A method for rapid detection of thyroxine T4 based on fluorescence resonance transfer

Abstract

The present invention discloses a method for detecting thyroxine T4 based on fluorescence resonance energy transfer. The method comprises: synthesizing carbon quantum dots containing amino groups on the surface and silicon dioxide with carboxyl groups modified on the surface respectively, modifying the amino carbon quantum dots on the carboxylated silicon dioxide through an amidation reaction, introducing bromide ions as initiators to participate in the subsequent atom transfer radical reaction after reacting with dibromoisobutyryl bromide, and finally synthesizing the molecular imprinting on the surface of silicon dioxide. Using the fluorescent monomer in the atom transfer radical polymerization reaction as an energy donor and the carbon quantum dots as an energy acceptor, a fluorescence resonance energy transfer system is constructed, and the fluorescence in the system is attenuated during use, thereby establishing a highly sensitive detection method for thyroxine T4. In addition, the applicant noticed the structural similarity between thyroxine and thyroxine interfering substances and applied it to the detection of thyroid interfering substances.

Description

Technical Field

[0001] The invention belongs to the field of environmental monitoring, and more specifically, relates to a method for detecting thyroxine. Background Art

[0002] Thyroid hormone is a hormone secreted by the thyroid gland, which acts on almost all cells in the human body. The most obvious effect of thyroid hormone in promoting growth and development is in infancy, and the greatest impact is within the first five months after birth. It mainly promotes the growth and development of bones, brain and other organs. Without thyroid hormone, the pituitary GH cannot function. Moreover, when thyroid hormone is deficient, the pituitary gland produces and secretes less GH. Therefore, congenital or childhood lack of thyroid hormone causes cretinism. Cretinism patients have stagnant bone growth and short stature, and the length ratio of the upper and lower body is abnormal, with the upper body accounting for a larger proportion than normal people. In addition, due to the growth disorders of nerve cell dendrites, axons, myelin sheaths and glial cells, the brain is not fully developed and the intelligence is low. When the thyroid function of newborns is hypothyroid, thyroid hormone should be supplemented in appropriate amounts within one year old, which is effective for the development of the central nervous system and the recovery of brain function. Later than this period, even if a large amount of T3 or T4 is supplemented later, normal function cannot be restored, and the treatment is often ineffective. Therefore, it is necessary to test thyroid hormone.

[0003] Thyroxine disruptors (TDCs) are a class of exogenous compounds that can affect the synthesis, transport, metabolism and utilization of THs. Many studies have pointed out that the interference effects of known TDCs only account for a small part of the total interference toxicity in the environment or biological samples, and there are a large number of undiscovered interferents. Screening for unknown compounds with thyroid-disrupting effects in the environment is also of great significance to environmental and health research. Summary of the invention

[0004] In view of the above defects or improvement needs of the prior art, the present invention provides a method for rapid detection of thyroxine T4 based on fluorescence resonance transfer, the purpose of which is to utilize the selective adsorption principle of molecular imprinting polymers, the principle of fluorescence quenching caused by the interaction between monomers and templates, and the principle of fluorescence resonance energy transfer, and organically combine the three. The first step is that the molecular imprinting polymer selectively adsorbs thyroxine, the second step is that the interaction between the monomers and the template causes the fluorescence of the monomers to weaken, and the fluorescence signal is amplified by the principle of fluorescence resonance energy transfer, and the rapid and highly sensitive detection of thyroxine interfering substances is achieved by measuring the fluorescence attenuation intensity. This provides a new detection technology method and idea for the rapid identification of thyroxine interfering substances.

[0005] In addition, the applicant noticed the structural similarities between thyroxine and thyroxine interfering substances and applied them to the detection of thyroid interfering substances. Therefore, the applicant tried to detect thyroid interfering substances using thyroxine molecular imprinting and found that it could also be used for the detection of thyroxine interfering substances.

[0006] A method for rapid detection of thyroxine T4 based on fluorescence resonance transfer, characterized in that it comprises the following steps:

[0007] Step 1: Synthesis of carbon quantum dots with amino groups;

[0008] Step 2: Synthesizing silica with surface modified carboxyl groups;

[0009] Step 3: mixing carbon quantum dots with carboxylated silica for amidation reaction;

[0010] Step 4: carrying out bromination reaction of SiO2-CD-NH3;

[0011] Step 5: Using SiO2-CD-Br as a substrate, add a template (thyroxine T4), a monomer (2-(3-indole)ethyl methacrylamide), a crosslinker (N-methylenebisacrylamide), and a catalyst (cuprous bromide, 2,2-bipyridine) to carry out an atom transfer radical reaction;

[0012] Step 6: Add the substance to be tested, detect the fluorescence decay in the system, and determine the content of thyroxine T4 based on the detected fluorescence decay.

[0013] In a preferred implementation, step 1 comprises: dissolving 300 mg of chitosan in an acetic acid aqueous solution containing 1 mM silver nitrate, stirring vigorously, transferring the mixture obtained after stirring to a high-pressure reactor, and reacting at 160° C. for a predetermined time; after the reaction is completed, cooling to room temperature and centrifuging, discarding the precipitate, and freeze-drying the obtained dark yellow solution to obtain carbon dots with amino groups.

[0014] In another preferred implementation, step 2 comprises: dispersing equimolar amounts of 3-aminopropyltriethoxysilane and succinic anhydride in N,N-dimethylformamide, stirring, then adding an N,N-dimethylformamide solution containing silica, and simultaneously adding deionized water, continuing the reaction at the same temperature for a predetermined time, centrifuging, washing the precipitate with ethanol for multiple times, and then vacuum drying to obtain carboxyl-modified silica.

[0015] In another preferred implementation, step 3 comprises: dissolving carboxylated silica in N, N-dimethylformamide, ultrasonically dispersing, dissolving 1 mmol N-hydroxysuccinimide and 1 mmol 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in 1 ml of deionized water, and adding dropwise to the previous solution, stirring the mixed solution at 40° C. for 1 hour, then slowly adding 15 ml of an aqueous solution containing 50 mg of amino carbon dots, stirring and reacting at 40° C. for 24 hours. The solution is centrifuged at 8000 rpm for 3 minutes, and the precipitate is washed with ethanol several times and then vacuum dried.

[0016] In another preferred implementation, step 4 comprises: taking 2 g of carbon dot-modified silica, adding 30 ml of tetrahydrofuran and 1 ml of triethylamine, stirring, adding 2 ml of 2-bromoisobutyryl bromide dropwise under ice bath conditions, passing nitrogen for 5 min, and then reacting in an ice bath for 12 h. The obtained product is washed several times with ethanol and then dried in vacuo.

[0017] In another preferred implementation, step 5 includes the following steps:

[0018] (1) Synthesis of 2-(3-indole)ethyl methacrylamide monomer: 2 g of tryptamine was added to 50 ml of tetrahydrofuran and ultrasonically mixed. 3.46 ml of triethylamine was added and 2.42 ml of methacryloyl chloride was added dropwise while stirring. The mixture was stirred at room temperature for 6 h. After centrifugation, the supernatant (yellow liquid) was distilled under reduced pressure to 2 ml. The mixture was purified by silica gel column chromatography using dichloromethane:methanol (V / V=99:1) as the eluent. The yellow oily liquid was evaporated to dryness and stored at -20°C.

[0019] (3) Synthesis of thyroxine molecular imprinting: Weigh 570 mg of 2-(3-indole)ethyl methacrylamide monomer, dissolve 973 mg of thyroxine in 125 ml of dimethyl sulfoxide, prepolymerize by ultrasonication for 30 min, add 3850 mg of N,N-methylenebisacrylamide, stir evenly, add 70 mg of cuprous bromide, 75 mg of 2,2-bipyridine, stir evenly, and finally add the brominated carbon dot silica obtained in step 4, pass nitrogen for 30 min, and react at 70°C with stirring for 18 h. The synthesized molecular imprinting was washed with ammonia: ethanol (V:V = 2:1) until the supernatant was free of thyroxine.

[0020] In another preferred implementation, step 6 includes the following steps:

[0021] 6.1. Preparation of thyroxine standard curve: Weigh 5 mg of thyroxine and dissolve it in 50 ml of methanol, and dilute it step by step to 50 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, and 1 ng / ml.

[0022] 6.2. Fluorescence detection: Weigh 5 mg of the synthesized thyroxine molecular imprinting powder respectively and use it to adsorb 1 ml of thyroxine standard solution with concentrations of 50 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml and 1 ng / ml. The adsorption time is 2 h. Use a fluorescence spectrophotometer to measure the fluorescence intensity of the supernatant after adsorption and calculate the value of ΔF / F0, where ΔF is the difference in fluorescence values ​​before and after adsorption and F0 is the fluorescence value before adsorption.

[0023] The present invention also provides a molecular imprinting synthesis method for thyroid hormone detection, comprising: weighing 570 mg of 2-(3-indole)ethyl methacrylamide monomer, dissolving 973 mg of thyroid hormone in 125 ml of dimethyl sulfoxide, prepolymerizing by ultrasonication for 30 minutes, adding 3850 mg of N, N-methylenebisacrylamide, stirring evenly, adding 70 mg of cuprous bromide, 75 mg of 2,2-bipyridine, stirring evenly, and finally adding the brominated carbon point silica obtained in step 4, passing nitrogen for 30 minutes, and stirring at 70°C for 18 hours. The synthesized molecular imprint is washed with ammonia: ethanol (V:V=2:1) ​​until the supernatant is free of thyroid hormone.

[0024] When conducting thyroxine testing, it is necessary to configure a thyroxine standard curve: weigh 5 mg of thyroxine and dissolve it in 50 ml of methanol, and then dilute it step by step to 50 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, and 1 ng / ml.

[0025] During the detection, the molecular imprinting adsorption of the thyroid hormone labeled curve: 5 mg of the synthetic thyroid hormone molecular imprinting powder was weighed separately and used to adsorb 1 ml of thyroid hormone standard solution with concentrations of 50 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, and 1 ng / ml, and the adsorption time was 2 h.

[0026] Finally, fluorescence measurement was performed: after adsorption, centrifugation was performed at 8000 rpm for 5 min, and the supernatant was taken to measure the fluorescence value under a spectrophotometer, and compared with the fluorescence value before adsorption to obtain the ΔF / F0 value corresponding to different concentrations.

[0027] (1) The advantages of the present invention are

[0028] The present invention applies fluorescent monomers and carbon quantum dots with excellent optical properties to fluorescence resonance energy transfer, uses fluorescent monomers as energy donors and carbon quantum dots as energy acceptors to construct a new energy transfer system, and the thyroxine standard solution can weaken the fluorescence in the system, thereby establishing a highly sensitive and selective detection method for thyroxine. The detection method of the present invention has the characteristics of good stability, high sensitivity, good selectivity, etc.

[0029] The present invention can significantly improve the detection limit and increase the detection sensitivity of thyroxine to 1 ng / ml. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the standard curve for thyroid.

[0031] Figure 2 The fluorescence emission spectrum of the fluorescent monomer at its maximum excitation wavelength (280nm) and the emission spectrum of the synthetic material at this excitation wavelength. As shown in the figure, the fluorescent monomer in the molecular imprinting system can produce a large emission fluorescence at 350nm under the excitation wavelength of 280nm, and 350nm is just close to the fluorescence excitation wavelength of NH3-CD, 370nm. Other materials did not produce emission fluorescence under 280nm excitation.

[0032] Figure 3 The fluorescence emission spectrum of amino carbon dots at their maximum excitation wavelength and the emission spectrum of related substances at the excitation wavelength. As shown in the figure, at an excitation wavelength of 370nm, amino carbon dots have a strong emission fluorescence at 457nm.

[0033] Figure 4 The fluorescence emission spectra of thyroxine MIP after adsorbing T4 series concentrations (1, 10, 50 ppb). As shown in the figure, the peaks of the emission spectrum at 350nm and 470nm just show that when the excitation wavelength is set to 280nm, the fluorescence emitted by the monomer at 370nm can just excite CD to produce fluorescence at 470nm. As the concentration of the target substance T4 increases, the fluorescence value at 470nm decreases. This is because the fluorescent monomer in the imprinting material has a binding effect with T4, which affects the emission of fluorescence from the fluorescent monomer, thereby affecting the emission of fluorescence from CD at 470nm.

[0034] Figure 5 This is the fluorescence emission spectrum of thyroxine NIP after adsorbing the thyroxine standard curve. Similarly, the monomer on the surface of NIP can also adsorb the target molecule T4, resulting in a decrease in the fluorescence value at 470nm. However, since NIP does not have a template, it cannot form a hole structure that matches the template molecule, so the binding force with T4 is relatively weak. Compared with MIP, the decrease in fluorescence at 470nm is also smaller.

[0035] Figure 6 When synthesizing thyroxine MIP, the monomer: template ratio was optimized. The results show that when the monomer: template ratio is 2:1, thyroxine MIP has the best adsorption capacity.

[0036] Figure 7When synthesizing thyroxine MIP, the cross-linker: template ratio was optimized. The results show that when the cross-linker: template ratio is 20:1, thyroxine MIP has the best adsorption capacity.

[0037] Figure 8 It is a schematic diagram of the reaction process of the present invention. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0039] The technical solution provided by the present invention is further described below through specific embodiments:

[0040] Example 1

[0041] This embodiment provides a method for rapid detection of thyroxine T4 based on fluorescence resonance transfer, comprising the following steps:

[0042] Step 1: Synthesis of carbon quantum dots with amino groups;

[0043] Step 2: Synthesizing silica with surface modified carboxyl groups;

[0044] Step 3: mixing carbon quantum dots with carboxylated silica for amidation reaction;

[0045] Step 4: carrying out bromination reaction of SiO2-CD-NH3;

[0046] Step 5: Using SiO2-CD-Br as the substrate, adding template, monomer, crosslinker and catalyst to carry out atom transfer radical reaction;

[0047] Step 6: The addition of the target substance thyroxine T4 causes the fluorescence in the system to decay.

[0048] The following is a detailed description of each of the above steps.

[0049] Step (1), synthesis of carbon quantum dots with amino groups

[0050] 300 mg of chitosan was dissolved in an acetic acid aqueous solution containing 1 mM silver nitrate (1:100, V / V), and stirred vigorously. The mixture obtained after stirring was transferred to a high-pressure reactor and reacted at 160°C for 3 hours. After the reaction was completed, it was cooled to room temperature and centrifuged at 11000 rpm for 20 minutes. After discarding the precipitate, the dark yellow solution was freeze-dried to obtain carbon dots with amino groups.

[0051] Step (2), synthesizing silica with surface modified carboxyl groups

[0052] Equimolar 3-aminopropyltriethoxysilane and succinic anhydride were dispersed in 100 ml of N, N-dimethylformamide and magnetically stirred at 40°C for 3 h. Then, 20 ml of N, N-dimethylformamide solution containing 2 g of silica was added, and 2 ml of deionized water was added at the same temperature. After the reaction was continued for 5 h, the mixture was centrifuged at 5000 rpm for 10 min. The precipitate was washed with ethanol several times and then vacuum dried to obtain carboxyl-modified silica.

[0053] Step 3: Mix the carbon quantum dots with carboxylated silica for amidation reaction

[0054] Dissolve carboxylated silica in N, N-dimethylformamide and disperse by ultrasonication. Dissolve 1 mmol N-hydroxysuccinimide and 1 mmol 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in 1 ml deionized water and add dropwise to the previous solution. Stir the mixed solution at 40°C for 1 hour, then slowly add 15 ml of an aqueous solution containing 50 mg of amino carbon dots and stir at 40°C for 24 hours. Centrifuge the solution at 8000 rpm for 3 minutes, wash the precipitate with ethanol several times and then dry it in vacuum.

[0055] Step 4, carrying out bromination reaction of SiO2-CD-NH3;

[0056] Take 2g of carbon dot-modified silica, add 30ml of tetrahydrofuran and 1ml of triethylamine, stir, add 2ml of 2-bromoisobutyryl bromide dropwise in an ice bath, pass nitrogen for 5min, then react in an ice bath for 12h. The obtained product is washed several times with ethanol and dried in vacuum.

[0057] Step 5: performing an atom transfer radical reaction, which comprises:

[0058] Step 5.1, synthesis of 2-(3-indole)ethyl methacrylamide monomer: add 2 g of tryptamine to 50 ml of tetrahydrofuran and mix by ultrasonication, add 3.46 ml of triethylamine, add 2.42 ml of methacryloyl chloride dropwise while stirring, stir at room temperature for 6 h, centrifuge, distill the supernatant (yellow liquid) under reduced pressure to 2 ml, purify by silica gel column chromatography, the eluent is dichloromethane:methanol (V / V=99:1), evaporate to dryness to obtain a yellow oily liquid, and store at -20°C.

[0059] Step 5.2, prepare thyroxine molecular imprint. This step includes: weighing 570 mg of 2-(3-indole)ethyl methacrylamide monomer, dissolving 973 mg of thyroxine in 125 ml of dimethyl sulfoxide, ultrasonically prepolymerizing for 30 minutes, adding 3850 mg of N, N-methylenebisacrylamide, stirring evenly, adding 70 mg of cuprous bromide, 75 mg of 2,2-bipyridine, stirring evenly, and finally adding the brominated carbon dot silica obtained in step (4), passing nitrogen for 30 minutes, and stirring at 70°C for 18 hours. The synthesized molecular imprint was washed with ammonia: ethanol (V:V = 2:1) until the supernatant was free of thyroxine.

[0060] The step 6, adding the substance to be tested, detecting the fluorescence attenuation in the system, and determining the content of thyroxine T4 based on the detected fluorescence attenuation. This step includes:

[0061] 6.1. Preparation of thyroxine standard curve: Weigh 5 mg of thyroxine and dissolve it in 50 ml of methanol, and dilute it step by step to 50 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, and 1 ng / ml.

[0062] 6.2 Fluorescence detection: Weigh 5 mg of the synthetic thyroxine molecular imprinting powder respectively and use it to adsorb 1 ml of thyroxine standard solution with concentrations of 50 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml and 1 ng / ml. The adsorption time is 2 h. Use a fluorescence spectrophotometer to measure the fluorescence intensity of the supernatant after adsorption and calculate the value of ΔF / F0, where ΔF is the difference in fluorescence values ​​before and after adsorption and F0 is the fluorescence value before adsorption.

[0063] The results are shown in Table 1. As the concentration of thyroxine increases, the value of ΔF / F0 increases. Based on this, the standard curve of thyroxine for ΔF / F0 is obtained ( Figure 1 ), the concentration of thyroxine in the solution can be determined according to the value of ΔF / F0. The scheme of the present invention is compared with the traditional enzyme-linked immunosorbent assay for measuring T4. The sensitivity of the traditional enzyme-linked immunosorbent assay is 5ng / ml, while the sensitivity of the scheme of the present invention is 1ng / ml. The sensitivity of the scheme of the present invention is better, which is much higher than other current detection methods.

[0064] According to the principle that structure leads to effect, many thyroid disruptors bind to thyroid transporter proteins because they have a similar structure to T4, thereby causing a certain impact on the human thyroid system. Therefore, the thyroid molecular imprinted material synthesized by the present invention can continue to be explored for its application in the identification of thyroid disruptors.

[0065] In the above molecular imprinting synthesis process, the ratio of template, monomer and cross-linking agent was changed, and different materials were synthesized. After 10ug / ml T4 was adsorbed, the optimal conditions for synthesis were determined according to the fluorescence difference (ΔF / F0) at 470nm. Through a large number of experimental comparisons, the inventors found that when the ratio of template, monomer and cross-linking agent was 1:2:20, the value of ΔF / F0 was the largest, and the effect of synthesizing the material was the best at this time. The results are as follows Figure 6 , 7 shown.

[0066]

[0067] Table 1 Relative changes in fluorescence intensity of thyroxine MIP / NIP after adsorption of thyroxine at concentration gradients.

[0068] In addition, the inventors selected two thyroid hormone disruptors (tetrabromobisphenol A (TBBPA) and 4-hydroxy-2',3,5,5'-tetrachlorobiphenyl (4-OH-PCB 72)) as test substances and conducted similar tests.

[0069]

[0070] Tetrabromobisphenol A

[0071]

[0072] 4-Hydroxy-2',3,5,5'-tetrachlorobiphenyl

[0073] Add thyroid hormone disruptors, detect the fluorescence attenuation in the system, and determine the content of the thyroid hormone disruptors based on the detected fluorescence attenuation. This step includes:

[0074] 6.1. Preparation of standard curve of thyroxine interferon: Weigh 5 mg of thyroxine interferon and dissolve it in 50 ml of methanol, and dilute it step by step to 50 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml and 1 ng / ml.

[0075] 6.2 Fluorescence detection: Weigh 5 mg of the synthetic thyroxine molecular imprinting powder and use it to adsorb 1 ml of thyroxine standard solution with concentrations of 50 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml and 1 ng / ml. The adsorption time is 2 h. Use a fluorescence spectrophotometer to measure the fluorescence intensity of the supernatant after adsorption.

[0076] The results confirmed that the molecular imprinting material prepared by the present invention also has a highly sensitive detection capability for the above-mentioned interferents.

[0077] Comparative experiment

[0078] The adsorption capacity of T4 of the same concentration in the same series was compared between the molecular imprinting material synthesized by the present invention and the molecular imprinting material prepared by the conventional method. The results show that the emission spectrum of the molecular imprinting material synthesized by the conventional method at 280nm has only one emission peak at 350nm after adsorbing T4. This is the emission fluorescence generated by the fluorescent monomer. Because there is no CD in the material as an energy acceptor for resonance fluorescence, it fails to continue to generate fluorescence at 470nm. Although the fluorescence at 350nm also decreases due to the action of the monomer and T4, the decrease is not as large as that at 470nm, as shown in Table 2, indicating that the fluorescence resonance system of the present invention plays a role in signal amplification.

[0079]

[0080] Table 2.

Claims

1. A method for rapid detection of thyroxine T4 based on fluorescence resonance transfer, characterized in that: The steps include: Step 1: Synthesis of carbon quantum dots with amino groups; Step 2: Synthesizing silica with surface modified carboxyl groups; Step 3: mixing carbon quantum dots with carboxylated silica for amidation reaction; Step 4: carrying out bromination reaction of SiO2-CD-NH3; Step 5: Using SiO2-CD-Br as the substrate, adding template, monomer, crosslinker and catalyst to carry out atom transfer radical reaction; Step 6: Add the substance to be tested, detect the fluorescence decay in the system, and determine the content of thyroxine T4 based on the detected fluorescence decay. The step 5 includes the following steps Synthesis of 2-(3-indole)ethyl methacrylamide monomer: add 2g tryptamine to 50ml tetrahydrofuran and mix by ultrasonic, add 3.46ml triethylamine, add 2.42ml methacryloyl chloride dropwise while stirring, stir at room temperature for 6h, centrifuge and distill the supernatant under reduced pressure to 2ml, purify by silica gel column chromatography, the eluent is dichloromethane:methanol V / V=99:1, evaporate to dryness to obtain a yellow oily liquid, store at -20℃; Synthesis of thyroxine molecular imprinting: weigh 570 mg of 2-(3-indole)ethyl methacrylamide monomer, dissolve 973 mg of thyroxine in 125 ml of dimethyl sulfoxide, prepolymerize by ultrasonication for 30 min, add 3850 mg of N, N-methylenebisacrylamide, stir evenly, add 70 mg of cuprous bromide and 75 mg of 2,2-bipyridine, stir evenly, finally add the brominated carbon dot silica obtained in step 4, pass nitrogen for 30 min, stir and react at 70 ° C for 18 h, and wash the synthesized molecular imprint with ammonia: ethanol V:V = 2:1 until the supernatant is free of thyroxine; The step 6 comprises the following steps: Preparation of thyroxine standard curve: Weigh 5 mg of thyroxine and dissolve it in 50 ml of methanol, and dilute it step by step to 50 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, and 1 ng / ml. Fluorescence detection: Weigh 5 mg of the synthesized thyroxine molecular imprinting powder respectively and use it to adsorb 1 ml of thyroxine standard solution with concentrations of 50 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml and 1 ng / ml. The adsorption time is 2 h. Use a fluorescence spectrophotometer to measure the fluorescence intensity of the supernatant after adsorption and calculate the value of ΔF / F0, where ΔF is the difference in fluorescence values ​​measured before and after adsorption, and F0 is the fluorescence value before adsorption.

2. The method according to claim 1, characterized in that The step 1 comprises: dissolving 300 mg of chitosan in an acetic acid aqueous solution containing 1 mM silver nitrate, stirring vigorously, transferring the mixture obtained after stirring to a high-pressure reactor, and reacting at 160° C. for a predetermined time; cooling to room temperature after the reaction is completed, centrifuging, discarding the precipitate, and freeze-drying the obtained dark yellow solution to obtain carbon dots with amino groups.

3. The method according to claim 1, characterized in that The step 2 comprises: dispersing equimolar amounts of 3-aminopropyltriethoxysilane and succinic anhydride in N,N-dimethylformamide, stirring, then adding an N,N-dimethylformamide solution containing silica, and simultaneously adding deionized water, continuing the reaction at the same temperature for a predetermined time, centrifuging, washing the precipitate with ethanol for multiple times, and then vacuum drying to obtain carboxyl-modified silica.

4. The method according to claim 2, characterized in that The step 3 comprises: dissolving carboxylated silica in N,N-dimethylformamide, dispersing by ultrasonication, dissolving 1 mmol N-hydroxysuccinimide and 1 mmol 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in 1 ml deionized water, and adding the mixture dropwise to the previous solution, stirring the mixed solution at 40° C. for reaction for 1 hour, then slowly adding 15 ml of an aqueous solution containing 50 mg of amino carbon dots, stirring the mixture at 40° C. for reaction for 24 hours, centrifuging the solution at 8000 rpm for 3 minutes, washing the precipitate with ethanol for multiple times, and then vacuum drying.

5. The method according to claim 4, characterized in that The step 4 comprises: taking 2 g of carbon dot-modified silica, adding 30 ml of tetrahydrofuran and 1 ml of triethylamine, stirring, adding 2 ml of 2-bromoisobutyryl bromide dropwise under ice bath conditions, passing nitrogen for 5 min, and then reacting in an ice bath for 12 h, and the obtained product is washed several times with ethanol and then vacuum dried.

6. A molecular imprinting synthesis method, applied to the method for rapid detection of thyroxine T4 based on fluorescence resonance transfer according to claim 1, characterized in that: include: Weigh 570 mg of 2-(3-indole)ethyl methacrylamide monomer and 973 mg of thyroxine, dissolve in 125 ml of dimethyl sulfoxide, prepolymerize by ultrasonication for 30 minutes, add 3850 mg of N,N-methylenebisacrylamide, stir evenly, add 70 mg of cuprous bromide and 75 mg of 2,2-bipyridine, stir evenly, finally add the brominated carbon-dot silica obtained in step 4, pass nitrogen for 30 minutes, stir and react at 70°C for 18 hours, and wash the synthesized molecular imprint with ammonia: ethanol V:V=2:1 until the supernatant is free of thyroxine.

7. A method for detecting thyroid hormone interfering substances, characterized in that: The method comprises: preparing molecular imprinting materials according to steps 1 to 5 of the method according to claim 1, adding a substance to be tested, detecting fluorescence attenuation in the system, and determining the content of thyroxine interfering substances based on the detected fluorescence attenuation.

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