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 PDFInfo
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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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- 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
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- 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
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- 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
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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
- 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
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- 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
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
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. a kind of pathogen immunologic detection method, which comprises the steps of:
(1) digital microcurrent-controlled chip is made；The digital microcurrent-controlled chip includes loading hole, the reagent electricity for forming reagent reservoir
Pole, 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.
2. detection method according to claim 1, which is characterized in that 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 on the base layer；The electrode layer includes the reagent electrode to form reagent reservoir, forms drop operation
The detecting electrode for transporting electrode and forming detection zone in channel；
(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, and loading hole is offered on the ITO electro-conductive glass, described
Loading hole is communicated with drop operation channel；
(2) reagent reservoir mouth is set on the upper layer micro-fluidic chip, and examination is collectively formed in the reagent reservoir mouth and reagent electrode
Agent reservoir；
Step 3: the upper layer micro-fluidic chip and lower layer's micro-fluidic chip are superimposed together up and down.
3. detection method according to claim 1, which is characterized in that set electrode layer includes in the step (1)
It forms the waste liquid electrode of waste liquid reservoir and forms the PBS electrode in the pond PBS, be provided with waste liquid reservoir on the upper layer micro-fluidic chip
Mouth and PBS Chi Kou, the waste liquid reservoir, the PBS Chi Kou and institute is collectively formed in the waste liquid reservoir mouth and the waste liquid electrode
It states PBS electrode and the pond PBS is collectively formed；The waste liquid reservoir, the pond PBS are communicated with liquid operation channel respectively.
4. detection method according to claim 2, which is characterized in that the detection zone in the digital microcurrent-controlled chip
Modified antigen, comprising:
(1) multilayer micro-nano technology technology, the surface of insulating layer deposited gold film on detecting electrode are used, and utilizes wet etching skill
Art is patterned；
(2) envelope antigen on the detecting electrode for be deposited with golden film.
5. detection method according to claim 4, which is characterized in that described to be coated on the detecting electrode for be deposited with golden film
Antigen or antibody, including,
(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 sulfhydrylization reagent that concentration is 2.0mg/ml；
(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 condition
Under 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 obtained by step (3)
Reaction solution dialyse 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, mercapto obtained in 3 μ l steps (4) is added dropwise on golden film surface to be coated with
Base antigenic solution makes it that golden film surface, 37 DEG C of incubation 1h be completely covered.
6. detection method according to claim 1, which is characterized in that the addition sample simultaneously runs digital microcurrent-controlled core
Piece is taken pictures by fluorescence detecting system CCD and detects fluorescence signal and carry out quantitative analysis, specifically:
(3.1) blood serum sample is added by the position in loading hole, blood serum sample runs logical via the liquid for transporting electrode formation
Road is delivered to one of detection zone, and blood serum sample moves back and forth between corresponding detection zone and neighbouring electrode, makes anti-in serum
Body is 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 the anti-of detection zone surface
Precursor reactant, the antibody are primary antibody, and drop intermittently moves back and forth between detection zone and adjacent electrode when incubation, promotes the identification of antibody
With combination；
(3.3) it is taken pictures by fluorescence detecting system CCD and detects fluorescence signal and carry out quantitative analysis.
7. detection method according to claim 6, which is characterized in that further include on the digital microcurrent-controlled chip to be formed it is useless
The waste liquid electrode of liquid reservoir and the PBS electrode for forming the pond PBS, the addition sample and the step of run digital microcurrent-controlled chip
(3.1) it between step (3.2), further comprises the steps of:
(a) PBS drop is generated from the pond PBS, cleans the residual sample at loading hole, waste liquid is discharged to waste liquid pool；
(b) waste liquid after reacting is transported to waste liquid pool through transporting electrode, extracts out manually through syringe；
(c) detection zone is transported to by the PBS drop that PBS reservoir generates, equally moves back and forth, washes away residual serum,
Waste liquid drains into waste liquid pool, repeats 2-3 times；
It is further comprised the steps of: between the step (3.2) and step (3.3)
(d) rinse of PBS drop is used after waste liquid discharge, repeated 2-3 times.
8. detection method according to claim 6, which is characterized in that the antigen of detection zone modification be core, NS3,
NS4 hybrid antigen, the secondary antibody mark IgG using Dylight-488.
9. a kind of digital microcurrent-controlled chip, which is characterized in that pathogen described in the claim 1-8 any one is immune
Detection method uses the digital microcurrent-controlled chip, and the digital microcurrent-controlled chip includes that the lower layer of upper and lower overlapping setting is micro-fluidic
Chip and upper layer micro-fluidic chip, lower layer's micro-fluidic chip are equipped with multiple electrodes (5), and the multiple electrode (5) includes
It is correspondingly formed the reagent electrode of reagent reservoir (10), form the detecting electrode of detection zone (12) and forms drop operation channel
Transport electrode (13)；The upper layer micro-fluidic chip is equipped with the loading hole (11) for penetrating through the upper layer micro-fluidic chip.
10. digital microcurrent-controlled chip according to claim 9, which is characterized in that the micro-fluidic chip includes basal layer
(1), the multiple electrode gap is laid in the substrate surface, is formed on the electrode insulating layer (2), the detection electricity
Surface of insulating layer on extremely is deposited with golden film (6).
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| WO2022134062A1 (en) * | 2020-12-25 | 2022-06-30 | 京东方科技集团股份有限公司 | Substrate, microfluidic device, driving method and manufacturing method |
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