Microchip for detecting multiple marine pathogenic bacteria based on gold film

Abstract

The invention provides a microchip for detecting multiple marine pathogenic bacteria based on a gold film. The microchip comprises a PCB (Printed Circuit Board), an electrode layer, a flow channel layer and a sealing layer which are sequentially arranged from bottom to top, a detection area and a bonding pad area are arranged on the upper surface of the electrode layer; the electrode layer, the flow channel layer and the sealing layer are all arranged on the detection area; a sample inlet, a detection cavity and a sample outlet which penetrate through the upper end and the lower end and are sequentially communicated are sequentially formed in the runner layer from left to right; a sample injection hole channel communicated with the sample injection port and a sample discharge hole channel communicated with the sample discharge port are formed in the sealing layer; a three-electrode structure is arranged on the upper surface of the electrode layer in the detection cavity, and the free ends of the three-electrode structure extend outwards and are used for being electrically connected with the bonding pad area; the structure is simple, and miniaturization and production manufacturing are facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection chips, and more particularly, to a microchip for detecting multiple marine pathogenic bacteria based on a gold thin film. Background Art

[0002] Marine bacteria are prokaryotic single-celled organisms that live in the ocean and do not contain chlorophyll and phycocyanin. Their individual diameters are usually less than 1 micron, and they are spherical, rod-shaped, spiral-shaped, and branched filamentous. Among them, marine bacteria that cause diseases in humans or marine organisms are called marine pathogenic bacteria. These pathogenic bacteria are commonly found in seafood aquaculture, such as fish, shellfish, crabs, shrimps, etc. The pathogenic bacteria can cause these marine animals to get sick. When humans eat seafood contaminated by pathogenic bacteria, it often causes diarrhea, vomiting, and in severe cases, death. Among them, Vibrio parahaemolyticus (VP) and Escherichia coli (E.coli) are both marine pathogenic bacteria. After humans are infected by these two marine pathogenic bacteria, they will cause symptoms such as abdominal pain, diarrhea, vomiting, and fever. The treatment methods for the two marine pathogenic bacteria are different. Therefore, the differentiation and on-site monitoring of the concentration of the two marine pathogenic bacteria are of great significance for treating infected people, preventing food poisoning caused by seafood, preventing diseases in marine animals, detecting contaminated seafood, and ensuring the safety of people's property and lives.

[0003] Currently, the detection of marine pathogenic bacteria is often completed on a microfluidic chip, which can reduce the experimental cost, improve portability, and increase the integration degree, and is suitable for on-site detection. Currently, the microfluidic chip analysis system mainly adopts two detection technologies: optical and electrochemical. Among them, the optical analysis detection system faces certain limitations in the development of miniaturization due to its complex structure and large volume; in contrast, the electrochemical analysis detection system shows greater application potential due to its significant advantages. This system not only has the characteristic of high sensitivity, but also the peripheral devices such as its signal processing system are relatively simple and are more easily matched with the chip microelectrodes. These characteristics make it have broad application prospects in the development of miniaturization, portability, and efficient and rapid detection. Therefore, the structure of the corresponding microfluidic chip also needs to be continuously miniaturized and the structure needs to be continuously streamlined to improve production efficiency to meet market needs. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a microchip for detecting multiple marine pathogenic bacteria based on a gold thin film, which has a simple structure and is convenient for miniaturization and production and manufacturing.

[0005] The present invention provides a microchip for detecting multiple marine pathogenic bacteria based on a gold thin film, which includes a PCB board, an electrode layer, a flow channel layer, and a sealing layer arranged in sequence from bottom to top; a detection area and a pad area are arranged on the upper surface of the electrode layer; the electrode layer, the flow channel layer, and the sealing layer are all arranged on the detection area; an injection port, a detection cavity, and a sample outlet that penetrate through the upper and lower ends and are sequentially communicated are arranged on the flow channel layer from left to right in sequence; an injection hole channel communicated with the injection port and a sample outlet hole channel communicated with the sample outlet are arranged on the sealing layer; a three-electrode structure is arranged on the upper surface of the electrode layer in the detection cavity, and free ends of the three-electrode structure all extend outwards for electrical connection with the pad area.

[0006] Compared with the prior art, the present application has the following advantages. The chip is designed through a four-layer structure, which greatly reduces the longitudinal space. The detection liquid can be input from the injection hole channel and extracted from the sample outlet hole channel, passing through the three-electrode structure in the detection cavity for electrochemical induction detection. The structure is very simple and there is no complex flow channel structure, which is convenient for miniaturization and production manufacturing.

[0007] In a possible implementation manner, the detection cavity includes a first detection cavity and a second detection cavity that are sequentially communicated from left to right. The first detection cavity is a circular structure with a center at point O1 and a radius of R1; the second detection cavity is a circular structure with a center at point O2 and a radius of R2; the three-electrode structure includes a first pair of electrodes, a first working electrode, a first reference electrode, a second pair of electrodes, a second working electrode, and a second reference electrode; the first pair of electrodes and the first reference electrode are both arc-shaped structures and are both located on the circumference of a virtual circle with a center at point O1 and a radius of r1, where r1 < R1; the first working electrode is a circular structure with a center at O1 and a radius of r2, where r2 < r1; the second pair of electrodes and the second reference electrode are both arc-shaped structures and are both located on the circumference of a virtual circle with a center at point O2 and a radius of r3, where r3 < R2; the first working electrode is a circular structure with a center at O2 and a radius of r4, where r4 < r3.

[0008] Compared with the prior art, adopting the above technical solution can detect two different bacteria simultaneously with two detection cavities, and the above three-electrode structure can make the detection liquid contact more fully, and it is not easy for the three electrodes to generate electrical interference with each other, affecting the accuracy.

[0009] In a possible implementation manner, the length of the first pair of electrodes is greater than the length of the first reference electrode; the length of the second pair of electrodes is greater than the length of the second reference electrode.

[0010] Compared with the prior art, the above technical solution is adopted because the current in the first reference electrode and the second reference electrode is small, and the interference generated can be ignored. Therefore, in order to save space, the space can be left for the first pair of electrodes and the second pair of electrodes to ensure the detection accuracy.

[0011] In a possible implementation, the extension parts of the first reference electrode and the second reference electrode are collinearly arranged.

[0012] Compared with the prior art, the above technical solution is adopted because the current in the first reference electrode and the second reference electrode is small, and the interference generated can be ignored. Therefore, the collinear arrangement can reduce the material consumption and cost, and is more convenient to manufacture.

[0013] In a possible implementation, the sample injection channel is coaxially arranged with the sample injection port; the sample output channel is coaxially arranged with the sample output port.

[0014] Compared with the prior art, adopting the above technical solution can shorten the flow path, facilitate the input and output of the detected liquid, and at the same time facilitate the input and output of the cleaning liquid.

[0015] In a possible implementation, a placement groove for placing and fixing the power supply electrode layer is provided on the PCB board.

[0016] Compared with the prior art, adopting the above technical solution can facilitate the installation and fixation of the electrode layer, and the installation is more stable.

[0017] In a possible implementation, the flow channel layer is made of PMMA material; the electrode layer is made of BF3 glass.

[0018] Compared with the prior art, adopting the above technical solution can be convenient to manufacture and has a low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is an exploded view of the present invention; Figure 2 is a top view schematic diagram of the flow channel layer of the present invention; Figure 3 is a top view schematic diagram of the electrode layer of the present invention; Figure 4 is a top view schematic diagram of the assembled flow channel layer and electrode layer of the present invention; Figure 5 is a schematic diagram of the assembled present invention; Figure 6 is a schematic diagram of the use of the chip of the present invention; Description of the reference numerals: 1 - PCB board, 2 - electrode layer, 3 - flow channel layer, 4 - sealing layer, 11 - detection area, 12 - pad area, 13 - three - electrode structure, 131 - first pair of electrodes, 132 - first working electrode, 133 - first reference electrode, 134 - second pair of electrodes, 135 - second working electrode, 136 - second reference electrode, 14 - placement groove, 31 - sample inlet, 32 - detection cavity, 33 - sample outlet, 41 - sample inlet channel, 42 - sample outlet channel, 100 - cassette, 321 - first detection cavity, 322 - second detection cavity. Detailed implementation manners

[0020] First of all, those skilled in the art should understand that these implementation manners are only used to explain the technical principles of the embodiments of the present application, and are not intended to limit the protection scope of the embodiments of the present application. Those skilled in the art can make adjustments according to needs to adapt to specific application scenarios.

[0021] In the description of the embodiments of the present application, it should be noted that unless otherwise clearly specified and limited, the terms "connected" and "coupled" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium. For those of ordinary skill in the art, the specific meanings of the above terms in the embodiments of the present application can be understood according to specific situations.

[0022] In the embodiments of the present application, unless otherwise clearly specified and limited, the first feature being "on" or "under" the second feature can be that the first and second features are in direct contact, or the first and second features are indirectly in contact through an intermediate medium. Moreover, the first feature being "above", "over" and "on top of" the second feature can be that the first feature is directly above or obliquely above the second feature, or simply means that the first feature has a higher horizontal height than the second feature. The first feature being "under", "beneath" and "underneath" the second feature can be that the first feature is directly below or obliquely below the second feature, or simply means that the first feature has a lower horizontal height than the second feature.

[0023] The present application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] An embodiment of the present application discloses a multi - marine - pathogenic - bacterium detection microchip based on a gold thin film, which includes a PCB board 1, an electrode layer 2, a flow channel layer 3, and a sealing layer 4 arranged in sequence from bottom to top; a detection area 11 and a pad area 12 are arranged on the upper surface of the electrode layer 1; the electrode layer 2, the flow channel layer 3, and the sealing layer 4 are all arranged on the detection area 11; an injection port 31, a detection cavity 32, and a sampling port 33 that penetrate through the upper and lower ends and are sequentially connected are arranged on the flow channel layer 3 from left to right in sequence; an injection hole channel 41 communicated with the injection port 31 and a sampling hole channel 42 communicated with the sampling port 33 are arranged on the sealing layer 4; a three - electrode structure 13 is arranged on the upper surface of the electrode layer 1 in the detection cavity 32, and free ends of the three - electrode structure 13 all extend outwards for electrical connection with the pad area 12.

[0025] Specifically, a four - inch glass wafer with single - side polishing is selected as the substrate for the electrode layer 2. Then, a photoresist ROL7133 is coated on the substrate. After lithography, metal sputtering, and wet photoresist removal in sequence, a three - electrode structure 13, namely a counter electrode, a working electrode, and a reference electrode, is sputtered on the substrate. The flow channel layer 3 can be an acrylic plate with a thickness of 1 mm. One - side protective film is torn off, and double - sided adhesive tape is pasted on this side. After squeezing out the excess air bubbles, the acrylic plate is placed on the cutting platform of a laser cutting machine for cutting the injection port 31, the detection cavity 32, and the sampling port 33 to complete the production. The sealing layer 4 is a silicon wafer with a flat surface. It is incubated in a fluorosilane vapor box for 4 h. The PDMS prepolymer and the curing agent are weighed with a weight ratio of 10:1. The two are mixed evenly and placed in a vacuum desiccator with a vacuum degree of 13 psi and left standing for 30 min to remove most of the air bubbles; the silicon wafer is placed on a horizontal platform, and the PDMS mixture is poured, left standing for 30 min to fill the microstructure with PDMS; it is placed in an 80 °C oven and heated for 1 h. After the PDMS is completely cured, the PDMS is peeled off from the silicon wafer; then it is cut according to the cutting line to obtain the sealing layer 4. The prepared electrode layer 2 is bonded to the detection area 11 on the PCB board 1 with glue. A first pad array 121 corresponding to the three - electrode structure 13 is arranged at the rear side of the pad area 12. The free ends of the three - electrode structure 13 are electrically connected to the first pad array 121 with aluminum wire leads by a wire bonder. Further soldering can be carried out if necessary. Attached Figure 1A second pad array 122 is provided on the front side of the middle pad region 12 and is used as an external wire access area to access external current to the PCB 1, such as accessing the current of a detection device so that the chip can operate. The sealing layer 4 covers the upper surface of the flow channel layer 3 and seals the gaps with sealant. When in use, the detection liquid is injected into the detection cavity 32 through the sample injection channel 41. When it needs to be taken out, the detection liquid is extracted through the sample extraction channel 42. The same applies to cleaning. When in use, before detection, a capture aptamer of Vibrio parahaemolyticus or Escherichia coli with an appropriate concentration is first added and incubated at 37 °C for 1 hour, then MCH is added and incubated at 37 °C for 1 hour, then the sample solution to be detected is added and incubated at 37 °C for 1 hour, and finally the graphene oxide complex of the bacteria to be detected is added and incubated at 37 °C for 1 hour. Then it is connected to the detection device and connected to an electrochemical workstation for measurement.

[0026] As shown in the Figure 4 accompanying drawings, in some embodiments, the detection cavity 32 includes a first detection cavity 321 and a second detection cavity 322 that are connected in sequence from left to right. The first detection cavity 321 is a circular structure with a center at point O1 and a radius of R1; the second detection cavity 322 is a circular structure with a center at point O2 and a radius of R2; the three-electrode structure 13 includes a first pair of electrodes 131, a first working electrode 132, a first reference electrode 133, a second pair of electrodes 134, a second working electrode 135, and a second reference electrode 136; both the first pair of electrodes 131 and the first reference electrode 133 are arc-shaped structures and are both located on the circumference of a virtual circle with a center at point O1 and a radius of r1, where r1 < R1; the first working electrode 132 is a circular structure with a center at O1 and a radius of r2, where r2 < r1; both the second pair of electrodes 134 and the second reference electrode 136 are arc-shaped structures and are both located on the circumference of a virtual circle with a center at point O2 and a radius of r3, where r3 < R2; the first working electrode 132 is a circular structure with a center at O2 and a radius of r4, where r4 < r3.

[0027] In this embodiment, both point O1 and point O2 are located on the same horizontal line. The first pair of electrodes 131 and the first reference electrode 133 are both in an arc structure, and the outer peripheries of the first pair of electrodes 131 and the first reference electrode 133 are both located on the circumference of a virtual circle with point O1 as the center and a radius of r1; the outer peripheries of the second pair of electrodes 134 and the second reference electrode 136 are both located on the circumference of a virtual circle with point O2 as the center and a radius of r3; the length and width of the electrode layer 2 are 20*14.4 mm. The diameters of the first working electrode 132 and the second working electrode 135 are both 3 mm. The distance between the centers of O1 and O2 is 7.5 mm. The width of the first pair of electrodes 131 and the second pair of electrodes 134 is 0.8 mm. The width of the first reference electrode 133 and the second reference electrode 136 is 0.8 mm. The extension parts at the free ends of the three-electrode structure 13 are all 0.2 mm wide, and the extension ends are electrically connected to a square pad with a length and width of 1.5 mm. The diameters of the first detection chamber 321 and the second detection chamber 322 in the flow channel layer 3 are 7 mm, and they are interconnected through a flow channel 0.5 mm wide. The diameters of the sample inlet 31 and the sample outlet 34 are 1 mm. The sample inlet 31 and the first detection chamber 321 are interconnected through a flow channel 0.2 mm wide. The sample outlet 34 and the second detection chamber 322 are interconnected through a flow channel 0.2 mm wide. The length and width of the PCB board 1 are 11*30 mm.

[0028] As shown in the Figure 3 accompanying drawings, in some embodiments, the length of the first pair of electrodes 131 is greater than the length of the first reference electrode 133; the length of the second pair of electrodes 134 is greater than the length of the second reference electrode 136. Since the current in the first reference electrode 133 and the first reference electrode 133 is smaller, this can save space and facilitate leaving space for the first pair of electrodes 131 and the second pair of electrodes 134.

[0029] As shown in the Figure 3 accompanying drawings, in some embodiments, the extension parts of the first reference electrode 133 and the second reference electrode 136 are arranged collinearly. Since the current in the first reference electrode 133 and the second reference electrode 136 is smaller, the interference generated can be ignored. By arranging them collinearly and connecting them out, the material consumption can be reduced, the cost can be lowered, and it is convenient for production and manufacturing.

[0030] As shown in the Figure 1 accompanying drawings, in some embodiments, the sample inlet channel 41 and the sample inlet 31 are coaxially arranged; the sample outlet channel 42 and the sample outlet 33 are coaxially arranged. This can facilitate the rapid input and output of the detection liquid.

[0031] As shown in the Figure 1 accompanying drawings, in some embodiments, a placement groove 14 for placing and fixing the electrode layer 2 is provided on the PCB board 1. The length and width of the placement groove 14 are 20*14.4 mm, and the depth can be flexibly adjusted according to the thickness of the electrode layer 2, which is convenient for placing and installing the electrode layer 2.

[0032] In some embodiments, the flow channel layer 3 is made of PMMA material; the electrode layer 2 is made of BF33 glass. It is convenient for miniaturization, production and manufacturing, and has low cost.

[0033] It should also be noted that when this application is used, as shown in the appendix Figure 6 The chip is set in the cassette 100. The cassette 100 is electrically connected to the electrochemical workstation terminal computer. In the cassette 100, there are leads for accessing current to the second pad array 122 and a photomultiplier tube for detecting the optical signal intensity of the three-electrode structure 13. When the optical signal value in the cassette 100 drops to zero, detection can be carried out. If there is light, it will cause errors. Then, the excitation method of cyclic voltammetry is used for detection, and the voltage range is 1V - 1.5V. The electrochemiluminescence intensity of the three-electrode structure 13 is detected by the photomultiplier tube. Since the luminescence intensity value has a linear relationship with the logarithm of the concentration of marine pathogenic bacteria, only by substituting the luminescence values into the standard curves for the detection of two marine pathogenic bacteria, namely Vibrio parahaemolyticus and Escherichia coli, respectively, the concentrations of the two marine pathogenic bacteria in the sample can be obtained. This detection method and the chip usage method belong to the prior art and will not be elaborated in this application.

[0034] In the description of the embodiments of this application, it should be noted that in the description of this application, terms such as "inside", "outside", etc., indicating directions or positional relationships are based on the directions or positional relationships shown in the drawings. This is only for the convenience of description and does not indicate or imply that the device or component must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation to this application.

[0035] In the description of this application, the description referring to terms such as "one embodiment", "some embodiments", "in this embodiment", "specific example", or "some examples", etc., means that the specific features, mechanisms, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, mechanisms, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0036] The above is only the specific implementation manner of this application, but the protection scope of this application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed in this application should be covered by the protection scope of this application. Therefore, the protection scope of this application should be subject to the protection scope of the claims.

Claims

1. A microchip for detecting multiple marine pathogenic bacteria based on a gold thin film, characterized in that, It includes a PCB board (1), an electrode layer (2), a flow channel layer (3), and a sealing layer (4) arranged successively from bottom to top; a detection area (11) and a pad area (12) are arranged on the upper surface of the electrode layer (1); the electrode layer (2), the flow channel layer (3), and the sealing layer (4) are all arranged on the detection area (11); a sample inlet (31), a detection cavity (32), and a sample outlet (33) that penetrate through the upper and lower ends and are successively connected are arranged on the flow channel layer (3) from left to right; a sample inlet channel (41) communicated with the sample inlet (31) and a sample outlet channel (42) communicated with the sample outlet (33) are arranged on the sealing layer (4); a three - electrode structure (13) is arranged on the upper surface of the electrode layer (1) in the detection cavity (32), and free ends of the three - electrode structure (13) all extend outwards for electrical connection with the pad area (12).

2. The microchip for detecting multiple marine pathogenic bacteria based on a gold thin film according to claim 1, wherein The detection cavity (32) includes a first detection cavity (321) and a second detection cavity (322) that are successively connected from left to right. The first detection cavity (321) is a circular structure with a center at point O1 and a radius of R1; the second detection cavity (322) is a circular structure with a center at point O2 and a radius of R2; the three - electrode structure (13) includes a first pair of electrodes (131), a first working electrode (132), a first reference electrode (133), a second pair of electrodes (134), a second working electrode (135), and a second reference electrode (136); the first pair of electrodes (131) and the first reference electrode (133) are both arc - shaped structures and are both located on the circumference of a virtual circle with a center at point O1 and a radius of r1, where r1 < R1; the first working electrode (132) is a circular structure with a center at O1 and a radius of r2, where r2 < r1; the second pair of electrodes (134) and the second reference electrode (136) are both arc - shaped structures and are both located on the circumference of a virtual circle with a center at point O2 and a radius of r3, where r3 < R2; the first working electrode (132) is a circular structure with a center at O2 and a radius of r4, where r4 < r3.

3. The microchip for detecting multiple marine pathogenic bacteria based on a gold thin film according to claim 2, wherein, The length of the first pair of electrodes (131) is greater than the length of the first reference electrode (133); the length of the second pair of electrodes (134) is greater than the length of the second reference electrode (136).

4. The microchip for detecting multiple marine pathogenic bacteria based on a gold thin film according to claim 3, characterized in that, The extending parts of the first reference electrode (133) and the second reference electrode (136) are collinearly arranged.

5. The microchip for detecting multiple marine pathogenic bacteria based on a gold thin film according to claim 1, 2, 3 or 4, characterized in that, The sample inlet channel (41) is coaxially arranged with the sample inlet (31); the sample outlet channel (42) is coaxially arranged with the sample outlet (33).

6. The microchip for detecting multiple marine pathogenic bacteria based on a gold thin film according to claim 1, 2, 3 or 4, characterized in that A placement groove (14) for placing and fixing the electrode layer (2) is arranged on the PCB board (1).

7. The microchip for detecting multiple marine pathogenic bacteria based on a gold thin film according to claim 1, 2, 3 or 4, characterized in that The flow channel layer (3) is made of PMMA material; the electrode layer (2) is made of BF33 glass.

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