Composite nanomaterial, electrochemical luminescence biosensor and preparation method and application of electrochemical luminescence biosensor

Abstract

The invention relates to the field of polymer chemical industry, and discloses a composite nanomaterial, an electrochemical luminescence biosensor and a preparation method and application thereof. The composite nano material comprises an inner core, a first shell layer and a second shell layer, wherein the outer periphery of the inner core is coated with the first shell layer, and the outer periphery of the first shell layer is coated with the second shell layer; wherein the inner core contains metal nanoparticles, the first shell layer contains an inorganic oxide I, and the second shell layer contains an inorganic oxide II and an iridium complex. The electrochemical luminescence biosensor comprises an electrode, and the electrode is modified with the composite nano material, a target substance aptamer and g-C3N4. The composite nano material has excellent electrochemical luminescence performance, has the advantages of high sensitivity and high analysis speed for detecting target substances in water when being used for preparing an electrochemical luminescence biosensor for detecting the target substances, and has a remarkable market prospect.

Description

Technical Field

[0001] The present invention relates to the field of polymer chemical industry, and in particular to a composite nano material, an electrochemical luminescent biosensor and a preparation method and application thereof. Background Art

[0002] Electrochemiluminescence (ECL) is an analytical technique that combines chemiluminescence and electrochemistry. It applies a specific electrical signal to the luminescent substance to cause a series of redox reactions of gaining and losing electrons at the electrode interface, ultimately achieving the process of luminescence. Due to the advantages of high sensitivity, low background noise, wide linear range, and fast analysis speed, ECL is widely used in immunoassay, biosensor, environmental monitoring, food safety and other fields. Although ECL has been used in many analytical detection fields, there are still many problems. For example, there are fewer types of luminescent reagents, especially commercial ECL reagents. The most widely used one is ruthenium complex. Although ruthenium complexes have good water solubility and stable luminescence performance, they also have problems such as single emission wavelength and low luminescence efficiency. Therefore, the development of more ECL reagents with efficient luminescence effects is of great practical significance for improving analytical sensitivity.

[0003] Iridium complexes are a class of substances formed by coordination bonds between metallic iridium and organic ligands. Due to the strong heavy atom effect of iridium complexes, multicolor luminescence can be achieved by designing and synthesizing iridium complexes with different organic ligands. In addition, wavelength resolution and potential resolution can also be achieved. In addition, iridium complexes have high quantum yields and long fluorescence lifetimes. Therefore, iridium complexes have more advantages in multicolor luminescence, bioimaging, and multiple immunoassays. However, iridium complexes also have some problems, especially the poor solubility of iridium complexes in water. They have good ECL performance in organic systems, but limited luminescence performance in the aqueous phase.

[0004] Therefore, there is an urgent need to provide a composite nanomaterial that can regulate and enhance the electrochemiluminescence properties of iridium complexes and expand their applications in analytical detection. Summary of the invention

[0005] The purpose of the present invention is to overcome the problem of poor water solubility of iridium complexes in water phase in the prior art, which leads to poor luminescence performance, and to provide a composite nanomaterial, an electrochemiluminescent biosensor and a preparation method and application thereof. The composite nanomaterial has excellent electrochemiluminescent performance. When the composite nanomaterial is used to prepare an electrochemiluminescent biosensor for detecting target substances, it has the advantages of high sensitivity and fast analysis speed for detecting target substances in water, and has significant market prospects.

[0006] The first aspect of the present invention provides a composite nanomaterial, which comprises an inner core, a first shell layer coated on the outer periphery of the inner core, and a second shell layer coated on the outer periphery of the first shell layer; wherein the inner core comprises metal nanoparticles, the first shell layer comprises inorganic oxide I, and the second shell layer comprises inorganic oxide II and an iridium complex.

[0007] Preferably, the inorganic oxide I and the inorganic oxide II are each independently silicon dioxide and / or titanium dioxide, and more preferably silicon dioxide.

[0008] Preferably, the particle size of the core is 20-60 nm, the thickness of the first shell layer is 4-10 nm, and the thickness of the second shell layer is 4-15 nm.

[0009] Preferably, the metal nanoparticles are gold nanoparticles and / or silver nanoparticles, more preferably gold nanoparticles.

[0010] Preferably, the iridium complex is at least one selected from bis(2-phenylpyridine)-(4-carboxypropyl-2-2'-bipyridine)iridium, tris(2-phenylpyridine)iridium and tris(1-phenylpyridine)iridium, more preferably bis(2-phenylpyridine)-(4-carboxypropyl-2-2'-bipyridine)iridium.

[0011] The second aspect of the present invention provides a method for preparing a composite nanomaterial, comprising the following steps: sequentially coating a first shell layer and a second shell layer on the outside of an inner core; wherein the inner core contains metal nanoparticles, the first shell layer contains inorganic oxide I, and the second shell layer contains inorganic oxide II and an iridium complex.

[0012] Preferably, the inorganic oxide I and the inorganic oxide II are each independently silicon dioxide and / or titanium dioxide, and more preferably silicon dioxide.

[0013] Preferably, the metal nanoparticles are gold nanoparticles and / or silver nanoparticles, more preferably gold nanoparticles.

[0014] Preferably, the iridium complex is at least one selected from bis(2-phenylpyridine)-(4-carboxypropyl-2-2'-bipyridine)iridium, tris(2-phenylpyridine)iridium and tris(1-phenylpyridine)iridium, more preferably bis(2-phenylpyridine)-(4-carboxypropyl-2-2'-bipyridine)iridium.

[0015] Preferably, the coating process of the first shell layer comprises: mixing the metal nanoparticles, 3-aminopropyltrimethoxysilane and silicate for reaction I.

[0016] Preferably, the molar ratio of the metal nanoparticles, the 3-aminopropyltrimethoxysilane and the silicate is 1:1×10 6 -1.3×10 6 :1.4×10 5 -3×10 5 .

[0017] Preferably, the conditions of the mixed reaction I at least include: a temperature of 80-100° C. and a time of 2-3 h.

[0018] Preferably, the coating process of the second shell layer comprises: subjecting the metal nanoparticles coated with the first shell layer, the iridium complex and tetraethoxysilane to a mixed reaction II.

[0019] Preferably, the molar ratio of the metal nanoparticles, the iridium complex and the tetraethoxysilane is 1:1.2×10 6 -2×10 6 :4.5×10 8 -7×10 8 .

[0020] Preferably, the conditions of the mixed reaction II at least include: pH 8-10, and time 20-30h.

[0021] The third aspect of the present invention provides the use of the composite nanomaterial described in the first aspect or the composite nanomaterial prepared by the preparation method described in the second aspect in the preparation of an electrochemiluminescent biosensor for detecting a target substance.

[0022] Preferably, the target substance is microcystin.

[0023] The fourth aspect of the present invention provides an electrochemiluminescent biosensor, the electrochemiluminescent biosensor comprising an electrode, the electrode being modified with the composite nanomaterial described in the first aspect or the composite nanomaterial prepared by the preparation method described in the second aspect, a target substance aptamer, and gC 3 N 4 .

[0024] Preferably, the target substance is microcystin.

[0025] Preferably, the electrode is selected from at least one of a glassy carbon electrode, an ITO electrode and a screen-printed electrode, and is more preferably a glassy carbon electrode.

[0026] Preferably, the target substance aptamer is a ferrocene-labeled target substance aptamer. More preferably, the nucleotide sequence of the target substance aptamer is as shown in SEQ ID NO:1.

[0027] Preferably, the composite nanomaterial, g-C 3 N 4 and the aptamer of the target substance are sequentially modified on the electrode.

[0028] The fifth aspect of the present invention provides a method for detecting the content of microcystin, including the following steps: after incubating a sample to be tested in contact with the electrochemiluminescence biosensor described in the fourth aspect or the electrochemiluminescence biosensor described in the fifth aspect of the present invention, electrochemiluminescence signal detection is performed.

[0029] By the above technical solution, the beneficial effects of the present invention are as follows:

[0030] The composite nanomaterial provided by the present invention has a first shell containing inorganic oxide I coated on the inner core containing metal nanoparticles, and a second shell containing inorganic oxide II and iridium complex. Through the cooperative action of the inner core, the first shell and the second shell, the electrochemiluminescence performance of the composite nanomaterial in the aqueous phase can be significantly enhanced. Moreover, by setting the first shell, the distance between the iridium complex in the second shell and the metal nanoparticles in the inner core can be adjusted, so that the electrochemical performance of the composite nanomaterial can be adjusted, further expanding its application in analytical detection.

[0031] The electrochemiluminescence biosensor provided by the present invention can realize the recognition of the target substance through the synergistic action of the composite nanomaterial, the aptamer of the target substance and g-C 3 N 4 It has high sensitivity and high selectivity for the detection of the target substance, and has significant market prospects.

[0032] Other features and advantages of the present invention will be described in detail in the subsequent specific implementation part. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the preparation process of the composite nanomaterial in the present invention and the schematic diagram of the preparation principle of the electrochemiluminescence biosensor;

[0034] Figure 2 is the TEM and EDS results of Au NPs@SiO 2 -Ir obtained in Example 1-1 of the present invention. Among them, (A) is the TEM image of AuNPs@SiO 2 -Ir, (B) is the EDS mapping image of Au element, (C) is the EDS mapping image of Si element, (D) is the EDS mapping image of O element, (E) is the EDS mapping image of N element, and (F) is the EDS mapping image of Ir element;

[0035] Figure 3 It is gC 3 N 4 The electron microscope scanning results;

[0036] Figure 4 It is gC 3 N 4 UV spectrum of

[0037] Figure 5 It is gC 3 N 4 Fluorescence emission spectrum of

[0038] Figure 6 It is gC 3 N 4 The fluorescence emission spectrum of and the UV spectrum of the iridium complex;

[0039] Figure 7 is the electrochemiluminescence response diagram of the electrode modified in different steps in Example 2-1;

[0040] Figure 8 is a differential pulse voltammetry curve of the electrode modified in different steps in Example 2-1;

[0041] Fig. 9 are the impedance results of the electrodes modified in different steps in Example 2-1;

[0042] Fig.10 The ECL test results of MC-LR standard solutions with different concentrations in Test Example 5;

[0043] Fig.11 is a graph showing the relationship between the ECL signal intensity and the concentration of MC-LR obtained in Test Example 5;

[0044] Fig.12 This is a graph showing the stability results of the electrochemiluminescent biosensor in Test Example 6;

[0045] Fig.13 This is a graph showing the specificity results of the electrochemiluminescence biosensor in Test Example 6. DETAILED DESCRIPTION

[0046] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0047] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0048] In the first aspect, the present invention provides a composite nanomaterial, which comprises an inner core, a first shell layer coated on the outer periphery of the inner core, and a second shell layer coated on the outer periphery of the first shell layer; wherein the inner core comprises metal nanoparticles, the first shell layer comprises inorganic oxide I, and the second shell layer comprises inorganic oxide II and an iridium complex.

[0049] The inventor of the present invention and his research group have rich research and development experience and research and development achievements in the fields of materials, sensors, etc. for many years. The inventor unexpectedly found in the process of research that the first shell containing inorganic oxide I and the second shell containing inorganic oxide II and iridium complex are coated outside the inner core containing metal nanoparticles, and the electrochemical luminescence performance of the composite nano material in the water phase can be significantly enhanced through the coordination of the inner core, the first shell and the second shell. Moreover, by setting the first shell, the distance between the iridium complex in the second shell and the metal nanoparticles in the inner core can be adjusted, so that the electrochemical performance of the composite nano material can be adjusted, and its application in analysis and detection can be further expanded.

[0050] According to the present invention, in order to further improve the electrochemiluminescence performance of the composite nanomaterial in the aqueous phase, preferably, the inorganic oxide I and the inorganic oxide II are each independently silicon dioxide and / or titanium dioxide, and silicon dioxide is more preferably used.

[0051] According to the present invention, preferably, the particle size of the core is 20-60nm, specifically 20nm, 40nm, 60nm, or any value between the two values; the thickness of the first shell is 4-10nm, specifically 4nm, 6nm, 8nm, 10nm, or any value between the two values; the thickness of the second shell is 4-15nm, specifically 4nm, 10nm, 15nm, or any value between the two values. The inventors found that under this preferred embodiment, the particle size of the core, the thickness of the first shell and the thickness of the second shell are controlled within the above range, which can further enhance the electrochemiluminescence performance of the composite nanomaterial in the aqueous phase; and the first shell within the above thickness range can adjust the electrochemiluminescence performance of the composite nanomaterial, thereby expanding its application in analytical detection.

[0052] According to the present invention, the metal nanoparticles can be metal nanoparticles conventionally selected in the art, for example, gold nanoparticles, silver nanoparticles, gold-silver composite nanoparticles, and noble metal nanoparticles of different morphologies. In order to further improve the electrochemiluminescence performance of the composite nanomaterial in the aqueous phase, preferably, the metal nanoparticles are gold nanoparticles and / or silver nanoparticles, and more preferably gold nanoparticles.

[0053] According to the present invention, the above-mentioned metal nanoparticles can be purchased commercially or prepared by oneself.

[0054] According to the present invention, in order to further improve the electrochemiluminescence performance of the composite nanomaterial in the aqueous phase, preferably, the iridium complex is selected from at least one of bis(2-phenylpyridine)-(4-carboxypropyl-2-2'-bipyridine)iridium, tris(2-phenylpyridine)iridium and tris(1-phenylpyridine)iridium, and more preferably bis(2-phenylpyridine)-(4-carboxypropyl-2-2'-bipyridine)iridium.

[0055] In a second aspect, the present invention provides a method for preparing a composite nanomaterial, comprising the following steps: sequentially coating a first shell layer and a second shell layer on the outside of an inner core; wherein the inner core contains metal nanoparticles, the first shell layer contains inorganic oxide I, and the second shell layer contains inorganic oxide II and an iridium complex.

[0056] According to the present invention, the preparation method of the composite nano material is simple, easy to operate and suitable for industrial production.

[0057] According to the present invention, in order to further improve the electrochemiluminescence performance of the composite nanomaterial in the aqueous phase, preferably, the inorganic oxide I and the inorganic oxide II are each independently silicon dioxide and / or titanium dioxide, and silicon dioxide is more preferably used.

[0058] According to the present invention, the metal nanoparticles can be metal nanoparticles conventionally selected in the art, for example, gold nanoparticles, silver nanoparticles, gold-silver composite nanoparticles, and noble metal nanoparticles of different morphologies. In order to further improve the electrochemiluminescence performance of the composite nanomaterial in the aqueous phase, preferably, the metal nanoparticles are gold nanoparticles and / or silver nanoparticles, and more preferably gold nanoparticles.

[0059] According to the present invention, the above-mentioned metal nanoparticles can be purchased commercially or prepared by oneself.

[0060] Exemplarily, the preparation process of gold nanoparticles includes: using sodium citrate as a reducing agent to reduce chloroauric acid to obtain gold nanospheres.

[0061] According to the present invention, in order to further improve the electrochemiluminescence performance of the composite nanomaterial in the aqueous phase, preferably, the iridium complex is selected from at least one of bis(2-phenylpyridine)-(4-carboxypropyl-2-2'-bipyridine)iridium, tris(2-phenylpyridine)iridium and tris(1-phenylpyridine)iridium, and more preferably bis(2-phenylpyridine)-(4-carboxypropyl-2-2'-bipyridine)iridium.

[0062] According to the present invention, preferably, the coating process of the first shell layer comprises: mixing the metal nanoparticles, 3-aminopropyltrimethoxysilane (APTMS) and silicate for reaction I. The inventors found that under this preferred embodiment, 3-aminopropyltrimethoxysilane is modified on the surface of the metal nanoparticles, and silicate is used as a silicon source to form a dense first shell layer on the surface of the metal nanoparticles, thereby enhancing the electrochemiluminescence performance of the composite nanoparticles.

[0063] According to the present invention, the silicate may be a silicate conventionally selected in the art, for example, sodium silicate, potassium silicate, calcium silicate, etc. In order to further improve the electrochemiluminescence performance of the composite nanomaterial, preferably, the silicate is sodium silicate.

[0064] According to the present invention, in order to further improve the electrochemiluminescence performance of the composite nanoparticles in the aqueous phase, preferably, the molar ratio of the metal nanoparticles, the 3-aminopropyltrimethoxysilane and the silicate is 1:1×10 6 -1.3×10 6 :1.4×10 5 -3×10 5 .

[0065] According to the present invention, in order to further improve the reaction efficiency and yield of the mixed reaction I, preferably, the conditions of the mixed reaction I at least include: the temperature is 80-100°C, specifically 80°C, 90°C, 100°C, or any value between the foregoing two values; the time is 2-3h, specifically 2h, 2.5h, 3h, or any value between the foregoing two values.

[0066] According to the present invention, in order to further improve the coverage of the first shell layer on the surface of the inner core and the electrochemiluminescence performance of the composite nanomaterial, preferably, the coating process of the first shell layer includes: reacting the metal nanoparticles and 3-aminopropyltrimethoxysilane at room temperature for 30-40 minutes, and then adding silicate to react at a temperature of 80-100°C for 2-3 hours.

[0067] According to the present invention, preferably, the coating process of the second shell layer comprises: mixing the metal nanoparticles coated with the first shell layer, the iridium complex and tetraethoxysilane (TEOS) to perform reaction II. The inventors found that under this preferred embodiment, the iridium complex is modified at the periphery of the first shell layer, and the second shell layer doped with inorganic oxide II and iridium complex is coated at the periphery of the first shell layer using tetraethoxysilane as a silicon source, thereby further improving the electrochemiluminescence performance of the composite nanomaterial.

[0068] According to the present invention, in order to further improve the electrochemiluminescence performance of the composite nanomaterial in the aqueous phase, preferably, the molar ratio of the metal nanoparticles, the iridium complex and the tetraethoxysilane is 1:1.2×10 6 -2×10 6 :4.5×10 8 -7×10 8 .

[0069] According to the present invention, in order to further improve the reaction efficiency and yield of the mixed reaction II, preferably, the conditions of the mixed reaction II include at least a pH of 8-10, specifically 8, 9, 10, or any value between the foregoing two values; a time of 20-30h, specifically 20h, 25h, 30h, or any value between the foregoing two values.

[0070] According to the present invention, in order to further improve the coverage of the second shell layer on the periphery of the first shell layer and the electrochemiluminescence performance of the composite nanomaterial, preferably, the coating process of the second shell layer includes: reacting the metal nanoparticles coated with the first shell layer and the iridium complex at a pH of 8-10 for 30-40 minutes, and then adding tetraethoxysilane to react for 20-30 hours.

[0071] In a third aspect, the present invention provides use of the composite nanomaterial described in the first aspect or the composite nanomaterial prepared by the preparation method described in the second aspect in preparing an electrochemiluminescent biosensor for detecting a target substance.

[0072] According to the present invention, in order to further improve the sensitivity of the electrochemiluminescent biosensor to the detection of the target substance, preferably, the target substance is microcystin. Further preferably, the target substance is MC-LR.

[0073] In a fourth aspect, the present invention provides an electrochemiluminescent biosensor, the electrochemiluminescent biosensor comprising an electrode, the electrode being modified with the composite nanomaterial described in the first aspect or the composite nanomaterial prepared by the preparation method described in the second aspect, a target substance aptamer, and gC 3 N 4 .

[0074] According to the present invention, in order to further improve the sensitivity of the electrochemiluminescent biosensor to the detection of the target substance, preferably, the target substance is microcystin. Further preferably, the target substance is MC-LR.

[0075] According to the present invention, in order to further improve the sensitivity of the electrochemiluminescent biosensor to the detection of the target substance, preferably, the electrode is selected from at least one of a glassy carbon electrode, an ITO electrode and a screen-printed electrode, and is more preferably a glassy carbon electrode.

[0076] According to the present invention, in order to further improve the sensitivity of the electrochemiluminescent biosensor to target substance detection, preferably, the target substance aptamer is a ferrocene-labeled target substance aptamer, and more preferably, the nucleotide sequence of the target substance aptamer is as shown in SEQ ID NO:1.

[0077] According to the present invention, in order to further improve the sensitivity of the electrochemiluminescent biosensor to the detection of the target substance, preferably, the electrode is modified with the composite nanomaterial, the gC 3 N 4 and the target substance aptamer.

[0078] According to the present invention, gC 3 N 4 It can be purchased commercially or prepared by yourself.

[0079] In the present invention, the electrochemiluminescent biosensor described in the fourth aspect can be prepared by a conventional preparation method in the art. Preferably, the preparation method of the electrochemiluminescent biosensor comprises the following steps: modifying the composite nanomaterial described in the first aspect or the composite nanomaterial prepared by the preparation method described in the second aspect, the target substance aptamer and gC on the surface of the electrode; 3 N 4 .

[0080] According to the present invention, preferably, the composite nanomaterial, the target substance aptamer and the gC 3 N 4 The inventors found that under this preferred embodiment, the composite nanomaterial, the target substance aptamer and gC 3 N 4 The modification effect on the electrode surface can further improve the sensitivity of the electrochemiluminescence biosensor to the detection of target substances.

[0081] According to the present invention, preferably, in the dispersion of the composite nanomaterial, the concentration of the composite nanomaterial is 1-2 mg / mL, specifically 1 mg / mL, 1.5 mg / mL, 2 mg / mL, or any value between the above two values; in the dispersion of the target substance aptamer, the concentration of the target substance aptamer is 0.2-1.8 μM, specifically 0.2 μM, 0.6 μM, 1.2 μM, 1.8 μM, or any value between the above two values; in the gC 3 N 4 In the dispersion, the gC 3 N 4 The concentration is 0.01-0.04 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, or any value between the foregoing two values.

[0082] According to the present invention, gC 3 N 4 It can be purchased commercially or prepared by yourself.

[0083] For example, gC 3 N 4 The preparation process includes: heating melamine to 550-600°C at a rate of 3-4°C / min, and after the heating reaction is complete, dispersing the product in water and ultrasonicating for 9 hours to make gC 3 N 4 Completely dispersed to obtain gC 3 N 4 Aqueous solution.

[0084] In a fifth aspect, the present invention provides a method for detecting the content of microcystin, comprising the following steps: contacting a sample to be tested with the electrochemiluminescent biosensor described in the fourth aspect for incubation, and then detecting an electrochemiluminescent signal.

[0085] According to the present invention, preferably, the incubation time is 20-120 min, specifically 20 min, 70 min, 120 min, or any value between the above two values. Preferably, the incubation time is 60-100 min.

[0086] According to the present invention, the method further comprises: measuring the electrochemiluminescence signal of the microcystin standard solution and drawing a standard curve, and calculating the content of the microcystin in the sample to be tested according to the standard curve.

[0087] According to the present invention, the preparation process of the microcystin standard solution includes: using 10mM PBS buffer to prepare MC-LR solutions of different concentrations, for example, 0.001, 0.01, 0.05, 0.5, 1, and 10 ng / mL.

[0088] According to the present invention, in order to improve the stability of the microcystin standard solution, preferably, the pH of the PBS buffer is 6-9.5, specifically 6, 7, 8, 9, 9.5, or any value between the above two values. Preferably, the pH of the PBS buffer is 8-8.5.

[0089] In the present invention, the process of measuring the electrochemiluminescence signal of the microcystin standard solution includes: setting scanning parameters on the electrochemiluminescence online analysis system, using a platinum wire electrode as a counter electrode, Ag / AgCl as a reference electrode, and an electrochemiluminescence biosensor after incubation with microcystin as a working electrode to form a three-electrode system, and measuring the electrochemiluminescence signal of the microcystin standard solution in a solution containing 0.1-0.15M K 2 S 2 O 8 ECL assays were performed in 0.1-0.2 M PBS buffer.

[0090] According to the present invention, in order to further improve the accuracy of the ECL test, preferably, the scanning parameters at least include: a CV scanning speed of 0.01-0.2V / s, and a scanning voltage of -1.5 to 0V.

[0091] According to a particularly preferred embodiment of the present invention, see Figure 1 , provides a method for preparing an electrochemiluminescent biosensor, comprising the following steps: modifying the surface of an electrode with a composite nanomaterial, gC 3 N 4 and target substance aptamers;

[0092] Preparation of composite nanomaterials:

[0093] The first shell layer and the second shell layer are sequentially coated on the outer side of the inner core; wherein the inner core contains metal nanoparticles, the first shell layer contains inorganic oxide I, and the second shell layer contains inorganic oxide II and iridium complex; the coating process of the first shell layer comprises: reacting the metal nanoparticles, 3-aminopropyltrimethoxysilane and silicate at a temperature of 80-100° C. for 2-3 hours; the coating process of the second shell layer comprises: reacting the metal nanoparticles coated with the first shell layer, the iridium complex and tetraethoxysilane at a pH of 8-10 for 20-30 hours;

[0094] Inorganic oxide I and inorganic oxide II are both silicon dioxide, the metal nanoparticles are gold nanoparticles, the iridium complex is bis(2-phenylpyridine)-(4-carboxypropyl-2-2'-bipyridine)iridium; the target substance is microcystin; the electrode is a glassy carbon electrode, and the nucleotide sequence of the target substance aptamer is shown in SEQ ID NO: 1;

[0095] The method for detecting the content of microcystin comprises the following steps: contacting a sample to be tested with an electrochemiluminescent biosensor for incubation, and then detecting an electrochemiluminescent signal; measuring the electrochemiluminescent signal of a microcystin standard solution and drawing a standard curve, and calculating the content of microcystin in the sample to be tested according to the standard curve.

[0096] The electrochemiluminescent biosensor prepared in the above particularly preferred embodiment is prepared by using composite nanomaterials, target substance aptamers and gC 3 N 4 The synergistic effect can realize the recognition of target substances, and the detection of target substances is highly sensitive and selective, and has significant market prospects.

[0097] The present invention is described in detail below with reference to the embodiments, but the scope of the present invention is not limited thereby.

[0098] In the following examples and comparative examples, the iridium complex is bis(2-phenylpyridine)-(4-carboxylpropyl-2-2'-bipyridine)iridium purchased from Suzhou Nakai Company; Fc-Apt was purchased from Shanghai Shenggong Group Co., Ltd., and the nucleotide sequence is shown in SEQ ID NO: 1; gC 3 N 4 All materials were homemade; APTMS was purchased from Sinopharm Chemical Reagent Co., Ltd.; TEOS was purchased from Sinopharm Chemical Reagent Co., Ltd.; unless otherwise specified, other raw materials were purchased from commercial sources.

[0099] Example 1-1

[0100] (1) Add 150 mL of ultrapure water to a clean three-necked flask, stir vigorously, heat and boil. After boiling, add 348 μL of 127 mM HAuCl 4 , 2 min later, 1 mL of 73.6 mM sodium citrate solution was quickly added, reacted for 40 min, and then transferred to a 90 °C oil bath and heated for 30 min;

[0101] (2) Add 660 μL of 180 mM sodium citrate, and 2 min later add 660 μL of 75 mM HAuCl 4 After heating for 40 min, the process was repeated. After the reaction was completed, the Au NPs were prepared by cooling to room temperature.

[0102] (3) Take 1 mL of the 0.1565 nM Au NPs dispersion obtained in step (2), dilute it ten-fold and place it in a clean glass bottle, add 200 μL of 1 mM APTMS solution, stir at room temperature for 30 min, and then add 800 μL of 44.24 μM Na 2 SiO 3The solution was stirred at 90°C for 2 h. After the reaction was completed, it was placed in ice water to cool down and stop the reaction, thus obtaining dense SiO 2 Coated Au NPs, denoted as Au NPs@SiO 2 ;

[0103] (4) Next, Au NPs@SiO 2 After washing once, the mixture was dispersed in water and 10 mL of Au NPs@SiO 2 0.1M NaOH solution was added to adjust the pH of the solution to 9, and then 250μL 1mM iridium complex methanol solution was added and stirred for 30min; finally, 100μL 0.8976M TEOS-methanol solution was added and stirred for 24h; after the reaction, the product was centrifuged and resuspended in water to obtain Au NPs@SiO 2 -Ir.

[0104] Example 1-2

[0105] (1) Add 150 mL of ultrapure water to a clean three-necked flask, stir vigorously, heat and boil. After boiling, add 348 μL of 127 mM HAuCl 4 , 2 min later, 1 mL of 73.6 mM sodium citrate solution was quickly added, reacted for 40 min, and then transferred to a 90 °C oil bath and heated for 30 min;

[0106] (2) Add 660 μL of 180 mM sodium citrate, and 2 min later add 660 μL of 75 mM HAuCl 4 After heating for 40 min, the process was repeated. After the reaction was completed, the Au NPs were prepared by cooling to room temperature.

[0107] (3) Take 1 mL of the 0.1565 nM Au NPs dispersion obtained in step (2) and dilute it ten-fold into a clean glass bottle, add 200 μL of 0.8 mM APTMS solution, stir at room temperature for 30 min, and then add 1000 μL of 44.24 μM Na 2 SiO 3 The solution was stirred at 80°C for 3 h. After the reaction was completed, it was placed in ice water to cool down and stop the reaction, thus obtaining dense SiO 2 Coated Au NPs, denoted as Au NPs@SiO 2 ;

[0108] (4) Next, Au NPs@SiO 2 After washing once, the mixture was dispersed in water and 10 mL of Au NPs@SiO 20.1M NaOH solution was added to adjust the pH of the solution to 9, and then 200μL 1mM iridium complex methanol solution was added and stirred for 30min; finally 80μL 0.8976M TEOS-methanol solution was added and stirred for 20h; after the reaction, the product was centrifuged and resuspended in water to obtain Au NPs@SiO 2 -Ir.

[0109] Examples 1-3

[0110] (1) Add 150 mL of ultrapure water to a clean three-necked flask, stir vigorously, heat and boil. After boiling, add 348 μL of 127 mM HAuCl 4 , 2 min later, 1 mL of 73.6 mM sodium citrate solution was quickly added, reacted for 40 min, and then transferred to a 90 °C oil bath and heated for 30 min;

[0111] (2) Add 660 μL of 180 mM sodium citrate, and 2 min later add 660 μL of 75 mM HAuCl 4 After heating for 40 min, the process was repeated. After the reaction was completed, the Au NPs were prepared by cooling to room temperature.

[0112] (3) Take 1 mL of the 0.1565 nM Au NPs dispersion obtained in step (2), dilute it tenfold and place it in a clean glass bottle, add 200 μL of 1.5 mM APTMS solution, stir at room temperature for 30 min, and then add 500 μL of 44.24 μM Na 2 SiO 3 The solution was stirred at 100°C for 2.5 hours. After the reaction was completed, it was placed in ice water to cool down and stop the reaction, thus obtaining dense SiO 2 Coated Au NPs, denoted as Au NPs@SiO 2 ;

[0113] (4) Next, Au NPs@SiO 2 After washing once, the mixture was dispersed in water and 10 mL of Au NPs@SiO 2 0.1M NaOH solution was added to adjust the pH of the solution to 9, and then 300μL 1mM iridium complex methanol solution was added and stirred for 30min; finally, 120μL 0.8976M TEOS-methanol solution was added and stirred for 30h; after the reaction, the product was centrifuged and resuspended in water to obtain Au NPs@SiO 2 -Ir.

[0114] Examples 1-4

[0115] The composite nanomaterial was prepared according to the method of Example 1-1, except that the Na 2 SiO 3 The volume of the solution was replaced to 100 μL.

[0116] Examples 1-5

[0117] The composite nanomaterial was prepared according to the method of Example 1-1, except that the concentration of APTMS in step (3) was replaced with 2 mM.

[0118] Examples 1-6

[0119] The composite nanomaterial was prepared according to the method of Example 1-1, except that the volume of the TEOS-methanol solution in step (4) was replaced with 50 μL.

[0120] Examples 1-7

[0121] The composite nanomaterial was prepared according to the method of Example 1-1, except that the volume of the TEOS-methanol solution in step (4) was replaced with 200 μL.

[0122] Comparative Example 1-1

[0123] The composite nanomaterial was prepared according to the method of Example 1-1, except that step (3) and step (4) were replaced by:

[0124] (3) Take 1 mL of the 0.1565 nM Au NPs dispersion obtained in step (2), dilute it tenfold and place it in a clean glass bottle, add 0.1 M NaOH solution to adjust the pH of the solution to 9, then add 250 μL of 1 mM iridium complex methanol solution, stir and react for 30 minutes; finally, add 100 μL of 0.8976 M TEOS-methanol solution, stir and react for 24 hours; after the reaction, centrifuge the product and resuspend it in water to obtain Au NPs@-Ir.

[0125] Comparative Example 1-2

[0126] The composite nanomaterial was prepared according to the method of Example 1-1, except that step (4) was replaced by:

[0127] (4) Next, Au NPs@SiO 2 After washing once, the mixture was dispersed in water and 10 mL of Au NPs@SiO 2 0.1 M NaOH solution was added to adjust the pH of the solution to weak alkalinity, and then 250 μL 1 mM iridium complex methanol solution was added and stirred for 30 min. After the reaction, the product was centrifuged and resuspended in water to obtain Au NPs@SiO 2 -Ir.

[0128] Test Example 1

[0129] The Au NPs@SiO obtained in Example 1-1 2 -Ir was tested by TEM and EDS, the results are as follows Figure 2 shown.

[0130] from Figure 2 It can be seen that (A) is Au NPs@SiO 2 TEM image of Au NPs@SiO 2 -Ir is a core-shell structure, with a spherical shape of gold nanoparticles Au NPs as the core, and a layer of SiO 2 Shell; (B) is the EDS mapping of Au element, (C) is the EDS mapping of Si element, (D) is the EDS mapping of O element, (E) is the EDS mapping of N element, and (F) is the EDS mapping of Ir element. It can be found that Au element is distributed in the core, Si and O elements are distributed in SiO 2 shell, therefore, the material has a core-shell structure.

[0131] Test Example 2

[0132] The particle size of the inner core and the thickness of the first shell layer and the second shell layer of the composite nanomaterials prepared in Examples 1-1 to 1-7 and Comparative Examples 1-1 to 1-2 were tested. The results are shown in Table 1.

[0133] Table 1

[0134] serial number Core(nm) First shell (nm) Second shell (nm) Example 1-1 20 5 7 Example 1-2 20 9 5 Examples 1-3 20 4 10 Examples 1-4 20 2 7 Examples 1-5 20 8 8 Examples 1-6 20 5 4 Examples 1-7 20 5 12 Comparative Example 1-1 20 - 7 Comparative Example 1-2 20 6 -

[0135] Example 2-1

[0136] The glassy carbon electrode was polished with 0.05 μm and 0.3 μm alumina powder, cleaned with ethanol and water ultrasonically, and dried with argon gas for later use; 10 μL of Au@SiO prepared in Example 1-1 was added dropwise on the surface of the electrode. 2 -Ir (concentration of 1 mg / mL), after drying, add 6 μL of 0.025 mg / mL gC 3 N 4 solution; after drying, 6 μL 1.2 μM Fc-Apt was added dropwise to the electrode surface, and after incubation for 80 min, the excess unincubated Fc-Apt was washed away with water to obtain an electrochemiluminescent biosensor.

[0137] gC used in Example 2-1 3 N 4 The electron microscope scanning results are as follows Figure 3 As shown, the UV spectrum is Figure 4 The fluorescence emission spectrum is shown in Figure 5 shown.

[0138] Example 2-2

[0139] The glassy carbon electrode was polished with 0.05 μm and 0.3 μm alumina powder, cleaned with ethanol and water ultrasonically, and dried with argon gas for later use; 10 μL of Au@SiO prepared in Example 1-2 was added dropwise on the surface of the electrode. 2 -Ir (concentration of 1 mg / mL), after drying, add 6 μL of 0.025 mg / mL gC 3 N 4 solution; after drying, 6 μL 1.2 μM Fc-Apt was added to the electrode surface, incubated for 80 min, and then the excess unincubated Fc-Apt was washed off with water.

[0140] Example 2-3

[0141] The glassy carbon electrode was polished with 0.05 μm and 0.3 μm alumina powder, cleaned with ethanol and water ultrasonically, and dried with argon gas for later use; 10 μL of Au@SiO prepared in Example 1-3 was added dropwise on the surface of the electrode. 2 -Ir (concentration of 1 mg / mL), after drying, add 6 μL of 0.025 mg / mL gC 3 N 4 solution; after drying, 6 μL 1.2 μM Fc-Apt was added to the electrode surface, incubated for 80 min, and then the excess unincubated Fc-Apt was washed off with water.

[0142] Embodiment 2-4

[0143] The sensor was subjected to ECL test according to the method in Example 2-1, except that Au@SiO 2 -Ir is replaced by the composite nanomaterial prepared in Example 1-4.

[0144] Embodiment 2-5

[0145] The sensor was subjected to ECL test according to the method in Example 2-1, except that Au@SiO 2 -Ir is replaced by the composite nanomaterial prepared in Example 1-5.

[0146] Embodiment 2-6

[0147] The sensor was subjected to ECL test according to the method in Example 2-1, except that Au@SiO 2 -Ir is replaced by the composite nanomaterial prepared in Example 1-6.

[0148] Embodiment 2-7

[0149] The sensor was subjected to ECL test according to the method in Example 2-1, except that Au@SiO 2 -Ir is replaced by the composite nanomaterial prepared in Example 1-7.

[0150] Embodiment 2-8

[0151] The sensor was subjected to ECL test according to the method in Example 2-1, except that Au@SiO 2 -Ir is replaced by the composite nanomaterial prepared in Example 1-8.

[0152] Comparative Example 2-1

[0153] The sensor was subjected to ECL test according to the method in Example 2-1, except that Au@SiO 2 -Ir is replaced by the composite nanomaterial prepared in Example 2-1.

[0154] Comparative Example 2-2

[0155] The sensor was subjected to ECL test according to the method in Example 2-1, except that Au@SiO 2 -Ir is replaced by the composite nanomaterial prepared in Example 2-2.

[0156] Test Example 3

[0157] Using gC 3 N 4 The feasibility of the resonance energy transfer (RET) process was studied by the fluorescence emission spectrum of iridium and the UV-visible absorption spectrum of the iridium complex.

[0158] For the raw material gC in Example 2-1 3 N 4 The fluorescence emission spectrum was tested, and the UV-visible absorption spectrum of the iridium complex was tested. The results are shown in Figure 6 .

[0159] like Figure 6 As shown, the UV-visible absorption peak of the iridium complex appears in the range of 250-500 nanometers (curve a), and the absorption peak is located at 440nm gC 3 N 4 The fluorescence spectra of (curve b) have a partial overlap, and this overlap can infer the possibility of resonance energy transfer between the donor (CCN) and the acceptor (RuSi nanoparticles).

[0160] Test Example 4

[0161] 6 μL of MC-LR solution (with a concentration of 1 ng / mL) was added dropwise to the electrochemiluminescent biosensors prepared in Examples 2-1 to 2-7, respectively. After incubation for 80 min, unbound MC-LR was washed away, and then ECL test was performed.

[0162] The ECL test process includes: setting the scanning parameters on the electrochemiluminescence online analysis system as follows: CV scanning speed of 0.1 V / s, scanning voltage: -1.5 to 0 V, photomultiplier tube high voltage of 500 V; using a platinum wire electrode as the counter electrode, Ag / AgCl as the reference electrode, and the electrochemiluminescence biosensor after incubation of MC-LR as the working electrode to form a three-electrode system, in a solution containing 0.1 MK 2 S 2 O 8 The ECL test was carried out in 0.1 M PBS buffer with a pH of 8.0. The results are shown in Table 1.

[0163] The electrochemiluminescence response of the electrodes modified in different steps in Example 2-1 was studied. Figure 7 The electrochemiluminescence response diagram of the electrode modified in different steps in Example 2-1; Figure 8 The differential pulse voltammetry curves of the electrodes modified in different steps in Example 2-1; Fig. 9 These are the impedance results of the electrodes modified in different steps in Example 2-1.

[0164] Figure 7 In the figure, a is Au NPs@SiO 2 -Ir, b is gC 3 N 4 , c is Au NPs@SiO 2 -Ir / gC 3 N 4 , d is Au NPs@SiO 2 -Ir / gC 3 N 4 / Fc-Apt, e is Au NPs@SiO 2 -Ir / gC 3 N 4 / Fc-Apt / MC-LR. Figure 8 and Fig. 9 In the figure, a is bare GCE (glassy carbon electrode before modification), b is Au NPs@SiO 2 -Ir, c is Au NPs@SiO 2 -Ir / gC 3 N 4 , d is Au NPs@SiO 2 -Ir / gC 3 N 4 / Fc-Apt, e is Au NPs@SiO 2 -Ir / gC 3 N 4 / Fc-Apt / MC-LR.

[0165] from Figure 8 It can be seen that when Au NPs@SiO 2 -Ir and gC 3 N 4 When modified onto the electrode together, it was found that the electrochemiluminescence response was significantly enhanced, which can be attributed to the efficient resonance energy transfer between the nanomaterials and the surface enhancement effect of the precious metal gold. After modifying Fc-Apt, the ECL signal was well quenched because ferrocene can quench the ECL signal. Finally, when MC-LR was incubated with Fc-Apt, the quenching probe was away from the electrode surface and the ECL signal was restored.

[0166] Depend on Fig. 9 It can be seen that the impedance of the bare glassy carbon electrode is very small; the Au NPs@SiO 2 -Ir, due to SiO 2 Hinder electron transfer, resulting in increased impedance; modify gC 3 N 4 After that, the impedance continued to increase; after Fc-Apt was modified, the impedance increased significantly because DNA greatly hindered electron transfer; finally, after MC-LR was incubated for a period of time, due to the binding of MC-LR to Fc-Apt, Fc-Apt moved away from the electrode surface and the impedance decreased.

[0167] Table 1

[0168] serial number ECL signal value Example 2-1 1742a.u. Example 2-2 1276a.u. Example 2-3 1329a.u. Embodiment 2-4 1105a.u. Embodiment 2-5 945a.u. Embodiment 2-6 826a.u. Embodiment 2-7 848a.u. Comparative Example 2-1 678a.u. Comparative Example 2-2 468a.u.

[0169] Test Example 5

[0170] First, 10mM PBS buffer was used to prepare MC-LR solutions of different concentrations. As the concentration of MC-LR increased, more Fc-Apt left the electrode surface, and the ECL signal recovery became more obvious. Therefore, this principle can be used to detect MC-LR of different concentrations.

[0171] Different concentrations of MC-LR standard solutions (0.001, 0.01, 0.05, 0.5, 1, 10 ng / mL) were added to the electrochemiluminescent biosensor prepared in Example 2-1, and the unbound MC-LR was washed away after incubation for 80 min. The ECL test was performed. The test results are as follows: Fig.10The ECL test process includes: setting the scanning parameters on the electrochemiluminescence online analysis system as follows: CV scanning speed of 0.1V / s, scanning voltage: -1.5 to 0V, photomultiplier tube high voltage of 500V; using the platinum wire electrode as the counter electrode, Ag / AgCl as the reference electrode, and the glassy carbon electrode after the MC-LR incubation as the working electrode to form a three-electrode system, in a solution containing 0.1MK 2 S 2 O 8 ECL assays were performed in 0.1 M PBS buffer, pH 8.0.

[0172] from Fig.10 It can be seen that as the concentration of MC-LR increases, the ECL signal gradually increases. Fig.11 It can be seen that the ECL signal intensity is linearly related to the concentration of MC-LR, y=1462.62lgC+5873.99, R 2 =0.972 (y value is the ECL signal intensity value, C is the concentration of MC-LR), and the detection limit is 0.86 pg / mL.

[0173] Test Example 6

[0174] In order to verify the stability of the electrochemiluminescent biosensor, the electrode in Example 2-1 was scanned eight times. Fig.12 As shown. Fig.12 It can be seen that after eight cycles of scanning, the ECL signal value is still relatively stable, with an RSD of 2.54%, indicating that the electrochemiluminescence biosensor has good stability.

[0175] In order to verify the specificity of the sensor, some interferences were selected for selectivity testing, including MC-RR, MC-YR, and AFB. 1 , CAP, Ca 2+ As interference substances, MC-RR and MC-YR belong to different subtypes of microcystin and have very similar structures as MC-LR, and are used as interference substances. The concentration of the interference substances is 5 ng / mL, and the 1 ng / mL MC-LR standard solution is used as the control group.

[0176] 6 μL of the above interfering substances were added to the electrochemiluminescent biosensor prepared in Example 2-1, and the unbound interfering substances were washed away after incubation for 80 min. The ECL test was performed using the method of Test Example 4. The results are as follows: Fig.13 As shown. Fig.13 It can be seen that even if the concentration of interfering substances is more than 5 times higher than that of MC-LR, their electrochemiluminescence (ECL) intensity is still lower than that of MC-LR, which indicates that the electrochemiluminescence biosensor exhibits excellent selectivity for MC-LR.

[0177] Test Example 7

[0178] Actual sample spike recovery test

[0179] The local lake water was taken as the actual sample, and after filtering it three times with a 0.45 μm microporous filter membrane to remove impurities, MC-LR was added thereto so that the concentrations of MC-LR were 0.5 ng / mL, 0.050 ng / mL, and 0.005 ng / mL, respectively, to obtain spiked samples. The spiked samples were subjected to ECL tests according to the method of Test Example 4, and the results are shown in Table 2. The results in Table 2 show that the electrochemiluminescence biosensor can detect the content of MC-LR in actual samples.

[0180] Table 2

[0181] Spiked amount (ng / mL) Measured concentration (ng / mL) Recovery rate (%) Relative standard deviation (%) 0.5 0.54 108 11.18 0.05 0.0586 117.3 9.179 0.005 0.0046 91.57 10.33

[0182] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A composite nanomaterial, characterized in that: The composite nanomaterial comprises a core, a first shell layer coated on the periphery of the core, and a second shell layer coated on the periphery of the first shell layer; The inner core contains metal nanoparticles, the first shell contains inorganic oxide I, and the second shell contains inorganic oxide II and an iridium complex.

2. The composite nanomaterial according to claim 1, characterized in that: The inorganic oxide I and the inorganic oxide II are each independently silicon dioxide and / or titanium dioxide, preferably silicon dioxide; Preferably, the particle size of the core is 20-60 nm, the thickness of the first shell layer is 4-10 nm, and the thickness of the second shell layer is 4-15 nm.

3. The composite nanomaterial according to claim 1, characterized in that: The metal nanoparticles are gold nanoparticles and / or silver nanoparticles, preferably gold nanoparticles; Preferably, the iridium complex is at least one selected from bis(2-phenylpyridine)-(4-carboxypropyl-2-2'-bipyridine)iridium, tris(2-phenylpyridine)iridium and tris(1-phenylpyridine)iridium, more preferably bis(2-phenylpyridine)-(4-carboxypropyl-2-2'-bipyridine)iridium.

4. A method for preparing a composite nanomaterial, characterized in that: The following steps are involved: The first shell layer and the second shell layer are sequentially coated on the outer side of the inner core; The inner core contains metal nanoparticles, the first shell contains inorganic oxide I, and the second shell contains inorganic oxide II and an iridium complex.

5. The preparation method according to claim 4, characterized in that: The inorganic oxide I and the inorganic oxide II are each independently silicon dioxide and / or titanium dioxide, preferably silicon dioxide; Preferably, the metal nanoparticles are gold nanoparticles and / or silver nanoparticles, more preferably gold nanoparticles; Preferably, the iridium complex is at least one selected from bis(2-phenylpyridine)-(4-carboxypropyl-2-2'-bipyridine)iridium, tris(2-phenylpyridine)iridium and tris(1-phenylpyridine)iridium, more preferably bis(2-phenylpyridine)-(4-carboxypropyl-2-2'-bipyridine)iridium.

6. The preparation method according to claim 4, characterized in that: The coating process of the first shell layer comprises: mixing the metal nanoparticles, 3-aminopropyltrimethoxysilane and silicate for reaction I; Preferably, the molar ratio of the metal nanoparticles, the 3-aminopropyltrimethoxysilane and the silicate is 1:1×10 6 -1.3×10 6 :1.4×10 5 -3×10 5 ; Preferably, the conditions of the mixed reaction I include at least: a temperature of 80-100° C. and a time of 2-3 h; Preferably, the coating process of the second shell layer comprises: performing a mixed reaction II on the metal nanoparticles coated with the first shell layer, the iridium complex and tetraethoxysilane; Preferably, the molar ratio of the metal nanoparticles, the iridium complex and the tetraethoxysilane is 1:1.2×10 6 -2×10 6 :4.5×10 8 -7×10 8 ; Preferably, the conditions of the mixed reaction II at least include: pH 8-10, and time 20-30h.

7. Use of the composite nanomaterial according to any one of claims 1 to 3 or the composite nanomaterial prepared by the preparation method according to any one of claims 4 to 6 in preparing an electrochemiluminescent biosensor for detecting a target substance; Preferably, the target substance is microcystin.

8. An electrochemiluminescent biosensor, characterized in that: The electrochemiluminescent biosensor comprises an electrode, on which the composite nanomaterial according to any one of claims 1 to 3 or the composite nanomaterial prepared by the preparation method according to any one of claims 4 to 6, a target substance aptamer and g-C3N4 are modified.

9. The electrochemiluminescent biosensor according to claim 8, characterized in that: The target substance is microcystin; Preferably, the electrode is selected from at least one of a glassy carbon electrode, an ITO electrode and a screen-printed electrode, and is further preferably a glassy carbon electrode; Preferably, the target substance aptamer is a ferrocene-labeled target substance aptamer, and more preferably, the nucleotide sequence of the target substance aptamer is as shown in SEQ ID NO: 1; Preferably, the electrode is modified with the composite nanomaterial, the g-C3N4 and the target substance aptamer in sequence.

10. A method for detecting the content of microcystin, characterized in that: The following steps are involved: After the sample to be tested is contacted with the electrochemiluminescent biosensor according to claim 8 or 9 for incubation, the electrochemiluminescent signal is detected.

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