A COF renewable detection device, a use method and a sensor using the same

Abstract

The application relates to a COF renewable detection device, a use method and a sensor applying the same, relates to the technical field of analytical chemistry, and wherein the COF renewable detection device comprises the following steps: synthesis of TAPB-DMTP covalent organic framework (COF), synthesis of COF-Au-MB, synthesis of COF-Au-MB-Apt, synthesis of PB / SPE, synthesis of ssDNA / PB / SPE, and synthesis of COF-Au-MB-Apt / ssDNA / PB / SPE. The COF renewable detection device, the use method and the sensor applying the same can be used in a complex background environment on site and realize repeated use of the detection device.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry, and in particular to a COF regenerative detection device, a use method and a sensor using the same. Background Art

[0002] Ochratoxin A (OTA) is a mycotoxin produced by various Aspergillus and Penicillium fungi, causing serious contamination of various grains, including corn, oats, and wheat. After entering the food chain, OTA can have serious impacts on human health, including kidney damage and immune impairment. Existing methods for detecting OTA primarily rely on chromatography, mass spectrometry, and LC-MS / MS. While these methods offer high specificity and sensitivity, they generally suffer from complex and time-consuming sample pretreatment, are unsuitable for field use, and are therefore becoming increasingly popular. Therefore, in resource-limited settings, micro-biosensors are becoming a powerful tool for detecting OTA.

[0003] However, they are somewhat unsatisfactory in field applications due to complex background environments and reuse requirements. Summary of the Invention

[0004] In order to be applicable in complex background environments on site and to achieve the reuse of the detection device, the present invention provides a COF regenerative detection device, a method of use and a sensor using the same.

[0005] In a first aspect, the present invention provides a COF regenerative detection device, which is manufactured by the following steps:

[0006] Synthesis of TAPB-DMTP covalent organic framework (COF):

[0007] S11. Add 10.5 mg of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 8.7 mg of 2,5-dimethoxy-p-benzaldehyde (DMTP) to 4.5 mL of a 4:4:1 volume ratio solution of 1,4-dioxane, 1-butanol, and methanol. Ultrasonicate for 20 min, and simultaneously add 50 μL of 12 M glacial acetic acid dropwise. After ultrasonication, let the mixture stand at 25°C for 2 h to obtain a mixed solution A.

[0008] S12, adding 450 μL of 12 M glacial acetic acid to the mixed solution A, and heating at 70° C. for 24 h. After the addition is complete, cooling to room temperature and centrifuging at 8000 rpm for 15 min, washing with tetrahydrofuran and acetone, and drying in vacuo at 70° C. to obtain COF;

[0009] S13. Add 30 mg of the dried product to 20 mL of methanol and sonicate for 3 minutes. The suspension is mixed with 160 μL of 1% chloroauric acid (HAuCl4) and stirred at 0°C for 5 hours. After adding 1 mL of 0.2 M sodium borohydride (NaBH4) dissolved in methanol solution dropwise, stir at 0°C for 3 hours, wash with methanol, centrifuge at 8000 rpm for 15 minutes, and dry at 60°C for 12 hours to obtain COF-Au.

[0010] Synthesis of COF-Au-MB: 5 mL of 0.1-2 mg / mL COF-Au and 2 mL of 1 mg / mL methylene blue (MB) were mixed and stirred until a precipitate formed. The mixture was then centrifuged at 10,000 rpm for 10 min and washed with deionized water to obtain COF-Au-MB.

[0011] Synthesis of COF-Au-MB-Apt: COF-Au-MB was redispersed in 2 mL of 10 mM tris-hydroxymethylaminomethane hydrochloride (Tris-HCl) buffer. At 4°C, 1 mL of COF-Au-MB suspension was mixed with 200 μL of 6 μM aptamer (Apt). The DNA sequence of the aptamer was SH-GATCGGTGTGGGTGGCGTAAAGGGAGCATCGGACA. After incubation with shaking for 10 h, the mixture was centrifuged at 5000 rpm for 10 min to obtain the COF-Au-MB-Apt probe.

[0012] Synthesis of PB / SPE: The detection base (SPE) was incubated with a solution of 3 mM potassium ferricyanide (K3[Fe(CN)]6), 3 mM ferric chloride (FeCl3), 0.1 M hydrochloric acid (HCl), and 0.1 M potassium chloride (KCl) at a constant potential of 0.35 V for 60 s to obtain PB / SPE.

[0013] Synthesis of ssDNA / PB / SPE: 6 μL of 10 μM semi-complementary strand (ssDNA) was drop-coated on PB / SPE and incubated for 60 min at room temperature. The DNA sequence of the ssDNA was: SH-CATGGACTGTCCGATGCT. After incubation, the ssDNA was passivated in 6 μL of 2 mM 6-mercapto-1-hexanol (MCH) for 30 min to obtain ssDNA / PB / SPE.

[0014] Synthesis of COF-Au-MB-Apt / ssDNA / PB / SPE: Incubate ssDNA / PB / SPE with 10 μL of 1 mg / mL COF-Au-MB-Apt for 80-100 min to obtain COF-Au-MB-Apt / ssDNA / PB / SPE.

[0015] By adopting the above technical solution, COF obtained from 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 2,5-dimethoxy-p-formaldehyde (DMTP) becomes an ideal carrier due to its high surface area, abundant active sites and feasible modification. The addition of Au can improve the MB capture ability. COF-Au is an efficient signal carrier. MB enters the hollow COF-Au skeleton and forms a stable host-guest complex through molecular interactions, so that MB is well retained in COF-Au-MB.

[0016] Aptamers (Apt), as an artificial oligonucleotide sequence, exhibit strong specificity and affinity for OTA due to the unique conformational changes of the secondary structure and the formation of G-quadruplex. Highly selective Apt can be immobilized on porous covalent organic frameworks (COFs) for OTA recognition. After COF is loaded with gold nanoparticles (AuNPs), COF-Au can absorb Apt through Au-S bonds, forming a stable COF-Au-Apt complex to capture the target OTA. The addition of aptamers is expected to support OTA sensing detection in complex backgrounds.

[0017] Using SPE as the working electrode, which can accommodate permanent on-chip modification of Prussian blue (PB) and ssDNA, by permanently fixing the semi-complementary chain (ssDNA) to the sensing interface, Apt-ssDNA interaction can be established due to the base pair stacking between Apt and ssDNA.

[0018] The flexible and folded Apt can cause changes in the microstructure and electrical behavior of the sensing surface and trigger readable signals.

[0019] Since the combination between Apt and ssDNA follows common helical unwinding rules at high temperatures, thermal regulation can achieve regeneration of the sensing surface and is expected to ensure multiplexed analysis.

[0020] Optionally, in S2, the amount of COF-Au added in the synthesis of COF-Au-MB is 5 mL 1 mg / mL.

[0021] By adopting the above technical solution, when the addition amount of COF-Au is 5mL / 1mg / mL, the content of AuNPs on the COF can be increased, resulting in a higher capture ability for MB and a higher absorption capacity for Apt. At the same time, as the amount of COF-Au increases, the fluorescence peak of the material under 600 nm excitation is regularly suppressed. Therefore, when the addition amount is 5mL / 1mg / mL, the fluorescence peak of the material under 600 nm excitation will not be excessively weakened due to the addition of excessive AuNPs, which will affect the experimental results.

[0022] Optionally, the PB / SPE is synthesized as follows: after incubation in the solution, the incubated SPE is plated with 10 mM HAuCl4 and 0.1 M KCl at a constant potential of -0.8 V for 80 seconds to obtain the PB / SPE.

[0023] By adopting the above technical solution, PB / SPE showed a stable peak in electrochemical analysis, and the addition of AuNPs, due to the high conductivity of AuNPs, PB / SPE with AuNPs showed a larger peak and consistent stability.

[0024] In a second aspect, the present invention provides a method for using a COF regenerative detection device, which adopts the following technical solution:

[0025] Sample preparation: Each natural sample was crushed and accurately weighed to 5 g, added to 20 mL of acetonitrile, and homogenized at 5000 rpm for 5 min. After homogenization, 2 g of sodium chloride was added, and ultrasonic treatment was performed for 40 min. After standing for 5 min, centrifugation was performed. After centrifugation, the supernatant was quantitatively diluted to 20 mL with tris-hydrochloride buffer to obtain the target OTA and stored at 4°C until analysis.

[0026] OTA drop coating: The target OTA was drop coated on COF-Au-MB-Apt / ssDNA / PB / SPE and incubated for 60 min to obtain the incubation solution, and then prepared for electrochemical analysis.

[0027] Electrochemical analysis: Differential pulse voltammetry (DPV) was used with a pulse amplitude of 50 mV, a pulse width of 16.7 ms, a pulse period of 100 ms, and a window setting of -0.5 to 0.5 V. The DPV was performed in 10 mM phosphate buffer (PBS) to measure the target signal (I MB ) and the reference signal (I PB ),

[0028] Judgment: Judgment I MB With the set threshold value, if I MB If it is greater than or equal to the set threshold, it is determined that there is no OTA. MB If it is less than the set threshold, it is determined that there is OTA.

[0029] Unwinding: At room temperature, use a heating device to bring the COF-Au-MB-Apt / ssDNA / PB / SPE filled with the target solution to a preset temperature of 70°C within 60 seconds and maintain it for 7-8 minutes. Upon completion, immediately empty the target solution and allow the ssDNA / PB / SPE to cool naturally to room temperature.

[0030] By adopting the above technical solution, during the use of COF-Au-MB-Apt / ssDNA / PB / SPE, the hybridized COF-Au-MB-Apt probe is captured by OTA and leaves the sensing interface. OTA provides a stronger affinity than ssDNA when binding to Apt. The bond between Apt and OTA is contributed by π-π stacking, halogen bonds and hydrophobic interactions, which is stronger than the semi-complementary bond between Apt and ssDNA, thus leading to the decomposition of dsDNA.

[0031] OTA alone has no signal, but it can effectively suppress the signal of Apt. PB peak and MB peak were obtained by electrochemical analysis. In the blank sample, SPE showed obvious PB and MB peaks. With the increase of OTA concentration, MB peak was significantly suppressed, while PB peak remained unchanged. At this time, the reference target signal (I MB ) value to determine whether OTA exists.

[0032] Before the presence of OTA, COF-Au-MB-Apt probes were assembled onto ssDNA / PB / SPE at room temperature through hybridization between Apt and ssDNA. In the presence of OTA, COF-Au-MB-Apt tended to detach from the ssDNA / PB / SPE, leading to self-regeneration of the ssDNA / PB / SPE assembly. Because Apt-ssDNA bonds loosen at high temperatures without disrupting the secondary structures of Apt and ssDNA, thermal conditioning on the electrode likely facilitates this regeneration behavior. This thermal conditioning allowed the COF-Au-MB-Apt probe to dissociate from the ssDNA / PB / SPE into solution. Afterward, the solution on the SPE was immediately emptied to prevent possible recombination between the ssDNA and the expired COF-Au-MB-Apt probe. The ssDNA / PB / SPE module was then naturally cooled to 25°C to allow for reassembly. The residual COF-Au-MB-Apt probes were successfully removed under the synergistic effect of OTA-induced Apt-ssDNA dissociation and on-chip thermal regulation function, while the ssDNA / PB remained on the sensing surface for the reassembly of new probes, regenerating the sensing surface for reuse.

[0033] At the same time, ssDNA / PB / SPE has high selectivity for other mycotoxins and metal ions. Compared with the OTA signal alone, 1000-fold concentrations of mycotoxins or metal ions have no significant effect on the OTA signal. ssDNA / PB / SPE also has good repeatability and long-term stability.

[0034] Optionally, there is a concentration analysis step after the electrochemical analysis step.

[0035] Concentration analysis: The concentration of ochratoxin A is calculated based on the target signal and the reference signal. The calculation model is as follows:

[0036]

[0037] Where, is the standard target signal, To detect target signals, is the difference between the standard target signal and the detection target signal, - , is the reference signal, is the concentration of ochratoxin A.

[0038] By adopting the above technical solution, this method can detect the specific concentration of OTA. As the OTA concentration increases, I MB Quantitative decrease, while I PB In the range of 0.2 pg / mL to 0.6 μg / mL, ΔI MB / I PB A good relationship is established between the logarithm of the OTA concentration, i.e. , the detection limit was calculated to be 0.12 pg / mL.

[0039] Optionally, the holding time during unwinding is 8 minutes.

[0040] By adopting the above technical scheme, the optimized heat treatment at 70 °C for 8 min can further expel more unreacted COF-Au-MB-Apt and further improve the regeneration efficiency while maintaining the retention of ssDNA / PB on the sensing surface.

[0041] In a third aspect, the present invention provides a sensor suitable for use with the COF regenerative detection device of the first aspect, employing the following technical solution:

[0042] A sensor includes a circuit board, on which a detection base is provided.

[0043] The detection base includes a counter electrode, a working electrode and a reference electrode. The counter electrode is arranged in a semicircular shape, and the reference electrode and the working electrode are arranged in an arc shape. The combination of the counter electrode, the reference electrode and the working electrode is arranged in a circular ring shape. A regenerable probe is arranged on the working electrode.

[0044] The counter electrode, working electrode and reference electrode are all connected to the circuit board electrical signal.

[0045] By employing this technical solution, the SPE, including pre-designed pads, is used as a semi-finished three-electrode system. A semicircular electrode is directly used as the counter electrode, while a circular electrode, after Ag / AgCl functionalization, is selected as the reference electrode. The circular electrode is designed with permanent on-chip modifications and a regenerable probe to serve as the working electrode. Optionally, the sensor also includes a heating module.

[0046] The heating module and the detection sensor are arranged on the circuit board opposite to each other.

[0047] The circuit board is connected to the heating module via electrical signals.

[0048] By adopting the above technical solution, the heating modules are assembled to the back of the SPE, just below the three electrodes. They work as a remote sensing heater and cooperate with the on-chip control to achieve thermal regulation regeneration.

[0049] In summary, the present invention includes at least one of the following beneficial technical effects:

[0050] The aptamer (Apt) in the present invention is an artificial oligonucleotide sequence. Due to the unique conformational change of the secondary structure and the formation of G-quadruplex, Apt shows strong specificity and affinity for OTA. The highly selective Apt can be immobilized on a porous covalent organic framework (COF) for OTA recognition.

[0051] The present invention successfully removes residual COF-Au-MB-Apt probes under the synergistic effect of OTA-induced Apt-ssDNA dissociation and on-chip thermal regulation function, while ssDNA / PB remains on the sensing surface for reassembly of new probes, regenerating the sensing surface for reuse.

[0052] The ssDNA / PB / SPE system of the present invention has high selectivity for other mycotoxins and metal ions. Compared with the signal of OTA alone, a 1000-fold concentration of mycotoxins or metal ions has no significant effect on the OTA signal. ssDNA / PB / SPE also has good reproducibility and long-term stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is a schematic structural diagram of the heating module of the present invention;

[0054] Figure 2 This is an electrochemical analysis diagram of the product of the synthesis step of COF-Au-MB-Apt / ssDNA / PB / SPE in Example 1;

[0055] Figure 3 The electrochemical comparison diagram of the co-regeneration and the dissociation of OTA alone is shown in Figure 2. Figure 3-ⅠThis is the analysis diagram obtained from the electrochemical analysis step in Example 1. Figure 3-Ⅱ This is the electrochemical analysis diagram of the product of the synthesis step of COF-Au-MB-Apt / ssDNA / PB / SPE in the comparative example, Figure 3-III This is the electrochemical analysis diagram of ssDNA / PB / SPE after unwinding in Example 3. Figure 3-IV This is an electrochemical analysis diagram of the product of the synthesis step of COF-Au-MB-Apt / ssDNA / PB / SPE in Example 3;

[0056] Figure 4 is the electrochemical test signal I MB and I PB Graph of repeated cycles before and after (re)assembly and the obtained I MB and I PB Error bars,

[0057] Explanation of the accompanying drawings: 1. First resistor; 2. Second resistor; 3. Third resistor; 4. Fourth resistor; First DC power supply; 6. Second DC power supply; 7. Operational amplifier; 8. Fifth resistor; 9. First transistor; 10. Second transistor; 11. Heating resistor; 12. Third DC power supply. DETAILED DESCRIPTION

[0058] The present invention is further described in detail below with reference to the examples.

[0059] Example 1: This example discloses a COF regenerative detection device, its application, and a sensor using the same, including the following steps.

[0060] A COF regenerative detection device is manufactured using the following steps:

[0061] Synthesis of TAPB-DMTP covalent organic framework (COF):

[0062] S11. Add 10.5 mg of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 8.7 mg of 2,5-dimethoxy-p-benzaldehyde (DMTP) to 4.5 mL of a 4:4:1 volume ratio solution of 1,4-dioxane, 1-butanol, and methanol. Ultrasonicate for 20 min, and simultaneously add 50 μL of 12 M glacial acetic acid dropwise. After ultrasonication, let the mixture stand at 25°C for 2 h to obtain a mixed solution A.

[0063] S12, adding 450 μL of 12 M glacial acetic acid to the mixed solution A, and heating at 70° C. for 24 h. After the addition is complete, cooling to room temperature and centrifuging at 8000 rpm for 15 min, washing with tetrahydrofuran and acetone, and drying in vacuo at 70° C. to obtain COF;

[0064] S13. Add 30 mg of the dried product to 20 mL of methanol and sonicate for 3 minutes. The suspension is mixed with 160 μL of 1% chloroauric acid (HAuCl4) and stirred at 0°C for 5 hours. After adding 1 mL of 0.2 M sodium borohydride (NaBH4) dissolved in methanol solution dropwise, stir at 0°C for 3 hours, wash with methanol, centrifuge at 8000 rpm for 15 minutes, and dry at 60°C for 12 hours to obtain COF-Au.

[0065] Synthesis of COF-Au-MB: 5 mL of 1 mg / mL COF-Au and 2 mL of 1 mg / mL methylene blue (MB) were mixed and stirred until a precipitate formed. The mixture was then centrifuged at 10,000 rpm for 10 min and washed with deionized water to obtain COF-Au-MB.

[0066] Synthesis of COF-Au-MB-Apt: COF-Au-MB was redispersed in 2 mL of 10 mM tris-hydroxymethylaminomethane hydrochloride (Tris-HCl) buffer. At 4°C, 1 mL of COF-Au-MB suspension was mixed with 200 μL of 6 μM aptamer (Apt). The DNA sequence of the aptamer was SH-GATCGGTGTGGGTGGCGTAAAGGGAGCATCGGACA. After incubation with shaking for 10 h, the mixture was centrifuged at 5000 rpm for 10 min to obtain the COF-Au-MB-Apt probe.

[0067] Synthesis of PB / SPE: Under a constant potential of 0.35 V, the detection base (SPE) was incubated with a solution of 3 mM potassium ferricyanide (K3[Fe(CN)]6), 3 mM ferric chloride (FeCl3), 0.1 M hydrochloric acid (HCl), and 0.1 M potassium chloride (KCl) for 60 s. The incubated detection base was then plated with 10 mM chloroauric acid4 and 0.1 M potassium chloride at a constant potential of -0.8 V for 80 s to obtain PB / SPE.

[0068] Synthesis of ssDNA / PB / SPE: 6 μL of 10 μM semi-complementary strand (ssDNA) was drop-coated on PB / SPE and incubated for 60 min at room temperature. The DNA sequence of the ssDNA was: SH-CATGGACTGTCCGATGCT. After incubation, the ssDNA was passivated in 6 μL of 2 mM 6-mercapto-1-hexanol (MCH) for 30 min to obtain ssDNA / PB / SPE.

[0069] Synthesis of COF-Au-MB-Apt / ssDNA / PB / SPE: Incubate ssDNA / PB / SPE with 10 μL of 1 mg / mL COF-Au-MB-Apt for 80-100 min to obtain COF-Au-MB-Apt / ssDNA / PB / SPE.

[0070] Here’s how to use it:

[0071] Sample preparation: Each natural sample was crushed and accurately weighed to 5 g, added to 20 mL of acetonitrile, and homogenized at 5000 rpm for 5 min. After homogenization, 2 g of sodium chloride was added, and ultrasonic treatment was performed for 40 min. After standing for 5 min, centrifugation was performed. After centrifugation, the supernatant was quantitatively diluted to 20 mL with tris-hydrochloride buffer to obtain the target OTA and stored at 4°C until analysis.

[0072] OTA drop coating: The target OTA was drop coated on COF-Au-MB-Apt / ssDNA / PB / SPE and incubated for 60 min to obtain the incubation solution, and then prepared for electrochemical analysis.

[0073] Electrochemical analysis: Differential pulse voltammetry (DPV) was used with a pulse amplitude of 50 mV, a pulse width of 16.7 ms, a pulse period of 100 ms, and a window setting of -0.5 to 0.5 V. The DPV was performed in 10 mM phosphate buffer (PBS) to measure the target signal (I MB ) and the reference signal (I PB ),

[0074] Judgment: Judgment I MB With the set threshold value, if I MB If it is greater than or equal to the set threshold, it is determined that there is no OTA. MB If it is less than the set threshold, it is determined that there is OTA.

[0075] Unwinding: At room temperature, use a heating device to bring the COF-Au-MB-Apt / ssDNA / PB / SPE filled with the target solution to a preset temperature of 70°C within 60 seconds and maintain it for 8 minutes. Upon completion, immediately empty the target solution and allow the ssDNA / PB / SPE to cool naturally to room temperature.

[0076] The above-mentioned sensor adopts the following technical solutions:

[0077] A sensor includes a circuit board, on which a detection base and a heating module are arranged.

[0078] The heating module is arranged back to back with the detection sensor on the circuit board. The detection base includes a counter electrode, a working electrode, a reference electrode and a hollow plastic reaction chamber. The counter electrode is arranged in a semicircular shape, and the reference electrode and the working electrode are both arranged in an arc shape. The combination of the counter electrode, the reference electrode and the working electrode is in a circular ring shape. Regenerable probes are provided on the counter electrode, the reference electrode and the working electrode.

[0079] During detection, connect the reference electrode on the detection base to the RE pole of the electrochemical workstation, connect the counter electrode on the detection base to the CE pole of the electrochemical workstation, and connect the working electrode on the detection base to the WE pole of the electrochemical workstation.

[0080] The above-mentioned heating module includes: a first resistor, a second resistor, a third resistor, a fourth resistor, a fifth resistor, a heating resistor, a first DC power supply, a second DC power supply, an operational amplifier, a first transistor, a second transistor, and a third DC power supply, wherein the fourth resistor is a temperature-sensitive resistor.

[0081] One end of the first resistor is connected to the positive electrode of the first DC power supply, and the other end is connected to one end of the second resistor, the other end of the second resistor is connected to the positive electrode of the second DC power supply, and the positive electrode of the second DC power supply is connected to one end of the third resistor, the other end of the third resistor is connected to one end of the fourth resistor, and the other end of the fourth resistor is connected to the positive electrode of the first DC power supply.

[0082] The positive input terminal of the first operational amplifier is connected between the first resistor and the second resistor, and the negative input terminal of the first operational amplifier is connected between the third resistor and the fourth resistor; the output terminal of the first operational amplifier is connected to one end of the fifth resistor, and the other end of the fifth resistor is connected to the B end of the first transistor. The first transistor is an NPN transistor, and the E end of the first transistor is connected to the E end of the second transistor. The C end of the first transistor is connected to the positive electrode of the third DC power supply. The second transistor is a PNP transistor, and the B end of the second transistor is connected between the fifth resistor and the B end of the first transistor. The C end of the second transistor is grounded.

[0083] One end of the heating resistor is connected between the E end of the first transistor and the E end of the second transistor, and the other end of the heating resistor is grounded.

[0084] Example 2: This example discloses a COF regenerative detection device, its application, and a sensor using the same, including the following steps.

[0085] Here’s how to use it:

[0086] After the unwinding step, the unwound ssDNA / PB / SPE was reapplied to the synthesis step of COF-Au-MB-Apt / ssDNA / PB / SPE, reassembled and used to obtain the test results.

[0087] The rest is exactly the same as in Example 1.

[0088] Comparative Example: This comparative example discloses a COF regenerable detection device, its application and a sensor using the same, including the following steps.

[0089] Here’s how to use it:

[0090] After the judgment step, the used ssDNA / PB / SPE was directly reapplied to the COF-Au-MB-Apt / ssDNA / PB / SPE synthesis step without unwinding, and was reassembled and used to obtain the test results.

[0091] The product of the synthesis step of COF-Au-MB-Apt / ssDNA / PB / SPE in Example 1 was subjected to electrochemical analysis to obtain Figure 2 ;

[0092] In Example 1, the analysis diagram obtained in the electrochemical analysis step is as follows Figure 3-Ⅰ ;

[0093] The products of the synthesis step of COF-Au-MB-Apt / ssDNA / PB / SPE in the comparative example were subjected to electrochemical analysis, and the analysis results were as follows: Figure 3-Ⅱ ;

[0094] The electrochemical analysis of the unwound ssDNA / PB / SPE in Example 3 was performed to obtain the analysis results. Figure 3-III ;

[0095] The product of the synthesis step of COF-Au-MB-Apt / ssDNA / PB / SPE in Example 3 was subjected to electrochemical analysis, and the analysis results were Figure 3-IV ;

[0096] pass Figure 2 and Figure 3-IV It can be seen from the comparison that in Example 3, after unwinding and re-synthesis of COF-Au-MB-Apt / ssDNA / PB / SPE, the synthesized product is MB The product of the synthesis step of COF-Au-MB-Apt / ssDNA / PB / SPE in Example 1 MB consistent.

[0097] pass Figure 3-Ⅰ 、 Figure 3-ⅡAnalysis shows that after completing an OTA test, I MB It can still be observed, revealing the presence of COF-Au-MB-Apt residues. If the COF-Au-MB-Apt / ssDNA / PB / SPE synthesis is carried out directly without unwinding, the unreacted residues will fuse with the newly assembled probe, and the MB content will increase, resulting in I MB Too many will affect subsequent detection.

[0098] And through Figure 3-III Analysis shows that after unwinding, there is no residual MB on the base, so after re-synthesis of COF-Au-MB-Apt / ssDNA / PB / SPE, the I MB The synthesis steps of COF-Au-MB-Apt / ssDNA / PB / SPE in Example 1 are as follows: MB To be consistent, at the same time I PB Compared with I in Figure X-1 PB Maintaining consistency means that the ssDNA / PB / SPE will not decompose during the unwinding process and remains intact.

[0099] As can be seen from Example 1, thermal regulation can well solve such a problem. After thermal regulation, MB The signal disappears ( Figure 3-III During this process, the Apt-ssDNA bond loosens at high temperature without destroying the secondary structure of Apt and ssDNA. With the help of heat treatment, the residual COF-Au-MB-Apt probe is successfully removed, while the ssDNA / AuNPs / PB remains on the sensing surface for the reassembly of new probes.

[0100] As can be seen from Example 2, COF-Au-MB-Apt can be reassembled in a stable and controllable manner. Figure 4 It can be seen that after 7 regeneration cycles, the reassembled I MB and I PB The continuous recording of readings shows that the reproduction efficiency is extremely high. The synergistic effect of Apt-ssDNA dissociation and on-chip thermal regulation performed by the present invention effectively erases expired signal probes and rebuilds new signal probes, thereby reasonably ensuring the multiple use of the sensor and allowing the sensor to be regenerated and applied.

[0101] Example 3: This example discloses a COF regenerative detection device, its application, and a sensor using the same, including the following steps.

[0102] Here’s how to use it:

[0103] Add a concentration analysis step after the unwinding step,

[0104] Concentration analysis: The concentration of ochratoxin A is calculated based on the target signal and the reference signal. The calculation model is as follows:

[0105]

[0106] Where, is the standard target signal, To detect target signals, is the difference between the standard target signal and the detection target signal, - , is the reference signal, is the concentration of ochratoxin A.

[0107] The rest is exactly the same as in Example 1.

[0108]

[0109] Multiple natural samples were tested using the method of Example 1. One type of sample was tested three times. The results of this method were consistent with those of the HPLC method, with a relative error of 3.06-4.82%, indicating that the accuracy of this method is basically consistent with that of the laboratory method.

[0110] The above are all preferred embodiments of the present invention, and are not intended to limit the scope of protection of the present invention. Therefore, any equivalent changes made based on the structure, shape, and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. A COF regenerative detection device, characterized in that: Made using the following steps: Synthesis of TAPB-DMTP covalent organic framework: S11. Add 10.5 mg of 1,3,5-tris(4-aminophenyl)benzene and 8.7 mg of 2,5-dimethoxy-p-benzaldehyde to 4.5 mL of a solution of 1,4-dioxane, 1-butanol, and methanol in a volume ratio of 4:4:

1. Ultrasonicate for 20 min, and simultaneously add 50 μL of 12 M glacial acetic acid dropwise. After ultrasonication, let the mixture stand at 25°C for 2 h to obtain a mixed solution A. S12, adding 450 μL of 12 M glacial acetic acid to the mixed solution A, and heating at 70° C. for 24 h. After the addition is complete, cooling to room temperature and centrifuging at 8000 rpm for 15 min, washing with tetrahydrofuran and acetone, and drying in vacuo at 70° C. to obtain COF; S13. 30 mg of the dried product was added to 20 mL of methanol and sonicated for 3 minutes. The suspension was mixed with 160 μL of 1% chloroauric acid and stirred at 0°C for 5 hours. 1 mL of 0.2 M sodium borohydride dissolved in methanol was added dropwise. The mixture was stirred at 0°C for 3 hours, washed with methanol, centrifuged at 8000 rpm for 15 minutes, and dried at 60°C for 12 hours to obtain COF-Au. Synthesis of COF-Au-MB: 5 mL of 0.1-2 mg / mL COF-Au and 2 mL of 1 mg / mL methylene blue were mixed and stirred until a precipitate formed. The mixture was then centrifuged at 10,000 rpm for 10 min and washed with deionized water to obtain COF-Au-MB. Synthesis of COF-Au-MB-Apt: COF-Au-MB was redispersed in 2 mL of 10 mM Tris-HCl buffer. 1 mL of the COF-Au-MB suspension was mixed with 200 μL of a 6 μM aptamer with the DNA sequence: SH-GATCGGTGTGGGTGGCGTAAAGGGAGCATCGGACA at 4°C. The mixture was incubated with shaking for 10 h and centrifuged at 5000 rpm for 10 min to obtain the COF-Au-MB-Apt probe. Synthesis of PB / SPE: The detection base was incubated with a solution of 3 mM potassium ferrocyanide, 3 mM ferric chloride, 0.1 M hydrochloric acid, and 0.1 M potassium chloride at a constant potential of 0.35 V for 60 s to obtain PB / SPE. Synthesis of ssDNA / PB / SPE: 6 μL of 10 μM semi-complementary strand was dropwise applied to PB / SPE and incubated for 60 min at room temperature. The DNA sequence of the semi-complementary strand was: SH-CATGGACTGTCCGATGCT. After incubation, the strand was passivated in 6 μL of 2 mM 6-mercapto-1-hexanol for 30 min to obtain ssDNA / PB / SPE. Synthesis of COF-Au-MB-Apt / ssDNA / PB / SPE: Incubate ssDNA / PB / SPE with 10 μL of 1 mg / mL COF-Au-MB-Apt for 80-100 min to obtain COF-Au-MB-Apt / ssDNA / PB / SPE.

2. A COF regenerative detection device according to claim 1, characterized in that: In the above S2, the amount of COF-Au added in the synthesis of COF-Au-MB was 5 mL 1 mg / mL.

3. A COF regenerative detection device according to claim 1, characterized in that: The synthesis of the PB / SPE is as follows: after incubation in the solution, the incubated detection base is plated in 10 mM chloroauric acid 4 and 0.1 M potassium chloride at a constant potential of -0.8 V for 80 seconds to obtain the PB / SPE.

4. A method for using a COF regenerative detection device, for using the COF regenerative detection device according to any one of claims 1 to 3, characterized in that: The following technical solutions are adopted: Sample preparation: Each natural sample was crushed and accurately weighed to 5 g, added to 20 mL of acetonitrile, and homogenized at 5000 rpm for 5 min. After homogenization, 2 g of sodium chloride was added, and ultrasonic treatment was performed for 40 min. After standing for 5 min, centrifugation was performed. After centrifugation, the supernatant was quantitatively diluted to 20 mL with tris-hydrochloride buffer to obtain the target OTA and stored at 4°C until analysis. OTA drop coating: The target OTA was drop coated on COF-Au-MB-Apt / ssDNA / PB / SPE and incubated for 60 min to obtain the incubation solution, and then prepared for electrochemical analysis. Electrochemical analysis: Differential pulse voltammetry was used with a pulse amplitude of 50 mV, a pulse width of 16.7 ms, a pulse period of 100 ms, and a window setting of -0.5 to 0.5 V. The differential pulse voltammetry was performed in 10 mM phosphate buffer to measure the target signal current and the reference signal current. Judgment: Determine the size of the target signal and the set threshold. If the target signal current is greater than or equal to the set threshold, it is determined that ochratoxin A is not present. If the target signal current is less than the set threshold, it is determined that ochratoxin A is present. Unwinding: At room temperature, use a heating device to bring the COF-Au-MB-Apt / ssDNA / PB / SPE filled with the target solution to a preset temperature of 70°C within 60 seconds and maintain it for 7-8 minutes. Upon completion, immediately empty the target solution and allow the ssDNA / PB / SPE to cool naturally to room temperature.

5. The method for using the COF regenerative detection device according to claim 4, characterized in that: After the electrochemical analysis step, there is a concentration analysis step. Concentration analysis: The concentration of ochratoxin A is calculated based on the target signal and the reference signal. The calculation model is as follows: ； Where, is the standard target signal, To detect target signals, is the difference between the standard target signal and the detection target signal, - , is the reference signal, is the concentration of ochratoxin A.

6. The method for using the COF regenerative detection device according to claim 4, characterized in that: The holding time during the unwinding is 8 minutes.

7. A sensor for a COF regenerable detection device, comprising: using the COF regenerable detection device according to any one of claims 1 to 3 to implement the method for using the COF regenerable detection device according to any one of claims 4 to 6, characterized in that: It includes a circuit board, on which a detection base is provided. The detection base includes a counter electrode, a working electrode, a reference electrode and a hollow plastic reaction chamber. The counter electrode is arranged in a semicircular shape, and the reference electrode and the working electrode are arranged in an arc shape. The counter electrode, the reference electrode and the working electrode are provided with the COF-Au-MB-Apt described in any one of claims 1 to 3. The counter electrode, working electrode and reference electrode are all connected to the circuit board electrical signal.

8. The sensor of the COF regenerative detection device according to claim 7, characterized in that: Also included is a heating module; The heating module and the detection base are arranged back to back on the circuit board.

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