CN103041876B - Preparation of electrochemical three-dimensional microfluidic paper chip and application of electrochemical three-dimensional microfluidic paper chip to field test - Google Patents
Preparation of electrochemical three-dimensional microfluidic paper chip and application of electrochemical three-dimensional microfluidic paper chip to field test Download PDFInfo
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- CN103041876B CN103041876B CN201210577821.2A CN201210577821A CN103041876B CN 103041876 B CN103041876 B CN 103041876B CN 201210577821 A CN201210577821 A CN 201210577821A CN 103041876 B CN103041876 B CN 103041876B
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Abstract
The invention discloses a preparation method of an electrochemical three-dimensional microfluidic paper chip with high flux, low cost and simplicity in operation, and an application method of the electrochemical three-dimensional microfluidic paper chip to a field test. According to the invention, hydrophobic wax patterns are printed on three pieces of common filter paper with the A4 size respectively in batches by a full-printing-level adhesion piling mode. The preparation process comprises the following steps: printing the hydrophobic wax patterns in batches; melting wax and forming; screen-printing an array working electrode, a reference electrode and a counter electrode on the filter paper in batches; printing horse radish peroxidase and oxidase on the surface, without the working electrode, of the filter paper sequentially in batches; cutting the electrochemical three-dimensional microfluidic paper chip; preparing a double-sided adhesive tape sheet; adhering the filter sheets through the double-sided adhesive tape sheet; and preparing an electrochemical three-dimensional microfluidic paper chip clip. A method for performing field test on the electrochemical three-dimensional microfluidic paper chip comprises the following steps: clamping the paper chip by using the electrochemical three-dimensional microfluidic paper chip clip; inserting into six copper finger slots; and connecting an electrochemical work station through a multiplexer.
Description
Technical field
The present invention relates to the instant detection technique field, scene of low cost, high flux, high sensitivity, high specific, more specifically say a kind of to be suitable for the structure of the micro-fluidic paper chip lab of high-flux electric enzyme analysis.
Background technology
Paper be a kind ofly dirt cheap, abundant material, and test strips is also widely used in the fields such as simple medical diagnosis on disease, environment measuring as a kind of analysis platform (such as immunochromatography), such as very early pregnancy test strips etc.The Whiteside seminar of Ha Fu university chemistry institute has proposed the new ideas of paper chip lab first, is referred to as again micro-fluidic refill sheet analysis device.Micro-fluidic refill sheet analysis device refers to by draw hydrophobic pattern on paper, and therefore hydrophilic very capable due to paper do not draw hydrophobic pattern part and just form hydrophilic channel/region, and by the REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power of paper, solution can flow at paper passage interior orientation.Based on this, the basic operation units such as the sample preparation of biological, chemistry, medical analysis process, reaction, separation, detection are integrated on paper, form micro-fluidic refill sheet analysis device.The fields such as the medical diagnosis on disease at the scene of this technology, on-the-spot food analysis and site environment monitoring have broad application prospects.On the other hand, compare with traditional Microfluid based Lab on a chip being based upon at the bottom of plastic sheet, sheet glass or silicon wafer-based, micro-fluidic paper chip lab has that cost is low, preparation method simply (utilizes simple printing technique, and without harsh conditions such as dust free rooms), (without additional equipment, as pump etc.) simple to operate, the advantage such as can process arbitrarily after using.
At present, the analytical method being based upon on micro-fluidic refill sheet analysis device is mainly colorimetric method, and because colorimetric method can only provide the signal response of " Yes/No ", and colorimetric method sensitivity is lower, selectively poor, is prone to result false positive.Therefore in micro-fluidic paper chip lab, set up the analytical method of high sensitivity, high selectivity, the on-the-spot instant analysis detection that realizes high sensitivity, high specific just becomes current this research field and needs one of problem of solution badly.In addition, in order to carry out more accurately disease early diagnosis, disorder in screening and assessment and medicine response, often need to monitor the content of various ingredients in sample simultaneously.Therefore explore and set up high-throughout micro-fluidic refill sheet and contribute to address this problem.
Summary of the invention
The technical problem to be solved in the present invention be on micro-fluidic refill sheet, set up have that sample treatment is simple, detection speed is fast, cost is low, the electrochemical enzymatic analyzing detecting method of the feature such as highly sensitive, high specificity.Further build the integrated three-dimensional microflow control refill of high flux sheet, and detect for sample glucose, cholesterol, uric acid and ferroheme time.
In order to solve the problems of the technologies described above, the present invention realizes by building the micro-fluidic refill sheet of a kind of novel three Dimensional Electrochemical, and the preparation method of the micro-fluidic refill sheet of this three Dimensional Electrochemical is:
(1) design on computers three hydrophobic wax bulk print patterns of the micro-fluidic refill sheet of three Dimensional Electrochemical, be respectively wax bulk print pattern A (pattern as shown in Figure 1), wax bulk print pattern B (pattern is as shown in Figure 2), wax bulk print pattern C (pattern is as shown in Figure 3).
(2) the array working electrode that design is mated with wax bulk print pattern C is on computers printed patterns (pattern as shown in Figure 4) in batches.
(3) the reference electrode batch printed patterns (pattern as shown in Figure 5) corresponding with wax bulk print pattern B of design on computers.
(4) design is on computers corresponding to hydrophobic wax pattern B electrode batch printed patterns (pattern as shown in Figure 6).
(5) the horseradish peroxidase bulk print pattern (pattern is with accompanying drawing 4) that design is mated with wax bulk print pattern C on computers.
(6) the oxidizing ferment bulk print pattern (pattern is respectively as shown in accompanying drawing 7, accompanying drawing 8, accompanying drawing 9 and accompanying drawing 10) that design is mated with wax bulk print pattern C on computers, a kind of oxidizing ferment bulk print pattern is only used for printing a kind of oxidizing ferment.
(7) filter paper is cut into the filter paper of the required conventional A4 size of three printers.
(8) the A4 filter paper cutting out in step (7) is placed in wax spray printer, three hydrophobic wax bulk print patterns of the design in step (1) are printed on three A4 filter paper in step (7), obtain respectively filter paper A, filter paper B, filter paper C.
(9) all A4 filter paper with wax pattern in step (8) are placed in panel heater or baking oven, under 60-150 ℃ degree Celsius, heat 0.5-2 minute.Wax melted and soak into the thickness of whole paper, forming hydrophobic wall (principle is as accompanying drawing 11).
(10) adopt method for printing screen, the array working electrode batch printed patterns according in step (2), is printed onto array working electrode on the filter paper C obtaining in step (9).
(11) adopt method for printing screen, the reference electrode batch printed patterns according in step (3), is printed onto reference electrode on the filter paper B obtaining in step (9).
(12) adopt method for printing screen, according in step (4) to electrode printed patterns in batches, will be printed onto on the filter paper B obtaining in step (11) electrode.
(13) the filter paper C of preparation in step (10) is put into ink-jet printer.According to the horseradish peroxidase bulk print pattern in step (5), by horseradish peroxidase China ink print to filter paper C in step (10) without on working electrode face.
(14) the filter paper C of preparation in step (13) is put into ink-jet printer.According to the oxidizing ferment bulk print pattern in step (6), successively four kinds of oxidizing ferment China inks are printed to respectively filter paper C in step (13) without on working electrode face.
(15) the filter paper C of preparation in the filter paper B of preparation in the filter paper A of preparation in step (9), step (12), step (14) is carried out to cutting along wax pattern outward flange, obtain respectively filter paper A (pattern as shown in Figure 12), filter paper B (pattern is as shown in Figure 13), filter paper C (pattern is as shown in Figure 14).
(16) get common double face plastic adhesive tape, be cut into the double faced adhesive tape strap A with the middle filter paper formed objects of step (15).Then, scratch four holes in relevant position, the size in hole is consistent with paper channel width in filter paper A, the end (pattern as shown in Figure 15) of four paper passages in four corresponding filter paper A of hole difference.
(17) by the double faced adhesive tape strap A obtaining in step (16), filter paper A and filter paper B are bonded together (stack manner as shown in Figure 16).
(18) get common double face plastic adhesive tape, be cut into the double faced adhesive tape strap B with the middle filter paper formed objects of step (15).Then, scratch 16 holes in relevant position, the size in hole is consistent with paper channel width in filter paper B, 16 Electrochemical Detection holes (pattern is with accompanying drawing 14) in 16 corresponding filter paper C of hole difference.
(19), by the double faced adhesive tape strap B obtaining in step (16), will in step (17), obtain multi-layer filter paper sheet and filter paper C is bonded together (stack manner as shown in Figure 16).
(20) get common one side plastic adhesive tape, be cut into the one side adhesive tape sheet with the middle filter paper formed objects of step (15).Then, in center, scratch a hole, the size in hole, position consistent with paper channel width in filter paper A (pattern as shown in Figure 17).
(21) the one side adhesive tape sheet obtaining in step (20) is attached on the filter paper A of multi-layer filter paper sheet in step (19) to the micro-fluidic refill sheet of three Dimensional Electrochemical (stack manner as shown in Figure 16) obtaining.
(22) design on computers the wire depiction (pattern as shown in Figure 18) of the micro-fluidic paper chip gripper of three Dimensional Electrochemical.
(23) utilize circuit board engraving machine, according to the pattern of design in step (22), the micro-fluidic paper chip gripper of engraving preparation three Dimensional Electrochemical.The micro-fluidic paper chip gripper of three Dimensional Electrochemical is used for connecting the micro-fluidic refill sheet of three Dimensional Electrochemical and electrochemical workstation.
As shown in Figure 12, wherein black is partly hydrophobic region to the hydrophobic wax pattern characteristics of designed filter paper A, and white portion is hydrophilic paper passage, and channel width is 1.0 ~ 4.0 mm; Passage length is 15 mm ~ 60 mm.
As shown in Figure 13, wherein black is partly hydrophobic region to the hydrophobic wax pattern characteristics of designed filter paper B, and white portion is hydrophilic paper passage, and channel width is identical with the channel width in filter paper A; Passage length is 6 mm ~ 24 mm.In addition, filter paper B respectively exposes at two ends an electrode contact, for by the reference electrode of printing with electrode is connected to electrochemical workstation.
As shown in Figure 14, wherein black is partly hydrophobic region to the hydrophobic wax pattern characteristics of designed filter paper C, and white portion is hydrophilic Electrochemical Detection hole, and the diameter in each hole is identical, and diameter is 2.0 ~ 8.0 mm.
As shown in Figure 15, wherein black part is divided expression adhesive tape to the feature of designed double faced adhesive tape strap A, and white portion represents the hole of scratching, and bore dia is identical with the channel width in filter paper A.
As shown in Figure 14, wherein black part is divided expression adhesive tape to the feature of designed double faced adhesive tape strap B, and white portion represents the hole of scratching, and bore dia is identical with the channel width in filter paper B.
As shown in Figure 17, wherein black part is divided expression adhesive tape to the feature of designed one side adhesive tape sheet, and white portion represents the hole of scratching, and bore dia is identical with the channel width in filter paper A.
The micro-fluidic refill sheet of described three Dimensional Electrochemical, is characterized in that: refill sheet overall size is 25.0 mm ~ 100.0, mm * 30.0 mm * 120.0 mm.
The micro-fluidic refill sheet of described three Dimensional Electrochemical, it is characterized in that: when filter paper A and filter paper B is bonding by double faced adhesive tape strap A, four holes on the paper channel end on filter paper A, double faced adhesive tape strap A and the crosspoint vertical alignment of upper four the paper passages of filter paper B.
The micro-fluidic refill sheet of described three Dimensional Electrochemical, it is characterized in that: when filter paper B and filter paper C is bonding by double faced adhesive tape strap B, the porose and upper all Electrochemical Detection of the filter paper C hole vertical alignment of institute on all paper channel ends on filter paper B, double faced adhesive tape strap B.
The micro-fluidic refill sheet of described three Dimensional Electrochemical, is characterized in that: when one side adhesive tape is attached on filter paper A, and the crosspoint vertical alignment of the upper paper passage of the hole on one side adhesive tape sheet and filter paper A.
The filter paper adopting is conventional filter paper or blotting paper.
The plastic adhesive tape adopting is conventional plastic adhesive tape.
Serigraphy array working electrode on described filter paper C, is characterized in that: the printing-ink adopting is conventional carbon ink (slurry).Each working electrode position and Electrochemical Detection hole consistent (pattern as shown in Figure 4) on size and filter paper C.
Described serigraphy reference electrode, is characterized in that: the printing-ink adopting is conventional silver/silver chlorate mixed ink.Serigraphy reference electrode is connected the paper passage (pattern is as shown in accompanying drawing 5 and accompanying drawing 16) on all filter paper B.
Described serigraphy, to electrode, is characterized in that: the printing-ink adopting is conventional carbon ink (slurry).Serigraphy is connected the paper passage (pattern is as shown in accompanying drawing 6 and accompanying drawing 16) on all filter paper B to electrode.
The wax spray printer adopting is conventional Fuji-Xerox's wax spray printer.
The noble metal nano particles solution that horseradish peroxidase China ink described in step (13) is modified for horseradish peroxidase.The noble metal nano particles adopting is common noble metal nano particles, can be golden nanometer particle, Nano silver grain.
The noble metal nano particles solution that oxidizing ferment China ink described in step (14) is modified for oxidizing ferment, the oxidizing ferment adopting is respectively glucose oxidase, cholesterol oxidase, urate oxidase and heme oxidase.The noble metal nano particles adopting is common noble metal nano particles, can be golden nanometer particle, Nano silver grain.
Oxidizing ferment bulk print pattern described in step (6), is characterized in that: accompanying drawing 7 is bulk print patterns of glucose oxidase; Accompanying drawing 8 is bulk print patterns of cholesterol oxidase; Accompanying drawing 9 is bulk print patterns of urate oxidase; Accompanying drawing 10 is bulk print patterns of heme oxidase.
The micro-fluidic paper chip gripper of described three Dimensional Electrochemical, it is characterized in that: the micro-fluidic paper chip gripper of three Dimensional Electrochemical is comprised of two circuit boards that (pattern as shown in Figure 18, circuit board A, circuit board B), the size of circuit board A is identical with the micro-fluidic refill chip size of three Dimensional Electrochemical.The upper all conductive copper layers of circuit board A are removed, and a sample holes is scratched in heart position therein, and size, position are consistent with the sample holes on one side adhesive tape sheet.With circuit board engraving machine, according to the pattern of design in step (22), circuit board B is carved to processing.Copper conductor engraving figure feature on circuit board B as shown in Figure 18, comprises 16 contact circles and ten six conductors.Array working electrode on size, position and the filter paper C of 16 contact circles always.Ten six conductors ends are arranged as 16 bronze medal fingers, and circuit board B grows 5 mm in 16 bronze medal finger one end than circuit board A, to insert 16 bronze medal finger slots.
Utilize the micro-fluidic refill sheet of three Dimensional Electrochemical of above-mentioned preparation to realize the step simultaneously detecting at multi-component scene to be:
(1) the reference electrode joint of filter paper B and the reference electrode wire of portable electrochemical work station in the micro-fluidic refill sheet of three Dimensional Electrochemical are connected; By electrode cable is connected (connected mode as shown in Figure 19) to electrode contact and portable electrochemical work station of filter paper B in the micro-fluidic refill sheet of three Dimensional Electrochemical.
(2) the micro-fluidic refill sheet of three Dimensional Electrochemical is clamped with electrochemistry three-dimensional microflow control paper chip gripper, 16 bronze medal fingers on circuit board B are inserted in 16 bronze medal finger slots, and this slot is connected (connected mode as shown in Figure 19) with the working electrode wire of portable electrochemical work station by multiplexer.
(3) sample solution after dilution is added drop-wise in the sample holes of the micro-fluidic refill sheet of three Dimensional Electrochemical continuously.Drip after 5 ~ 10 times continuously, open electrochemical workstation, start to detect successively the current strength relevant to measured object content in 16 Electrochemical Detection holes, obtain reflecting the shape appearance figure of electric current power.
In the present invention, first sample solution by the sample holes on one side adhesive tape sheet, enters the paper passage on filter paper A, under the driving of capillary force, through filter paper A four-way, shunts; Then by four holes on double faced adhesive tape strap A, enter four split channels on filter paper B; Under the driving of capillary force, sample solution continues to be split into 16 tunnels by filter paper B.Finally by 16 holes on double faced adhesive tape strap B, enter 16 Electrochemical Detection holes on filter paper C, then complete Electrochemical Detection.
beneficial effect of the present invention:
1. in micro-fluidic paper chip lab, introduce the electrochemical enzymatic analyzing detecting method of high sensitivity, high specific, expanded the detection range of micro-fluidic paper chip lab, the detection sensitivity and the degree of accuracy that have improved micro-fluidic paper chip lab.
2. adopt the preparation mode of all print, simplified the preparation process of the micro-fluidic refill sheet of three Dimensional Electrochemical, reduced preparation cost, improved the repeatability of preparation and the detection of micro-fluidic refill sheet.
3. adopt three-dimensional stacked shunt mode, the extensive detecting unit that can realize on micro-fluidic paper chip lab is integrated, improves detection flux and the detectability of micro-fluidic paper chip lab.
4. the print pattern that adopted all can carry out integrated print, realizes on each page and prints a plurality of patterns for prepare the micro-fluidic refill sheet of a plurality of three Dimensional Electrochemical simultaneously.
5. in oxidizing ferment stamping ink, contain noble metal nano particles, by the catalytic action of noble metal nano particles, can improve enzyme identification catalytic capability and efficiency, the Electrochemical Detection sensitivity that further improves the micro-fluidic refill sheet of this three Dimensional Electrochemical.
6. due to the porous character of filter paper, so filter paper A also has the separated function of paper chromatography with the paper passage in filter paper B, and large granular impurity is filtered out.The dual specificity identification catalytic capability of co-oxidation enzyme and horseradish peroxidase molecule, the direct sample that the micro-fluidic refill sheet of this three Dimensional Electrochemical can be realized without pre-treatment adds detection, has simplified detecting step, has saved sample pre-treatments cost.
7. the micro-fluidic paper chip gripper of three Dimensional Electrochemical can be reused, and has reduced use cost.
Accompanying drawing explanation
Fig. 1. be the hydrophobic wax bulk print pattern of filter paper A in the micro-fluidic refill sheet of three Dimensional Electrochemical.
Fig. 2. be the hydrophobic wax bulk print pattern of filter paper B in the micro-fluidic refill sheet of three Dimensional Electrochemical.
Fig. 3 is the hydrophobic wax bulk print pattern of filter paper C in the micro-fluidic refill sheet of three Dimensional Electrochemical.
Fig. 4 is the silk screen batch printed patterns of array working electrode on filter paper C, also for filter paper C, goes up the bulk print pattern of horseradish peroxidase.
Fig. 5 is the batch screen printed pattern of the upper reference electrode of filter paper B.
Fig. 6 is the upper batch screen printed pattern to electrode of filter paper B.
Fig. 7 is the bulk print pattern of the upper glucose oxidase of filter paper C.
Fig. 8 is the bulk print pattern of the upper cholesterol oxidase of filter paper C.
Fig. 9 is the bulk print pattern of the upper urate oxidase of filter paper C.
Figure 10 is the bulk print pattern of the upper heme oxidase of filter paper C.
Figure 11 is that wax spray is printed the principle schematic that builds hydrophilic channel, and A figure is the schematic diagram of blank filter paper; B figure is the schematic diagram of printing wax pattern on filter paper, and wherein a is the wax layer of printing; C figure is that after heating in baking oven or dull and stereotyped firing equipment, wax pattern melts and soak into the thickness of whole filter paper, forms hydrophobic wall.
Figure 12 is filter paper A hydrophobic pattern schematic diagram, and wherein white portion is paper passage, and black part is divided into wax.
Figure 13 is filter paper B hydrophobic pattern schematic diagram, and wherein white portion is paper passage, and black part is divided into wax.Two ends projection is used separately as reference electrode and the electrode contact to electrode.
Figure 14 is filter paper C hydrophobic pattern schematic diagram, and wherein white portion is paper passage, and black part is divided into wax.It is 1. wherein glucose detection hole; 2. be cholesterol detection hole; 3. be that uric acid detects hole; 4. hematin test hole.This figure is also double faced adhesive tape strap B schematic diagram, and black part is divided into adhesive tape, and white portion is the hole of scratching.
Figure 15 is double faced adhesive tape strap A schematic diagram, and black part is divided into adhesive tape, and white portion is the hole of scratching.
Figure 16 is the chip-stacked process schematic diagram of the micro-fluidic paper of three Dimensional Electrochemical.
Figure 17 is one side adhesive tape sheet schematic diagram, and black part is divided into adhesive tape, and white portion is the sample holes of scratching.
Figure 18 is the micro-fluidic paper chip gripper of three Dimensional Electrochemical schematic diagram, and black part is divided into the circuit board that engraving removes conductive copper layer; White portion is the sample holes of scratching; Ash color part is conductive copper layer.
Figure 19 is the schematic top plan view of the micro-fluidic refill sheet of three Dimensional Electrochemical and electrochemical workstation method of attachment.It is 1. wherein electrochemical workstation; 2. be multiplexer; 3. 16 bronze medals are pointed slots; 4. 16 bronze medals on the micro-fluidic paper chip gripper of three Dimensional Electrochemical are pointed; 5. the reference electrode joint on filter paper B in the micro-fluidic refill sheet of three Dimensional Electrochemical; 6. in the micro-fluidic refill sheet of three Dimensional Electrochemical on filter paper B to electrode contact; 7. the wire that connects reference electrode joint; 8. connect the wire to electrode contact; 9. the micro-fluidic refill sheet of three Dimensional Electrochemical that paper chip gripper is clamped.
The specific embodiment
Below in conjunction with drawings and Examples, the present invention will be further described.
embodiment 1in human blood, the scene of glucose, cholesterol, uric acid and ferroheme is detected fast simultaneously.
(1) utilize on computers three hydrophobic wax bulk print patterns of the micro-fluidic refill sheet of Adobe Illustrator CS4 Software for Design three Dimensional Electrochemical, be respectively wax bulk print pattern A (pattern as shown in Figure 1), wax bulk print pattern B (pattern is as shown in Figure 2), wax bulk print pattern C (pattern is as shown in Figure 3).Paper channel width on wax pattern A is 2 mm, and length is 30 mm.Paper channel width on wax pattern B is 2 mm, and length is 12 mm.Electrochemical Detection bore dia on wax pattern C is 4 mm.
(2) the array working electrode batch printed patterns (pattern as shown in Figure 4) that utilizes on computers Adobe Illustrator CS4 Software for Design to mate with wax bulk print pattern C, each working electrode diameter is 4 mm.
(3) utilize on computers the reference electrode batch screen printed pattern (pattern as shown in Figure 5) that Adobe Illustrator CS4 Software for Design is corresponding with wax bulk print pattern B.
(4) utilize on computers Adobe Illustrator CS4 Software for Design corresponding with hydrophobic wax pattern B to electrode screen printed pattern (pattern as shown in Figure 6) in batches.
(5) the horseradish peroxidase bulk print pattern (pattern as shown in Figure 4) that utilizes on computers Adobe Illustrator CS4 Software for Design to mate with wax pattern C.
(6) utilize on computers the oxidizing ferment bulk print pattern that Adobe Illustrator CS4 Software for Design mates with wax pattern C (glucose oxidase bulk print patterns as shown in Figure 7, cholesterol carbohydrate oxidase bulk print patterns as shown in Figure 8, urate oxidase bulk print patterns as shown in Figure 9, heme oxidase bulk print patterns as shown in Figure 10)
(7) common qualitative filter paper is cut into the filter paper of three A4 sizes.
(8) filter paper cutting out in step (7) is placed in wax spray printer, three hydrophobic wax bulk print patterns of the design in step (1) are printed on three filter paper in step (7), obtain respectively filter paper A, filter paper B, filter paper C.
(9) filter paper A, filter paper B, filter paper C in step (8) are placed in panel heater or baking oven, under 100 ℃ degrees Celsius, heat 1 minute.
(10) adopt method for printing screen and use carbon ink, the array working electrode batch screen printed pattern according in step (2), is printed onto array working electrode on the filter paper C obtaining in step (9).
(11) adopt method for printing screen and use silver/silver chlorate China ink, the reference electrode batch screen printed pattern according in step (3), is printed onto reference electrode on the filter paper B obtaining in step (9).
(12) adopt method for printing screen and use carbon ink, according to electrode being printed to silk screen map brushing case in batches in step (4), will be printed onto on the filter paper B obtaining in step (11) electrode.
(13) prepare oxidizing ferment stamping ink: get 0.5 milliliter of solution of streptavidin (1.0 milligrams every milliliter), join (golden nanometer particle diameter is 12.0 nm) in 20.0 milliliters of solution of gold nanoparticles, under room temperature, stir 0.5 hour.Then add the bovine serum albumin solution that 0.5 milliliter of mass fraction is 5 percent, under room temperature, stir 5 minutes.Then by the centrifugation 20 minutes under the rotating speed of 12500 rpms of above-mentioned reaction solution.The precipitation obtaining is distributed in biotinylated horseradish peroxidase/glucose oxidase/cholesterol oxidase/urate oxidase/heme oxidase solution, under 37 degrees Celsius, stirs 1 hour.By the centrifugation 10 minutes under the rotating speed of 12500 rpms of above-mentioned reaction solution.The precipitation obtaining is distributed to (pH=8.0,0.05 mole every liter) in Tris-HCl cushioning liquid, prepares oxidizing ferment stamping ink.
(14) the filter paper C of preparation in step (10) is put into the ink-jet printer that horseradish peroxidase print cartridge is installed.According to the horseradish peroxidase bulk print pattern in step (5), by horseradish peroxidase China ink print to filter paper C in step (10) without on working electrode face.
(15) the filter paper C of preparation in step (14) is put into the ink-jet printer that four oxidizing ferment print cartridges are installed.According to the oxidizing ferment bulk print pattern in step (6), successively four kinds of oxidizing ferment China inks are printed to filter paper C in step (14) without on working electrode face.
(16) with scissors along the filter paper A of preparation in step (9), in step (12) the filter paper B of preparation, in step (15) the filter paper C wax pattern outward flange of preparation carry out cutting, obtain respectively filter paper A (pattern as shown in Figure 12), filter paper B (pattern is as shown in Figure 13), filter paper C (pattern is as shown in Figure 14).
(17) get common double face plastic adhesive tape, by its with scissors be cut into step (16) in the double faced adhesive tape strap A of filter paper formed objects.Then, scratch four holes in relevant position, the size in hole is consistent with paper channel width in filter paper A, the end (pattern as shown in Figure 15) of four paper passages in four corresponding filter paper A of hole difference.
(18) by the double faced adhesive tape strap A obtaining in step (17), filter paper A and filter paper B are bonded together (stack manner as shown in Figure 16).
(19) get common double face plastic adhesive tape, by its with scissors be cut into step (16) in the double faced adhesive tape strap B of filter paper formed objects.Then, scratch 16 holes in relevant position, the size in hole, position are consistent with hydrophilic pores in filter paper C, and 16 Electrochemical Detection holes (pattern is with accompanying drawing 14) in corresponding filter paper C are distinguished in 16 holes.
(20), by the double faced adhesive tape strap B obtaining in step (16), will in step (18), obtain multi-layer filter paper sheet and filter paper C is bonded together (stack manner as shown in Figure 16).
(21) get common one side plastic adhesive tape, be cut into the one side adhesive tape sheet with the middle filter paper formed objects of step (16).Then, in center, scratch a hole, the size in hole consistent with paper channel width in filter paper A (pattern as shown in Figure 17).
(22) the one side adhesive tape sheet obtaining in step (21) is attached on the filter paper A of multi-layer filter paper sheet in step (20) to the micro-fluidic refill sheet of three Dimensional Electrochemical (stack manner as shown in Figure 16) obtaining.
(23) utilize on computers the wire depiction (pattern as shown in Figure 18) of the micro-fluidic paper chip gripper of Adobe Illustrator CS4 Software for Design three Dimensional Electrochemical.
(24) utilize circuit board engraving machine, according to the pattern of design in step (23), the micro-fluidic paper chip gripper of engraving preparation three Dimensional Electrochemical.
With three high patients' blood sample, as analytic sample, in blood sample, contain glucose, cholesterol, uric acid and ferroheme.Utilize the micro-fluidic refill sheet of three Dimensional Electrochemical of above-mentioned preparation to realize multi-component scene detects simultaneously:
(25) the reference electrode joint of filter paper B and the reference electrode wire of portable electrochemical work station in the micro-fluidic refill sheet of three Dimensional Electrochemical are connected; By electrode cable is connected (as shown in Figure 19) to electrode contact and portable electrochemical work station of filter paper B in the micro-fluidic refill sheet of three Dimensional Electrochemical.
(26) the micro-fluidic refill sheet of three Dimensional Electrochemical is clamped with electrochemistry three-dimensional microflow control paper chip gripper, 16 bronze medal fingers on circuit board B are inserted in 16 bronze medal finger slots, and this slot is connected (connected mode as shown in Figure 19) with the working electrode wire of portable electrochemical work station by multiplexer.
(27) get 0.5 milliliter of patient's blood sample, join (pH=8.0,0.05 mole every liter) in 5 milliliters of Tris-HCl cushioning liquid and dilute.Sample solution by after dilution, is added drop-wise to continuously with common plastics dropper in the sample holes of the micro-fluidic refill sheet of three Dimensional Electrochemical.Drip after 10 times continuously, open electrochemical workstation, start to detect successively 16 current strength of first closing with measured object content that detect in holes, obtain reflecting the shape appearance figure of electric current power.
embodiment 2the scene of the glucose in human urine, polyphenol, xanthine, cholesterol, uric acid and ferroheme is detected fast simultaneously.
" utilizing Adobe Illustrator CS4 Software for Design " in embodiment 1 step (1) (2) (3) (4) (5) (6) (23) changed into " utilizing Photoshop CS4 Software for Design "; " common qualitative filter paper " in embodiment 1 changed into " common quantitative filter paper "; " under 100 ℃ degrees Celsius, heating 1 minute " in embodiment 1 step (9) changed into " under 150 degrees Celsius, heating 30 seconds "; By embodiment 1 step 13) in " in solution of gold nanoparticles " change " silver nano-particle solution " into; " three high patient's blood samples " in embodiment 1 changed into " three high urine samples "; Embodiment 1 step (27) is changed into and " gets 5 milliliters of urine samples, be added drop-wise to continuously in the sample holes of the micro-fluidic refill sheet of three Dimensional Electrochemical with common plastics dropper.Drip after 10 times continuously, open electrochemical workstation, start to detect successively 16 current strength of first closing with measured object content that detect in holes, obtain reflecting the shape appearance figure of electric current power.”。
Claims (20)
1. a preparation method for the micro-fluidic refill sheet of simple to operate, low-cost, multichannel three Dimensional Electrochemical, is characterized in that comprising the following steps:
1.1 design three hydrophobic wax bulk print patterns of the micro-fluidic refill sheet of three Dimensional Electrochemical on computers, be respectively the paper channel pattern A of two intersections of wax bulk print, 16 paper passages of wax bulk print, intersect between two pattern B, wax bulk print black is partly hydrophobic region, and white portion is hydrophilic Electrochemical Detection sectional hole patterns C;
The 1.2 array working electrodes that design is mated with wax bulk print pattern C are on computers printed patterns in batches;
The reference electrode batch printed patterns that 1.3 designs are on computers corresponding with wax bulk print pattern B;
1.4 designs are on computers corresponding to hydrophobic wax pattern B electrode batch printed patterns;
The 1.5 horseradish peroxidase bulk print patterns that design is mated with wax bulk print pattern C on computers;
The 1.6 oxidizing ferment bulk print patterns that design is mated with wax bulk print pattern C on computers, a kind of oxidizing ferment bulk print pattern is only used for printing a kind of oxidizing ferment;
1.7 are cut into the filter paper of the required conventional A4 size of three printers by filter paper;
1.8 are placed into the A4 filter paper cutting out in step 1.7 in wax spray printer, and three hydrophobic wax bulk print patterns of the design in step 1.1 are printed on three A4 filter paper in step 1.7, obtain respectively filter paper A, filter paper B, filter paper C;
1.9 are placed into all A4 filter paper with wax pattern in step 1.8 in panel heater or baking oven, under 60-150 ℃ degree Celsius, heat 0.5-2 minute;
1.10 adopt method for printing screen, and the array working electrode batch printed patterns according in step 1.2, is printed onto array working electrode on the filter paper C obtaining in step 1.9;
1.11 adopt method for printing screen, and the reference electrode batch printed patterns according in step 1.3, is printed onto reference electrode on the filter paper B obtaining in step 1.9;
1.12 adopt method for printing screen, according in step 1.4 to electrode printed patterns in batches, will electrode be printed onto on the filter paper B obtaining in step 1.11;
1.13 put into ink-jet printer by the filter paper C of preparation in step 1.10, according to the horseradish peroxidase bulk print pattern in step 1.5, by horseradish peroxidase China ink print to filter paper C in step 1.10 without on working electrode face;
1.14 put into ink-jet printer by the filter paper C of preparation in step 1.13, according to the oxidizing ferment bulk print pattern in step 1.6, successively four kinds of oxidizing ferment China inks are printed to respectively filter paper C in step 1.13 without on working electrode face;
In 1.15 pairs of steps 1.9, in the filter paper A of preparation, step 1.12, in the filter paper B of preparation, step 1.14, the filter paper C of preparation carries out cutting along wax pattern outward flange, obtains respectively filter paper A, filter paper B, filter paper C;
1.16 get common double face plastic adhesive tape, be cut into step 1.15 in the double faced adhesive tape strap A of filter paper formed objects, then, in relevant position, scratch four holes, the size in hole is consistent with paper channel width in filter paper A, the end of four paper passages in four corresponding filter paper A of hole difference;
1.17 by the double faced adhesive tape strap A obtaining in step 1.16, and filter paper A and filter paper B are bonded together;
1.18 get common double face plastic adhesive tape, be cut into step 1.15 in the double faced adhesive tape strap B of filter paper formed objects, then, in relevant position, scratch 16 holes, the size in hole is consistent with paper channel width in filter paper B, 16 Electrochemical Detection holes in 16 corresponding filter paper C of hole difference;
1.19 by the double faced adhesive tape strap B obtaining in step 1.16, will in step 1.17, obtain multi-layer filter paper sheet and filter paper C is bonded together;
1.20 get common one side plastic adhesive tape, be cut into step 1.15 in the one side adhesive tape sheet of filter paper formed objects, then, in center, scratch a hole, the size in hole, position are consistent with paper channel width in filter paper A;
1.21 are attached to the one side adhesive tape sheet obtaining in step 1.20 on the filter paper A of multi-layer filter paper sheet in step 1.19, the micro-fluidic refill sheet of three Dimensional Electrochemical obtaining;
1.22 design the wire depiction of the micro-fluidic paper chip gripper of three Dimensional Electrochemical on computers;
1.23 utilize circuit board engraving machine, according to the pattern of design in step 1.22, and the micro-fluidic paper chip gripper of engraving preparation three Dimensional Electrochemical, the micro-fluidic paper chip gripper of three Dimensional Electrochemical is used for connecting the micro-fluidic refill sheet of three Dimensional Electrochemical and electrochemical workstation.
2. the preparation method of the micro-fluidic refill sheet of a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, the hydrophobic wax pattern of filter paper A is characterized in that, wax pattern forms the paper passage of two intersections, paper channel width is 1.0 ~ 4.0 mm, and passage length is 15 mm ~ 60 mm.
3. the preparation method of the micro-fluidic refill sheet of a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, the hydrophobic wax pattern of filter paper B is characterized in that, wax pattern forms 16 paper passages, intersect between two, paper channel width is identical with the channel width in filter paper A, and passage length is 6 mm ~ 24 mm, in addition, filter paper B respectively exposes at two ends an electrode contact, for by the reference electrode of printing with electrode is connected to electrochemical workstation.
4. the preparation method of the micro-fluidic refill sheet of a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, the hydrophobic wax pattern of filter paper C is characterized in that, and the diameter in each hole is identical, and diameter is 2.0 ~ 8.0 mm.
5. the preparation method of the micro-fluidic refill sheet of a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, double faced adhesive tape strap A is characterized in that, bore dia is identical with the channel width in filter paper A.
6. the preparation method of the micro-fluidic refill sheet of a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, double faced adhesive tape strap B is characterized in that, bore dia is identical with the channel width in filter paper B.
7. the preparation method of the micro-fluidic refill sheet of a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, one side adhesive tape sheet is characterized in that, bore dia is identical with the channel width in filter paper A.
8. the preparation method of the micro-fluidic refill sheet of a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, refill sheet overall size is 25.0 mm ~ 100.0, mm * 30.0 mm * 120.0 mm.
9. the preparation method of the micro-fluidic refill sheet of a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, filter paper A and filter paper B be by double faced adhesive tape strap A when bonding, four holes on the paper channel end on filter paper A, double faced adhesive tape strap A and the crosspoint vertical alignment of upper four the paper passages of filter paper B.
10. the preparation method of the micro-fluidic refill sheet of a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, when filter paper B and filter paper C are bonding by double faced adhesive tape strap B, the porose and upper all Electrochemical Detection of the filter paper C hole vertical alignment on all paper channel ends on filter paper B, double faced adhesive tape strap B.
The preparation method of the micro-fluidic refill sheet of 11. a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, when one side adhesive tape is attached on filter paper A, the crosspoint vertical alignment of the upper paper passage of the hole on one side adhesive tape sheet and filter paper A.
The preparation method of the micro-fluidic refill sheet of 12. a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, the filter paper adopting is conventional filter paper or blotting paper.
The preparation method of the micro-fluidic refill sheet of 13. a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, the plastic adhesive tape adopting is conventional plastic adhesive tape.
The preparation method of the micro-fluidic refill sheet of 14. a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, serigraphy array working electrode on filter paper C is characterized in that, the printing-ink adopting is conventional carbon ink, and each working electrode position is consistent with the Electrochemical Detection hole on size and filter paper C.
The preparation method of the micro-fluidic refill sheet of 15. a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, serigraphy reference electrode is characterized in that, the printing-ink adopting is conventional silver/silver chlorate mixed ink, and serigraphy reference electrode is connected the paper passage on all filter paper B.
The preparation method of the micro-fluidic refill sheet of 16. a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, serigraphy is characterized in that electrode, the printing-ink adopting is conventional carbon ink, and serigraphy is connected the paper passage on all filter paper B to electrode.
The preparation method of the micro-fluidic refill sheet of 17. a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, horseradish peroxidase China ink is characterized in that, golden nanometer particle or silver nano-particle solution that horseradish peroxidase China ink is modified for horseradish peroxidase.
The preparation method of the micro-fluidic refill sheet of 18. a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, oxidizing ferment China ink is characterized in that, golden nanometer particle, silver nano-particle solution that oxidizing ferment China ink is modified for oxidizing ferment, the oxidizing ferment adopting is respectively glucose oxidase, cholesterol oxidase, urate oxidase and heme oxidase.
The preparation method of the micro-fluidic refill sheet of 19. a kind of three Dimensional Electrochemical simple to operate, low-cost, multichannel according to claim 1, it is characterized in that, the micro-fluidic paper chip gripper of three Dimensional Electrochemical is comprised of circuit board A and two circuit boards of circuit board B, and the size of circuit board A is identical with the micro-fluidic refill chip size of three Dimensional Electrochemical; The upper all conductive copper layers of circuit board A are removed, and a sample holes is scratched in heart position therein, and size, position are consistent with the sample holes on one side adhesive tape sheet; With circuit board engraving machine, according to the pattern of design in step (1.22), circuit board B is carved to processing; Copper conductor engraving figure feature on circuit board B is to comprise 16 contact circles and ten six conductors; Size, the position of 16 contact circles are consistent with the array working electrode on filter paper C; Ten six conductors ends are arranged as 16 bronze medal fingers, and circuit board B grows 5 mm in 16 bronze medal finger one end than circuit board A, to insert 16 bronze medal finger slots.
The instant application detecting in scene of the micro-fluidic refill sheet of three Dimensional Electrochemical that 20. preparation methods as claimed in claim 1 obtain:
The reference electrode joint of filter paper B and the reference electrode wire of portable electrochemical work station in the micro-fluidic refill sheet of three Dimensional Electrochemical are connected, by electrode cable is connected to electrode contact and portable electrochemical work station of filter paper B in the micro-fluidic refill sheet of three Dimensional Electrochemical;
The micro-fluidic refill sheet of three Dimensional Electrochemical is clamped with electrochemistry three-dimensional microflow control paper chip gripper, copper conductor on circuit board B comprises 16 contact circles and ten six conductors, size, the position of 16 contact circles are consistent with the array working electrode on filter paper C, 16 bronze medal fingers on circuit board B are inserted in 16 bronze medal finger slots, and this slot is connected with the working electrode wire of portable electrochemical work station by multiplexer;
Sample solution after dilution is added drop-wise in the sample holes of the micro-fluidic refill sheet of three Dimensional Electrochemical continuously, drip continuously after 5 ~ 10 times, open electrochemical workstation, start to detect successively the current strength relevant to measured object content in 16 Electrochemical Detection holes, obtain reflecting the shape appearance figure of electric current power.
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