.
  • the microfluidic chip-based bacteria detection method also includes using the pathogenic bacteria to be tested as a standard sample, using the above detection method to collect background signals and chemiluminescence spectra, taking the concentration of the standard sample as the abscissa, and taking its The peak value of the chemiluminescent spectrum is drawn as the ordinate to draw the standard curve, and the linear fitting is used to obtain the standard equation;
  • the peak value of the chemiluminescence spectrum of the sample to be tested is substituted into the standard equation to obtain the concentration of pathogenic bacteria in the sample to be tested.
  • the buffer solution is a Tris-HCl buffer solution
  • the volume ratio of the luminol and hydrogen peroxide is 1:1;
  • the volumes of the luminol and hydrogen peroxide are both 10 ⁇ L;
  • the luminol and hydrogen peroxide are injected into the mixing microchannel at a flow rate of 10 ⁇ L/min;
  • the pathogenic bacteria are food-borne pathogenic bacteria.
  • an ultra-weak chemiluminescence analyzer is used to collect background signals and chemiluminescence spectra
  • the ultra-weak chemiluminescence analyzer is preheated for 30 minutes before collecting the background signal;
  • the chemiluminescence spectrum is collected within 900 s from the start of injecting the luminol and hydrogen peroxide.
  • microfluidic chip based on catalyzed hairpin self-assembly (CHA) and chemiluminescent biosensing provided by the present invention is used to detect food-borne pathogenic bacteria, compared with traditional food-borne pathogenic bacteria detection methods , the required sample and reagent consumption are small, the degree of automation is high, and the detection speed is fast.
  • CHA catalyzed hairpin self-assembly
  • the narrow and elongated characteristics of the serpentine mixing microchannel allow the chemiluminescent reagents to be fully mixed to a greater extent in terms of hydrodynamics, which can quickly generate a higher chemiluminescent signal, and folding the mixing channel into a serpentine shape can save space. effect.
  • the boat-shaped reaction microchannel adopts a streamlined design with gradual expansion at the entrance and tapering at the exit, which avoids the generation of dead angles and enables the mixed reagents to be distributed more evenly after entering the boat-shaped channel.
  • the diameter of the micro-pillar array is 200 ⁇ m, the depth is 150 ⁇ m, and the spacing between the micro-pillars is 200 ⁇ m, which increases the inner surface area of the boat-shaped reaction microchannel, provides more space for its inner surface modification, and can increase the hydrodynamics to a greater extent.
  • the probability of collision between the priming strand (I) that catalyzes the hairpin self-assembly reaction and the micropillar increases the binding efficiency of the priming strand (I) and the hairpin oligonucleotide 1 (H1).
  • the material of the microfluidic chip is polydimethylsiloxane (PDMS), which has good biocompatibility and strong light transmission, and is suitable for the detection of biomarkers.
  • the detection method of pathogenic bacteria is to trigger two hairpin oligonucleotides (H1, H2) to catalyze the hairpin self-assembly reaction (CHA) through the competition of pathogenic bacteria, wherein the hairpin
  • the hairpin oligonucleotide 1 (H1) was modified on the micropillar array in the boat-shaped reaction microchannel of the microfluidic chip, and the hairpin oligonucleotide 2 (H2) and horseradish peroxidase (HRP) were co-modified on the On the surface of gold nanoparticles, the catalyzed hairpin self-assembly reaction (CHA) not only amplifies the concentration of pathogenic bacteria, but also immobilizes horseradish peroxidase (HRP) on the micropillar array, thereby catalyzing luminol and peroxidase.
  • HRP horseradish peroxidase
  • the hydrogen peroxide undergoes a chemiluminescent reaction, and the pathogenic bacteria are quantified by the signal intensity of the chemical reaction.
  • the method has good specificity, low detection limit and wide linear range, and provides more accurate data for the quantitative detection of pathogenic bacteria. This method has important application prospects in the field of detection of foodborne pathogens.
  • the present invention can detect the concentration of food-borne pathogenic bacteria in a sample in a relatively short period of time, greatly improving the efficiency of quantitative detection of food-borne pathogenic bacteria.
  • the microfluidic chip platform has a small amount of reagents, can be integrated and automated, which greatly reduces the material cost and labor cost of quantitative detection of food-borne pathogens, and provides a new technical platform for the detection of food-borne pathogens. It has a good application prospect in food safety testing.
  • Fig. 1 is the three-dimensional structural diagram of the microfluidic chip of embodiment 1; 1- serpentine mixing microchannel, 2- boat-shaped reaction microchannel, 3- microcolumn array;
  • Fig. 2 is the size design drawing of cover slip channel mask of embodiment 2;
  • Fig. 3 is the microchannel modification result figure of embodiment 2;
  • Fig. 4 is the detection system based on the microfluidic chip of embodiment 3; a-micro-injection pump, b-syringe, c-PTFE catheter, d-microfluidic chip, e-waste liquid pool, f-PMT detector, g-ultra-weak chemiluminescence analyzer;
  • Fig. 5 is the chemiluminescence spectrum and standard curve of Escherichia coli O157:H7 standard sample in embodiment 4.
  • the microfluidic chip is composed of a bonded substrate and a cover sheet.
  • the material of the substrate is glass
  • the material of the cover is polydimethylsiloxane (PDMS).
  • the cover sheet is provided with a serpentine mixing microchannel 1 and a boat-shaped reaction microchannel 2, and the micropillar arrays 3 are evenly distributed in the boat-shaped reaction microchannel 2.
  • the boat-shaped reaction microchannel 2 includes two bows and a hull between the two bows, one bow is used as an inlet, and the other is used as an outlet.
  • the mixing microchannel includes 2 inlets and 1 outlet, and the 2 inlets are used as the inlets of luminol and hydrogen peroxide respectively.
  • the outlet of the mixing microchannel is connected to the bow inlet of the boat-shaped reaction microchannel.
  • the serpentine mixing microchannel 1 has a width of 200 ⁇ m, a depth of 150 ⁇ m, and a length of 20 cm. Its narrow and elongated characteristics allow the chemiluminescent reagents to be fully mixed to a greater extent in terms of hydrodynamics, and can quickly generate higher chemiluminescent signals. Folding the mixing channel into a serpentine shape can save space.
  • the length of the boat-shaped reaction microchannel 2 is 16 mm, and the width is 3 mm.
  • the streamlined design of gradual expansion at the entrance and tapering at the exit avoids the generation of dead angles, so that the mixed reagents can be distributed more evenly after entering the boat-shaped channel.
  • the micropillar array 3 of the chip has a diameter of 200 ⁇ m, a depth of 150 ⁇ m, and a micropillar spacing of 200 ⁇ m, which increases the inner surface area of the boat-shaped microchannel and provides more space for its inner surface modification.
  • the microcolumn is modified with hairpin oligonucleotide 1 (H1), and the hairpin oligonucleotide is used to catalyze the hairpin self-assembly reaction.
  • the microcolumn was modified with streptavidin, and the hairpin oligonucleotide 1 (H1) was modified on the microcolumn by combining biotin and streptavidin.
  • the silicon wafer was firstly ultrasonicated with ethanol for 5 minutes, and then deionized water for 5 minutes, and then heated on a heating plate for 120 minutes after performing three times of ultrasonication.
  • a thin layer of SU-82050 negative photoresist was spin-coated on the silicon wafer, and the thickness of the adhesive layer was about 50 ⁇ m by controlling the rotating speed.
  • Lay the mask with the structure shown in Figure 2 on the silicon wafer expose the photoresist through the ultraviolet light through the photomask, and dissolve the unexposed part with a developer.
  • the raised SU-8 structure on the silicon wafer surface serves as a positive mold for the PDMS cover slip. The silicon positive mold was then silanized overnight.
  • the channel was treated with 4% (v/v) MPTS in absolute ethanol solution at room temperature, and allowed to stand for 30 minutes. Then wash with absolute ethanol, repeat three times or until completely washed. After washing, the chips were dried in an oven at 100 °C for 1 h.
  • the hairpin oligonucleotide 1 (H1) was successfully modified by using biotin-labeled hairpin oligonucleotide 1 (H1) modified with a FAM group to verify the microchannel, and used Washing with PBS buffer removed unbound capture aptamers. Photographs of the microchannels were taken using a fluorescence microscope, as shown in Figure 3.
  • Embodiment 3 detection system based on microfluidic chip
  • This embodiment provides a detection system based on a microfluidic chip, as shown in FIG. The inlet connection of the mixing microchannel of the fluidic chip.
  • the detection and analysis unit includes a PMT detector f and an ultra-weak chemiluminescence analyzer g.
  • the size of the microfluidic chip matches the card slot in the box of the PMT detector (25*25mm), and after it is placed, the boat-shaped reaction microchannel just matches the position of the light inlet of the PMT detector, and the chemical produced
  • the luminescence signal was collected by PMT and analyzed by ultra-weak chemiluminescence analyzer g.
  • the sampling unit includes a micro-injection pump a, a PTFE catheter c and a syringe b.
  • the microfluidic chip d can be connected to the syringe b through the PTFE catheter c, and the flow rate and flow rate can be precisely controlled by the micro-injection pump a, and the chemical perfusion is automated after programming.
  • Luminescence reagent a micro-injection pump a
  • PTFE catheter c a PTFE catheter c
  • syringe b a syringe b
  • the outlet of the microfluidic chip needs to be connected with the waste liquid pool e by a PTFE catheter to prevent the liquid at the outlet from causing damage to the PMT detector.
  • Example 4 Detection kit based on microfluidic chip
  • Example 1 Including the microfluidic chip described in Example 1, the hybrid strand formed by the specific nucleic acid aptamer of pathogenic bacteria and the priming strand, hairpin oligonucleotide 2 and gold nanoparticles modified by horseradish peroxidase , luminol and hydrogen peroxide.
  • the 3' end of the specific nucleic acid aptamer of the pathogenic bacterium has several complementary paired bases with the priming strand, wherein the number of complementary paired bases accounts for about 1/3 of the number of bases in the specific nucleic acid aptamer sequence,
  • the specific nucleic acid aptamer of the pathogenic bacteria forms a hybrid chain (I/Apt) with the priming chain, and the pathogenic bacteria can be combined into the specific nucleic acid adapter of the pathogenic bacteria in the hybrid chain (I/Apt).
  • the initiator strand (I) that catalyzes the hairpin self-assembly reaction is competed.
  • the 5' end of the priming strand is paired with the exposed bases at the 3' end of the hairpin oligonucleotide 1.
  • the 5' end of the hairpin oligonucleotide 1 is complementary to the exposed base at the 3' end of the hairpin oligonucleotide 2.
  • the initiator strand (I) catalyzing the hairpin self-assembly reaction can trigger the self-assembly of hairpin oligonucleotide 1 (H1) and hairpin oligonucleotide 2 (H2) to form a hybrid strand (H1/H2).
  • the 5' end of the priming strand (I) interacts with the exposed base at the 3' end of the hairpin oligonucleotide 1 (H1), thereby opening the hairpin oligonucleotide 1 (H1), and opening
  • the 5' end of the hairpin oligonucleotide 1 (H1) is complementary paired with the exposed base of the 3' end of the hairpin oligonucleotide 2 (H2), thereby opening the hairpin oligonucleotide 2 ( H2), as the hairpin oligonucleotide 2 (H2) binds to the hairpin oligonucleotide 1 (H1) to form a hybrid strand (H1/H2)
  • the priming strand (I) is competed and
  • the descending priming strand (I) continues to open hairpin oligo 1 (H1), and so on cycle until all hairpin oligo 2 (H2) is bound to hairpin oligo 1 (H1) superior.
  • H2 hairpin oligonucleotide 2
  • H1 hairpin oligonucleotide 1
  • HRP modified horseradish peroxidase
  • Embodiment 4 Bacterial detection method based on microfluidic chip
  • microfluidic chip prepared in Example 2 was used to detect Escherichia coli O157:H7. Specific steps are as follows:
  • the nucleotide sequences of Escherichia coli O157:H7-specific nucleic acid aptamer, priming strand, hairpin oligonucleotide 1 and hairpin oligonucleotide 2 in this embodiment are shown in Table 2.
  • the 5' end of hairpin oligonucleotide 1 was modified with biotin, and the 5' end of hairpin oligonucleotide 2 was labeled with a sulfhydryl group.

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Abstract

一种微流控芯片、基于微流控芯片的检测系统及细菌的检测方法，该微流控芯片由键合在一起的基片与盖片组成，盖片上设有混合微通道(1)和反应微通道(2)，混合微通道(1)和反应微通道(2)相互连接，反应微通道(2)内具有微柱阵列(3)，微柱上修饰有发夹状寡核苷酸1。该检测方法是通过致病性细菌竞争结合引发两条发夹状寡核苷酸(H1,H2)发生催化发夹自组装反应，该反应不仅对致病性细菌的浓度进行放大，还将辣根过氧化物酶固定在微柱阵列上，进而催化鲁米诺和过氧化氢发生化学发光反应，通过化学反应信号强度来对致病性细菌进行定量。该检测方法具有良好的特异性，低的检测限和宽的线性范围，为定量检测食源性致病菌提供了较为准确的数据。

Description

微流控芯片、基于微流控芯片的检测系统及细菌的检测方法 技术领域
本发明属于微流控芯片技术领域，具体涉及微流控芯片、基于微流控芯片的检测系统及细菌的检测方法，特别是涉及一种用于致病性细菌的微流控芯片、基于微流控芯片的检测系统及致病性细菌的检测方法。
背景技术
食源性致病菌是可以引起食物中毒或以食品为传播媒介的致病性细菌。致病性细菌直接或间接污染食品及水源，可导致畜禽传染病或人肠道传染病及食物中毒，是食品安全问题的根源之一。常见食源性致病菌主要有致病性大肠埃希氏菌、沙门氏菌、金黄葡萄球菌、单核细胞增生李斯特氏菌等。大肠埃希氏菌O157：H7(E.coil O157:H7)是主要的食源性致病细菌之一，它是肠出血性大肠埃希氏菌(EHEC)的代表菌株，感染剂量极低，潜伏期为3-10天，病程2-9天，可导致出血性结肠炎(HC)，通常是突然发生剧烈腹痛和水样腹泻，数天后出现出血性腹泻，部分患者可发展为溶血性尿毒综合症(HUS)和血栓性血小板减少性紫癜(TTP)等，严重者可导致死亡。食源性致病菌引发的食物中毒给公共卫生带来了沉重负担，也给人们的生命健康带来了严重威胁。因此，食品中致病菌的限量将有助于预防和控制此类事件的发生与传播。根据《食品安全国家标准食品中致病菌限量》规定，除金黄葡萄球菌的可接受水平的限量值为100CFU/g(mL)外，大肠埃希氏菌O157:H7、沙门氏菌和单核细胞增生李斯特氏菌的可接受水平的限量值为0。食源性致病菌的限量必须依靠准确和快速的检测方法，所以，采用有效方法检测食源性致病菌至关重要。
目前，检测食源性致病菌的方法可分为间接法和直接法。间接法主要是对食源性致病菌的分泌物如内外毒素以及核酸进行检测，间接获得致病菌是否存在，往往需要复杂的样品前处理。直接法则是直接对样本中食源性致病菌的数量或浓度进行定量检测，包括平板计数法和生物传感器法。其中，平板计数法是食源性致病菌检测的金标准，拥有高的准确性和可靠性，但是需要专业的技术人员对样品进行长时间的培养和预富集，且检测时间长达2-3天，耗费大量时间和人力物力。
微流控芯片技术是一种将生物或者化学实验(如样品的反应制备、分析提纯，检测分析等)微型化并集成到具有微米甚至纳米尺度微通道的芯片上，通过对流体进行精确控制完成复杂的分析流程。由于微流控芯片技术具有样品和试剂消耗量小，多样本可并行检测，高通量，分析速度快，以及易于集成化和自动化等优点，使其被广泛应用到分析检测领域。越来 越多的研究开发了基于微流控芯的食源性致病菌检测方法，例如比色法、荧光法、电化学法、表面拉曼散射法、表面等离子体共振法以及侧向流动条等方法。这些方法各自存在一定的不足，其中比色法和荧光法灵敏度不高，电化学法重现性较差，表面拉曼散射法、表面等离子体共振法需要复杂且昂贵的信号检测仪器。因此需要开发一种检测灵敏度高，信号检测仪器简单廉价的食源性致病菌检测方法。
发明内容
为了解决现有技术中的不足，本发明基于催化发夹自组装和化学发光法，提出微流控芯片、基于微流控芯片的检测系统及细菌的检测方法。
化学发光法是一种将检测目标的浓度信号转换为化学发光强度的方法，主要的信号产生机制是基于通过酶催化底物和氧化剂(如鲁米诺-过氧化氢体系)发生化学发光反应。化学发光法依靠痕量分析物的化学反应产生化学发光，因此灵敏度高。此外，不需要激发光源，从而避免了光散射的干扰，并且化学发光法的仪器和操作简单，校准范围宽，其小型化的适用性为食源性致病菌的检测提供了巨大的潜力。
催化发夹自组装(catalysed hairpin assembly,CHA)是一种优越的信号放大策略，其基本原理是由精心设计的两条发夹状寡核苷酸和一条链状寡核苷酸，利用茎环结构上“立足点”，能够被序列互补的裸露核酸通过碱基互补配对原则及拓扑反应动力学作用改变结构，从而完成寡核苷酸之间的自组装及去组装过程。该反应的净结果是产生两条发夹状寡核苷酸的杂交链，通过在发夹状寡核苷酸中引入特定基团，将杂交链固定在基底上和产生分析信号。由于链状寡核苷酸被多次重复使用，只要将链状寡核苷酸和目标分析物建立关联，就可以通过上述反应，将目标分析物的浓度信号进行放大。
本发明具体技术方案如下：
本发明第一方面提供一种微流控芯片，所述微流控芯片由键合在一起的基片与盖片组成，所述盖片上设有混合微通道和反应微通道，所述混合微通道和所述反应微通道相互连接，所述反应微通道内具有微柱阵列。
进一步地，所述混合微通道为蛇形混合微通道，所述反应微通道为船形反应微通道；
所述船形反应微通道包括两个船头和位于两个船头之间的船身，一个船头作为进口，另一个作为出口；
所述混合微通道包括进口和出口；
优选地，所述混合微通道包括2个进口；
优选地，所述混合微通道出口与所述船形反应微通道的船头进口相互连接；
优选地，所述反应微通道的长为16mm，宽为3mm，进口处渐扩和出口处渐缩的流线形设计，避免了死角的产生，使混合后的试剂进入船形通道后可以分布的更加均匀；
优选地，所述蛇形混合微通道的宽度为200μm，深度为150μm，长度为20cm，其狭窄延长的特点在流体力学上更大程度地让化学发光试剂充分混合，能够快速产生较高的化学发光信号，且将混合通道折叠成蛇形可以直到节省空间的作用。
优选地，所述微柱的直径为200μm，深度为150μm，微柱间距为200μm，提高船形反应微通道的内表面积，为其内表面修饰提供了更多的空间，在流体力学上能更大程度上增加催化发夹自组装反应的引发链与微柱的碰撞几率，增加引发链与发夹状寡核苷酸1的结合效率；
优选地，所述盖片的材料为聚二甲基硅氧烷(PDMS)，其生物兼容性强，易于表面修饰，透光性好，适合化学发光检测。
进一步地，所述微柱上修饰有发夹状寡核苷酸1(H1)，所述发夹状寡核苷酸1用于发生催化发夹自组装反应；优选地，所述H1进行荧光标记来验证H1的成功修饰，正式实验使用无荧光标记的H1。
优选地，微柱进行链霉亲和素修饰，发夹状寡核苷酸1通过生物素和链霉亲和素的结合修饰在微柱上。
本发明第二方面提供一种基于微流控芯片的检测系统，包括所述微流控芯片、进样单元和检测分析单元，所述进样单元与所述微流控芯片混合微通道的进口连通；
优选地，所述检测分析单元包括PMT探测器和超微弱化学发光分析仪，所述PMT探测器的进光口与反应微通道相匹配；
优选地，所述进样单元包括微量注射泵、导管和注射器，微流控芯片可以通过导管与注射器相连，通过微量注射泵精确的控制流量和流速，编程后自动化灌注化学发光试剂；
优选地，所述检测系统还包括与微流控芯片出口连通的废液池。
本发明第三方面提供一种基于微流控芯片的检测试剂盒，包括所述微流控芯片、致病性细菌的特异性核酸适配体(Apt)与引发链(I)形成的杂交链(I/Apt)、发夹状寡核苷酸2(H2)与辣根过氧化物酶(HRP)修饰的金纳米颗粒(H2-AuNP-HRP)、鲁米诺以及过氧化氢；
所述致病性细菌的特异性核酸适配体、引发链、发夹状寡核苷酸1和发夹状寡核苷酸2满足下述条件(1)或(2)；
(1)所述致病性细菌的特异性核酸适配体具有m个碱基，所述致病性细菌的特异性核酸适配体3’端与引发链有n个互补配对碱基，其中n/m为1/4～1/3；
所述引发链的5’端与发夹状寡核苷酸1的3’端6～10个裸露的碱基互配对；
所述发夹状寡核苷酸1的5’端与发夹状寡核苷酸2的3’端6～10个裸露的碱基互补配对；
优选地，所述发夹状寡核苷酸1的5’端用生物素修饰；所述发夹状寡核苷酸2的5’端用巯基标记，辣根过氧化物酶上有氨基酸残基巯基，所述发夹状寡核苷酸2和辣根过氧化物酶通过金硫键修饰到金纳米颗上，形成发夹状寡核苷酸2与辣根过氧化物酶修饰的金纳米颗粒；
(2)所述致病性细菌的特异性核酸适配体具有m个碱基，所述致病性细菌的特异性核酸适配体5’端与引发链有n个互补配对碱基，其中n/m为1/4～1/3；
所述引发链的3’端与发夹状寡核苷酸1的5’端6～10个裸露的碱基互配对；
所述发夹状寡核苷酸1的3’端与发夹状寡核苷酸2的5’端6～10个裸露的碱基互补配对；
优选地，所述发夹状寡核苷酸1的3’端用生物素修饰；所述发夹状寡核苷酸2的3’端用巯基标记，辣根过氧化物酶上有氨基酸残基巯基，所述发夹状寡核苷酸2和辣根过氧化物酶通过金硫键修饰到金纳米颗上，形成发夹状寡核苷酸2与辣根过氧化物酶修饰的金纳米颗粒。
优选地，所述发夹状寡核苷酸2(H2)进行荧光标记来验证发夹状寡核苷酸1(H1)的成功修饰，正式实验使用无荧光标记的适配体2。
优选地，所述辣根过氧化物酶在420nm处有特征吸收峰，通过金纳米颗粒修饰前后在420nm处有无特征吸收峰，来验证辣根过氧化物酶。
进一步地，所述致病性细为大肠埃希氏菌O157：H7时，致病性细菌的特异性核酸适配体的序列如SEQ ID NO.1所示，所述引发链的序列如SEQ ID NO.2所示，所述发夹状寡核苷酸1的序列如SEQ ID NO.3所示，所述发夹状寡核苷酸2的序列如SEQ ID NO.4所示。
本发明第四方面提供所述微流控芯片、所述基于微流控芯片的检测系统或所述基于微流控芯片的检测试剂盒在致病性细菌检测中的应用；
优选地，所述致病性细为食源性致病菌。
以致病性细菌的特异性核酸适配体3’端与引发链有若干互补配对碱基为例，本发明进行致病性细菌检测的原理为：致病性细菌可以结合到所述杂交链(I/Apt)中致病性细菌的特异性核酸适配体(Apt)上，从而将催化发夹自组装反应的引发链(I)竞争下来。催化发夹自组装反应的引发链(I)可以引发发夹状寡核苷酸1(H1)和发夹状寡核苷酸2(H2)自组装形成杂交链(H1/H2)，具体地，引发链(I)的5’端与发夹状寡核苷酸1(H1)的3’端的裸露的碱基互配对，从而打开发夹状寡核苷酸1(H1)，打开的发夹状寡核苷酸1(H1)的5’端与发夹状寡核苷酸2(H2)的3’端的裸露的碱基互补配对，从而打开发夹状寡核苷酸2(H2)，随着发夹状寡核苷酸2(H2)结合到发夹状寡核苷酸1(H1)上形成杂交链(H1/H2)，引发链(I)被竞争下来，被竞争下来的引 发链(I)继续打开发夹状寡核苷酸1(H1)，如此循环直到所有发夹状寡核苷酸2(H2)都结合到发夹状寡核苷酸1(H1)上。随着发夹状寡核苷酸2(H2)结合到发夹状寡核苷酸1(H1)上形成杂交链(H1/H2)，从而固定在微柱阵列上，金纳米颗粒(AuNP)及其上修饰的辣根过氧化物酶(HRP)也固定在了微柱阵列上。在辣根过氧化物酶的催化下可以发生化学发光反应，产生化学发光信号，从而实现对致病性细菌的检测。
本发明第五方面提供一种基于微流控芯片的细菌检测方法，其特征在于，包括如下步骤：
将待测样品、致病性细菌的特异性核酸适配体与引发链形成的杂交链、发夹状寡核苷酸2混合均匀，注入所述微流控芯片的反应微通道中，静置，发生致病性细菌竞争结合引发的催化发夹自组装反应；
用缓冲溶液冲洗微流控芯片的反应微通道，采集反应微通道的本底信号；
向所述微流控芯片的混合微通道内分别注入鲁米诺和过氧化氢，在混合微通道内混合后注入反应微通道，在辣根过氧化物酶催化下发生化学发光反应；
采集反应微通道的化学发光图谱，用化学发光图谱判定待测样品中致病性细菌的有无及其浓度。
进一步地，所述基于微流控芯片的细菌检测方法还包括以待测致病性细菌为标准样品，采用上述检测方法采集本底信号和化学发光图谱，以标准样品浓度为横坐标，以其化学发光图谱的峰值为纵坐标绘制标准曲线，线性拟合得到标准方程；
优选地，将待测样品的化学发光图谱的峰值代入标准方程，得到待测样品中致病性细菌的浓度。
进一步地，所述缓冲溶液为Tris-HCl缓冲溶液；
所述鲁米诺和过氧化氢的体积比为1：1；
优选地，所述鲁米诺和过氧化氢的体积均为10μL；
优选地，所述鲁米诺和过氧化氢以10μL/min的流速注入混合微通道；
优选地，所述致病性细为食源性致病菌。
进一步地，采用超微弱化学发光分析仪采集本底信号和化学发光图谱；
优选地，超微弱化学发光分析仪预热30min后，再采集本底信号；
优选地，从开始注入鲁米诺和过氧化氢后的900s内采集化学发光图谱。
本发明的有益效果为：
1.本发明提供的基于催化发夹自组装(CHA)和化学发光生物传感的微流控芯片用于检测食源性致病菌时，与传统的食源性致病菌检测方法相比，所需样品、试剂消耗量少，自动 化程度高，检出速度快。
进一步地，蛇形混合微通道狭窄延长的特点在流体力学上更大程度地让化学发光试剂充分混合，能够快速产生较高的化学发光信号，且将混合通道折叠成蛇形可以直到节省空间的作用。船形反应微通道采用进口处渐扩和出口处渐缩的流线形设计，避免了死角的产生，使混合后的试剂进入船形通道后可以分布的更加均匀。微柱阵列的直径为200μm，深度为150μm，微柱间距为200μm，提高了船形反应微通道的内表面积，为其内表面修饰提供了更多的空间，在流体力学上能更大程度上增加催化发夹自组装反应的引发链(I)与微柱的碰撞几率，增加引发链(I)与发夹状寡核苷酸1(H1)的结合效率。微流控芯片的材料采用聚二甲基硅氧烷(PDMS)，其生物兼容性好，透光性强，适用于生物标志物的检测。
2.本发明提供的致病性细菌的检测方法，通过致病性细菌竞争结合引发两条发夹状寡核苷酸(H1,H2)发生催化发夹自组装反应(CHA)，其中发夹状寡核苷酸1(H1)修饰在微流控芯片船形反应微通道内的微柱阵列上，发夹状寡核苷酸2(H2)和辣根过氧化物酶(HRP)共同修饰在金纳米颗粒表面，催化发夹自组装反应(CHA)不仅对致病性细菌的浓度进行放大，还将辣根过氧化物酶(HRP)固定在微柱阵列上，进而催化鲁米诺和过氧化氢发生化学发光反应，通过化学反应信号强度来对致病性细菌进行定量。该方法具有良好的特异性，低的检测限和宽的线性范围，为定量检测致病性细菌提供了较为准确的数据。该方法在食源性致病菌检测领域具有重要的应用前景。此外，本发明在较短时间内即可检测出样品中食源性致病菌的浓度，大大提高了定量检测食源性致病菌的效率。并且微流控芯片平台具有试剂用量小，可集成化与自动化，大大减少了定量检测食源性致病菌的材料成本和人力成本，为食源性致病菌检测提供了新的技术平台，具有较好的食品安全检测应用前景。
附图说明
图1为实施例1的微流控芯片的立体结构图；1-蛇形混合微通道，2-船形反应微通道，3-微柱阵列；
图2为实施例2的盖片通道掩膜尺寸设计图；
图3为实施例2的微通道修饰结果图；
图4为实施例3的基于微流控芯片的检测系统；a-微量注射泵，b-注射器，c-PTFE导管，d-微流控芯片，e-废液池，f-PMT探测器，g-超微弱化学发光分析仪；
图5为实施例4中大肠埃希氏菌O157:H7标准样品的化学发光图谱和标准曲线。
具体实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于 解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1：微流控芯片的结构
本实施例提供一种微流控芯片，其立体结构如图1所示。该微流控芯片由键合在一起的基片和盖片组成。在本实施例中基片的材料为玻璃，盖片的材料为聚二甲基硅氧烷(PDMS)。盖片上设有蛇形混合微通道1和船形反应微通道2，船形反应微通道2内均匀分布微柱阵列3。所述船形反应微通道2包括两个船头和位于两个船头之间的船身，一个船头作为进口，另一个作为出口。所述混合微通道包括2个进口和1个出口，2个进口分别作为鲁米诺和过氧化氢的进口。混合微通道出口与船形反应微通道的船头进口相互连接。
蛇形混合微通道1的宽度为200μm，深度为150μm，长度为20cm，其狭窄延长的特点在流体力学上更大程度地让化学发光试剂充分混合，能够快速产生较高的化学发光信号，且将混合通道折叠成蛇形可以起到节省空间的作用。
船形反应微通道2的长为16mm，宽为3mm，进口处渐扩和出口处渐缩的流线形设计，避免了死角的产生，使混合后的试剂进入船形通道后可以分布的更加均匀。
芯片的微柱阵列3的直径为200μm,深度为150μm,微柱间距为200μm，提高船形微通道的内表面积，为其内表面修饰提供了更多的空间。
进一步地，微柱上修饰有发夹状寡核苷酸1(H1)，此发夹状寡核苷酸用于发生催化发夹自组装反应。微柱进行链霉亲和素修饰，发夹状寡核苷酸1(H1)通过生物素和链霉亲和素的结合修饰在微柱上。
实施例2：微流控芯片的制备
将硅片先用乙醇超声5分钟后，再用去离子水超声5分钟，进行三次后，在加热板上加热120分钟。
在硅片上甩涂一薄层SU-82050负性光刻胶，通过控制转速使胶层厚度大约为50μm。将具有图2结构的掩膜与硅片贴合，紫外线通过光掩模使光刻胶曝光，未曝光的部分用显影液溶解。硅片表面上凸起的SU-8结构作为PDMS盖片的阳模。然后将硅阳模硅烷化过夜。
PDMS预聚体(单体/固化剂＝10/1混合)浇注在硅阳模上并真空脱气，然后放入75℃烘箱中固化2小时左右，然后将PDMS从阳模剥离，形成了PDMS的盖片。将PDMS盖片切割，并进行打孔。
将玻璃基片与PDMS盖片一起放入氧等离子体真空管中，抽真空90秒，打开高频电源，90秒后取出基片和盖片，立即键合。
键合后在室温下用4％(v/v)MPTS的无水乙醇溶液处理通道，静置30min。再用无水乙醇进行洗涤，重复三次或至完全洗涤干净。洗涤干净后将芯片放置于100℃的烘箱中干燥1h。
在室温下将芯片用新鲜制备的0.01μmol/mL GMBS的无水乙醇溶液在室温下处理，同样静置30min。再用无水乙醇进行洗涤，重复三次或至完全洗涤干净。洗涤干净后将芯片放置于100℃的烘箱中干燥1h。
在室温下用10μg/mL链霉亲和素的PBS溶液填充通道，静置1h，之后引入浓度为1μM的生物素标记的发夹状寡核苷酸1(H1)溶液，并在室温下孵育30min，然后用PBS缓冲液洗涤以除去未结合的发夹状寡核苷酸1(H1)。修饰完成后将该芯片保存在4℃，备用。
在一个具体的实施方案中，使用修饰有FAM基团的生物素标记的发夹状寡核苷酸1(H1)修饰微通道来验证成功修饰发夹状寡核苷酸1(H1)，并用PBS缓冲液洗涤除去了未结合的捕获适配体。使用荧光显微镜拍摄微通道的照片，如图3。
实施例3：基于微流控芯片的检测系统
本实施例提供一种基于微流控芯片的检测系统，如图4所示，包括实施例1所述微流控芯片d、进样单元和检测分析单元，所述进样单元与所述微流控芯片混合微通道的进口连通。
在一个具体的实施方案中，所述检测分析单元包括PMT探测器f和超微弱化学发光分析仪g。微流控芯片大小与PMT探测器的匣子中的卡槽相匹配(25*25mm)，并使其放置后所述船形反应微通道正好与PMT探测器的进光口位置相匹配，产生的化学发光信号由PMT收集并由超微弱化学发光分析仪g分析处理。
所述进样单元包括微量注射泵a、PTFE导管c和注射器b，微流控芯片d可以通过PTFE导管c与注射器b相连，通过微量注射泵a精确的控制流量和流速，编程后自动化灌注化学发光试剂。
微流控芯片出口需由PTFE导管与废液池e相连，防止出口处的液体对PMT探测器造成损伤。
实施例4：基于微流控芯片的检测试剂盒
包括实施例1所述微流控芯片、致病性细菌的特异性核酸适配体与引发链形成的杂交链、发夹状寡核苷酸2与辣根过氧化物酶修饰的金纳米颗粒、鲁米诺以及过氧化氢。
所述致病性细菌的特异性核酸适配体3’端与引发链有若干个互补配对碱基，其中互补配对碱基数量占特异性核酸适配体序列碱基数量的1/3左右，致病性细菌的特异性核酸适配体与引发链形成杂交链(I/Apt)，致病性细菌可以结合到所述杂交链(I/Apt)中致病性细菌的特异性 核酸适配体(Apt)上，从而将催化发夹自组装反应的引发链(I)竞争下来。
所述引发链的5’端与发夹状寡核苷酸1的3’端裸露的碱基互配对。所述发夹状寡核苷酸1的5’端与发夹状寡核苷酸2的3’端裸露的碱基互补配对。催化发夹自组装反应的引发链(I)可以引发发夹状寡核苷酸1(H1)和发夹状寡核苷酸2(H2)自组装形成杂交链(H1/H2)。具体地，引发链(I)的5’端与发夹状寡核苷酸1(H1)的3’端的裸露的碱基互配对，从而打开发夹状寡核苷酸1(H1)，打开的发夹状寡核苷酸1(H1)的5’端与发夹状寡核苷酸2(H2)的3’端的裸露的碱基互补配对，从而打开发夹状寡核苷酸2(H2)，随着发夹状寡核苷酸2(H2)结合到发夹状寡核苷酸1(H1)上形成杂交链(H1/H2)，引发链(I)被竞争下来，被竞争下来的引发链(I)继续打开发夹状寡核苷酸1(H1)，如此循环直到所有发夹状寡核苷酸2(H2)都结合到发夹状寡核苷酸1(H1)上。
随着发夹状寡核苷酸2(H2)结合到发夹状寡核苷酸1(H1)上形成杂交链(H1/H2)，从而固定在微柱阵列上，金纳米颗粒(AuNP)及其上修饰的辣根过氧化物酶(HRP)也固定在了微柱阵列上。在辣根过氧化物酶的催化下可以发生化学发光反应，产生化学发光信号，从而实现对致病性细菌的检测。
实施例4：基于微流控芯片的细菌检测方法
利用实施例2制得的微流控芯片检测大肠埃希氏菌O157：H7。具体步骤如下：
(1)将待测样品，大肠埃希氏菌O157:H7特异性核酸适配体与CHA引发链形成的复合物，发夹状寡核苷酸2(H2)与辣根过氧化物酶修饰的金纳米颗粒，三者混合均匀后，注入微流控芯片的船形反应微通道内，静置，发生大肠埃希氏菌O157:H7竞争结合引发的催化发夹自组装反应。
(2)用Tris-HCl缓冲溶液冲洗微流控芯片的船型反应微通道三次。
(3)打开超微弱化学发光分析仪及分析软件，预热30min后，采集本底信号。
(4)将微流控芯片放置在带有PMT探测器的匣子中的卡槽里，用两只注射器分别吸取鲁米诺和过氧化氢，用PTFE导管与微流控芯片相连，通过精密微量注射器以10μL/min的流速向微流控芯片中灌注10μL鲁米诺和10μL过氧化氢，经混合微通道混合后流入船形反应微通道，在辣根过氧化物酶的催化下发生化学发光反应。
(5)采集从开始灌注化学发光试剂后的900s内的化学发光图谱。
(6)按照上述步骤(1)～(5)采集500，10 3，10 4，10 5，10 6，10 6，10 7，10 8CFU/mL的大肠埃希氏菌O157:H7标准样品的化学发光图谱。
(7)以大肠埃希氏菌O157:H7标准样品浓度为横坐标，以其化学发光图谱的峰值为纵 坐标绘制标准曲线，线性拟合得到标准方程，如图5。
(8)按照上述步骤(1)～(5)采集待测样品的化学发光图谱，将其化学发光图谱的峰值代入步骤(7)的标准方程，得到待测样品中大肠埃希氏菌O157:H7的浓度，如表1。
本实施例中大肠埃希氏菌O157:H7特异性核酸适配体、引发链、发夹状寡核苷酸1和发夹状寡核苷酸2的核苷酸序列如表2所示。发夹状寡核苷酸1的5’端用生物素修饰，发夹状寡核苷酸2的5’端用巯基标记。
表格1 在加标牛奶样品中检测E.coli O157:H7(N＝3)
Figure PCTCN2022080095-appb-000001
表格2 实施案例4中使用的核苷酸序列
Figure PCTCN2022080095-appb-000002
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种微流控芯片，其特征在于，所述微流控芯片由键合在一起的基片与盖片组成，所述盖片上设有混合微通道和反应微通道，所述混合微通道和所述反应微通道相互连接，所述反应微通道内具有微柱阵列。
  2. 根据权利要求1所述的微流控芯片，其特征在于，所述混合微通道为蛇形混合微通道，所述反应微通道为船形反应微通道；
    所述船形反应微通道包括两个船头和位于两个船头之间的船身，一个船头作为进口，另一个作为出口；
    所述混合微通道包括进口和出口；
    优选地，所述混合微通道包括2个进口；
    优选地，所述混合微通道出口与所述船形反应微通道的船头进口相互连接；
    优选地，所述反应微通道的长为16mm，宽为3mm；
    优选地，所述蛇形混合微通道的宽度为200μm，深度为150μm，长度为20cm；
    优选地，所述微柱的直径为200μm，深度为150μm，微柱间距为200μm；
    优选地，所述盖片的材料为聚二甲基硅氧烷。
  3. 根据权利要求1所述的微流控芯片，其特征在于，所述微柱上修饰有发夹状寡核苷酸1，所述发夹状寡核苷酸1用于发生催化发夹自组装反应；
    优选地，微柱进行链霉亲和素修饰，发夹状寡核苷酸1通过生物素和链霉亲和素的结合修饰在微柱上。
  4. 一种基于微流控芯片的检测系统，其特征在于，包括权利要求1-3任一项所述微流控芯片、进样单元和检测分析单元，所述进样单元与所述微流控芯片混合微通道的进口连通；
    优选地，所述检测分析单元包括PMT探测器和超微弱化学发光分析仪，所述PMT探测器的进光口与反应微通道相匹配；
    优选地，所述进样单元包括微量注射泵、导管和注射器；
    优选地，所述检测系统还包括与微流控芯片出口连通的废液池。
  5. 一种基于微流控芯片的检测试剂盒，其特征在于，包括权利要求1-3任一项所述微流控芯片、致病性细菌的特异性核酸适配体与引发链形成的杂交链、发夹状寡核苷酸2与辣根过氧化物酶修饰的金纳米颗粒、鲁米诺以及过氧化氢；
    所述致病性细菌的特异性核酸适配体、引发链、发夹状寡核苷酸1和发夹状寡核苷酸2满足下述条件(1)或(2)；
    (1)所述致病性细菌的特异性核酸适配体具有m个碱基，所述致病性细菌的特异性核酸适配体3’端与引发链有n个互补配对碱基，其中n/m为1/4～1/3；
    所述引发链的5’端与发夹状寡核苷酸1的3’端6～10个裸露的碱基互配对；
    所述发夹状寡核苷酸1的5’端与发夹状寡核苷酸2的3’端6～10个裸露的碱基互补配对；
    优选地，所述发夹状寡核苷酸1的5’端用生物素修饰；所述发夹状寡核苷酸2的5’端用巯基标记，辣根过氧化物酶上有氨基酸残基巯基，所述发夹状寡核苷酸2和辣根过氧化物酶通过金硫键修饰到金纳米颗上，形成发夹状寡核苷酸2与辣根过氧化物酶修饰的金纳米颗粒；
    (2)所述致病性细菌的特异性核酸适配体具有m个碱基，所述致病性细菌的特异性核酸适配体5’端与引发链有n个互补配对碱基，其中n/m为1/4～1/3；
    所述引发链的3’端与发夹状寡核苷酸1的5’端6～10个裸露的碱基互配对；
    所述发夹状寡核苷酸1的3’端与发夹状寡核苷酸2的5’端6～10个裸露的碱基互补配对；
    优选地，所述发夹状寡核苷酸1的3’端用生物素修饰；所述发夹状寡核苷酸2的3’端用巯基标记，辣根过氧化物酶上有氨基酸残基巯基，所述发夹状寡核苷酸2和辣根过氧化物酶通过金硫键修饰到金纳米颗上，形成发夹状寡核苷酸2与辣根过氧化物酶修饰的金纳米颗粒。
  6. 根据权利要求5所述的基于微流控芯片的检测试剂盒，其特征在于，所述致病性细为大肠埃希氏菌O157：H7时，所述致病性细菌的特异性核酸适配体的序列如SEQ ID NO.1所示，所述引发链的序列如SEQ ID NO.2所示，所述发夹状寡核苷酸1的序列如SEQ ID NO.3所示，所述发夹状寡核苷酸2的序列如SEQ ID NO.4所示。
  7. 权利要求1-3任一项所述微流控芯片、权利要求4所述基于微流控芯片的检测系统或权利要求5或6所述基于微流控芯片的检测试剂盒在致病性细菌检测中的应用；
    优选地，所述致病性细为食源性致病菌。
  8. 一种基于微流控芯片的细菌检测方法，其特征在于，包括如下步骤：
    将待测样品、致病性细菌的特异性核酸适配体与引发链形成的杂交链、发夹状寡核苷酸2混合均匀，注入权利要求1所述微流控芯片的反应微通道中，静置，发生致病性细菌竞争结合引发的催化发夹自组装反应；
    用缓冲溶液冲洗微流控芯片的反应微通道，采集反应微通道的本底信号；
    向权利要求1所述微流控芯片的混合微通道内分别注入鲁米诺和过氧化氢，在混合微通道内混合后注入反应微通道，在辣根过氧化物酶催化下发生化学发光反应；
    采集反应微通道的化学发光图谱，用化学发光图谱判定待测样品中致病性细菌的有无及其浓度。
  9. 根据权利要求8所述的检测方法，其特征在于，所述检测方法还包括以待测致病性细菌为标准样品，采用权利要求8所述检测方法采集本底信号和化学发光图谱，以标准样品浓度为横坐标，以其化学发光图谱的峰值为纵坐标绘制标准曲线，线性拟合得到标准方程；
    优选地，将待测样品的化学发光图谱的峰值代入标准方程，得到待测样品中致病性细菌的浓度。
  10. 根据权利要求8所述的检测方法，其特征在于，所述缓冲溶液为Tris-HCl缓冲溶液；
    所述鲁米诺和过氧化氢的体积比为1：1；
    优选地，所述鲁米诺和过氧化氢的体积均为10μL；
    优选地，所述鲁米诺和过氧化氢以10μL/min的流速注入混合微通道；
    优选地，所述致病性细为食源性致病菌；
    优选地，采用超微弱化学发光分析仪采集本底信号和化学发光图谱；
    优选地，超微弱化学发光分析仪预热30min后，再采集本底信号；
    优选地，从开始注入鲁米诺和过氧化氢后的900s内采集化学发光图谱。
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