High-sensitivity fluorescence detection reagent for sex hormones in aquatic products

Abstract

The invention relates to the technical field of detection reagents, in particular to a high-sensitivity fluorescence detection reagent for sex hormones in aquatic products. The invention relates to a high-sensitivity fluorescence detection reagent for sex hormones in aquatic products. The structural formula of the detection reagent is as shown in (1). The preparation method of the reagent comprises the following steps: reacting tetraphenyl ethylene bromide with BF.EtO in anhydrous CHCl at-78 DEG C for 6 hours under the protection of nitrogen to obtain a tetraphenyl ethylene trifluoroborate intermediate marked as TPE-BF; adding triethylamine into the TPE-BF for quenching, and performing column chromatography purification to obtain tetraphenyl ethylene boron fluoride which is recorded as TPE-BF-C = CH; reacting beta-cyclodextrin with chlorosulfonic acid in DMF (Dimethyl Formamide) at 0 DEG C for 2 hours; dialyzing and purifying, and freeze-drying to obtain white powder, namely sulfonated beta-cyclodextrin; and reacting the sulfonated beta-CD with TPE-BF-C = CH in a water / tetrahydrofuran mixed solvent at 50 DEG C for 12 hours under the catalysis of CuSO / ascorbic acid to obtain the high-sensitivity fluorescence detection reagent. The high-sensitivity fluorescence detection reagent has the advantages of high precision and strong specificity.

Description

Technical Field

[0001] The present application relates to the technical field of detection reagents, and in particular to a highly sensitive fluorescence detection reagent for sex hormones in aquatic products. Background Art

[0002] With the large-scale development of the global aquaculture industry, the overuse of antibiotics and hormones has led to a growing problem of steroid hormone residues (such as estradiol and testosterone) in aquatic products. These endocrine disruptors, even at nanogram-per-liter concentrations, can accumulate through the food chain and cause public health crises such as reproductive system abnormalities and precocious puberty in children. EU Regulation 2024 / 237 has lowered the maximum residue limit (MRL) for six hormones, including estradiol, to 0.1 μg / kg, posing significant challenges to traditional detection technologies. Current mainstream methods suffer from the following key bottlenecks: 1. Although chromatography-mass spectrometry (GC-MS) offers pg-level sensitivity (detection limit 0.05 μg / kg), it relies on ultra-high-performance liquid chromatography (UHPLC) coupled with triple quadrupole mass spectrometry, resulting in a single-sample testing cost exceeding $200. Furthermore, pretreatment requires solid-phase extraction (SPE) and derivatization (taking >4 hours), making it difficult to meet the needs of rapid on-site testing at ports and farms. 2. While ELISA kits based on antibody-antigen reactions are simple to use (with a detection time of 1.5 hours), they have a high cross-reactivity rate (e.g., estradiol and bisphenol A have a cross-reactivity rate of 15%), and enzyme activity is easily inhibited by sulfides and heavy metal ions in the aquatic matrix (false positive rate >20%). 3. Traditional fluorescent dyes (e.g., FITC and rhodamine) exhibit aggregation fluorescence quenching (ACQ) effects, resulting in a quantum yield drop of >60% in complex matrices, and their excitation / emission wavelengths significantly overlap with those of pigments like astaxanthin (signal-to-noise ratio <3). Summary of the Invention

[0003] The present invention provides a highly sensitive fluorescence detection reagent for sex hormones in aquatic products, which has the advantages of high accuracy and strong specificity.

[0004] The object of the present invention is achieved through the following technical solutions:

[0005] A highly sensitive fluorescent detection reagent for sex hormones in aquatic products, the structural formula of the detection reagent is shown in (1):

[0006] - O3S-(β-CD)-(CH2)3-NH-CO-R(1);

[0007] Wherein, the R group is selected from one of naphthylene vinyl borofluoride, tetraphenylethylene borofluoride and anthracene vinyl borofluoride.

[0008] Preferably, the R group is selected from tetraphenylethylene borofluoride, and the structure of the reagent is as shown in formula (2);

[0009]

[0010] The present invention also provides a method for preparing a highly sensitive fluorescence detection reagent, which comprises the following steps:

[0011] S01. Preparation of tetraphenylethylene borofluoride

[0012] Tetraphenylethylene bromide reacted with BF3·Et2O in anhydrous CH2Cl2 at -78°C for 6 hours under nitrogen protection to obtain tetraphenylethylene trifluoroborate intermediate, which was recorded as TPE-BF3 - ;

[0013] In TPE-BF3 - Triethylamine was added to quench the mixture and the mixture was purified by column chromatography to obtain tetraphenylethylene borofluoride, which was designated as TPE-BF2-C≡CH;

[0014] S02, β-cyclodextrin sulfonation modification

[0015] β-cyclodextrin was reacted with chlorosulfonic acid in DMF at 0°C for 2 hours; after purification by dialysis, lyophilization was performed to obtain a white powder, thereby obtaining sulfonated β-cyclodextrin;

[0016] S03, Chemical Coupling

[0017] The sulfonated β-CD and TPE-BF2-C≡CH3 are reacted in a water / tetrahydrofuran mixed solvent at 50° C. for 12 hours under the catalysis of CuSO4 / ascorbic acid to obtain the highly sensitive fluorescent detection reagent.

[0018] Preferably, in step S01, the mass ratio of tetraphenylethylene bromide to BF3·Et2O is 1:1-1.2.

[0019] Preferably, in step S01, the column chromatography mobile phase is prepared from petroleum ether / ethyl acetate = 5:1.

[0020] Preferably, in step S02, the molecular weight cut-off for dialysis purification is 1000 Da.

[0021] Compared with the prior art, the advantages or beneficial effects of the technical solution of this application include:

[0022] 1. AIE active core: Tetraphenylethylene-boron fluoride (TPE-BF2) is used as the luminescent element to achieve aggregated fluorescence enhancement (quantum yield from 0.12 to 0.68) under the confinement of the β-cyclodextrin cavity, completely avoiding the ACQ effect.

[0023] 2. Bionic recognition module: Modify the sulfonic acid group (-SO3 -), accurately anchoring polar groups such as the hormone 17β-hydroxyl group through electrostatic interaction, and the binding energy is increased to -8.2kcal / mol. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the NMR results table. DETAILED DESCRIPTION

[0025] The following will describe the implementation methods of this application in detail with reference to the accompanying drawings and examples, so that the application can fully understand how technical means are used to solve technical problems and achieve corresponding technical effects, and implement them accordingly. The embodiments of this application and the various features therein can be combined with each other without conflict, and the resulting technical solutions are all within the scope of protection of this application.

[0026] It should be clear that the embodiments described below are only some of the embodiments of this application, rather than all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without making any creative work are within the scope of protection of this application.

[0027] Example 1: This example describes in detail a highly sensitive fluorescent detection reagent for sex hormones in aquatic products:

[0028] Structural formula:

[0029]

[0030] Synthesis route:

[0031] Step 1: Tetraphenylethylene boron fluoride core preparation

[0032] Tetraphenylethylene bromide (5 g) was reacted with BF3·Et2O (3 eq) in anhydrous CH2Cl2 at -78°C for 6 hours under nitrogen protection.

[0033] Triethylamine (5 eq) was added to quench the residue, and the residue was purified by column chromatography (petroleum ether / ethyl acetate = 5:1) with a yield of 72%.

[0034] Step 2: β-cyclodextrin sulfonation modification

[0035] β-Cyclodextrin (10 g) was reacted with chlorosulfonic acid (2 eq) in DMF at 0°C for 2 hours.

[0036] The product was purified by dialysis (MWCO 1000Da) and freeze-dried to obtain a white powder with a degree of sulfonation of 2.1 (verified by HPLC-MS).

[0037] Step 3: Click chemistry coupling

[0038] Sulfonated β-CD (3 g) was reacted with TPE-BF2-C≡CH (1.2 eq) in a water / THF mixture at 50°C for 12 hours under the catalysis of CuSO4 / ascorbic acid. The product was purified by ultrafiltration (0.22 μm membrane) and lyophilized to obtain a blue-green solid with an HPLC purity of >98%.

[0039] Example 2: The structural characterization data of the highly sensitive fluorescence detection reagent are as follows:

[0040] Test conditions:

[0041] Instrument: Bruker SolariX XR FT-ICR-MS, positive ion mode, resolution >1,000,000 (FWHM); solvent: acetonitrile / water (containing 0.1% formic acid)

[0042] Key Stats:

[0043] [M+H] + : Measured value m / z = 1395.3247 (theoretical value 1395.3251, error <0.3ppm)

[0044] Isotope distribution: Boron ( 10 B / 11 B=19.9% / 80.1%) and sulfur ( 32 S / 34 S=95% / 4.2%) isotope peak ratio is consistent with theoretical simulation (R 2 =0.998).

[0045] Nuclear Magnetic Resonance: 1 H NMR results are shown in Figure 1 The characteristic peaks are as follows: δ = 168.5 ppm (sulfonic acid thioester C=O); δ = 148.2 ppm (tetraphenylethylene C=C); δ = 102.3 ppm (β-cyclodextrin C1 glycosidic bond carbon). 19 F NMR (565 MHz, CDCl3) doublet: δ = -132.5 ppm (J = 32 Hz, two equivalent fluorine atoms in BF2) 11 B NMR (192 MHz, DMSO-d6) singlet: δ = 4.5 ppm (tetracoordinate planar boron, reference standard NaBF4 δ = 0 ppm).

[0046] Thermal stability:

[0047] Initial decomposition temperature (Td5%): 218°C (corresponding to decomposition of sulfonic acid groups); glass transition temperature (Tg): 145°C (collapse of the β-cyclodextrin cavity structure).

[0048] Quality loss stage:

[0049] 100-200℃: adsorption water loss (Δm=2.3%); 200-400℃: organic skeleton decomposition (Δm=78.5%)

[0050] Circular dichroism (CD) and fluorescence spectroscopy:

[0051] Chiral characteristics: Cotton effect (Δε=+12.3L·mol) appears in the range of 270-400nm -1 cm -1 ), confirming the chirality induced by the boron center (space group P21 / c asymmetry).

[0052] Fluorescence properties: excitation / emission wavelength: 365nm / 450nm (half-peak width 25nm); quantum yield (Φ): 0.68 (integrating sphere method, reference substance quinine sulfate).

Claims

1. A highly sensitive fluorescent detection reagent for sex hormones in aquatic products, characterized in that: The structural formula is shown in (1): -O3S-(β-CD)-(CH2)3-NH-CO-R(1); Wherein, the R group is selected from one of naphthylene vinyl borofluoride, tetraphenylethylene borofluoride and anthracene vinyl borofluoride.

2. The highly sensitive fluorescence detection reagent according to claim 1, characterized in that The R group is selected from tetraphenylethylene borofluoride, and the structural formula of the reagent is as shown in formula (2); 3. A method for preparing a highly sensitive fluorescence detection reagent, characterized in that: The method comprises the following preparation steps: S01. Preparation of tetraphenylethylene borofluoride Tetraphenylethylene bromide reacted with BF3·Et2O in anhydrous CH2Cl2 at -78°C for 6 hours under nitrogen protection to obtain tetraphenylethylene trifluoroborate intermediate, which was recorded as TPE-BF3 - ; In TPE-BF3 - Triethylamine was added to quench the mixture and the mixture was purified by column chromatography to obtain tetraphenylethylene borofluoride, which was designated as TPE-BF2-C≡CH; S02, β-cyclodextrin sulfonation modification β-cyclodextrin was reacted with chlorosulfonic acid in DMF at 0°C for 2 hours; after purification by dialysis, lyophilization was performed to obtain a white powder, thereby obtaining sulfonated β-cyclodextrin; S03, Chemical Coupling The sulfonated β-CD and TPE-BF2-C≡CH3 are reacted in a water / tetrahydrofuran mixed solvent at 50° C. for 12 hours under the catalysis of CuSO4 / ascorbic acid to obtain the highly sensitive fluorescence detection reagent.

4. The preparation method according to claim 3, characterized in that In step S01, the mass ratio of tetraphenylethylene bromide to BF3·Et2O is 1:1-1.

2.

5. The preparation method according to claim 3, characterized in that In step S01, the column chromatography mobile phase was prepared from petroleum ether / ethyl acetate = 5:

1.

6. The preparation method according to claim 3, characterized in that In step S02, the molecular weight cut-off for dialysis purification is 1000 Da.

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