A fluorescence resonance energy transfer sensor based on gold nanoflowers and time-resolved fluorescent microspheres, its fabrication method and application.

Abstract

This invention provides a fluorescence resonance energy transfer sensor based on gold nanoflowers and time-resolved fluorescent microspheres, its preparation method, and its application. The method includes using gold nanoflowers with a multi-branched structure labeled with a monoclonal antibody against cadmium as the receptor, and time-resolved fluorescent microspheres with time-resolved fluorescence properties labeled with a cadmium antigen. Relying on the specific recognition between the antigen and antibody, the gold nanoflower-antibody conjugate can quench the fluorescence of the time-resolved fluorescent microsphere-antigen conjugate through the fluorescence resonance energy transfer principle. By preparing cadmium ion standard solutions of different concentrations, a standard curve is established to show the change in fluorescence intensity with cadmium ion concentration. Tea samples are pretreated and added to the system as the test solution. By measuring the time-resolved fluorescence signal intensity, the cadmium ion concentration in the tea is calculated based on the standard curve. This achieves higher sensitivity and faster high-throughput detection of cadmium ions, providing a basis for the development of commercial reagents.

Description

Technical Field

[0001] This invention belongs to the field of rapid detection technology of heavy metal ions, specifically relating to the construction and application of a fluorescence resonance energy transfer (FRET) sensor based on gold nanoflowers and time-resolved fluorescent microspheres, that is, using the FRET sensor of gold nanoflowers and time-resolved fluorescent microspheres to rapidly detect cadmium ions in food. Background Technology

[0002] Cadmium is a common heavy metal, usually existing in the form of cadmium ions. Affected by human activities such as industrial development, agricultural activities, and the combustion of fossil fuels, heavy metals released into the environment can further enter the biosphere cycle through the atmosphere, soil, and water. Since tea trees thrive in acidic soil, if the soil where tea trees are planted is contaminated with heavy metals, the acidic environment further facilitates the conversion of heavy metals into bioavailable forms, increasing the absorption of heavy metals by the tea tree roots and making it easier for heavy metals to migrate from the soil to the tea leaves. Cadmium ion pollution in tea not only leads to a decline in tea yield and quality but also endangers human health through direct contact or bioaccumulation in the food chain. Studies have shown that heavy metals can cause damage to organs such as the lungs, kidneys, liver, blood, gastrointestinal tract, and bones, thereby inducing hepatotoxicity, nephrotoxicity, carcinogenicity, teratogenicity, and reproductive toxicity. Tea contaminated with heavy metals not only weakens the competitiveness of tea-producing areas but also has a certain impact on the import and export market for tea. Traditional heavy metal detection techniques include atomic absorption spectrometry, atomic fluorescence spectrometry, and inductively coupled plasma atomic absorption spectrometry, which require complex and expensive large-scale instruments and experienced operators. In contrast, fluorescence immunoassay is simple to operate, highly specific, and has a short detection time, making it an important rapid detection method.

[0003] Time-resolved fluorescence immunoassay (TRFI) combines time-resolved fluorescence with immunological techniques to achieve quantitative labeling and fluorescence reading of antigens and antibodies. It leverages the advantages of time-resolved fluorescent materials, such as long fluorescence lifetime (typically in the μs-ms range), high quantum yield, narrow emission bandwidth, and large Stokes shift, while also possessing strong immunological specificity, high sensitivity, and fast response. Gold nanoflowers and time-resolved fluorescent microspheres exhibit excellent optical properties and tunable size, facilitating conjugation with proteins such as antigens and antibodies. Furthermore, the UV absorption spectrum of gold nanoflowers and the emission spectrum of time-resolved fluorescent microspheres show significant overlap, providing conditions for FRET (Fluorescence Resonance Energy Transfer) to occur, thus making them suitable for constructing FRET detection systems. Currently, there are no reports on the construction of a fluorescence resonance energy transfer system based on gold nanoflowers and time-resolved fluorescent microspheres, nor its application in the detection of cadmium ions in tea. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres, along with its preparation method and applications. This detection system uses a gold nanoflower-labeled monoclonal antibody against cadmium as the receptor, and time-resolved fluorescent microspheres with time-resolved fluorescence properties to label the cadmium antigen. Relying on the specific recognition between the antigen and antibody, a standard curve can be constructed based on the relationship between cadmium ion concentration and fluorescence intensity under the principle of fluorescence resonance energy transfer. This detection method can effectively shorten the detection time of heavy metal ions and has high sensitivity, achieving high-throughput detection of cadmium ions in tea.

[0005] To achieve the above objectives, the solution of the present invention is:

[0006] The method for fabricating the FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres of the present invention includes the following steps:

[0007] (1) Preparation of gold nanoflower-cadmium antibody conjugate;

[0008] (2) Preparation of time-resolved fluorescent microsphere-cadmium antigen conjugate;

[0009] (3) Add gold nanoflower-cadmium antibody conjugate and time-resolved fluorescent microsphere-cadmium antigen conjugate to a black 96-well plate, mix well, and construct a FRET sensor based on gold nanoflower-time-resolved fluorescent microsphere.

[0010] As a preferred embodiment of the present invention, in step (1), the preparation method of the gold nanoflower-cadmium antibody conjugate includes:

[0011] Add sodium citrate to boiling chloroauric acid solution. When the color turns orange-red, keep heating until the solution color no longer changes. Take this solution and mix it with filtered water, chloroauric acid solution, sodium citrate solution and hydroquinone solution to obtain gold nanoflowers.

[0012] After concentrating the gold nanoflowers, the pH was adjusted to obtain a concentrated gold nanoflower solution. Then, a certain amount of heavy metal cadmium monoclonal antibody was added to carry out the conjugation reaction. Bovine serum albumin (BSA) solution was added to block the unbound sites. The conjugate was centrifuged and dissolved in ultrapure water for storage, thus obtaining the gold nanoflower-heavy metal cadmium antibody conjugate.

[0013] In a preferred embodiment of the present invention, the particle size of the gold nanoflowers is 80-100 nm.

[0014] In a preferred embodiment of the present invention, the OD value of the concentrated gold nanoflower is 10-30.

[0015] In a preferred embodiment of the present invention, the pH value of the gold nanoflower concentrated solution is 7-9.

[0016] In a preferred embodiment of the present invention, the immunogen Cd-ITCBE-BSA is emulsified and injected into mice. After polyclonal antibody titer determination, cell fusion, and hybridoma cell screening, the hybridoma cells are injected into the peritoneal cavity of unimmunized mice to obtain ascites. After dialysis, the heavy metal cadmium monoclonal antibody is obtained. The amount of heavy metal cadmium monoclonal antibody added is 5-20 μg.

[0017] In a preferred embodiment of the present invention, the coupling reaction temperature is 20-40°C and the reaction time is 0.5-5h.

[0018] In a preferred embodiment of the present invention, the centrifugation speed is 3000-8000 rpm and the time is 2-10℃.

[0019] In a preferred embodiment of the present invention, in step (2), the time-resolved fluorescent microspheres are europium time-resolved fluorescent microspheres with a diameter of 50-350 nm.

[0020] As a preferred embodiment of the present invention, in step (2), the preparation method of the time-resolved fluorescent microsphere-cadmium antigen conjugate includes:

[0021] Time-resolved fluorescent microspheres were diluted with phosphate buffer and sonicated. Carbodiimide hydrochloride solution was added for activation. After centrifugation, the supernatant was discarded. Cadmium antigen was added for coupling. After centrifugation, the supernatant was discarded. BSA blocking solution was added to block unbound sites. After constant temperature and shaking, the reaction was centrifuged and the supernatant was discarded. The mixture was dissolved in phosphate buffer and stored under cold for later use.

[0022] In a preferred embodiment of the present invention, the concentration of the phosphate buffer is 0.01-0.05 mol / L; the pH of the phosphate buffer is 6-8.

[0023] In a preferred embodiment of the present invention, the final concentration of the carbodiimide hydrochloride solution in the reaction solution is 0.2-0.4 μg / μL.

[0024] In a preferred embodiment of the present invention, the activation reaction time is 20-45 min and the reaction temperature is 30-40℃.

[0025] In a preferred embodiment of the present invention, the centrifugation speed is 12000-16000 rpm; the centrifugation time is 30-50 min.

[0026] In a preferred embodiment of the present invention, after mixing and reacting cadmium nitrate solution with ITCBE solution, it is coupled with ovalbumin, and after dialysis, the heavy metal cadmium antigen is obtained. The amount of heavy metal cadmium antigen added is 12-80 μg / mL.

[0027] In a preferred embodiment of the present invention, the coupling time is 30-45 minutes and the coupling temperature is 2-8°C.

[0028] In a preferred embodiment of the present invention, the sealing time is 20-45 minutes and the sealing temperature is 30-40°C.

[0029] In a preferred embodiment of the present invention, in step (3), the amount of gold nanoflower-cadmium antibody conjugate added is 30-48 μL, and the amount of time-resolved fluorescent microsphere-cadmium antigen conjugate added is 0.5-15 μL.

[0030] This invention provides a FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres, which is obtained by the above-described preparation method.

[0031] The present invention also provides an application of the above-mentioned FRET sensor based on gold nanoflowers-time-resolved fluorescent microspheres in the detection of cadmium ions in food.

[0032] As a preferred embodiment of the present invention, the process of detecting cadmium ions in tea leaves using a FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres includes the following steps:

[0033] (a) Prepare standard solutions of cadmium ions of different concentrations using reaction buffer, mix them with a FRET sensor based on gold nanoflower-time-resolved fluorescent microspheres, measure the fluorescence values, and construct a standard curve.

[0034] (b) The tea leaves were pretreated and added to the FRET sensor based on gold nanoflower time-resolved fluorescent microspheres, and the concentration of cadmium ions was calculated according to the standard curve.

[0035] As a preferred embodiment of the present invention, in step (a), the method for constructing the standard curve is as follows: a cadmium ion standard solution is prepared into a stock solution using a reaction buffer, and the stock solution is gradient diluted to different concentrations of cadmium ion standard solutions using the reaction buffer. The stock solution is then mixed with a gold nanoflower-time-resolved fluorescent microsphere FRET sensor at 30-40°C for 20-60 min, and the fluorescence signal is detected. The fluorescence signal value without the addition of cadmium ion standard solution is F0, and the fluorescence signal value with the addition of cadmium ion standard solution is F. The standard curve is fitted using (F-F0) / F0 as the ordinate.

[0036] As a preferred embodiment of the present invention, the different concentrations of the cadmium ion standard solution are 0 ng / mL, 0.25 ng / mL, 1 ng / mL, 5 ng / mL, 25 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL.

[0037] In a preferred embodiment of the present invention, the reaction buffer is tris(hydroxymethyl)aminomethane hydrochloride buffer.

[0038] In a preferred embodiment of the present invention, the pH of the reaction buffer is 7.0-9.0.

[0039] In a preferred embodiment of the present invention, the concentration of the reaction buffer is 0.01-0.2 mol / L.

[0040] In a preferred embodiment of the present invention, the concentration of sodium chloride in the reaction buffer is 0-100 mmol / L.

[0041] In a preferred embodiment of the present invention, the concentration of ethylenediaminetetraacetic acid in the reaction buffer is 0.5-5 mmol / L.

[0042] In a preferred embodiment of the present invention, in step (b), 1g of dried tea powder is mixed with acid solution for digestion, the supernatant is collected by centrifugation, the pH is adjusted and diluted to obtain the tea sample to be tested. The tea sample is added to the FRET sensor of gold nanoflower-time-resolved fluorescent microspheres and the fluorescence signal is detected. The fluorescence signal value is recorded as F1, and the fluorescence signal value of the reaction buffer is F2. The cadmium ion content in the tea is calculated by (F1-F2) / F2 and the standard curve of step (a).

[0043] In a preferred embodiment of the present invention, the acid solution is a mixture of concentrated nitric acid and 30% hydrogen peroxide in a volume ratio of 5-3:1, and the digestion time is 0.5-10h.

[0044] Due to the adoption of the above solution, the beneficial effects of the present invention are:

[0045] (1) The time-resolved fluorescence used in this invention has the characteristics of long fluorescence lifetime and high quantum yield. After setting a certain delay time, the short-lived background fluorescence cannot be detected and only the long-lived time-resolved fluorescence can be detected, thus effectively filtering out background interference. At the same time, the surface of the time-resolved fluorescent microspheres can be modified with carboxyl groups to couple with proteins to prepare conjugates, so that they have both the optical properties of fluorescent materials and the immune properties of immune proteins.

[0046] (2) Gold nanoflowers can couple with proteins via gold-sulfur bonds. Their multi-branched surface increases the contact area with proteins, thus improving coupling efficiency. The structure of gold nanoflowers is tunable, and their ultraviolet absorption spectrum can highly overlap with the fluorescence spectrum of time-resolved fluorescent microspheres. After coupling, under the specific action of antigen and antibody, the distance between the two conjugates can be effectively shortened, which is conducive to the occurrence of fluorescence resonance energy transfer.

[0047] (3) The FRET sensor method based on gold nanoflower-time-resolved fluorescent microspheres constructed in this invention is simple. By establishing the relationship between the concentration of the analyte and the fluorescence intensity, it can detect cadmium ions in food. The detection speed is fast and the sensitivity is high. With the help of a 96-well plate, a large number of samples can be detected simultaneously, which helps to quickly screen heavy metals in the food industry. Specifically, it can be applied to the detection of cadmium ions in tea, which has important practical significance and application value in tea quality control.

[0048] (4) By preparing cadmium ion standard solutions of different concentrations, a standard curve of fluorescence intensity versus cadmium ion concentration was established. Tea samples were pretreated to replace the cadmium ion standard solutions. The concentration of cadmium ions in the tea was calculated based on the standard curve by measuring the time-resolved fluorescence signal intensity, achieving higher sensitivity and faster high-throughput detection of the heavy metal cadmium ions. This invention utilizes the immunoreaction and fluorescence resonance energy transfer between gold nanoflowers and time-resolved fluorescent microspheres to achieve rapid detection of heavy metal cadmium ions, effectively improving the sensitivity of fluorescence immunoassay and overcoming the drawbacks of cumbersome operation and long detection time of traditional fluorescence-linked immunoassay reactions, providing a basis for the development of commercial reagents. Attached Figure Description

[0049] Figure 1 This is a schematic diagram of the FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres according to the present invention.

[0050] Figure 2 The images are transmission electron microscopy (TEM) images of gold nanoflowers before (A) and after (B) coupling with a heavy metal cadmium antibody in Example 1 of this invention.

[0051] Figure 3 The images are transmission electron microscopy (TEM) images of the time-resolved fluorescent microspheres in Example 1 of this invention before (A) and after (B) coupling with the heavy metal cadmium antigen.

[0052] Figure 4 The images show the zeta potentials of gold nanoflowers before and after coupling with cadmium antibody and time-resolved fluorescent microspheres before and after coupling with cadmium antigen in Example 1 of this invention.

[0053] Figure 5 The images show the ultraviolet absorption spectrum of the gold nanoflowers and the fluorescence spectrum of the time-resolved fluorescent microspheres in Example 1 of this invention.

[0054] Figure 6 This is a graph showing the change in time-resolved fluorescence intensity of the FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres in Example 1 of the present invention as the cadmium ion concentration changes.

[0055] Figure 7 This is a fluorescence standard curve for detecting cadmium ions in Example 1 of the present invention. Detailed Implementation

[0056] This invention provides a FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres, its preparation method, and its application.

[0057] like Figure 1 As shown, the method for constructing the FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres of the present invention includes the following steps:

[0058] (1) Preparation of gold nanoflower-cadmium antibody conjugate;

[0059] (2) Preparation of time-resolved fluorescent microsphere-cadmium antigen conjugate;

[0060] (3) Add gold nanoflower-cadmium antibody conjugate and time-resolved fluorescent microsphere-cadmium antigen conjugate to a black 96-well plate, mix well, and construct a FRET sensor based on gold nanoflower-time-resolved fluorescent microsphere.

[0061] In step (1), the preparation method of gold nanoflower-cadmium antibody conjugate includes: adding sodium citrate to boiling chloroauric acid solution, maintaining heating until the color of the solution no longer changes after the color turns orange-red, and mixing the solution with filtered water, chloroauric acid solution, sodium citrate solution and hydroquinone solution to obtain gold nanoflower.

[0062] Take 1-5 mL of concentrated gold nanoflower, adjust the pH of the solution with 0.1 mol / L K2CO3, then add a certain amount of heavy metal cadmium monoclonal antibody, carry out the coupling reaction, and add 100 μL of 10% (w / v) BSA solution to block the unbound sites. Centrifuge the conjugate at 5000 rpm and 4℃, dissolve it in 1-5 mL of ultrapure water, and store it for later use.

[0063] Specifically, the OD value of concentrated gold nanoflowers can be 10-30, preferably 20.

[0064] The pH value of the concentrated solution of gold nanoflower can be 7-9, preferably 8.5.

[0065] Immunogen Cd-ITCBE-BSA is emulsified and injected into mice. After polyclonal antibody titer determination, cell fusion, and hybridoma cell screening, the hybridoma cells are injected into the peritoneal cavity of non-immunized mice to obtain mouse ascites. After dialysis, heavy metal cadmium monoclonal antibody is obtained. The amount of heavy metal cadmium monoclonal antibody added can be 5-20 μg, preferably 10 μg.

[0066] The coupling reaction temperature can be 20-40℃, preferably 36℃; the reaction time can be 0.5-5h, preferably 2h.

[0067] The particle size of gold nanoflowers can be 80-100 nm, preferably 83 nm.

[0068] In step (2), the preparation method of the time-resolved fluorescent microsphere-heavy metal cadmium antigen conjugate includes: diluting 20 μL of time-resolved fluorescent microspheres to 200 μL with phosphate buffer, sonicating for 2-5 min to mix, adding carbodiimide hydrochloride solution, activating the reaction, centrifuging and discarding the supernatant, adding heavy metal cadmium antigen, coupling reaction, centrifuging and discarding the supernatant, adding 200 μL of 1% (w / v) BSA blocking solution to block unbound sites, shaking and reacting at constant temperature, centrifuging and discarding the supernatant, dissolving in 200 μL of phosphate buffer, and storing for later use.

[0069] The concentration of the phosphate buffer can be 0.01-0.05 mol / L, preferably 0.05 mol / L; the pH of the phosphate buffer can be 6-8, preferably 7.4.

[0070] The final concentration of the carbodiimide hydrochloride solution in the reaction solution can be 0.2-0.4 μg / μL, preferably 0.3 μg / μL.

[0071] The activation reaction time can be 20-45 min, preferably 30 min; the activation temperature can be 30-40℃, preferably 36℃.

[0072] The centrifugation speed can be 12,000-16,000 rpm, preferably 14,000 rpm; the centrifugation time can be 30-50 min, preferably 45 min.

[0073] After the cadmium nitrate solution is mixed and reacted with the ITCBE solution, it is coupled with ovalbumin and then dialyzed to obtain the heavy metal cadmium antigen. The amount of heavy metal cadmium antigen added can be 12-80 μg / mL, preferably 30 μg / mL.

[0074] The coupling reaction time can be 30-45 min, preferably 30 min; the coupling temperature can be 2-8℃, preferably 4℃.

[0075] The sealing time can be 20-45 minutes, preferably 30 minutes; the sealing temperature can be 30-40℃, preferably 36℃.

[0076] The time-resolved fluorescent microspheres are europium time-resolved fluorescent microspheres, with a diameter of 50-350 nm, preferably 210 nm. These time-resolved fluorescent microspheres emit red fluorescence under ultraviolet light irradiation, with an excitation wavelength of 349 nm and an emission wavelength of 616 nm.

[0077] In step (3), the construction method of the fluorescence resonance energy transfer detection system based on gold nanoflower-time-resolved fluorescent microspheres includes: adding gold nanoflower-cadmium heavy metal antibody conjugate and time-resolved fluorescent microsphere-cadmium heavy metal antigen conjugate to a black 96-well plate, and then mixing and reacting at 37°C for 30-60 min to construct the FRET sensor based on gold nanoflower-time-resolved fluorescent microspheres.

[0078] The amount of gold nanoflower-cadmium antibody conjugate added can be 30-48 μL, preferably 45 μL; the amount of time-resolved fluorescent microsphere-cadmium antigen conjugate added can be 0.5-15 μL, preferably 5 μL.

[0079] The present invention also provides an FRET sensor based on gold nanoflower-time-resolved fluorescent microspheres obtained by the above preparation method.

[0080] Finally, this invention provides an application of the aforementioned FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres in the detection of cadmium ions in food. The FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres, constructed using gold nanoflowers and cadmium antibody conjugates and time-resolved fluorescent microspheres and cadmium antigen conjugates, can achieve rapid, high-throughput, and highly sensitive detection of cadmium ions in tea samples.

[0081] The process of detecting cadmium ions in tea leaves using a FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres includes the following steps:

[0082] (a) Prepare standard solutions of cadmium ions of different concentrations using reaction buffer, mix them with a FRET sensor based on gold nanoflower-time-resolved fluorescent microspheres, measure the fluorescence values, and construct a standard curve.

[0083] (b) The tea leaves were pretreated and added to the FRET sensor based on gold nanoflower time-resolved fluorescent microspheres, and the concentration of cadmium ions was calculated according to the standard curve.

[0084] In fact, in step (a), the standard curve for cadmium ions is constructed as follows: a 1000 mg / mL cadmium ion standard solution is prepared into a stock solution using a reaction buffer. The stock solution is then serially diluted with the reaction buffer to obtain cadmium ion standard solutions of different concentrations (0 ng / mL, 0.25 ng / mL, 1 ng / mL, 5 ng / mL, 25 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL). 100 μL of this solution is mixed with a FRET sensor of gold nanoflower-time-resolved fluorescent microspheres at 30-40℃ for 20-60 min. The fluorescence signal is detected. The fluorescence signal value of the solution without cadmium ion standard is F0, and the fluorescence signal value of the solution with cadmium ion standard is F. The standard curve is fitted using (F-F0) / F0 as the ordinate.

[0085] Specifically, the reaction buffer is tris(hydroxymethyl)aminomethane hydrochloride buffer.

[0086] The pH of the reaction buffer can be 7.0-9.0, preferably 8.0.

[0087] The concentration of the reaction buffer can be 0.01-0.2 mol / L, preferably 0.05 mol / L.

[0088] The concentration of sodium chloride in the reaction buffer can be 0-100 mmol / L, preferably 20 mmol / L.

[0089] The concentration of ethylenediaminetetraacetic acid in the reaction buffer can be 0.5-5 mmol / L, preferably 1 mmol / L.

[0090] In step (b), 1g of dried tea powder is mixed with acid solution for digestion, centrifuged at 2000rpm and 25℃, and the supernatant is collected. The pH is adjusted to 8.0 with 1mol / L sodium hydroxide solution and diluted 50-100 times with reaction buffer as the tea sample to be tested. 100μL of this tea sample is added to the FRET sensor of gold nanoflower-time-resolved fluorescent microspheres, and the fluorescence signal is detected. The fluorescence signal value is recorded as F1, and the fluorescence signal value of the reaction buffer is F2. The cadmium ion content in the tea is calculated by (F1-F2) / F2 and the standard curve of step (a).

[0091] Specifically, the acid solution is a mixture of concentrated nitric acid and 30% hydrogen peroxide, with a volume ratio of 5-3:1, preferably 3:1; the digestion time is 0.5-10h, preferably 2h.

[0092] The present invention will now be described in detail with reference to specific embodiments.

[0093] Example 1:

[0094] The process of detecting cadmium ions in tea leaves using the FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres in this embodiment includes the following steps:

[0095] (1) Preparation of gold nanoflower-cadmium antibody conjugate:

[0096] Take 1 mL of concentrated gold nanoflowers with an OD value of 20 and a particle size of 83 nm. Adjust the pH of the solution to 8.5 with 0.1 mol / L K2CO3, then add 10 μg of heavy metal cadmium monoclonal antibody. Perform the coupling reaction at 36 °C for 2 h, and add 100 μL of 10% (w / v) BSA solution to block unbound sites. Centrifuge the conjugate at 5000 rpm and 4 °C, dissolve it in 1 mL of ultrapure water, and store it for later use to obtain the gold nanoflower-heavy metal cadmium antibody conjugate.

[0097] (2) Preparation of time-resolved fluorescent microsphere-cadmium antigen conjugate:

[0098] 20 μL of europium time-resolved fluorescent microspheres (210 nm in diameter) were diluted to 200 μL with 0.05 mol / L phosphate buffer (pH 7.4) and sonicated for 2 min. Carbodiimide hydrochloride solution was added to bring the final concentration in the reaction solution to 0.3 μg / μL. After activation at 36 °C for 30 min, the supernatant was discarded after centrifugation at 14000 rpm for 45 min. Cadmium antigen with a final concentration of 30 μg / mL was added. After coupling reaction, the supernatant was discarded after centrifugation at 14000 rpm for 45 min. 200 μL of 1% (w / v) BSA solution was added to block unbound sites. After incubation and shaking, the mixture was centrifuged at 14000 rpm for 45 min and the supernatant was discarded. The mixture was dissolved in 200 μL of phosphate buffer and stored for later use.

[0099] (3) Constructing a FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres:

[0100] Add 45 μL of gold nanoflower-cadmium antibody conjugate and 5 μL of time-resolved fluorescent microsphere-cadmium antigen conjugate to a black 96-well microplate, and then mix and react at 37 °C for 30 min to obtain a gold nanoflower-time-resolved fluorescent microsphere FRET sensor.

[0101] (4) Fitting the cadmium ion standard curve:

[0102] A 1000 mg / mL cadmium ion standard solution was prepared as a stock solution using 0.05 mol / L tris(hydroxymethyl)aminomethane hydrochloride buffer (containing 20 mmol / L sodium chloride and 1 mmol / L ethylenediaminetetraacetic acid) at pH 8.0. The stock solution was then serially diluted with the same tris(hydroxymethyl)aminomethane hydrochloride buffer to prepare cadmium ion standard solutions of different concentrations (0 ng / mL, 0.25 ng / mL, 1 ng / mL, 5 ng / mL, 25 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL). 100 μL of this solution was mixed with a FRET sensor of gold nanoflower-time-resolved fluorescent microspheres at 37 °C for 30 min, and the fluorescence signal was detected. The fluorescence signal value of the solution without cadmium ion standard was F0, and the fluorescence signal value of the solution with cadmium ion standard was F. A standard curve was fitted using (F-F0) / F0 as the ordinate.

[0103] (5) Detection of cadmium ion content in tea:

[0104] 1g of dried tea powder was mixed with concentrated nitric acid and 30% hydrogen peroxide (volume ratio 3:1) acid solution and digested for 2h. The supernatant was collected by centrifugation at 2000rpm and 25℃. The pH was adjusted to 8.0 with 1mol / L sodium hydroxide solution and diluted 50 times with 0.05mol / L tris(hydroxymethyl)aminomethane hydrochloride buffer (containing 20mmol / L sodium chloride and 1mmol / L ethylenediaminetetraacetic acid) at pH 8.0 as the tea sample to be tested. 100μL of the tea sample was added to the FRET sensor of gold nanoflower-time-resolved fluorescent microspheres and the fluorescence signal was detected. The measured fluorescence signal value was recorded as F1 and the fluorescence signal value of the tris(hydroxymethyl)aminomethane hydrochloride buffer was F2. The cadmium ion content in the tea was calculated by (F1-F2) / F2 and the standard curve of step (4).

[0105] from Figure 2 It can be seen that the morphology of the gold nanoflowers in this embodiment is different before and after coupling with the heavy metal cadmium antibody. After coupling, the surface morphology becomes uneven and irregular.

[0106] from Figure 3 As can be seen, the time-resolved fluorescent microspheres in this embodiment have different morphologies before and after coupling with the heavy metal cadmium antigen, and the surface becomes irregular after coupling.

[0107] from Figure 4As can be seen, the zeta potentials of the gold nanoflowers in this embodiment changed significantly before and after conjugation with the heavy metal cadmium antibody, and the time-resolved fluorescent microspheres changed significantly before and after conjugation with the heavy metal cadmium antigen. This is because, generally speaking, a zeta potential of approximately (±) 30 mV for a colloidal solution is considered to indicate good dispersion stability, at which point the surface charge of the colloid can prevent particle aggregation in the solution. The absolute values ​​of the zeta potentials of the two conjugates were lower than those of the unconjugated protein, indicating that the presence of the protein altered the surface charge distribution, thus confirming the successful preparation of the two conjugates.

[0108] from Figure 5 It can be seen that the maximum ultraviolet absorption peak of the gold nanoflower is located at 620 nm, which highly overlaps with the fluorescence emission peak of the time-resolved fluorescent microspheres.

[0109] from Figure 6 It can be seen that as the cadmium ion concentration increases from 0 ng / mL to 1000 ng / mL, the fluorescence intensity of the FRET sensor based on gold nanoflower-time-resolved fluorescent microspheres increases from weak to strong. Figure 7 It can be seen that the constructed system has a linear range of 1-500 ng / mL and a limit of detection of 0.29 ng / mL, indicating that the method has a wide linear range and high sensitivity.

[0110] Example 2:

[0111] The process of detecting cadmium ions in tea leaves using the FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres in this embodiment includes the following steps:

[0112] (1) Preparation of gold nanoflower-cadmium antibody conjugate:

[0113] Take 2 mL of concentrated gold nanoflowers with an OD value of 20 and a particle size of 80 nm. Adjust the pH of the solution to 8.5 with 0.1 mol / L K2CO3, then add 15 μg of cadmium monoclonal antibody, and perform a coupling reaction at 36 °C for 1 h. Add 100 μL of 10% (w / v) BSA solution to block unbound sites. Centrifuge the conjugate at 5000 rpm at 2 °C, dissolve it in 2 mL of ultrapure water, and store it for later use to obtain the gold nanoflower-cadmium antibody conjugate.

[0114] (2) Preparation of time-resolved fluorescent microsphere-cadmium antigen conjugate:

[0115] 20 μL of europium time-resolved fluorescent microspheres (56 nm in diameter) were diluted to 200 μL with 0.05 mol / L phosphate buffer (pH 7.4) and sonicated for 2 min. Carbodiimide hydrochloride solution was added to bring the final concentration in the reaction solution to 0.2 μg / μL. After activation at 36 °C for 35 min, the mixture was centrifuged at 12000 rpm for 50 min and the supernatant was discarded. Cadmium antigen was added to a final concentration of 15 μg / mL. After the coupling reaction, the mixture was centrifuged at 12000 rpm for 50 min and the supernatant was discarded. 200 μL of 1% (w / v) BSA solution was added to block unbound sites. After incubation and shaking, the mixture was centrifuged at 12000 rpm for 50 min and the supernatant was discarded. The conjugate was dissolved in 200 μL of phosphate buffer and stored for later use.

[0116] (3) Constructing a FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres:

[0117] Add 35 μL of gold nanoflower-cadmium antibody conjugate and 15 μL of time-resolved fluorescent microsphere-cadmium antigen conjugate to a black 96-well microplate, and then mix and react at 37 °C for 40 min to obtain a gold nanoflower-time-resolved fluorescent microsphere FRET sensor.

[0118] (4) Fitting the cadmium ion standard curve:

[0119] A 1000 mg / mL cadmium ion standard solution was prepared as a stock solution using 0.01 mol / L tris(hydroxymethyl)aminomethane hydrochloride buffer (containing 20 mmol / L sodium chloride and 1 mmol / L ethylenediaminetetraacetic acid) at pH 8.5. The stock solution was then serially diluted with the same tris(hydroxymethyl)aminomethane hydrochloride buffer to prepare cadmium ion standard solutions of different concentrations (0 ng / mL, 0.25 ng / mL, 1 ng / mL, 5 ng / mL, 25 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL). 100 μL of this solution was mixed with a FRET sensor of gold nanoflower-time-resolved fluorescent microspheres at 30 °C for 50 min, and the fluorescence signal was detected. The fluorescence signal value of the solution without cadmium ion standard was F0, and the fluorescence signal value of the solution with cadmium ion standard was F. A standard curve was fitted using (F-F0) / F0 as the ordinate.

[0120] (5) Detection of cadmium ion content in tea:

[0121] 1g of dried tea powder was mixed with concentrated nitric acid and 30% hydrogen peroxide (volume ratio 3.5:1) acid solution and digested for 1h. The supernatant was collected by centrifugation at 2000rpm and 25℃. The pH was adjusted to 8.0 with 1mol / L sodium hydroxide solution and diluted 60 times with 0.01mol / L tris(hydroxymethyl)aminomethane hydrochloride buffer (containing 20mmol / L sodium chloride and 2mmol / L ethylenediaminetetraacetic acid) at pH 8.5 as the tea sample to be tested. 100μL of the tea sample was added to the FRET sensor of gold nanoflower-time-resolved fluorescent microspheres and the fluorescence signal was detected. The measured fluorescence signal value was recorded as F1 and the fluorescence signal value of the tris(hydroxymethyl)aminomethane hydrochloride buffer was F2. The cadmium ion content in the tea was calculated by (F1-F2) / F2 and the standard curve of step (4).

[0122] Example 3:

[0123] The process of detecting cadmium ions in tea leaves using the FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres in this embodiment includes the following steps:

[0124] (1) Preparation of gold nanoflower-cadmium antibody conjugate:

[0125] Take 1 mL of concentrated gold nanoflowers with an OD value of 15 and a particle size of 83 nm. Adjust the pH of the solution to 8.5 with 0.1 mol / L K2CO3, then add 12 μg of cadmium monoclonal antibody, and carry out the coupling reaction at 40 °C for 0.5 h. Add 100 μL of 10% (w / v) BSA solution to block unbound sites. Centrifuge the conjugate at 4000 rpm and 8 °C, dissolve it in 1 mL of ultrapure water, and store it for later use to obtain the gold nanoflower-cadmium antibody conjugate.

[0126] (2) Preparation of time-resolved fluorescent microsphere-cadmium antigen conjugate:

[0127] 20 μL of europium time-resolved fluorescent microspheres (92 nm in diameter) were diluted to 200 μL with 0.05 mol / L phosphate buffer (pH 6) and sonicated for 3 min to mix. Carbodiimide hydrochloride solution was added to bring the final concentration in the reaction solution to 0.2 μg / μL. After activation at 40 °C for 25 min, the mixture was centrifuged at 14000 rpm for 45 min and the supernatant was discarded. Cadmium antigen with a final concentration of 30 μg / mL was added. After coupling reaction, the mixture was centrifuged at 15000 rpm for 40 min and the supernatant was discarded. 200 μL of 1% (w / v) BSA solution was added to block unbound sites. After incubation and shaking, the mixture was centrifuged at 15000 rpm for 40 min and the supernatant was discarded. The mixture was dissolved in 200 μL of phosphate buffer and stored for later use.

[0128] (3) Constructing a FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres:

[0129] Add 40 μL of gold nanoflower-cadmium antibody conjugate and 10 μL of time-resolved fluorescent microsphere-cadmium antigen conjugate to a black 96-well microplate, and then mix and react at 37 °C for 45 min to obtain a gold nanoflower-time-resolved fluorescent microsphere FRET sensor.

[0130] (4) Fitting the cadmium ion standard curve:

[0131] A 1000 mg / mL cadmium ion standard solution was prepared as a stock solution using 0.02 mol / L tris(hydroxymethyl)aminomethane hydrochloride buffer (containing 10 mmol / L sodium chloride and 2 mmol / L ethylenediaminetetraacetic acid) at pH 8.5. The stock solution was then serially diluted with the same tris(hydroxymethyl)aminomethane hydrochloride buffer to prepare cadmium ion standard solutions of different concentrations (0 ng / mL, 0.25 ng / mL, 1 ng / mL, 5 ng / mL, 25 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL). 100 μL of this solution was mixed with a FRET sensor of gold nanoflower-time-resolved fluorescent microspheres at 40 °C for 25 min, and the fluorescence signal was detected. The fluorescence signal value of the solution without cadmium ion standard was F0, and the fluorescence signal value of the solution with cadmium ion standard was F. A standard curve was fitted using (F-F0) / F0 as the ordinate.

[0132] (5) Detection of cadmium ion content in tea:

[0133] 1g of dried tea powder was mixed with concentrated nitric acid and 30% hydrogen peroxide (volume ratio 4:1) acid solution and digested for 5h. The supernatant was collected by centrifugation at 2000rpm and 25℃. The pH was adjusted to 8.0 with 1mol / L sodium hydroxide solution and diluted 60 times with 0.02mol / L tris(hydroxymethyl)aminomethane hydrochloride buffer (containing 10mmol / L sodium chloride and 2mmol / L ethylenediaminetetraacetic acid) at pH 8.5 as the tea sample to be tested. 100μL of the tea sample was added to the FRET sensor of gold nanoflower-time-resolved fluorescent microspheres and the fluorescence signal was detected. The measured fluorescence signal value was recorded as F1 and the fluorescence signal value of the tris(hydroxymethyl)aminomethane hydrochloride buffer was F2. The cadmium ion content in the tea was calculated by (F1-F2) / F2 and the standard curve of step (4).

[0134] Example 4:

[0135] The process of detecting cadmium ions in tea leaves using the FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres in this embodiment includes the following steps:

[0136] (1) Preparation of gold nanoflower-cadmium antibody conjugate:

[0137] Take 1 mL of concentrated gold nanoflowers with an OD value of 30 and a particle size of 90 nm. Adjust the pH of the solution to 8.5 with 0.1 mol / L K2CO3, then add 15 μg of cadmium monoclonal antibody, and perform a coupling reaction at 25 °C for 5 h. Add 100 μL of 10% (w / v) BSA solution to block unbound sites. Centrifuge the conjugate at 8000 rpm and 6 °C, dissolve it in 1 mL of ultrapure water, and store it for later use to obtain the gold nanoflower-cadmium antibody conjugate.

[0138] (2) Preparation of time-resolved fluorescent microsphere-cadmium antigen conjugate:

[0139] 20 μL of europium time-resolved fluorescent microspheres (155 nm in diameter) were diluted to 200 μL with 0.05 mol / L phosphate buffer (pH 8) and sonicated for 5 min to mix. Carbodiimide hydrochloride solution was added to bring the final concentration in the reaction solution to 0.2 μg / μL. After activation at 40 °C for 20 min, the supernatant was discarded after centrifugation at 16000 rpm for 30 min. Cadmium antigen with a final concentration of 25 μg / mL was added. After coupling reaction, the supernatant was discarded after centrifugation at 16000 rpm for 30 min. 200 μL of 1% (w / v) BSA solution was added to block unbound sites. After incubation and shaking, the mixture was centrifuged at 16000 rpm for 30 min and the supernatant was discarded. The mixture was dissolved in 200 μL of phosphate buffer and stored for later use.

[0140] (3) Constructing a FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres:

[0141] Add 47.5 μL of gold nanoflower-cadmium antibody conjugate and 0.5 μL of time-resolved fluorescent microsphere-cadmium antigen conjugate to a black 96-well microplate, and then mix and react at 37 °C for 60 min to obtain a gold nanoflower-time-resolved fluorescent microsphere FRET sensor.

[0142] (4) Fitting the cadmium ion standard curve:

[0143] A 1000 mg / mL cadmium ion standard solution was prepared as a stock solution using 0.04 mol / L tris(hydroxymethyl)aminomethane hydrochloride buffer (containing 0 mmol / L sodium chloride and 1 mmol / L ethylenediaminetetraacetic acid) at pH 8.5. The stock solution was then serially diluted with this tris(hydroxymethyl)aminomethane hydrochloride buffer to prepare cadmium ion standard solutions of different concentrations (0 ng / mL, 0.25 ng / mL, 1 ng / mL, 5 ng / mL, 25 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL). 100 μL of this solution was mixed with a FRET sensor of gold nanoflower-time-resolved fluorescent microspheres at 37 °C for 50 min, and the fluorescence signal was detected. The fluorescence signal value of the solution without cadmium ion standard was F0, and the fluorescence signal value of the solution with cadmium ion standard was F. A standard curve was fitted using (F-F0) / F0 as the ordinate.

[0144] (5) Detection of cadmium ion content in tea:

[0145] 1g of dried tea powder was mixed with concentrated nitric acid and 30% hydrogen peroxide (volume ratio 4.5:1) acid solution and digested for 4.5h. The supernatant was collected by centrifugation at 2000rpm and 25℃. The pH was adjusted to 8.0 with 1mol / L sodium hydroxide solution and diluted 80 times with 0.04mol / L tris(hydroxymethyl)aminomethane hydrochloride buffer (containing 0mmol / L sodium chloride and 1mmol / L ethylenediaminetetraacetic acid) at pH 8.5 as the tea sample to be tested. 100μL of the tea sample was added to the FRET sensor of gold nanoflower-time-resolved fluorescent microspheres and the fluorescence signal was detected. The measured fluorescence signal value was recorded as F1 and the fluorescence signal value of the tris(hydroxymethyl)aminomethane hydrochloride buffer was F2. The cadmium ion content in the tea was calculated by (F1-F2) / F2 and the standard curve of step (4).

[0146] Example 5:

[0147] The process of detecting cadmium ions in tea leaves using the FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres in this embodiment includes the following steps:

[0148] (1) Preparation of gold nanoflower-cadmium antibody conjugate:

[0149] Take 1 mL of concentrated gold nanoflowers with an OD value of 30 and a particle size of 100 nm. Adjust the pH of the solution to 8.5 with 0.1 mol / L K2CO3, then add 20 μg of cadmium monoclonal antibody, and perform a coupling reaction at 36 °C for 1.5 h. Add 100 μL of 10% (w / v) BSA solution to block unbound sites. Centrifuge the conjugate at 8000 rpm and 8 °C, dissolve it in ultrapure water, and store it for later use to obtain the gold nanoflower-cadmium antibody conjugate.

[0150] (2) Preparation of time-resolved fluorescent microsphere-cadmium antigen conjugate:

[0151] 20 μL of europium time-resolved fluorescent microspheres (316 nm in diameter) were diluted to 200 μL with 0.05 mol / L phosphate buffer (pH 7.4) and sonicated for 2 min. Carbodiimide hydrochloride solution was added to bring the final concentration in the reaction solution to 0.4 μg / μL. After activation at 30 °C for 45 min, the mixture was centrifuged at 13000 rpm for 50 min and the supernatant was discarded. Cadmium antigen with a final concentration of 80 μg / mL was added. After coupling reaction, the mixture was centrifuged at 13000 rpm for 50 min and the supernatant was discarded. 200 μL of 1% (w / v) BSA solution was added to block unbound sites. After incubation and shaking, the mixture was centrifuged at 13000 rpm for 50 min and the supernatant was discarded. The mixture was dissolved in 200 μL of phosphate buffer and stored for later use.

[0152] (3) Constructing a FRET sensor based on gold nanoflowers and time-resolved fluorescent microspheres:

[0153] Add 43 μL of gold nanoflower-cadmium antibody conjugate and 7 μL of time-resolved fluorescent microsphere-cadmium antigen conjugate to a black 96-well microplate, and then mix and react at 37 °C for 60 min to obtain a gold nanoflower-time-resolved fluorescent microsphere FRET sensor.

[0154] (4) Fitting the cadmium ion standard curve:

[0155] A 1000 mg / mL cadmium ion standard solution was prepared as a stock solution using 0.03 mol / L tris(hydroxymethyl)aminomethane hydrochloride buffer (containing 30 mmol / L sodium chloride and 1.5 mmol / L ethylenediaminetetraacetic acid) at pH 8.3. The stock solution was then serially diluted with this tris(hydroxymethyl)aminomethane hydrochloride buffer to prepare cadmium ion standard solutions of different concentrations (0 ng / mL, 0.25 ng / mL, 1 ng / mL, 5 ng / mL, 25 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL). 100 μL of this solution was mixed with a FRET sensor of gold nanoflower-time-resolved fluorescent microspheres at 37 °C for 60 min, and the fluorescence signal was detected. The fluorescence signal value of the solution without cadmium ion standard was F0, and the fluorescence signal value of the solution with cadmium ion standard was F. A standard curve was fitted using (F-F0) / F0 as the ordinate.

[0156] (5) Detection of cadmium ion content in tea:

[0157] 1g of dried tea powder was mixed with concentrated nitric acid and 30% hydrogen peroxide (volume ratio 5:1) acid solution and digested for 8h. The supernatant was collected by centrifugation at 2000rpm and 25℃. The pH was adjusted to 8.0 with 1mol / L sodium hydroxide solution and diluted 80 times with 0.03mol / L tris(hydroxymethyl)aminomethane hydrochloride buffer (containing 30mmol / L sodium chloride and 1.5mmol / L ethylenediaminetetraacetic acid) at pH 8.3 as the tea sample to be tested. 100μL of the tea sample was added to the FRET sensor of gold nanoflower-time-resolved fluorescent microspheres and the fluorescence signal was detected. The measured fluorescence signal value was recorded as F1 and the fluorescence signal value of the tris(hydroxymethyl)aminomethane hydrochloride buffer was F2. The cadmium ion content in the tea was calculated by (F1-F2) / F2 and the standard curve of step (4).

[0158] The above description of the embodiments is intended to enable those skilled in the art to understand and use the present invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention, without departing from the scope of the invention, should be within the protection scope of the present invention.

Claims

1. A method for fabricating a fluorescence resonance energy transfer sensor based on gold nanoflowers and time-resolved fluorescent microspheres, characterized in that: It comprises the following steps: (1) preparation of gold nanoflower-heavy metal cadmium antibody conjugate; the preparation method of the gold nanoflower-heavy metal cadmium antibody conjugate comprises: Boiling chloroauric acid solution is added with sodium citrate, and when the color turns into orange red, the heating state is kept until the solution color no longer changes, and the solution is mixed with filtered water, chloroauric acid solution, sodium citrate solution and hydroquinone solution to obtain gold nanoflower; After the gold nanoflower is concentrated, the pH of the gold nanoflower is adjusted to obtain a gold nanoflower concentrated solution, then heavy metal cadmium monoclonal antibody is added for coupling reaction, and bovine serum albumin solution is added to block the unbound sites, the coupling product is centrifuged and dissolved in ultrapure water, and the gold nanoflower-heavy metal cadmium antibody conjugate is obtained and stored for use. (2) preparation of time-resolved fluorescent microsphere-heavy metal cadmium antigen conjugate; the preparation method of the time-resolved fluorescent microsphere-heavy metal cadmium antigen conjugate comprises: The time-resolved fluorescent microspheres are diluted with phosphate buffer solution, ultrasonically mixed, and then carbonic acid imide hydrochloride solution is added for activation reaction, and then the supernatant is discarded after centrifugation, heavy metal cadmium antigen is coupled, the supernatant is discarded after centrifugation, and bovine serum albumin is added to block the unbound sites, and then the reaction is carried out after constant temperature shaking, the supernatant is discarded after centrifugation, and the time-resolved fluorescent microsphere-heavy metal cadmium antigen conjugate is dissolved in phosphate buffer solution and stored in cold storage. (3) the gold nanoflower-heavy metal cadmium antibody conjugate and the time-resolved fluorescent microsphere-heavy metal cadmium antigen conjugate are mixed to obtain a gold nanoflower-time-resolved fluorescent microsphere-based fluorescence resonance energy transfer sensor.

2. The method of claim 1, wherein: The particle size of the gold nanoflower is 80-100 nm; and / or, The OD value of the concentrated gold nanoflower is 10-30; and / or, The pH value of the gold nanoflower concentrated solution is 7-9; and / or, The preparation process of the heavy metal cadmium monoclonal antibody comprises: emulsifying Cd-ITCBE-BSA and then injecting into the body of a mouse, determining the titer of the polyclonal serum, cell fusion, hybridoma cell screening, injecting the hybridoma cells into the abdominal cavity of a non-immunized mouse to obtain mouse ascites, dialyzing, and obtaining the heavy metal cadmium monoclonal antibody; and / or, The addition amount of the heavy metal cadmium monoclonal antibody is 5-20 μg; and / or, The coupling reaction temperature is 20-40℃, and the reaction time is 0.5-5 h; and / or, The centrifugal speed is 3000-8000 rpm, and the time is 2-10℃.

3. The method of claim 1, wherein: In step (2), the time-resolved fluorescent microspheres are europium time-resolved fluorescent microspheres, and the microsphere diameter is 50-350 nm.

4. The method of claim 3, wherein: The concentration of the phosphate buffer solution is 0.01-0.05 mol / L; the pH of the phosphate buffer solution is 6-8; and / or, The final concentration of the carbonic acid imide hydrochloride solution in the reaction solution is 0.2-0.4 μg / μL; and / or, The activation reaction time is 20-45 min, and the temperature is 30-40℃; and / or, The centrifugal speed is 12000-16000 rpm; and the centrifugal time is 30-50 min; and / or, The preparation process of the heavy metal cadmium antigen comprises: mixing cadmium nitrate solution with ITCBE solution, coupling with ovalbumin, and then dialyzing to obtain the heavy metal cadmium antigen; and / or, The heavy metal cadmium antigen is added in an amount of 12-80 μg / mL; and / or, The coupling time is 30-45 min, and the coupling temperature is 2-8℃; and / or, The blocking time is 20-45 min, and the blocking temperature is 30-40℃; and / or, In step (3), the gold nanoflower-heavy metal cadmium antibody conjugate is added in an amount of 30-48 μL, and the time-resolved fluorescent microsphere-heavy metal cadmium antigen conjugate is added in an amount of 0.5-15 μL.

5. A fluorescent resonance energy transfer sensor based on gold nanoflower-time-resolved fluorescent microspheres, characterized in that: It is obtained by the preparation method of any one of claims 1-4.

6. Use of the gold nanoflower-time-resolved fluorescent microsphere-based fluorescence resonance energy transfer sensor of claim 5 in detecting cadmium ions in food.

7. Use according to claim 6, characterized in that: The process for detecting cadmium ions in tea by the gold nanoflower-time-resolved fluorescent microsphere-based fluorescence resonance energy transfer sensor comprises the following steps: (a) preparing cadmium ion standard solution of different concentrations with a reaction buffer, mixing with the gold nanoflower-time-resolved fluorescent microsphere-based fluorescence resonance energy transfer sensor, determining the fluorescence value, and constructing a standard curve; (b) pretreating tea, adding to the gold nanoflower-time-resolved fluorescent microsphere-based fluorescence resonance energy transfer sensor, and calculating the concentration of cadmium ions according to the standard curve.

8. Use according to claim 7, characterized in that: In step (a), the method for constructing the standard curve: preparing a cadmium ion standard solution mother liquor with a reaction buffer, gradient diluting the mother liquor into cadmium ion standard solution of different concentrations with the reaction buffer, mixing with the gold nanoflower-time-resolved fluorescent microsphere-based fluorescence resonance energy transfer sensor at 30-40℃ for 20-60 min, detecting the fluorescence signal, taking the fluorescence signal value without adding the cadmium ion standard solution as F0, taking the fluorescence signal value after adding the cadmium ion standard solution as F, and taking (F-F0) / F0 as the ordinate to fit the standard curve; and / or, The different concentrations of the cadmium ion standard solution are 0 ng / mL, 0.25 ng / mL, 1 ng / mL, 5 ng / mL, 25 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL; and / or, The reaction buffer is a tris-hydroxymethyl aminomethane hydrochloride buffer; and / or, The pH of the reaction buffer is 7.0-9.0; and / or, The concentration of the reaction buffer is 0.01-0.2 mol / L; and / or, The concentration of sodium chloride contained in the reaction buffer is 0-100 mmol / L; and / or, The concentration of ethylenediaminetetraacetic acid contained in the reaction buffer is 0.5-5 mmol / L.

9. Use according to claim 7, characterized in that: In step (b), the dried tea powder is mixed with acid solution for digestion, centrifuged to take the supernatant, adjusted for pH and diluted to obtain a tea sample to be tested, the tea sample is added to the gold nanoflower-time-resolved fluorescent microsphere-based fluorescence resonance energy transfer sensor, the fluorescence signal value is recorded as F1, the fluorescence signal value of the reaction buffer is recorded as F2, and the content of cadmium ions in the tea is calculated by (F1-F2) / F2 and the standard curve of step (a); and / or, The acid liquid is a mixture of concentrated nitric acid and 30% hydrogen peroxide, the volume ratio is 5-3:1, and the digestion time is 0.5-10h.

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