A biosensor for detecting kanamycin based on aptamer

Abstract

This invention relates to the field of kanamycin detection technology, and in particular to a biosensor for kanamycin detection based on aptamers. It includes an upper cover and a lower cover located on the lower end face of the upper cover. A first recess is formed on the lower end face of the upper cover, and a second recess is formed on the upper end face of the lower cover corresponding to the first recess. A substrate is positioned between the first and second recesses, and an aptamer sheet covers the upper end face of the substrate. A signal probe and a display bar are positioned on the upper end face of the aptamer sheet. A placement opening is formed on the inner wall of the observation port, and a transparent sheet is positioned at the placement opening. This invention, through the combined use of the aptamer sheet, signal probe, and display bar, can effectively improve detection sensitivity, accuracy, and efficiency. Simultaneously, the transparent sheet positioned within the observation port provides good protection for the device, thus solving the problems of complex operation, long processing time, insufficient sensitivity, and detection area failure due to the lack of a protective structure.

Description

Technical Field

[0001] This utility model relates to a biological detection sensor, and more particularly to a biological sensor based on aptamer detection of kanamycin, belonging to the field of kanamycin detection technology. Background Technology

[0002] Kanamycin (KAN) is a widely used aminoglycoside antibiotic. Due to its effectiveness against Gram-negative bacteria and some Gram-positive bacteria, it is widely used in human and veterinary clinical treatment as well as in animal husbandry. In order to protect consumer health and address the problem of drug resistance, regulatory agencies around the world have set strict standards for the maximum residue limits of kanamycin in animal-derived foods.

[0003] Aptamers are a class of single-stranded DNA or RNA molecules obtained by screening random oligonucleotide libraries through in vitro screening techniques. They can fold into specific three-dimensional structures with high affinity and specificity, thereby recognizing and binding to specific target molecules. Biosensors are usually composed of biorecognition elements and signal transducers. With the above-mentioned significant advantages, aptamers have become ideal biorecognition elements for building a new generation of biosensors, especially for rapid detection of small molecules.

[0004] Existing kanamycin detection methods suffer from drawbacks such as complex operation, long processing time, or insufficient sensitivity. Furthermore, the exposed detection area of ​​the biosensors used lacks protective structures, making it susceptible to failure due to environmental contact or operational errors. Utility Model Content

[0005] To address the shortcomings of existing technologies, this invention provides a highly efficient and sensitive biosensor for detecting kanamycin based on aptamers.

[0006] The technical solution adopted by this utility model to solve the above-mentioned technical problems is as follows: A biosensor for detecting kanamycin based on aptamers includes an upper cover and a lower cover located on the lower end face of the upper cover. The lower end face of the upper cover has a first recess, and the upper end face of the lower cover has a second recess corresponding to the first recess. A substrate is disposed between the first and second recesses. An aptamer sheet covers the upper end face of the substrate. A signal probe and a display bar are disposed on the upper end face of the aptamer sheet. The upper end face of the upper cover has a drip port communicating with the interior of the first recess. An observation port communicating with the interior of the first recess is located on the upper end face of the upper cover next to the drip port. The signal probe and display bar are located below the observation port. A placement opening is located on the inner wall of the observation port, and a transparent sheet is disposed at the placement opening.

[0007] Furthermore, in this invention, a positioning frame is fixed to the periphery of the transparent sheet at the placement opening, and a plurality of plug-in rods are fixed to the lower end face of the positioning frame.

[0008] Furthermore, in this invention, the upper end of the mounting opening is provided with insertion holes at the positions corresponding to the insertion rods, and multiple insertion rods are respectively inserted into the corresponding insertion holes.

[0009] Furthermore, in this invention, a buffer pad is fixed to the top surface of the notch, and the buffer pad abuts against the upper surface of the adapter piece.

[0010] Furthermore, in this invention, an auxiliary frame is fixed to the inner wall of the first recess and inserted into the inner position of the second recess, and the auxiliary frame abuts against the inner wall of the second recess.

[0011] Furthermore, the present invention provides mounting holes at the four opposite corners of the upper surface of the lower cover, and mounting rods inserted into the mounting holes are fixed at the four opposite corners of the lower surface of the upper cover.

[0012] Furthermore, the present invention provides positioning openings at the four opposite corners of the lower end face of the substrate, and a number of positioning blocks are fixed on the bottom surface inside the second recess, which are respectively inserted into the positioning openings.

[0013] Compared with the prior art, the beneficial effects of this utility model are as follows: (1) This application pours the test sample into the dropper from the dropper opening so that the test sample is in contact with the adapter. When KAN is present in the test sample, the adapter preferentially binds to KAN, causing cDNA to separate from the adapter. At the same time, the signal probe is supported by heme chloride. Therefore, after the adapter reacts with KAN, it will catalyze the display strip composed of TMB substrate to develop color. At this time, it can be determined that KAN is present in the sample. When KAN is not present in the sample, cDNA does not separate from the adapter, the adapter cannot bind to heme chloride, and cannot catalyze the display strip to develop color. At the same time, this method can improve the detection sensitivity, accuracy and reduce the detection time. (2) By placing the transparent sheet in the observation port, the transparent sheet is positioned at the resting port. The resting port supports and limits the transparent sheet, ensuring its stable position. The transparent sheet also blocks the observation port, preventing damage to the display strip and signal probe located below the observation port. Attached Figure Description

[0014] Figure 1 This is an exploded view of the overall structure of this utility model; Figure 2 This is an overall structural assembly diagram of this utility model; Figure 3 This is a top view of the upper cover of this utility model; Figure 4 This is a bottom structural diagram of the upper cover in this utility model; Figure 5 This is a structural diagram of the lower cover of this utility model; Figure 6 This is a schematic diagram of the substrate structure in this utility model; Figure 7 This is a schematic diagram of the bottom structure of the substrate in this utility model; Figure 8 This is a structural diagram of the transparent sheet in this utility model.

[0015] In the diagram: 1. Upper cover; 101. Drip nozzle; 102. Observation port; 103. Placement port; 104. Insertion hole; 105. Buffer pad; 106. Assembly rod; 107. Auxiliary frame; 108. Recess one; 2. Lower cover; 201. Recess two; 202. Positioning block; 203. Assembly hole; 3. Substrate; 301. Adapter sheet; 302. Signal probe; 303. Display bar; 304. Positioning port; 4. Transparent sheet; 401. Positioning frame; 402. Insertion rod. Detailed Implementation

[0016] The technical solution of this utility model will be further described in a non-limiting manner below with reference to preferred embodiments and accompanying drawings.

[0017] like Figure 1-8 As shown, this utility model provides a technical solution: a biosensor for detecting kanamycin based on aptamers, comprising an upper cover 1 and a lower cover 2 located at the lower end face of the upper cover 1. A notch 108 is formed on the lower end face of the upper cover 1, and a notch 201 is formed on the upper end face of the lower cover 2 corresponding to the notch. A substrate 3 is disposed between the notch 108 and the notch 201. An aptamer 301 is covered on the upper end face of the substrate 3, and the upper end face of the aptamer 301 is provided with... The device includes a signal probe 302 and a display bar 303. The upper end face of the upper cover 1 is provided with a drip port 101 that communicates with the inside of the recess 108. The upper end face of the upper cover 1 located to the right of the drip port 101 is provided with an observation port 102 that communicates with the inside of the recess 108. The signal probe 302 and the display bar 303 are located below the observation port 102. A placement opening 103 is provided on the inner wall of the observation port 102. A transparent sheet 4 is provided at the placement opening 103.

[0018] The sample to be tested is poured into the dropper 101, allowing it to bind to the adapter 301. When KAN is present in the sample, the adapter 301 preferentially binds to KAN, causing the cDNA to separate from the adapter 301. Simultaneously, the signal probe 302 is supported by heme chloride. After the adapter 301 reacts with KAN, it catalyzes the color development of the display strip 303 composed of TMB substrate, indicating the presence of KAN in the sample. When there is no target in the sample, the cDNA does not separate from the adapter 301, the adapter 301 cannot bind to heme chloride, and the display strip 303 does not catalyze color development. This method can improve detection sensitivity, accuracy, and detection efficiency.

[0019] It should be noted that the adapter 301 is a component modularly prepared using conventional methods in the field, and is a conventional component in the field of KAN detection. Specifically, the preferred preparation method for adapter 301 is as follows: biotin-modified aptamers are connected to avidin-modified magnetic nanoparticles (SA-MNPs) using the method described in patent publication number CN103852460A, or similar methods are used to prepare magnetic nanoparticles as described in documents such as "Aptamer-functionalized magnetic nanoparticle-based bioassay for the detection of ochratoxin using upconversion nanoparticles as labels, Shijia Wu"; "Aptamer-functionalized magnetic nanoparticles for simultaneous fluorometric determination of oxytetracycline and kanamycin, Changbin Liu"; then the aptamer is hybridized with the complementary strand, which is often made of biotinylated complementary aptamer DNA (cDNA) to obtain functionalized magnetic nanomaterials. A certain volume of magnetic nanobeads-cDNA was taken and added to a kanamycin aptamer with a G-quadruplex structure (Xing. et al., ScientficReports, 2015, 5:8125). After the reaction, the mixture was coated onto a sheet to obtain aptamer sheet 301.

[0020] The signal probe 302 used is composed of heme chloride. The supernatant containing adapter 301 is collected by magnetic separation. Then, the adapter 301 bound to KAN is eluted by heating. Since the adapter 301 has a single-stranded DNA with a G-quadruplex structure, the addition of heme chloride forms a heme chloride / G-quadruplex DNAase with peroxidase activity.

[0021] The signal probe 302 and the display strip 303 are located at the observation port 102. The transparent sheet 4 is placed in the observation port 102, and the transparent sheet 4 is located at the support port 103. The support port 103 supports and limits the transparent sheet 4 to ensure the stability of the transparent sheet 4. The transparent sheet 4 also blocks the observation port 102 to prevent damage to the display strip 303 and the signal probe 302 located below the observation port 102.

[0022] The substrate 3 is placed in the first recess 108 and the second recess 201. After the upper cover 1 and the lower cover 2 are assembled together, the substrate 3, the adapter 301, the signal probe 302 and the display strip 303 are limited in the first recess 108 and the second recess 201 to ensure the assembly stability of the structure.

[0023] A positioning frame 401 is fixed on the periphery of the transparent sheet 4 at the position of the placement opening 103. A plug-in rod 402 is fixed on the lower end face of the positioning frame 401. A plug-in hole 104 is opened on the upper end face of the placement opening 103 at the position corresponding to the plug-in rod 402. Multiple plug-in rods 402 are inserted into the corresponding plug-in holes 104 respectively. After the transparent sheet 4 is placed into the observation port 102, the positioning frame 401 abuts against the end face of the resting port 103. The insertion rod 402 fixed to the end face of the positioning frame 401 is inserted into the corresponding insertion hole 104. Through the tight cooperation between the insertion rod 402 and the insertion hole 104, the positioning frame 401 can be stably placed in the resting port 103, ensuring that the transparent sheet 4 is stably placed in the observation port 102. At the same time, after the upper cover 1 and the lower cover 2 are separated, the transparent sheet 4 is pushed upward from the lower position of the upper cover 1, which can cause the transparent sheet 4 to drive the positioning frame 401 to move upward, so that the insertion rod 402 is removed from the insertion hole 104, thereby realizing the disassembly of the transparent sheet 4.

[0024] A buffer pad 105 is fixed on the top surface of the first recess 108, and the buffer pad 105 abuts against the upper end surface of the adapter 301. An auxiliary frame 107 is fixed on the inner wall of the first recess 108 and inserted into the inner position of the second recess 201, and the auxiliary frame 107 abuts against the inner wall of the second recess 201. Assembly holes 203 are opened at the diagonal positions of the upper end surface of the lower cover 2. Assembly rods 106 are fixed at the diagonal positions of the lower end surface of the upper cover 1 and inserted into the positions of the assembly holes 203. Positioning holes 304 are opened at the diagonal positions of the lower end surface of the substrate 3. Multiple positioning blocks 202 are fixed on the bottom surface inside the second recess 201 and inserted into the positioning holes 304 respectively. After the substrate 3 is placed in the first recess 108 and the second recess 201, the positioning port 304 at the lower end of the substrate 3 will be directly fitted into the positioning block 202. The positioning block 202 limits the substrate 3 to prevent the substrate 3 from shaking freely in the second recess 201. When assembling the upper cover 1 and the lower cover 2, the upper cover 1 and the lower cover 2 are aligned and connected. The auxiliary frame 107 fixed on the lower end face of the upper cover 1 is inserted into the second recess 201, and the assembly rod 106 is inserted into the corresponding assembly hole 203 so that the upper cover 1 and the lower cover 2 can be assembled. At the same time, after the upper cover 1 and the lower cover 2 are assembled, the buffer pad 105 in the first recess 108 is connected to the upper end face of the adapter 301. The buffer pad 105 pushes the adapter 301, making the buffer pad 105 more stable.

[0025] The foregoing description illustrates and describes preferred embodiments of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. Modifications and variations made by those skilled in the art without departing from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A biosensor for detecting kanamycin based on aptamers, comprising an upper cover (1) and a lower cover (2) located on the lower end face of the upper cover (1), characterized in that: The lower end face of the upper cover (1) has a notch 1 (108), and the upper end face of the lower cover (2) has a notch 2 (201) corresponding to the notch. A substrate (3) is disposed between the notch 1 (108) and the notch 2 (201). The upper end face of the substrate (3) is covered with an adapter sheet (301). The upper end face of the adapter sheet (301) is provided with a signal probe (302) and a display bar (303). The upper end face of the upper cover (1) An infusion port (101) is provided that communicates with the inside of the recess (108). An observation port (102) that communicates with the inside of the recess (108) is provided on the upper end face of the upper cover (1) located on one side of the infusion port (101). The signal probe (302) and the display bar (303) are located below the observation port (102). A placement opening (103) is provided on the inner wall of the observation port (102). A transparent sheet (4) is provided at the position of the placement opening (103).

2. The biosensor for detecting kanamycin based on aptamers according to claim 1, characterized in that: The transparent sheet (4) has a positioning frame (401) fixed on its periphery at the placement opening (103), and a plug rod (402) is fixed on the lower end face of the positioning frame (401).

3. The biosensor for detecting kanamycin based on aptamers according to claim 2, characterized in that: The upper end of the mounting opening (103) is provided with a plug hole (104) at the position corresponding to the plug rod (402), and multiple plug rods (402) are respectively inserted into the corresponding plug hole (104).

4. The biosensor for detecting kanamycin based on aptamers according to claim 1, characterized in that: A buffer pad (105) is fixed on the top surface of the notch (108), and the buffer pad (105) abuts against the upper surface of the adapter (301).

5. The biosensor for detecting kanamycin based on aptamers according to claim 1, characterized in that: An auxiliary frame (107) is fixed to the inner wall of the first recess (108) and inserted into the inner position of the second recess (201), and the auxiliary frame (107) abuts against the inner wall of the second recess (201).

6. The biosensor for detecting kanamycin based on aptamers according to claim 5, characterized in that: The lower cover (2) has mounting holes (203) at opposite corners on its upper end face, and the upper cover (1) has mounting rods (106) at opposite corners on its lower end face that are inserted into the mounting holes (203).

7. The biosensor for detecting kanamycin based on aptamers according to claim 6, characterized in that: The substrate (3) has positioning openings (304) at the diagonal positions of the lower end face. Multiple positioning blocks (202) are fixed inside the bottom surface of the recess (201) and inserted into the positioning openings (304).

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