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CN109557150A - Digital microcurrent-controlled chip and pathogen immunologic detection method based on it - Google Patents

Digital microcurrent-controlled chip and pathogen immunologic detection method based on it Download PDF

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CN109557150A
CN109557150A CN201910030200.4A CN201910030200A CN109557150A CN 109557150 A CN109557150 A CN 109557150A CN 201910030200 A CN201910030200 A CN 201910030200A CN 109557150 A CN109557150 A CN 109557150A
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electrode
microfluidic chip
detection
pbs
waste liquid
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王云华
郑国侠
卢玲
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Dalian University
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Dalian University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept

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  • Health & Medical Sciences (AREA)
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  • Chemical Kinetics & Catalysis (AREA)
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  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Electrochemistry (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Dispersion Chemistry (AREA)
  • Hematology (AREA)
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Abstract

The present invention relates to micro fluidic chip technical fields, and in particular to a kind of digital microcurrent-controlled chip and the pathogen immunologic detection method based on it include the following steps: that (1) makes digital microcurrent-controlled chip；The digital microcurrent-controlled chip includes loading hole, the reagent electrode for forming reagent reservoir, the detecting electrode for transporting electrode and forming detection zone for forming drop operation channel；(2) in the detection zone modified antigen of the digital microcurrent-controlled chip；(3) sample is added and runs digital microcurrent-controlled chip, is taken pictures by fluorescence detecting system CCD and detects fluorescence signal and carry out quantitative analysis.The digital microcurrent-controlled chip and pathogen immunologic detection method based on it, the reaction time is short, and detection sensitivity is high, and peripheral equipment is simple, and strong flexibility, low energy consumption；It can satisfy the demand of the occasions such as community hospital, remote diagnosis, be suitable for building portable analysis platform；Both single sample can also be can analyze, to the adaptable of sample size with batch quantity analysis.

Description

Digital microcurrent-controlled chip and pathogen immunologic detection method based on it
Technical field
The present invention relates to micro fluidic chip technical fields, and in particular to a kind of digital microcurrent-controlled chip and the cause of disease based on it Body immunologic detection method.
Background technique
Immune detection is one of most important detection means in current clinical detection, is applied to medical diagnosis on disease, pathogen is sieved It looks into, drug test etc., is also widely used in fields such as environmental monitoring, food safeties.Since the eighties in last century, automatically The introduction and improvement for changing detection technique, make it possible automation, high-throughput immunoassay, and immune detection skill is greatly facilitated The application of art.
There are cumbersome, reactions for current automation immune detection instrument slowly, time-consuming, reagent and sample consumption Greatly, the disadvantages of testing cost is high.Meanwhile assay device structures are complicated, rely on high-accuracy manufacturing technology, expensive, volume is huge Greatly, trained professional is needed to operate.In addition, current automation immune detection instrument is mainly mass detection And design, most instruments only provide 2-3 emergent detection passage, and flexibility is poor, are not suitable for the detection of field sample.With bedside Detect (point-of-care), family health care detects (Home health tests), remote diagnosis (tele-diagnosis) Etc. the proposition of concepts and the development of community hospital, existing automation immune detection instrument have been unable to meet modern medical service hair The demand of exhibition mode.And using colloidal gold technique as representative test strips detect, although easy to use, its detect target finite and Detection sensitivity is lower, and detection effect is limited.
Summary of the invention
Exempt from order to solve the above technical problems, The present invention provides a kind of digital microcurrent-controlled chip and based on its pathogen Epidemic disease detection method, the reaction time is short, and detection sensitivity is high, at low cost.
In order to reach above-mentioned technical effect, the present invention includes following technical scheme: in a first aspect, the present invention provides one kind Pathogen immunologic detection method, includes the following steps:
(1) make digital microcurrent-controlled chip: the digital microcurrent-controlled chip includes loading hole, the reagent for forming reagent reservoir Electrode, the detecting electrode for transporting electrode and forming detection zone for forming drop operation channel；
(2) in the detection zone modified antigen of the digital microcurrent-controlled chip；
(3) sample is added and runs digital microcurrent-controlled chip, is taken pictures by fluorescence detecting system CCD and detects fluorescence signal simultaneously Carry out quantitative analysis.
Further, the specific steps of the digital microcurrent-controlled chip of production include:
Step 1: production lower layer's micro-fluidic chip:
(1) electrode layer is set in substrate；The electrode layer includes the reagent electrode to form reagent reservoir, forms drop fortune The detecting electrode for transporting electrode and forming detection zone of row of channels；
(2) SiO is set on the electrode layer2Insulating layer；
Step 2: production upper layer micro-fluidic chip:
(1) the upper layer micro-fluidic chip uses ITO electro-conductive glass, offers loading hole on the ITO electro-conductive glass, The loading hole is communicated with drop operation channel；
(2) reagent reservoir mouth, the reagent reservoir mouth and reagent electrode common shape are set on the upper layer micro-fluidic chip At reagent reservoir；
Step 3: the upper layer micro-fluidic chip and lower layer's micro-fluidic chip are superimposed together up and down.
The detection zone modified antigen in the digital microcurrent-controlled chip, comprising:
(1) multilayer micro-nano technology technology, the surface of insulating layer deposited gold film on detecting electrode are used, and is carved using wet process Erosion technology is patterned；
(2) envelope antigen on the detecting electrode for be deposited with golden film.
Further, the addition sample and digital microcurrent-controlled chip is run, is taken pictures detection by fluorescence detecting system CCD Fluorescence signal simultaneously carries out quantitative analysis, specifically:
(3.1) blood serum sample is added by the position in loading hole, blood serum sample is transported via the liquid for transporting electrode formation Row of channels is delivered to one of detection zone, and blood serum sample moves back and forth between corresponding detection zone and neighbouring electrode, makes in serum Antibody sufficiently reacted with the antigen that detection zone is modified；
(3.2) detection zone is transported to by the fluorescent marker secondary antibody drop that reagent reservoir generates, and is adsorbed on detection zone surface Antibody response, which is primary antibody, and drop intermittently moves back and forth between detection zone and adjacent electrode when incubation, promotes antibody Identification and combination；
(3.3) it is taken pictures by fluorescence detecting system CCD and detects fluorescence signal and carry out quantitative analysis.
In the detection, since the mixing of reversing current acts on, the antibody in serum not will form anti-in usual ELISA detection Bulk concentration gradient difference improves the antibody concentration of liquid Yu antigen contact face, and the combination speed and joint efficiency of antigen-antibody will It greatly improves, shortens the reaction time, improve detection sensitivity.
Preferably, the antigen of the detection zone modification is core, NS3, NS4 hybrid antigen, and the secondary antibody uses Dylight-488 marks IgG.With stronger fluorescence intensity and higher stability, the use of fluorescence antibody is conducive to enhance Signal is detected, detection sensitivity is improved.
Second aspect, the present invention provides a kind of digital microcurrent-controlled chip, above-mentioned pathogen immunologic detection method is used The digital microcurrent-controlled chip, the digital microcurrent-controlled chip include that lower layer's micro-fluidic chip that upper and lower overlapping is arranged and upper layer are micro- Fluidic chip, lower layer's micro-fluidic chip are equipped with multiple electrodes, and the multiple electrode includes being correspondingly formed reagent reservoir It reagent electrode, the detecting electrode for forming detection zone and forms drop operation channel and transports electrode；The micro-fluidic core in upper layer On piece is equipped with the loading hole for penetrating through the upper layer micro-fluidic chip.
Further, the micro-fluidic chip includes basal layer, and the multiple electrode gap is laid in the basal layer table Face is formed with insulating layer on the electrode, and the surface of insulating layer on the detecting electrode is deposited with golden film.
By adopting the above technical scheme, including it is following the utility model has the advantages that digital microcurrent-controlled chip provided by the present invention and being based on Its pathogen immunologic detection method, the reaction time is short, and detection sensitivity is high, and peripheral equipment is simple, and strong flexibility, low energy consumption； Since mechanical part is few, maintenance is small, can satisfy the demand of the occasions such as community hospital, remote diagnosis, is suitable for constructing portable Analysis platform；Both single sample can also be can analyze, to the adaptable of sample size with batch quantity analysis.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of the digital microcurrent-controlled chip of pathogen immune detection provided by the present invention；
Fig. 2 is the electrode arrangement schematic diagram of the digital microcurrent-controlled chip of pathogen immune detection provided by the present invention.
In figure,
1, basal layer；2, insulating layer；3, Teflon layer；4, conductive glass layer；5, electrode；6, golden film；7, teflon pipe；8, The pond PBS；9, waste liquid reservoir；10, reagent reservoir；11, loading hole；12, detection zone；13, electrode is transported；14, first passage；15, Second channel；16, third channel.
Specific embodiment
The present invention is described in further detail below by specific embodiment and in conjunction with attached drawing.
Embodiment:
A kind of pathogen immunologic detection method is present embodiments provided, the detection method uses digital microcurrent-controlled chip, The micro-fluidic chip includes the lower layer's micro-fluidic chip and upper layer micro-fluidic chip of upper and lower overlapping setting, and the lower layer is micro-fluidic Chip is equipped with multiple electrodes, and specifically, lower layer's micro-fluidic chip includes basal layer 1, the multiple electrode gap tiling In being formed with insulating layer 2 on the substrate surface, the electrode, 2 surface of insulating layer on the detecting electrode is deposited with gold Film 6.In the present embodiment, it is preferable that the basal layer 1 is glass substrate layers, and the insulating layer is SiO2Insulating layer, the conduction Glass is ITO electro-conductive glass.
The multiple electrode include the reagent electrode for being correspondingly formed reagent reservoir 10, formed waste liquid reservoir 9 waste liquid electrode, Form the PBS electrode in the pond PBS 8, the detecting electrode for forming detection zone 12 and formation drop operation channel transports electrode 13；Institute It states upper layer micro-fluidic chip and is equipped with the loading hole 11 for penetrating through the upper layer micro-fluidic chip, the loading hole 11 covers described One on 8 place liquid of the pond PBS operation channel transports electrode.Entire lower layer's micro-fluidic chip is coated with Teflon layer 3, described The Teflon layer on 6 surface of golden film is detached from after eluting.Golden film pan coating at detection zone has HCV antigen.
The upper layer micro-fluidic chip includes conductive glass layer 4, and the conductive glass layer bottom surface is coated with Teflon layer 3, The loading hole penetrates through the conductive glass layer.Teflon pipe is inserted in the loading hole 11.In upper and lower layer micro-fluidic chip Upper coating Teflon layer can reduce sample sticking on the electrode, reduce the resistance of liquid drop movement, while advantageously reducing sample Between product a possibility that cross contamination.
Drop operation channel includes two first passage 14 for setting up two column separately, second channel 15 and connection channels Third channel 16, the first passage are that liquid where the pond PBS runs channel, and the second channel is liquid fortune where reagent Row of channels.
In order to improve the adaptability to sample size, the detection zone has 24 and respectively four column, 6 is often shown, wherein two Column are located at the two sides of the first channel, and in addition two column are located at the second channel two sides, the loading hole, the pond PBS, reagent Pond, waste liquid pool and detection zone are connected to by transporting electrode.
Pathogen immunologic detection method using above-mentioned digital microcurrent-controlled chip includes the following steps:
S1, the digital microcurrent-controlled chip of production；
Step 1: production lower layer's micro-fluidic chip:
(1) electrode layer is set in substrate；The electrode layer includes the reagent electrode to form reagent reservoir, forms drop fortune The detecting electrode for transporting electrode and forming detection zone of row of channels；
Set electrode layer includes the waste liquid electrode to form waste liquid reservoir and the PBS electrode for forming the pond PBS, the upper layer Waste liquid reservoir mouth and PBS Chi Kou are provided on micro-fluidic chip, institute is collectively formed in the waste liquid reservoir mouth and the waste liquid electrode Waste liquid reservoir is stated, the pond PBS is collectively formed in the PBS Chi Kou and the PBS electrode；The waste liquid reservoir, the pond PBS respectively with Liquid operation channel communicates.
(2) SiO is set on the electrode layer2Insulating layer；
It silica membrane insulating properties and has good stability, film layer is secured, is widely used.The present embodiment uses magnetron sputtering Method prepares SiO2 film；Cathode size (target size) can be scaled up in magnetron sputtering, and production technology is easy to amplify, and be fitted In being commercially produced.
Step 2: production upper layer micro-fluidic chip:
(1) the upper layer micro-fluidic chip uses ITO electro-conductive glass, offers loading hole on the ITO electro-conductive glass, The loading hole is communicated with drop operation channel；
The spin coating 1.5%Teflon AF1600 on ito glass, then 175 DEG C of baking 30min, keep Teflon coating solid Change；Teflon layer can reduce the resistance of liquid drop movement, while reduce the absorption of sample or reagent on the electrode, reduce pollution Possibility.
(2) reagent reservoir mouth is set on the upper layer micro-fluidic chip, is injected for reagent；The reagent reservoir mouth and examination Reagent reservoir is collectively formed in agent electrode；
Step 3: the upper layer micro-fluidic chip and lower layer's micro-fluidic chip are superimposed together up and down.
S2, in the detection zone modified antigen of the digital microcurrent-controlled chip, comprising:
S201, using multilayer micro-nano technology technology, surface of insulating layer deposited gold film on detecting electrode, and utilize wet process Lithographic technique is patterned；
Sputtering SiO2The chip surface spin coating positive photoresist of insulating layer covers the area other than detecting electrode with exposure mask Domain, uv-exposure 15s wash away the positive photoresist of exposure area (electrode) with 5%NaOH；Sputter the golden film of 300nm thickness；Whole ultraviolet exposure Light 15s washes away the positive photoresist of exposure area (detecting electrode is with exterior domain) with 5%NaOH, and the golden film sputtered in positive photoresist also can be same When washed away, only leave the golden film on electrode.
Further include processing Teflon layer: in chip surface spin coating positive photoresist, covering detecting electrode region with exposure mask, it is purple Outer exposure 15s, the positive photoresist of exposure area (detecting electrode is with exterior domain) is washed away with 5%NaOH；Spin coating 1.5%Teflon AF1600, then 175 DEG C of baking 30min, solidify Teflon coating；Whole uv-exposure 15s washes away detection with 5%NaOH Remaining positive photoresist on electrode can also be washed away simultaneously coated in the Teflon in positive photoresist, leave the golden film not covered by Teflon Surface.
S202, the envelope antigen on the detecting electrode for be deposited with golden film, specifically:
(1) antigen is diluted to 0.1mg/ml with PBS buffer solution；
(2) it takes 2.0mg hydrochloric acid mercaptan imine to be dissolved in 1.0ml distilled water, obtains the sulfhydrylation that concentration is 2.0mg/ml and try Agent；
(3) antigenic solution of 1.0ml above-mentioned steps (1) and the sulfhydrylization reagent of 25 μ l above-mentioned steps (2) are mixed, room temperature Under the conditions of be stirred to react 0.5h；
(4) PBS buffer solution of 20mM is used, wherein NaCl containing 0.15M and 1.0mM EDTA, pH 7.2, to step (3) institute Obtained reaction solution dialysis 48h, during which every 2h replaces a dialyzate, obtains sulfhydrylation antigenic solution；
(5) chip for being coated with golden film is cleaned and is dried, be added dropwise obtained in 3 μ l steps (4) on golden film surface to be coated with Sulfhydrylation antigenic solution, make it that golden film surface, 37 DEG C of incubation 1h be completely covered.
S3, sample is added and runs digital microcurrent-controlled chip, taken pictures by fluorescence detecting system CCD and detect fluorescence signal simultaneously Carry out quantitative analysis.
The addition sample simultaneously runs digital microcurrent-controlled chip, is taken pictures by fluorescence detecting system CCD and detects fluorescence signal And quantitative analysis is carried out, specifically:
S301, blood serum sample is added by the position in loading hole, blood serum sample is transported via the liquid for transporting electrode formation Row of channels is delivered to one of detection zone, and blood serum sample moves back and forth between corresponding detection zone and neighbouring electrode, makes in serum Antibody sufficiently reacted with the antigen that detection zone is modified；
S302, PBS drop is generated from the pond PBS, cleans the residual sample at loading hole, waste liquid is discharged to waste liquid pool；
Waste liquid after S303, reaction is transported to waste liquid pool through transporting electrode, extracts out manually through syringe；
S304, detection zone is transported to by the PBS drop that PBS reservoir generates, equally moves back and forth, washes away residual Serum, waste liquid drain into waste liquid pool, repeat 2-3 times；
S305, detection zone is transported to by the fluorescent marker secondary antibody drop that reagent reservoir generates, and is adsorbed on detection zone surface Antibody response, which is primary antibody, and drop intermittently moves back and forth between detection zone and adjacent electrode when incubation, promotes antibody Identification and combination；
The antigen of the detection zone modification is core, NS3, NS4 hybrid antigen, and the secondary antibody is marked using Dylight-488 Remember IgG.
The rinse of PBS drop is used after S306, waste liquid discharge, is repeated 2-3 times；
S307, it is taken pictures by fluorescence detecting system CCD and detects fluorescence signal and carry out quantitative analysis.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

Claims (10)

1.一种病原体免疫检测方法，其特征在于，包括如下步骤：1. a pathogen immune detection method, is characterized in that, comprises the steps: (1)制作数字微流控芯片；所述数字微流控芯片包括上样孔、形成试剂储池的试剂电极、形成液滴运行通道的输运电极以及形成检测区的检测电极；(1) making a digital microfluidic chip; the digital microfluidic chip includes a sample loading hole, a reagent electrode forming a reagent reservoir, a transport electrode forming a droplet running channel, and a detection electrode forming a detection area; (2)在所述数字微流控芯片的检测区修饰抗原；(2) modifying the antigen in the detection area of the digital microfluidic chip; (3)加入样本并运行数字微流控芯片，通过荧光检测系统CCD拍照检测荧光信号并进行定量分析。(3) Add the sample and run the digital microfluidic chip. The fluorescence signal is detected and quantitatively analyzed by taking pictures of the fluorescence detection system CCD. 2.根据权利要求1所述的检测方法，其特征在于，所述制作数字微流控芯片的具体步骤包括：2. The detection method according to claim 1, wherein the specific steps of making a digital microfluidic chip comprise: 步骤1：制作下层微流控芯片：Step 1: Fabrication of the lower microfluidic chip: (1)在基底层上设置电极层；所述电极层包括形成试剂储池的试剂电极、形成液滴运行通道的输运电极以及形成检测区的检测电极；(1) An electrode layer is arranged on the base layer; the electrode layer includes a reagent electrode forming a reagent reservoir, a transport electrode forming a droplet running channel, and a detection electrode forming a detection area; (2)所述电极层上设置SiO2绝缘层；(2) a SiO2 insulating layer is arranged on the electrode layer; 步骤2：制作上层微流控芯片：Step 2: Fabrication of the upper microfluidic chip: (1)所述上层微流控芯片采用ITO导电玻璃，在所述ITO导电玻璃上开设有上样孔，所述上样孔与所述液滴运行通道相通；(1) The upper-layer microfluidic chip adopts ITO conductive glass, and a sample loading hole is opened on the ITO conductive glass, and the sample loading hole is communicated with the droplet running channel; (2)所述上层微流控芯片上设置试剂储池口，所述试剂储池口和试剂电极共同形成试剂储池；(2) A reagent reservoir port is provided on the upper microfluidic chip, and the reagent reservoir port and the reagent electrode together form a reagent reservoir; 步骤3：将所述上层微流控芯片和下层微流控芯片上下叠合在一起。Step 3: Lay the upper-layer microfluidic chip and the lower-layer microfluidic chip on top of each other. 3.根据权利要求1所述的检测方法，其特征在于，所述步骤(1)中所设置的电极层包括形成废液储池的废液电极和形成PBS池的PBS电极，所述上层微流控芯片上设置有废液储池口和PBS池口，所述废液储池口和所述废液电极共同形成所述废液储池，所述PBS池口和所述PBS电极共同形成所述PBS池；所述废液储池、PBS池分别与所述液体运行通道相通。3. detection method according to claim 1, is characterized in that, the electrode layer that is set in the described step (1) comprises the waste liquid electrode that forms waste liquid storage tank and the PBS electrode that forms PBS pool, and described upper layer micro The fluid control chip is provided with a waste liquid storage pool port and a PBS pool port, the waste liquid storage pool port and the waste liquid electrode together form the waste liquid storage pool, and the PBS pool port and the PBS electrode together form the PBS pool ; The waste liquid storage tank and the PBS tank are respectively communicated with the liquid running channel. 4.根据权利要求2所述的检测方法，其特征在于，所述在所述数字微流控芯片的检测区修饰抗原，包括：4. The detection method according to claim 2, wherein the modified antigen in the detection area of the digital microfluidic chip comprises: (1)采用多层微纳加工技术，在检测电极上的绝缘层表面沉积金膜，并利用湿法刻蚀技术进行图案化；(1) Using multi-layer micro-nano processing technology, a gold film is deposited on the surface of the insulating layer on the detection electrode, and patterned by wet etching technology; (2)在沉积有金膜的检测电极上包被抗原。(2) Coat the antigen on the detection electrode deposited with the gold film. 5.根据权利要求4所述的检测方法，其特征在于，所述在沉积有金膜的检测电极上包被抗原或抗体，包括，5. detection method according to claim 4 is characterized in that, described coating antigen or antibody on the detection electrode deposited with gold film, comprises, (1)将抗原用PBS缓冲液稀释至0.1mg/ml；(1) Dilute the antigen to 0.1 mg/ml with PBS buffer; (2)取2.0mg盐酸硫醇亚胺溶于1.0ml蒸馏水中，得到浓度为2.0mg/ml的巯基化试剂；(2) get 2.0mg of thiolimide hydrochloride and dissolve it in 1.0ml of distilled water to obtain a sulfhydrylation reagent with a concentration of 2.0mg/ml; (3)将1.0ml上述步骤(1)的抗原溶液和25μl上述步骤(2)的巯基化试剂混合，室温条件下搅拌反应0.5h；(3) Mix 1.0 ml of the antigen solution in the above step (1) with 25 μl of the thiolation reagent in the above step (2), and stir for 0.5 h at room temperature; (4)用20mM的PBS缓冲液，其中含0.15M NaCl和1.0mM EDTA，pH为7.2，对步骤(3)所得到的反应液透析48h，期间每2h更换一次透析液，得到巯基化抗原溶液；(4) with 20mM PBS buffer containing 0.15M NaCl and 1.0mM EDTA, the pH is 7.2, dialyze the reaction solution obtained in step (3) for 48h, during which the dialysate is replaced every 2h to obtain a thiolated antigen solution ; (5)将镀有金膜的芯片洗净并晾干，在待包被金膜表面滴加3μl步骤(4)中所获得的巯基化抗原溶液，使其完全覆盖金膜表面，37℃温育1h。(5) Wash and dry the chip coated with gold film, drop 3 μl of the thiolated antigen solution obtained in step (4) on the surface of the gold film to be coated, so that it completely covers the surface of the gold film, and the temperature is 37°C. Breed for 1h. 6.根据权利要求1所述的检测方法，其特征在于，所述加入样本并运行数字微流控芯片，通过荧光检测系统CCD拍照检测荧光信号并进行定量分析，具体为：6. detection method according to claim 1, is characterized in that, described adding sample and running digital microfluidic chip, by fluorescence detection system CCD photographing to detect fluorescence signal and carry out quantitative analysis, be specifically: (3.1)将血清样品由上样孔的位置处加入，血清样品经由输运电极形成的液体运行通道输送至其中一个检测区，血清样品在相应检测区与邻近电极间往复运动，使血清中的抗体与检测区修饰的抗原充分反应；(3.1) The serum sample is added from the position of the sample loading hole, and the serum sample is transported to one of the detection areas through the liquid running channel formed by the transport electrode, and the serum sample reciprocates between the corresponding detection area and the adjacent electrode to make the serum The antibody fully reacts with the antigen modified in the detection zone; (3.2)由试剂储池产生的荧光标记二抗液滴输运至检测区，与吸附在检测区表面的抗体反应，该抗体为一抗，孵育时液滴在检测区与相邻电极间间歇往复运动，促进抗体的识别与结合；(3.2) The fluorescently labeled secondary antibody droplets generated by the reagent reservoir are transported to the detection area, and react with the antibody adsorbed on the surface of the detection area. Reciprocating motion to promote the recognition and binding of antibodies; (3.3)通过荧光检测系统CCD拍照检测荧光信号并进行定量分析。(3.3) The fluorescence signal was detected and quantitatively analyzed by taking pictures of the fluorescence detection system CCD. 7.根据权利要求6所述的检测方法，其特征在于，所述数字微流控芯片上还包括形成废液储池的废液电极和形成PBS池的PBS电极，所述加入样本并运行数字微流控芯片的步骤(3.1)和步骤(3.2)之间，还包括步骤：7. The detection method according to claim 6, wherein the digital microfluidic chip further comprises a waste liquid electrode forming a waste liquid storage tank and a PBS electrode forming a PBS tank, and the sample is added and the digital microfluidic chip is run. Between the step (3.1) and the step (3.2) of the microfluidic chip, further steps are included: (a)从PBS池生成PBS液滴，清洗上样孔处的残留样品，废液排出到废液池；(a) PBS droplets are generated from the PBS pool, the residual sample at the loading hole is washed, and the waste liquid is discharged to the waste liquid pool; (b)反应后的废液经输运电极运至废液池，经注射器手动抽出；(b) The reacted waste liquid is transported to the waste liquid pool through the transport electrode, and is manually drawn out by a syringe; (c)由PBS储池产生的一个PBS液滴输运至检测区，同样进行往复运动，洗去残留血清，废液排至废液池，重复2-3次；(c) A PBS droplet generated by the PBS storage tank is transported to the detection area, and the reciprocating motion is also performed to wash away the residual serum, and the waste liquid is discharged to the waste liquid pool, and repeat 2-3 times; 所述步骤(3.2)和步骤(3.3)之间还包括步骤：The step (3.2) and the step (3.3) also include steps: (d)废液排出后用PBS液滴润洗，重复2-3次。(d) Rinse with PBS droplets after draining the waste liquid, repeat 2-3 times. 8.根据权利要求6所述的检测方法，其特征在于，所述检测区修饰的抗原为core、NS3、NS4混合抗原，所述二抗使用Dylight-488标记IgG。8 . The detection method according to claim 6 , wherein the antigen modified in the detection area is a mixed antigen of core, NS3 and NS4, and the secondary antibody is labeled with IgG with Dylight-488. 9 . 9.一种数字微流控芯片，其特征在于，所述权利要求1-8任意一项中所述的病原体免疫检测方法采用所述数字微流控芯片，所述数字微流控芯片包括上下叠合设置的下层微流控芯片和上层微流控芯片，所述下层微流控芯片上设有多个电极(5)，所述多个电极(5)包括对应形成试剂储池(10)的试剂电极、形成检测区(12)的检测电极以及形成液滴运行通道的输运电极(13)；所述上层微流控芯片上设有贯通所述上层微流控芯片的上样孔(11)。9. A digital microfluidic chip, characterized in that, the method for immune detection of pathogens described in any one of claims 1-8 adopts the digital microfluidic chip, and the digital microfluidic chip comprises an upper and lower A lower-layer microfluidic chip and an upper-layer microfluidic chip are superimposed, wherein a plurality of electrodes (5) are arranged on the lower-layer microfluidic chip, and the plurality of electrodes (5) comprise correspondingly formed reagent reservoirs (10) The reagent electrode, the detection electrode that forms the detection area (12), and the transport electrode (13) that forms the droplet running channel; the upper microfluidic chip is provided with a sample loading hole ( 11). 10.根据权利要求9所述的数字微流控芯片，其特征在于，所述微流控芯片包括基底层(1)，所述多个电极间隔平铺于所述基底层表面，所述电极上形成有绝缘层(2)，所述检测电极上的绝缘层表面沉积有金膜(6)。10 . The digital microfluidic chip according to claim 9 , wherein the microfluidic chip comprises a base layer ( 1 ), the plurality of electrodes are spread on the surface of the base layer at intervals, and the electrodes An insulating layer (2) is formed thereon, and a gold film (6) is deposited on the surface of the insulating layer on the detection electrode.
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