Microfluidic chip and its manufacturing method, method and device for testing biochemical oxygen demand

Abstract

The present disclosure provides a microfluidic chip and a method for making the same, as well as a method and device for testing biochemical oxygen demand. The microfluidic chip includes: a flow channel layer and an electrochemical detection layer integrated below the flow channel layer. A biochemical reaction zone, a microchannel, a sample inlet zone, and a sample outlet zone are arranged in the flow channel layer. The biochemical reaction zone has an inner surface in the form of a three-dimensional structure array, on which microorganisms for metabolizing organic matter are fixed. The sample inlet zone is connected to one side of the biochemical reaction zone through a first microchannel, and the sample outlet zone is connected to the other side of the biochemical reaction zone through a second microchannel. There is a accommodating space between the electrochemical detection layer and the biochemical reaction zone, and the accommodating space is used to hold a buffer solution or a sample solution to be tested. The electrochemical detection layer includes: a detection electrode corresponding to the position of the biochemical reaction zone, which realizes the miniaturization and integration of biochemical oxygen demand sensing detection, and can improve the microorganism fixation efficiency and fixation stability, thereby improving the signal conversion efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of biological or chemical micro-nano sensors, and relates to a microfluidic chip for electrochemical detection of biochemical oxygen demand and a manufacturing method thereof, and also relates to a method and device for testing the biochemical oxygen demand of a sample based on the microfluidic chip. Background Art

[0002] Biochemical oxygen demand (BOD) refers to the amount of dissolved oxygen consumed by microorganisms in the biochemical process of decomposing certain oxidizable substances in water, especially organic matter, under specified conditions. The unit is mg / L. The higher the BOD value, the higher the amount of dissolved oxygen consumed by microorganisms in metabolizing organic pollutants, the higher the content of organic pollutants in water, and the more serious the pollution.

[0003] The standard method for measuring biochemical oxygen demand in my country is the dilution and inoculation method. Current commercial instruments usually use the pressure measurement method, such as Hach's BODTrak TM Biochemical oxygen demand analyzer, OxiTop BOD meter from Global Water, USA, etc. The main disadvantages of the above methods are that the measurement cycle is long, requiring a 5-day or (2+5)-day culture phase, making it difficult to conduct online and real-time monitoring, and there are also problems such as cumbersome operation process and poor reproducibility.

[0004] In response to the problem of long BOD measurement cycle, the BOD microbial sensor developed in recent years has received extensive attention and research. At present, although a large number of studies have been carried out on microbial sensors for rapid BOD detection, the rapidity of BOD detection still needs to be further improved. In addition, although the detection time of the sample is greatly shortened, the detection process is complicated. First, the determination of BOD value is the determination of dissolved oxygen consumption, and the dissolved oxygen content in the control group and the sample needs to be determined separately, and the test liquid needs to be repeatedly replaced during the determination; secondly, microorganisms are used in the BOD detection process. Buffer solution is needed to activate the microorganisms before each test, and buffer solution is needed to reactivate the microorganisms after each test to ensure that the state of the microorganisms is consistent before each test. Microorganisms need to be immersed in buffer solution when not in use. These detection and operation processes are very complicated and require high operator requirements.

[0005] Therefore, it is an urgent need in the field of BOD detection to research and develop an integrated BOD detection system that can realize rapid detection of biochemical oxygen demand. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] The present disclosure provides a microfluidic chip and a method for manufacturing the same, and a method and device for testing biochemical oxygen demand, so as to at least partially solve the technical problems raised above.

[0008] (II) Technical solution

[0009] The first aspect of the present disclosure provides a microfluidic chip for electrochemical detection of biochemical oxygen demand. The microfluidic chip includes: a flow channel layer, and an electrochemical detection layer integrated below the flow channel layer. A biochemical reaction zone, a microchannel, a sample inlet zone and a sample outlet zone are arranged in the flow channel layer. The biochemical reaction zone has an inner surface in the form of a three-dimensional structure array, and microorganisms for metabolizing organic matter are fixed on the inner surface. The microchannel includes a first microchannel and a second microchannel. The sample inlet zone is connected to one side of the biochemical reaction zone through the first microchannel, and the sample outlet zone is connected to the other side of the biochemical reaction zone through the second microchannel. There is a accommodating space between the electrochemical detection layer and the biochemical reaction zone, and the accommodating space is used to hold a buffer solution or a sample solution to be tested. The electrochemical detection layer includes: a detection electrode corresponding to the position of the biochemical reaction zone.

[0010] According to an embodiment of the present disclosure, the above-mentioned flow channel layer includes a flow channel layer body, the above-mentioned flow channel layer body has the above-mentioned accommodating space and the microchannel connected to the above-mentioned accommodating space, the top of the above-mentioned accommodating space is a biochemical reaction area, and the bottom of the above-mentioned accommodating space is a detection electrode; the above-mentioned sample inlet area and the above-mentioned sample outlet area are arranged on the above-mentioned flow channel layer body, for realizing the connection between the above-mentioned microchannel and the outside.

[0011] According to an embodiment of the present disclosure, the electrochemical detection layer further comprises a detection layer body, the detection electrode is arranged on the detection layer body, and the detection layer body is bonded to the flow channel layer body.

[0012] According to an embodiment of the present disclosure, the three-dimensional structure array includes a pyramid array.

[0013] According to an embodiment of the present disclosure, microorganisms for metabolizing organic matter are fixed to the inner surface of the three-dimensional structure array by chemical cross-linking to form a three-dimensional microbial sensitive surface for biochemical signal conversion.

[0014] According to an embodiment of the present disclosure, the above-mentioned detection electrode is a three-electrode system, including: a working electrode, a reference electrode and a counter electrode, the above-mentioned working electrode is an ultramicroelectrode array, the ultramicroelectrode units in the above-mentioned ultramicroelectrode array are ultramicrocircular electrodes, and the above-mentioned reference electrode and the above-mentioned counter electrode are arc-shaped and are spaced around the periphery of the ultramicroelectrode array.

[0015] According to an embodiment of the present disclosure, the radius of the biochemical reaction zone is greater than the radius of the detection electrode.

[0016] According to an embodiment of the present disclosure, the above-mentioned microfluidic chip further includes: a PCB circuit board integrated below the above-mentioned electrochemical detection layer, and the PCB circuit board is electrically connected to the detection electrodes of the electrochemical detection layer.

[0017] The second aspect of the present disclosure provides a method for manufacturing a microfluidic chip for electrochemical detection of biochemical oxygen demand as described above. The above-mentioned manufacturing method includes: manufacturing a flow channel layer, wherein the above-mentioned flow channel layer is provided with a biochemical reaction zone, a microchannel, a sample inlet zone and a sample outlet zone, the above-mentioned biochemical reaction zone has an inner surface in the form of a three-dimensional structure array, the above-mentioned inner surface is fixed with microorganisms for metabolizing organic matter, the above-mentioned microchannel includes a first microchannel and a second microchannel, the above-mentioned sample inlet zone is connected to one side of the above-mentioned biochemical reaction zone through the above-mentioned first microchannel, and the above-mentioned sample outlet zone is connected to the other side of the above-mentioned biochemical reaction zone through the above-mentioned second microchannel; manufacturing an electrochemical detection layer, the above-mentioned electrochemical detection layer includes: detection electrodes corresponding to the position of the above-mentioned biochemical reaction zone; and integrating the electrochemical detection layer below the above-mentioned flow channel layer to form a containing space between the above-mentioned electrochemical detection layer and the above-mentioned biochemical reaction zone, and the containing space is used to hold a buffer solution or a sample solution to be tested.

[0018] According to an embodiment of the present disclosure, the manufacturing method further includes: integrating a PCB circuit board below the electrochemical detection layer, the PCB circuit board being electrically connected to the detection electrodes of the electrochemical detection layer.

[0019] According to an embodiment of the present disclosure, the above-mentioned manufacturing of the flow channel layer includes: forming a patterned mask on a clean silicon substrate, the pattern of the patterned mask is used to expose the position of the three-dimensional structure to be formed in the first three-dimensional structure array; performing anisotropic wet etching on the position of the three-dimensional structure to be formed in the first three-dimensional structure array to form a surface with the first three-dimensional structure array on the silicon substrate; removing the patterned mask on the surface with the first three-dimensional structure array; etching a preset depth in the area outside the first three-dimensional structure array of the silicon substrate based on a photolithography process to form an inverted microchannel. A mold structure is provided; a non-stick coating is grown on the upper surface of a silicon substrate of an inverted mold structure forming a microfluidic channel and a glass sheet is bonded to the lower surface to obtain a mold; a PDMS structure complementary to the surface of the silicon substrate is formed on the mold based on an injection molding method, a second three-dimensional structure array and a microfluidic channel are formed on the PDMS structure, and the second three-dimensional structure array is a three-dimensional structure array on the surface of a biochemical reaction zone; a through hole connecting the upper surface of the PDMS structure and the microfluidic channel is formed in an area outside the second three-dimensional structure array of the PDMS structure, and the through hole includes: a sample inlet area and a sample outlet area, to obtain a flow channel layer.

[0020] According to an embodiment of the present disclosure, the above-mentioned manufacturing method also includes: fixing microorganisms for metabolizing organic matter on the inner surface of the above-mentioned three-dimensional structure array form by chemical crosslinking method to form a microbial three-dimensional sensitive surface for biochemical signal conversion. Among them, the above-mentioned fixing microorganisms for metabolizing organic matter on the inner surface of the above-mentioned three-dimensional structure array form by chemical crosslinking method includes: subjecting the lower surface of the above-mentioned flow channel layer to oxygen plasma etching treatment, and the above-mentioned flow channel layer is a PDMS layer; using EDC and NHS to activate the surface of the biochemical reaction zone of the above-mentioned PDMS layer; and dripping the microbial solution into the biochemical reaction zone of the above-mentioned PDMS layer, and letting it stand for a preset time to achieve crosslinking and fixation of the microorganisms and PDMS.

[0021] According to an embodiment of the present disclosure, the above-mentioned preparation of the electrochemical detection layer includes: forming a preliminary working electrode and its leads and welding points on a clean glass substrate based on a photolithography process, and the preliminary working electrode is circular; forming a reference electrode and its leads and welding points, and a counter electrode and its leads and welding points on the substrate formed with the preliminary working electrode and its leads and welding points based on a photolithography process, and the reference electrode and the counter electrode are arc-shaped and spaced around the periphery of the preliminary working electrode; forming an insulating layer on the substrate formed with the preliminary working electrode and its leads and welding points, the reference electrode and its leads and welding points, and the counter electrode and its leads and welding points; and processing the above-mentioned insulating layer based on a photolithography process to expose a partial area of ​​the preliminary working electrode, which is a working electrode in the form of an ultramicroelectrode array, and the ultramicroelectrode units in the ultramicroelectrode array are ultramicrocircular ring electrodes, and at the same time, the reference electrode and the counter electrode, as well as the corresponding welding points of the three electrodes of the preliminary working electrode, the reference electrode and the counter electrode are also exposed, and the leads connected between the three electrodes and the corresponding welding points are covered by the insulating layer, thereby obtaining an electrochemical detection layer. In the electrochemical detection layer, the detection function is performed by the detection electrode, which refers to the working electrode, the reference electrode and the counter electrode. The detection electrode is exposed on the surface of the detection layer body, and the surface of the detection layer body also exposes welding points. An insulating layer is distributed around the detection electrode and the welding point, and the detection electrode and the welding point are connected by a lead shielded by the insulating layer.

[0022] According to the embodiments of the present disclosure, the above-mentioned manufacturing method also includes: integrating the PCB circuit board (lower layer), the electrochemical detection layer (middle layer) and the flow channel layer (upper layer). Specifically, it includes: slicing the prepared electrochemical detection layer and fixing it on the PCB circuit board with AB glue, and the solder joints of the electrodes of the electrochemical detection layer are pressure-welded and encapsulated with AB glue to complete the connection between the electrochemical detection layer and the PCB circuit board. The integrated PCB circuit board and the electrochemical detection layer are cleaned and dried, and the flow channel layer is cleaned with oxygen plasma for a preset time (for example, 20s) to generate silicon hydroxyl groups on its surface; the cleaned flow channel layer is aligned with the electrochemical detection layer, and gently pressed to make the two tightly combined to achieve the integration of the PCB circuit board, the electrochemical detection layer and the flow channel layer.

[0023] The third aspect of the present disclosure also provides a method for testing biochemical oxygen demand based on the above-mentioned microfluidic chip. The above-mentioned method includes: injecting a buffer solution for blank control into the flow channel layer, so that the above-mentioned buffer solution enters the accommodation space through the first microchannel, and obtaining a first dissolved oxygen concentration by testing the detection electrode; injecting a sample solution to be tested into the flow channel layer, so that the above-mentioned sample solution to be tested fills the accommodation space, and after a preset time, obtaining a second dissolved oxygen concentration by testing the above-mentioned detection electrode; calculating the difference between the above-mentioned second dissolved oxygen concentration and the above-mentioned first dissolved oxygen concentration, and the difference is used to characterize the biochemical oxygen demand of the sample solution to be tested.

[0024] The fourth aspect of the present disclosure also provides a device for biochemical oxygen demand detection. The device includes the microfluidic chip as described above; a syringe pump connected to the sample injection area of ​​the microfluidic chip, used to inject a buffer solution for a blank control or a sample solution to be tested into the flow channel layer; and an analysis detector, electrically connected to the PCB circuit board, used to apply a voltage to the detection electrode through the PCB board and measure the current to achieve the detection of dissolved oxygen, thereby achieving the detection of biochemical oxygen demand.

[0025] (III) Beneficial effects

[0026] It can be seen from the above technical solutions that the microfluidic chip and its manufacturing method, and the method and device for testing biochemical oxygen demand provided by the present disclosure have the following beneficial effects:

[0027] (1) The microfluidic chip integrates the biochemical reaction area, electrochemical detection layer, sample inlet and outlet areas, and microchannels into a single microchip, realizing the miniaturization and integration of biochemical oxygen demand sensing detection.

[0028] (2) The biochemical reaction zone of the flow channel layer of the microfluidic chip has an inner surface in the form of a three-dimensional structure array, which can improve the microbial fixation efficiency and fixation stability, thereby improving the biochemical signal conversion efficiency.

[0029] (3) In the biochemical reaction zone of the flow channel layer of the microfluidic chip, the microorganisms are fixed on the inner surface of the three-dimensional structure array by chemical cross-linking, thereby achieving firm fixation of the microorganisms without the need for a semipermeable membrane or embedding material, etc., effectively reducing the mass transfer resistance of the substance and increasing the mass transfer rate, which is conducive to the rapid detection of biochemical oxygen demand.

[0030] (4) In the electrochemical detection layer of the microfluidic chip, the working electrode is configured as an ultramicroelectrode array, so that the working electrode has both the nonlinear diffusion characteristics of the ultramicroelectrode and a sufficiently large output signal, thereby improving the sensitivity and anti-interference ability of the detection.

[0031] (5) The microfluidic chip can be prepared by micromachining technology, which can achieve batch production and the prepared chips have good consistency. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1A Schematic diagram of the structure of a microfluidic chip according to an embodiment of the present disclosure.

[0033] Figure 1B Schematic diagram of the structure of a microfluidic chip according to another embodiment of the present disclosure.

[0034] Figure 2 It is a schematic structural diagram of a flow channel layer observed from obliquely above according to an embodiment of the present disclosure.

[0035] Figure 3 It is a schematic structural diagram of the flow channel layer observed from the bottom according to an embodiment of the present disclosure.

[0036] Figure 4 for Figure 3 The structure diagram of the flow channel layer shown is observed from top to bottom after being cut along the AA line.

[0037] Figure 5 Schematic diagram of the structure of an electrochemical detection layer according to an embodiment of the present disclosure.

[0038] Figure 6 Flow chart of a method for manufacturing a microfluidic chip according to an embodiment of the present disclosure.

[0039] Figure 7 It is a schematic diagram of a detailed manufacturing process of a flow channel layer according to an embodiment of the present disclosure.

[0040] Figure 8 It is a top view of a patterned mask used to form a reverse mold structure of a microfluidic channel in a manufacturing method according to an embodiment of the present disclosure.

[0041] Fig. 9 Schematic diagram of the detailed manufacturing process of the electrochemical detection layer according to one embodiment of the present disclosure.

[0042] Fig.10 It is a top view of a graphically prepared working electrode and its leads and solder joints obtained according to the manufacturing method of an embodiment of the present disclosure.

[0043] Fig.11 It is a top view of a reference electrode and its lead wire and solder joint, and a counter electrode and its lead wire and solder joint obtained according to the manufacturing method of an embodiment of the present disclosure.

[0044] Fig.12 It is a top view of the exposure area of ​​the patterned photoresist obtained according to the manufacturing method of the embodiment of the present disclosure.

[0045] Fig.13 The present invention is a flowchart of a method for testing biochemical oxygen demand based on a microfluidic chip according to an embodiment of the present invention.

[0046] Fig.14 It is a structural block diagram of a device for biochemical oxygen demand detection according to an embodiment of the present disclosure.

[0047]

Explanation of symbols

[0048] 1- Microfluidic chip;

[0049] 11- flow channel layer;

[0050] 111- flow channel layer body; 110- accommodating space;

[0051] 1111-biochemical reaction area; 1112-three-dimensional structure array;

[0052] 112-microfluidic channel;

[0053] 1121 - first microchannel; 1122 - second microchannel;

[0054] 113 - sample inlet area; 114 - sample outlet area;

[0055] 301- a base for making a reverse mold structure of a flow channel layer;

[0056] 302-a patterned mask for forming a first three-dimensional structure array;

[0057] 303-a first three-dimensional structure array;

[0058] 304-a patterned mask for forming a reverse mold structure of a microfluidic channel;

[0059] 3041-biochemical reaction area pattern mask part;

[0060] 3042-first microfluidic channel pattern mask portion;

[0061] 3043-second microfluidic channel pattern mask portion;

[0062] 305-etched area;

[0063] 306-protruding structure;

[0064] 307-Platform;

[0065] 308-non-stick coating;

[0066] 309- glass pieces;

[0067] 310-PDMS structure;

[0068] 12-electrochemical detection layer;

[0069] 120- detection layer body; 401- substrate for making electrochemical detection layer;

[0070] 402-patterned photoresist for forming a preliminary working electrode and its leads and solder joints;

[0071] 121- detection electrode;

[0072] 1211-working electrode;

[0073] 1212-reference electrode;

[0074] 1213- counter electrode;

[0075] 403'-deposited prepared working electrode;

[0076] 403-graphically prepared working electrodes and their leads and solder joints;

[0077] 403a-preparing a working electrode;

[0078] 403b-prepare working electrode lead;

[0079] 403c-preparation of working electrode welding points;

[0080] 404-patterned reference electrode and its leads and solder joints;

[0081] 404a - reference electrode;

[0082] 404b - reference electrode lead;

[0083] 404c-reference electrode welding point;

[0084] 405-patterned counter electrode and its leads and solder joints;

[0085] 405a - counter electrode;

[0086] 405b - electrode lead;

[0087] 405c- electrode welding point;

[0088] 406-insulation layer;

[0089] 407a-retained patterned photoresist;

[0090] 407b - exposed area of ​​photoresist;

[0091] 4071b- ultramicroelectrode array pattern photoresist portion;

[0092] 4072b- working electrode solder joint pattern photoresist portion;

[0093] 4073b-reference electrode pattern photoresist portion;

[0094] 4074b-reference electrode pad pattern photoresist portion;

[0095] 4075b-photoresist portion for electrode pattern;

[0096] 4076b-photoresist portion of electrode solder joint pattern;

[0097] 13-PCB circuit board;

[0098] 500-Biochemical oxygen demand detection device;

[0099] 501-microfluidic chip;

[0100] 502-syringe pump;

[0101] 503-Analytical detector. DETAILED DESCRIPTION

[0102] The embodiments of the present disclosure provide a microfluidic chip for electrochemical detection of biochemical oxygen demand and a method for making the same, a method for testing the biochemical oxygen demand of a sample based on the microfluidic chip, and a device for detecting biochemical oxygen demand. The microfluidic chip integrates a biochemical reaction area, an electrochemical detection layer, a sample inlet and outlet area, and a microchannel into a microchip, thereby realizing miniaturization and integration of biochemical oxygen demand sensing detection. At the same time, the biochemical reaction area of ​​the flow channel layer of the microfluidic chip has an inner surface in the form of a three-dimensional structure array, which can improve the fixation efficiency and fixation stability of microorganisms, thereby improving the signal conversion efficiency.

[0103] In order to make the objectives, technical solutions and advantages of the present disclosure more clearly understood, the present disclosure is further described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0104] A first exemplary embodiment of the present disclosure provides a microfluidic chip for electrochemical detection of biochemical oxygen demand.

[0105] Figure 1A Schematic diagram of the structure of a microfluidic chip according to an embodiment of the present disclosure. Figure 2 It is a schematic structural diagram of a flow channel layer observed from obliquely above according to an embodiment of the present disclosure. Figure 3 It is a schematic structural diagram of the flow channel layer observed from the bottom according to an embodiment of the present disclosure. Figure 4 for Figure 3 The structure diagram of the flow channel layer shown is observed from top to bottom after being cut along the AA line. Figure 4 The section lines are indicated by oblique lines. Figure 2 The main purpose is to show the overall flow channel structure, and its internal structure is indicated by dotted lines. Figure 3 and Figure 4 Middle Pair Figure 2 The internal structure is shown in detail.

[0106] Reference Figure 1A As shown, the microfluidic chip 1 provided in the embodiment of the present disclosure includes: a flow channel layer 11 , and an electrochemical detection layer 12 integrated under the flow channel layer 11 .

[0107] Figure 1B Schematic diagram of the structure of a microfluidic chip according to another embodiment of the present disclosure.

[0108] According to the embodiments of the present disclosure, referring to Figure 1B As shown, the microfluidic chip 1 , in addition to integrating the flow channel layer 11 and the electrochemical detection layer 12 , further includes: a PCB circuit board 13 integrated below the electrochemical detection layer 12 .

[0109] Reference Figure 2 to Figure 4 As shown, a biochemical reaction zone 1111, a microchannel 112, a sample inlet zone 113 and a sample outlet zone 114 are arranged in the flow channel layer 11. The biochemical reaction zone 1111 has an inner surface in the form of a three-dimensional structure array 1112, on which microorganisms for metabolizing organic matter are fixed, the microchannel 112 includes a first microchannel 1121 and a second microchannel 1122, the sample inlet zone 113 is connected to one side of the biochemical reaction zone 1111 through the first microchannel 1121, and the sample outlet zone 114 is connected to the other side of the biochemical reaction zone 1111 through the second microchannel 1122.

[0110] Combination Figure 2 to Figure 4As shown, the flow channel layer 11 includes a flow channel layer body 111, the flow channel layer body 111 has a containing space 110 inside, and the top of the containing space 110 is a biochemical reaction area 1111. A microchannel 112 connected to the containing space 110 is arranged in the flow channel layer body 111; a sample inlet area 113 and a sample outlet area 114 for connecting the microchannel 112 with the outside are arranged on the flow channel layer body 111. The containing space 110 is used to hold a buffer solution or a sample solution to be tested.

[0111] In one embodiment, referring to Figure 4 As shown, the top wall of the accommodating space 110 of the flow channel layer body 111 is a circular biochemical reaction zone 1111, and a three-dimensional structure array 1112 is arranged on the surface of the biochemical reaction zone 1111. The three-dimensional structure array 1112 is circular or rectangular as a whole. The three-dimensional structure array 1112 can be distributed in the entire area or part of the biochemical reaction zone 1111. The microchannel 112 serves as a channel connecting the above-mentioned accommodating space 110 and the inlet and outlet sample areas, wherein the first microchannel 1121 connects the sample inlet area 113 and the biochemical reaction zone 1111 at the top of the accommodating space 110, and the second microchannel 1122 connects the sample outlet area 114 and the biochemical reaction zone 1111 at the top of the accommodating space 110.

[0112] Reference Figure 5 As shown, the detection electrode 121 is a three-electrode system, including: a working electrode 1211, a reference electrode 1212 and a counter electrode 1213. In an exemplary embodiment, the working electrode 1211 is an ultra-microelectrode array, and each ultra-microelectrode unit in the array is an ultra-micro circular electrode. In an exemplary embodiment, the ultra-microelectrode array is arranged as a circle or a polygon as a whole, for example, a hexagon. The reference electrode 1212 and the counter electrode 1213 are arranged at intervals on the periphery of the working electrode 1211 in the form of an ultra-microelectrode array. The reference electrode 1212 and the counter electrode 1213 are arc-shaped and are spaced around the periphery of the ultra-microelectrode array. In one example, the arc length of the counter electrode 1213 is greater than the arc length of the reference electrode 1212, and the sum of the arc lengths of the counter electrode 1213 and the reference electrode 1212 is less than the total circumference, that is, an unclosed gap is left between the counter electrode 1213 and the reference electrode 1212, and the gap is used to make the lead of the working electrode 1211. The working electrode 1211 extends to the external solder joint through the above-mentioned lead. After bonding between the flow channel layer 11 and the electrochemical detection layer 12, the solder joint will be exposed to the outside to facilitate subsequent electrical connection with the PCB circuit board.

[0113] Ultramicro-electrode (UME) refers to an electrode that reaches the micrometer or nanometer scale in one dimension.

[0114] The PCB circuit board 13 is electrically connected to the three electrodes of the detection electrode 121 of the electrochemical detection layer 12. When the biochemical oxygen demand is detected, the PCB board can be connected to an external analysis and detection instrument, and the analysis and detection instrument can apply voltage to the detection electrode through the PCB board and measure the current to achieve the detection of biochemical oxygen demand. Specifically, the electrical connection method is: the PCB circuit board 13 is electrically connected to the external solder joints of the electrochemical detection layer 12.

[0115] Figure 5 Schematic diagram of the structure of an electrochemical detection layer according to an embodiment of the present disclosure.

[0116] In one embodiment of the present disclosure, referring to Figure 5 As shown, the electrochemical detection layer 12 includes: a detection layer body 120, and a detection electrode 121 disposed on the detection layer body 120. The detection electrode 121 is disposed at a position corresponding to the position of the biochemical reaction zone 1111. The detection layer body 120 is a part that carries the detection electrode 121. The detection layer body includes an insulating layer located around the detection electrode 121. The surface of the detection layer body 120 also exposes welding points. Figure 5 As shown in the small squares in the figure. An insulating layer is distributed around the detection electrode 121 and the solder joint, and the detection electrode and the solder joint are connected by a lead wire. Figure 5 The center lead is hidden by the insulation layer, so the lead cannot be seen.

[0117] Reference Figure 4 and Figure 5 As shown, the flow channel layer 11 and the electrochemical detection layer 12 are bonded through the flow channel layer body 111 and the detection layer body 120 to achieve the encapsulation between the lower electrochemical detection layer 12 and the upper flow channel layer 11. In one embodiment, the detection electrode 121 is arranged directly below the biochemical reaction area 1111. Figure 2 to Figure 5 As shown, the hollow area between the electrochemical detection layer 12 and the biochemical reaction area 1111 is the above-mentioned accommodation space 110. That is, the top of the above-mentioned accommodation space 110 is the biochemical reaction area 1111, and the bottom of the accommodation space 110 is the detection electrode 121.

[0118] When the microfluidic chip is not in use, a buffer solution is injected into the accommodating space 110 so that the buffer solution fills the accommodating space 110 and the microchannel 112 to maintain the biological activity of the microorganisms fixed in the biochemical reaction area of ​​the microfluidic chip 1. When the microfluidic chip 1 is used for biochemical oxygen demand detection, a sample solution to be tested and a buffer solution for a blank control are respectively injected into the accommodating space 110.

[0119] The biochemical reaction area 1111 is used to realize the biochemical signal conversion process, that is, through the metabolism of microorganisms, the amount of metabolizable organic pollutants in the sample is converted into the change of dissolved oxygen concentration in the water. When the biochemical oxygen demand is detected, the sample solution to be tested reacts with the biochemical reaction area 1111 fixed with microorganisms at the top of the accommodating space 110, and the microorganisms fixed on the surface of the biochemical reaction area 1111 at the top of the accommodating space 110 metabolize the organic matter in the sample solution to be tested, and the change of dissolved oxygen concentration generated by the metabolic reaction is detected by the detection electrode 121 at the bottom of the accommodating space 110.

[0120] In order to achieve efficient conversion of biochemical signals, a large number of microorganisms are fixed in the biochemical reaction zone. The embodiment of the present disclosure provides a biochemical reaction zone 1111 having an inner surface in the form of a three-dimensional structure array. The surface area of ​​the three-dimensional structure is greatly increased compared to the planar structure, which increases the effective area for microbial fixation; at the same time, the three-dimensional structure increases the surface roughness, which is beneficial to improve the stability of microbial fixation. Since the inner surface of the biochemical reaction zone of the flow channel layer of the microfluidic chip is an inner surface in the form of a three-dimensional structure array, the microbial fixation efficiency and fixation stability can be improved, thereby improving the signal conversion efficiency.

[0121] In one embodiment, the three-dimensional structure array includes a pyramid array. For example, in a specific example, the three-dimensional structure is a quadrangular pyramid structure, the side length of the quadrangular pyramid is between 80 μm and 120 μm, for example, 100 μm, and each quadrangular pyramid is aligned horizontally and vertically, and maintains a spacing of 15 μm to 25 μm, for example, the spacing is 20 μm. In one example, the three-dimensional structure is a quadrangular pyramid, and the degree of the bottom angle of the side view of the quadrangular pyramid is 54.74°.

[0122] According to an embodiment of the present disclosure, microorganisms for metabolizing organic matter are fixed to the inner surface of the three-dimensional structure array by chemical cross-linking to form a three-dimensional microbial sensitive surface for biochemical signal conversion. When no detection is performed, the microchannels and the accommodating space are filled with a buffer solution to maintain the activity of the microorganisms. When used for the test of biochemical oxygen demand, the microchannels and the accommodating space are filled with a sample solution to be tested or a buffer solution for a blank control.

[0123] For example, the three-dimensional surface with a tetrahedral pyramid structure in the above-mentioned biochemical reaction zone is fixed with Bacillus subtilis, which has strong ability to metabolize organic matter, is non-toxic and widely distributed, by chemical cross-linking to form a three-dimensional microbial sensitive surface for the conversion of biochemical signals.

[0124] In the sensor chip used in the related art to realize electrochemical detection of biochemical oxygen demand, the embedding method is used to fix microorganisms, and the mass transfer resistance of organic matter and dissolved oxygen is large, which increases the mass transfer resistance of organic matter; the PE-PP film is used to increase the mass transfer resistance of dissolved oxygen, which is not conducive to the rapid detection of biochemical oxygen demand. In the microfluidic chip provided by the embodiment of the present disclosure, the inner surface of the three-dimensional structure array of the biochemical reaction zone of the flow channel layer is fixed with microorganisms by chemical cross-linking, which realizes the firm fixation of microorganisms, and no longer needs to use semipermeable membranes or embedding substances, etc., which effectively reduces the mass transfer resistance of substances, improves the mass transfer rate, and is conducive to the rapid detection of biochemical oxygen demand.

[0125] In the related art, some microfluidic chips are suitable for optical detection methods, which have problems such as difficulty in integrating optical detection equipment and high equipment cost, which is not conducive to the integration and miniaturization of the detection system. In the microfluidic chip provided by the embodiment of the present disclosure, the working electrode of the electrochemical detection layer is set as an ultra-microelectrode array, so that the working electrode has both the nonlinear diffusion characteristics of the ultra-microelectrode and a sufficiently large output signal, which improves the sensitivity and anti-interference ability of the detection, and also realizes the integration and miniaturization of the overall chip structure.

[0126] In the embodiment of the present disclosure, in order to ensure that the chemical signal after the microbial metabolic reaction, that is, the change in dissolved oxygen concentration, can be detected by the detection electrode 121, the radius of the biochemical reaction zone is set to be larger than the radius of the detection electrode (three-electrode system) to ensure that the change in dissolved oxygen concentration can be sensed by the working electrode.

[0127] In one example, the biochemical reaction zone 1111 is circular with a radius of 3600 μm, and the corresponding accommodation space 110 is a hollow cylinder. The microchannels 112 (including the first microchannel 1121 and the second microchannel 1122) on both sides of the biochemical reaction zone 1111 are used for sample entry and exit. Here, the first microchannel 1121 and the second microchannel 1122 are set as symmetrical structures, and the width (along the y direction) of both is 400 μm, the depth (along the z direction) is 150 μm, and the length (along the x direction) is 5 mm. The sample inlet area 113 and the sample outlet area 114 at both ends are hollow cylindrical structures, and the diameter of both is 600 μm. The outside of the flow channel layer 11 is a rectangular parallelepiped, and the length × width along the x direction and the y direction is 25 mm × 12.5 mm.

[0128] The electrochemical detection layer 12 is used to realize the electrochemical detection of dissolved oxygen in water. The electrochemical detection layer integrates a detection electrode 121 in the form of a three-electrode system. The detection electrode 121 corresponds to the position of the biochemical reaction zone 1111 in the upper flow channel layer 11. In one embodiment, the working electrode is a gold (Au) ultra-micro circular ring electrode array, the width of a single ring is designed to be 10μm, the average radius is designed to be 25μm, and the area of ​​a single ring is 0.00157mm 2 The spacing between the ultrafine circular ring electrodes in the working electrode: the average radius of the working electrode = 10:1. The ultrafine circular ring electrode array is arranged in a hexagonal shape (an example of a polygon) with a total area of ​​0.2 mm 2 There are 127 circular electrodes in total. The counter electrode and reference electrode are both platinum electrodes, surrounding the working electrode. The inner diameter of the counter electrode is 2.0mm, the outer diameter is 2.8mm, and the surrounding angle is 210°. The total area of ​​the counter electrode is 7.0mm 2 , meeting the requirement of being much larger than the working electrode area. The reference electrode has an inner diameter of 2.0mm, an outer diameter of 2.8mm, and a wrap angle of 90°. The total area of ​​the reference electrode is 3.0mm 2 To match the size of the chip flow channel layer 11 , the length×width dimensions of the electrochemical detection layer 12 are 27 mm×16 mm.

[0129] A second exemplary embodiment of the present disclosure provides a method for manufacturing a microfluidic chip.

[0130] Figure 6 Flow chart of a method for manufacturing a microfluidic chip according to an embodiment of the present disclosure.

[0131] Reference Figure 6 As shown in the solid line frame, the microfluidic chip manufacturing method provided in the embodiment of the present disclosure includes the following operations: S21, S22, and S23.

[0132] In operation S21, a flow channel layer is manufactured, in which a biochemical reaction zone, a microfluidic channel, a sample inlet zone and a sample outlet zone are arranged. The biochemical reaction zone has an inner surface in the form of a three-dimensional structure array. The microfluidic channel includes a first microfluidic channel and a second microfluidic channel. The sample inlet zone is connected to one side of the biochemical reaction zone through the first microfluidic channel, and the sample outlet zone is connected to the other side of the biochemical reaction zone through the second microfluidic channel.

[0133] In operation S22, an electrochemical detection layer is manufactured. The electrochemical detection layer includes: detection electrodes corresponding to the positions of the biochemical reaction zones, and detection electrode leads and detection electrode welding points connected to the detection electrodes.

[0134] In operation S23, an electrochemical detection layer is integrated below the flow channel layer to form a containing space between the electrochemical detection layer and the biochemical reaction area, and the containing space is used to contain a buffer solution or a sample solution to be tested.

[0135] Based on the above operations S21 to S23, the Figure 1A The structure of the microfluidic chip 1 shown in the figure includes: a flow channel layer 11 and an electrochemical detection layer 12 integrated under the flow channel layer 11 .

[0136] According to the embodiments of the present disclosure, referring to Figure 6 As shown in the dotted box, in addition to the operations S21 to S23, the manufacturing method may further include an operation S24: integrating a PCB circuit board below the electrochemical detection layer.

[0137] The PCB circuit board 13 is electrically connected to three electrodes of the detection electrodes 121 of the electrochemical detection layer 12 via solder joints.

[0138] The structure of the microfluidic chip 1 prepared based on the above operations S21 to S24 can be referred to Figure 1B As shown, the PCB circuit board 13 is electrically connected to the three electrodes of the detection electrode 121 of the electrochemical detection layer 12 .

[0139] According to the embodiments of the present disclosure, the above-mentioned manufacturing method also includes: integrating the PCB circuit board (lower layer), the electrochemical detection layer (middle layer) and the flow channel layer (upper layer). Specifically, it includes: slicing the prepared electrochemical detection layer, fixing it on the PCB circuit board with AB glue, and pressing and encapsulating the solder joints of the electrodes of the electrochemical detection layer with the conductive points on the PCB circuit board with AB glue to complete the connection between the electrochemical detection layer and the PCB circuit board. The integrated PCB circuit board and the electrochemical detection layer are cleaned and dried, and the flow channel layer is cleaned with oxygen plasma for a preset time (for example, 20s) to generate silicon hydroxyl groups on its surface; the cleaned flow channel layer is aligned with the electrochemical detection layer, and gently pressed to make the two tightly combined to achieve the integration of the PCB circuit board, the electrochemical detection layer and the flow channel layer.

[0140] Figure 7 It is a schematic diagram of a detailed manufacturing process of a flow channel layer according to an embodiment of the present disclosure.

[0141] According to the embodiments of the present disclosure, referring to Figure 7 As shown, the operation S21 of manufacturing the flow channel layer includes the following sub-operations: S211 to S216.

[0142] In sub-operation S211, a substrate 301 is prepared, such as Figure 7As shown in (a). In this embodiment, the substrate is a silicon substrate, and the sub-operation S211 includes: cleaning the silicon substrate. For example, a 3-inch silicon wafer ((100) crystal orientation) with a thickness of 500 μm is selected. The silicon wafer is cleaned according to a standard cleaning process.

[0143] In sub-operation S212, a reverse mold structure of the biochemical reaction area is manufactured to obtain a silicon substrate including a pattern structure of the biochemical reaction area.

[0144] A patterned mask 302 is formed on a clean silicon substrate 301, such as Figure 7 As shown in (b), the pattern of the above-mentioned patterned mask is used to expose the position of the three-dimensional structure to be formed in the first three-dimensional structure array. In one embodiment, the above-mentioned patterned mask is a hard mask, for example, the material of the hard mask is Si3N4. For example, by low pressure chemical vapor deposition (LPCVD) growth Then, positive photoresist AZ1500 is applied, photolithography is performed, and development is performed to expose the area to be etched away; then, the exposed silicon nitride is etched away by plasma etching to form a patterned mask 302 required for wet etching.

[0145] Based on the patterned mask 302, anisotropic wet etching is performed on the positions of the three-dimensional structures to be formed in the first three-dimensional structure array, such as Figure 7 As shown in (c), a surface having a first three-dimensional structure array 303 is formed on the silicon substrate 301. For example, a KOH solution with a concentration of 33 wt% (mass fraction) is prepared, and a silicon wafer is placed in a water bath at 85° C., and anisotropic wet etching is performed for a controlled time (30 to 90 minutes).

[0146] Remove the patterned mask 302 on the surface having the first three-dimensional structure array 303, such as Figure 7 As shown in (d), for example, the remaining silicon nitride mask is etched away by plasma etching, thereby obtaining a silicon substrate including an inverted mold structure of the biochemical reaction zone, based on which a complementary shape can be subsequently formed as a three-dimensional structure array.

[0147] In sub-operation S213, a reverse mold structure of the microfluidic channel is manufactured.

[0148] Based on the photolithography process, a preset depth is etched in the area outside the first three-dimensional structure array of the silicon substrate to form a reverse mold structure of the microfluidic channel, that is, in the reverse mold structure of the microfluidic channel, the position where the microfluidic channel is to be formed is a solid structure, and the structure outside the microfluidic channel position is a hollow structure, for example Figure 7In the example of (e), another patterned mask 304 is formed on the silicon substrate 301 having the surface of the first three-dimensional structure array 303 , wherein the patterned mask 304 is a photoresist.

[0149] Figure 8 FIG. 1 is a top view of a patterned mask for forming a reverse mold structure of a microchannel in a manufacturing method according to an embodiment of the present disclosure. Figure 8 As shown, the patterned mask 304 includes: a biochemical reaction area pattern mask portion 3041, a first microfluidic channel pattern mask portion 3042 and a second microfluidic channel pattern mask portion 3043. In this embodiment, the portion below the patterned mask 304 is used to be retained, and the area outside the mask is etched to form a reverse mold structure of the microfluidic channel.

[0150] For example, by spinning positive photoresist AZ4620, the pattern of the microfluidic channel is etched on the surface of the silicon substrate containing the pattern structure of the biochemical reaction area through pre-baking, exposure, development, and post-baking; then, based on the above-mentioned patterned mask 304, the reverse mold structure of the microfluidic channel is deeply etched, and the etching depth is 150μm. Finally, the etched silicon wafer is immersed in an acetone solution for lift-off to remove the photoresist to form the reverse mold structure of the microfluidic channel, which is repeatedly washed with deionized water and dried in an oven at 80°C. That is, a convex structure 306 (corresponding to the solid area of ​​the microfluidic channel position, as a reverse mold) with a surface having a first three-dimensional structure array 303 protruding upward relative to the etched platform 307 is formed in the silicon substrate 301, and the etched area is 305 (corresponding to the hollow structure outside the microfluidic channel position), such as Figure 7 As shown in (f).

[0151] In sub-operation S214, a non-stick coating is grown on the upper surface of the silicon substrate forming the reverse mold structure of the microfluidic channel and a glass sheet is bonded to the lower surface to obtain a mold.

[0152] The above sub-operation can be divided into two steps and implemented separately. For example, a non-stick coating 308 is first grown on the upper surface of the silicon substrate 301 forming the microchannel. Figure 7 As shown in (g), a glass sheet 309 is then bonded to the lower surface of the silicon substrate 301. Figure 7 As shown in (h), for example, a 200 nm thick polytetrafluoroethylene is grown on the surface of the silicon substrate forming the microfluidic channel as a non-stick layer; a clean 3-inch glass sheet is adhered to the back of the silicon substrate (silicon wafer) using PDMS and dried in an oven at 80°C.

[0153] In operation S215, a PDMS structure complementary to the surface of the silicon substrate is formed on the mold based on an injection molding method, and a second three-dimensional structure array is formed at a position corresponding to the first three-dimensional structure array of the PDMS structure. The second three-dimensional structure array forms a three-dimensional structure array on the surface of the biochemical reaction area of ​​the subsequent flow channel layer. The position of the PDMS structure corresponding to the reverse mold structure of the microfluidic channel forms a microfluidic channel.

[0154] For example, first prepare the PDMS mixture: fully mix the two components of PDMS (curing agent and resin) in a mass ratio of 10:1, and degas in a vacuum environment. Then, on a horizontal laboratory table, place the mold in a disposable culture dish, and slowly cast the degassed PDMS onto the mold surface to achieve the desired thickness (1-2mm). During the casting process, bubbles will be generated, and it is necessary to degas again and let it stand for 30 minutes. Next, when there are no bubbles on the surface of the silicon wafer, place the cast PDMS culture dish in a 70°C oven for three hours to solidify it and obtain a solidified PDMS structure 310 as shown in FIG. Figure 7 As shown in (i). During this process, the culture dish must be kept level, otherwise the PDMS thickness will be uneven. Finally, a clean surgical blade is used to remove the solidified PDMS from the silicon mold. The cross-sectional structure of the demolded PDMS structure 310 along the central axis is shown in FIG. Figure 7 As shown in (j), the oblique lines are used to indicate the section lines, combined with Figure 8 and Figure 7 As shown in (j), the PDMS mold corresponding to the biochemical reaction area pattern mask portion 3041 is a hollow portion, forming an accommodation space 110, the top of which is a biochemical reaction area 1111, and the inner surface of the biochemical reaction area 1111 is a three-dimensional structure array 1112. The PDMS mold corresponding to the first microfluidic channel pattern mask portion 3042 and the second microfluidic channel pattern mask portion 3043 is a hollow portion, forming a first microfluidic channel 1121 and a second microfluidic channel 1122.

[0155] Next, in operation S216, a sample inlet region and a sample outlet region connecting the microchannel with the outside are formed on the PDMS structure.

[0156] For example, refer to Figure 7As shown in (k), the PDMS structure 310 is cut according to the position marking line, and a flat-headed hollow needle is used to make a sample inlet area 113 and a sample outlet area 114 at the fluid inlet and outlet. The PDMS structure as a whole is a flow channel layer body 111, and a biochemical reaction area 1111 and a microchannel 112 are made inside the flow channel layer body 111. The flow channel layer body 111 is provided with a sample inlet area 113 and a sample outlet area 114 for connecting the microchannel 112 with the outside, thereby realizing the production of the flow channel layer 11. The above-mentioned accommodating space 110 is used to hold a buffer solution or a sample solution to be tested. The top wall of the accommodating space 110 of the flow channel layer body 111 is a circular biochemical reaction area 1111, and a three-dimensional structure array 1112 is provided on the surface of the biochemical reaction area 1111. The shape of the periphery of the three-dimensional structure array can be circular or rectangular (including square). The microchannel 112 serves as a channel connecting the above-mentioned accommodating space and the sample inlet and outlet areas. PDMS debris will be generated during demolding and drilling, and the PDMS structure needs to be cleaned with acetone and ethanol.

[0157] According to an embodiment of the present disclosure, the above-mentioned manufacturing method further includes the following operation S25: using a chemical cross-linking method to fix microorganisms for metabolizing organic matter on the inner surface of the above-mentioned three-dimensional structure array form to form a microbial three-dimensional sensitive surface for biochemical signal conversion. The operation S25 can be performed after the operation S21 and before the operation S23, and the execution order of the operation S25 and the operation S22 is not limited.

[0158] The method of fixing the microorganisms for metabolizing organic matter on the inner surface of the three-dimensional structure array by chemical crosslinking includes: subjecting the lower surface of the flow channel layer to oxygen plasma etching, wherein the flow channel layer is a PDMS layer; activating the surface of the biochemical reaction zone of the PDMS layer by EDC and NHS; and applying a microbial solution (e.g., a concentration of 6×10 7 CFU / ml~6×10 9 CFU / ml) is dripped into the biochemical reaction area of ​​the PDMS layer and allowed to stand for a preset time (2h to 12h) to achieve cross-linking and fixation of the microorganisms and PDMS.

[0159] Fig. 9 Schematic diagram of the detailed manufacturing process of the electrochemical detection layer according to one embodiment of the present disclosure.

[0160] According to an embodiment of the present disclosure, the above-mentioned operation S22 of manufacturing the electrochemical detection layer includes the following sub-operations: S221 to S224.

[0161] In sub-operation S221, a preliminary working electrode and its leads and solder joints are formed on a clean substrate based on a photolithography process.

[0162] In sub-operation S222, a reference electrode and its lead wires and solder joints, and a counter electrode and its lead wires and solder joints are formed on the substrate on which the preliminary working electrode and its lead wires and solder joints are formed based on a photolithography process.

[0163] In sub-operation S223, an insulating layer is formed on the substrate on which the preliminary working electrode and its lead wire and pad, the reference electrode and its lead wire and pad, and the counter electrode and its lead wire and pad are formed.

[0164] In sub-operation S224, the insulating layer is processed based on a photolithography process to expose a portion of the prepared working electrode as a working electrode, expose a reference electrode, a counter electrode and corresponding welding points of each electrode, and obtain an electrochemical detection layer.

[0165] In a specific example, the substrate 401 is prepared first. For example, a 3-inch glass sheet is selected and the glass sheet is cleaned using a standard microelectronics process. Fig. 9 As shown in (a).

[0166] Then, the photolithography step is performed: spin coating positive photoresist AZ1500, and the pattern of the mask is transferred to the photoresist on the surface of the substrate 401 through the steps of pre-baking, exposure, development, and post-baking. Then, the substrate surface not protected by the photoresist is cleaned by an oxygen plasma etching process to remove the residual photoresist after development, and a patterned photoresist 402 is obtained. Fig. 9 As shown in (b).

[0167] Next, a growth step is performed to deposit a tantalum / gold composite layer on the patterned photoresist 402 using a magnetron sputtering process, for example, Ta / Au The composite layer is formed into a deposited preliminary working electrode 403', referring to Fig. 9 As shown in (c), Fig. 9 In the figure, different reference numerals are used to distinguish the deposited preliminary working electrode 403 ′ from the subsequently prepared patterned preliminary working electrode and its leads and solder joints 403 .

[0168] Then the stripping step is performed. Fig. 9 The substrate 401 with the deposited preliminary working electrode 403' in (c) is immersed in an acetone solution, and the photoresist is stripped off to form a patterned preliminary working electrode and its leads and solder joints 403, as shown in FIG. Fig. 9 Then, the surface is dried and cleaned by oxygen plasma etching to remove the residual photoresist.

[0169] Fig.10 It is a top view of a graphically prepared working electrode and its leads and solder joints obtained according to the manufacturing method of an embodiment of the present disclosure.

[0170] Reference Fig.10As shown, a patterned preliminary working electrode and its lead wire and solder joint 403 are formed on a substrate 401 through a photolithography process. The patterned preliminary working electrode and its lead wire and solder joint 403 include a preliminary working electrode 403a, a preliminary working electrode lead wire 403b and a preliminary working electrode solder joint 403c connected in sequence. Exemplarily, the preliminary working electrode 403a is circular, the preliminary working electrode lead wire 403b is in the form of an extended thin wire, and the preliminary working electrode solder joint 403c is rectangular. Fig.10 The prepared working electrode 403a described in the process is different from the working electrode (in Figure 5 1211) are not the same, and the preliminary working electrode formed in this operation step needs to deposit an insulating layer and etch off the patterned insulating layer later so that a part of the area of ​​the preliminary working electrode is exposed, thereby obtaining a working electrode in the form of an ultra-microelectrode array. The preliminary working electrode lead 403b in this step is subsequently shielded by the insulating layer, and the preliminary working electrode solder joint 403c in this step is subsequently exposed after depositing an insulating layer and etching off the patterned insulating layer, and is used as a solder joint for electrically connecting the PCB circuit board with the working electrode.

[0171] Then repeat the above-mentioned photolithography and growth steps, and change the magnetron sputtering Au (gold) in the growth step to magnetron sputtering Pt (platinum). Repeat the stripping step to obtain a patterned reference electrode and its lead and solder joint 404 and a patterned counter electrode and its lead and solder joint 405, as shown in FIG. Fig. 9 As shown in (e).

[0172] Fig.11 It is a top view of a reference electrode and its lead wires and solder joints and a counter electrode and its lead wires and solder joints obtained according to the manufacturing method of an embodiment of the present disclosure.

[0173] Reference Fig.11As shown, through photolithography, a patterned reference electrode and its lead wire and solder joint 404 and a patterned counter electrode and its lead wire and solder joint 405 are formed on a substrate 401. The patterned reference electrode and its lead wire and solder joint 404 include a reference electrode 404a, a reference electrode lead wire 404b and a reference electrode solder joint 404c connected in sequence. The patterned counter electrode and its lead wire and solder joint 405 include a counter electrode 405a, a counter electrode lead wire 405b and a counter electrode solder joint 405c connected in sequence. The above-mentioned reference electrode 404a and counter electrode 405a are both arc-shaped and spaced apart from each other. The reference electrode 404a and counter electrode 405a are used to surround the periphery of the prepared working electrode 403a. The reference electrode 404a and counter electrode 405a correspond to the same circle and the total arc length of the reference electrode 404a and counter electrode 405a is smaller than the circumference of the circle, so as to reserve a gap between the reference electrode 404a and the counter electrode 405a to place the prepared working electrode lead 403b, and the prepared working electrode lead 403b also serves as the lead of the working electrode formed subsequently.

[0174] Next, the insulating layer is prepared: a 1 μm thick silicon nitride SiNx is deposited by plasma enhanced chemical vapor deposition (PECVD) process, which is used as the insulating layer 406 between the ultra-microelectrode array units and between the working electrode and the counter electrode and the reference electrode. Fig. 9 As shown in (f).

[0175] Another photolithography step is then performed: the pattern of the ultra-fine circular electrode array, the reference electrode, the counter electrode and the welding points of the three electrodes are etched on the front side, and the developed photoresist is used as a mask for reactive ion etching to obtain another patterned photoresist 407a. The specific details can refer to the aforementioned photolithography steps.

[0176] The etching step is then performed: silicon nitride (SiN) is processed using trifluoromethane (CHF3) plasma reactive ion etching (RIE) x ) insulating layer to expose the working electrode in the form of a gold (Au) ultra-fine circular ring electrode array, as well as the counter electrode and reference electrode. The structure can refer to Fig. 9 The structure shown in (g) has another form of patterned photoresist 407a thereon.

[0177] Fig.12 Schematic diagram of the exposure area of ​​the patterned photoresist obtained by the manufacturing method according to the embodiment of the present disclosure, wherein the reference numeral 407b is used to indicate the exposure area of ​​the photoresist, and the exposure area 407b is subsequently removed. Fig. 9 The patterned photoresist 407a shown in (g) is the photoresist portion remaining after the exposure area 407b is removed.

[0178] Reference Fig.12As shown, an exemplary patterned photoresist exposure area 407b includes: ultra-microelectrode array pattern photoresist portion 4071b, working electrode solder joint pattern photoresist portion 4072b, reference electrode pattern photoresist portion 4073b, reference electrode solder joint pattern photoresist portion 4074b, counter electrode pattern photoresist portion 4075b and counter electrode solder joint pattern photoresist portion 4076b. After the photoresist is exposed and developed, Fig.12 The exposed area 407b of the graphical photoresist shown in the diagram is dissolved, and the insulating layer below the dissolved position is etched to expose the area below the photoresist part 4071b corresponding to the ultra-microelectrode array pattern in the prepared working electrode 403a. The exposed area is the working electrode in the final microfluidic chip. The prepared working electrodes 403a in other areas are blocked by the insulating layer and are not used as working electrodes for detection in the final microfluidic chip. The arc-shaped reference electrode 404a, the arc-shaped counter electrode 405a and the welding points of the three electrodes are also exposed: the working electrode welding point 403c, the reference electrode welding point 404c and the counter electrode welding point 405c.

[0179] Finally, the degumming and cleaning step is performed: the glass sheet is immersed in an acetone solution to remove the photoresist, and the residual photoresist is removed by oxygen plasma etching. Fig. 9 As shown in (h).

[0180] The microfluidic chip provided in this embodiment can be prepared by micromachining technology, and can be prepared in batches, and the prepared chips have good consistency.

[0181] The third exemplary embodiment of the present disclosure also provides a method for testing biochemical oxygen demand based on the above-mentioned microfluidic chip.

[0182] Fig.13 The present invention is a flowchart of a method for testing biochemical oxygen demand based on a microfluidic chip according to an embodiment of the present invention.

[0183] Reference Fig.13 As shown, the method for testing biochemical oxygen demand based on a microfluidic chip in this embodiment includes the following operations: S31, S32 and S33.

[0184] In operation S31, a buffer solution for a blank control is injected into the flow channel layer so that the buffer solution enters the containing space through the first microchannel, and a first dissolved oxygen concentration is obtained by testing with the detection electrode.

[0185] In operation S32, the sample solution to be tested is injected into the flow channel layer so that the sample solution to be tested fills the containing space, and after a preset time, the second dissolved oxygen concentration is measured by the detection electrode.

[0186] In operation S33, a difference between the second dissolved oxygen concentration and the first dissolved oxygen concentration is calculated, and the difference is used to characterize the biochemical oxygen demand of the sample solution to be tested.

[0187] A fourth exemplary embodiment of the present disclosure also provides a device for biochemical oxygen demand detection.

[0188] Fig.14 It is a structural block diagram of a device for biochemical oxygen demand detection according to an embodiment of the present disclosure.

[0189] Reference Fig.14 As shown, the device 500 for biochemical oxygen demand detection in this embodiment includes: a microfluidic chip 501, a syringe pump 502 and an analysis detector 503. The microfluidic chip 501 can be the microfluidic chip 1 described in the first embodiment.

[0190] The injection pump 502 is connected to the sample injection area of ​​the microfluidic chip 501 and is used to inject a buffer solution for blank control or a sample solution to be tested into the flow channel layer and the accommodating space.

[0191] The above-mentioned analysis detector 503 is electrically connected to the PCB circuit board of the microfluidic chip 501, and is used to apply voltage to the microfluidic chip 501 and measure current to achieve the detection of dissolved oxygen and further the detection of biochemical oxygen demand.

[0192] In summary, the embodiments of the present disclosure provide a microfluidic chip and a method for making the same, and a method and device for testing biochemical oxygen demand. The microfluidic chip integrates a biochemical reaction zone, an electrochemical detection layer, an inlet and outlet sample zone, and a microchannel into a microchip, thereby realizing miniaturization and integration of biochemical oxygen demand sensing detection. The biochemical reaction zone of the flow channel layer of the microfluidic chip has an inner surface in the form of a three-dimensional structure array, which can improve the microbial fixation efficiency and fixation stability, thereby improving the signal conversion efficiency. In the biochemical reaction zone of the flow channel layer of the microfluidic chip, microorganisms are fixed on the inner surface in the form of a three-dimensional structure array by chemical crosslinking, thereby realizing firm fixation of the microorganisms, and no longer needing to use a semipermeable membrane or an embedded substance, etc., effectively reducing the mass transfer resistance of the substance, improving the mass transfer rate, and facilitating the rapid detection of biochemical oxygen demand. In the detection electrode of the electrochemical detection layer of the microfluidic chip, the working electrode is set as an ultramicroelectrode array, so that the working electrode has both the nonlinear diffusion characteristics of the ultramicroelectrode and a sufficiently large output signal, thereby improving the sensitivity and anti-interference ability of the detection. The microfluidic chip can be prepared by micromachining technology, can be prepared in batches, and the prepared chips have good consistency.

[0193] It should be noted that although the present disclosure is described in conjunction with the accompanying drawings, the embodiments disclosed in the accompanying drawings are intended to exemplify the preferred embodiments of the present disclosure and should not be construed as a limitation on the present disclosure. The dimensional ratios in the accompanying drawings are merely schematic and should not be construed as limitations on the present disclosure. Directional terms mentioned in the embodiments, such as "up", "down", "front", "back", "left", "right", etc., are only references to the directions of the accompanying drawings and are not intended to limit the scope of protection of the present disclosure. Throughout the accompanying drawings, the same elements are represented by the same or similar figure marks. Conventional structures or constructions will be omitted when they may cause confusion in the understanding of the present disclosure.

[0194] Moreover, the shapes and sizes of the components in the figures do not reflect the real size and proportion, but only illustrate the contents of the embodiments of the present disclosure. In addition, in the claims, any reference symbols between brackets shall not be constructed as limiting the claims.

[0195] The ordinal numbers used in the specification and claims, such as "first", "second", "third", etc., to modify the corresponding elements, do not themselves mean that the elements have any ordinal numbers, nor do they represent the order of one element and another element, or the order in the manufacturing method. The use of these ordinal numbers is only used to clearly distinguish a component with a certain name from another component with the same name.

[0196] Furthermore, the word "comprising" or "including" does not exclude the presence of elements or steps not listed in a claim. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

[0197] Unless there are technical obstacles or contradictions, the above-mentioned various embodiments of the present disclosure can be freely combined to form additional embodiments, and these additional embodiments are all within the protection scope of the present disclosure.

[0198] The specific embodiments described above further illustrate the purpose, technical solutions and beneficial effects of the present disclosure. It should be understood that the above description is only a specific embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure should be included in the protection scope of the present disclosure.

Claims

1. A microfluidic chip for electrochemical detection of biochemical oxygen demand, characterized in that: include: A flow channel layer, wherein a biochemical reaction zone, a microfluidic channel, a sample inlet zone and a sample outlet zone are arranged in the flow channel layer, wherein the biochemical reaction zone has an inner surface in the form of a three-dimensional structure array, wherein microorganisms for metabolizing organic matter are fixed on the inner surface, wherein the microfluidic channel comprises a first microfluidic channel and a second microfluidic channel, wherein the sample inlet zone is connected to one side of the biochemical reaction zone through the first microfluidic channel, and the sample outlet zone is connected to the other side of the biochemical reaction zone through the second microfluidic channel; An electrochemical detection layer integrated under the flow channel layer, wherein there is a containing space between the electrochemical detection layer and the biochemical reaction zone, wherein the containing space is used to hold a buffer solution or a sample solution to be tested, and the electrochemical detection layer comprises: detection electrodes arranged corresponding to the position of the biochemical reaction zone; The top of the accommodating space is the biochemical reaction zone, the bottom of the accommodating space is the detection electrode, and the detection electrode is a three-electrode system including: a working electrode, a reference electrode and a counter electrode.

2. The microfluidic chip according to claim 1, characterized in that: The channel layer comprises a channel layer body, wherein the channel layer body has the accommodating space and the microchannel connected with the accommodating space, and the sample inlet area and the sample outlet area are arranged on the channel layer body to realize the connection between the microchannel and the outside.

3. The microfluidic chip according to claim 2, characterized in that: The electrochemical detection layer further comprises: a detection layer body, the detection electrode is arranged on the detection layer body, and the detection layer body is bonded to the flow channel layer body.

4. The microfluidic chip according to claim 1, characterized in that: The three-dimensional structure array includes a pyramid array; The microorganisms are fixed on the inner surface of the three-dimensional structure array by chemical cross-linking to form a three-dimensional sensitive surface of microorganisms for biochemical signal conversion.

5. The microfluidic chip according to claim 1, characterized in that: The working electrode is an ultramicroelectrode array, the ultramicroelectrode units in the ultramicroelectrode array are ultramicrocircular electrodes, and the reference electrode and the counter electrode are arc-shaped and surround the periphery of the ultramicroelectrode array at intervals.

6. The microfluidic chip according to claim 1, characterized in that: The radius of the biochemical reaction zone is greater than the radius of the detection electrode.

7. The microfluidic chip according to any one of claims 1 to 6, characterized in that: Also includes: A PCB circuit board is integrated below the electrochemical detection layer, and the PCB circuit board is electrically connected to the detection electrodes of the electrochemical detection layer.

8. A device for detecting biochemical oxygen demand, characterized in that: include: The microfluidic chip according to claim 7; A syringe pump connected to the sample injection area of ​​the microfluidic chip, and used for injecting a buffer solution for a blank control or a sample solution to be tested into the flow channel layer and the accommodating space; as well as The analytical detector is electrically connected to the PCB circuit board and is used to apply voltage to the microfluidic chip and measure current to detect dissolved oxygen and further detect biochemical oxygen demand.

9. A method for manufacturing a microfluidic chip according to any one of claims 1 to 7, characterized in that: include: A flow channel layer is prepared, wherein a biochemical reaction zone, a microfluidic channel, a sample inlet zone and a sample outlet zone are arranged in the flow channel layer, wherein the biochemical reaction zone has an inner surface in the form of a three-dimensional structure array, wherein microorganisms for metabolizing organic matter are fixed on the inner surface, wherein the microfluidic channel comprises a first microfluidic channel and a second microfluidic channel, wherein the sample inlet zone is connected to one side of the biochemical reaction zone through the first microfluidic channel, and the sample outlet zone is connected to the other side of the biochemical reaction zone through the second microfluidic channel; Producing an electrochemical detection layer, the electrochemical detection layer comprising: a detection electrode corresponding to the position of the biochemical reaction zone; as well as An electrochemical detection layer is integrated below the flow channel layer to form a containing space between the electrochemical detection layer and the biochemical reaction area, and the containing space is used to contain a buffer solution or a sample solution to be tested.

10. A method for testing biochemical oxygen demand based on the microfluidic chip according to any one of claims 1 to 7, characterized in that: include: A buffer solution for blank control is injected into the flow channel layer so that the buffer solution enters the containing space through the first microchannel, and a first dissolved oxygen concentration is obtained by testing the detection electrode; Injecting a sample solution to be tested into the flow channel layer so that the sample solution to be tested fills the containing space, and after a preset time, measuring and obtaining a second dissolved oxygen concentration by the detection electrode; as well as The difference between the second dissolved oxygen concentration and the first dissolved oxygen concentration is calculated, and the difference is used to characterize the biochemical oxygen demand of the sample solution to be tested.

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