A portable microfluidic chip based on electrochemical detection and a use method thereof

Abstract

The application provides a portable micro-fluidic chip based on electrochemical detection and a use method, and belongs to the field of micro-nano manufacturing. The portable micro-fluidic chip is composed of a channel layer and a sensing electrode layer to form a detection device, and can be used in the field of biochemical detection. The sensing electrode layer is located above the channel layer, and the two can be packaged through thermal bonding or adhesive bonding or other ways. The channel layer is used for forming a fluid channel and plays a role of an insulation layer, and includes a pretreatment area, a reagent storage area, a detection area, a reference electrode chamber and a waste pool, so that various functions such as separation, mixing and detection can be realized. The sensing electrode layer is a cover sheet layer with a sensing electrode, and a three-electrode working system is integrated on the cover sheet layer, including a working electrode, a reference electrode and a counter electrode. The application can guarantee the stability of electrochemical sensing through the simple structure of the substrate and the cover sheet structure, effectively reduce the assembly difficulty, reduce the process cost, improve the production efficiency, and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of micro-nano manufacturing, and particularly relates to a micro-fluidic chip based on an electrochemical detection principle and a use method. BACKGROUND

[0002] Micro-fluidic technology is a means for precisely controlling micro-fluids, especially for controlling fluid at the level of microliters or nanoliters. A micro-fluidic chip is a chip on which functions of a biological and chemical laboratory, including sampling, dilution, reagent addition, reaction, separation and detection, are integrated. The chip is generally composed of various liquid storage pools and a micro-channel network connected with each other, can greatly shorten sample processing time, and realizes maximum utilization efficiency of reagents and consumables through precise control of liquid flow. The micro-fluidic chip system has the characteristics of low reagent consumption (the analysis reagent sample amount is only microliters), high efficiency (analysis and detection are mostly completed within seconds), miniaturization and integration, but its sensor.

[0003] Laboratory detection commonly uses mass spectrometers, ion chromatographs, gas chromatographs, atomic fluorescence photometers and other equipment, has wide application range and high accuracy, but is complex to operate and must be completed by professionals, and is large in size and inconvenient to move, and cannot meet the needs of on-site detection. In electrochemical detection, according to the types of output electrical signals, there are amperometric detection, conductometric detection and potentiometric detection. The amperometric detection is a method for quantitatively detecting a to-be-detected substance according to an oxidation current or a reduction current generated by an electrochemical reaction of the to-be-detected substance on a working electrode under a constant (or pulse) potential condition, has high sensitivity and certain selectivity. Compared with other methods, the amperometric detection is not affected by the light transmittance, background fluorescence, reflectivity and other detection conditions of the chip material, and the detection process is not disturbed by environmental light; when there are multiple active substances for interference, the potential at which the peak current appears can be used to highly selectively identify the current signal of the to-be-detected substance.

[0004] Because the sensing electrode can be miniaturized and integrated through MEMS technology, the electrochemical detection system is integrated into the micro-fluidic chip, and this has become a hot research direction of instant detection. SUMMARY

[0005] The present application aims to provide an amperometric electrochemical sensing micro-fluidic chip which is simple in preparation method, convenient to carry and has universality, and use the chip for detection of heavy metal ions.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0007] The application discloses a portable micro-fluidic chip based on electrochemical detection, which is a simple electrochemical micro-fluidic sensing chip 1, which is composed of a channel layer 2 and a sensing electrode layer 3 to form a detection device, and the sensing electrode layer 3 is located above the channel layer 2, and the two can be packaged by thermal bonding or adhesive bonding.

[0008] The channel layer 2 is used for forming a fluid channel, the channel has a multifunctional unit inside, and after bonding, the liquid can only flow along a fixed route, and since the polymer material is not conductive, the liquid sample and the electrodes in non-detection areas can be isolated, thus playing a role of an insulating layer, and an additional insulation operation is not needed, and the consistency of the effective area of the electrodes passing through the interdigital electrode part of the working electrode is ensured. The channel layer 2 comprises a pretreatment area 5, a reagent storage area 6, a detection area 7, a reference electrode chamber 8 and a waste liquid pool 9, and all the components are of an open structure on the upper surface. The height of the pretreatment area 5 is slightly lower than that of the reagent storage area 6 and the detection area 7, and a porous filter membrane is arranged in the pretreatment area 5, which is mainly used for removing large particles in the sample to be detected, so as to prevent the large particles from blocking the channel and to reduce the obstacles to the electrode surface reaction; the detection area 7 is an elongated straight channel, and the upper surface thereof is of an open structure; the liquid flows through the detection area 7 under the action of capillary force and contacts a counter electrode 11 and a working electrode 12 in the sensing electrode layer 3; the reagent storage area 6 is a connecting transition area between the pretreatment area 5 and the detection area 7, and is used for storing dry powder reagents or dry test strips used for electrochemical detection reaction; the waste liquid pool 9 is a large storage pool at the end of the straight channel of the detection area 7, and is used for containing the solution after reaction. The reference electrode chamber 8 contains KCl gel, and the reference electrode chamber 8 is connected with the detection area 7 through a gap, so that the stability of the reference electrode potential is maintained by maintaining the liquid loop formed by the reference electrode and the working electrode and reducing the ion diffusion in the liquid to be detected.

[0009] The sensing electrode layer 3 is a cover layer of the electrochemical micro-fluidic sensing chip 1, and a three-electrode system for electrochemical detection is integrated on the sensing electrode layer 3, which comprises a working electrode 12, a counter electrode 11 and a reference electrode 10. The working electrode 12 and the counter electrode 11 are arranged in an interdigital electrode structure above the detection area 7, and the interdigital length is 1.5-5 times the width of the channel of the detection area 7. The reference electrode 10 is located above the reference electrode chamber 8 and is covered by KCl gel (after the channel layer 2 and the sensing electrode layer 3 are packaged), and an exhaust hole is arranged at the connecting position of the reference electrode chamber 8 and the detection area 7, so as to avoid the formation of a "gas dead zone" after the detection liquid flows through the channel and to ensure the contact between the gel and the detection liquid. The sensing electrode layer 3 is provided with a liquid inlet 4 located above the pretreatment area 5. The sensing electrode layer 3 is provided with a plurality of exhaust holes 13, which are respectively located at the positions close to the corners of the pretreatment area 5, the connecting position of the reference electrode chamber 8 and the detection area 7 and the position above the waste liquid pool 9.

[0010] The use method is as follows: when detection, the electrochemical microfluidic sensing chip 1 is connected with the external conductive area and the electrochemical workstation interface, the sample to be detected is injected from the liquid inlet 4, and flows forward under the capillary force, first enters the pretreatment area 5, the large particles are intercepted in the area, the filtered sample flows out from below the filter membrane to realize solid-liquid separation, enters the reagent storage area 6 and mixes with the medicine attached in advance, then enters the detection area 7 and contacts with the counter electrode 11 and the working electrode 12 in the sensing electrode layer 3, after the liquid flows through the reference electrode chamber 8, the three-electrode system forms a loop, the electrochemical workstation is used to apply an excitation signal to the three-electrode system and read the response current, and the sample solution after detection finally flows into the waste liquid pool 9. Here, the reference electrode 10 is an electrode coated with Ag / AgCl, in order to maintain the stability of the relative potential in the reaction process, the KCl gel in the reference electrode chamber 8 needs to cover the reference electrode 10. In addition, the whole system needs to form a current loop, therefore, there is a gap between the reference electrode chamber 8 and the detection area 7, when the sample to be detected flows, contacts with the gel in the reference electrode chamber 8, and the whole reaction system can be connected. Because the sample concentration is proportional to the peak response current, different concentrations of samples are used to draw a regression curve in advance. For the concentration of the sample to be detected, the electrochemical workstation is stopped, the peak response current is read, and the concentration of the sample to be detected can be obtained by bringing the regression curve.

[0011] Further, the working electrode 12 is composed of 3-20 interdigital electrodes, and the total area of the working electrode 12 in the channel is 0.1-15 mm 2 ; the number of interdigital electrodes of the counter electrode 11 is consistent with that of the working electrode 12, but the total area of the counter electrode 11 in the channel is 2-8 times that of the working electrode 12.

[0012] Further, the channel layer 2 is characterized by including a pretreatment unit for rapid filtration, an electrochemical detection area and a waste liquid storage area, the channel layer 2 is used to form a fluid channel and plays an insulating role.

[0013] Further, the sensing electrode layer 3 has the functions of a sensing electrode and a channel cover plate, wherein the sensing electrode integrates a three-electrode system, and the channel cover plate includes a liquid inlet and a plurality of exhaust holes above the waste liquid pool.

[0014] Further, the reference electrode chamber 8 contains KCl gel, and the gel is configured by agarose and KCl solution, wherein the mass fraction of agarose is 3-6%, and the concentration of KCl solution is 1 mol / L to saturation.

[0015] Further, the connecting port between the reference electrode chamber 8 and the detection area 7 is separated by a plurality of gaps with a cylindrical, square column, elliptical column and other column structures with a width of 10-200 μm.

[0016] Further, the assembly between the channel layer 2 and the sensing electrode layer 3 can adopt a hot bonding, ultrasonic bonding or rapid adhesive bonding method.

[0017] The bonding of the channel layer 2 and the sensing electrode layer 3 to obtain the electrochemical sensing chip 1 comprises the following steps:

[0018] Firstly, the channel layer 2 and the sensing electrode layer 3 are prepared.

[0019] Secondly, a glue layer drawing paper is drawn according to the profile of the channel layer 2, and a laser cutting double-sided adhesive film is adopted;

[0020] Thirdly, the adhesive film is placed on the lower pressing plate of a sealing machine, and the sensing electrode layer 3 is aligned with the adhesive film, and then the sealing pressure and sealing time of the sealing machine are set, so that the upper pressing plate of the sealing machine moves downward to make the sensing electrode layer 3 completely adhere to the adhesive film.

[0021] Fourthly, the sensing electrode layer 3 with the adhesive is placed on the lower pressing plate, and the channel layer 2 is aligned with the sensing electrode layer 3, and then the sealing pressure and sealing time of the sealing machine are set, so that the upper pressing plate of the sealing machine moves downward to completely adhere to the upper surface of the cover sheet, thereby completing the sealing of the chip.

[0022] Further, the adhesive film is selected from PET double-sided adhesive film, 3M double-sided adhesive film or PMMA double-sided adhesive film, and the thickness of the adhesive layer is not greater than 50 μm, so as to prevent part of the adhesive film from entering the channel during the bonding process and hindering the liquid flow and electrochemical reaction.

[0023] Further, in order to ensure the flatness of the adhesive film during the pasting, the cut pattern should be connected with the whole without "cantilever" and the minimum line width should be greater than 1 mm.

[0024] Further, the alignment symbols of the double-sided adhesive film, the channel layer 2 and the sensing electrode layer 3 are arranged on the diagonal lines, and the alignment symbol is a "T" shape with a length and width of 50 μm-1 mm. The minimum distance between the alignment symbol and the boundary is 2 mm, so as to prevent the boundary from being broken.

[0025] Further, the sealing pressure and sealing time of the sealing machine are set to 0.1 MPa and 5-15 s.

[0026] The beneficial effects of the present application are as follows:

[0027] (1) The structure uses the channel as an insulating mask layer, which simplifies the process flow of the electrode insulation operation on the non-detection area.

[0028] (2) The fork-shaped design of the working electrode and the counter electrode realizes sensitive detection and signal amplification, and does not need to be precisely aligned with the cover sheet layer, thereby reducing the influence of assembly errors on the three-electrode electrochemical system.

[0029] (3) Structure is simple, more easily batch production, can be used to avoid the systematic error caused by incomplete cleaning. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Axonometric view of the electrochemical sensing chip of the present application.

[0031] Figure 2 Top view of the channel layer of the present application.

[0032] Figure 3 Top view of the sensing electrode layer of the present application.

[0033] Figure 4 Cutting pattern of the adhesive layer of the present application.

[0034] Figure 5 Cyclic voltammetry test results of the specific implementation application of the present application.

[0035] In the figure: 1 electrochemical sensing chip; 2 channel layer; 3 sensing electrode layer; 4 liquid inlet; 5 pretreatment area; 6 reagent storage area; 7 detection area; 8 reference electrode chamber; 9 waste liquid area; 10 reference electrode; 11 counter electrode; 12 working electrode; 13 exhaust hole. DETAILED DESCRIPTION

[0036] The specific embodiments of the present application are further explained in conjunction with the examples and drawings, but are not used to limit the present application.

[0037] An electrochemical sensing chip 1, comprising a channel layer 2 and a sensing electrode layer 3. The channel layer 2 is made of PMMA material, including a pretreatment area 5, a reagent storage area 6, a detection area 7, a reference electrode chamber 8 and a waste liquid pool 9. The sensing electrode layer 3 is made of 300μm thick gold screen printed on PET material, having a working electrode 12 area of 8mm 2 , a reference electrode 10 area of 1.5mm 2 , a counter electrode 11 area of 16mm 2 , and each of the working electrode and the counter electrode includes 6 interdigital electrodes.

[0038] The sealing step of the channel layer 2 and the sensing electrode layer 3 is as follows:

[0039] 1) PET double-sided adhesive with a thickness of 10μm is used, and the shape of the adhesive layer is cut as Figure 4 .

[0040] 2) Place the PET adhesive film on the lower pressing plate of the sealing machine, then align the channel layer 2 with the adhesive film, set the sealing machine to 0.1MPa, 10s, so that the upper pressing plate of the sealing machine moves down and completely adheres to the upper surface of the channel layer 2.

[0041] 3) Put the sealed channel layer 2 and PET adhesive film on the lower pressing plate, and then align the sensing electrode layer 3 with the channel layer 2, and use the sealing machine to seal the chip under the same sealing pressure and sealing time as in the previous step.

[0042] During detection, the sample is injected from the liquid inlet 4, passes through the pretreatment area 5 with water filter membrane, and enters the detection area 7 under the action of capillary force. The reference electrode chamber 8 is filled with 3% agarose and 3 mol / L KCl solution gel, which is used as a supporting electrolyte and covers the reference electrode 10 to ensure the stability of the potential. After the liquid flows through the reference electrode chamber 8, the electrochemical workstation is used for detection.

[0043] The test sample is a mixed solution of 5 mmol / L potassium ferricyanide and potassium ferrocyanide and 0.1 mol / L potassium chloride solution, and the cyclic voltammetry test effect is as follows Figure 5 : The test sample is a mixed solution of 5 mmol / L potassium ferricyanide and potassium ferrocyanide and 0.1 mol / L potassium chloride solution, and the cyclic voltammetry test effect is as follows Figure 5 It can be seen that the repeatability of the five groups of experiments is good, and the curves almost completely coincide. The oxidation peak appears at about 0.38V in the five groups of experiments, and the position of the oxidation peak in the five experiments has almost no deviation, indicating that the reference electrode developed can provide a stable reference potential, and the oxidation peak is about 1.9×10 -5 A, and the difference level is analyzed by t-test, P<0.01 (n=5), indicating that there is no significant difference between the results, and the microfluidic electrochemical detection electrode designed and manufactured by the method has good repeatability.

[0044] The above examples only express the embodiments of the present application, but cannot be understood as limiting the scope of the patent of the present application. For those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which all belong to the protection scope of the present application.

Claims

1. A portable microfluidic chip based on electrochemical detection, characterized in that, The portable microfluidic chip includes a channel layer (2) and a sensing electrode layer (3), with the sensing electrode layer (3) encapsulated above the channel layer (2); The channel layer (2) is used to form a fluid channel and acts as an insulating layer to ensure the consistency of the effective area of ​​the working electrode interdigitated part as the fluid passes through. The channel layer (2) includes a pretreatment area (5), a reagent storage area (6), a detection area (7), a reference electrode chamber (8), and a waste liquid pool (9). All components have an open structure on the upper surface. The pretreatment area (5) is equipped with a porous filter membrane to filter large particles in the sample. The detection area (7) is a long and narrow straight channel through which liquid flows by capillary action to interact with the counter electrode (11) and the working electrode (12) in the sensing electrode layer (3). Contact; The reagent storage area (6) is used to store dry powder reagents or dry test strips for electrochemical detection reactions, and also serves as a connecting transition area between the pretreatment area (5) and the detection area (7); The waste liquid pool (9) is a storage pool at the end of the straight channel of the detection area (7), used to contain the solution after the reaction; The reference electrode chamber (8) contains KCl gel, which serves as the supporting electrolyte for the reference electrode. There is a gap at the connection between the reference electrode chamber (8) and the detection area (7), which maintains the formation of a liquid circuit between the reference electrode and the working electrode, reduces ion diffusion in the test liquid, and maintains the stability of the reference electrode potential; The sensing electrode layer (3) is the cover layer of the electrochemical microfluidic sensing chip (1), on which a three-electrode system for electrochemical detection is integrated, including a working electrode (12), a counter electrode (11), and a reference electrode (10). The working electrode (12) and the counter electrode (11) are arranged in an interdigitated electrode structure and are located directly above the detection area (7). The reference electrode (10) is located above the reference electrode chamber (8) and is covered by KCl gel. An exhaust hole is provided at the connection between the reference electrode chamber (8) and the detection area (7) to ensure contact between the gel and the detection liquid. The surface of the sensing electrode layer (3) is provided with a liquid inlet (4) located directly above the pretreatment area (5). The sensing electrode layer (3) is provided with multiple sets of exhaust holes (13), located near the corner of the pretreatment area (5), at the connection between the reference electrode chamber (8) and the detection area (7), and directly above the waste liquid pool (9).

2. The portable microfluidic chip based on electrochemical detection according to claim 1, characterized in that, The length of the interdigitated electrode structure is 1.5-5 times the width of the detection area (7) channel.

3. A portable microfluidic chip based on electrochemical detection according to claim 1, characterized in that, The working electrode (12) consists of 3-20 interdigitated fingers, and the total area of ​​the working electrode (12) in the channel is 0.1-15 mm². 2 The cross-index of the counter electrode (11) is consistent with that of the working electrode (12), but the total area of ​​the counter electrode (11) in the channel is 2-8 times the total area of ​​the working electrode (12).

4. A portable microfluidic chip based on electrochemical detection according to claim 1, characterized in that, The channel layer (2) and the sensing electrode layer (3) are encapsulated by thermal bonding, ultrasonic bonding or adhesive bonding.

5. A portable microfluidic chip based on electrochemical detection according to claim 1, characterized in that, The height of the pretreatment zone (5) is lower than that of the reagent storage zone (6) and the detection zone (7).

6. A portable microfluidic chip based on electrochemical detection according to claim 1, characterized in that, The reference electrode chamber (8) contains KCl gel, which is prepared with agarose and KCl solution, wherein the mass fraction of agarose is 3-6% and the concentration of KCl solution is 1 mol / L or saturated.

7. A portable microfluidic chip based on electrochemical detection according to claim 1, characterized in that, The connection between the reference electrode chamber (8) and the detection area (7) is divided into multiple gaps by a cylindrical structure, with the width of the gaps being 10~200μm.

8. A method of using the portable microfluidic chip based on electrochemical detection as described in any one of claims 1-7, characterized in that, The sample to be tested is injected from the inlet (4) and flows forward autonomously under the action of capillary force. It first enters the pretreatment zone (5), where large particles in the sample are filtered and separated. The sample then enters the reagent storage zone (6) to be mixed with the reagents and then flows into the detection zone (7). An excitation signal is applied to the three-electrode working system using an electrochemical workstation, and the response current is read. The sample solution after detection finally flows into the waste liquid pool (9).

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