A detachable biosensor and its preparation method and application

Abstract

The present invention belongs to the field of electrochemical detection technology, and specifically relates to a detachable biosensor and its preparation method and application. The detachable biosensor includes an electrode plate and a single electrode detachably arranged on the electrode plate; a sample inlet, a sample outlet and an electrode socket corresponding to the single electrode are provided on the electrode plate, a sample passage connecting the sample inlet and the sample outlet is provided inside the electrode plate, and the electrode socket is cross-arranged with the sample passage; the single electrode is a screen-printed electrode, and a reagent layer is provided on the single electrode, and the reagent layer at least covers the position where the sample passage passes. The detachable biosensor in the present invention is simple to prepare and has high reliability. When different reagent layers are used, its high sensitivity and high selectivity can be guaranteed, which is conducive to reducing the cost of detection and has important significance and good application value for improving existing detection technology means.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical detection, and in particular relates to a detachable biosensor and a preparation method and application thereof. Background Art

[0002] Biosensors are analytical tools or systems that consist of immobilized biosensitive materials as recognition elements (including enzymes, antibodies, antigens, microorganisms, cells, tissues, nucleic acids and other bioactive substances), appropriate physical and chemical transducers (such as oxygen electrodes, photosensitive tubes, field effect tubes, piezoelectric crystals, etc.) and signal amplification devices. Compared with traditional methods such as LC-MS and GC-MS, biosensors have good selectivity, high sensitivity, fast analysis speed and low cost, and can be used for online continuous monitoring in complex systems. In particular, they are highly automated, miniaturized and integrated, which has enabled them to develop rapidly in recent decades.

[0003] However, in the prior art, biosensors related to electrochemical detection mostly use an independent solid-state three-electrode system for detection, and the working electrode mostly uses an independent single electrode. Therefore, after a test, the working electrode needs to be re-polished and activated, which not only greatly reduces the detection efficiency and reproducibility between different batches, but also increases the labor cost and technical difficulty requirements. These defects have limited the promotion and application of electrochemical biosensors to a certain extent. Summary of the invention

[0004] In order to solve the above technical problems, the present invention first provides a detachable biosensor, comprising an electrode plate and a single electrode detachably arranged on the electrode plate;

[0005] The electrode plate is provided with a sample inlet, a sample outlet and an electrode socket corresponding to the single electrode, a sample passage connecting the sample inlet and the sample outlet is provided inside the electrode plate, and the electrode socket is cross-arranged with the sample passage;

[0006] The single electrode is a screen-printed electrode, on which a reagent layer is arranged, and the reagent layer at least covers the position where the sample passage passes.

[0007] The present invention finds that, through the above-mentioned improvements, only a single electrode needs to be replaced each time a test is performed, which is not only beneficial to improving the test efficiency and the reproducibility between batches, but also reduces the labor cost of the test and the difficulty of handling the electrode.

[0008] Preferably, one sample inlet is connected to two or more sample passages, and each sample passage is cross-arranged with the electrode socket.

[0009] The above settings not only help simplify operations during batch screening, but also ensure the consistency of other variables, further reducing the impact of factors such as environmental or operational errors on the accuracy of test results.

[0010] Preferably, in order to match different detection requirements, when corresponding to different electrode sockets, the reagent layers of the single electrodes are the same or different.

[0011] The reagent layer in the present invention includes one or a mixture of biological tissue, microorganism, organelle, enzyme, antibody, antigen, nucleic acid. When detecting acetamiprid, the reagent layer preferably includes one or a mixture of nucleic acids with sequences such as SEQ ID NO1, 2, and 3; preferably, the nucleic acid with a sequence such as SEQ ID NO1.

[0012] In some embodiments, the opening of the sample outlet faces the distal side, so as to facilitate the incubation of the sample to be tested with the reagent layer for a longer period of time.

[0013] Preferably, when the single electrode is inserted into the electrode insertion port with the reagent layer facing the ground-proximal side, it is more conducive to an effective reaction between the sample to be tested and the reagent layer.

[0014] Preferably, the sample passage is divided into an incubation point passage and other position passages, the incubation point passage is located at the intersection of the sample passage and the electrode socket; the aperture of the other position passage < the aperture of the incubation point passage < the aperture of the sample inlet ≤ the aperture of the sample outlet.

[0015] Preferably, the electrode plate is made of polymethyl methacrylate.

[0016] The present invention further provides a method for preparing the detachable biosensor. When the reagent layer includes nucleic acid, the preparation process of the single electrode includes:

[0017] a) dissolving the nucleic acid in a buffer, placing it at 70-90°C for activation treatment for 1-10 minutes, and then cooling it naturally to room temperature to prepare a nucleic acid solution with a concentration of 0.2-10 μM; the first T base at the 5' end of the nucleic acid is modified with HO-(CH 2 ) 6 -SS-(CH 2 ) 6 Group;

[0018] b) mixing the nucleic acid solution prepared in step a) with tri(2-carbonylethyl)phosphine hydrochloride to control the final concentration of tri(2-carbonylethyl)phosphine hydrochloride to be 0.8-1.2 mM, and incubating at room temperature for 25-35 minutes;

[0019] c) applying the nucleic acid solution prepared in step b) to the electrode interface and incubating at room temperature for 1.8 to 2.5 hours.

[0020] In some embodiments, a blocking agent such as 6-mercapto-1-hexanol (1 mM) may be coated on the surface of the reagent layer.

[0021] The buffer can be composed of Tris, HClO 4 , HCl, NaHCO 3 、Na 2 CO 3 , NaOH, NaH 2 PO 4 , Na 2 HPO 4 , phosphoric acid, acetic acid, sodium acetate, preferably 20 mM Tris-HClO 4 The buffer solution has a pH value of 7 to 9, more preferably 7.5.

[0022] In order to remove the nucleic acid not bound to the electrode, after the incubation, a buffer solution may be used to rinse the electrode interface, which is not further limited herein.

[0023] The present invention further provides an application of the detachable biosensor in the field of electrochemical detection; preferably, when the reagent layer includes one or a mixture of nucleic acids with sequences such as SEQ ID NOs 1, 2, and 3, the detachable biosensor is applied to the detection of acetamiprid.

[0024] The present invention further provides a method for using the detachable biosensor, comprising:

[0025] 1) inserting the single electrode into the electrode socket to assemble a biosensor;

[0026] 2) preparing standard solutions of the analyte with different concentrations, adding them into the sample inlet, performing electrochemical signal detection after the incubation, and constructing a standard curve of the analyte concentration and the electrochemical signal intensity response;

[0027] 3) Preparing a sample to be tested for electrochemical signal detection to obtain the electrochemical signal intensity, and obtaining the concentration of the sample to be tested based on the measured electrochemical signal intensity and the standard curve.

[0028] In some schemes, when preparing the standard solution of the substance to be tested and the sample to be tested, the buffer solution used can be Tris, HClO 4 , HCl, NaHCO 3 、Na 2 CO 3 , NaOH, NaH 2PO 4 , Na 2 HPO 4 , phosphoric acid, acetic acid, sodium acetate, preferably 20 mM Tris-HClO 4 The buffer solution has a pH value of 7 to 9, more preferably 7.5.

[0029] The present invention has the following advantages:

[0030] The detachable biosensor of the present invention is simple to prepare and has high reliability. When different reagent layers are used, it can ensure high sensitivity and high selectivity, which is conducive to reducing the cost of detection. In addition, it is not only simple to operate, fast in response, small in sample usage, and pollution-free, but also has the characteristics of high efficiency and high throughput, which is of great significance and good application value for improving existing detection technology means. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is a schematic top view of the detachable biosensor in Example 1;

[0032] Figure 2 for Figure 1 A magnified view of point A;

[0033] Figure 3 A physical diagram of the detachable biosensor in Example 1;

[0034] Figure 4 The electrochemical signal measurement results before and after modification of the SEQ ID NO1 nucleic acid in Example 2; Figure 4 In the figure, a represents the electrochemical impedance spectrum of the screen-printed electrode without nucleic acid modification; b represents the electrochemical impedance spectrum of the screen-printed electrode with nucleic acid modification.

[0035] Among them, 1. electrode plate; 11. sample inlet; 12. sample outlet; 13. electrode socket; 14. sample passage; 141. incubation point passage; 142. other position passage; 2. single electrode. DETAILED DESCRIPTION

[0036] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0037] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0038] The nucleic acids used in the present invention are shown in SEQ ID NOs 1, 2, and 3, and the first T base at the 5' end is modified with HO-(CH2 ) 6 -SS-(CH 2 ) 6 Group.

[0039] Example 1

[0040] This embodiment provides a detachable biosensor (schematic diagram as shown in FIG. Figures 1-2 As shown in the actual picture Figure 3 As shown), comprising an electrode plate 1 and a single electrode 2 detachably arranged on the electrode plate 1;

[0041] The electrode plate 1 is provided with a sample inlet 11 and a sample outlet 12 which are opened toward the distal side, and an electrode socket 13 which is arranged corresponding to the single electrode 2. A sample passage 14 which connects the sample inlet 11 and the sample outlet 12 is arranged inside the electrode plate 1, and the electrode socket 13 is arranged crosswise with the sample passage 14; 8 sample passages 14 are connected to one sample inlet 11, and the electrode socket 13 is arranged crosswise on each sample passage 14. The sample passage 14 is divided into an incubation point passage 141 and other position passages 142, and the incubation point passage 141 is located at the intersection of the sample passage 14 and the electrode socket 13. The aperture of the other position passage 142 is 1 mm, the aperture of the incubation point passage 141 is 4 mm, the aperture of the sample inlet 11 is 5 mm, and the aperture of the sample outlet 12 is 5 mm.

[0042] The single electrode 2 is a screen-printed electrode, and a reagent layer is disposed on the single electrode 2, and the reagent layer at least covers the position where the sample passage 14 passes. The reagent layer contains a nucleic acid with a sequence as shown in SEQ ID NO1.

[0043] Example 2

[0044] This example provides a preparation process of the detachable biosensor in Example 1, which is as follows:

[0045] (1) The solid nucleic acid powder shown in SEQ ID NO1 was treated with an appropriate amount of Tris-HClO 4 Dissolve the buffer solution (pH=7.5, 20mM), mix well with a vortex oscillator, and heat and activate it in a water bath at 80-95°C for 3-15min, then cool naturally to room temperature for use.

[0046] Use Tris-HClO 4A 2 μM solution of the nucleic acid shown in SEQ ID NO1 was prepared with a buffer solution (pH=7.5, 20 mM), and then an appropriate amount of TCEP (tris(2-carbonylethyl)phosphine hydrochloride) was added to a final concentration of 1 mM. The mixture was mixed using a vortex oscillator and then allowed to stand at room temperature for 30 min to obtain a DNA assembly solution for later use.

[0047] (2) Apply 10 μL of the nucleic acid assembly solution prepared in step (1) above to the electrode interface, let it stand for 2 h at room temperature in a dust-free environment, and then use Tris-HClO 4 The interface was washed with a buffer solution (pH = 7.5, 20 mM), and then 10 μL of 6-mercapto-1-hexanol (1 mM) was applied dropwise to the electrode interface for 1 h, and then washed again with Tris-HClO 4 The interface was washed with a buffer solution (pH=7.5, 20 mM) and dried with nitrogen gas to obtain a screen-printed single electrode for later use.

[0048] The electrochemical signal measurement results before and after modification of SEQ ID NO1 nucleic acid are as follows Figure 4 shown. Figure 4 In the figure, a represents the electrochemical impedance spectrum of the screen-printed electrode without nucleic acid modification; b represents the electrochemical impedance spectrum of the screen-printed electrode with nucleic acid modification.

[0049] (3) Insert the screen-printed single electrodes prepared in the above step (2) into the corresponding electrode sockets of the electrode plate, seal them with a sealant, and let them stand for 24 hours to obtain a detachable biosensor, such as Figure 1 and Figure 3 shown.

[0050] Test Example 1

[0051] The method in Example 2 was used to prepare screen-printed single electrodes containing nucleic acids shown in SEQ ID NOs 2 and 3, respectively, and the same method was used to fix them in different electrode sockets of the same electrode plate.

[0052] 100 nM of the pesticide acetamiprid was added to the sample inlet to react with the above single electrodes. After incubation at room temperature for 30 min, Tris-HClO 4 The interface was washed with a buffer solution (pH=7.5, 20 mM) and then electrochemical impedance spectroscopy was performed. The electrochemical impedance spectroscopy signals R measured by different nucleic acid sensors are shown in Table 1.

[0053] Table 1 Electrochemical impedance spectroscopy signals generated by different nucleic acid sensors

[0054]

[0055] The results showed that the electrochemical impedance spectroscopy signal intensity of the nucleic acid represented by SEQ ID NO1 was the strongest.

[0056] Test Example 2

[0057] Use Tris-HClO 4 (pH=7.5, 20 mM) prepared different concentrations of the pesticide acetamiprid, and then added the different concentrations of the pesticide acetamiprid solutions to the detachable biosensor in Example 1, incubated at room temperature for 1 hour, and then the electrochemical impedance spectroscopy signal was measured. The electrochemical signals R generated by the different concentrations of the pesticide acetamiprid are shown in Table 2.

[0058] Table 2 Changes in electrochemical impedance spectroscopy signals produced by different concentrations of the pesticide acetamiprid

[0059]

[0060] The results showed that the electrochemical impedance spectroscopy signal intensity generated by the screen-printed array electrode electrochemical nucleic acid sensor after the action of the pesticide acetamiprid increased with the increase of the concentration of the pesticide acetamiprid, and the two were linearly correlated. According to the linear relationship, the detection limit was calculated to be 1nM.

[0061] Although the present invention has been described in detail above by means of general description, specific implementation methods and tests, it is obvious to those skilled in the art that some modifications or improvements may be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention. Sequence Listing <110> Beijing Agricultural Quality Standard and Testing Technology Research Center <120> A detachable biosensor and its preparation method and application <130> KHP191115027.7F <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 42 <212> DNA <213> Artificial Sequence <400> 1 tttttgcagc ggttcttgat cgctgacacc atattatgaa ga 42 <210> 2 <211> 42 <212> DNA <213> Artificial Sequence <400> 2 tttttgtggt actgtgcctg gtccaattcc tttttcccga tt 42 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <400> 3 tttttgcagc ggttcttgat cgctgacacc 30

Claims

1. A detachable biosensor, characterized in that: It includes an electrode plate and a single electrode detachably arranged on the electrode plate; The electrode plate is provided with a sample inlet, a sample outlet and an electrode socket corresponding to the single electrode, a sample passage connecting the sample inlet and the sample outlet is provided inside the electrode plate, and the electrode socket is cross-arranged with the sample passage; The single electrode is a screen-printed electrode, on which a reagent layer is disposed, the reagent layer at least covering the position where the sample passage passes; the reagent layer includes one or a mixture of nucleic acids with sequences as shown in SEQ ID NOs: 1-3.

2. The detachable biosensor according to claim 1, characterized in that: One sample inlet is connected with two or more sample passages, and each sample passage is cross-arranged with the electrode socket.

3. The detachable biosensor according to claim 2, characterized in that: Corresponding to different electrode sockets, the reagent layers of the single electrodes are the same or different.

4. The detachable biosensor according to any one of claims 1 to 3, characterized in that: The reagent layer also includes one of biological tissue, microorganism, cell organelle, enzyme, antibody, antigen or a mixture thereof.

5. The detachable biosensor according to any one of claims 1 to 3, characterized in that: The opening of the sample outlet faces the distal side; And / or, the single electrode is inserted into the electrode insertion port with the reagent layer facing the ground-proximal side.

6. The detachable biosensor according to any one of claims 1 to 3, characterized in that: The sample passage is divided into an incubation point passage and other position passages, and the incubation point passage is located at the intersection of the sample passage and the electrode socket; The aperture of the passage at the other position < the aperture of the passage at the incubation point < the aperture of the sample inlet ≤ the aperture of the sample outlet.

7. The detachable biosensor according to any one of claims 1 to 3, characterized in that: The electrode plate is made of polymethyl methacrylate.

8. The method for preparing a detachable biosensor according to any one of claims 1 to 7, characterized in that: The preparation process of the single electrode includes: a) dissolving the nucleic acid in a buffer, placing it at 70-90°C for activation treatment for 1-10 minutes, and then naturally cooling it to room temperature to prepare a nucleic acid solution with a concentration of 0.2-10 μM; the first T base at the 5' end of the nucleic acid is modified with a HO-(CH2)6-SS-(CH2)6 group; b) mixing the nucleic acid solution prepared in step a) with tri(2-carbonylethyl)phosphine hydrochloride to control the final concentration of tri(2-carbonylethyl)phosphine hydrochloride to be 0.8-1.2 mM, and incubating at room temperature for 25-35 minutes; c) Apply the nucleic acid solution prepared in step b) to the electrode interface and incubate at room temperature for 1.8 to 2.5 hours.

9. Use of the detachable biosensor according to any one of claims 1 to 7 in the field of electrochemical detection; applying the detachable biosensor to the detection of acetamiprid.

10. The method for using the detachable biosensor according to any one of claims 1 to 7, characterized in that: include: 1) inserting the single electrode into the electrode socket to assemble a biosensor; 2) preparing standard solutions of the analyte with different concentrations, adding them into the sample inlet, performing electrochemical signal detection after the incubation, and constructing a standard curve of the analyte concentration and the electrochemical signal intensity response; 3) Preparing a sample to be tested for electrochemical signal detection to obtain the electrochemical signal intensity, and obtaining the concentration of the sample to be tested based on the measured electrochemical signal intensity and the standard curve.

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