CN103041876B - Preparation of electrochemical three-dimensional microfluidic paper chip and application of electrochemical three-dimensional microfluidic paper chip to field test - Google Patents

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

Preparation of 3D microfluidic paper chip and its application in field electrochemical detection

technical field

The invention relates to the field of low-cost, high-throughput, high-sensitivity, high-specificity on-site instant detection technology, more specifically a construction of a microfluidic paper-on-a-chip laboratory suitable for high-throughput electrochemical enzyme analysis . the

Background technique

Paper is a very cheap and abundant material, and test strips have also been widely used as an analysis platform (such as immunochromatography) for simple disease diagnosis and environmental testing, such as early pregnancy test strips, etc. . The Whiteside research group of the Harvard University School of Chemistry first proposed a new concept of a paper chip laboratory, also known as a microfluidic paper chip analysis device. The microfluidic paper chip analysis device refers to drawing hydrophobic patterns on paper. Since the paper has a strong hydrophilic ability, the part without hydrophobic patterns will form a hydrophilic channel/region. With the help of the capillary driving force of the paper, the solution can be in the Oriented flow in the paper channel. Based on this, the basic operation units such as sample preparation, reaction, separation, and detection in the biological, chemical, and medical analysis processes are integrated on the paper to form a microfluidic paper chip analysis device. This technology has broad application prospects in the fields of on-site disease diagnosis, on-site food analysis, and on-site environmental monitoring. On the other hand, compared with the traditional microfluidic lab-on-a-chip based on plastic sheets, glass sheets or silicon wafers, the microfluidic paper lab-on-a-chip has low cost and simple preparation method (using simple printing technology, and No need for harsh conditions such as clean room), simple operation (no need for additional equipment, such as pumps, etc.), and can be disposed of after use. the

At present, the analysis methods based on microfluidic paper chip analysis devices are mainly colorimetric methods, because colorimetric methods can only give a "yes/no" signal response, and colorimetric methods have low sensitivity and poor selectivity , prone to false positive results. Therefore, establishing a high-sensitivity and high-selectivity analysis method in the microfluidic paper-on-a-chip laboratory to achieve high-sensitivity and high-specificity on-site analysis and detection has become one of the problems that need to be solved urgently in this research field. In addition, in order to carry out early disease diagnosis, disease screening and evaluation, and drug response more accurately, it is often necessary to monitor the content of multiple components in the sample at the same time. Therefore, exploring the establishment of high-throughput microfluidic paper chips will help to solve this problem. the

Contents of the invention

The technical problem to be solved by the present invention is to establish an electrochemical enzyme analysis and detection method with the characteristics of simple sample processing, fast detection speed, low cost, high sensitivity and strong specificity on the microfluidic paper chip. A high-throughput integrated three-dimensional microfluidic paper chip was further constructed and used for the simultaneous detection of glucose, cholesterol, uric acid and hemoglobin in samples. the

In order to solve the above technical problems, the present invention is achieved by constructing a novel electrochemical three-dimensional microfluidic paper chip, the preparation method of the electrochemical three-dimensional microfluidic paper chip is as follows:

(1) Design three hydrophobic wax batch printing patterns of the electrochemical three-dimensional microfluidic paper chip on the computer, respectively wax batch printing pattern A (style shown in Figure 1), wax batch printing pattern B (style as attached Shown in Figure 2), wax batch printing pattern C (style as shown in Figure 3).

(2) Design the array working electrode batch printing pattern matching the wax batch printing pattern C on the computer (the style is shown in Figure 4). the

(3) Design the reference electrode batch printing pattern corresponding to the wax batch printing pattern B on the computer (the style is shown in Figure 5). the

(4) Design the counter electrode batch printing pattern corresponding to the hydrophobic wax pattern B on the computer (the style is shown in Figure 6). the

(5) Design a horseradish peroxidase batch printing pattern matching the wax batch printing pattern C on the computer (the style is the same as Figure 4). the

(6) Design an oxidase batch printing pattern matching the wax batch printing pattern C on the computer (the styles are shown in accompanying drawings 7, 8, 9 and 10 respectively), a batch printing pattern of oxidase Used to print only one oxidase. the

(7) Cut the filter paper into three commonly used A4 size filter papers required by the printer. the

(8) Put the cut A4 filter paper in step (7) into the wax spray printer, and print the three hydrophobic wax batch printing patterns designed in step (1) to the three A4 filter paper in step (7) On, get filter paper A, filter paper B, filter paper C respectively. the

(9) Place all the A4 filter papers with wax patterns in step (8) into a flat heater or oven, and heat at 60-150°C for 0.5-2 minutes. The wax is melted and soaked through the entire thickness of the paper to form a hydrophobic wall (the principle is shown in Figure 11). the

(10) Using the screen printing method, print the pattern of the array working electrode in batches according to the array working electrode in step (2), and print the array working electrode on the filter paper C obtained in step (9). the

(11) Using the screen printing method, follow the batch printing pattern of the reference electrode in step (3), and print the reference electrode on the filter paper B obtained in step (9). the

(12) Using the screen printing method, print the counter electrode on the filter paper B obtained in step (11) according to the batch printing pattern of the counter electrode in step (4). the

(13) Put the filter paper C prepared in step (10) into the inkjet printer. According to the horseradish peroxidase batch printing pattern in step (5), the horseradish peroxidase ink is printed on the non-working electrode surface of filter paper C in step (10). the

(14) Put the filter paper C prepared in step (13) into the inkjet printer. According to the oxidase batch printing pattern in step (6), print the four kinds of oxidase inks in turn on the non-working electrode surface of filter paper C in step (13). the

(15) Cut the filter paper A prepared in step (9), the filter paper B prepared in step (12), and the filter paper C prepared in step (14) along the outer edge of the wax pattern to obtain filter paper sheet A (the style is as attached) Shown in Figure 12), filter paper sheet B (pattern as shown in accompanying drawing 13), filter paper sheet C (pattern as shown in accompanying drawing 14). the

(16) Take ordinary double-sided plastic tape and cut it into a double-sided tape piece A of the same size as the filter paper piece in step (15). Then, cut four holes at the corresponding positions, the size of the holes is consistent with the width of the paper passage in the filter paper sheet A, and the four holes correspond to the ends of the four paper passages in the filter paper sheet A respectively (the pattern is as shown in Figure 15). the

(17) Use the double-sided tape sheet A obtained in step (16) to bond the filter paper sheet A and the filter paper sheet B together (the stacking method is shown in Figure 16). the

(18) Take ordinary double-sided plastic tape, and cut it into double-sided tape B of the same size as the filter paper in step (15). Then, cut sixteen holes at the corresponding positions, the size of the holes is consistent with the width of the paper channel in the filter paper sheet B, and the sixteen holes correspond to the sixteen electrochemical detection holes in the filter paper sheet C respectively (the pattern is the same as accompanying drawing 14). the

(19) Through the double-sided adhesive tape sheet B obtained in step (16), the multi-layer filter paper sheet and filter paper sheet C obtained in step (17) are bonded together (the stacking method is shown in Figure 16). the

(20) Take an ordinary single-sided plastic tape and cut it into a single-sided tape of the same size as the filter paper in step (15). Then, cut a hole at the central position, the size and position of the hole are consistent with the width of the paper channel in the filter paper sheet A (the pattern is as shown in Figure 17). the

(21) Paste the single-sided adhesive tape sheet obtained in step (20) on the filter paper sheet A of the multilayer filter paper sheet in step (19), and obtain the electrochemical three-dimensional microfluidic paper chip (stacking method as shown in Figure 16) Show). the

(22) Design the wire engraving pattern of the electrochemical three-dimensional microfluidic paper chip clip on the computer (the style is shown in Figure 18). the

(23) Use a circuit board engraving machine to engrave and prepare electrochemical three-dimensional microfluidic paper chip clips according to the pattern designed in step (22). The electrochemical three-dimensional microfluidic paper chip clip is used to connect the electrochemical three-dimensional microfluidic paper chip and the electrochemical workstation. the

The characteristics of the hydrophobic wax pattern of the designed filter paper sheet A are shown in Figure 12, where the black part is the hydrophobic area, and the white part is the hydrophilic paper channel, the channel width is 1.0-4.0 mm; the channel length is 15 mm-60 mm. the

The characteristics of the hydrophobic wax pattern of the designed filter paper sheet B are shown in Figure 13, wherein the black part is the hydrophobic area, and the white part is the hydrophilic paper channel, and the channel width is the same as that in the filter paper sheet A; the channel length is 6 mm ~24 mm. In addition, an electrode joint is exposed at both ends of the filter paper sheet B, which is used to connect the printed reference electrode and counter electrode to the electrochemical workstation. the

The features of the hydrophobic wax pattern of the designed filter paper sheet C are shown in Figure 14, in which the black part is the hydrophobic area, and the white part is the hydrophilic electrochemical detection hole. The diameter of each hole is the same, and the diameter is 2.0-8.0 mm. the

The features of the designed double-sided adhesive tape sheet A are shown in Figure 15, wherein the black part represents the adhesive tape, and the white part represents the hole that has been cut out, and the diameter of the hole is the same as the channel width in the filter paper sheet A. the

The features of the designed double-sided adhesive tape sheet B are shown in Figure 14, wherein the black part represents the adhesive tape, and the white part represents the hole cut out, and the diameter of the hole is the same as the channel width in the filter paper sheet B. the

The features of the designed single-sided adhesive tape sheet are shown in Figure 17, wherein the black part represents the adhesive tape, and the white part represents the hole cut out, and the diameter of the hole is the same as the channel width in the filter paper sheet A. the

The electrochemical three-dimensional microfluidic paper chip is characterized in that: the total size of the paper chip is 25.0 mm×30.0 mm~100.0 mm×120.0 mm. the

The electrochemical three-dimensional microfluidic paper chip is characterized in that: when the filter paper sheet A and the filter paper sheet B are bonded through the double-sided tape sheet A, the end of the paper channel on the filter paper sheet A and the end of the double-sided tape sheet A are The four holes are aligned vertically with the intersection of the four paper channels on filter paper sheet B. the

The electrochemical three-dimensional microfluidic paper chip is characterized in that: when the filter paper sheet B and the filter paper sheet C are bonded through the double-sided adhesive tape sheet B, all the paper channel ends on the filter paper sheet B and the double-sided adhesive tape sheet B are All the holes of the filter paper and all the electrochemical detection holes on the filter paper sheet C are vertically aligned. the

The electrochemical three-dimensional microfluidic paper chip is characterized in that: when the single-sided tape is pasted on the filter paper sheet A, the holes on the single-sided tape sheet are vertically aligned with the intersections of the paper channels on the filter paper sheet A. the

The filter paper used is commonly used filter paper or absorbent paper. the

The plastic tapes used are commonly used plastic tapes. the

The screen printing array working electrode on the filter paper sheet C is characterized in that the printing ink used is commonly used carbon ink (paste). The position and size of each working electrode are consistent with the electrochemical detection hole on the filter paper sheet C (the style is shown in Figure 4). the

The screen printing reference electrode is characterized in that: the printing ink used is commonly used silver/silver chloride mixed ink. The screen-printed reference electrode is connected to the paper passages on all filter paper sheets B (the patterns are shown in Figure 5 and Figure 16). the

The screen printing counter electrode is characterized in that: the printing ink used is commonly used carbon ink (paste). The screen printing counter electrode is connected to the paper passages on all the filter paper sheets B (the patterns are shown in Figure 6 and Figure 16). the

The wax spray printer adopted is the commonly used Fuji Xerox wax spray printer. the

The horseradish peroxidase ink described in step (13) is a solution of noble metal nanoparticles modified by horseradish peroxidase. The noble metal nanoparticles used are common noble metal nanoparticles, such as gold nanoparticles and silver nanoparticles. the

The oxidase ink described in step (14) is a noble metal nanoparticle solution modified by oxidase, and the oxidase used are glucose oxidase, cholesterol oxidase, urate oxidase and heme oxidase respectively. The noble metal nanoparticles used are common noble metal nanoparticles, such as gold nanoparticles and silver nanoparticles. the

The oxidase batch printing pattern described in step (6) is characterized in that: accompanying drawing 7 is the batch printing pattern of glucose oxidase; Accompanying drawing 8 is the batch printing pattern of cholesterol oxidase; Accompanying drawing 9 is the urate oxidase Batch printing pattern; accompanying drawing 10 is the batch printing pattern of heme oxidase. the

The electrochemical three-dimensional microfluidic paper chip clip is characterized in that: the electrochemical three-dimensional microfluidic paper chip clip is made up of two circuit boards (style shown in Figure 18, circuit board A, circuit board B), the circuit The size of plate A is the same as that of the electrochemical three-dimensional microfluidic paper chip. All the conductive copper layers on the circuit board A are removed, and a sampling hole is cut in its center, the size and position of which are consistent with the sampling hole on the single-sided adhesive tape. Use a circuit board engraving machine to engrave circuit board B according to the pattern designed in step (22). The engraving features of the copper wires on the circuit board B are shown in Figure 18, including sixteen contact circles and sixteen wires. The size and position of the sixteen contact circles are consistent with the array working electrodes on the filter paper sheet C. The ends of the sixteen wires are arranged as sixteen copper fingers, and the circuit board B is 5 mm longer than the circuit board A at one end of the sixteen copper fingers, so as to be inserted into the sixteen copper finger slots. the

Using the electrochemical three-dimensional microfluidic paper chip prepared above, the steps to realize simultaneous detection of multiple components on site are as follows:

(1) Connect the reference electrode connector of the filter paper sheet B in the electrochemical three-dimensional microfluidic paper chip to the reference electrode wire of the portable electrochemical workstation; connect the counter electrode connector of the filter paper sheet B in the electrochemical three-dimensional microfluidic paper chip It is connected with the counter electrode wire of the portable electrochemical workstation (the connection method is shown in Figure 19).

(2) Clamp the electrochemical three-dimensional microfluidic paper chip with the electrochemical three-dimensional microfluidic paper chip clip, and insert the sixteen copper fingers on the circuit board B into the sixteen copper finger slots, which are passed through multiple The multiplexer is connected to the working electrode wire of the portable electrochemical workstation (the connection method is shown in Figure 19). the

(3) Continuously drop the diluted sample solution into the injection hole of the electrochemical three-dimensional microfluidic paper chip. After 5-10 continuous drops, the electrochemical workstation was turned on, and the current intensity related to the content of the analyte in the 16 electrochemical detection holes was sequentially detected, and a topography map reflecting the current strength was obtained. the

In the present invention, the sample solution first passes through the sampling hole on the single-sided tape sheet, enters the paper channel on the filter paper sheet A, and is driven by capillary force to shunt through the four channels of the filter paper sheet A; The four holes of the filter paper B enter into the four shunt channels on the filter paper B; driven by the capillary force, the sample solution continues to be shunted into 16 channels by the filter paper B. Finally, enter the 16 electrochemical detection holes on the filter paper sheet C through the 16 holes on the double-sided tape sheet B, and then complete the electrochemical detection. the

Beneficial effects of the present invention:

1. Introduced a high-sensitivity and high-specificity electrochemical enzyme analysis and detection method in the microfluidic paper-on-a-chip laboratory, expanded the detection range of the microfluidic paper-on-a-chip laboratory, and improved the performance of the microfluidic paper-on-a-chip laboratory. Detection sensitivity and accuracy.

2. Adopting the full-printing preparation mode simplifies the preparation steps of the electrochemical three-dimensional microfluidic paper chip, reduces the preparation cost, and improves the repeatability of the preparation and detection of the microfluidic paper chip. the

3. The three-dimensional stacking and shunting mode can realize the integration of large-scale detection units on the microfluidic paper-on-a-chip laboratory, and improve the detection throughput and detection ability of the microfluidic paper-on-a-chip laboratory. the

4. All the printing patterns used can be integrated and printed, so that multiple patterns can be printed on each page to prepare multiple electrochemical three-dimensional microfluidic paper chips at the same time. the

5. The oxidase printing ink contains noble metal nanoparticles. With the help of the catalysis of noble metal nanoparticles, the ability and efficiency of enzyme recognition and catalysis can be improved, and the electrochemical detection sensitivity of the electrochemical three-dimensional microfluidic paper chip can be further improved. the

6. Due to the porous nature of the filter paper, the paper channel in the filter paper sheet A and filter paper sheet B also has the function of paper chromatographic separation to filter out large particles of impurities. Combined with the dual specific recognition and catalytic capabilities of oxidase and horseradish peroxidase molecules, the electrochemical three-dimensional microfluidic paper chip can realize direct sample addition detection without pretreatment, simplify the detection steps, and save the cost of sample pretreatment . the

7. The electrochemical three-dimensional microfluidic paper chip clip can be reused, which reduces the cost of use. the

Description of drawings

Figure 1. The hydrophobic wax batch printing pattern of the filter paper sheet A in the electrochemical three-dimensional microfluidic paper chip. the

Figure 2. The hydrophobic wax batch printing pattern of the filter paper sheet B in the electrochemical three-dimensional microfluidic paper chip. the

Fig. 3 is the hydrophobic wax batch printing pattern of the filter paper sheet C in the electrochemical three-dimensional microfluidic paper chip. the

Fig. 4 is the screen batch printing pattern of the array working electrode on the filter paper C, and also the batch printing pattern of horseradish peroxidase on the filter paper C. the

Figure 5 is the batch screen printing pattern of the reference electrode on the filter paper sheet B. the

Figure 6 is the batch screen printing pattern of the counter electrode on the filter paper sheet B. the

Figure 7 is the batch printing pattern of glucose oxidase on the filter paper sheet C. the

Fig. 8 is the batch printing pattern of cholesterol oxidase on the filter paper sheet C. the

Fig. 9 is a batch printing pattern of urate oxidase on the filter paper sheet C. the

Figure 10 is the batch printing pattern of heme oxidase on the filter paper sheet C. the

Figure 11 is a schematic diagram of the principle of wax spray printing to build a hydrophilic channel. Figure A is a schematic diagram of blank filter paper; Figure B is a schematic diagram of printing wax patterns on filter paper, where a is the printed wax layer; Figure C is heating in an oven or a flat plate When heated in the device, the wax pattern melts and saturates the entire thickness of the filter paper, forming a hydrophobic wall. the

Fig. 12 is a schematic diagram of the hydrophobic pattern of the filter paper sheet A, wherein the white part is the paper channel, and the black part is the wax. the

Figure 13 is a schematic diagram of the hydrophobic pattern of the filter paper sheet B, in which the white part is the paper channel, and the black part is the wax. The protruding parts at both ends are respectively used as electrode joints for the reference electrode and the counter electrode. the

Fig. 14 is a schematic diagram of the hydrophobic pattern of the filter paper sheet C, wherein the white part is the paper channel, and the black part is the wax. Among them, ① is the detection hole for glucose; ② is the detection hole for cholesterol; ③ is the detection hole for uric acid; ④ the detection hole for hemoglobin. This figure is also a schematic diagram of the double-sided adhesive tape sheet B, the black part is the adhesive tape, and the white part is the hole cut out. the

Figure 15 is a schematic diagram of double-sided adhesive tape sheet A, the black part is the adhesive tape, and the white part is the hole cut out. the

Fig. 16 is a schematic diagram of the electrochemical three-dimensional microfluidic paper chip stacking process. the

Figure 17 is a schematic diagram of a single-sided adhesive tape sheet, the black part is the adhesive tape, and the white part is the sample injection hole cut out. the

Figure 18 is a schematic diagram of an electrochemical three-dimensional microfluidic paper chip holder. The black part is the circuit board with the conductive copper layer removed; the white part is the sample hole cut out; the gray part is the conductive copper layer. the

Fig. 19 is a schematic top view of the connection method between the electrochemical three-dimensional microfluidic paper chip and the electrochemical workstation. Among them, ① is an electrochemical workstation; ② is a multiplexer; ③ sixteen copper finger slots; ④ sixteen copper fingers on the electrochemical three-dimensional microfluidic paper chip clip; The reference electrode connector on the filter paper B; ⑥ the counter electrode connector on the filter paper B in the electrochemical three-dimensional microfluidic paper chip; ⑦ the wire connecting the reference electrode connector; ⑧ the wire connecting the counter electrode connector; ⑨ the paper chip clip Sandwiched electrochemical three-dimensional microfluidic paper chip. the

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments. the

Example 1 On-site rapid and simultaneous detection of glucose, cholesterol, uric acid and hemoglobin in human blood.

(1) Use Adobe Illustrator CS4 software on the computer to design three hydrophobic wax batch printing patterns of the electrochemical three-dimensional microfluidic paper chip, respectively wax batch printing pattern A (the style is shown in Figure 1), wax batch printing pattern B (style shown in Figure 2), wax batch printing pattern C (style shown in Figure 3). The paper channel on wax pattern A has a width of 2 mm and a length of 30 mm. The paper channel on wax pattern B has a width of 2 mm and a length of 12 mm. The diameter of the electrochemical detection hole on the wax pattern C is 4 mm. the

(2) Use Adobe Illustrator CS4 software on the computer to design a batch printing pattern of the array working electrodes that matches the wax batch printing pattern C (the style is shown in Figure 4), and the diameter of each working electrode is 4 mm. the

(3) Use Adobe Illustrator CS4 software on the computer to design the reference electrode batch screen printing pattern corresponding to the wax batch printing pattern B (the style is shown in Figure 5). the

(4) Use Adobe Illustrator CS4 software on the computer to design the batch screen printing pattern of the counter electrode corresponding to the hydrophobic wax pattern B (the style is shown in Figure 6). the

(5) Use Adobe Illustrator CS4 software on the computer to design a horseradish peroxidase batch printing pattern that matches the wax pattern C (the style is shown in Figure 4). the

(6) Use Adobe Illustrator CS4 software on the computer to design a batch printing pattern of oxidase that matches the wax pattern C (the batch printing pattern of glucose oxidase is shown in Figure 7, and the batch printing pattern of cholesterol sugar oxidase is shown in Figure 8 As shown, the urate oxidase batch printing pattern style is shown in Figure 9, and the heme oxidase batch printing pattern style is shown in Figure 10),

(7) Cut ordinary qualitative filter paper into three A4 size filter papers.

(8) Place the cut filter paper in step (7) into the wax spray printer, and print the three hydrophobic wax batch printing patterns designed in step (1) onto the three filter papers in step (7), Filter paper A, filter paper B, and filter paper C were obtained respectively. the

(9) Place filter paper A, filter paper B, and filter paper C in step (8) in a flat heater or oven, and heat at 100°C for 1 minute. the

(10) Using the screen printing method and using carbon ink, according to the batch screen printing pattern of the array working electrode in step (2), print the array working electrode on the filter paper C obtained in step (9). the

(11) Using the screen printing method and using silver/silver chloride ink, follow the batch screen printing pattern of the reference electrode in step (3), and print the reference electrode onto the filter paper B obtained in step (9). the

(12) Using the screen printing method and using carbon ink, print the screen printing pattern on the counter electrode in batches according to the step (4), and print the counter electrode on the filter paper B obtained in step (11). the

(13) Preparation of oxidase printing ink: Take 0.5 ml of streptavidin solution (1.0 mg per ml), add it to 20.0 ml of gold nanoparticle solution (the diameter of gold nanoparticles is 12.0 nm), and stir at room temperature for 0.5 hours. Then add 0.5 ml of bovine serum albumin solution with a mass fraction of 5%, and stir at room temperature for 5 minutes. Then the above reaction solution was centrifuged at 12500 rpm for 20 minutes. The resulting precipitate was dispersed into a biotinylated horseradish peroxidase/glucose oxidase/cholesterol oxidase/uric acid oxidase/heme oxidase solution and stirred at 37°C for 1 hour. The above reaction solution was centrifuged at 12500 rpm for 10 minutes. The obtained precipitate was dispersed into a Tris-HCl buffer solution (pH=8.0, 0.05 moles per liter) to prepare an oxidase printing ink. the

(14) Put the filter paper C prepared in step (10) into an inkjet printer equipped with a horseradish peroxidase cartridge. According to the horseradish peroxidase batch printing pattern in step (5), the horseradish peroxidase ink is printed on the non-working electrode surface of filter paper C in step (10). the

(15) Put the filter paper C prepared in step (14) into an inkjet printer equipped with four oxidase cartridges. According to the oxidase batch printing pattern in step (6), sequentially print four kinds of oxidase inks on the non-working electrode surface of filter paper C in step (14). the

(16) Use scissors to cut along the outer edge of the wax pattern of filter paper A prepared in step (9), filter paper B prepared in step (12), and filter paper C prepared in step (15), to obtain filter paper sheet A (style as Shown in accompanying drawing 12), filter paper sheet B (style as shown in accompanying drawing 13), filter paper sheet C (style as shown in accompanying drawing 14). the

(17) Take ordinary double-sided plastic tape and cut it with scissors into a double-sided tape piece A of the same size as the filter paper piece in step (16). Then, cut four holes at the corresponding positions, the size of the holes is consistent with the width of the paper passage in the filter paper sheet A, and the four holes correspond to the ends of the four paper passages in the filter paper sheet A respectively (the pattern is as shown in Figure 15). the

(18) Use the double-sided tape sheet A obtained in step (17) to bond the filter paper sheet A and the filter paper sheet B together (the stacking method is shown in Figure 16). the

(19) Take ordinary double-sided plastic tape and cut it into a double-sided tape B of the same size as the filter paper in step (16) with scissors. Then, cut sixteen holes at the corresponding positions, the size and position of the holes are consistent with the hydrophilic holes in the filter paper sheet C, and the sixteen holes correspond to the sixteen electrochemical detection holes in the filter paper sheet C respectively (the pattern is the same as that of accompanying drawing 14) . the

(20) Through the double-sided adhesive tape sheet B obtained in step (16), the multi-layer filter paper sheet and filter paper sheet C obtained in step (18) are bonded together (the stacking method is shown in Figure 16). the

(21) Take an ordinary single-sided plastic tape and cut it into a single-sided tape of the same size as the filter paper in step (16). Then, cut a hole at the central position, the size of the hole is consistent with the width of the paper channel in the filter paper sheet A (the pattern is as shown in Figure 17). the

(22) Paste the single-sided adhesive tape sheet obtained in step (21) on the filter paper sheet A of the multi-layer filter paper sheet in step (20), and obtain the electrochemical three-dimensional microfluidic paper chip (stacking method as shown in Figure 16) Show). the

(23) Use Adobe Illustrator CS4 software on the computer to design the wire engraving pattern of the electrochemical three-dimensional microfluidic paper chip clip (the style is shown in Figure 18). the

(24) Use a circuit board engraving machine to engrave and prepare electrochemical three-dimensional microfluidic paper chip clips according to the pattern designed in step (23). the

The blood sample of the three high patients is used as the analysis sample, which contains glucose, cholesterol, uric acid and heme. The electrochemical three-dimensional microfluidic paper chip prepared above is used to realize the simultaneous detection of multiple components on site: 

(25) Connect the reference electrode connector of the filter paper sheet B in the electrochemical three-dimensional microfluidic paper chip to the reference electrode wire of the portable electrochemical workstation; connect the counter electrode connector of the filter paper sheet B in the electrochemical three-dimensional microfluidic paper chip It is connected with the opposite electrode wire of the portable electrochemical workstation (as shown in Figure 19).

(26) Clamp the electrochemical three-dimensional microfluidic paper chip with the electrochemical three-dimensional microfluidic paper chip clip, and insert the sixteen copper fingers on the circuit board B into the sixteen copper finger slots, which are passed through multiple The multiplexer is connected to the working electrode wire of the portable electrochemical workstation (the connection method is shown in Figure 19). the

(27) Take 0.5 ml of the patient's blood sample and add it to 5 ml of Tris-HCl buffer solution (pH=8.0, 0.05 mol per liter) for dilution. The diluted sample solution was continuously dripped into the injection hole of the electrochemical three-dimensional microfluidic paper chip with an ordinary plastic dropper. After 10 consecutive drops, the electrochemical workstation was turned on, and the current intensity related to the content of the analyte in the 16 detection holes was sequentially detected, and a topography map reflecting the current strength was obtained. the

Example 2 On-site rapid simultaneous detection of glucose, polyphenols, xanthine, cholesterol, uric acid and heme in human urine.

"Utilize Adobe Illustrator CS4 software design" in embodiment 1 step (1) (2) (3) (4) (5) (6) (23) to " utilize Photoshop CS4 software design "; "Ordinary qualitative filter paper" in "ordinary qualitative filter paper" is changed to "ordinary quantitative filter paper"; "heating at 100°C for 1 minute" in step (9) of Example 1 is changed to "heating at 150°C for 30 seconds"; the embodiment 1 step 13) in the "gold nanoparticle solution" into "silver nanoparticle solution"; change the "three high patient blood sample" in Example 1 to "three high patient urine sample"; change the embodiment 1 step ( 27) Change to "Take 5 ml of the patient's urine sample, and use an ordinary plastic dropper to continuously drop it into the sampling hole of the electrochemical three-dimensional microfluidic paper chip. After 10 consecutive drops, turn on the electrochemical workstation and start sequential detection The current intensity related to the content of the analyte in the 16 detection holes is obtained, and the topography map reflecting the current intensity is obtained.". the

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.