CN100401048C - A multi-channel microfluidic chip and its preparation method - Google Patents

Abstract

The microfluidic chip has the characteristics of small size and fast analysis speed, and can realize high automation and integration. It is the frontier of the development of the field of analytical chemistry. Although microfluidic chips combined with laser-induced fluorescence detection have been able to perform high-throughput detection of 384 channels, multi-channel electrochemical detection has not been reported so far. The invention provides a multi-channel microfluidic chip. The chip uses polymer material polymethyl methacrylate as a material, and a single crystal silicon positive mold is made by using technologies such as photolithography and etching. The microchannel adopts hot pressing method or Made by in situ polymerization. The detection electrode channel is integrated on the substrate, the electrode can be filled with functional materials, and the distance between the working electrode and the separation channel can be effectively and precisely controlled, which simplifies the experimental operation. The present invention fully utilizes the advantages of electrochemical detection in microfluidic chips, develops a multi-channel microfluidic chip for electrochemical detection for the first time, and can analyze samples with high efficiency and high throughput.

Description

A multi-channel microfluidic chip and its preparation method

technical field

The invention relates to the field of microfluidic chips, and develops a multi-channel microfluidic chip based on polymethyl methacrylate as a material and applied to electrochemical detection. The invention also provides a preparation method of the chip.

Background technique

Since the early 1990s when Manz et al. put forward the concept of "micro-total analysis system", the integration and portability of the analysis system has become the main development trend of modern analytical instruments. Compared with traditional capillary electrophoresis technology, microfluidic chip has the advantages of less sample consumption and fast separation speed, but at the same time, it has higher requirements for detection sensitivity and response speed. The most commonly used detection method is laser-induced fluorescence detection (LIF), but the instruments required for detection by this method are expensive, bulky and cumbersome. Electrochemical detection (ED) has high sensitivity, and the required instrument is small in size and easy to integrate, which matches the concept of lab-on-a-chip in size. Printed thick-film electrodes, carbon paste, and carbon fibers have also been widely used in electrochemical detection of microfluidic chips as electrode materials.

The most common materials from which chips are made are glass and quartz. Glass and quartz have good electroosmotic and optical properties, and can be processed by standard etching technology, which is easy to modify the surface of the channel, but it is difficult to obtain a channel with a large width and depth, high processing cost, and difficult sealing. High molecular polymers have attracted more and more attention as materials for microfluidic chips. Various types of polymers, including polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polycarbonate, etc., have been widely used in chip fabrication.

At the same time, the integration level of the chip has also been greatly improved, and the design forms of the channels are very diverse. Patent applications related to microfluidic chips are also increasing. The United States patents from 2001 to 2005 were retrieved using microfluidic and chip as keywords, and there were 11 patents in total. Among them, there is one patent on multi-channel chip (200401133068, Multi-channel microfluidic chip for electrosprayionization), which designed a chip for mass spectrometry analysis; one patent on electrochemical detection (20020079219, Microfluidic chip having integrated electrodes), the patent designs a chip that integrates electrodes. The material of the electrodes can be metal or conductive ink, and the electrodes will be made on the plastic film by electrodeposition, printing or other methods. This patent mainly focuses on new Electrode fabrication.

Searching Chinese patents from 1985 to 2005 with the combination of keywords "multi-channel" and "chip" yielded 4 patents. Among them, there are 2 microfluidic chip patents, namely: application number 02256483, and the invention name is a multi-channel chip amperometric detector and detection system. This patent designs a parallel constant potential current-voltage converter, mainly an amperometric detector Design; application number: 02353129, multi-channel microfluidic chip, patented for appearance design, designed a multi-channel chip for laser-induced fluorescence. A total of 28 patents were retrieved by using "microfluidics" and "chip" as keywords, of which 1 was applied to electrochemical detection, application number 03145053, and the name of the invention was electrochemical detection-microfluidic chip and its manufacturing method and regeneration method. The patent invented an electrochemical detection-microfluidic chip, which aligns the silicon rubber polydimethylsiloxane sheet containing microchannels with the electrode strips on the glass slide to form a microfluidic channel, and the microchannel outlet The distance between the detection electrode and the detection electrode is 20-60 microns, and the electrochemical detection is the column end detection mode. The invention uses commercial chrome plate glass as the material, and produces a glass hard template through photolithography and wet etching processes, which is used for molding of PDMS, and the microchannels required for the composition of the microfluidic chip are obtained and used for making Soft template for electrodes. The glass hard template can be used repeatedly in operation, and the fabricated PDMS microchannel has uniform size and good shape reproducibility. Zhou Xiaomian, Lin Bingcheng and others invented a microfluidic chip (application number 02274236, a microfluidic chip). The chip has a width of 4-6cm, a length of 7-9cm, a thickness of 1-3mm, and a cross structure. type microchannel, the chip is composed of a polymer plate A with a cross-shaped microchannel integrated on one side and a polymer sealing plate B; a closed channel is formed in the middle of the two plates, and on any one of the plates The entrance and exit of the channel are provided, and the feature is that the thickness of the plate B is 100 μm to 600 μm. The cross-shaped microchannels can be arranged alternately with one forward and one reverse.

From the patent retrieval of microfluidic chips, it can be seen that although the chip materials and electrode materials used in the electrochemical detection of microfluidic chips are various, and the manufacturing method of the chip has made great progress compared with the traditional glass chip, it is still The limitation of single channel has not been broken through, therefore, the advantages of electrochemical detection in microfluidic chips have not been really brought into play.

Contents of the invention

An object of the present invention is to provide a multi-channel microfluidic chip applied to electrochemical detection.

Another object of the present invention is to provide a method for preparing the above-mentioned multi-channel microfluidic chip.

The invention provides a multi-channel microfluidic chip. The chip is composed of a buffer pool, a sample pool, a detection pool, a sample injection channel, a separation channel, a working electrode channel, electrodes in the working electrode channel, and the like. The buffer pool of the chip protrudes from several separation channels, and each separation channel has a sample pool, a detection pool, a sampling channel, a working electrode channel and an electrode in the working electrode channel, and the detection pool is at one end of the separation channel; The sample pool communicates with the separation channel through the sampling channel; one end of the working electrode channel communicates with the external power supply, and the other end is close to the detection pool. Different separation channels, sampling channels, sample pools, and detection pools are not connected to each other.

The microfluidic chip of the present invention can be made of organic glass. For example, high molecular polymers such as polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), and polycarbonate are used as materials. In one embodiment of the present invention, polymethyl methacrylate Esters are used as materials for microfluidic chips.

In the microfluidic chip of the present invention, the height of the separation channel, sampling channel and working electrode channel of the microfluidic chip is 25-100 microns, the width is 25-100 microns, and the length of the separation channel is 40-80 mm; The length of the sampling channel is 1-5 mm, and the distance from the intersection point of the sampling channel and the separation channel to the end point of the detection pool of the separation channel is 35-75 mm.

In the microfluidic chip of the present invention, the distance between the working electrode channel and the edge of the detection pool is 25-50 microns.

In the microfluidic chip of the present invention, the diameter of the sample pool is 1-3 mm, the diameter of the detection pool is 3-6 mm, and the distance between the centers of the detection pools is 8-15 mm.

In the microfluidic chip of the present invention, the separation channels are parallel, and the distance between two adjacent separation channels is 7 to 20 millimeters; the working electrode channels of the microfluidic chip are parallel, and the distance between two adjacent working electrode channels The distance between them is 7-20mm.

In the microfluidic chip of the present invention, the angle between the sampling channel and the separation channel is 30-90 degrees.

In the microfluidic chip of the present invention, a waste liquid pool can also be added on each detection channel (i.e. separation channel), and the waste liquid pool communicates with the separation channel through another section of sampling channel, and the diameter of the waste liquid pool is 1 to 3 mm.

In the microfluidic chip of the present invention, the cross section of the buffer pool can be a parallelogram with a width of 4-10 mm, a length of 24-49 mm, and an angle between the length and width of 30-90 degrees.

In the microfluidic chip of the present invention, the heights of the buffer pool, the sample pool, the detection pool and the waste liquid pool are 1-3 mm.

The number of channels of the microfluidic chip of the present invention can be determined according to the actual situation, such as the nature of the material used, the number of samples to be tested and the difference in properties of each component, the wiring of an external voltage control instrument, and so on. In an embodiment of the present invention, a 4-channel multi-channel microfluidic chip is prepared.

In another aspect, the present invention provides a method for preparing the above-mentioned multi-channel microfluidic chip, comprising the following steps:

(1) Make separation channels, sample injection channels and electrode channels on the substrate by hot pressing or in-situ polymerization, and fill the electrode channels with functional carbon paste materials as working electrodes;

(2) Use diamond drilling to make buffer pools on the cover slip, and make sample pools, detection pools and waste liquid pools by laser drilling;

(3) Seal the substrate and the cover.

The above-mentioned preparation method can be specifically operated as follows:

(1) Manufacture of monocrystalline silicon positive mold

The chip design graphics are drawn with CAD software, the line width of the graphics is 25-100 μm, and the high-definition laser phototypesetting system is used to output it on a transparent film (the background is transparent, the required image is black), and the photolithography mask is obtained.

Deposit a layer of silicon nitride film on the substrate by chemical vapor deposition (CVD) as a sacrificial layer, and coat a layer of SU-8 photosensitive adhesive (positive photoresist) on the substrate by spin coating technology, and treat it in an oven at 60 ° C for 10 ~15min; cover the photoresist mask on the substrate, and transfer the pattern on the photoresist mask to the photoresist layer on the surface of the substrate through the principle of exposure imaging; The plane two-dimensional graphics are processed into a three-dimensional structure with a certain depth. A male monocrystalline silicon mold with raised channels can be obtained. The fabricated positive mold is washed with H ₂ O ₂ :H ₂ SO ₄ solution (volume ratio 1:4), acetone and double distilled water in order to remove oxides on the surface.

(2) Replicate channels by hot pressing or in situ polymerization

Cut the PMMA plexiglass sheet to a certain size, ultrasonically clean it, and dry it naturally. The PMMA substrate is heated to the softening temperature (106 ° C) in a hot press device, and a certain pressure (4 inches) is applied on the single crystal silicon positive mold. 20~30kN on the area), and keep it for 30~60s, the microchannel complementary to the male mold can be pressed on the PMMA substrate, and then under the condition of pressurization, the male mold and the PMMA substrate engraved with the microchannel After the sheets are cooled together, they are released from the mold to obtain the desired microchannels.

The instrument required by the hot pressing method is simple and easy to operate, but the channel is easily deformed. We tried to use ultraviolet light to initiate in situ polymerization of methyl methacrylate to prepare microfluidic chip, mix polymer monomer methyl methacrylate with initiator, and add a certain amount of functional monomer (methacrylic acid or 4-vinylpyridine, etc.). The mixture is poured into a mold, and ultraviolet light is used to initiate polymerization. After the chip is released from the mold, a PMMA substrate engraved with microchannels is obtained. This method can effectively eliminate the problem of channel deformation.

Carefully fill the electrode channel with carbon paste (or other functional materials) as a working electrode, scrape off excess carbon paste, and cure under infrared light for 12 to 16 hours.

(3) Make a liquid reservoir on the cover slip

Conventional chips are mostly drilled with diamond drills, and the buffer pool in the present invention is drilled with diamond drills. Since the invention develops a multi-channel chip, the relative positions of the sample pool, the waste liquid pool and the detection pool are required to be very precise, so laser drilling is used for the production of the sample pool, the waste liquid pool, and the detection pool. PMMA can be degraded into volatile small molecules under the action of ultraviolet laser. Prepare a photolithographic mask with copper foil to determine the hole position, use an ultraviolet laser to pass through a microscope objective lens and a photolithographic mask, and focus the laser energy on the photolyzable PMMA cover sheet, so that the photolithographic mask is placed on the PMMA cover sheet. Laser sputtering occurs in a defined area. The depth of laser ablation can be controlled by adjusting the laser intensity and the number of pulses received by the coverslip surface. Through the method provided by the invention, the liquid reservoir holes with precise size and position can be obtained.

(4) Chip sealing

Align the PMMA substrate and the cover under the microscope so that the distance between the electrode channel and the edge of the detection cell is about 25-50 μm (the distance between the electrode channel and the end of the separation channel), and fix the substrate and the cover with two coverslips. , placed in an oven at 108° C. for 10 to 15 minutes, and then the desired PMMA multi-channel microfluidic chip can be obtained.

The present invention also provides the application of the above-mentioned microchip, that is, connecting the multi-channel microfluidic chip with a control device, adding a buffer and a sample, and controlling the voltage to complete the separation, analysis and detection of the components to be tested.

Connect the chip to the control equipment, add the separation liquid and samples, and control the voltage to complete the separation, analysis and detection of the samples. The principle is that under the action of an electric field, the separation is realized by using the different mobility of each component in the sample in the buffer solution, and the components to be measured in the separated sample arrive at the detection cell in turn and are detected. Since the plurality of detection cells designed in the present invention are mutually independent, high-throughput detection of the same substance in the sample can be carried out, and multiple substances in the sample can also be measured by using different electrochemical detection methods.

The specific measurement steps are as follows:

First, modify and modify the channels of the chip according to actual needs.

Secondly, in the schematic diagram of the multi-channel chip shown in Figure 3, the platinum wire is inserted into the buffer pool 1 as the positive electrode for high-pressure separation, the platinum wire is inserted into the sample pool 2 as the positive electrode for high-pressure sampling, and the platinum wire is inserted into the waste pool 3. The wire was used as the negative electrode for sampling. 4 is the detection cell, in which there is a three-electrode system: working electrode, reference electrode and counter electrode. The working electrode was led out with silver glue and copper wire, the reference electrode was self-made Ag/AgCl electrode, and the counter electrode was platinum wire. The relative positions among the three electrodes are shown in Fig. 4 . The diameter of the copper wire is 0.5-1 mm, and the length is 1-2 cm. The diameter of the platinum wire is 0.5-1 mm, and the length is 1-2 cm. The reference electrode is a self-made Ag/AgCl reference electrode. The length of Ag is about 0.5-1mm, and the length is 1-2cm, and a layer of AgCl with a thickness of about 100-300nm is deposited on it by electroplating to obtain the Ag/AgCl reference electrode.

Again, connect the lead wires of all electrodes on the chip to the corresponding working electrode wiring, counter electrode wiring and reference electrode wiring ports of the multi-channel electrochemical analyzer.

Then, connect the voltage-applying wires of the high-voltage power supply of the microfluidic chip to the liquid reservoirs respectively.

Subsequently, the method setting and parameter setting of the electrochemical analyzer are set according to actual needs; the voltage application method of the high-voltage power supply of the microfluidic chip is set in the program.

Finally, the sampling channel and separation channel of the multi-channel chip are filled with buffer solution by vacuum decompression method. Add the sample into the sample pool 2, apply high pressure, the sample solution is filled with the sampling channel 5, when the high pressure between the liquid storage pool 1 and the detection pool 4 is started, the sample solution at the intersection of the separation channel and the sampling channel is brought Enter the separation channel, start the set program, and measure the sample.

The invention fully utilizes the advantages of electrochemical detection in microfluidic chips, and successfully applies multi-channel microfluidic chips to electrochemical detection for the first time. The microfluidic chip of the present invention is used to identify samples in chemical, biological, and pharmaceutical industries, and can simultaneously separate, analyze, and detect components in complex samples, and avoids the high cost and inconvenience caused by fluorescent reagents in existing conventional analysis methods. Interference problems, with the advantages of high throughput and high sensitivity. In addition, the channel width-depth ratio of the microfluidic chip of the present invention is large, the preparation process is simple, and the cost is low. Therefore, the present invention will have very broad application prospects in the field of electrochemical detection.

Description of drawings

Figure 1 is a design diagram of a multi-channel microfluidic chip mask. The radius of the circle is 4 inches (the same size as the monocrystalline silicon mold); 5 is the sampling channel, the length is 1 to 5 mm, and the three line segments below 5 on the same straight line are sampling channels, and the size is the same as 5 ; 6 is a separation channel, the length of which is 40-80mm. The angle between 5 and 6 is 30-60 degrees, the effective degree of the sampling channel is 35-75mm (the distance between the intersection of 5 and 6 and the right end of the separation channel), and the three parallel lines above 6 are also separation The channel has the same size as 6, the vertical distance between two adjacent separation channels is 8-15 mm, and the horizontal distance between the ends of two adjacent separation channels is 6-10 mm. The line width of all channels is 25-100 μm; 7 is the working electrode channel, the length is 7-9 mm, and there are three parallel line segments on the right side of 7, which are all working electrode channels, and the lengths are 13-15, 19-21 , 25 ~ 27mm, the horizontal distance between two adjacent working electrodes is 6 ~ 10mm.

Fig. 2 is a schematic diagram of a single crystal silicon positive mold after dicing.

Figure 3 is a schematic diagram of a multi-channel microfluidic chip applied to electrochemical detection. Among them, 1: buffer pool; 2: sample pool; 3: waste liquid pool; 4: detection pool. 5 is the sampling channel; 6 is the separation channel; 7 is the working electrode channel.

Fig. 4 is a schematic diagram of the relative positions of the outlet of the separation channel and the working electrode. Among them, 4 is the detection cell; 6 is the separation channel; 8 is the filled carbon paste as the working electrode; 9 is the Pt wire as the counter electrode; 10 is the Ag/AgCl reference electrode.

Detailed ways

The making of embodiment 1 A sheet

Sheet A is the substrate, and its production includes the production of the monocrystalline silicon positive mold, the separation and injection microchannel, and the working electrode. The specific production process is as follows:

(1) Fabrication of the mask: use CAD software to draw the design diagram shown in Figure 1, the length of the sampling channel (referring to the distance between the sample pool and the edge of the waste liquid pool) is 2.828mm, and the lengths of the four working electrode channels are respectively 7, 13, 19, 25mm, the horizontal distance between two adjacent working electrodes is 6mm, the length of the separation channel is 50mm, the distance between the intersection point of the sampling channel and the separation channel is 45mm from the right end of the separation channel, and the vertical distance between two adjacent separation channels is 7mm, all line widths are 50μm. Output this design drawing on a transparent film using a high-definition laser phototypesetting system to obtain a photolithography mask.

(2) Manufacture of single crystal silicon positive mold: Deposit a layer of silicon nitride film on the substrate as a sacrificial layer by chemical vapor deposition (CVD) at 550-700 ° C, and coat a layer of SU on the substrate by spin coating technology. -8 Photosensitive adhesive (positive photoresist), process it in an oven at 60°C for 10 minutes; cover the photoresist mask on the substrate, and transfer the pattern on the photoresist mask to the photoresist layer on the surface of the substrate through the principle of exposure imaging ; and then process the plane two-dimensional graphics on the photoresist layer into a three-dimensional structure with a certain depth by dry etching. A male monocrystalline silicon mold with raised channels can be obtained, the depth and width of which are both 50 μm. The fabricated positive mold is washed with H ₂ O ₂ :H ₂ SO ₄ solution (volume ratio 1:4), acetone and double distilled water in order to remove oxides on the surface.

(3) Replicating microchannels by hot pressing

The thickness of the substrate is 0.5-2 mm, and in this embodiment, the thickness of the selected substrate is 1 mm. Cut a 1mm thick PMMA plexiglass sheet into a rectangle with a size of 4.0×8.0cm, ultrasonically clean it, and dry it naturally. Heat the PMMA substrate to 106°C in a hot pressing device, and apply a certain The pressure (20kN on the 4-inch area) is maintained for 30s, and the microchannel complementary to the male mold can be pressed on the PMMA substrate, and then the male mold and the PMMA with the microchannel are engraved The substrates are cooled together and released from the mold to obtain the required microchannels.

(4) Fabrication of the working electrode

The lengths of the four working electrode channels are 7, 13, 19, and 25 mm respectively. The electrode channels are electrode grooves with a width of 50 μm and a depth of 50 μm. Scrape off the excess carbon paste, and cure it under an infrared lamp for 12 hours to complete the production of the substrate.

The making of embodiment 2B sheet

Sheet B is a cover sheet, and its structure includes various reservoir holes, including 1 buffer pool, 4 sample pools, 4 waste liquid pools and 4 independent electrochemical detection pools. The specific production process is:

(1) The size of sheet B is 4.0×8.0 cm, and the thickness is 1-3 mm. In this embodiment, PMMA with a thickness of 2 mm is selected. The coverslip was washed with distilled water, soaked in 0.1mol/L NaOH solution for 30min, washed with distilled water and dried with nitrogen.

(2) Fabrication of the buffer pool: the cross section of the buffer pool is a parallelogram with a width of 5 mm and a length of 34 mm, and the angle between the length and width is 45 degrees. Since the requirement for the size of the buffer pool is not particularly precise, it is sufficient to use a diamond drill to drill holes in this embodiment. The height of the buffer pool is the thickness of slice B, ie 2mm.

(3) Fabrication of sample pool, waste liquid pool and electrochemical detection pool: LAM66 laser ablation machine was used for laser drilling. Prepare a photolithography mask with copper foil, and accurately determine the position of the required punching hole. Use an ultraviolet laser to pass through a microscope objective lens and a photolithographic mask to focus the laser energy on the photolyzable PMMA cover sheet, select the laser frequency to be 50Hz, and set the corresponding unit length of the laser pulse to be 500 pulses/mm. Laser sputtering occurs in the area defined by the engraved mask. The sample pool and waste liquid pool are round holes with a diameter of 1mm, the electrochemical detection pool is a round hole with a diameter of 3mm, and the height of the liquid storage pool is the thickness of the B sheet. , ie 2mm.

Embodiment 3 The sealing of sheet A and sheet B and the making of electrodes

Use hot pressing method to seal A piece and B piece. The specific production process is:

(1) Ultrasonic cleaning of slices A and B, and air-dried.

(2) Align slice A and slice B accurately under the microscope so that the distance between the working electrode and the edge of the reservoir is about 30 μm. After aligning the position, fix and clamp the substrate and the cover with two pieces of glass and put them into the oven, program the temperature to 108°C, keep it warm for 2 minutes, and then program to cool down to 45°C.

(3) Production of each electrode: the electrode used to provide high voltage in the buffer pool 1 is a Pt wire with a length of 10 mm and a diameter of 0.5 mm; the electrodes of the sample pool 2 and the waste liquid pool 3 are 5 mm long and 0.5 mm in diameter The detection cell 4 is a three-electrode working system, and the working electrode is led out with silver glue and copper wire. The diameter of the copper wire is 0.5 mm, and the length is 1 cm. The opposite electrode is 5 mm long, and the diameter is 0.5 mm. The reference electrode is self-made Ag/AgCl Reference electrode. The length of Ag is about 1.5cm, and the diameter is 0.5mm. By electroplating, a layer of AgCl with a thickness of about 300nm is deposited on it to obtain the Ag/AgCl reference electrode.

Embodiment 4 Multi-channel microfluidic chip production example 2

1A production

Sheet A is the substrate, and its production includes the production of the monocrystalline silicon positive mold, the separation and injection microchannel, and the working electrode. The specific production process is as follows:

(1) Fabrication of the mask: use CAD software to draw the design diagram shown in Figure 1, the length of the sampling channel is 4 mm, the lengths of the four working electrode channels are 8, 15, 22, and 29 mm, and the distance between two adjacent working electrodes The horizontal spacing is 8mm, the length of the separation channel is 40mm, the distance between the intersection point of the sampling channel and the separation channel is 35mm from the right end of the separation channel, and the angle between the sampling channel and the separation channel is 30 degrees. The vertical distance between two adjacent separation channels is 7 mm, and all line widths are 100 μm. Output this design drawing on a transparent film using a high-definition laser phototypesetting system to obtain a photolithography mask.

(2) Manufacture of single crystal silicon positive mold: Deposit a layer of silicon nitride film on the substrate as a sacrificial layer by chemical vapor deposition (CVD) at 550-700 ° C, and coat a layer of SU on the substrate by spin coating technology. -8 Photosensitive adhesive (positive photoresist), process it in an oven at 60°C for 10 minutes; cover the photoresist mask on the substrate, and transfer the pattern on the photoresist mask to the photoresist layer on the surface of the substrate through the principle of exposure imaging ; and then process the plane two-dimensional graphics on the photoresist layer into a three-dimensional structure with a certain depth by dry etching. A male monocrystalline silicon mold with raised channels can be obtained, and the depth and width of the channels are both 100 μm. The fabricated positive mold is washed with H ₂ O ₂ :H ₂ SO ₄ solution (volume ratio 1:4), acetone and double distilled water in order to remove oxides on the surface.

(3) In situ initiated polymerization to replicate microchannels on PMMA substrates

After slicing, the size of the silicon mold is 4×8cm. The PMMA substrate (thickness 0.5mm) of the same size as the silicon mold is ultrasonically cleaned and dried, and the microchannel is replicated by in-situ polymerization. The conditions for photo-initiated in-situ polymerization were obtained through a series of optimization experiments, and the microfluidic chip technology was established using methyl methacrylate UV-induced in-situ polymerization. The formula of the polymerization solution is: 100ml of methyl methacrylate monomer, 0.2g of photothermal mixed initiator azobisisobutyronitrile, 0.2g of photoinitiator benzoin and 1-2g of bulk modifier such as methacrylic acid. The polymerization conditions are: thermal prepolymerization temperature 80-90°C, prepolymerization time 15-20min. The mixture was poured into a mold and polymerized using UV light. The photopolymerization conditions after injection molding are: temperature 20-35°C, ultraviolet light wavelength 356nm, power 20w, distance from the ultraviolet lamp 5-6cm, polymerization time about 30-60min. The composite rapid photoinitiation system composed of azobisisobutyronitrile and benzoin can greatly shorten the photopolymerization time. It takes 4-6h to use the traditional photoinitiator benzoin methyl ether, while thermal polymerization takes 8-10h. The photopolymerization system can also Sunlight is used to initiate polymerization, because it is a rapid polymerization, which avoids the excessive irradiation of ultraviolet light on the chip and prevents the problem of yellowing of the chip.

(4) Fabrication of the working electrode

The lengths of the four working electrode channels are 8, 15, 22, and 29 mm respectively. The electrode channels are electrode grooves with a width of 100 μm and a depth of 100 μm. As the working electrode, the carbon fiber is fixed with conductive silver glue, that is, the production of the working electrode is completed.

Production of 2B films

Sheet B is a cover sheet, and its structure includes various reservoir holes, including 1 buffer pool, 4 sample pools, 4 waste liquid pools and 4 independent electrochemical detection pools. The specific production process is:

(1) The size of sheet B is 4.0×8.0 cm, and PMMA with a thickness of 3 mm is selected in this embodiment. The coverslip was washed with distilled water, soaked in 0.1mol/L NaOH solution for 30min, washed with distilled water and dried with nitrogen.

(2) Fabrication of the buffer pool: the cross section of the buffer pool is a parallelogram with a width of 5 mm and a length of 46 mm, and the angle between the length and width is 30 degrees. Since the requirement for the size of the buffer pool is not particularly precise, it is sufficient to use a diamond drill to drill holes in this embodiment. The height of the buffer pool is the thickness of slice B, ie 3mm. The distance between the buffer pool and the upper and lower edges of the coverslip is 7mm and 10mm, respectively.

(3) Fabrication of sample pool, waste liquid pool and electrochemical detection pool: LAM66 laser ablation machine was used for laser drilling. Prepare a photolithography mask with copper foil, and accurately determine the position of the required punching hole. Use an ultraviolet laser to pass through a microscope objective lens and a photolithography mask to focus the laser energy on the photolyzable PMMA cover sheet, select the laser frequency as 50 Hz, and set the corresponding unit length of the received laser pulse to 800 pulses/mm. Laser sputtering occurs in the area defined by the engraving mask, the sample pool and the waste liquid pool are round holes with a diameter of 2mm, and the electrochemical detection pool is a round hole with a diameter of 5mm. The center of the electrochemical detection cell on the far right is 4.5 mm from the right edge of the cover, and 11 mm from the upper edge of the cover. The height of the liquid reservoir is the thickness of the B sheet, that is, 3mm.

3. Sealing of sheet A and sheet B and fabrication of electrodes

Use hot pressing method to seal A piece and B piece. The specific production process is:

(1) Ultrasonic cleaning of slices A and B, and air-dried.

(2) Align slice A and slice B accurately under the microscope so that the distance between the working electrode and the edge of the reservoir is about 50 μm. After aligning the position, fix and clamp the substrate and the cover with two pieces of glass and put them into the oven, program the temperature to 108°C, keep it warm for 2 minutes, and then program to cool down to 45°C.

(3) The making of each electrode: the electrode that is used to provide high voltage in the buffer pool 1 is a Pt wire, with a diameter of 1mm and a length of 45mm, wherein the length of the part inserted into the buffer pool is 40mm; the sample pool 2 and the waste liquid pool 3 The electrode is 2cm long and 0.5mm in diameter; the detection cell 4 is a three-electrode working system, and the working electrode is drawn out with silver glue and copper wire. The diameter of the copper wire is 0.5mm, and the length is 2cm. The reference electrode is a self-made Ag/AgCl reference electrode. The length of Ag is about 1.5cm, and the diameter is 0.5mm. By electroplating, a layer of AgCl with a thickness of about 100nm is deposited on it to obtain the Ag/AgCl reference electrode.

Example 5 Application of multi-channel microfluidic chip in high-throughput detection of organophosphorus pesticides in seawater

(1) Fabrication of multi-channel microfluidic chip

Make 4-channel microfluidic electrochemical detection chip according to the method of embodiment 1, 2, 3

(2) Preparation of simulated phosphoester hydrolase

The catalytic active center of organophosphate hydrolase is a cluster composed of two divalent metal ions and four histidine residues. In natural phosphoester hydrolase, the metal ion is Zn ²⁺ . When Zn ²⁺ is replaced by other divalent metal ions such as Co ²⁺ , Ni ²⁺ , and Cd ²⁺ , the cluster is still resistant to organophosphates. Hydrolysis is catalytic. We use a series of divalent metal ions as functional monomers in order to obtain molecularly imprinted phosphohydrolase with the best catalytic performance, and modify the imprinted polymer into the channel. Since organophosphate pesticides can generate p-nitrophenol under the catalysis of simulated phosphoester hydrolase, it can be detected by amperometric method.

Mock phosphoesterases were prepared using standard methods. That is, the functional monomers are arranged around the imprinted molecules in the solution, and after cross-linking and drying, they are ground, crushed, and sieved to obtain molecularly imprinted media with a certain particle size, and finally the template molecules are eluted to remove them. A typical preparation process is as follows: 1 mmol 4(5) vinylimidazole, 4 mmol diethyl (4-nitrobenzyl) phosphonate (D4NP), 2 mmol CoCl ₂ are dissolved in 250 ml chloroform. After vacuum degassing for 5 minutes, 0.1 mmol of azobisisobutyronitrile was added to initiate polymerization, and reacted in a water bath at 60° C. for 24 hours to obtain a blue polymer. After the polymer is mechanically ground, it is sieved. Take a granular product with a particle size of about 25 μm, extract it with methanol and acetic acid to remove D4NP, and then dry it to obtain the molecularly imprinted simulated phosphoester hydrolase.

The catalytic performance of imprinted polymers is described in terms of catalytic activity (reaction speed), selectivity, etc. The traditional polymer characterization methods are used, such as the determination of specific surface area, pore volume measurement, pore size and distribution measurement, pore shape measurement and other structural characterization of molecularly imprinted polymers through adsorption experiments. The chemical composition of molecularly imprinted polymers was analyzed by elemental analysis, gravimetric analysis, and nuclear magnetic resonance.

(3) Modification and modification of multi-channel microfluidic chip

The interior of the PMMA chip channel is hydrophobic, and it needs to be modified before the molecularly imprinted phospholipase can be modified into the channel.

By modifying the simulated phosphoester hydrolase into the channel through the sol-gel method, the catalytic specific surface area can be greatly increased, and the catalytic efficiency can be greatly improved.

(4) Detection by electrochemical method

First, connect the lead wires of the three electrodes on the 4-channel chip to the corresponding working electrode, counter electrode and reference electrode connecting wire ports of the electrochemical workstation (CHI1030, Shanghai Chenhua Instrument Co., Ltd.).

Secondly, the voltage-applying wiring of the microfluidic chip high-voltage power supply (CDY-500L, Institute of Instrumentation, Shandong Provincial Research Institute of Chemical Industry) was connected to each liquid storage pool respectively.

Again, set the method setting and parameter setting of the electrochemical analyzer according to actual needs: the method is set to amperometric it curve, the sampling time is set to 300s, and the detection potential is set to 0.9V (vs.Ag/ AgCl), the sensitivity was set to 1×10 ^-6 A. The voltage application method of the high-voltage power supply of the microfluidic chip is set in the program. The injection voltage was set to 300V, and the injection time was 20s; the separation voltage was set to 300V, and the high voltage application time was 240s.

Finally, a vacuum pump is used to fill each separation and sampling microchannel with solution, and the channels of the microfluidic chip are washed with 0.1M HCl, double distilled water, 0.1M NaOH, double distilled water, and buffer solution in sequence. Add 20mmol/L borax-NaOH buffer solution (pH is 9.6) in buffer pool 1, add organophosphorus pesticide paraoxon standard solution (or actual sample, that is, seawater filtered through 0.22 μm filter membrane) in sample pool 2 ), add 20mmol/L borax-NaOH damping solution (pH is 9.6) in the waste liquid pool 3, apply sample injection high pressure, the standard (sample) solution promptly fills the sampling channel 5, when start buffer pool 1 and detection pool When the high pressure between 4 and 4, the standard (sample) solution at the intersection of the separation channel and the sampling channel is brought into the separation channel, the electrochemical workstation program that has been set is opened, and the organophosphorus pesticide hydrolyzate p-nitrophenol Detection can be used for quantitative determination of organophosphorus pesticides in samples. Add different concentrations of organophosphorus pesticide paraoxon standard solutions in the sample cell 2, and measure the peak current of the paraoxon hydrolyzate p-nitrophenol oxidation peak, and then establish a linear correlation between the peak current and the concentration of the organophosphorus pesticide relation. Under the set conditions, the peak time of p-nitrophenol is about 52s. The measurement results show that the relative standard deviation of the detection results between different channels is less than 10% (n=3), and the three measurement results of the same channel are relatively standard. The deviation is less than 5%. Injecting 1×10 ^-5 mmol/L paraoxon, the average concentration obtained in 4 channels was 9.6×10 ^-6 mmol/L, with a relative deviation of 4%. The above results show that the multi-channel microfluidic electrochemical chip developed by the present invention can be successfully applied to the efficient and rapid determination of actual samples.

Claims (9)

1. A multi-channel microfluidic chip is characterized in that the buffer pool of the chip stretches out some separation channels, and on each separation channel, there are respectively a sample pool, a detection pool, a sampling channel and a working electrode channel and For the electrodes in the working electrode channel, the detection cell is at one end of the separation channel; the sample cell communicates with the separation channel through the sampling channel; one end of the working electrode channel communicates with an external power supply, and the other end is close to the detection cell. 2. The microfluidic chip according to claim 1, characterized in that, the height of the separation channel, sampling channel and working electrode channel of the microfluidic chip is 25-100 microns, and the width is 25-100 microns. The length of the channel is 40-80 mm; the length of the sampling channel is 1-5 mm, and the distance from the intersection of the sampling channel and the separation channel to the detection pool side end of the separation channel is 35-75 mm. 3. The microfluidic chip according to claim 1, wherein the distance between the channel of the working electrode of the microfluidic chip and the edge of the detection cell is 25-50 microns. 4. The microfluidic chip according to claim 1, wherein the diameter of the microfluidic chip sample pool is 1 to 3 mm, the diameter of the detection pool is 3 to 6 mm, and the distance between the centers of each detection pool is The distance is 8-15 mm. 5. The microfluidic chip according to claim 1, wherein the separation channels of the microfluidic chip are parallel to each other, and the distance between two adjacent separation channels is 7 to 20 millimeters; the microfluidic chip The working electrode channels are parallel to each other, and the distance between two adjacent working electrode channels is 7-20 mm. 6. The microfluidic chip according to claim 1, wherein the included angle between the sampling channel and the separation channel of the microfluidic chip is 30-90 degrees. 7. The microfluidic chip according to claim 1, characterized in that, each separation channel of the microfluidic chip also has a waste liquid pool, and the waste liquid pool communicates with the separation channel through another section of the sampling channel, The diameter of the waste liquid pool is 1-3 mm. 8. The microfluidic chip according to claim 1, characterized in that it is made of polymethyl methacrylate. 9. A method for preparing a multi-channel microfluidic chip as claimed in claim 1, characterized in that it comprises the following steps: (1) Make separation channels, sample injection channels and working electrode channels on the substrate by hot pressing or in-situ polymerization, and fill the electrode channels with functional carbon paste materials as working electrodes; (2) Make a buffer pool on the cover slip by diamond drilling, and make a sample pool, a detection pool, and a waste liquid pool by laser drilling; (3) Seal the substrate and the cover.

Images