TiO 2 Electrochemiluminescent immunosensor of nanoflower loaded with CdTe quantum dots and its preparation method and application

Abstract

The present invention discloses an electrochemiluminescent immunosensor of TiO2 nanoflowers loaded with CdTe quantum dots and its preparation method and application. The CdTe / TiO2 NFs composite material is prepared and used as an electrochemiluminescent signal marker. The surface and interior of TiO2NFs can load a large amount of CdTe QDs, and at the same time can catalyze the CdTe QDs / S2O8 2‑ The co-reaction process of the system significantly improves the electrochemiluminescence intensity of CdTe / TiO2 NFs. The magnetic material Fe3O4@SiO2 NPs is used as a magnetic separation carrier, which has the advantages of superparamagnetism, good biocompatibility, and easy biomodification. The present invention constructs an electrochemiluminescence immunosensor based on the interaction between antigen and antibody. The sensor has the characteristics of high sensitivity, strong specificity, good stability, low detection limit, wide linear range, and simple operation.

Description

Technical Field

[0001] The invention belongs to the field of electrochemiluminescence analysis, and in particular relates to an electrochemiluminescence immunosensor of TiO2 nanoflowers loaded with CdTe quantum dots, and a preparation method and application thereof. Background Art

[0002] At present, various methods of immunoassay markers have been proposed, such as enzyme-linked immunosorbent assay, capillary electrophoresis combined with chemiluminescence, fluorescence, electrochemical method, colorimetry, etc. Among the many immunoassay techniques, electrochemiluminescence immunoassay has attracted much attention due to its high sensitivity and specificity, low detection limit, and rapid detection process.

[0003] Luminescent markers are one of the key factors affecting the performance of ECL immunosensors and are essential for achieving ultrasensitive detection of antigens. Since Bard first studied the ECL phenomenon of Si nanocrystals, semiconductor nanocrystals (also known as quantum dots, QDs) with excellent ECL performance have gradually come into people's view. In the QDs family, II-VI cadmium-based QDs (CdS, CdTe, CdSe) are widely used as ECL luminescent markers due to their excellent ECL efficiency and narrow emission spectrum. However, due to their inherent physicochemical properties, the luminescence efficiency of II-VI cadmium-based QDs still lags behind traditional ECL reagents such as ruthenium bipyridine and luminol, which greatly limits the sensitivity of immunoassays.

[0004] Recently, a novel "co-reaction promoter" concept has been proposed to improve the efficiency of the co-reaction process and has been widely used in many ECL biosensor systems. Based on this concept, loading II-VI cadmium QDs in certain "co-reaction promoter" materials with high specific surface area is beneficial to promote ECL emission, thereby improving the sensitivity of immunosensors. Products based on this concept are urgently needed to be developed. Summary of the invention

[0005] Purpose of the invention: One of the purposes of the present invention is to provide an electrochemiluminescent immunosensor of CdTe quantum dots loaded with TiO2 nanoflowers to solve the problem of weak electrochemiluminescence intensity of quantum dots, using a quantum dot-based composite material (CdTe / TiO2 NFs) with significantly enhanced electrochemiluminescence efficiency. 2- Due to the catalytic effect of the co-reaction system, the electrochemiluminescence intensity of CdTe / TiO2 NFs composite materials is significantly improved.

[0006] The second object of the present invention is to provide a method for preparing the electrochemiluminescent immunosensor of the above-mentioned TiO2 nanoflower loaded with CdTe quantum dots.

[0007] The third object of the present invention is to provide the application of the electrochemiluminescent immunosensor of the above-mentioned TiO2 nanoflower loaded with CdTe quantum dots in detecting antigens. Based on CdTe / TiO2 NFs, a CdTe / TiO2NFs / K2S2O8 system is constructed, and a strong and stable cathode electrochemiluminescent signal of CdTe / TiO2 NFs can be collected.

[0008] Technical solution: In order to achieve the above purpose, the present invention is implemented through the following solutions:

[0009] 1. An electrochemiluminescent immunosensor of TiO2 nanoflowers loaded with CdTe quantum dots, characterized in that: the sensor comprises a signal unit CdTe / TiO2 NFs-Ab2, Fe3O4@SiO2-Ab1 with blocked non-specific binding sites, and antigens respectively connected to CdTe / TiO2 NFs-Ab2 and Fe3O4@SiO2-Ab1 through antigen-antibody interaction; CdTe / TiO2 NFs-Ab2 is a second antibody corresponding to the antigen labeled by CdTe / TiO2 NFs; CdTe / TiO2 NFs is TiO2 NFs loaded with CdTeQDs; Fe3O4@SiO2-Ab1 is a first antibody Fe3O4@SiO2 with surface chemical modification corresponding to the antigen, and Fe3O4@SiO2 is Fe3O4 coated with SiO2.

[0010] The preparation method of CdTe / TiO2 NFs-Ab2 comprises the following steps:

[0011] 1) Activate the carboxylic acid groups on the surface of CdTe / TiO2 NFs;

[0012] 2) The second antibody is reacted with the carboxylic acid groups on the surface and inside of the activated CdTe / TiO2 NFs to obtain the second antibody corresponding to the antigen labeled by the CdTe / TiO2 NFs.

[0013] Wherein, the particle size of CdTe / TiO2 NFs is 1.34 μm to 1.41 μm. In order to obtain excellent electrochemical luminescence performance. Preferably, the preparation method of the above CdTe / TiO2 NFs is:

[0014] 1) Preparation of CdTe QDs:

[0015] 35mL~45mL H2O and 90mg~95mg CdCl2 were added to a 250mL three-necked flask and ultrasonicated until completely dissolved. Then, 120mg~125mg HMP and 65μL~70μL MPA were added successively under magnetic stirring and the pH value of the solution was adjusted to 10.0~11.0. Subsequently, 0.8mL~1.0mL of the pre-made NaHTe precursor solution (0.04M) was quickly injected into the solution, and the resulting mixture was continuously heated to reflux at 100℃ for 8h~10h under vigorous stirring. The preparation method of NaHTe precursor is as follows: Under N2 protection, 50.8mg NaBH4 was added to 10mL oxygen-free aqueous solution containing 50.8mg Te powder, and heated to reflux at 80℃ for 1h.

[0016] 2) Preparation of TiO2 nanoflowers (TiO2 NFs):

[0017] Take 0.8mL~1.2mL of TBOT and add 25mL~35mL of acetic acid, and dissolve it completely by ultrasound. Then, transfer the mixture to a 100mL single-necked flask and stir vigorously until a milky homogeneous solution is formed. Subsequently, transfer the solution to a 100mL polytetrafluoroethylene-lined reactor, place it in an oven, and react at 130℃~150℃ for 10h~14h. Wash it by centrifugation several times and dry it to obtain TiO2 NFs.

[0018] 3) Amination of TiO2 nanoflowers (TiO2 NFs):

[0019] Take 20mg TiO2 NFs powder in a 100mL three-necked flask, add 28mL~32mL deionized water and 1.5mL~2.5mL ethanol, and dissolve by ultrasonic. Then, add 200μL APTES and 2mL concentrated ammonia water to the mixed solution in sequence under vigorous stirring, stir at room temperature overnight and heat at 70℃ for 1h~2h. Finally, centrifuge, wash and dry to obtain the aminated TiO2 NFs.

[0020] 4) Preparation of TiO2 nanoflower loaded CdTe QDs composite materials (CdTe / TiO2 NFs):

[0021] Take a certain amount of CdTe QDs and dissolve it in distilled water (the concentration of CdTe QDs is about 4 mg mL -1 ~6 mg mL -1 ), weigh 0.05 mM EDC and 0.05 mM NHS respectively and add them to the above solution, and activate for 0.5 h to 1 h under stirring. Then add the aminated TiO2 NFs (2 mg mL -1 ~3mg mL -1), stirred vigorously for 24 h. After repeated centrifugal washing, it was redispersed in water.

[0022] The preparation method of Fe3O4@SiO2-Ab1 with blocked non-specific binding sites comprises the following steps:

[0023] 1) reacting glutaraldehyde with surface-amino-functionalized Fe3O4@SiO2 to obtain glutaraldehyde-functionalized Fe3O4@SiO2;

[0024] 2) reacting the first antibody with Fe3O4@SiO2 functionalized with glutaraldehyde, connecting the first antibody to Fe3O4@SiO2 through a cross-linking reaction of glutaraldehyde, and obtaining Fe3O4@SiO2 with the first antibody modified on the surface;

[0025] 3) Bovine serum albumin was used to block the non-specific binding sites of Fe3O4@SiO2 with the first antibody modified on the surface, thereby obtaining Fe3O4@SiO2-Ab1 with blocked non-specific binding sites.

[0026] The particle size of Fe3O4@SiO2 is 310nm-370nm, so as to obtain excellent electrochemical luminescence performance.

[0027] Preferably, the preparation method of the above-mentioned surface amino-treated Fe3O4@SiO2 is:

[0028] 1) Preparation of Fe3O4:

[0029] 0.6g-0.7g FeCl3·6H2O and 0.2g-0.25g trisodium citrate are first dissolved in 20mL-25mL ethylene glycol, and then 1.2g-1.5g sodium acetate is added under stirring. The mixture is vigorously stirred for 0.5h-1h, and then sealed in an autoclave, and the autoclave is heated at 200°C and maintained for 10h-12h, cooled to room temperature, washed, and dried to obtain Fe3O4.

[0030] 2) Preparation of amination Fe3O4@SiO2:

[0031] Ultrasonic dispersion of 0.04g-0.06g Fe3O4 in 35mL-40mL ethanol and 5mL-8mL water, pH adjusted to 9.0-10.0 with dilute ammonia, then 0.2mL-0.3mL TEOS was added under stirring, and the reaction was continued for 2.5h-3.5h. Then 0.1mL-0.15mL APTES was added, and the mixture was stirred at room temperature for 7.5h-9.5h. Finally, the product was washed and vacuum dried to obtain surface-amino-treated Fe3O4@SiO2.

[0032] 2. A method for preparing an electrochemiluminescent immunosensor of TiO2 nanoflowers loaded with CdTe quantum dots, characterized in that it comprises the following steps:

[0033] 1) adding an aqueous solution of EDC / NHS to a dispersion of CdTe / TiO2 NFs to activate the carboxylic acid groups on the surface and inside of CdTe / TiO2 NFs to obtain an activated CdTe / TiO2 NFs dispersion;

[0034] 2) adding the second antibody to the activated CdTe / TiO2 NFs dispersion, shaking and reacting at 35°C to 37°C for 1.5h to 2.5h, removing the unattached second antibody and byproducts by centrifugation to obtain CdTe / TiO2 NFs-Ab2;

[0035] 3) Mix the aminated Fe3O4@SiO2 and glutaraldehyde in water, stir and react for 5 to 7 hours, and then separate the product using an external magnet, wash and remove excess glutaraldehyde to obtain glutaraldehyde-functionalized Fe3O4@SiO2;

[0036] 4) The first antibody and the glutaraldehyde-functionalized Fe3O4@SiO2 were mixed evenly in a PBS buffer solution, and the mixture was shaken at 35°C to 37°C for 1.5h to 2.5h. The product was separated by an external magnet, and the unreacted first antibody was washed away to obtain Fe3O4@SiO2 with the first antibody modified on the surface;

[0037] 5) Incubate the Fe3O4@SiO2 surface modified with the first antibody in a PBS buffer containing bovine serum albumin at 35°C to 37°C for 0.5h to 1h to block the nonspecific binding sites, and separate the products using an external magnet to obtain Fe3O4@SiO2-Ab1 with blocked nonspecific binding sites;

[0038] 6) Fe3O4@SiO2-Ab1 and antigen are mixed in PBS buffer, oscillated at 35°C to 37°C for 1.5h to 2.5h, the product is separated by an external magnet, and the antigen that is not specifically bound to Fe3O4@SiO2-Ab1 is washed away. The obtained product is mixed with CdTe / TiO2 NFs-Ab2 obtained in step 2), and incubated at 35°C to 37°C for 1.5h to 2.5h. The product is separated by an external magnet and washed to obtain an electrochemiluminescent immunosensor of TiO2 nanoflowers loaded with CdTe quantum dots.

[0039] Among them, the preparation method of the aminated Fe3O4@SiO2 in step 3) is: disperse Fe3O4 in a mixed solvent of ethanol and water, adjust the pH to 9.0-10.0 with dilute ammonia water, add TEOS under stirring, react for 2.5h-3.5h, add APTES, stir for 7.5h-9.5h, wash and dry the product to obtain aminated Fe3O4@SiO2; the stirring reaction is carried out at room temperature for 5h-7h, and the washing liquid for washing to remove excess glutaraldehyde is PBS buffer.

[0040] The method of obtaining CdTe / TiO2 NFs-Ab2 in step 1) and step 2) is specifically as follows:

[0041] Take a certain amount of CdTe / TiO2 NFs and dissolve it in distilled water (the concentration of CdTe / TiO2 NFs is about 2 mg mL -1 ~3mg mL -1 ), weigh 0.05mM EDC and 0.05mM NHS respectively and add them to the above solution, and activate for 0.5h~1h under stirring. Then, add 0.8mL~1.2mL of the secondary antibody solution corresponding to the antigen to be detected to the obtained solution (the concentration of the second antibody in the solution is 10μg mL -1 ), gently shake in the dark, and react at 35°C to 37°C for 1.5h to 2.5h. Finally, remove the unattached second antibody and byproducts by centrifugation to obtain CdTe / TiO2 NFs-Ab2.

[0042] The method of obtaining Fe3O4@SiO2-Ab1 with blocked non-specific binding sites in step 3) to step 5) is specifically as follows:

[0043] The first antibody corresponding to the antigen to be tested is connected to Fe3O4@SiO2 through a cross-linking reaction of glutaraldehyde. In brief, 10 mg to 20 mg of aminated Fe3O4@SiO2 is fully dissolved in 8 mL to 10 mL of ultrapure water, and then 3.0 mL to 4.0 mL of 2.5 wt% glutaraldehyde solution is added to the aqueous solution of aminated Fe3O4@SiO2 and stirred at room temperature for 5 h to 7 h. After that, the product is separated by an external magnet and washed with 10 mM PBS (pH 7.4) to remove excess glutaraldehyde to obtain glutaraldehyde-functionalized Fe3O4@SiO2. The obtained product is redispersed in 8 mL to 10 mL of PBS (10 mM, pH 7.4). Subsequently, 450 μL to 500 μL of a 10 μg mL -1The first antibody solution corresponding to the antigen to be tested is added to 450 μL to 500 μL of glutaraldehyde-functionalized Fe3O4@SiO2, and the mixture is gently shaken at 35°C to 37°C for 1.5h to 2.5h. The product is separated by an external magnet, and the unreacted first antibody is washed with 10mM PBS (pH 7.4) to remove the first antibody, and Fe3O4@SiO2 with the first antibody modified on the surface is obtained. Finally, Fe3O4@SiO2 is incubated in a PBS solution containing BSA (100 μL to 120 μL, 1.0wt%) at 35°C to 37°C for 0.5h to 1h to block nonspecific binding sites, and the product is separated by an external magnet, washed with PBS, and Fe3O4@SiO2-Ab1 with blocked nonspecific binding sites is obtained, namely Fe3O4@SiO2 / Ab1 / BSA. The obtained Fe3O4@SiO2 / Ab1 / BSA was redispersed in 500 μL PBS to obtain Fe3O4@SiO2 / Ab1 / BSA solution, which was stored at 4°C for future use.

[0044] Among them, the method of obtaining the electrochemiluminescent immunosensor of TiO2 nanoflowers loaded with CdTe quantum dots in step 6) is specifically as follows: 50 μL to 70 μL of the Fe3O4@SiO2 / Ab1 / BSA solution prepared above is added to the PBS solution containing the antigen, and oscillated at 35°C to 37°C for 1.5h to 2.5h. The obtained antigen-antibody complex is carefully washed with 1mL to 2mL of 10mM PBS (pH 7.4) to remove the physically adsorbed antigen. Then, CdTe / TiO2 NFs-Ab2 is mixed with the antigen-antibody complex and incubated at 35°C to 37°C for 1.5h to 2.5h to construct a sandwich immunosensor. Finally, the obtained immunosensor is washed with PBS to remove the unbound CdTe / TiO2 NFs-Ab2, and then dispersed in 90μL to 100μL PBS solution and stored at 4°C for use.

[0045] 3. Application of electrochemiluminescent immunosensor based on TiO2 nanoflowers loaded with CdTe quantum dots in detecting antigens.

[0046] Among them, when detecting antigens, a three-electrode system is used, including a working electrode, a counter electrode and a reference electrode; K2S2O8 is used as a co-reaction reagent, and an electrochemiluminescence signal is collected by an electrochemiluminescence analyzer, which specifically includes the following steps:

[0047] 1) The working electrode polished on the suede containing aluminum oxide powder was ultrasonically cleaned in ethanol and double distilled water;

[0048] 2) adding the electrochemiluminescent immunosensor solution dropwise onto the washed working electrode surface and drying naturally at room temperature;

[0049] 3) Prepare K2S2O8 in PBS buffer as the electrolyte of the three-electrode system;

[0050] 4) Set the potential window to -1.6 V to 0 V and the scan rate to 100 mV s -1 , the high voltage of the photomultiplier tube was set to 800 V, and an electrochemiluminescence analyzer was used to collect electrochemiluminescence signals, and a linear relationship between electrochemiluminescence intensity and different concentrations of antigens was established;

[0051] 5) The antigen detection result is obtained based on the linear relationship calculation, which provides a method for quantitative detection of antigens in actual samples.

[0052] Furthermore, another aspect of the present invention provides a method for detecting antigen concentration using the above-mentioned TiO2 nanoflower-loaded CdTe quantum dot electrochemiluminescence immunosensor, the specific steps of which are as follows:

[0053] 1) Construct an electrochemiluminescent immunosensor containing carcinoembryonic antigen (CEA) of different known concentrations, using K2S2O8 as a co-reactant, and use an electrochemiluminescence analyzer to collect electrochemiluminescence signals in a three-electrode system. The potential window is set at -1.6V to 0V, and a linear relationship between electrochemiluminescence intensity and CEA concentration is established. The linear equation is I = 3127.9lg (C CEA )+698.1, and the linear correlation coefficient R is 0.995.

[0054] 2) By replacing the first antibody and the second antibody, different cancer markers can be detected. An electrochemiluminescence immunosensor containing different cancer markers with different known concentrations was constructed, and K2S2O8 was used as a co-reactant. In a three-electrode system, an electrochemiluminescence analyzer was used to collect electrochemiluminescence signals, and the potential window was set at -1.6V~0V to establish a linear relationship between the electrochemiluminescence intensity and the concentration of different cancer markers.

[0055] Preferably, in the above three-electrode system, the working electrode is a glassy carbon electrode modified by an electrochemiluminescence immunosensor, the counter electrode is a platinum wire electrode, and the reference electrode is a saturated calomel electrode; the high voltage of the photomultiplier tube of the electrochemiluminescence analyzer is set to 800 V, the applied potential is -1.6 V to 0 V, and the scanning rate is 100 mV s -1 ; The specific method of collecting electrochemiluminescence signals using an electrochemiluminescence analyzer in a three-electrode system using K2S2O8 as a co-reaction reagent is as follows: in a PBS buffer solution containing 20 mM K2S2O8 at a pH of 7 to 8, the electrochemiluminescence response of the antigen is detected by an electrochemiluminescence analyzer.

[0056] Beneficial effects: Compared with the prior art, the advantages of the present invention are as follows:

[0057] (1) The present invention loads CdTe QDs onto the surface and interior of TiO2 nanoflowers to prepare CdTe / TiO2NFs composite materials. Due to the large specific surface area of ​​TiO2 NFs on the enrichment effect of CdTe QDs and the effect of TiO2NFs on CdTe QDs / S2O8 2- Due to the catalytic effect of the co-reaction system, the electrochemiluminescence intensity of CdTe / TiO2 NF composite materials is significantly improved.

[0058] (2) The present invention uses CdTe / TiO2 NFs composite materials as ECL signal markers to construct a signal amplification immunosensor, which significantly improves the sensitivity of antigen detection.

[0059] (3) The present invention prepares magnetic Fe3O4@SiO2 nanomaterials and uses the magnetic Fe3O4@SiO2 nanomaterials as carriers of electrochemiluminescent immunosensors. The magnetic Fe3O4@SiO2 nanomaterials have the advantages of large specific surface area, superparamagnetism and good biocompatibility, which is conducive to fixing a large amount of luminescent materials on its surface and amplifying the electrochemiluminescent signals.

[0060] (4) The present invention can achieve simple and rapid separation with the help of an external magnet, avoid nonspecific adsorption, and simplify the experimental steps.

[0061] (5) The present invention constructs an electrochemiluminescence immunosensor based on the interaction between antigen and antibody. The sensor has the characteristics of high sensitivity, strong specificity, good stability, low detection limit, wide linear range and simple operation.

[0062] (6) The detection range of the signal amplification electrochemiluminescence immunosensor prepared by the present invention for carcinoembryonic antigen (CEA) is 1 pg mL -1 ~100ng mL -1 , the detection limit was 0.32 pg mL -1 . BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 A process diagram for preparing CdTe / TiO2 NFs-Ab2 of the present invention;

[0064] Figure 2 This is a diagram showing the enhancement mechanism of the CdTe QDs ECL signal by TiO2 NFs of the present invention;

[0065] Figure 3 A flow chart of preparing an electrochemiluminescent immunosensor according to the present invention;

[0066] Figure 4It is an electrochemiluminescent signal diagram of the electrochemiluminescent biosensor of the present invention in the presence of different concentrations of CEA;

[0067] Figure 5 is a linear relationship diagram of the electrochemiluminescence intensity of the present invention and the logarithmic value of the CEA concentration;

[0068] Figure 6 A is the TEM characterization image of the CdTe quantum dots of the present invention, Figure 6 B is the SEM characterization image of TiO2 NFs of the present invention, Figure 6 C is the TEM characterization image of TiO2 NFs of the present invention, Figure 6 D is a TEM characterization image of the CdTe / TiO2 NFs composite material of the present invention, Figure 6 E is a high-magnification TEM characterization image of the CdTe / TiO2 NFs composite material of the present invention; Figure 6 F is a HRTEM characterization image of CdTe quantum dots on the edge flakes of the CdTe / TiO2 NFs composite material of the present invention;

[0069] Figure 7 A is the SEM characterization picture of Fe3O4 of the present invention, Figure 7 B is the SEM characterization picture of Fe3O4@SiO2 of the present invention, Figure 7 C TEM characterization diagram of Fe3O4 of the present invention, Figure 7 D TEM characterization image of Fe3O4@SiO2 of the present invention. DETAILED DESCRIPTION

[0070] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods.

[0071] Figure 4 The electrochemiluminescent signal diagram of the electrochemiluminescent biosensor of the present invention in the presence of different concentrations of CEA; wherein the concentrations of CEA are 1 pg mL -1 , 5pg mL -1 , 10pg mL -1 , 50pg mL -1 , 100pg mL -1 ,1ng mL -1 ,10ng mL -1 , 100ng mL -1 , the test was carried out in 0.1 M PBS buffer containing 20 mM K2S2O8.

[0072] Figure 5 The figure is a linear relationship diagram of the electrochemiluminescence intensity of the present invention and the logarithmic value of the CEA concentration; it can be seen that there is a good linear relationship between the two, and the correlation coefficient is 0.995.

[0073] Reagents and instruments used in the examples of the present invention: trisodium citrate dihydrate (Na3Cit·2H2O), ferric chloride hexahydrate (FeCl3·2H2O), ethylene glycol (EG), cadmium chloride (CdCl2·2.5H2O), anhydrous sodium acetate (NaAc), sodium borohydride (NaBH4), tellurium powder (Te), tetraethyl orthosilicate (TEOS), potassium persulfate (K2S2O8), concentrated ammonia water and tetrabutyl titanate (TBOT) were purchased from Shanghai Sinopharm Chemical Reagent Co., Ltd., 3-aminopropyltriethoxysilane (APTES) and 3-mercaptopropionic acid (MPA) were purchased from Aladdin Reagent, sodium hexametaphosphate (HMP) was purchased from Xilong Reagent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich. Carcinoembryonic antigen (CEA) and antibodies (Ab1 and Ab2) were purchased from Bosai Biotechnology Co., Ltd., bovine serum albumin (BSA) was purchased from Biotech Co., Ltd., human serum samples were provided by Nanjing Zhongda Hospital, and MPI-E multifunctional electrochemical and chemiluminescence analysis system was purchased from Xi'an Ruimai Analytical Instrument Co., Ltd.

[0074] For omitted parts in the embodiments of the present invention, please refer to the invention content of the specification and will not be described again.

[0075] Embodiment 1:

[0076] The sensor includes a signal unit CdTe / TiO2 NFs-Ab2, Fe3O4@SiO2-Ab1 with blocked nonspecific binding sites, and antigens connected to CdTe / TiO2 NFs-Ab2 and Fe3O4@SiO2-Ab1 respectively through antigen-antibody interactions; CdTe / TiO2 NFs-Ab2 is the second antibody corresponding to the antigen labeled with CdTe / TiO2 NFs; CdTe / TiO2 NFs are TiO2 NFs loaded with CdTe QDs; Fe3O4@SiO2-Ab1 is the first antibody Fe3O4@SiO2 with surface chemical modification corresponding to the antigen, and Fe3O4@SiO2 is Fe3O4 coated with SiO2.

[0077] 1. Preparation of a second antibody corresponding to an antigen labeled with CdTe quantum dots loaded on TiO2 nanoflowers (CdTe / TiO2 NFs-Ab2), comprising the following steps:

[0078] 1) Preparation of CdTe QDs: 40mLH2O and 92mgCdCl2 were added to a 250mL three-necked flask and ultrasonicated until completely dissolved. Then, 124mgHMP and 67.5μLMPA were added successively under magnetic stirring and the pH value of the solution was adjusted to 10. Subsequently, 0.8mL of the previously prepared NaHTe precursor solution (0.04M) was quickly injected into the solution, and the resulting mixture was heated to reflux at 100°C for 10h under vigorous stirring. Among them, the preparation method of the NaHTe precursor is as follows: Under N2 protection, 50.8mg NaBH4 was added to 10mL of oxygen-free aqueous solution containing 50.8mg Te powder, and heated to reflux at 80°C for 1h.

[0079] 2) Preparation of TiO2 nanoflowers (TiO2 NFs): Take 1 mL of TBOT and add 30 mL of acetic acid, and dissolve it completely by ultrasound. Then, transfer the mixture to a 100 mL single-necked flask and stir vigorously until a milky homogeneous solution is formed. Subsequently, transfer the solution to a 100 mL polytetrafluoroethylene-lined reactor, place it in an oven, and react at 140°C for 12 hours. Wash by centrifugation several times and dry to obtain TiO2 NFs.

[0080] 3) Preparation of aminated TiO2 NFs: 20 mg of TiO2 NFs powder was placed in a 100 mL three-necked flask, 30 mL of deionized water and 2 mL of ethanol were added, and the mixture was dissolved by ultrasonication. Then, 200 μL of APTES and 2 mL of concentrated ammonia were added to the mixed solution in sequence under vigorous stirring, and the mixture was stirred at room temperature overnight and heated at 70°C for 2 h. Finally, the mixture was centrifuged, washed, and dried to obtain aminated TiO2 NFs.

[0081] 4) Preparation of TiO2 nanoflower loaded CdTe QDs composite material (CdTe / TiO2 NFs): Take a certain amount of CdTe QDs and dissolve it in distilled water (CdTe QDs concentration is about 5 mg mL -1 ), 0.05 mM EDC and 0.05 mM NHS were weighed and added to the above solution, and activated for 1 h under stirring. Then, the aminated TiO2NFs (3 mg mL -1 ), stirred vigorously for 24 h. After repeated centrifugal washing, it was redispersed in water.

[0082] 5) Preparation of CdTe / TiO2 NFs-Ab2: Take a certain amount of CdTe / TiO2 NFs and dissolve it in distilled water (the concentration of CdTe / TiO2NFs is about 3 mg mL -1), weigh 0.05 mM EDC and 0.05 mM NHS respectively and add them to the above solution, and activate for 1 hour under stirring. Then, add 1 mL of the second antibody solution corresponding to the antigen to be tested to the obtained solution (the concentration of the second antibody in the solution is 10 μg mL -1 ), gently shake in the dark, and react at 37°C for 2h. Finally, remove the unattached secondary antibody and byproducts by centrifugation to obtain CdTe / TiO2 NFs-Ab2. The complete preparation process of CdTe / TiO2 NFs-Ab2 is shown in the attached Figure 1 The enhancement mechanism of TiO2NFs on the electrochemiluminescence of CdTe QDs is shown in the attached Figure 2 The electron microscopy images of CdTe QDs, TiO2 NFs and CdTe / TiO2 NFs are shown in the attached Figure 6 .

[0083] 2. Preparation of magnetic separation carrier Fe3O4@SiO2 / Ab1 / BSA:

[0084] 1) Preparation of Fe3O4: 0.64 g FeCl3·6H2O and 0.2 g trisodium citrate were first dissolved in 20 mL ethylene glycol, and then 1.2 g sodium acetate was added under stirring. The mixture was vigorously stirred for 0.5 h, and then sealed in an autoclave, and the autoclave was heated at 200°C and maintained for 12 h, cooled to room temperature, washed, and dried to obtain Fe3O4.

[0085] 2) Preparation of amination-modified Fe3O4@SiO2: 0.05 g Fe3O4 was ultrasonically dispersed in 35 mL ethanol and 5 mL water, the pH was adjusted to 9.0 with dilute ammonia, and then 0.2 mL TEOS was added under stirring, and the reaction was continued for 3 h. Then 0.15 mL APTES was added, and the mixture was stirred at room temperature for 8 h. Finally, the product was washed and vacuum dried to obtain surface-modified Fe3O4@SiO2.

[0086] 3) Preparation of Fe3O4@SiO2-Ab1: The first antibody corresponding to the antigen to be tested is connected to Fe3O4@SiO2 through a cross-linking reaction of glutaraldehyde. In brief, 20 mg of amino-treated Fe3O4@SiO2 was fully dissolved in 10 mL of ultrapure water, and then 3 mL of 2.5 wt% glutaraldehyde solution was added to the aqueous solution of amino-treated Fe3O4@SiO2 and stirred at room temperature for 7 h. After that, the product was separated with an external magnet and washed with 10 mM PBS (pH 7.4) to remove excess glutaraldehyde to obtain glutaraldehyde-functionalized Fe3O4@SiO2. The obtained product was redispersed in 10 mL of PBS (10 mM, pH 7.4). Subsequently, 500 μL of a 10 μg mL -1The first antibody solution corresponding to the antigen to be tested was added to 500 μL of glutaraldehyde-functionalized Fe3O4@SiO2, and the mixture was gently shaken for 2 h at 37°C. The product was separated by an external magnet, and the unreacted first antibody was removed by washing with 10 mM PBS (pH 7.4) to obtain Fe3O4@SiO2 with the first antibody modified on the surface (Fe3O4@SiO2-Ab1).

[0087] 4) Blocking non-specific sites: Incubate Fe3O4@SiO2-Ab1 in a PBS solution containing BSA (100 μL, 1.0 wt%) at 37°C for 1 h to block non-specific binding sites. Use an external magnet to separate the product, wash with PBS, and obtain Fe3O4@SiO2-Ab1 with blocked non-specific binding sites, i.e., Fe3O4@SiO2 / Ab1 / BSA. Redisperse the obtained Fe3O4@SiO2 / Ab1 / BSA in 500 μL PBS to obtain Fe3O4@SiO2 / Ab1 / BSA solution, store at 4°C for later use. See the attached electron microscopy characterization diagram of Fe3O4 and Fe3O4@SiO2 for details. Figure 7 .

[0088] 3. Preparation of electrochemiluminescent immunosensor based on TiO2 nanoflowers loaded with CdTe quantum dots:

[0089] 60 μL of Fe3O4@SiO2 / Ab1 / BSA solution was added to the solution containing different concentrations of CEA (1 pg mL -1 , 5pgmL -1 , 10pg mL -1 , 50pg mL -1 , 100pg mL -1 , 1ng mL -1 ,10ng mL -1 , 100ng mL -1 ) in a PBS solution and shaken at 37°C for 2h. The obtained antigen-antibody complex was carefully washed with 10mM PBS (pH 7.4) to remove the physically adsorbed antigen. Then, CdTe / TiO2 NFs-Ab2 was mixed with the antigen-antibody complex and incubated at 37°C for 2h to construct a sandwich immunosensor. Finally, the obtained immunosensor was washed with PBS to remove unbound CdTe / TiO2 NFs-Ab2, and then dispersed in 100μL PBS solution and stored at 4°C for future use. The preparation of this immunosensor is shown in the attached Figure 3 .

[0090] 4. Detection of CEA at different concentrations by electrochemiluminescence immunosensor loaded with TiO2 nanoflowers and CdTe quantum dots:

[0091] The detection adopted a three-electrode system, with the working electrode being a glassy carbon electrode modified by an electrochemiluminescence immunosensor, the counter electrode being a platinum wire electrode, and the reference electrode being a saturated calomel electrode. 20 mM K2S2O8 was used as a co-reactant, and the high voltage of the photomultiplier tube of the electrochemiluminescence analyzer was set to 800 V, with a scan rate of 100 mV s -1 , the potential window is set to -1.6~0V, and the electrochemiluminescence signal is collected by an electrochemiluminescence analyzer. The specific steps are:

[0092] 1) The glassy carbon electrode (GCE) polished on the suede containing 0.3μm and 0.05μm alumina powder was ultrasonically cleaned in ethanol and double distilled water; 2) 6μL of biosensor solution was added to the polished GCE electrode surface and dried naturally at room temperature; 3) 0.1M PBS buffer containing 20mM K2S2O8 was prepared as the electrolyte of the three-electrode system. 4) The potential window was set to -1.6V~0V and the scan rate was set to 100mV s -1 The high voltage of the photomultiplier tube was set to 800 V, and the electrochemiluminescence signal was collected by an electrochemiluminescence analyzer to obtain the ECL response of the immunosensor under different CEA concentrations, as shown in the attached figure. Figure 3 The linear relationship between electrochemiluminescence signal and CEA is shown in the attached Figure 4 , where the linear equation is I = 3127.9lg (C CEA )+698.1, the linear correlation coefficient R was 0.995, and the detection limit was 0.32pg mL -1 .

[0093] Five: Effect analysis:

[0094] The present invention loads CdTe QDs onto the surface and interior of TiO2 nanoflowers to prepare CdTe / TiO2NFs composite materials. Due to the enrichment effect of TiO2 NFs with a larger specific surface area on CdTe QDs and the effect of TiO2NFs on CdTe QDs / S2O8 2-Due to the catalytic effect of the co-reaction system, the electrochemiluminescence intensity of the CdTe / TiO2 NF composite material is significantly improved. Furthermore, the present invention uses the CdTe / TiO2 NFs composite material as an ECL signal marker to construct a signal amplification immunosensor, which significantly improves the sensitivity of antigen detection. In addition, the present invention prepares magnetic Fe3O4@SiO2 nanomaterials as carriers of electrochemiluminescence immunosensors. The magnetic Fe3O4@SiO2 nanomaterials have the advantages of large specific surface area, superparamagnetism and good biocompatibility, which are conducive to fixing a large amount of luminescent materials on its surface and amplifying electrochemiluminescence signals. In the process of constructing the biosensor, the present invention can achieve simple and rapid separation with the help of external magnets, avoid nonspecific adsorption, and simplify the experimental steps. Based on this, the present invention uses the interaction between antigens and antibodies to construct an electrochemiluminescence immunosensor, which has the characteristics of high sensitivity, strong specificity, good stability, low detection limit, wide linear range, simple operation, etc. Its detection range for CEA is 1pg mL -1 ~100ng mL -1 , the detection limit was 0.32 pg mL -1 .

Claims

1. An electrochemiluminescent immunosensor of TiO2 nanoflowers loaded with CdTe quantum dots, characterized in that: The sensor includes a signal unit CdTe / TiO2 NFs-Ab2, Fe3O4@SiO2-Ab1 with blocked non-specific binding sites, and antigens connected to CdTe / TiO2 NFs-Ab2 and Fe3O4@SiO2-Ab1 respectively through antigen-antibody interactions; the CdTe / TiO2 NFs-Ab2 is a second antibody corresponding to the antigen labeled by CdTe / TiO2 NFs; the CdTe / TiO2 NFs is TiO2 NFs loaded with CdTe QDs; the Fe3O4@SiO2-Ab1 is a first antibody Fe3O4@SiO2 with a surface chemically modified with the antigen corresponding to the Fe3O4@SiO2, and the Fe3O4@SiO2 is Fe3O4 coated with SiO2; The preparation method of CdTe / TiO2 NFs-Ab2 comprises the following steps: 1) Activate the carboxylic acid groups on the surface of CdTe / TiO2 NFs; 2) reacting the second antibody with the carboxylic acid groups on the surface and inside of the activated CdTe / TiO2 NFs to obtain the second antibody corresponding to the antigen labeled by the CdTe / TiO2 NFs; The preparation method of Fe3O4@SiO2-Ab1 with blocked non-specific binding sites comprises the following steps: 1) reacting glutaraldehyde with surface-amino-functionalized Fe3O4@SiO2 to obtain glutaraldehyde-functionalized Fe3O4@SiO2; 2) reacting the first antibody with Fe3O4@SiO2 functionalized with glutaraldehyde, connecting the first antibody to Fe3O4@SiO2 through a cross-linking reaction of glutaraldehyde, and obtaining Fe3O4@SiO2 with the first antibody modified on the surface; 3) Bovine serum albumin was used to block the non-specific binding sites of Fe3O4@SiO2 with the first antibody modified on the surface, thereby obtaining Fe3O4@SiO2-Ab1 with blocked non-specific binding sites.

2. The electrochemiluminescent immunosensor of TiO2 nanoflowers loaded with CdTe quantum dots according to claim 1, characterized in that: The particle size of the CdTe / TiO2 NFs is 1.34 μm to 1.41 μm.

3. The electrochemiluminescent immunosensor of TiO2 nanoflowers loaded with CdTe quantum dots according to claim 1, characterized in that: The particle size of the Fe3O4@SiO2 is 310nm-370nm.

4. A method for preparing an electrochemiluminescent immunosensor of TiO2 nanoflowers loaded with CdTe quantum dots according to claim 1, characterized in that: The following steps are involved: 1) adding an aqueous solution of EDC / NHS to a dispersion of CdTe / TiO2 NFs to activate the carboxylic acid groups on the surface and inside of CdTe / TiO2 NFs to obtain an activated CdTe / TiO2 NFs dispersion; 2) adding the second antibody to the activated CdTe / TiO2 NFs dispersion, shaking and reacting at 35°C to 37°C for 1.5h to 2.5h, removing the unattached second antibody and byproducts by centrifugation to obtain CdTe / TiO2 NFs-Ab2; 3) Mix the aminated Fe3O4@SiO2 and glutaraldehyde in water, stir and react for 5 to 7 hours, and then separate the product using an external magnet, wash and remove excess glutaraldehyde to obtain glutaraldehyde-functionalized Fe3O4@SiO2; 4) The first antibody and the glutaraldehyde-functionalized Fe3O4@SiO2 were mixed evenly in a PBS buffer solution, and the mixture was shaken at 35°C to 37°C for 1.5h to 2.5h. The product was separated by an external magnet, and the unreacted first antibody was washed away to obtain Fe3O4@SiO2 with the first antibody modified on the surface; 5) Incubate the Fe3O4@SiO2 surface modified with the first antibody in a PBS buffer containing bovine serum albumin at 35°C to 37°C for 0.5h to 1h to block the nonspecific binding sites, and separate the products using an external magnet to obtain Fe3O4@SiO2-Ab1 with blocked nonspecific binding sites; 6) Fe3O4@SiO2-Ab1 and antigen are mixed in PBS buffer, oscillated at 35°C to 37°C for 1.5h to 2.5h, the product is separated by an external magnet, and the antigen that is not specifically bound to Fe3O4@SiO2-Ab1 is washed away. The obtained product is mixed with CdTe / TiO2 NFs-Ab2 obtained in step 2), and incubated at 35°C to 37°C for 1.5h to 2.5h. The product is separated by an external magnet and washed to obtain an electrochemiluminescent immunosensor of TiO2 nanoflowers loaded with CdTe quantum dots.

5. The preparation method according to claim 4, characterized in that: The preparation method of the aminated Fe3O4@SiO2 in the step 3) is as follows: disperse Fe3O4 in a mixed solvent of ethanol and water, adjust the pH to 9.0-10.0 with dilute ammonia water, add TEOS under stirring, react for 2.5h-3.5h, add APTES, stir for 7.5h-9.5h, wash and dry the product to obtain the aminated Fe3O4@SiO2; the stirring reaction is carried out at room temperature for 5h-7h, and the washing solution for washing to remove excess glutaraldehyde is PBS buffer.

6. An application of the electrochemiluminescent immunosensor comprising TiO2 nanoflowers loaded with CdTe quantum dots according to claim 1 in detecting antigens.

7. The use in detecting antigens according to claim 6, characterized in that: When detecting antigens, a three-electrode system is used, including a working electrode, a counter electrode and a reference electrode; K2S2O8 is used as a co-reactant, and an electrochemiluminescence analyzer is used to collect electrochemiluminescence signals, which specifically includes the following steps: 1) The working electrode polished on the suede containing aluminum oxide powder was ultrasonically cleaned in ethanol and double distilled water; 2) adding the electrochemiluminescent immunosensor solution dropwise onto the washed working electrode surface and drying naturally at room temperature; 3) Prepare K2S2O8 in PBS buffer as the electrolyte of the three-electrode system; 4) Set the potential window to -1.6 V to 0 V and the scan rate to 100 mV s -1 , the high voltage of the photomultiplier tube was set to 800 V, and an electrochemiluminescence analyzer was used to collect electrochemiluminescence signals, and a linear relationship between electrochemiluminescence intensity and different concentrations of antigens was established; 5) Obtain the antigen detection result based on the linear relationship calculation.

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