Hybrid synergistic electrochemiluminescence quencher, preparation method and application thereof

Abstract

The application discloses a hybrid synergistic electrochemiluminescence quencher, a preparation method and application thereof. Dopamine, zinc ions and 2-methyl imidazole are used as raw materials to synthesize polydopamine and a hybrid material of a zeolitic imidazolate framework by one-pot synthesis under alkaline conditions. The introduction of the electropositive zeolitic imidazolate framework can greatly eliminate the strong electronegativity of the polydopamine and simultaneously endow the quencher with good porosity. The quencher prepared by the application is nanospheres, has a large specific surface area and weak electronegativity, and exhibits good scavenging capacity for free radicals, especially electrogenerated anionic radicals, and has a high quenching efficiency on electrochemiluminescence. After the quencher is labeled with an antibody, the quencher is used for constructing an electrochemiluminescence immunosensor for ochratoxin A. The electrochemiluminescence immunosensor prepared by the application has a wide detection range, a low detection limit and good stability in the detection of ochratoxin A, and has a wide application prospect in the fields of electrochemiluminescence technology and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to a preparation method of a hybrid synergistic quencher for electrochemiluminescence and its application in an electrochemiluminescence immunosensor, in particular to a strategy for improving the free radical scavenging ability of a commonly used quencher, polydopamine, and thus improving the electrochemiluminescence quenching efficiency and the sensitivity of an immunosensor, belonging to the technical field of immunoassay. BACKGROUND

[0002] Electrochemiluminescence (ECL) is a chemical luminescence phenomenon excited by electrochemistry, which has the advantages of low background, high sensitivity, wide detection range and good controllability. According to the luminescence mechanism, ECL has annihilation type and co-reactant type, among which the co-reactant type ECL can occur in aqueous solution, and thus is suitable for the field of immunoassay technology. The luminescence principle of co-reactant type ECL is that under the excitation of oxidation or reduction, the luminophore and the co-reactant are simultaneously oxidized or reduced to free radicals, and then the oxidation or reduction products of the two immediately react to generate an excited state luminophore, which emits light in the process of returning from the excited state to the ground state. According to the excitation potential and the nature of the co-reactant, the co-reactant type ECL can be divided into oxidation-reduction type (anodic ECL) and reduction-oxidation type (cathodic ECL). Some mycotoxins are prone to be oxidized at a relatively high oxidation potential, which in turn affects the stability of immunoassay, therefore, the immunoassay method based on cathodic ECL is more suitable for mycotoxins.

[0003] Mycotoxins are a class of small molecules, common mycotoxins include aflatoxins, vomitoxin and ochratoxin, etc., usually only one antigenic epitope, therefore, in ECL immunoassay, a competitive immunoassay mode is often used. Among them, ECL immunosensor based on luminescence quenching principle is quite common, which usually prepares an ECL immunosensor interface first, then adds quencher-labeled immunomolecules to react with it, and establishes the response relationship between ECL signal and target concentration. In the competitive immunoassay mode, the ECL signal output of this type of sensor is "turn on". Polydopamine (PDA) is a commonly used ECL quencher, which is currently known to have a quenching effect on a variety of ECL luminophores, such as carbon nitride nanosheets (CNNS), Ru(bpy)3 2+and quantum dots. In addition, PDA has been reported to have quenching effects on both cathodic ECL and anodic ECL systems, and the quenching mechanism is generally believed to be energy transfer between PDA and the excited state of the luminophore (ACS Applied Materials and Interfaces, 2020, 12, 8006-8015; Analytica Chimica Acta, 2017, 963, 17-23; Science Bulletin, 2016, 61, 931-938). It has been reported that PDA has a strong free radical scavenging ability (Biomacromolecules, 2011, 12, 625-632), so the scavenging mechanism of free radicals of the co-reactant and the luminophore should also be included in the quenching of ECL. However, PDA has a strong electronegativity (Langmuir, 2013, 29, 10539-10548), and in the quenching mode of cathodic ECL analysis, a large number of co-reactant radical anions generated on the electrode surface have a strong electrostatic repulsion with PDA, making it difficult for them to diffuse away from the electrode surface to PDA, resulting in that this quenching mechanism is not significant in cathodic ECL. In addition, PDA does not have a porous structure, and its capture and scavenging of free radicals mainly rely on the quinone or phenol groups on its surface, and the efficiency needs to be improved. Therefore, there is still a lot of room for improvement in applying PDA as a quencher in ECL immunoassay.

[0004] Zeolitic imidazolate frameworks (ZIFs) are framework structures formed by coordination hybridization of imidazole organic ligands and transition metal ions, have high porosity, stability and good biocompatibility, and also show electropositivity. In summary, in view of the applicability of cathodic ECL in mycotoxin detection and the need to improve the quenching efficiency of PDA on cathodic ECL, the present application proposes a hybrid structure of PDA and ZIFs (PDA@ZIFs), in which ZIFs endow PDA with good porosity and can effectively eliminate the strong electronegativity of PDA, thereby improving the quenching effect on cathodic ECL, and ultimately improving the detection performance of the ECL immunosensor for mycotoxins. The present application is expected to make up for some shortcomings of the prior art. SUMMARY

[0005] One of the purposes of the present application is to provide a hybrid synergistic electrochemiluminescence quencher and a preparation method thereof, which can improve the quenching efficiency of electrochemiluminescence by strengthening the mechanism of scavenging co-reactant radical anions.

[0006] The second purpose of the present application is to provide the application of the hybrid synergistic electrochemiluminescence quencher, i.e. to provide a sensitive electrochemiluminescence immunosensor detection method, which provides a technical basis for the application of electrochemiluminescence analysis method in mycotoxin detection.

[0007] The above-mentioned object of the present application is achieved by the following technical means:

[0008] The hybrid synergistic electrochemiluminescence quencher relates to the present application, which is prepared by the following method:

[0009] (1) First, zinc acetate (1-10 mM) and dopamine (2.65-10.6 mM) are dissolved in an aqueous ethanol solution (ethanol / deionized water = 3:1), and stirred to fully dissolve, then PVP and 2-methylimidazole (10-40 mM) are sequentially added under continuous stirring, and finally Tris or NaOH is added to adjust the pH of the reaction solution to above 8.5, and the reaction is continuously stirred for 24-48 h. In the basic aqueous ethanol solution, zeolitic imidazolate framework self-assembly and dopamine oxidation polymerization occur simultaneously, and 2-methylimidazole and polydopamine compete with zinc ions for coordination, and a stable porous structure is formed after 24 h. The whole process is carried out in the dark.

[0010] (2) After the reaction is completed, large particles are removed by centrifugation at a lower speed (5,000-8,000 rpm), and the product is collected by high-speed centrifugation (13,000 rpm or above). After the product is washed with deionized water and ethanol, a hybrid synergistic electrochemiluminescence quencher is obtained.

[0011] The hybrid synergistic electrochemiluminescence quencher prepared by the above-mentioned method is a hybrid structure material PDA@ZIFs of PDA and ZIFs, which is a monodisperse nanosphere with a microporous structure and weak electronegativity. The hybrid synergistic electrochemiluminescence quencher has better scavenging ability for neutral free radicals and anion free radicals than polydopamine due to its good porous structure and weak electronegativity, thereby showing better quenching ability for cathode electrochemiluminescence.

[0012] Further, the diameter distribution of the monodisperse microspheres is 40-120 nm, and the average diameter is 80 nm; the average pore size of the micropores is 1.6 nm; and in addition, the BET specific surface area of the PDA@ZIFs microspheres is greatly increased compared with that of polydopamine PDA.

[0013] The electrochemiluminescence immunosensor for detecting mycotoxins relates to the present application, which is prepared by the following method:

[0014] (1) First, the hybrid synergistic electrochemiluminescence quencher prepared by one of the objects of the present application is labeled with OTA antibody, i.e. an appropriate concentration of OTA antibody is added to the quencher, and the labeling of the OTA antibody is realized by physical adsorption between the antibody and the quencher. Then, the blank sites on the surface of the quencher are blocked with BSA, and an ECL-quenchable immunoprobe is obtained.

[0015] (2) The Au@CNNS modified by nano-gold is used as a luminous body to modify a glassy carbon electrode, and after drying at room temperature, the luminous body modified electrode surface is added with an OTA artificial antigen (OTA-BSA), and after 12 hours of incubation at 4 DEG C, the empty sites are blocked by BSA, and thus the ECL immunosensing interface is obtained.

[0016] (3) The prepared immunoprobe is mixed with the to-be-detected OTA, and reacts with the ECL immunosensing interface, the electrode surface is washed thoroughly after the reaction is completed, and the ECL test is immediately performed, and the test conditions are as follows: the test electrolyte is 0.1M PBS (pH 8.0), the concentration of the co-reactant K2S2O8 is 80mM, the potential scanning window is 0-(-1.3) V, the scanning rate is 0.1V / s, PMT is 600, and then the response relationship between the ECL signal intensity and the OTA concentration is established, so that the quantitative detection of the OTA is realized.

[0017] Compared with the prior art, the present application has the beneficial effects including:

[0018] (1) The hybrid synergistic quencher PDA@ZIFs proposed in the present application has a microporous structure (average pore size 1.6nm), a large specific surface area (1067.6m 2 / g) and weak electronegativity (zeta potential-13.1mV), while PDA itself does not have a porous structure, a small specific surface area (2.1m 2 / g) and strong electronegativity (zeta potential 40mV), so that the hybrid synergistic quencher PDA@ZIFs proposed in the present application has better free radical scavenging capacity, and further, PDA@ZIFs can more efficiently quench the cathode ECL than PDA.

[0019] (2) The mycotoxin ECL immunosensing detection method established by using the hybrid synergistic quencher PDA@ZIFs proposed in the present application has high sensitivity, a wide detection range and good stability, wherein the linear range of detection is 10.0fg / mL-1.0ng / mL, and the minimum detection limit is 4.8fg / mL. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 A is a scanning electron microscope picture of PDA@ZIFs, Figure 1 B-F are transmission electron microscope combined energy spectrum element analysis diagrams of PDA@ZIFs.

[0021] Figure 2 is a nitrogen isothermal adsorption-desorption curve of PDA@ZIFs, Zn-PDA and PDA.

[0022] Figure 3is the x-ray photoelectron spectroscopy (XPS) results of PDA@ZIFs, wherein Figure 3 A is the elemental scan total spectrum of PDA@ZIFs, Figure 3 B is the Zn LMM spectrum, Figure 3 C is the O1s spectrum.

[0023] Figure 4 is the zeta potential test results of PDA@ZIFs and PDA.

[0024] Figure 5 A is the result comparison of DPPH free radical scavenging experiments of PDA@ZIFs and PDA, Figure 5 B is the cyclic voltammetry curve and ECL-potential curve diagram comparing the quenching effect of PDA@ZIFs and PDA on cathode ECL.

[0025] Figure 6 is the preparation process of the immunosensor characterized by electrochemical impedance spectroscopy (EIS) and ECL.

[0026] Figure 7 A is the response relationship between the ECL signal intensity of the immunosensor prepared by the present application and different OTA concentrations, Figure 7 B is the linear fitting working curve of ECL signal intensity and OTA concentration.

[0027] Figure 8 is the ECL curve of the ECL immunosensor prepared by the present application in the test, which is continuously scanned for 16 cycles.

[0028] Figure 9 is the preparation process and application schematic diagram of the hybrid synergistic electrochemiluminescence quencher of the present application. DETAILED DESCRIPTION

[0029] The present application will be further explained in combination with specific embodiments below, but the embodiments do not make any form of limitation on the present application. Those skilled in the art should understand that the details and forms of the technical solutions of the present application can be modified or replaced without departing from the spirit and scope of the present application, and these modifications and replacements all fall within the protection scope of the present application.

[0030] Example 1 is a preparation method of a hybrid synergistic electrochemiluminescence quencher, and the method steps are as shown in Figure 9

[0031] ​(1) 45 mg zinc acetate dihydrate and 20 mg dopamine hydrochloride were dissolved in 20 mL of 75% ethanol aqueous solution, stirred at room temperature for 20 min, then 0.4 g PVP (average molecular weight 24,000) and 32.8 mg 2-methylimidazole were added successively under stirring, finally, the pH of the reaction solution was adjusted to more than 8.5 by adding appropriate amount of Tris, and the reaction was continuously stirred for 24 h, and the whole process was carried out in the dark.

[0032] (2) After the reaction was completed, large particles were removed by centrifugation at 8,000 rpm, and then the product was collected by centrifugation at 13,000 rpm for 30 min, and then the product was washed with deionized water and ethanol for 6 times to obtain a hybrid synergistic electrochemiluminescence quencher PDA@ZIFs.

[0033] Figure 1 is a morphology diagram of a hybrid synergistic quencher, wherein Figure 1 A is a scanning electron microscope diagram, showing that the obtained quencher is a nanometer microsphere with good dispersity, the diameter is mainly distributed in 40 nm-120 nm, and the average diameter is 80 nm, Figure 1 B-F are transmission electron microscope combined energy spectrum element analysis diagrams, showing that the carbon, nitrogen, oxygen and zinc elements in the nanometer microsphere are uniformly distributed.

[0034] Figure 2 is a nitrogen isothermal adsorption-desorption curve of a hybrid synergistic quencher, from which it can be seen that the hybrid synergistic quencher PDA@ZIFs has a good porous structure, and the BET specific surface area is calculated as 1067.6 m 2 / g, while the synthesized product (Zn-PDA) without 2-methylimidazole and PDA alone have no obvious porous structure, and the BET specific surface areas are 7.8 m 2 / g and 2.1 m 2 / g, respectively.

[0035] Figure 3 is an XPS energy spectrum diagram of a hybrid synergistic quencher, and the results show that there are carbon, nitrogen, oxygen and zinc elements, and the zinc ions are coordinated with 2-methylimidazole and PDA.

[0036] Figure 4 is the zeta potential test result of a hybrid synergistic quencher, wherein the zeta potential of PDA@ZIFs is -13.1 mV, and the electronegativity is obviously weaker than that of PDA.

[0037] Figure 5A is the evaluation of the scavenging ability of hybrid synergistic quencher to neutral DPPH free radicals, from the figure, the scavenging ability of quencher PDA@ZIFs to DPPH free radicals is better than that of PDA alone, but it is not very significant, the reason may be that DPPH is a nonpolar molecule, which is difficult to enter the hydrophilic PDA@ZIFs channel structure, Figure 5 B is the evaluation of the quenching effect of hybrid synergistic quencher on cathode ECL. The evaluation system first modifies the luminophore Au@CNNS on the surface of GCE, then adds 1% PVP, and after drying at room temperature, the quencher PDA@ZIFs to be evaluated is added. From the figure, it can be seen that the quenching effect of PDA@ZIFs on cathode ECL is obviously better than that of PDA. The reason is that PDA@ZIFs has a large surface area and weak electronegativity, which is more likely to remove the co-reactant anion radicals generated in the ECL process.

[0038] Example 2 is a preparation method of an electrochemiluminescence immunosensor for detecting mycotoxins, the method steps are as shown in Figure 9

[0039] (1) Add an appropriate concentration of anti-OTA monoclonal antibody solution to 2 mg / mL PDA@ZIFs prepared in Example 1 to make the final concentration of the antibody 25 μg / mL, and shake at room temperature for 3 h. Then add BSA with a final concentration of 1% to the reaction solution, continue to shake for 2 h, and then centrifuge to remove excess BSA to obtain an immunoprobe.

[0040] (2) Drop 4 μL of luminophore Au@CNNS on the surface of a clean GCE, dry at room temperature, wash the electrode surface with deionized water to remove the luminophore that is not firmly combined, then add 6 μL of OTA-BSA with a concentration of 16 μg / mL to the electrode surface, incubate at 4°C for 12 h, then wash the electrode surface with deionized water, and then block the blank sites on the electrode surface with 1% BSA to obtain an ECL immunosensor interface. EIS and ECL are used to monitor the preparation process, respectively. Figure 6 A can be seen that after adding luminophore Au@CNNS to the surface of GCE, the interface impedance increases. After adding artificial antigen OTA-BSA to the interface, the artificial antigen is fixed to the interface through Au-N or Au-S bond, and the impedance further increases. When BSA is used to block the blank sites, the impedance continues to increase, Figure 6 B is the monitoring of the preparation process of the immunosensor interface by ECL. After modifying Au@CNNS on GCE, a strong ECL signal is generated, and there is a significant overload phenomenon. After further modifying the electrode with OTA-BSA and BSA, the ECL signal gradually decreases. The above monitoring results of EIS and ECL show that the preparation of the ECL immunosensor interface is successful.

[0041] ​(3) Mix 100 μg / mL of the immunoprobe with an equal volume of OTA standard of appropriate concentration, and react it with the ECL immunosensing interface at room temperature for 45 min. The OTA to be tested will compete with OTA-BSA to bind to the immunoprobe. After the reaction, clean the electrode surface, and then test the ECL intensity. The ECL test conditions are: test electrolyte is 0.1M PBS (pH 8.0), co-reactant K2S2O8 concentration is 80mM, potential scan window is 0~1.3V, scan rate is 0.1V / s, and PMT is 600. Establish the response relationship between ECL signal intensity and OTA concentration, and then realize the quantitative detection of OTA in the test sample. Figure 7 The linear relationship between ECL signal intensity and OTA concentration was investigated. The results showed that the ECL signal intensity increased with increasing OTA concentration, exhibiting a good linear relationship within the range of 10.0 fg / mL to 1.0 ng / mL. The linear equation was: I ECL =1615.5log C oTA +14509(R 2 =0.9984), where c is the OTA concentration (ng / mL), and the limit of detection is 4.8 fg / mL, indicating that the ECL immunosensor prepared in this invention has a wide detection range and high sensitivity. Figure 8 The ECL immune sensor was continuously scanned for 16 cycles in the ECL test. The results show that the ECL immune sensor prepared in this invention has good test stability.

[0042] (5) Different concentrations of OTA standard were added to OTA-negative rice and flour samples, respectively, and then detected using the ECL immunosensor proposed in this invention. The recovery rate was calculated. The results are shown in Table 1:

[0043] Table 1

[0044]

[0045]

[0046] The above embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

Claims

1. The application of a hybrid synergistic electrochemiluminescence quencher, characterized in that: The hybrid synergistic electrochemiluminescent quencher is used as an electrochemiluminescent immunosensor for the detection of ochratoxin A; The preparation method of the hybrid-enhanced electrochemiluminescence quencher includes the following steps: S1. Zinc acetate and dopamine are dissolved in an ethanol aqueous solution in sequence and stirred until fully dissolved. Then, polyvinylpyrrolidone (PVP) and 2-methylimidazole are added in sequence under continuous stirring. Finally, the pH of the reaction solution is adjusted to be greater than 8.5, and stirring is continued for 24-48 hours. In the alkaline ethanol aqueous solution, the self-assembly of the zeolite imidazole ester skeleton and the oxidative polymerization of dopamine occur simultaneously. 2-methylimidazole and polydopamine compete with zinc ions for coordination, and a stable porous structure is formed after the reaction. The entire reaction process is carried out in a light-protected environment. S2. After the reaction is complete, large particles are removed by low-speed centrifugation, and the product is collected by high-speed centrifugation. The product is then washed several times with deionized water and ethanol. The obtained product is the hybrid synergistic electrochemiluminescence quencher PDA@ZIFs.

2. The application of the hybrid synergistic electrochemiluminescence quencher according to claim 1, characterized in that: The synthesis was carried out using a one-pot method, in which the zinc ion concentration was 1 mM - 10 mM, the dopamine concentration was 2.65 mM - 10.6 mM, the mass fraction of PVP was 0.5% - 2%, the concentration of 2-methylimidazole was 10 mM - 40 mM, and the pH was adjusted to be greater than 8.5 using 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) or sodium hydroxide.

3. The application of the hybrid synergistic electrochemiluminescence quencher according to claim 1, characterized in that: The hybrid-enhanced electrochemiluminescence quencher is a hybrid structure material of PDA and ZIFs, namely PDA@ZIFs, which is a monodisperse nanosphere with a microporous structure and has weak electronegativity and large BET specific surface area.

4. The application of the hybrid synergistic electrochemiluminescence quencher according to claim 3, characterized in that: The microspheres have a diameter ranging from 40 to 120 nm, with an average diameter of 80 nm; the micropores have an average pore size of 1.6 nm.

5. The application of the hybrid synergistic electrochemiluminescence quencher according to claim 1, characterized in that, The method for detecting ochratoxin A includes the following steps: (1) Add ochratoxin A antibody, i.e. OTA antibody, to the hybrid enhanced electrochemiluminescence quencher. The OTA antibody is labeled by relying on the physical adsorption between the antibody and the quencher. Then, the blank sites on the surface of the quencher are blocked by BSA to obtain an immune probe that can quench ECL. (2) Gold nano-modified carbon nitride nanosheets (Au@CNNS) were drop-coated onto the surface of a glassy carbon electrode. Au@CNNS was then used as a luminescent material to modify the glassy carbon electrode. After drying at room temperature, OTA artificial antigen (OTA-BSA) was drop-coated onto the surface of the luminescent material-modified electrode. After incubation for a period of time, the vacancy sites were blocked with BSA to obtain the electrochemiluminescent ECL immunosensing interface. (3) The immune probe prepared in step (1) is mixed with an appropriate concentration of OTA standard and reacted with the ECL immunosensing interface prepared in step (2). The OTA to be tested will compete with OTA-BSA to bind to the immune probe. After the reaction is complete, the electrode surface is cleaned and then the ECL intensity test is performed. Then the response relationship between ECL signal intensity and OTA concentration is established, thereby realizing the quantitative detection of OTA.

6. The application of the hybrid synergistic electrochemiluminescence quencher according to claim 5, characterized in that, The test conditions in step (3) are as follows: PBS with pH = 8.5 is used as the test electrolyte, K2S2O8 is used as the co-reactant, the potential scan window is 0 ~ -1.3 V, the scan rate is 0.1 V / s, and PMT 600.

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