CN106423313A - Method for detecting lactic acid by visual electrochemical luminescence sensor based on bipolar electrode array-micro-fluidic chip - Google Patents
Method for detecting lactic acid by visual electrochemical luminescence sensor based on bipolar electrode array-micro-fluidic chip Download PDFInfo
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Abstract
The invention discloses a method for detecting lactic acid by a visual electrochemical luminescence sensor based on a bipolar electrode array-micro-fluidic chip, and belongs to the technical field of biosensors. Based on lactate oxidase/carbon nano tube/chitosan modified electrode, bipolar electrode and micro-fluidic chip technology, the lactic acid can be visually detected by electrochemical luminescence imaging. As a bipolar electrode is a conductive material capable of performing electrochemical reaction at the tail end without external electric contact, bulk array analysis is conveniently performed, mass production is facilitated, and detection cost is reduced. The electrochemical luminescence imaging technology has the advantages of high sensitivity, simple light path and the like and is used for visually detecting the lactic acid, the method is simple and convenient, and results are visual.
Description
Technical field
The present invention relates to biosensor technology field, it is based on bipolar electrode array-micro-fluidic chip particularly to a kind of
Visualization electrochemical luminous sensor detect lactic acid method.
Background technology
Lactic acid is a kind of organic base, is the constituent of acetolactic precursor and lecithin, exists in neurolemma phosphorus
In fat.Lactic acid in adjustment animal body fat metabolism, control cholesterol savings, prevent fatty liver, ensure somatic normal
The aspects such as the normal operation of vital movement, promotion cartilage development and nervous system all play an important role, and its detection has weight
The biological significance wanted.
Because lactic acid does not have UV absorption and electro-chemical activity, so become the weight measuring lactic acid using the catalytic reaction of enzyme
Want method.Most lactic acid sensors use peroxidase, simultaneously by means of electron mediator or nano material.The use of enzyme can
So that the selectivity of sensor is ensured, but the fixing means of enzyme and its activity are still one to be needed to continue asking of research
Topic.The performance of sensor can be made to greatly improve additionally, introducing medium, but have also been introduced complexity and limitation simultaneously.Mesh
Front in human serum lactic acid concn detection biology sensor it has been reported that but because anti-interference is poor, the range of linearity
The weak point such as narrow, service life is short, they are restricted on clinical detection is applied.
Therefore, a kind of development selectivity is good, electrode structure is easy, with low cost novel lactic sensor will have important
Research Significance and wide market value.
Content of the invention
In order to make up the deficiencies in the prior art, the invention provides one kind is selectively good, electrode structure is easy, with low cost
Lactate detection method, the visualization electrochemical luminescence that the method for the present invention is constituted based on bipolar electrode array-micro-fluidic chip
Sensor.
The technical scheme is that：
A kind of preparation method of the visualization electrochemical luminous sensor based on bipolar electrode array-micro-fluidic chip, including step
Suddenly：
1) prepare cover plate
Balance, then above the PDMS formpiston being poured on silanization after the PDMS mother liquor degassing that PDMS monomer and curing agent mix
Cover preservative film, be heating and curing；After solidification, PDMS is peeled off from the PDMS formpiston of silanization, obtains containing channel groove
PDMS cover plate；Respectively process a duct at PDMS cover plate passage two ends, for sample introduction and placement driving electrodes；
2) prepare substrate
The preparation of A.ITO bipolar electrode substrate
Apply the thick positive optical cement glue-line of 5-15 micron in the conductive surface of ITO electro-conductive glass, by photoetching drawn for laser photocomposing machine
Mask is fitted tightly with the ITO electro-conductive glass scribbling positive optical cement glue-line, then carries out mercury lamp exposure, after exposure puts electro-conductive glass
Develop in the NaOH solution of 0.5-1wt% and after manifesting completely to electrode pattern, take out electro-conductive glass, cleaning, it is dried；Continuing will
Electro-conductive glass is placed in nitric acid and etching in the mixed aqueous solution of hydrochloric acid, after etching finishes, cleaning, and it is dried, obtain surface and be carved with three
The substrate of ITO bipolar electrode；
B. prepare LO/Carbon Nanotubes/Chitosan compound
Shitosan is dissolved in the aqueous acetic acid of 0.5-2wt%, obtains the chitosan solution of 5-15mg/mL, adds multi-wall carbon nano-tube
Manage and be dispersed in and in chitosan solution, obtain black suspension, the addition of CNT is 1-3mg/mL；Concentration is taken to be 3-
The LO solution of 8mg/mL is mixed according to volume ratio 1: 1-3 with described black suspension, obtains LO/carbon nanometer
The mixed liquor of pipe/Chitosan Composites；
The surface modification of C.ITO bipolar electrode
With hydrophobic adhesive tape, one rectangle for preventing the diffusion of decorative material solution is pasted on the anode tap of described ITO bipolar electrode
Region, pipettes 10-30 microlitre of LO/Carbon Nanotubes/Chitosan solution and drips in rectangular area surface；Place the substrate in 4
In DEG C refrigerator, dried overnight is to form the ITO bipolar electrode that LO/Carbon Nanotubes/Chitosan complex thin film is modified；
3) chip sealing
Clean substrate and the cover plate of above-mentioned preparation with plasma cleaning device, three ITO bipolar electrode be aligned cover plates on substrate are led to
The middle part of road groove, then fits tightly, and obtains lactic acid electrochemical luminous sensor chip.
Preferably, step 1) in, in PDMS mother liquor, PDMS monomer and the mass ratio of curing agent are 8: 1-12: 1；
Place 5-20min under 4 DEG C of environment before the degassing of PDMS mother liquor；Degassing time is 20-40min.
Preferably, step 1) in, PDMS mother liquor equilibration time above the PDMS formpiston of silanization is 5-
15min.
Preferably, step 1) in, the duct at PDMS cover plate passage two ends is circular duct.
Preferably, step 2) in, before ITO conductive glass surface applies positive optical cement, ITO electro-conductive glass is carried out
Pretreatment；Described pretreatment is specially and for ITO electro-conductive glass to be placed in soaked overnight in the ethanol solution of 0.8-1.2M NaOH, it
It is cleaned by ultrasonic 10-20min in acetone, absolute ethyl alcohol and deionized water respectively afterwards, clean ITO electro-conductive glass nitrogen is blown
Dry.
Preferably, step 2) in A, with glue head dropper draw after positive optical cement uniformly drop coating in ITO electro-conductive glass
Conductive surface, using photoresist spinner spin coating under 400-600rpm rotating speed, then rotates 30-50s under 2500-3500rpm rotating speed
Whirl coating；Afterwards ITO electro-conductive glass is placed under 105-115 DEG C of environment and dries, then slowly cool on 25-30 DEG C of hot plate
Room temperature, forms positive optical cement glue-line in uniform thickness.
Preferably, step 2) in A, the time of mercury lamp exposure is 2-6min；Electro-conductive glass is placed in nitric acid and salt
6-15min is etched in the mixed aqueous solution of acid, wherein, in the mixed aqueous solution of nitric acid and hydrochloric acid, the quality of nitric acid, hydrochloric acid and water
Than for 2-3: 4-6: 4-6.
Using described, lactic acid is detected based on the visualization electrochemical luminous sensor of bipolar electrode array-micro-fluidic chip
Method, places a platinum filament, as driving electrodes on the duct at cover plate two ends respectively；Driving electrodes pass through D.C. regulated power supply
Driving voltage is provided；The cushioning liquid containing lactic acid solution to be measured and luminol for the injection into channel groove；Bipolar electrode surface
LO catalysis dissolved oxygen and the lactic acid modified react generation H2O2, and under alive driving outside, occur with luminol
Electrochemical luminescence, the electrochemical luminescence signals of generation pass through the ccd image sensing above lactic acid electrochemical luminous sensor chip
Device captures；Lactic acid concn in lactic acid solution to be measured is determined according to the luminous signal intensity of ccd image sensor capture.
Preferably, into channel groove, 15-30 microlitre of injection contains the buffering of lactic acid solution to be measured and luminol
Solution；Described cushioning liquid is PBS cushioning liquid；The concentration of PBS in cushioning liquid containing lactic acid solution to be measured and luminol
For 0.05-0.15mol/L, pH is 7.2-7.5；The concentration of lactic acid is 0.01mM-1mM；The concentration of luminol is 1.2-
1.4mmol/L.Liquor capacity used by present invention detection is only 15-30 microlitre, and achievable trace detection greatly improves detection
Sensitivity, and reduce testing cost.
Preferably, described driving voltage is 4.0-9.0V.Overtension may ito film can be destroyed, therefore electricity
Pressure selects this scope.
Idea of the invention is that：
With the complex film modified ITO bipolar electrode of LO/Carbon Nanotubes/Chitosan as sensing interface, by micro-fluidic
Chip system, platinum filament driving electrodes, D.C. regulated power supply and ccd image sensor realize the visualization electrochemical luminescence inspection of lactic acid
Survey.Driving electrodes provide driving voltage by D.C. regulated power supply.Into lactic acid electrochemical luminous sensor chip channel groove
Inject lactic acid solution to be measured and the cushioning liquid containing luminol, the LO of bipolar electrode surface modification can be catalyzed dissolving
Oxygen and lactic acid react generation H2O2, and under alive driving outside, there is electrochemical luminescence with luminol.The electrochemistry producing
Luminous signal is captured by the ccd image sensor above sensing chip.Within the specific limits, lactic acid concn is higher, enzymatic reaction
The hydrogen peroxide generating is more, and corresponding luminous signal is also stronger, thus can realize the Visual retrieval of lactic acid.
The present invention visualizes electrochemical luminous sensor and detects that the measuring principle of lactic acid is：
Catalytic reaction on bipolar electrode anode tap modified membrane：
Lactic acid and oxygen generate pyruvic acid and hydrogen peroxide in the presence of LO；
The electrochemical reaction of bipolar electrode anode tap：
Luminol+H2O2→3-Aminophthalate+hv
Luminol and hydrogen peroxide react generation 3- amino-repefral and with luminous；
The electrochemical reaction of bipolar electrode cathode terminal：
4H2O+4e-→2H2+4OH-
The present invention is based on LO/Carbon Nanotubes/Chitosan modified electrode, bipolar electrode and microfluidic chip technology, profit
Electricity consumption chemiluminescence imaging is realizing the Visual retrieval of lactic acid.Because bipolar electrode is that one kind does not need external electrical contact
There is the conductive material of electrochemical reaction in its end, this characteristic makes it easy to carry out a large amount of array analysis, beneficial to high-volume
Produce, thus reducing testing cost.
Using electrochemical luminescence imaging technique have the advantages that sensitivity is high, light path is simple, for the visualization of lactic acid
Detection, method is easy, visual result.
Electrode modified material LO/Carbon Nanotubes/Chitosan in the present invention, LO LO,
CNT, shitosan are indispensable, have LO that catalytic reaction just can be had to produce hydrogen peroxide, and then send out with luminol
Hair tonic light reaction；CNT is to promote electrochemical reaction, not having CNT electrochemical reaction to be obstructed, so also not having
Light produces；Shitosan is filmogen, and without shitosan, LO, carbon nano-tube solution cannot stably be attached to
Electrode surface.
Beneficial effects of the present invention are：
Preparation method of the present invention is simple and convenient, and LO/Carbon Nanotubes/Chitosan composite membrane can make LO fix
In electrode surface, there are preferable film forming and biocompatibility；CNT promotes Electron Transfer, and shitosan promotes film forming；
This sensor has that specificity is good, sensitivity is high, selective advantage, and material used is cheap and easy to get.
Open bipolar electrode due to there is no external electrical contact, in a microchannel, luminescence reagent and detected material
Can be blended in a solution, large scale array analysis can be carried out, fast high-flux detects lactic acid.
Microfluidic chip structure is simple, and preparation is simple, and with low cost, integrated micro, beneficial to production in enormous quantities, substantially reduces
Testing cost, is expected to be applied to the exploitation of lactic acid test paper.
The high sensitivity of electrochemical luminescence imaging technique, time and space is differentiated, the optics of simplification, and visual imaging energy
Power visualizes, and the content for detecting lactic acid provides a sensitive, easy and intuitively Sparklet testing method.
This preparation method can be generalized to other types enzyme, for the detection of other biochemical moleculars, can develop multicomponent
The bipolar electrode sensor of detection.
Brief description
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
Have technology description in required use accompanying drawing be briefly described it should be apparent that, drawings in the following description be only this
Some embodiments of invention, for those of ordinary skill in the art, on the premise of not paying creative work, acceptable
Other accompanying drawings are obtained according to these accompanying drawings.
Fig. 1 is the preparation based on the visualization electrochemical luminous sensor of bipolar electrode array-micro-fluidic chip for the present invention
The device of method and reaction principle schematic diagram；
Fig. 2 is the structural representation of ITO bipolar electrode array of the present invention；
Fig. 3 is the machining sketch chart of ITO bipolar electrode array chip of the present invention
Fig. 4 is the electrochemical luminescence figure of lactate detection of the present invention；Wherein, a corresponding concentration 0rnM；B corresponding concentration 0.01mM；C pair
Answer concentration 0.05mM；D corresponding concentration 0.1mM；E corresponding concentration 0.2mM；F corresponding concentration 0.5mM；G corresponding concentration 1.0mM.
Fig. 5 is the linear graph of lactate detection of the present invention.
Specific embodiment
The preparation method based on the visualization electrochemical luminous sensor of bipolar electrode array-micro-fluidic chip for the embodiment 1
(preparation of the visualization lactic acid sensor based on bipolar electrode array)
As shown in Figure 1, Figure 2, Figure 3 shows, a kind of visualization electrochemical luminous sensor based on bipolar electrode array-micro-fluidic chip
The method of detection lactic acid, including step：
1) prepare cover plate
By PDMS monomer and curing agent with 8: 1-12: 1 mass ratio is mixed and stirred for uniform PDMS mother liquor, by PDMS mother liquor in
5-20min is placed, then deaerate 20-40min under 4 DEG C of environment；It is poured on balance 5- above the PDMS formpiston of silanization after degassing
15min, then covers preservative film, the 70-80 DEG C of 1-2 hour that is heating and curing；After solidification, by PDMS from the PDMS formpiston of silanization
Peel off, be cut into the cover plate of 3.0cm*1.2cm, this cover plate contains channel groove；With the flat mouth syringe needle of 14G in PDMS cover plate passage
Two ends respectively process the circular duct of an a diameter of 0.5cm, for sample introduction and placement driving electrodes；
2) prepare substrate
A. there is the preparation of three ITO bipolar electrode substrates
ITO electro-conductive glass is cut into the small pieces of 3.0cm*1.2cm；The ITO electro-conductive glass of well cutting is placed in 0.8-1.2M
Soaked overnight in the ethanol solution of NaOH, is cleaned by ultrasonic 10-20min afterwards respectively in acetone, absolute ethyl alcohol and deionized water,
Clean ITO electro-conductive glass nitrogen is dried up；Determine conducting surface with universal meter, conduction faces up and is placed on photoresist spinner disk；
With glue head dropper draw after the positive optical cement of AZP4620 uniformly drop coating in the conductive surface of ITO electro-conductive glass, in 400-600rpm rotating speed
Lower spin coating, then rotates 30-50s whirl coating under 2500-3500rpm rotating speed；The conductive surface staying in ITO electro-conductive glass applies 5-15
After the thick red light glue-line of micron, ITO electro-conductive glass is placed under 105-115 DEG C of environment and dries, then in 25-30 DEG C of hot plate
On be slowly cooled to room temperature, formed positive optical cement glue-line in uniform thickness.
Photo etched mask drawn for laser photocomposing machine is fitted tightly with the ITO electro-conductive glass scribbling positive optical cement glue-line, so
After carry out selective mercury lamp exposure 2-6min, after exposure, electro-conductive glass is placed in the NaOH solution of 0.5-1wt% and develops to electricity
Pole figure case takes out electro-conductive glass after manifesting completely；Cleaning removes remaining developer solution in deionized water, is subsequently blown with hair-dryer
Dry electro-conductive glass, then dries 30min, baking terminates for hot plate temperature to be adjusted to 20-25 DEG C, slow on hot plate under 110 DEG C of hot plates
Slowly it is down to room temperature.
Continue by electro-conductive glass be placed in nitric acid and hydrochloric acid mixed aqueous solution (mass ratio of nitric acid, hydrochloric acid and water is 2-3:
Etching 6-15min in 4-6: 4-6), after etching finishes, deionized water, acetone, deionized water are cleaned by ultrasonic each 10min successively,
It is dried, obtain the substrate that three ITO bipolar electrodes are carved with surface；
B. prepare LO/Carbon Nanotubes/Chitosan compound
Shitosan is dissolved in the aqueous acetic acid of 0.5-2wt%, ultrasonic dissolution, obtains the chitosan solution of 5-15mg/mL, adds many
Wall carbon nano tube ultrasonic disperse in chitosan solution black suspension, the addition of CNT is 1-3mg/mL；Take
Concentration is that the LO solution of 3-8mg/mL is mixed according to volume ratio 1: 1-3 with described black suspension, and ultrasonic dissolution obtains
The mixed liquor of LO/Carbon Nanotubes/Chitosan Composites, 4 DEG C of preservations；
The surface modification of C.ITO bipolar electrode
With hydrophobic adhesive tape, one rectangle for preventing the diffusion of decorative material solution is pasted on the anode tap of described ITO bipolar electrode
Region, pipettes 10-30 microlitre of LO/Carbon Nanotubes/Chitosan solution and drips in rectangular area surface；Chip is placed in 4
In DEG C refrigerator, dried overnight is to form the ITO bipolar electrode that LO/Carbon Nanotubes/Chitosan complex thin film is modified；
3) chip sealing
The cover plate modified and substrate are respectively placed in plasma clean interior, each process 90 seconds and 25 seconds.By on substrate three
Root ITO bipolar electrode is directed at the middle part of cover plate channel groove, then fits tightly, obtains lactic acid electrochemical luminous sensor chip.
Embodiment 2 is used for lactic acid based on the visualization electrochemical luminous sensor of bipolar electrode array-micro-fluidic chip
The method using detecting lactic acid based on the visualization electrochemical luminous sensor of bipolar electrode array-micro-fluidic core for the detection, in lid
A piece platinum filament is placed on the duct at piece two ends, as driving electrodes respectively；Driving electrodes provide 8V's by D.C. regulated power supply
Driving voltage；Into channel groove, 20 microlitres of injection contains the cushioning liquid of lactic acid solution to be measured and luminol, wherein, PBS's
Concentration is 0.01mol/L, and pH is 7.4, and the concentration of lactic acid is 0.02mM-5mM, and the concentration of luminol is 1.3mmol/L；Bipolar electric
LO catalysis dissolved oxygen and the lactic acid of pole surface modification react generation H2O2, and under alive driving outside, with Rumi
There is electrochemical luminescence in promise, the electrochemical luminescence signals of generation pass through the CCD figure above lactic acid electrochemical luminous sensor chip
As sensor capture；Lactic acid concn in lactic acid solution to be measured is determined according to the luminous signal intensity of ccd image sensor capture.
Respectively the lactic acid of 0mM, 0.01mM, 0.05mM, 0.1mM, 0.2mM, 0.5mM and 1.0mM series concentration is examined
Survey, testing result is as shown in Figure 4 and Figure 5；The inventive method detects that the minimum detectability of lactic acid is 8.25 μM；With lactic acid concn
Rising, the gray value of luminescent image is also gradually increased.By linear fit, gray value and lactic acid concn are in 0.01-
There is good linear relationship in the range of 1.0mM.Gained fit equation is：
G=854+13626 [lactate] (mM) (R=0.9705).
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to aforementioned reality
Apply example the present invention has been described in detail, those skilled in the art are said, it still can be to aforementioned each enforcement
Technical scheme described in example is modified, or carries out equivalent to wherein some technical characteristics.All essences in the present invention
Within god and principle, any modification of being made, equivalent.Improve etc., should be included within the scope of the present invention.
Claims (10)
1. a kind of preparation method of the visualization electrochemical luminous sensor based on bipolar electrode array-micro-fluidic chip, it is special
Levy and be, including step：
1) prepare cover plate
Balance, then above the PDMS formpiston being poured on silanization after the PDMS mother liquor degassing that PDMS monomer and curing agent mix
Cover preservative film, be heating and curing；After solidification, PDMS is peeled off from the PDMS formpiston of silanization, obtains containing channel groove
PDMS cover plate；Respectively process a duct at PDMS cover plate passage two ends, for sample introduction and placement driving electrodes；
2) prepare substrate
The preparation of A.ITO bipolar electrode substrate
Apply the thick positive optical cement glue-line of 5-15 micron in the conductive surface of ITO electro-conductive glass, by photoetching drawn for laser photocomposing machine
Mask is fitted tightly with the ITO electro-conductive glass scribbling positive optical cement glue-line, then carries out mercury lamp exposure, after exposure puts electro-conductive glass
Develop in the NaOH solution of 0.5-1wt% and after manifesting completely to electrode pattern, take out electro-conductive glass, cleaning, it is dried；Continuing will
Electro-conductive glass is placed in nitric acid and etching in the mixed aqueous solution of hydrochloric acid, after etching finishes, cleaning, and it is dried, obtain surface and be carved with three
The substrate of ITO bipolar electrode array；
B. prepare LO/Carbon Nanotubes/Chitosan compound
Shitosan is dissolved in the aqueous acetic acid of 0.5-2wt%, obtains the chitosan solution of 5-15mg/mL, adds multi-wall carbon nano-tube
Manage and be dispersed in and in chitosan solution, obtain black suspension, the addition of CNT is 1-3mg/mL；Concentration is taken to be 3-
The LO solution of 8mg/mL is mixed according to volume ratio 1: 1-3 with described black suspension, obtains LO/carbon nanometer
The mixed liquor of pipe/Chitosan Composites；
The surface modification of C.ITO bipolar electrode
With hydrophobic adhesive tape, one rectangle for preventing the diffusion of decorative material solution is pasted on the anode tap of described ITO bipolar electrode
Region, pipettes 10-30 microlitre of LO/Carbon Nanotubes/Chitosan solution and drips in rectangular area surface；Place the substrate in 4
In DEG C refrigerator, dried overnight is to form the ITO bipolar electrode that LO/Carbon Nanotubes/Chitosan complex thin film is modified；
3) chip sealing
Clean substrate and the cover plate of above-mentioned preparation with plasma cleaning device, three ITO bipolar electrode be aligned cover plates on substrate are led to
The middle part of road groove, then fits tightly, and obtains lactic acid electrochemical luminous sensor chip.
2. the system of the visualization electrochemical luminous sensor based on bipolar electrode array-micro-fluidic chip as claimed in claim 1
Preparation Method it is characterised in that：Step 1) in, in PDMS mother liquor, PDMS monomer and the mass ratio of curing agent are 8: 1-12: 1；PDMS
Place 5-20min under 4 DEG C of environment before mother liquor degassing；Degassing time is 20-40min.
3. as claimed in claim 1 or 2 visualization electrochemical luminous sensor based on bipolar electrode array-micro-fluidic chip
Preparation method it is characterised in that：Step 1) in, PDMS mother liquor equilibration time above the PDMS formpiston of silanization is 5-15min.
4. as claimed in claim 1 or 2 visualization electrochemical luminous sensor based on bipolar electrode array-micro-fluidic chip
Preparation method it is characterised in that：Step 1) in, the duct at PDMS cover plate passage two ends is circular duct.
5. the system of the visualization electrochemical luminous sensor based on bipolar electrode array-micro-fluidic chip as claimed in claim 1
Preparation Method it is characterised in that：Step 2) in, before ITO conductive glass surface applies positive optical cement, ITO electro-conductive glass is carried out pre-
Process；Described pretreatment is specially and ITO electro-conductive glass is placed in soaked overnight in the ethanol solution of 0.8-1.2M NaOH, afterwards
It is cleaned by ultrasonic 10-20min in acetone, absolute ethyl alcohol and deionized water respectively, clean ITO electro-conductive glass nitrogen is blown
Dry.
6. the visualization electrochemical luminous sensor based on bipolar electrode array-micro-fluidic chip as described in claim 1 or 5
Preparation method it is characterised in that：Step 2) in A, draw after positive optical cement uniformly drop coating leading in ITO electro-conductive glass with glue head dropper
Ammeter face, using photoresist spinner spin coating under 400-600rpm rotating speed, then rotates 30-50s under 2500-3500rpm rotating speed and gets rid of
Glue；Afterwards ITO electro-conductive glass is placed under 105-115 DEG C of environment and dries, then room is slowly cooled on 25-30 DEG C of hot plate
Temperature, forms positive optical cement glue-line in uniform thickness.
7. the visualization electrochemical luminous sensor based on bipolar electrode array-micro-fluidic chip as described in claim 1 or 5
Preparation method it is characterised in that：Step 2) in A, the time of mercury lamp exposure is 2-6min；Electro-conductive glass is placed in nitric acid and hydrochloric acid
Mixed aqueous solution in etch 6-15min, wherein, in the mixed aqueous solution of nitric acid and hydrochloric acid, the mass ratio of nitric acid, hydrochloric acid and water
For 2-3: 4-6: 4-6.
8. using the visualization electrochemical luminous sensor based on bipolar electrode array-micro-fluidic chip as claimed in claim 1
Detection lactic acid method it is characterised in that：A piece platinum filament is placed on the duct at cover plate two ends, as driving electrodes respectively；Drive
Moving electrode provides driving voltage by D.C. regulated power supply；Into channel groove, injection is containing lactic acid solution to be measured and luminol
Cushioning liquid；LO catalysis dissolved oxygen and the lactic acid of bipolar electrode surface modification react generation H2O2, and in applied voltage
Driving under, there is electrochemical luminescence with luminol, the electrochemical luminescence signals of generation pass through lactic acid electrochemical luminous sensor
Ccd image sensor capture above chip；Determine that lactic acid to be measured is molten according to the luminous signal intensity of ccd image sensor capture
Lactic acid concn in liquid.
9. the visualization electrochemical luminous sensor detection based on bipolar electrode array-micro-fluidic chip as claimed in claim 8
The method of lactic acid it is characterised in that：Into channel groove, 15-30 microlitre of injection contains the buffering of lactic acid solution to be measured and luminol
Solution；Described cushioning liquid is PBS cushioning liquid；The concentration of PBS in cushioning liquid containing lactic acid solution to be measured and luminol
For 0.05-0.15mol/L, pH is 7.2-7.5；The concentration of lactic acid is 0.01mM-1mM；The concentration of luminol is 1.2-
1.4mmol/L.
10. the visualization electrochemical luminous sensor based on bipolar electrode array-micro-fluidic chip as described in claim 8 or 9
Detection lactic acid method it is characterised in that：Described driving voltage is 4.0-9.0V.
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