Preparation method of photoelectrochemical sensor based on methylene blue composite material loaded on Ni-MOF and application thereof

Abstract

The application belongs to the technical field of biosensing detection, and particularly relates to a preparation method of a photoelectrochemical sensor based on a methylene blue composite material loaded by Ni-MOF and application thereof. The application introduces a green Ni-MOF to fix a photosensitive molecule methylene blue, and SA-ALP catalyzes an electron donor ascorbic acid (AA) generated by ascorbic acid phosphate, thereby constructing a spatially confined aptamer sensor of the methylene blue composite material loaded by Ni-MOF, realizing the improvement of the sensitivity of the detection of MC-LR, and the sensor has good selectivity and stability. The spatially confined photoelectrochemical aptamer sensor developed by the application can realize the rapid, high-selectivity and sensitive analysis of the detection of MC-LR by adjusting the content of AA, and a good recovery rate is obtained.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of aptamer sensing and detection, and particularly relates to a preparation method of a photoelectrochemical sensor based on a methylene blue composite material loaded on Ni-MOF and application thereof. BACKGROUND

[0002] Due to the discharge of industrial wastewater, the flooding of chemical fertilizers and other reasons, water eutrophication has become an important water environmental problem faced by the world. Microcystin-LR (MC-LR) is one of the blue-green algae toxins with high risk and wide distribution, which can cause various toxic effects, including damage to the immune, urinary, digestive and reproductive systems. The World Health Organization stipulates that 1 μg / L is the threshold of MC-LR in water. At present, the detection of MC-LR has advantages and disadvantages. High performance liquid chromatography and enzyme-linked immunosorbent assay have good accuracy, but are expensive, complex to operate and time-consuming, which limits their application. Many new methods have been developed due to their simple equipment, easy operation and high sensitivity, including colorimetric method, fluorescence method, surface enhanced Raman spectroscopy and photoelectrochemical method, but the accuracy of these analysis methods needs to be improved. Therefore, it is of great significance to develop a sensitive, efficient, accurate and easy-to-promote MC-LR analysis method to protect water safety and human health.

[0003] In order to obtain better analysis performance, more and more researches are committed to establish various advanced photoelectrochemical (PEC) systems. Generally speaking, ideal photosensitive materials and suitable electron donors are important factors affecting the performance of PEC sensors. Photosensitive materials convert light energy into electrical signals, while electron donors provide electrons for photosensitive materials to output stable photocurrent. Commonly used photosensitive materials include organic semiconductors (such as methylene blue (MB), polyaniline), inorganic semiconductors (such as zinc oxide), and heterojunctions (such as CdTe-BiOBr). In addition to photosensitive materials, the analysis performance of PEC sensors can also be improved by introducing electron donors (such as ascorbic acid (AA)). It is worth noting that the AA produced by enzyme catalysis tends to diffuse into the solution, which may limit the enhancement of photocurrent on the sensing interface.

[0004] Therefore, the combination of the preparation of high-performance photosensitive materials and the effective use of confined electron donors can effectively improve the sensitivity of PEC sensors. Metal-organic frameworks (MOFs) have attracted considerable attention in the field of PEC sensing due to their large specific surface area, high stability and uniform pore size, so the unique functions of MOFs (such as high absorption capacity, spatial confinement) can be effectively used to improve the analysis performance of sensors. SUMMARY

[0005] The present application aims at the problem of insufficient analysis performance of MC-LR by existing sensors, and aims to combine the green and simple synthesis of MOFs, the spatial confinement and the photoelectrochemical sensing technology of providing electron donor strategy to construct a photoelectrochemical aptamer sensor of Ni-MOF loaded methylene blue composite material spatial confinement, and realize sensitive detection of MC-LR.

[0006] In order to realize the above technical purposes, the present application scheme is as follows:

[0007] A preparation method of a photoelectrochemical sensor based on a Ni-MOF loaded methylene blue composite material, comprising the following steps:

[0008] (1) Preparation of Ni-MOF / MB composite material:

[0009] S1, first, dissolve nickel acetate tetrahydrate in ultrapure water, ultrasonic dissolution to obtain a light green solution, denoted as solution A; dissolve 1.4-terephthalic acid in a mixed solution of N,N-dimethylacetamide and dichloroethane, ultrasonic dissolution to obtain a transparent solution, denoted as solution B; add solution A to solution B, collect the precipitate by centrifugation after a period of reaction, then dry the product to obtain Ni-MOF;

[0010] Preferably, in S1 of step (1), the amount ratio of nickel acetate tetrahydrate to ultrapure water is 223.2 mg:6 mL; the amount ratio of 1.4-terephthalic acid, N,N-dimethylacetamide and dichloroethane is 24.9 mg:6 mL:6 mL; the reaction time is 30 min; the volume ratio of ultrapure water in solution A to dichloroethane in solution B is 1:1 when solution A is added to solution B;

[0011] S2, weigh Ni-MOF and dissolve it in ultrapure water to obtain a Ni-MOF solution, then add MB and stir to react, after the reaction is completed, centrifuge, wash and dry the product to obtain Ni-MOF / MB;

[0012] Preferably, in S2 of step (1), the amount ratio of Ni-MOF, ultrapure water and MB is 30 mg:5 mL:1.05 mg; the reaction temperature is 25℃, the stirring speed is 300 rpm, and the reaction time is 3h;

[0013] S3, add HAuCl4·3H2O to ultrapure water, heat to boil, then add trisodium citrate solution, maintain the heating temperature and continue to react for a certain period of time, after the reaction, cool the solution to room temperature to form an Au NPs solution;

[0014] Preferably, in S3 of step (1), the amount ratio of HAuCl4·3H2O, ultrapure water and trisodium citrate solution is 0.1 mol:25 mL:0.25 mL, the concentration of the trisodium citrate solution is 100 mg / ml; the temperature of heating is maintained at 95-100℃; and the reaction is continuously heated for 10-15 min;

[0015] (2) The ITO glass electrode is first cleaned with anhydrous ethanol and ultrapure water by ultrasonic cleaning, then boiled with a NaOH solution, and cleaned again with ethanol and ultrapure water by ultrasonic cleaning. After drying at room temperature, a treated ITO electrode is obtained;

[0016] Preferably, in step (2), the diameter of the ITO electrode is d=6 mm; the concentration of the NaOH solution used is 0.3 mol / L, and the boiling time is 20 min; and the ultrasonic cleaning time is 15 min.

[0017] (3) The Ni-MOF / MB prepared in step (1) is added to water to obtain a Ni-MOF / MB solution; then the Ni-MOF / MB solution is added dropwise to the surface of the ITO electrode treated in step (2), and dried at room temperature to obtain a product marked as Ni-MOF / MB / ITO;

[0018] Preferably, in step (3), the amount of the Ni-MOF / MB solution added dropwise is 20 μL, and the concentration of the Ni-MOF / MB solution is 1 mg / mL.

[0019] (4) The Au NPs solution is modified on the surface of the Ni-MOF / MB / ITO sensor prepared in step (3) and dried; at this time, a film structure is formed on the surface of the sensor, which is marked as Au NPs / Ni-MOF / MB / ITO;

[0020] Preferably, in step (4), the concentration of the Au NPs solution is 5.2 nmol / L; and the amount of the Au NPs solution added dropwise is 20 μL.

[0021] (5) According to the known sequence of MC-LR aptamer (Apt) and the principle of base complementary pairing, MC-LR aptamer complementary DNA (cDNA) and double-stranded amplification are designed, which are denoted as cDNA, S1 and S2, respectively; Apt, cDNA, S1, S2 and streptavidin-alkaline phosphatase (SA-ALP) are added into a DNA amplifier for base complementary pairing and DNA double-stranded amplification, and the obtained product is denoted as DNA complex; the DNA complex is modified on the sensor surface obtained in step (4) (Apt is fixed on the electrode surface through gold-sulfur bond between Apt and Au NPs), and the sensor is rinsed with tris buffer, and the rinsed sensor is a photoelectrochemical sensor of Ni-MOF loaded methylene blue composite material, which is denoted as SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO.

[0022] Preferably, in step (5), the concentration of Apt is 10 μM, the concentration of cDNA is 15 μM, the concentration of S1 is 37.5 μM, the concentration of S2 is 37.5 μM and the concentration of SA-ALP is 1 mg / mL;

[0023] The volume ratio of Apt, cDNA, S1, S2 and SA-ALP is 1:1:1:1:1.5, and the amount of DNA complex modified on the sensor surface obtained in step (4) is 20 μL; the concentration of tris buffer is 0.1 M, pH = 8.0; the composition of tris buffer is: 10 mM tris, 5 mM MgCl2, 0.2 mM NaCl;

[0024] The sequences of aptamer of MC-LR, cDNA, S1 and S2 are as follows:

[0025] Apt: 5'-SH-GGC CGG AAA CAG GAC CAC CAT GAC AAT TAC CCA TAC CAC CTC ATT ATG CCC CAT CTC CGC-3'

[0026] cDNA: 5'-CCC GCG GCA GCG GAG ATG GGG CAT AAT-3'

[0027] S1: 5'-TGC CGC GGG AGG CGG ATT C biotin-3'

[0028] S2: 5'-CCC GCG GCA CGA ATC CGC C biotin-3'.

[0029] The application also relates to a use of a photoelectrochemical aptamer sensor based on a methylene blue composite material loaded with Ni-MOF for detecting MC-LR, and the steps are as follows:

[0030] (1) First, prepare MC-LR standard solutions with different concentrations, and then modify the MC-LR standard solutions with different concentrations and V1 volumes on the surface of the prepared sensor (SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO), one concentration of the MC-LR solution corresponds to one sensor, and the concentration and the sensor are in one-to-one correspondence; after incubation for a period of time, tris buffer is used for elution, so that an aptamer sensor based on the methylene blue composite material loaded with Ni-MOF is obtained, which is marked as MC-LR / SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO;

[0031] Preferably, in step (1), the concentration of the MC-LR standard solution is 10 fM to 100 pM;

[0032] Preferably, in step (1), the temperature of the incubation is 37 DEG C, and the period of time is 20 to 60 min;

[0033] Preferably, in step (1), the concentration of the tris buffer is 0.1 M, and the pH value is 8.0; the composition of the tris buffer is: 10 mM tris, 5 mM MgCl2 and 0.2 mM NaCl.

[0034] (2) The MC-LR / SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO obtained in step (1) is used as a working electrode, an Ag / AgCl electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode; then the working electrode is placed in an electrolyte for hydrolysis for a period of time, and then electrochemical detection is carried out, and the corresponding current value is obtained through photoelectrochemical testing; the current value is used as the ordinate, and the logarithmic value of the corresponding MC-LR concentration is used as the abscissa, and a corresponding standard curve based on the aptamer is established;

[0035] (3) Detection of MC-LR in a sample: first, obtain a sample liquid, modify V1 volume of the sample liquid on the surface of the sensor, and then perform electrochemical detection according to the operation methods of steps (1) and (2), and the corresponding current value is obtained through photoelectrochemical testing; the current value is substituted into the standard curve established in step (2), so that the concentration of MC-LR in the sample can be obtained; and the use of the detection of MC-LR in an unknown sample is achieved.

[0036] Preferably, in steps (1) and (3), the modified V1 volume is 20 muL.

[0037] Preferably, in steps (2) and (3), the electrolyte is a tris buffer containing L-ascorbic acid-2-phosphoric acid trisodium salt (AAP), wherein the final concentration of L-ascorbic acid-2-phosphoric acid trisodium salt is 10 mM, the concentration of tris buffer is 0.1 M, pH = 8.0, the composition of the tris buffer is: 10 mM tris, 5 mM MgCl2, 0.2 mM NaCl; the applied bias voltage is 0 V; and the hydrolysis time is 40 min.

[0038] The present application has the following advantages:

[0039] (1) The present application proposes a MOF spatial confinement method for detecting MC-LR for the first time to solve the problem of insufficient analysis performance of MC-LR by existing sensors. The present application combines the green and simple synthesis of MOFs, the spatial confinement, and the sensing technology of the photoelectrochemical electron donor strategy to construct a photoelectrochemical aptamer sensor of a Ni-MOF loaded methylene blue composite material spatial confinement, so that the sensor has the advantages of low background signal of photoelectrochemistry and sensitive spatial confinement, and realizes sensitive detection of MC-LR.

[0040] (2) The present application develops a green Ni-MOF synthesis method, which is used as an efficient reaction platform of MB and AA.

[0041] (3) The present application introduces a specific recognition aptamer of MC-LR to improve the selectivity of the photoelectrochemical sensor, reduce the interference of similar toxins in the water environment, and realize specific analysis of MC-LR.

[0042] (4) The photoelectrochemical aptamer sensor constructed by the present application has high sensitivity, good selectivity, good stability, and a wide linear range of 10 fM to 100 pM for detecting MC-LR. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 It is a schematic diagram of the construction process and working principle of the PEC aptamer sensor for detecting MC-LR.

[0044] Figure 2 A is a SEM image of Ni-MOF; B is a TEM image of Ni-MOF;

[0045] C is a SEM image of Ni-MOF / MB; D is a TEM image of Ni-MOF / MB;

[0046] E is an element map of Ni-MOF / MB; F is an EDX map of Ni-MOF / MB.

[0047] Figure 3A is the PEC response of the aptamer sensor to different concentrations (0, 1 and 10 pM) of MC-LR;

[0048] B is the Nyquist plot of (a) ITO, (b) Ni-MOF / MB / ITO, (c) Au NPs / Ni-MOF / MB / ITO, (d) SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO, (e) MC-LR / SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO, the inset in B is the Randles equivalent circuit used to fit the impedance spectra;

[0049] C is the PEC response of the aptamer sensor to different concentrations of MC-LR; D is the corresponding linear calibration curve; PEC tests were performed in tris (pH = 8.0) containing AAP at a bias potential of 0 V; Error bars: ± SD, n = 3.

[0050] Figure 4 A is the selectivity of the appropriate sensor to 100 pM MC-LR and 1 nM interferents MC-YR, MC-RR, L-cysteine and L-tyrosine;

[0051] B is the reproducibility of the aptamer sensor for 1 pM MC-LR, 7 replicates were performed;

[0052] C is the stability of the aptamer sensor for 1 pM MC-LR over 10 days of storage. DETAILED DESCRIPTION

[0053] Various illustrative embodiments of the present application are described in detail below. This detailed description is not intended to restrict the application unless so indicated, but rather to explain certain aspects, features and embodiments of the application.

[0054] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in further detail the methods and / or materials associated with the documents. In the case of conflict between the present specification and any document incorporated by reference, the present specification will control.

[0056] Many modifications and variations to the illustrative embodiments described herein will be apparent to those of ordinary skill in the art from this specification, which is to be construed as exemplary only. Other embodiments of the application will be apparent to those of ordinary skill in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples provided should be considered exemplary only.

[0057] The MC-LR aptamer, MC-LR aptamer complementary DNA (cDNA) and DNA amplification chain (S1 and S2) used in the present application are purchased from Shenguo Bioengineering (Shanghai) Co., Ltd.; the present application does not involve a sequence listing invention, and the used are conventional commercially available primers.

[0058] The composition of the tris buffer solution is: 10 mM tris, 5 mM MgCl2, 0.2 mM NaCl; the electrolyte is a tris buffer solution containing L-ascorbic acid-2-phosphoric acid trisodium salt (AAP), wherein the final concentration of L-ascorbic acid-2-phosphoric acid trisodium salt is 10 mM, the concentration of the tris buffer solution is 0.1 M, and the pH is 8.0.

[0059] Screening and optimization of conditions:

[0060] (1) Preparation of the composite material:

[0061] First, 223.2 mg of nickel acetate tetrahydrate was dissolved in 6 mL of ultrapure water, and ultrasonic was used to dissolve it, and the solution was light green, which was recorded as solution A; 24.9 mg of 1.4-terephthalic acid was dissolved in 6 mL of N, N-dimethylacetamide and 6 mL of dichloroethane mixed solution, and ultrasonic was used to dissolve it, and the solution was transparent, which was recorded as solution B. Solution A was added dropwise to solution B, and Ni-MOF was immediately formed at the interface of the two layers, which was allowed to stand for 30 min. Ni-MOF was collected by centrifugation, and then dried and ground into fine powder for subsequent use;

[0062] Then, 30 mg of Ni-MOF was dissolved in 5 mL of ultrapure water. 1.05 mg of MB was dissolved in the above solution. The mixed solution was stirred at a uniform speed of 300 rpm for 3 h. Then the mixed solution was centrifuged, washed and dried to obtain Ni-MOF / MB. After drying, the solid was ground into powder and dissolved in 5 mL of ultrapure water for storage;

[0063] Finally, 0.1 mol of HAuCl4·3H2O was added to 5 mL of ultrapure water, and heated to boiling. Then 0.25 ml of 100 mg / ml trisodium citrate was injected into the above solution, and finally reacted at this temperature for 15 min. When the mixture changed from colorless to wine red, spherical Au NPs were formed. When the aqueous solution cooled to room temperature, gold colloid was formed. It was stored in the refrigerator for freezing;

[0064] (2) Indium tin oxide glass (ITO) electrode was first ultrasonically cleaned with absolute ethanol and ultrapure water for 15 min, respectively, and then boiled with 0.3 mol / L NaOH solution for 20 min. The ITO electrode was cleaned again with ethanol and ultrapure water, dried at room temperature, and then fixed the modified area on the electrode surface to obtain the treated indium tin oxide glass electrode;

[0065] (3) 20 μL of the Ni-MOF / MB solution prepared in step (1) was added to the surface of the indium tin oxide glass electrode treated in step (2), and the product was dried at room temperature, and was marked as Ni-MOF / MB / ITO;

[0066] (4) 20 μL of the Au NPs solution was modified on the surface of the Ni-MOF / MB / ITO sensor prepared in step (3), and was dried. At this time, a film structure was formed on the surface of the sensor, and was marked as Au NPs / Ni-MOF / MB / ITO;

[0067] (5) According to the known sequence of the MC-LR aptamer and the base complementary pairing principle, MC-LR aptamer complementary DNA and amplification double-stranded were designed, and were marked as cDNA, S1 and S2. The aptamer, cDNA, S1, S2 and streptavidin-alkaline phosphatase were added to a DNA amplifier for base complementary pairing and DNA double-strand amplification, and were marked as DNA complex. 20 μL of the DNA complex was modified on the surface of the sensor obtained in step (4), and the gold-sulfur bond between the aptamer and the Au NPs was used to fix it on the electrode surface. The sensor was rinsed with tris buffer, and the rinsed sensor was marked as SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO;

[0068] (6) Different concentrations of MC-LR standard solution were modified on the surface of the sensor prepared in step (5). 20 μL of MC-LR solution of one concentration corresponded to one sensor, and the concentration and the sensor were in one-to-one correspondence. After incubation for a period of time, the sensor was rinsed with tris buffer, thereby obtaining a spatially confined aptamer sensor based on Ni-MOF loaded methylene blue composite material, which was marked as MC-LR / SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO.

[0069] (7) Concentration, pH, SA-ALP hydrolysis time and hybridization time optimization;

[0070] (a) Concentration optimization:

[0071] The DNA complex obtained in step (5) was optimized, and the amount of aptamer was optimized to affect the content of streptavidin-alkaline phosphatase; 20 μL MC-LR aptamer concentration was gradually increased from 0.05 μM to 0.15 μM, and the amount of streptavidin-alkaline phosphatase anchored thereon was increased, so that the photocurrent was enhanced. When the Apt concentration exceeds 0.15 μM, the photocurrent tends to be flat, indicating that the hybridization of MC-LR and aptamer reaches saturation; therefore, 0.15 μM is selected as the optimal Apt concentration for subsequent experiments.

[0072] (b) pH optimization:

[0073] MC-LR / SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO obtained in step (6) was used as the working electrode, Ag / AgCl electrode was used as the reference electrode, and platinum wire electrode was used as the counter electrode, and electrochemical detection was carried out in a tris buffer solution containing 10 mM L-ascorbic acid-2-phosphoric acid trisodium salt; when the pH value of the detection solution increased from 7.5 to 8.0, the solution gradually became weakly alkaline, which gradually increased the activity of SA-ALP, resulting in an increase in photocurrent. When the pH value of the detection solution gradually increased from 8.0 to 9.5, the increase in alkalinity led to a decrease in SA-ALP activity, resulting in a decrease in photocurrent. Therefore, the optimal pH value of the detection solution is 8.0.

[0074] (c) SA-ALP hydrolysis time optimization:

[0075] MC-LR / SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO obtained in step (6) was used as the working electrode, and SA-ALP hydrolysis was carried out in an electrolyte (tris buffer solution containing 10 mM L-ascorbic acid-2-phosphoric acid trisodium salt); a certain amount of AA was generated in the electrolyte to improve the photoelectric signal.

[0076] The hydrolysis time of SA-ALP affects the performance of the aptamer sensor. Under a certain AAP (L-ascorbic acid-2-phosphoric acid trisodium salt) concentration, the hydrolysis time is extended from 10 minutes to 40 minutes, and the photocurrent is gradually enhanced due to the generation of AA. As the hydrolysis time continues to be extended, the change in photocurrent is negligible. Therefore, 40 minutes is selected as the optimal hydrolysis time.

[0077] (d) Hybridization time optimization:

[0078] The MC-LR on the sensor obtained in step (5) was modified, and the incubation binding time of MC-LR and aptamer was optimized; as the binding time of MC-LR and aptamer increased from 20 minutes to 50 minutes, the recorded photocurrent decreased, and further prolonging the binding time, no obvious change in photocurrent could be observed. Therefore, 50 minutes was selected as the optimal incubation binding time.

[0079] Example 1:

[0080] The construction method flow of the space-limited photoelectrochemical aptamer sensor based on the methylene blue-loaded Ni-MOF composite material of the application is shown in FIG. 1, and the specific steps are as follows: Figure 1

[0081] (1) Preparation of composite material:

[0082] First, 223.2 mg of nickel acetate tetrahydrate was dissolved in 6 mL of ultrapure water, and ultrasonic was used to dissolve it. The solution was light green and was recorded as solution A. 24.9 mg of 1, 4-terephthalic acid was dissolved in a mixed solution of 6 mL of N, N-dimethylacetamide and 6 mL of dichloroethane, and ultrasonic was used to dissolve it. The solution was transparent and was recorded as solution B. Solution A was added dropwise to solution B, and Ni-MOF was immediately formed at the interface of the two layers. The Ni-MOF was collected by centrifugation, then dried and ground into fine powder for subsequent use;

[0083] Then, 30 mg of Ni-MOF was dissolved in 5 mL of ultrapure water. 1.05 mg of MB was dissolved in the above solution. The mixed solution was stirred at a speed of 300 rpm for 3 h. Then the mixed solution was centrifuged, washed and dried to obtain Ni-MOF / MB. After drying, the solid was ground into powder and dissolved in 5 mL of ultrapure water for storage;

[0084] Finally, 0.1 mol of HAuCl4·3H2O was added to 5 mL of ultrapure water, and heated to boiling. Then 0.25 ml of 100 mg / ml trisodium citrate was injected into the above solution, and finally reacted at this temperature for 15 min. During the process, the mixture changed from colorless to wine red to form spherical Au NPs. When the aqueous solution cooled to room temperature, gold colloid was formed. It was stored in the refrigerator for freezing;

[0085] (2) The indium tin oxide glass (ITO) electrode was first ultrasonically cleaned with anhydrous ethanol and ultrapure water for 15 min respectively, then boiled in 0.3 mol / L NaOH solution for 20 min, and then ultrasonically cleaned with ethanol and ultrapure water again. After drying at room temperature, the electrode surface modification area was fixed to obtain the treated indium tin oxide glass electrode;

[0086] ​(3) The Ni-MOF / MB solution prepared in step (1) was added dropwise to the surface of the indium tin oxide glass electrode treated in step (2), and dried at room temperature to obtain a product marked as Ni-MOF / MB / ITO;

[0087] (4) The Au NPs solution 20 μL was modified on the surface of the Ni-MOF / MB / ITO sensor prepared in step (3), and dried; at this time, a film structure was formed on the surface of the sensor, marked as Au NPs / Ni-MOF / MB / ITO;

[0088] (5) According to the known sequence of the MC-LR aptamer and the base complementary pairing principle, the MC-LR aptamer complementary DNA and amplification double-strand were designed, marked as cDNA, S1 and S2, and the aptamer, cDNA, S1, S2 and streptavidin-alkaline phosphatase were added into a DNA amplifier for base complementary pairing and DNA double-strand amplification, marked as DNA complex, and the DNA complex 20 μL was modified on the surface of the sensor obtained in step (4), and the gold-sulfur bond between the aptamer and Au NPs was used to fix the electrode surface, and the sensor was rinsed with tris buffer, and the rinsed sensor was marked as SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO;

[0089] (6) The sensor surface prepared in step (5) was modified with 20 μL of MC-LR standard solution with concentrations of 10 fM, 100 fM, 1 pM, 10 pM and 100 pM, one concentration of MC-LR solution corresponding to one sensor, and the concentration and the sensor were in one-to-one correspondence; after incubation at 37 °C for 50 min, the tris buffer was used for rinsing to remove the MC-LR not combined with the Apt, thereby obtaining a spatially confined photoelectrochemical aptamer sensor based on Ni-MOF loaded methylene blue composite material, marked as MC-LR / SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO.

[0090] In addition, 20 μL of MC-LR solution with different concentrations (10 fM, 100 fM, 1 pM, 10 pM, 100 pM) was modified on the surface of the prepared sensor (one concentration of MC-LR solution corresponds to one sensor, and the concentration and the sensor are in one-to-one correspondence); after incubation at 37 °C for 50 min, the electrode was washed with tris buffer solution; the prepared sensor (MC-LR / SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO) was used as the working electrode, Ag / AgCl (saturated KCl) electrode was used as the reference electrode, and platinum wire electrode was used as the counter electrode, and the photoelectrochemical signal was detected and recorded by the electrochemical workstation with the model CHI660E. All tests were carried out in the electrolyte, and the photoelectric current signal value was used as the vertical coordinate, and the lg value of the corresponding MC-LR concentration was used as the horizontal coordinate, and the corresponding standard curve was established for the detection of MC-LR concentration.

[0091] The performance of the photoelectrochemical aptamer sensor based on the Ni-MOF loaded methylene blue composite material was analyzed.

[0092] Figure 4 (A) is the selectivity of the photoelectrochemical aptamer sensor, wherein blank refers to the photoelectrochemical aptamer sensor without MC-LR, i.e. the sensor prepared in step (5) in Example 1, defined as blank;

[0093] MC-LR refers to the photoelectrochemical aptamer sensor modified with 20 μL of 100 pM MC-LR, i.e. the sensor obtained according to the operation of step (6) in Example 1;

[0094] In addition, MC-RR, MC-YR, L-Cysteine, L-Tyrosine as interference, respectively, according to the operation of step (6), the difference is that 20 μL of 1 nM interference is modified on the surface of the obtained photoelectrochemical aptamer sensor, wherein MC-RR is microcystin-RR, MC-YR is microcystin-YR, L-Cysteine is L-cysteine, L-Tyrosine is L-tyrosine, and Mix is a mixed solution of MC-RR, MC-YR, L-Cysteine, and L-Tyrosine);

[0095] From Figure 4In (A), it can be seen that the photocurrent caused by the interferents (MC-RR, MC-YR, L-Cysteine, L-Tyrosine and the mixed solution of the four) is almost consistent with that of the blank sample, while when MC-LR is present, including MC-LR and other interferents mixed, similar results are shown, indicating that the sensor has good selectivity and can specifically detect MC-LR.

[0096] From Figure 4 In (B), it can be seen that the photoelectrochemical aptamer sensor for MC-LR is detected for 10 consecutive days, and the RSD value is 2.2%, indicating that the sensor has good stability.

[0097] At the same time, the actual sample is analyzed using the spatially confined photoelectrochemical aptamer sensor based on the Ni-MOF loaded methylene blue composite material, and the steps are as follows:

[0098] (1) The sensor surface prepared in step (5) of Example 1 is modified with 20 μL of MC-LR standard solution of different concentrations (10 fM, 100 fM, 1 pM, 10 pM, 100 pM); one concentration of MC-LR solution corresponds to one sensor, and the concentration and the sensor are in one-to-one correspondence; after modification, incubate at 37°C for 50 min, and then wash the electrode with tris buffer; obtain the sensor MC-LR / SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO;

[0099] (2) Construct a standard curve

[0100] The above prepared sensor (MC-LR / SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO) is used as the working electrode, Ag / AgCl (saturated KCl) electrode as the reference electrode, and platinum wire electrode as the counter electrode, and photoelectrochemical test is carried out; the photoelectrochemical signal is detected and recorded by the electrochemical workstation with model CHI 660E. All tests are carried out in 0.1 M tris buffer solution containing 10 mM AAP, and the photocurrent signal value is used as the vertical coordinate, and the corresponding log value of the MC-LR concentration is used as the horizontal coordinate, to establish the corresponding standard curve (I=-45.1 lgC MC-LR +447.2), which is used for the detection of MC-LR concentration in actual samples.

[0101] (3) Detection of MC-LR in samples, and the samples are selected as culture water and cultured fish;

[0102] (a) First, the sample liquid was obtained, and the aquaculture water sample and the aquaculture fish sample for actual detection were collected in a breeding pond (Zhenjiang, China). The sample was treated using the method reported in the literature [1] Eissa S, Zourob M. A graphene-based electrochemical competitive immunosensor for the sensitive detection of okadaic acid in shellfish [J]. Nanoscale, 2012, 4(23): 7593-7599. [2] Moreno I M, Molina R, Jos A, et al. Determination of microcystins in fish by solvent extraction and liquid chromatography [J]. J Chromatogr A, 2005, 1080(2): 199-203.

[0103] (b) The 20 μL sample liquid was modified on the surface of the sensor, and the corresponding current value was obtained by photoelectrochemical test according to the operation method of steps (1) and (2). Finally, the current value was substituted into the standard curve constructed in step (2), so that the concentration of MC-LR in the sample could be obtained (as shown in Table 2). MC-LR was detected in the blank aquaculture water sample, and the recovery rate of the photoelectrochemical aptamer sensor (PEC sensor) constructed by the present application after spiking was between 99.3 and 103.5%, which was basically consistent with the determination result of the national standard method. It is shown that the method of the present application has reliability and accuracy, and can be used for the purpose of detecting MC-LR in unknown samples.

[0104] Table 1: The content of MC-LR in aquaculture water and aquaculture fish was determined using the constructed photoelectrochemical aptamer sensing method (n = 3) and the national standard method

[0105]

[0106] It should be understood by those skilled in the art that the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application.

Claims

1. A preparation method of a photoelectrochemical sensor based on a methylene blue composite material loaded on Ni-MOF, characterized in that, The method comprises the following steps: (1) Preparation of Ni-MOF / MB composite material: S1, first, dissolve nickel acetate tetrahydrate in ultrapure water, and ultrasonically dissolve it to obtain a light green solution, which is denoted as solution A; dissolve 1.4-terephthalic acid in a mixed solution of N,N-dimethylacetamide and dichloroethane, and ultrasonically dissolve it to obtain a transparent solution, which is denoted as solution B; add solution A dropwise into solution B, collect the precipitate by centrifugation after a period of reaction, and then dry the obtained product to obtain Ni-MOF; S2, weigh Ni-MOF and dissolve it in ultrapure water to obtain a Ni-MOF solution, and then add MB to perform stirring reaction, and after reaction, centrifugation, washing and drying, the obtained product is denoted as Ni-MOF / MB; S3, add HAuCl4·3H2O into ultrapure water, heat to boil, then add trisodium citrate solution, maintain the heating temperature, continue to react for a certain period of time, and after reaction, cool the solution to room temperature to form an Au NPs solution; (2) First, ultrasonically clean the indium tin oxide glass electrode (ITO electrode) with anhydrous ethanol and ultrapure water respectively, then boil the ITO electrode with NaOH solution, ultrasonically clean it with ethanol and ultrapure water again, dry it at room temperature, and obtain the treated ITO electrode; (3) Add the Ni-MOF / MB prepared in step (1) into water to obtain a Ni-MOF / MB solution; then add the Ni-MOF / MB solution dropwise onto the surface of the ITO electrode treated in step (2), and dry it at room temperature to obtain a product, which is denoted as a Ni-MOF / MB / ITO sensor; (4) Modify the Au NPs solution on the surface of the Ni-MOF / MB / ITO sensor prepared in step (3), and dry it; at this time, a film structure is formed on the surface of the sensor, which is denoted as an Au NPs / Ni-MOF / MB / ITO sensor; (5) According to the known sequence of MC-LR aptamer and the base complementary pairing principle, design MC-LR aptamer complementary DNA and double-stranded amplification, which are denoted as cDNA, S1 and S2 respectively; add Apt, cDNA, S1, S2 and streptavidin-alkaline phosphatase into a DNA amplifier to perform base complementary pairing and DNA double-stranded amplification, and the obtained product is denoted as a DNA complex; modify the DNA complex on the surface of the sensor obtained in step (4), and rinse the sensor with tris buffer solution; the rinsed sensor is a photoelectrochemical sensor based on Ni-MOF loaded methylene blue composite material, which is denoted as SA−ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO.

2. The preparation method of the photoelectrochemical sensor based on the Ni-MOF loaded methylene blue composite material according to claim 1, characterized in that, In S1 of step (1), the amount of nickel acetate tetrahydrate and ultrapure water was 223.2 mg:6 mL; the amount of 1,4-terephthalic acid, N,N-dimethylacetamide and dichloroethane was 24.9 mg:6 mL:6 mL; the reaction time was 30 min; solution A was added dropwise into solution B, and the volume ratio of ultrapure water in solution A to dichloroethane in solution B was 1:

1.

3. The preparation method of the photoelectrochemical sensor based on Ni-MOF loaded methylene blue composite material according to claim 1, characterized in that, In S2 of step (1), the amount of Ni-MOF, ultrapure water and MB was 30 mg:5 mL:1.05 mg; the reaction temperature was 25℃, the stirring speed was 300 rpm, and the reaction time was 3 h.

4. The preparation method of the photoelectrochemical sensor based on the Ni-MOF loaded methylene blue composite material according to claim 1, characterized in that, In S3 of step (1), the amount of HAuCl4·3H2O, ultrapure water and trisodium citrate solution was 0.1 mol:25 mL:0.25 mL, and the concentration of trisodium citrate solution was 100 mg / ml; the heating temperature was maintained at 95-100℃, and the heating time was 10-15 min.

5. The method for preparing a photoelectrochemical sensor based on Ni-MOF supported methylene blue composite material according to claim 1, characterized in that, In step (2), the diameter of the indium tin oxide glass electrode was d = 6 mm; the concentration of the NaOH solution used was 0.3 mol / L, and the boiling time was 20 min; the ultrasonic time was 15 min.

6. The method for preparing a photoelectrochemical sensor based on Ni-MOF supported methylene blue composite material according to claim 1, characterized in that, In step (3), the amount of Ni-MOF / MB solution added dropwise was 20 μL, and the concentration of Ni-MOF / MB solution was 1 μg / mL.

7. The method according to claim 1, wherein the photoelectrochemical sensor based on Ni-MOF supported methylene blue composite material is prepared by the following steps: 1) synthesizing Ni-MOF; 2) synthesizing methylene blue modified Ni-MOF; 3) synthesizing the photoelectrochemical sensor based on Ni-MOF supported methylene blue composite material. In step (4), the concentration of Au NPs solution was 5.2 nmol / L, the modification method was dropwise addition, and the amount of Au NPs solution added dropwise was 20 μL.

8. The preparation method of the photoelectrochemical sensor based on Ni-MOF loaded methylene blue composite material according to claim 1, characterized in that, In step (5), the concentration of Apt was 10 μM, the concentration of cDNA was 15 μM, the concentration of S1 was 37.5 μM, the concentration of S2 was 37.5 μM, and the concentration of streptavidin-alkaline phosphatase was 1 mg / mL; the volume ratio of Apt, cDNA, S1, S2 and streptavidin-alkaline phosphatase was 1:1:1:1:1.5, the amount of DNA complex modified on the sensor surface obtained in step (4) was 20 μL; the concentration of tris buffer was 0.1 M, pH = 8.0; the composition of tris buffer was: 10 mM tris, 5 mM MgCl2, 0.2 mM NaCl. The sequences of aptamer, cDNA, S1 and S2 of MC-LR are as follows: Apt: 5'-SH-GGC CGG AAA CAG GAC CAC CAT GAC AAT TAC CCA TAC CAC CTC ATTATG CCC CAT CTC CGC-3' cDNA: 5'-CCC GCG GCA GCG GAG ATG GGG CAT AAT-3' S1: 5'-TGC CGC GGG AGG CGG ATT C biotin-3' S2: 5'-CCC GCG GCA CGA ATC CGC C biotin-3'.

9. Use of the photoelectrochemical aptamer sensor prepared according to the method of any one of claims 1 to 8 for detecting MC-LR, characterized in that, The steps are as follows: (1) First, prepare MC-LR standard solutions with different concentrations, and then modify the prepared sensor surface with MC-LR standard solutions with different concentrations and V1 volume. One concentration of MC-LR solution corresponds to one sensor, and the concentration and the sensor are in one-to-one correspondence. After incubation for a period of time, tris buffer is used for elution, thereby obtaining a space-limited aptamer sensor based on Ni-MOF loaded methylene blue composite material, marked as MC-LR / SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO; (2) Take the MC-LR / SA-ALP / S1-S2 / cDNA / Apt / Au NPs / Ni-MOF / MB / ITO obtained in step (1) as the working electrode, Ag / AgCl electrode as the reference electrode, and platinum wire electrode as the counter electrode. Then, the working electrode is placed in the electrolyte for hydrolysis for a period of time, and electrochemical detection is performed. The corresponding current value is obtained through photoelectrochemical testing. Take the current value as the vertical coordinate, and the logarithmic value of the corresponding MC-LR concentration as the horizontal coordinate, to establish the corresponding standard curve based on aptamer; (3) Detection of MC-LR in the sample: First, obtain the sample liquid, modify V1 volume of the sample liquid on the surface of the sensor, and then perform electrochemical detection according to the operation methods of steps (1) and (2). The corresponding current value is obtained through photoelectrochemical testing. The current value is substituted into the standard curve constructed in step (2), and the concentration of MC-LR in the sample can be obtained, realizing the purpose of detecting MC-LR in unknown samples.

10. Use according to claim 9, characterized in that, In step (1), the concentration of the MC-LR standard solution is 10 fM to 100 pM; the incubation temperature is 37°C, and the period of time is 20 to 60 min; the concentration of the tris buffer is 0.1 M, and the pH is 8.0; the composition of the tris buffer is: 10 mM tris, 5 mM MgCl2, and 0.2 mM NaCl; In steps (1) and (3), V1 volume is 20 μL; In steps (2) and (3), the electrolyte is tris buffer containing L-ascorbic acid-2-phosphate trisodium salt, wherein the final concentration of L-ascorbic acid-2-phosphate trisodium salt is 10 mM, the concentration of the tris buffer is 0.1 M, and the pH is 8.

0. The composition of the tris buffer is: 10 mM tris, 5 mM MgCl2, and 0.2 mM NaCl. The additional bias voltage is 0 V; and the period of time for hydrolysis is 40 min.

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