Electrochemiluminescent sensor and method of making and use thereof

Abstract

The application belongs to the technical field of electrochemiluminescence analysis and detection, and relates to an electrochemiluminescence sensor and a preparation method and application thereof. The electrochemiluminescence sensor comprises the following steps: dropping PSA-HOF-dWO3·H2O dispersion liquid on a GCE electrode, adding microcystin-RR aptamer after drying, and finally obtaining an aptamer modified PSA-HOF-dWO3·H2O electrode as a working electrode, Ag / AgCl as a reference electrode, and Pt as a counter electrode, and then introducing a PBS phosphate buffer solution containing potassium persulfate, so that the PSA-HOF-dWO3·H2O system electrochemiluminescence sensor is used for microcystin-RR detection. The prepared PSA-HOF-dWO3·H2O system electrochemiluminescence sensor has the characteristics of more simple and flexible operation, wide detection range, low detection limit and low detection cost.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of electrochemiluminescence analysis and detection, and relates to an electrochemiluminescence sensor and a preparation method and application thereof, in particular to an electrochemiluminescence sensor based on dWO3·H2O nanomaterial enhanced pyrene self-assembly hydrogen bond organic framework (PSA-HOF) and a preparation method and application thereof in detection of microcystin-RR (MC-RR). BACKGROUND

[0002] The increase in global cyanobacterial biomass levels and the release of secondary metabolites in aquatic ecosystems is a problem worldwide. Cyanobacteria mainly include Synechococcus, Anabaena, Microcystis and Planktothrix, and these pollutants will reduce water quality and pose a serious threat to human health and ecosystems, which urgently requires scientists, governments and multilateral agencies to work together to ensure timely detection and removal of these pollutants.

[0003] Microcystin is a biologically active cyclic heptapeptide toxin, which is the most widely distributed hepatotoxin, and is produced as a secondary metabolite of Microcystis cyanobacteria during algal blooms in freshwater and eutrophic waters worldwide. It is mainly produced by the freshwater alga Microcystis aeruginosa, has relative stability, can strongly inhibit the activity of protein phosphatase, and is a strong liver tumor promoter. There is a toxic variant in its structure, in which both X and Z positions are arginine amino acids, which is called microcystin-RR (MC-RR), as shown in formula (I). Figure 7

[0004] Currently, the commonly used methods for detecting and quantifying microcystin-RR in related technologies include protein phosphatase inhibition tests and enzyme-linked immunosorbent assays in biochemistry, high-performance liquid chromatography and liquid chromatography-mass spectrometry in physical chemistry, and biosensor-based strategies. These methods have the disadvantages of high technical requirements, time-consuming, expensive instruments and equipment, or low detection sensitivity. Therefore, it is of great significance to find a high-efficiency and convenient analysis method for detecting the content of microcystin-RR in the environment. SUMMARY

[0005] To solve the problems in the related art, the application provides an electrochemiluminescence sensor based on dWO3·H2O nanomaterial enhanced pyrene self-assembly hydrogen bond organic framework (PSA-HOF) and a preparation method and application thereof, which is used for detecting microcystin-RR. The electrochemiluminescence sensor has high detection sensitivity, wide detection range, low detection limit, and is low in detection cost and can be applied to detection of microcystin-RR.

[0006] ​In a first aspect, the application provides an electrochemiluminescence sensor, comprising a glassy carbon electrode apt / PSA-HOF-dWO3·H2O / GCE co-modified by a PSA-HOF-dWO3·H2O complex and an aptamer with specific recognition.

[0007] In some optional embodiments, the aptamer with specific recognition is an MC-RR aptamer, and the nucleotide sequence of the MC-RR aptamer is: 5'-CAG CTC AGAAGC TTG ATC CTA CTG CCC TTC AAT GTT CACTCC TGT TTC CTG ATC TTT GTC GAC TCG AAG TCG TGC ATC TG-3', which can specifically recognize microcystin-RR.

[0008] In a second aspect, the application further provides a preparation method of the electrochemiluminescence sensor, comprising:

[0009] A certain amount of PSA-HOF and dWO3·H2O are dispersed in chitosan respectively to obtain PSA-HOF dispersion liquid and dWO3·H2O dispersion liquid, and then the two kinds of dispersion liquids are mixed, ultrasonically homogenized to obtain a PSA-HOF-dWO3·H2O complex;

[0010] The PSA-HOF-dWO3·H2O complex is drop-coated on the surface of a clean glassy carbon electrode for modification to obtain a PSA-HOF-dWO3·H2O / GCE modified electrode;

[0011] The aptamer is drop-coated and incubated on the PSA-HOF-dWO3·H2O / GCE modified electrode to obtain the electrochemiluminescence sensor apt / PSA-HOF-dWO3·H2O / GCE.

[0012] In some optional embodiments, the mass ratio of the PSA-HOF to the dWO3·H2O is 1:1, the mass-volume ratio of the PSA-HOF or the dWO3·H2O to the chitosan is 1:1 (g / L), and the mass fraction of the chitosan is 0.5%.

[0013] In some optional embodiments, the drop-coating amount of the PSA-HOF-dWO3·H2O complex on the glassy carbon electrode is 3.0 μL, and the concentration is 0.5-2.0 mg / mL.

[0014] In some alternative embodiments, the concentration of the aptamer is 0.5-3.0 μmol / L, and the drop-casting amount is 6.0 μL.

[0015] In a third aspect, the application further provides the use of the electrochemiluminescence sensor or the electrochemiluminescence sensor obtained by the preparation method of any one of the electrochemiluminescence methods in the electrochemiluminescence detection of microcystin-RR. After the aptamer specifically recognizing microcystin-RR specifically binds with microcystin-RR to form a complex, the complex leaves the surface of the sensing interface, the ECL performance is improved, and the light intensity has a linear relationship with the concentration of microcystin-RR, thereby being used for quantitative detection and analysis of the content of microcystin-RR.

[0016] In some alternative embodiments, the use comprises the following steps:

[0017] A series of microcystin-RR standard solutions with different concentrations are prepared;

[0018] The apt / PSA-HOF-dWO3·H2O / GCE is used as a working electrode, a platinum electrode is used as a counter electrode, and an Ag / AgCl electrode is used as a reference electrode to form a three-electrode system; the three-electrode system is placed in a PBS phosphate buffer solution containing potassium persulfate; in an electrochemical window range of -2.0-0 V, a photomultiplier high voltage of 600-800 V, a scanning speed of 0.1-0.15 V / s, and a recording time-light intensity curve is recorded;

[0019] The series of microcystin-RR standard solutions with different concentrations are respectively added dropwise to the electrochemiluminescence sensor apt / PSA-HOF-dWO3·H2O / GCE, and cyclic voltammetry scanning is continued in an electrochemical window range of -2.0-0 V, a photomultiplier high voltage of 600-800 V, a scanning speed of 0.1-0.15 V / s, and a recording time-light intensity curve before and after the combination of microcystin-RR and the aptamer is recorded;

[0020] The concentration of microcystin-RR in the sample solution is calculated according to the linear regression equation obtained according to the linear relationship between the light intensity difference and the logarithmic value of the concentration of microcystin-RR.

[0021] In some alternative embodiments, the PBS phosphate buffer solution containing potassium persulfate has a pH value of 5-9, and the concentration of PBS is 0.1 mol / L.

[0022] In some alternative embodiments, the combination time of microcystin-RR and the aptamer in the electrochemiluminescence sensor apt / PSA-HOF-dWO3·H2O / GCE is 10-50 min.

[0023] The application has specific recognition effect by taking dWO3·H2O composite material as a co-reaction accelerator, aptamer (apt) as a recognition element, and PSA-HOF-dWO3·H2O and apt being jointly modified on a glassy carbon electrode (GCE) through chitosan (CS) (amino groups on CS form phosphoramide bonds with phosphate groups of aptamer). Pyrene self-assembled hydrogen-bonded organic framework (PSA-HOF) is taken as a luminescent body and introduced into a PBS phosphate buffer solution containing potassium persulfate, and then the apt / PSA-HOF-dWO3·H2O / GCE modified electrode is taken as a working electrode for electrochemiluminescence detection. The introduction of dWO3·H2O can greatly promote the reaction of persulfate in the PBS phosphate buffer solution, so as to obtain a strong and stable ECL signal.

[0024] The electrochemiluminescence sensor provided by the application is based on dWO3·H2O nanomaterial enhanced PSA-HOF electrochemiluminescence sensor. Compared with common electrochemiluminescence sensors, the dWO3·H2O nanomaterial can promote the reaction of persulfate, so as to improve the ECL signal.

[0025] The application is used for detecting microcystin-RR and realizing quantitative detection of microcystin-RR. The high-performance dWO3·H2O nanomaterial enhanced PSA-HOF electrochemiluminescence sensor has high sensitivity, wide linear range, simple operation and other advantages of electrochemiluminescence analysis, and has important practical significance for specific detection of microcystin-RR in river water by using the high-performance PSA-HOF-dWO3·H2O system ECL system. BRIEF DESCRIPTION OF DRAWINGS

[0026] The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and serve to explain the principles of the application, but do not limit the application. In the drawings:

[0027] Figure 1 is a brief flowchart of preparation of the electrochemiluminescence sensor of the exemplary embodiment of the application and detection of microcystin-RR.

[0028] Figure 2 is a linear curve of the luminescence intensity after adding different concentrations of microcystin-RR to the three-electrode system of the exemplary embodiment of the application, a-i: 0 mol / L (a), 1.0×10 -16 mol / L (b), 1.0×10 -15 mol / L (c), 1.0×10 -14 mol / L (d), 1.0×10 -13 mol / L (e), 1.0×10 -12mol / L(f), 1.0×10 -11 mol / L(g), 1.0×10 - 10 mol / L(h), 1.0×10 -9 mol / L(i).

[0029] Figure 3 This is a standard curve of the luminescence intensity and the logarithm of the microcystin-RR concentration after adding microcystin-RR to a three-electrode system according to an exemplary embodiment of this application.

[0030] Figure 4 This is an exemplary embodiment of the present application showing the effect of microcystin-RR aptamer concentration on luminescence intensity.

[0031] Figure 5 This is an exemplary embodiment of the present application showing the effect of the binding time of microcystin-RR to the aptamer on the luminescence intensity.

[0032] Figure 6 This relates to the effect of the pH of the PBS solution on the ECL luminescence intensity in the exemplary embodiment of the application.

[0033] Figure 7 This is a schematic diagram of the structure of microcystin in the prior art. Detailed Implementation

[0034] This application is not limited to the specific embodiments listed below. Those skilled in the art can implement this application using various other specific embodiments based on the disclosure of this application. Any simple changes or modifications made to the design structure and concept of this application fall within the protection scope of this application. It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other.

[0035] The dWO3·H2O nanomaterials and pyrene self-assembled hydrogen-bonded organic frameworks (PSA-HOF) in the specific embodiments of this application can be obtained using methods already available in the prior art. For example, they can be prepared using the following method, see [link to relevant documentation]. Figure 1 :

[0036] (1) Preparation of dWO3·H2O nanomaterials

[0037] First, 0.5 mM NaWO4·H2O, 0.75 mM citric acid monohydrate (CA) and 0.5 mM glucose were dissolved in 15 mL ultrapure water. Then the mixture solution was stirred at room temperature for half an hour until it became transparent. After that, 1.5 mL 6M HCl was added to the mixture solution, and then stirred for 30 minutes. Finally, the mixture solution was transferred into a hydrothermal reactor at 120°C for 24 hours. After natural cooling, the precipitate was centrifuged at 8000 rpm for 10 minutes and washed with ultrapure water and ethanol at least three times, respectively. Subsequently, the precipitate was collected and vacuum dried at 60°C for 12h.

[0038] (2) Preparation of PSA-HOF

[0039] First, 90 mM 1,3,6,8-tetra(p-carboxybenzoic acid)pyrene (H4TBAPy) was dissolved in 10 mL N,N-dimethylformamide (DMF) and ultrasonically dissolved. Then, CH3OH (36 mL) was added to the above solution and stirred for 5 minutes. Finally, the mixed solution was left at room temperature for 12 hours, and the product was washed by centrifugation and with CH3OH and acetone three times, respectively, to remove impurities. The product was placed in a vacuum drying oven and dried at 60°C for 12 hours, finally obtaining a PSA-HOF yellow powder.

[0040] The preparation method of the electrochemiluminescence sensor of the specific embodiment of the present application comprises:

[0041] A certain amount of PSA-HOF and dWO3·H2O were dissolved in 0.5% chitosan by mass fraction according to a mass ratio of 1:1, wherein the mass-volume ratio of PSA-HOF or dWO3·H2O to chitosan was 1:1 (g / L); PSA-HOF dispersion and dWO3·H2O dispersion were obtained, respectively, and then the two dispersions were mixed to obtain a PSA-HOF-dWO3·H2O composite after ultrasonic homogenization.

[0042] The PSA-HOF-dWO3·H2O composite was drop-coated on the surface of a clean glassy carbon electrode to modify it, and a PSA-HOF-dWO3·H2O / GCE modified electrode was obtained.

[0043] The aptamer was drop-coated and incubated on the PSA-HOF-dWO3·H2O / GCE modified electrode to obtain the electrochemiluminescence sensor apt / PSA-HOF-dWO3·H2O / GCE.

[0044] The specific detection method of the specific embodiment of the present application is as follows:

[0045] Step 1, prepare a series of microcystin-RR standard solutions with different concentrations, the concentration range is 1.0 × 10- 16 1.0 x 10 -9 mol / L;

[0046] Step 2, the modified electrode apt / PSA-HOF-dWO3·H2O / GCE was used as the working electrode, a platinum electrode was used as the auxiliary electrode, and Ag / AgCl was used as the reference electrode to form a three-electrode system. The three-electrode system was placed in a PBS phosphate buffer solution containing potassium persulfate. A series of different concentrations of microcystin-RR standard solution prepared in step 1 was added dropwise to the GCE. In the electrochemical window range of -2.0-0 V, the photomultiplier high voltage was 600 V, the scanning speed was 0.1 V, and cyclic voltammetry scanning was performed. The time-luminous intensity curve was recorded, and the linear relationship between the luminous intensity difference before and after the addition of microcystin-RR and the logarithmic value of the concentration of microcystin-RR was established, and the corresponding linear regression equation was obtained.

[0047] Step 3, sample detection, the test was carried out according to the above step 2 and the luminous intensity was obtained. The obtained luminous intensity was calculated by the linear regression equation obtained in step 2, and the concentration of microcystin-RR in the sample was obtained.

[0048] The technical solutions of the present application will be further described below in conjunction with examples, but the present application is not limited to the following examples.

[0049] Example 1

[0050] Preparation of an electrochemiluminescence sensor for high-sensitivity detection of microcystin-RR:

[0051] (1) Preparation of dWO3·H2O nanomaterials

[0052] The dWO3·H2O nanocomposite was prepared by one-pot hydrothermal method. First, 0.5 mM NaWO4·H2O, 0.75 mM citric acid monohydrate (CA) and 0.5 mM glucose were dissolved in 15 mL ultrapure water. Then the mixture solution was stirred at room temperature for half an hour until it became transparent. After that, 1.5 mL of 6M HCl was added to the mixture solution, and then stirred for 30 minutes. Finally, the mixture solution was transferred to a hydrothermal reactor at 120℃ for 24 hours. After natural cooling, the precipitate was centrifuged at 8000 rpm for 10 minutes and washed with ultrapure water and ethanol for at least 3 times respectively. Subsequently, the precipitate was collected and vacuum dried at 60℃ for 12h.

[0053] (2) Preparation of PSA-HOF

[0054] First, 90 mM 1, 3, 6, 8-tetra (p-toluic acid) pyrene (H4TBAPy) was dissolved in 10 mL N, N-dimethylformamide (DMF) and ultrasonically dissolved. Then, CH3OH (36 mL) was added to the above solution and stirred for 5 minutes. Finally, the mixed solution was left to stand at room temperature for 12 hours, centrifuged, and the product was washed with CH3OH and acetone three times, respectively, to remove impurities. The product was placed in a vacuum drying oven and dried at 60°C for 12 hours, finally obtaining a yellow powder.

[0055] (3) Preparation of PSA-HOF-dWO3·H2O composite

[0056] Take 1 mg of prepared PSA-HOF powder and 1 mg of prepared dWO3·H2O, respectively, and dissolve in 1 mL of chitosan, mix uniformly, and ultrasonically disperse to obtain two dispersions with a concentration of 1 mg / mL. Then mix the two dispersions and ultrasonically disperse for 40 min to obtain the PSA-HOF-dWO3·H2O composite.

[0057] (4) Preparation of MC-RR detection PSA-HOF-dWO3·H2O electrochemical sensor

[0058] The glassy carbon electrode was cleaned by the following method, but not limited to: the glassy carbon electrode was polished with Al2O3 on a suede, ultrasonically cleaned with anhydrous ethanol and ultrapure water for 15 min, respectively, to remove surface contaminants, and naturally air-dried for use.

[0059] 3 μL of 1 mg / mL PSA-HOF-dWO3·H2O was modified on the surface of a clean GCE to obtain PSA-HOF-dWO3·H2O / GCE. Then, the modified electrode was incubated with 6 μL of 2 μmol / mL MC-RR aptamer for 12 hours.

[0060] The sequence of the aptamer in the above sensor is as follows: (ordered from Shenguo Bioengineering (Shanghai) Co., Ltd.)

[0061] Aptamer: 5'-CAG CTC AGA AGC TTG ATC CTA CTG CCC TTC AAT GTT CAC TCC TG TTT C CTG ATC TTT GTC GAC TCG AAG TCG TGC ATC TG-3'.

[0062] (5) Drawing of standard curve

[0063] A series of microcystin-RR standard solutions with different concentrations were prepared, with the concentration of microcystin-RR being: 1.0 × 10 -16 mol / L, 1.0 × 10 -151.0 x 10 -14 1.0 x 10 -13 1.0 x 10 -12 1.0 x 10 -11 1.0 x 10 -10 1.0 x 10 -9 1.0 x 10

[0064] The modified electrode apt / PSA-HOF-dWO3-H2O / GCE is used as the working electrode, a platinum electrode is used as the auxiliary electrode, and Ag / AgCl is used as the reference electrode to form a three-electrode system. The three-electrode system is placed in a PBS phosphate buffer solution containing potassium persulfate. In the electrochemical window range of -2.0-0 V, the high voltage of the photomultiplier tube is 600 V, the scanning speed is 0.1 V / s, and the time-luminous intensity curve (T-ECL) is recorded. See Figure 2 ;

[0065] A series of microcystin-RR standard solutions of different concentrations are further added dropwise to the modified electrode apt / PSA-HOF-dWO3-H2O / GCE, so as to obtain MC-RR / apt / PSA-HOF-dWO3-H2O / GCE corresponding to a series of microcystin-RR standard solutions of different concentrations.

[0066] The MC-RR / apt / PSA-HOF-dWO3-H2O / GCE continues to be used as the working electrode, a platinum electrode is used as the auxiliary electrode, and Ag / AgCl is used as the reference electrode to form a three-electrode system. The three-electrode system is placed in a PBS phosphate buffer solution containing potassium persulfate. In the electrochemical window range of -2.0-0 V, the high voltage of the photomultiplier tube is 600 V, the scanning speed is 0.1 V / s, and the time-luminous intensity curve (T-ECL) is recorded.

[0067] A linear relationship between the luminous intensity difference before and after the addition of microcystin-RR and the logarithmic value of the concentration of microcystin-RR is established. See Figure 3 , and the corresponding linear regression equation is: △I ECL = -14702.12808 + 822.87892 LogC (mol / L), and the correlation coefficient (R) is 0.9943. The detection range of the linear regression equation is 1.0 x 10 -16 -1.0 x 10 -9 mol / L, and the lowest detection limit is 1.31 x 10 -17 mol / L.

[0068] (6) Detection of samples

[0069] The three-electrode system was immersed in the filtered river water for 20 min, and electrochemiluminescence detection was performed according to the standard solution detection method described above. The sample was detected in parallel for 3 times, and the concentration of microcystin-RR in the sample was calculated according to the regression equation corresponding to the standard curve, and the results are shown in Table 1.

[0070] Example 2

[0071] Effect of different aptamer concentrations on the ECL performance of the PSA-HOF enhanced by dWO3·H2O nanomaterials:

[0072] (1) Preparation of dWO3·H2O nanomaterials: same as Example 1

[0073] (2) Preparation of PSA-HOF materials: same as Example 1

[0074] (3) Preparation of PSA-HOF-dWO3·H2O composite materials: same as Example 1

[0075] (4) Preparation of MC-RR detection PSA-HOF-dWO3·H2O electrochemical sensor:

[0076] The glassy carbon electrode was polished and ultrasonically cleaned with nitric acid and anhydrous ethanol, deionized water, respectively, and naturally air-dried for use. At room temperature, 6.0 μL of 0.01M PBS buffer solution (pH 7.4) containing 0.005M NHS and 0.01M EDC was dropped on the clean bare glassy carbon electrode for 1 h to activate the electrode. Then, 3.0 μL of PSA-HOF-dWO3·H2O CS solution was added to the GCE to obtain a PSA-HOF-dWO3·H2O / GCE modified electrode. Then, 6 μL of MC-RR aptamer Tris-HCl solution with concentrations of 0.5 μmol / L, 1 μmol / L, 1.5 μmol / L, 2 μmol / L, 2.5 μmol / L, and 3 μmol / L, respectively, was added to the electrode to obtain apt / PSA-HOF-dWO3·H2O / GCE modified electrode, which was marked as apt0 / PSA-HOF-dWO3·H2O / GCE, apt1 / PSA-HOF-dWO3·H2O / GCE, apt2 / PSA-HOF-dWO3·H2O / GCE, apt3 / PSA-HOF-dWO3·H2O / GCE, respectively. 0.5 1.5 2.5

[0077] ​​​The aptamer sequence is the same as that in Example 1.

[0078] (5) Electrochemiluminescence detection: in the electrochemical window range of -2.0-0V, the high voltage of the photomultiplier tube is 600V, the scanning speed is 0.1V / s, and the luminescence intensity is recorded.

[0079] As shown in Figure 4 , as the concentration of the aptamer increases, the detection substance pulls the aptamer away from the electrode surface, the luminescence recovers, and the ECL response gradually increases. When the concentration of the aptamer increases from 2μmol / L to 3μmol / L, the change in ECL intensity gradually stabilizes, and therefore the concentration of the aptamer is preferably 2μmol / L.

[0080] Example 3

[0081] Effect of the difference in the binding time of microcystin-RR and the aptamer on the ECL luminescence performance of the PSA-HOF enhanced by the dWO3·H2O nanomaterial electrochemiluminescence sensor:

[0082] (1) Preparation of the dWO3·H2O nanomaterial: the same as in Example 1

[0083] (2) Preparation of the PSA-HOF material: the same as in Example 1

[0084] (3) Preparation of the PSA-HOF-dWO3·H2O composite material: the same as in Example 1

[0085] (4) Preparation of the MC-RR detection PSA-HOF-dWO3·H2O electrochemical sensor:

[0086] The glassy carbon electrode is polished and ultrasonically cleaned with nitric acid and anhydrous ethanol and deionized water, respectively, and naturally air-dried for use. At room temperature, 6.0μL of 0.01M PBS buffer solution (pH 7.4) containing 0.005M NHS and 0.01M EDC is coated and dropped on the clean bare glassy carbon electrode for 1h to activate the electrode. Then, 3.0μL of the PSA-HOF-dWO3·H2O CS solution is added to the GCE to obtain a PSA-HOF-dWO3·H2O / GCE modified electrode. Then, 6μL of a Tris-HCl solution with a concentration of 2μmol / L is added to the electrode to obtain an apt / PSA-HOF-dWO3·H2O / GCE modified electrode. A microcystin-RR standard solution with a concentration of 1.0×10 -10 mol / L is prepared, and 1.0×10 -10 mol / L of the microcystin-RR standard solution is added to the apt / PSA-HOF-dWO3·H2O / GCE modified electrode, and the modification is performed for 10, 20, 30, 40 and 50min. The aptamer sequence is the same as that in Example 1.

[0087] (5) Electrochemiluminescence detection: In the electrochemical window range of -2.0-0V, the high voltage of photomultiplier tube is 600V, the scanning speed is 0.1V / s, and the luminescence intensity is recorded.

[0088] As shown in Figure 5 , with the increase of the binding time of microcystin-RR and aptamer, the detector pulls more aptamer away from the electrode surface, and the ECL luminescence recovers, but when the binding time of microcystin-RR and aptamer increases from 30min to 50min, the change of ECL intensity gradually stabilizes, therefore the binding time of microcystin-RR and aptamer is 30min as preferred.

[0089] Example 4

[0090] Effect of PBS phosphate buffer solution with different pH values on the ECL luminescence performance of the PSA-HOF enhanced by dWO3·H2O nanomaterials electrochemiluminescence sensor:

[0091] (1) Preparation of dWO3·H2O nanomaterials: same as Example 1

[0092] (2) Preparation of PSA-HOF materials: same as Example 1

[0093] (3) Preparation of PSA-HOF-dWO3·H2O composite materials: same as Example 1

[0094] (4) Preparation of PBS phosphate buffer solution containing persulfate:

[0095] 8.4758g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) and 0.2028g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) were added to the above-mentioned 250mL ultrapure water containing potassium persulfate and dissolved, and the pH was adjusted to 5, 6, 7.4, 8, and 9, respectively, to obtain PBS phosphate buffer solution containing potassium persulfate with different pH values, which was finally stored for standby;

[0096] (5) Preparation of MC-RR detection PSA-HOF-dWO3·H2O electrochemical sensor:

[0097] The glassy carbon electrode was polished, and then ultrasonically treated with nitric acid, anhydrous ethanol and deionized water in sequence, and naturally air-dried for use. At room temperature, 6.0 μL of 0.01M PBS buffer solution (pH 7.4) containing 0.005M NHS and 0.01M EDC was dropped on a clean bare glassy carbon electrode for 1 h to activate the electrode. Subsequently, 3.0 μL of PSA-HOF-dWO3·H2O CS solution was dropped on the GCE to obtain a PSA-HOF-dWO3·H2O / GCE modified electrode. Then, 6 μL of 2 μmol / L Tris-HCl solution was dropped on the electrode to obtain an apt / PSA-HOF-dWO3·H2O / GCE modified electrode. A microcystin-RR standard solution with a concentration of 1.0×10 -10 mol / L was prepared, and 1.0×10 -10 mol / L of the microcystin-RR standard solution was dropped on the apt / PSA-HOF-dWO3·H2O / GCE modified electrode, which was modified for 30 min. The aptamer sequence was the same as in Example 1.

[0098] (6) Electrochemiluminescence detection: The modified electrode apt / PSA-HOF-dWO3·H2O / GCE was used as the working electrode, a platinum electrode was used as the auxiliary electrode, and Ag / AgCl was used as the reference electrode to form a three-electrode system. The three-electrode system was placed in PBS phosphate buffer solution containing potassium persulfate with pH values of 5, 6, 7.4, 8 and 9, respectively. The electrochemical window was -2.0-0 V, the high voltage of the photomultiplier tube was 600 V, the scanning speed was 0.1 V / s, and the luminescence intensity was recorded.

[0099] As shown in Figure 6 , the ECL response gradually increased with the increase of pH value, but the ECL intensity decreased when the pH value increased from 7.4 to 9. Therefore, the pH value of 7.4 was preferred.

[0100] Comparative Example 1

[0101] (1) Preparation of PSA-HOF-dWO3·H2O / GCE modified electrode

[0102] The glassy carbon electrode was polished, and then ultrasonically treated with nitric acid, anhydrous ethanol and deionized water in sequence, and naturally air-dried for use. 3.0 μL of 0.5 mg / mL PSA-HOF-dWO3·H2O composite material CS solution (the preparation method of the PSA-HOF-dWO3·H2O composite material is as described in Example 1) was transferred to the surface of a clean glassy carbon electrode, which was dried at room temperature to obtain a PSA-HOF-dWO3·H2O / GCE modified electrode, which was used as the working electrode for electrochemiluminescence test. The electrode was placed in PBS phosphate buffer solution (0.1 mol / L) for test.

[0103] (2) Preparation of standard curve

[0104] The apt / PSA-HOF-dWO3-H2O / GCE was used as the working electrode, a platinum electrode was used as the auxiliary electrode, and Ag / AgCl was used as the reference electrode to form a three-electrode system. The three-electrode system was placed in a PBS phosphate buffer solution containing potassium persulfate, and was used to detect a series of microcystin-RR standard solutions with different concentrations. The detection method was the same as in Example 1. The results are shown in Table 1.

[0105] Comparative Example 2

[0106] (1) Preparation of apt / PSA-HOF-dWO3-H2O / GCE / GCE modified electrode

[0107] The glassy carbon electrode was polished and then ultrasonically cleaned with nitric acid, anhydrous ethanol, and deionized water, respectively, and was naturally air-dried for use. 3.0 μL of a 0.5 mg / mL CS solution of PSA-HOF-dWO3-H2O composite material (the PSA-HOF-dWO3-H2O / GCE composite material was prepared according to the method of Example 1) was transferred to the surface of a clean glassy carbon electrode, and was dried at room temperature. Then, 6.0 μL of apt was added dropwise to obtain an apt / PSA-HOF-dWO3-H2O / GCE / GCE modified electrode, which was used as the working electrode for electrochemiluminescence testing. The electrode was placed in a PBS phosphate buffer solution (0.1 mol / L) containing potassium persulfate for testing.

[0108] (2) Preparation of standard curve

[0109] The apt / PSA-HOF-dWO3-H2O / GCE was used as the working electrode, a platinum electrode was used as the auxiliary electrode, and Ag / AgCl was used as the reference electrode to form a three-electrode system. The three-electrode system was placed in a PBS phosphate buffer solution containing potassium persulfate, and was used to detect a series of microcystin-RR standard solutions with different concentrations. The detection method was the same as in Example 1. The results are shown in Table 1.

[0110] Comparative Example 3

[0111] Comparative Example 3 differs from Example 1 in that the apt / PSA-HOF-dWO3-H2O / GCE modified electrode was used as the working electrode, a platinum electrode was used as the auxiliary electrode, and Ag / AgCl was used as the reference electrode to form a three-electrode system. The three-electrode system was placed in a PBS phosphate buffer solution without potassium persulfate, and the detection method was the same as in Example 1. The detection results show that the sensor prepared in Comparative Example 3 has very low light intensity when used for microcystin-RR detection, and it is difficult to achieve microcystin-RR detection. The results are shown in Table 1.

[0112] Table 1 Determination results of microcystin-RR in river water

[0113]

[0114]

[0115] Note: a is the average value of three determinations.

[0116] As shown in Table 1, the sample was detected in parallel for 3 times, the relative standard deviation was less than 5%, and the recovery of standard addition was in the range of 96% to 104%. The above results show that without the apt / PSA-HOF-dWO3·H2O / GCE modification and placed in the PBS phosphate buffer solution containing potassium persulfate, the PSA-HOF-dWO3·H2O / GCE alone or the apt / PSA-HOF-dWO3·H2O / GCE modification but placed in the PBS phosphate buffer solution without potassium persulfate cannot detect microcystin-RR. The apt / PSA-HOF-dWO3·H2O / GCE electrochemiluminescence sensor provided in the specific embodiment of the present application is effective and feasible for detecting microcystin-RR in river water, and is simple, flexible, wide in detection range and low in detection limit.

[0117] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can make equivalent replacement or change according to the technical solution and concept of the present application within the technical range disclosed by the present application, which should be covered in the protection scope of the present application.

Claims

1. An electrochemiluminescence sensor, characterized in that, The invention includes a glassy carbon electrode apt / PSA-HOF-dWO3·H2O / GCE, co-modified with a PSA-HOF-dWO3·H2O complex and an aptamer with specific recognition properties; wherein the PSA-HOF-dWO3·H2O complex and the aptamer are connected by a phosphatamide bond formed by an amino group on chitosan and a phosphate group on the aptamer; PSA-HOF represents a pyrene self-assembled hydrogen-bonded organic framework; and apt represents an aptamer. The electrochemiluminescence sensor was obtained by the following method: A certain amount of PSA-HOF and dWO3·H2O were dispersed in chitosan to obtain PSA-HOF dispersion and dWO3·H2O dispersion, respectively. The two dispersions were then mixed and ultrasonicated to obtain PSA-HOF-dWO3·H2O complex. The PSA-HOF-dWO3·H2O composite was drop-coated onto a clean glassy carbon electrode surface to obtain a PSA-HOF-dWO3·H2O / GCE modified electrode. The aptamer was drop-coated and incubated on the PSA-HOF-dWO3·H2O / GCE modified electrode to obtain apt / PSA-HOF-dWO3·H2O / GCE.

2. The electrochemiluminescence sensor according to claim 1, characterized in that, The aptamer for the specific recognition function is the MC-RR aptamer; the nucleotide sequence of the MC-RR aptamer is: 5'-CAG CTC AGA AGC TTG ATC CTACTG CCC TTC AAT GTT CAC TCC TGT TTC CTG ATC TTT GTC GAC TCG AAG TCG TGC ATCTG-3'.

3. An electrochemiluminescence sensor according to claim 1 or 2, characterized in that, The mass ratio of PSA-HOF to dWO3·H2O is 1:1; the mass-to-volume ratio of PSA-HOF or dWO3·H2O to chitosan is 1:1 (g / L); and the mass fraction of chitosan is 0.5%.

4. The electrochemiluminescence sensor according to claim 3, characterized in that, The PSA-HOF-dWO3·H2O composite was applied to a glassy carbon electrode at a rate of 3.0 μL and a concentration of 0.5–2.0 mg / mL.

5. The electrochemiluminescence sensor according to claim 3, characterized in that, The concentration of the aptamer is 0.5~3.0 μmol / L, and the drop volume is 6.0 μL.

6. The application of an electrochemiluminescence sensor according to any one of claims 1 to 5 in the electrochemiluminescence detection of microcystin-RR.

7. The application of the electrochemiluminescence sensor according to claim 6 in the detection of microcystin-RR, characterized in that, Includes the following steps: A series of microcystin-RR standard solutions of different concentrations were prepared; An electrochemiluminescence sensor apt / PSA-HOF-dWO3·H2O / GCE was used as the working electrode, a platinum electrode as the counter electrode, and Ag / AgCl as the reference electrode to form a three-electrode system. The three-electrode system was placed in a PBS phosphate buffer solution containing potassium persulfate. Within the electrochemical window range of -2.0 to 0 V, a photomultiplier tube voltage of 600 to 800 V was used, and a scan rate of 0.1 to 0.15 V / s was used to record the time-luminescence intensity curve. Then, a series of microcystin-RR standard solutions of different concentrations were added dropwise to the apt / PSA-HOF-dWO3·H2O / GCE, and cyclic voltammetry was performed within the electrochemical window range of -2.0 to 0 V, with a photomultiplier tube voltage of 600 to 800 V and a scan rate of 0.1 to 0.15 V / s. The time-luminescence intensity curves before and after the binding of microcystin-RR to the aptamer were recorded. The concentration of microcystin-RR in the sample solution was calculated based on the linear regression equation obtained from the linear relationship between the difference in luminescence intensity and the logarithm of the microcystin-RR concentration.

8. The application of the electrochemiluminescence sensor according to claim 7 in the detection of microcystin-RR, characterized in that, The pH of the PBS phosphate buffer solution containing potassium persulfate is 5-9, and the concentration of PBS is 0.1 mol / L.

9. The application of the electrochemiluminescence sensor according to claim 7 in the detection of microcystin-RR, characterized in that, The binding time between the microcystin-RR and the aptamer in the electrochemiluminescence sensor apt / PSA-HOF-dWO3·H2O / GCE is 10~50 min.

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