CN104198469A - Bipolar electrode electrogenerated chemiluminescence paper based micro-fluidic chip and imaging sensing application thereof - Google Patents
Bipolar electrode electrogenerated chemiluminescence paper based micro-fluidic chip and imaging sensing application thereof Download PDFInfo
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- CN104198469A CN104198469A CN201410494915.2A CN201410494915A CN104198469A CN 104198469 A CN104198469 A CN 104198469A CN 201410494915 A CN201410494915 A CN 201410494915A CN 104198469 A CN104198469 A CN 104198469A
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Abstract
The invention discloses a bipolar electrode electrogenerated chemiluminescence paper based micro-fluidic chip and the application thereof to imaging sensing. The preparation method of the chip comprises the following steps: designing patterns of a micro-fluidic channel and an electrode through software and then manufacturing screens, clinging the electrode screen onto filter paper, printing the shape and the size of the electrode on the filter paper by brushing conductive carbon paste on the electrode screen, taking the filter paper on which the electrode is printed, clinging the channel screen onto filter paper, performing grinding and brushing on the channel screen through a wax pencil, then performing heating, so as to ensure that wax penetrates through the filter paper to form the micro-fluidic channel and a solution storage tank, and obtaining the bipolar electrode electrogenerated chemiluminescence paper based micro-fluidic chip. The chip takes the carbon paste as bipolar electrode material on paper for the first time, an electrogenerated chemiluminescence chip manufactured by taking the chip as a basis performs imaging sensing detection on tripropylamine, and the chip has the advantages of bipolar electrode electrochemistry and paper based microfluidics.
Description
Technical field
The invention belongs to micro-fluidic chip sensory field, be specifically related to a kind of paper substrate micro-fluidic chip and application in imaging sensing thereof of bipolar electrode electrogenerated chemiluminescence.
Background technology
In recent years, for micro-fluidic chip biochemical, that medical science detects, can significantly reduce reagent consumption, cheap, and can greatly improve analysis efficiency.Micro-fluidic chip technology obtains very fast development owing to having these advantages, in fields such as analytical chemistry, food hygiene, environmental monitoring, biology, medical science, has shown great application potential.Demand along with market, that researchist more and more seeks is cheap, small volume and less weight, simple to operate, good stability, fast, the highly sensitive micro-fluidic chip of detection speed, especially can be used in the relative poverty area of developing country and the medical treatment in resource-constrained area and detects micro-fluidic chip.
At present, Whiteside seminar proposes to make paper using as the concept of micro-fluidic backing material (being paper substrate micro-fluidic chip), to cause micro-fluidic researchers' very big concern for 2007.Paper substrate micro-fluidic chip is to utilize capillary force guiding liquids in hydrophilic paper passage, flow and distribute, and this has been avoided extra outside sample adding system, makes the operation of chip simpler and easy, facilitates.In addition, paper substrates material price is cheap, surface is easy to micro-patterning and it is compatible to have good biochemistry.Therefore, paper substrate micro-fluidic chip is expected to become light and handy portable biometric chip.In recent years, researchers can realize the analyses such as colorimetric, galvanochemistry, electrogenerated chemiluminescence, chemiluminescence on paper substrate micro-fluidic chip, and traditional expensive micro-fluidic chip is transformed into novel cheap paper substrate micro-fluidic chip.
Bipolar electrode galvanochemistry is because its unique advantage is used widely in a plurality of fields.In bipolar electrode galvanochemistry, working electrode does not directly contact with external power supply, can use a pair of drive electrode to control a plurality of working electrodes simultaneously, realizes enrichment and the screening of biochemical substances, and experimental facilities is simple.In recent years, the bipolar electrode galvanochemistry in micro-fluidic system has also obtained many researchers' concern.Up to the present, only capital seminar adopts the integrated next three kinds of Ammonia materials of separated detection of gold electrode bipolar electrode and electrophoretic techniques on paper.Yet it is expensive that this sensing detection technology has gold copper-base alloy, electrode preparation is complicated, not environmental protection, and checkout equipment requires the shortcomings such as high and expensive.
Summary of the invention
In order to overcome the defect of prior art, primary and foremost purpose of the present invention is to provide a kind of preparation method of paper substrate micro-fluidic chip of bipolar electrode electrogenerated chemiluminescence, its chip manufacture and cheap, easy, the environmental protection of electrode preparation.
Another object of the present invention is to provide the paper substrate micro-fluidic chip of the bipolar electrode electrogenerated chemiluminescence being prepared by said method, this chip combines the advantage of bipolar electrode galvanochemistry and the micro-fluidic two kinds of technology of paper substrate, its chip substrate and electrode material cheap.
The application of the paper substrate micro-fluidic chip of the bipolar electrode electrogenerated chemiluminescence that a further object of the present invention is to provide above-mentioned in imaging sensing, the desired checkout equipment of this chip is cheap, simple to operate, and applicability is wide.
Object of the present invention is achieved through the following technical solutions:
A preparation method for the paper substrate micro-fluidic chip of bipolar electrode electrogenerated chemiluminescence, comprises the following steps:
(1) use mapping software to design the shape and size of microfluidic channel and solution storage pool, then make passage web plate; Use mapping software to design the shape and size of drive electrode and working electrode, then make electrode web plate;
(2) get filter paper, electrode web plate is close on filter paper, by brush conductive carbon paste on electrode web plate, the shape and size of electrode are printed on filter paper; Brush completely, electrode web plate is toasted to 7～10min together with filter paper at 70～100 ℃, electrode resistance is tended towards stability, and then filter paper is separated with electrode web plate;
(3) get the filter paper that is printed on electrode, passage web plate is close on filter paper, with wax crayon, on passage web plate, grind brushing; After having brushed, filter paper and passage web plate are together heated to 4～6s at 70～90 ℃, wax infiltration forms microfluidic channel and solution storage pool in filter paper; Then filter paper is separated with passage web plate, obtain the paper substrate micro-fluidic chip of bipolar electrode electrogenerated chemiluminescence.
In step (1), the preferred Adobe Illustrate CS5 of described mapping software;
Described microfluidic channel is rectangle, to form uniform Electric Field Distribution in passage under drive electrode effect; Two solution storage pools are positioned at the two ends of microfluidic channel, by microfluidic channel, are communicated with; Drive electrode is positioned at solution storage pool, reduces its electric conductivity to avoid being covered by wax in step (3); Working electrode is arranged in microfluidic channel;
In step (2), filter paper should be cut into the size consistent with passage web plate, and described filter paper is No. 1 Whatman filter paper preferably.
The paper substrate micro-fluidic chip of the bipolar electrode electrogenerated chemiluminescence being prepared by said method can be used for imaging sensing, especially can be used for Ru (bpy)
3 2+the ECL system of (three-(2,2 '-dipyridine) rutheniums (II)) detects TPrA (tripropyl amine (TPA)); When it is during for detection of TPrA, comprise the following steps:
(1) with the PBS damping fluid of pH value 6.9, prepare the TPrA solution of gradient concentration and the Ru (bpy) that concentration is 2.0mM respectively
3 2+solution; Then by TPrA solution and Ru (bpy)
3 2+solution mixes with volume ratio 1:1, obtains containing Ru (bpy)
3 2+mixed solution with TPrA;
(2) micro-fluidic chip is placed in the camera bellows that includes CCD (Charge Coupled Device charge-coupled image sensor) camera, with conducting adhesive cloth, the drive electrode of chip is connected with power supply, and makes micro-fluidic chip unsettled with clip;
(3) with liquid-transfering gun, toward what drip 15～35 μ L in the microfluidic channel of micro-fluidic chip, contain Ru (bpy)
3 2+with the mixed solution of TPrA, wait for that 0.5～1.5min makes reactant liquor be full of passage; The electrode of micro-fluidic chip is applied to 12V voltage, and the electromotive force between drive electrode forms the consistent electric field of some strength in solution; Under this electric field action, the solution at working electrode and place, its two ends forms different electric potential differences, thereby working electrode two ends and solution carry out electron exchange; At the anode place of working electrode, emersion wave is about the Exocarpium Citri Rubrum coloured light into 610nm, after 3～5s luminescent image is stable, gathers image and analysis.
In step (3), question response liquid is opened constant voltage source after being full of microchannel, and voltage is transferred to 12V in advance.A little less than brownout can cause ECL luminous; Too high meeting causes working electrode anode to occur bubble and to affect ECL luminous.After 3～5s, luminescent image is stable, and operation computing machine is taken and preserved image.For each TPrA concentration, experiment at least repeats to do 5 times; Described collection image and analysis are, with Image J software, the main light emitting of every illuminated diagram is carried out to gray-scale value analysis.
The present invention has following advantage and effect with respect to prior art:
The inventive method is simple to the processing technology of chip, and experiment material used, equipment price are cheap.The present invention first on paper using carbon slurry as bipolar electrode material, and the electrogenerated chemiluminescence chip of making based on this carries out imaging sensing detection to tripropyl amine (TPA), possesses the micro-fluidic advantage of bipolar electrode galvanochemistry and paper substrate.In addition, this method is not to adopt expensive PMT, but cheap CCD is as detecting device.Based on this detecting device, the background signal on drive electrode can artificial removal, reduces object signal is disturbed.Method of the present invention is applicable to common laboratory and is convenient to promote.
Accompanying drawing explanation
Fig. 1 is the dimensional drawing of micro-fluidic chip.
Fig. 2 is the structural representation of micro-fluidic chip; Wherein, 1-chip hydrophobic region, 2-chip hydrophilic area (comprising microfluidic channel and solution storage pool), 3-paper substrates, 4, anode and the negative electrode of 5-drive electrode, 6-working electrode enlarged drawing.
Fig. 3 is imaging sensing assembling schematic diagram; Wherein, 1-chip operation electrode, 2-chip drives electrode, 3-conducting adhesive cloth, 4-cooling type CCD camera.
Fig. 4 is the image of the luminous method for sensing of the present invention.
Fig. 5 is ECL intensity TPrA concentration curve figure.
Fig. 6 is the corresponding Lg figure of the curve of Fig. 5.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1
A preparation method for the paper substrate micro-fluidic chip of bipolar electrode electrogenerated chemiluminescence, comprises the following steps:
(1) adopt Adobe Illustrate CS5 Software for Design to go out the pattern of microfluidic channel (comprising solution storage pool) and electrode (comprising working electrode and drive electrode), Bing You producer is processed to form microfluidic channel web plate and drive electrode web plate.
Microfluidic channel is of a size of 10 * 4mm, and drive electrode is of a size of 5 * 5mm, and working electrode is of a size of 3 * 1mm, and whole paper substrate micro-fluidic chip size is 30 * 12mm.Chip detailed dimensions as shown in Figure 1.
(2) first No. 1 Whatman filter paper is cut into the size that is applicable to passage web plate.Then take conductive carbon paste as material, by electrode web plate screen printing electrode on filter paper.After being completed for printing, electrode is placed on 100 ℃ of oven for baking 7～10min to electrode resistance is tended towards stability.
(3) adopt wax crayon on the filter paper of electrode, to print microfluidic channel and solution storage pool through passage web plate being printed on; After being completed for printing, filter paper and web plate are together placed on the heating plate of 85 ℃ and heat 4～6s, make wax infiltration in filter paper, form microfluidic channel and solution storage pool.
(4) by a plurality of unit chip cutting forming in batches on paper.Obtain the paper substrate micro-fluidic chip of bipolar electrode electrogenerated chemiluminescence, as shown in Figure 2.
Embodiment 2
The paper substrate micro-fluidic chip that embodiment 1 makes is for Ru (bpy)
3 2+eCL system detect TPrA, comprise the following steps:
(1) micro-fluidic chip is placed in the camera bellows that includes CCD camera, as shown in Figure 3; With conducting adhesive cloth, drive electrode is connected with power supply, and uses clip to clamp chip two ends to make it unsettled to detect;
(2) use the PBS damping fluid of pH value 6.9 by Ru (bpy)
3 2+be mixed with the solution that concentration is 2mM, with PBS damping fluid, TPrA be made into variable concentrations simultaneously, comprise 2000,1000,500,200,100,50,20 μ M.Before experiment detects, by TPrA solution and Ru (bpy)
3 2+solution mixes with volume ratio 1:1, obtains containing Ru (bpy)
3 2+mixed solution with TPrA;
(3) liquid-transfering gun is drawn the reactant liquor that 15 μ L prepare and (is contained Ru (bpy)
3 2+mixed solution with TPrA), drip in chip channel, after 0.5min, reactant liquor is full of whole microchannel.
(4) adjust constant voltage source to 12V, and be switched to drive electrode.After 3～5s, luminescent image is stable, and operation computing machine is taken and preserved image.For each TPrA concentration, at least do and repeat experiment for 5 times.
(9) luminescent image collecting (as shown in Figure 4) is analyzed by image J software, chooses every pictures main light emitting and carries out gray-scale value measurement.The data importing Origin that analysis is obtained carries out matching, obtains at certain density Ru (bpy)
3 2+lower electrogenerated chemiluminescence intensity is along with the change curve (as shown in Fig. 5-6) of TPrA concentration.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under Spirit Essence of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (7)
1. a preparation method for the paper substrate micro-fluidic chip of bipolar electrode electrogenerated chemiluminescence, is characterized in that comprising the following steps:
(1) use mapping software to design the shape and size of microfluidic channel and solution storage pool, then make passage web plate; Use mapping software to design the shape and size of drive electrode and working electrode, then make electrode web plate
(2) get filter paper, electrode web plate is close on filter paper, by brush conductive carbon paste on electrode web plate, the shape and size of electrode are printed on filter paper; Brush completely, electrode web plate is toasted to 7～10min together with filter paper at 70～100 ℃, and then filter paper is separated with electrode web plate;
(3) get the filter paper that is printed on electrode, passage web plate is close on filter paper, with wax crayon, on passage web plate, grind brushing; After having brushed, filter paper and passage web plate are together heated to 4～6s at 70～90 ℃, wax infiltration forms microfluidic channel and solution storage pool in filter paper; Then filter paper is separated with passage web plate, obtain the paper substrate micro-fluidic chip of bipolar electrode electrogenerated chemiluminescence;
Described microfluidic channel is rectangle, and two solution storage pools are positioned at the two ends of microfluidic channel, by microfluidic channel, are communicated with;
Drive electrode is positioned at solution storage pool, and working electrode is arranged in microfluidic channel.
2. the preparation method of the paper substrate micro-fluidic chip of bipolar electrode electrogenerated chemiluminescence according to claim 1, is characterized in that: described mapping software is Adobe Illustrate CS5.
3. a paper substrate micro-fluidic chip for bipolar electrode electrogenerated chemiluminescence, is characterized in that: be to be prepared by the method described in claim 1 or 2.
4. the application of paper substrate micro-fluidic chip claimed in claim 3 in imaging sensing or biochemistry detection.
5. the application of paper substrate micro-fluidic chip claimed in claim 3 in detecting TPrA.
6. the application of paper substrate micro-fluidic chip according to claim 5 in detecting TPrA, is characterized in that comprising the following steps:
(1) with the PBS damping fluid of pH value 6.9, prepare the TPrA solution of gradient concentration and the Ru (bpy) that concentration is 2.0mM respectively
3 2+solution; Then by TPrA solution and Ru (bpy)
3 2+solution mixes with volume ratio 1:1, obtains containing Ru (bpy)
3 2+mixed solution with TPrA;
(2) micro-fluidic chip is placed in the camera bellows that includes CCD camera, with conducting adhesive cloth, the drive electrode of chip is connected with power supply, and make micro-fluidic chip unsettled with clip;
(3) toward what drip 15～35 μ L in the microfluidic channel of micro-fluidic chip, contain Ru (bpy)
3 2+with the mixed solution of TPrA, wait for that 0.5～1.5min makes reactant liquor be full of passage; The electrode of micro-fluidic chip is applied to 12V voltage, and at the anode place of working electrode, emersion wave is about the Exocarpium Citri Rubrum coloured light into 610nm, after 3～5s luminescent image is stable, gathers image and analysis.
7. the application of paper substrate micro-fluidic chip according to claim 6 in detecting TPrA, is characterized in that: described collection image and analysis are, with Image J software, the main light emitting of every illuminated diagram is carried out to gray-scale value analysis.
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