COF (chip on film) reproducible detection device, using method and sensor applying COF reproducible detection device

Abstract

The invention relates to a COF (chip on film) reproducible detection device, a use method and a sensor applying the COF reproducible detection device, and relates to the technical field of analytical chemistry. Comprising the steps of synthesis of a 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. According to the COF reproducible detection device, the use method and the sensor pair applying the COF reproducible detection device, recycling of the detection device can be achieved in a complex background environment on site.

Description

Technical Field

[0001] The present invention relates to the technical field of analytical chemistry, and in particular to a COF regenerable 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, which can cause serious contamination to a variety of grains such as corn, oats and wheat. After invading the food chain, OTA has serious effects on human health, including kidney damage and immune damage. The existing detection methods for OTA mainly include chromatography, mass spectrometry, LC-MS / MS, etc. Although these methods have high specificity and sensitivity, they generally have shortcomings such as complicated and time-consuming sample pretreatment, and are not suitable for field operations. Therefore, under the condition of limited resources, 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: Synthesis of TAPB-DMTP covalent organic framework (COF): S11, 10.5 mg of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 8.7 mg of 2,5-dimethoxy-p-benzaldehyde (DMTP) were added to 4.5 mL of a solution of 1,4-dioxane, 1-butanol and methanol in a volume ratio of 4:4:1, and ultrasonically treated for 20 min, and 50 μL of 12 M glacial acetic acid was added dropwise at the same time. After the ultrasonic treatment, the mixture was allowed to 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 completed, cooling to room temperature and centrifuging at 8000 rpm for 15 min, washing with tetrahydrofuran and acetone, and vacuum drying at 70° C. to obtain COF; S13. Add 30 mg of the dried product into 20 mL of methanol and ultrasonicate for 3 minutes. Mix the suspension with 160 μL of 1% chloroauric acid (HAuCl4) at 0°C and stir for 5 hours. Add 1 mL of 0.2 M sodium borohydride (NaBH4) dissolved in methanol solution and 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.

[0006] 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 was formed, centrifuged at 10000 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. 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 under shaking for 10 h, the mixture was centrifuged at 5000 rpm for 10 min to obtain the COF-Au-MB-Apt probe.

[0007] Synthesis of PB / SPE: Under constant potential of 0.35 V, the detection base (SPE) was incubated with a solution of 3 mM potassium ferrocyanide (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 to obtain PB / SPE.

[0008] Synthesis of ssDNA / PB / SPE: At room temperature, 6 μL of 10 μM semi-complementary chain (ssDNA) was drop-coated on PB / SPE and incubated for 60 min. The DNA sequence of the ssDNA was: SH-CATGGACTGTCCGATGCT. After incubation, it was passivated in 6 μL of 2 mM 6-mercapto-1-hexanol (MCH) for 30 min to obtain ssDNA / PB / SPE. Synthesis of COF-Au-MB-Apt / ssDNA / PB / SPE: Incubate ssDNA / PB / SPE with 10 μL 1 mg / mL COF-Au-MB-Apt for 80-100 min to obtain COF-Au-MB-Apt / ssDNA / PB / SPE.

[0009] By adopting the above technical scheme, 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. As an artificial oligonucleotide sequence, aptamer (Apt) shows strong specificity and affinity for OTA due to the unique conformational change of the secondary structure and the formation of G-quadruplex. Highly selective Apt can be fixed on a porous covalent organic framework (COF) for OTA recognition. After COF is loaded with gold nanoparticles (AuNPs), COF-Au can absorb Apt through Au-S bonds to form 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. Using SPE as the working electrode, which accommodates permanent on-chip modification of Prussian blue (PB) and ssDNA, Apt-ssDNA interactions can be established due to the base pair stacking between Apt and ssDNA by permanently fixing the semi-complementary strand (ssDNA) to the sensing interface. The flexibly folded Apt can induce changes in the microstructure and electrical behavior of the sensing surface and trigger readable signals. 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.

[0010] Optionally, in S2, the amount of COF-Au added in the synthesis of COF-Au-MB is 5 mL 1 mg / mL.

[0011] By adopting the above technical solution, when the addition amount of COF-Au is 5mL 1mg / mL, the content of AuNPs on COF can be increased, and the MB capture ability and Apt absorption ability can be higher. At the same time, with the increase of the amount of COF-Au, 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 excessive addition of AuNPs, which will affect the experimental effect.

[0012] Optionally, the synthesis of the PB / SPE is 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.

[0013] 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 added showed a larger peak and consistent stability.

[0014] In a second aspect, the present invention provides a method for using a COF regenerable detection device, which adopts the following technical solution: Sample treatment: Each natural sample was crushed and accurately weighed 5 g, and 20 mL of acetonitrile was added. The mixture was homogenized at 5000 rpm for 5 min. After homogenization, 2 g of sodium chloride was added. The mixture was ultrasonically treated for 40 min, and centrifuged after standing for 5 min. After centrifugation, the supernatant was quantitatively diluted to 20 mL with tris(hydroxymethyl)aminomethane hydrochloride buffer to obtain the target OTA, and stored at 4 °C until analysis. OTA drop coating: drop the target OTA onto COF-Au-MB-Apt / ssDNA / PB / SPE, incubate for 60 min, obtain the incubation solution, and then prepare for electrochemical analysis. 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) and the target signal (I MB ) and the reference signal (I PB ), Judgment: Judgment I MB and set the 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 OTA exists. 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. After completion, immediately empty the target solution and allow the ssDNA / PB / SPE to cool naturally to room temperature.

[0015] 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. OTA alone has no signal, but it can effectively suppress the signal of Apt. The 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, the MB peak was significantly suppressed, while the PB peak remained unchanged. At this time, the reference target signal (I MB ) value to determine whether OTA exists.

[0016] Before the presence of OTA, COF-Au-MB-Apt probes were assembled onto ssDNA / PB / SPE at room temperature through hybridization of Apt and ssDNA. After the presence of OTA, COF-Au-MB-Apt tended to fall off from ssDNA / PB / SPE, resulting in self-regeneration of ssDNA / PB / SPE tissue. Since Apt-ssDNA bonds loosen at high temperatures without destroying the secondary structures of Apt and ssDNA, thermal regulation on the electrode may contribute to the regeneration behavior, through which COF-Au-MB-Apt probes can dissociate from ssDNA / PB / SPE into the solution, after which the solution on the SPE was immediately emptied to prevent possible recombination between ssDNA and expired COF-Au-MB-Apt probes, and then the ssDNA / PB / SPE module was naturally cooled to 25°C 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 ssDNA / PB remained on the sensing surface for the reassembly of new probes, regenerating the sensing surface for reuse.

[0017] At the same time, ssDNA / PB / SPE has high selectivity for other mycotoxins and metal ions. Compared with the OTA signal alone, 1000-fold concentration of mycotoxins or metal ions has no obvious effect on the OTA signal. ssDNA / PB / SPE also has good repeatability and long-term stability.

[0018] Optionally, there is a concentration analysis step after the electrochemical 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:

[0019] In the formula, 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.

[0020] By adopting the above technical solution, the method can detect the specific concentration of OTA. As the concentration of OTA increases, I MB The quantitative decrease, while I PB In the range of 0.2pg / mL to 0.6μg / mL, ΔI MB / I PB A good relationship is established between the logarithm of the OTA concentration, namely , the detection limit was calculated to be 0.12 pg / mL.

[0021] Optionally, the holding time during the detwisting is 8 minutes.

[0022] By adopting the above technical scheme, the optimized heat treatment at 70 °C for 8 min can expel more unreacted COF-Au-MB-Apt and further improve the regeneration efficiency, while maintaining the retention of ssDNA / PB on the sensing surface.

[0023] In a third aspect, the present invention provides a sensor suitable for using the COF regenerative detection device of the first aspect, and adopts the following technical solution: A sensor includes a circuit board, on which a detection base is arranged. 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. The counter electrode, working electrode and reference electrode are all connected to the circuit board electrical signal.

[0024] By adopting the above technical scheme, the SPE includes a pre-designed pad as a semi-finished three-electrode system, wherein the semicircular electrode is directly used as the counter electrode, and a circular arc electrode is selected as the reference electrode after Ag / AgCl functionalization. A permanent on-chip modification and a regenerable probe are designed on the circular electrode, which is used as a working electrode. Optionally, the sensor also includes a heating module.

[0025] The heating module is arranged opposite to the detection sensor on the circuit board.

[0026] The circuit board is connected with the heating module via electrical signals.

[0027] By adopting the above technical solution, the heating modules are assembled to the back of the SPE, just below the three electrodes, where they work as a remote sensing heater and cooperate with the on-chip control to achieve thermal regulation regeneration.

[0028] In summary, the present invention includes at least one of the following beneficial technical effects: 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 fixed on a porous covalent organic framework (COF) for OTA recognition.

[0029] The present invention successfully removes the residual COF-Au-MB-Apt probe 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 repeated use.

[0030] The ssDNA / PB / SPE in the present invention has high selectivity for other mycotoxins and metal ions. Compared with the signal of OTA alone, 1000 times the content of mycotoxins or metal ions has no obvious effect on the OTA signal. ssDNA / PB / SPE also has good repeatability and long-term stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the heating module structure of the present invention; 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; Figure 3 The electrochemical comparison diagram of the co-regeneration and the dissociation of OTA alone is shown in Figure 2. Figure 3-Ⅰ This is the analysis diagram obtained from the electrochemical analysis step in Example 1, Figure 3-ⅡThis 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; Figure 4 is the electrochemical test signal I MB and I PB Plot of the repeated cycles before and after (re)assembly and the obtained I MB and I PB Error bars, 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 triode; 10. second triode; 11. heating resistor; 12. third DC power supply. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below with reference to the embodiments.

[0033] Embodiment 1: This embodiment discloses a COF regenerable detection device, its application and a sensor using the same, including the following steps.

[0034] A COF regenerable detection device is manufactured by the following steps: Synthesis of TAPB-DMTP covalent organic framework (COF): S11, 10.5 mg of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 8.7 mg of 2,5-dimethoxy-p-benzaldehyde (DMTP) were added to 4.5 mL of a solution of 1,4-dioxane, 1-butanol and methanol in a volume ratio of 4:4:1, and ultrasonically treated for 20 min, and 50 μL of 12 M glacial acetic acid was added dropwise at the same time. After the ultrasonic treatment, the mixture was allowed to 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 completed, cooling to room temperature and centrifuging at 8000 rpm for 15 min, washing with tetrahydrofuran and acetone, and vacuum drying at 70° C. to obtain COF; S13. Add 30 mg of the dried product into 20 mL of methanol and ultrasonicate for 3 minutes. Mix the suspension with 160 μL of 1% chloroauric acid (HAuCl4) at 0°C and stir for 5 hours. Add 1 mL of 0.2 M sodium borohydride (NaBH4) dissolved in methanol solution and 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.

[0035] 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 was formed, centrifuged at 10000 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. 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 under shaking for 10 h, the mixture was centrifuged at 5000 rpm for 10 min to obtain the COF-Au-MB-Apt probe.

[0036] 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 ferrocyanide (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, and then the incubated detection base was plated with 10 mM chloroauric acid 4 and 0.1 M potassium chloride at a constant potential of -0.8 V for 80 s to obtain PB / SPE.

[0037] Synthesis of ssDNA / PB / SPE: At room temperature, 6 μL of 10 μM semi-complementary chain (ssDNA) was drop-coated on PB / SPE and incubated for 60 min. The DNA sequence of the ssDNA was: SH-CATGGACTGTCCGATGCT. After incubation, it was passivated in 6 μL of 2 mM 6-mercapto-1-hexanol (MCH) for 30 min to obtain ssDNA / PB / SPE. Synthesis of COF-Au-MB-Apt / ssDNA / PB / SPE: Incubate ssDNA / PB / SPE with 10 μL 1 mg / mL COF-Au-MB-Apt for 80-100 min to obtain COF-Au-MB-Apt / ssDNA / PB / SPE.

[0038] Here’s how to use it: Sample treatment: Each natural sample was crushed and accurately weighed 5 g, and 20 mL of acetonitrile was added. The mixture was homogenized at 5000 rpm for 5 min. After homogenization, 2 g of sodium chloride was added. The mixture was ultrasonically treated for 40 min, and centrifuged after standing for 5 min. After centrifugation, the supernatant was quantitatively diluted to 20 mL with tris(hydroxymethyl)aminomethane hydrochloride buffer to obtain the target OTA, and stored at 4 °C until analysis. OTA drop coating: drop the target OTA onto COF-Au-MB-Apt / ssDNA / PB / SPE, incubate for 60 min, obtain the incubation solution, and then prepare for electrochemical analysis. 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) and the target signal (I MB ) and the reference signal (I PB ), Judgment: Judgment I MB and set the 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 OTA exists. Unwinding: At room temperature, use a heating device to make the COF-Au-MB-Apt / ssDNA / PB / SPE filled with the target solution reach the preset 70°C within 60 seconds and maintain it for 8 minutes. After completion, immediately empty the target solution and cool the ssDNA / PB / SPE naturally to room temperature.

[0039] The above sensor adopts the following technical solutions: A sensor includes a circuit board, on which a detection base and a heating module are arranged. The heating module is arranged on the circuit board back to back with the detection sensor. 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 combination of the counter electrode, the reference electrode and the working electrode is in a circular ring shape. Regenerable probes are arranged on the counter electrode, the reference electrode and the working electrode.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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, 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, the B end of the second transistor is connected between the fifth resistor and the B end of the first transistor, and the C end of the second transistor is grounded.

[0044] 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.

[0045] Embodiment 2: This embodiment discloses a COF regenerable detection device, its application and a sensor using the same, including the following steps.

[0046] Here’s how to use it: After the unwinding step, the unwinding ssDNA / PB / SPE was reapplied to the synthesis step of COF-Au-MB-Apt / ssDNA / PB / SPE for reassembly and use to obtain the test results.

[0047] The rest is exactly the same as in Example 1.

[0048] Comparative example: This comparative example discloses a COF regenerable detection device, its application and a sensor using the same, including the following steps.

[0049] Here’s how to use it: After the judgment step, the used ssDNA / PB / SPE is directly reapplied to the synthesis step of COF-Au-MB-Apt / ssDNA / PB / SPE without unwinding, and is reassembled and used to obtain the test results.

[0050] 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 ; In Example 1, the analysis diagram obtained in the electrochemical analysis step is as follows Figure 3-Ⅰ ; 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 Figure 3-Ⅱ ; The unwinding ssDNA / PB / SPE in Example 3 was subjected to electrochemical analysis to obtain the analysis Figure 3-III ; 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 ; pass Figure 2 and Figure 3-IV It can be seen from the comparison that in Example 3, after unwinding and re-synthesizing COF-Au-MB-Apt / ssDNA / PB / SPE, the synthesized product has MB The product of the synthesis step of COF-Au-MB-Apt / ssDNA / PB / SPE in Example 1 MB Consistent.

[0051] pass Figure 3-Ⅰ , Figure 3-Ⅱ Analysis shows that after completing an OTA test, I MB It can still be observed that the presence of COF-Au-MB-Apt residues is revealed. 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.

[0052] And through Figure 3-III Analysis shows that after unwinding, there will be no residual MB on the base, so after re-synthesis of COF-Au-MB-Apt / ssDNA / PB / SPE, the I MB The same as the synthesis step of COF-Au-MB-Apt / ssDNA / PB / SPE in Example 1 MB To be consistent, at the same time I PB Compared with I in Figure X-1 PB Keeping it consistent means that the ssDNA / PB / SPE will not decompose during the unwinding process and will remain intact.

[0053] 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 bonds loosened at high temperatures without destroying the secondary structures of Apt and ssDNA, and with the help of heat treatment, the residual COF-Au-MB-Apt probes were successfully removed, while the ssDNA / AuNPs / PB remained on the sensing surface for the reassembly of new probes.

[0054] 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, so that multiple uses of the sensor are reasonably guaranteed and the sensor can be regenerated and applied.

[0055] Embodiment 3: This embodiment discloses a COF regenerable detection device, its application and a sensor using the same, including the following steps.

[0056] Here’s how to use it: Add a concentration analysis step after the unwinding 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:

[0057] In the formula, 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.

[0058] The rest is exactly the same as in Example 1.

[0059]

[0060] Multiple natural samples were tested in the manner 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.

[0061] The above are all preferred embodiments of the present invention, and are not intended to limit the protection scope 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 protection scope of the present invention.

Claims

1. A COF reproducible detection device, characterized in that: Made using the following steps: Synthesis of TAPB-DMTP covalent organic framework: S11, 10.5 mg of 1,3,5-tris(4-aminophenyl)benzene and 8.7 mg of 2,5-dimethoxy-p-benzaldehyde were added to 4.5 mL of a solution of 1,4-dioxane, 1-butanol and methanol in a volume ratio of 4:4:1, and ultrasonicated for 20 min, and 50 μL of 12 M glacial acetic acid was added dropwise at the same time. After the ultrasonication, the solution was allowed to 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 completed, cooling to room temperature and centrifuging at 8000 rpm for 15 min, washing with tetrahydrofuran and acetone, and vacuum drying at 70° C. to obtain COF; S13, 30 mg of the dried product was added to 20 mL of methanol, ultrasonicated for 3 minutes, the suspension was mixed with 160 μL of 1% chloroauric acid at 0°C and stirred for 5 hours, 1 mL of 0.2 M sodium borohydride dissolved in methanol solution was added dropwise, 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 was formed, then centrifuged at 10000 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. At 4°C, 1 mL of COF-Au-MB suspension was mixed with 200 μL of 6 μM aptamer, the DNA sequence of which was: SH-GATCGGTGTGGGTGGCGTAAAGGGAGCATCGGACA. After incubation under shaking for 10 h, the mixture was 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 for 60 s at a constant potential of 0.35 V to obtain PB / SPE; Synthesis of ssDNA / PB / SPE: At room temperature, 6 μL of 10 μM semi-complementary chain was dropped on PB / SPE and incubated for 60 min. The DNA sequence of the semi-complementary chain was: SH-CATGGACTGTCCGATGCT. After incubation, it 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 1 mg / mL COF-Au-MB-Apt for 80-100 min to obtain COF-Au-MB-Apt / ssDNA / PB / SPE.

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

3. A COF reproducible 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 regenerable detection device, for using the COF regenerable detection device according to any one of claims 1 to 3, characterized in that: The following technical solutions are adopted: Sample treatment: Each natural sample was crushed and accurately weighed 5 g, and 20 mL of acetonitrile was added. The mixture was homogenized at 5000 rpm for 5 min. After homogenization, 2 g of sodium chloride was added. The mixture was ultrasonically treated for 40 min, and centrifuged after standing for 5 min. After centrifugation, the supernatant was quantitatively diluted to 20 mL with tris(hydroxymethyl)aminomethane hydrochloride buffer to obtain the target OTA, and stored at 4 °C until analysis. OTA drop coating: drop the target OTA onto COF-Au-MB-Apt / ssDNA / PB / SPE, incubate for 60 min, obtain the incubation solution, and then prepare 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 a 10 mM phosphate buffer solution to measure the target signal current and the reference signal current. Judgment: judging the magnitude of the target signal and the set threshold, if the target signal current is greater than or equal to the set threshold, it is judged that ochratoxin A does not exist, if the target signal current is less than the set threshold, it is judged that ochratoxin A exists; 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. After 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 regenerable detection device according to claim 4, characterized in that: After the electrochemical analysis step, there is also 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: In the formula, 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 a COF regenerable 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, which is used to implement the method for using the COF regenerable detection device according to any one of claims 4 to 8 by using the COF regenerable detection device according to any one of claims 1 to 3, characterized in that: It includes a circuit board, on which a detection base is arranged. The detection base comprises a counter electrode, a working electrode, a reference electrode and a hollow plastic reaction chamber, the counter electrode is arranged in a semicircular shape, the reference electrode and the working electrode are arranged in an arc shape, and 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 regenerable 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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