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CN106832296B - Preparation method and application of saxitoxin molecularly imprinted nano fluorescent material - Google Patents

Preparation method and application of saxitoxin molecularly imprinted nano fluorescent material Download PDF

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CN106832296B
CN106832296B CN201710015262.9A CN201710015262A CN106832296B CN 106832296 B CN106832296 B CN 106832296B CN 201710015262 A CN201710015262 A CN 201710015262A CN 106832296 B CN106832296 B CN 106832296B
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saxitoxin
fluorescent material
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imprinted nano
shellfish
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CN106832296A (en
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史西志
肖婷婷
孙爱丽
王琳
李德祥
陈炯
李逊甲
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Ningbo University
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Abstract

本发明公开了一种石房蛤毒素分子印迹纳米荧光材料的制备方法及应用，特点制备方法包括以下步骤：以石房蛤毒素为模板分子，加入量子点荧光纳米材料，在交联剂正硅酸乙酯和功能单体3‑氨丙基三乙氧基硅烷或者甲基丙烯酰氧丙基三(三甲基硅氧烷基)硅烷存在条件下，引发聚合后，采用超声辅助萃取法除去所得聚合物中模板分子，即获得可特异识别石房蛤毒素的石房蛤毒素分子印迹纳米荧光材料，可将其用于检测贝类样品中石房蛤毒素的含量，优点是粒径均一、选择性好和荧光稳定性优，能实现对贝类样品中石房蛤毒素的快速、高灵敏度检测。

Figure 201710015262

The invention discloses a preparation method and application of a saxitoxin molecularly imprinted nano-fluorescent material. The characteristic preparation method comprises the following steps: using saxitoxin as a template molecule, adding quantum dot fluorescent nano-material, and adding a cross-linking agent ortho-silicon In the presence of ethyl acid ester and functional monomer 3-aminopropyltriethoxysilane or methacryloyloxypropyltris(trimethylsiloxane) silane, after initiating polymerization, remove by ultrasonic-assisted extraction The template molecule in the obtained polymer is to obtain a saxitoxin molecularly imprinted nano-fluorescent material that can specifically recognize saxitoxin, which can be used to detect the content of saxitoxin in shellfish samples. It has good properties and excellent fluorescence stability, and can achieve rapid and high-sensitivity detection of saxitoxin in shellfish samples.

Figure 201710015262

Description

Preparation method and application of saxitoxin molecularly imprinted nano fluorescent material
Technical Field
The invention relates to the technical field of analytical chemistry and material science, in particular to a preparation method of saxitoxin molecularly imprinted nano fluorescent material.
Background
Saxixin dihydrochloride (STX) is one of the most toxic marine toxins currently known, and was first discovered in mussels, california, and substances similar in chemical structure to STX were later isolated from gonyaulax catenella. STX is a derivative of tetrahydropurine, belonging to the marine toxamines. STX blocks Na on neuronal cells and cell sarcolemmas＋Passage, block Na＋The internal flow and thus the neuromuscular transmission are disturbed, and the voluntary muscle is relaxed and paralyzed, so the STX is classified as paralytic shellfish poison. Saxixin dihydrochloride (STX), one of the currently known marine toxins with the highest toxicity, inhibits nerve conduction by affecting sodium ion channels, and clinically mainly causes nervous system and innervation disorders, which makes toxic diagnosis difficult. In particular, STX has a strong resistance to high temperature and acidic environment, and cannot be damaged by general processing, thereby seriously affecting food safety. Therefore, in order to ensure the safety of aquatic products and the health of human beings, the enhancement of the detection of saxitoxin is urgently needed, and particularly, a rapid detection technology is developed.
At present, the rapid detection technology for saxitoxin is mainly an immunoassay technology, and particularly, an enzyme-linked immunosorbent assay (ELISA) is widely applied by the advantages of strong specificity, high sensitivity, low cost and the like, but has the defects of long antibody preparation period, animal experiment requirement, low stability and the like. The molecular imprinting artificial antibody has the advantages of better specificity with biological antibodies, simple synthesis, good stability and the like, and is highly valued. However, since saxitoxin is expensive, the conventional molecular imprinting technique requires a large amount of template molecules, and STX structural analogs are not easily available, resulting in high synthesis cost and unsuitability for the preparation of STX molecularly imprinted polymers. Therefore, the development of an STX molecularly imprinted nano fluorescent material is urgently needed, so that the usage amount of template molecules is effectively reduced, the preparation cost is obviously reduced, and the rapid detection of STX in shellfish samples is realized.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of saxitoxin molecularly imprinted nano fluorescent material with uniform particle size, good selectivity and excellent fluorescence stability and a detection method for building residual saxitoxin in shellfish samples based on the molecularly imprinted nano fluorescent material, thereby realizing rapid and high-sensitivity detection of saxitoxin in shellfish samples.
The technical scheme adopted by the invention for solving the technical problems is as follows: a preparation method of saxitoxin molecularly imprinted nano fluorescent material comprises the following steps: taking saxitoxin as a template molecule, adding a quantum dot fluorescent nano material, initiating polymerization in the presence of a cross-linking agent tetraethyl orthosilicate (TEOS) and a functional monomer 3-Aminopropyltriethoxysilane (APTES) or methacryloxypropyltris (trimethylsiloxy) silane (MPTES), and removing the template molecule in the obtained polymer by using an ultrasonic extraction method to obtain the saxitoxin molecularly imprinted nano fluorescent material capable of specifically identifying saxitoxin.
The method comprises the following specific steps:
(1) stirring 7.5mL of cyclohexane and 1.8mL of Triton X-100 for 15min, then adding 500 mu g of quantum dot fluorescent nano material, 50 mu L of tetraethyl orthosilicate TEOS and 100 mu L of ammonia water, stirring for 2h, finally adding 156 mu L of saxitoxin solution with the concentration of 1mg/mL and 22.8 mu L of functional monomer 3-Aminopropyltriethoxysilane (APTES) or methacryloxypropyl tris (trimethylsiloxy) silane (MPTES), and stirring for room temperature polymerization reaction for 12h to obtain a mixed solution;
(2) adding 10mL of acetone into the mixed solution obtained in the step (1), centrifuging to remove supernatant after precipitation, adding 6mL of double distilled water for dispersion, centrifuging to remove supernatant, adding 5mL of solution consisting of ethanol and acetonitrile in a volume ratio of 8:2, uniformly dispersing by ultrasonic waves, standing for 40min, and centrifuging;
(3) and (3) repeating the step (2) until the saxitoxin is completely removed, thus obtaining the saxitoxin molecularly imprinted nano fluorescent material.
The quantum dot fluorescent nano material is CdSe/ZnS, and the particle size is between 2.5nm and 6 nm.
The application of the saxitoxin molecularly imprinted nano fluorescent material is a method for detecting the content of saxitoxin in shellfish samples by using the saxitoxin molecularly imprinted nano fluorescent material, and the method comprises the following specific steps: dissolving saxitoxin molecularly imprinted nano fluorescent material in ethanol to prepare saxitoxin molecularly imprinted nano fluorescent material ethanol solution with concentration of 15 mug/mL, taking 500 mul of saxitoxin molecularly imprinted nano fluorescent material ethanol solution in a quartz cuvette, adding 500 mul of shellfish sample solution to be detected, and measuring fluorescence value F0Then, after 5min, the fluorescence value F was measured, and the fluorescence measurement conditions: the slit width of the excitation wavelength and the emission wavelength are both 5.0nm, the excitation wavelength is set to 270nm, the emission wavelength is set to 618nm, the voltage is 700eV, and finally the concentration of the saxitoxin is calculated according to the following formula: [ Q ]]=（（F0/F)-1)/Ksv，[Q]The concentration of saxitoxin in the shellfish sample to be tested, F0The fluorescence value when the saxitoxin molecularly imprinted nano fluorescent material and the sample to be detected are just mixed, F is the fluorescence value after 5min of reaction, and Ksv is 0.56.
The preparation method of the shellfish sample solution to be detected comprises the following specific steps: accurately weighing 1.00g of shellfish meat, adding 2mL of acetonitrile water extract containing 0.1wt% of formic acid, performing vortex oscillation for 1min, performing ultrasonic extraction for 10min in an ice water state, centrifuging at 4500rpm below 15 ℃ for 10min, performing ultrasonic extraction on the precipitate for 2 times repeatedly by using acetonitrile water extract containing 0.1wt% of formic acid, combining the supernatants obtained after centrifugation, freezing for 1h at-20 ℃, taking out, rapidly discarding the upper organic phase within 1min, and freeze-drying the lower layer to be nearly dry; redissolving with 0.1wt% formic acid aqueous solution and fixing the volume to 1mL, adding 50mg octadecylsilane chemically bonded silica C18And 50mg of acidic alumina adsorbent, performing vortex oscillation for 1min, centrifuging at the temperature below 15 ℃ at 4500rpm for 10min, and filtering supernatant with 0.22 mu m nylon filter membrane to obtain shellfish sample solution to be detected.
The volume ratio of acetonitrile to water in the acetonitrile water extracting solution is 80: 20.
compared with the prior art, the invention has the advantages that:
1) according to the preparation method of the molecularly imprinted nano fluorescent material, the molecularly imprinted nano fluorescent material taking saxitoxin as the template molecule is synthesized by adopting a reverse microemulsion method for the first time, the problem of high template consumption in the preparation process of the conventional molecularly imprinted-quantum dot nano fluorescent polymer is solved through method optimization, and the method has the advantages of simple and scientific process, capability of effectively reducing the use amount of the template molecule and experiment cost reduction.
2) The STX-MIP-QDs prepared by the method have the advantages of uniform particle size, good selectivity, good fluorescence stability and the like, and can be used for detecting saxitoxin.
3) The invention also provides an STX detection method based on saxitoxin molecularly imprinted nano fluorescent materials (STX-MIP-QDs), which has better fluorescence stability and specific selectivity to saxitoxin; the method is combined with a corresponding sample pretreatment method, can realize the rapid specific identification and detection of the STX in the shellfish sample, and has the advantages of high sensitivity, good stability, high selectivity, simple method and convenient operation.
4) The method can be popularized to the preparation and detection of other water-soluble shellfish toxin molecularly imprinted nano fluorescent materials.
In conclusion, the invention provides a preparation method of STX molecularly imprinted nano fluorescent materials (STX-MIP-QDs), which effectively reduces the usage amount of template molecules and obviously reduces the preparation cost. Meanwhile, the rapid detection of the STX in the shellfish sample is realized by utilizing the STX-MIP-QDs in combination with a corresponding sample pretreatment method, and the method has the advantages of rapidness, simplicity, better practicability and the like.
Drawings
FIG. 1 is a transmission electron microscope image of saxitoxin molecularly imprinted nano fluorescent material of the present invention;
FIG. 2 is a graph showing the comparison of fluorescence quenching results of saxitoxin and its structural analogs on MIP-QDs;
FIG. 3 is a graph showing the fluorescence response of saxitoxin molecularly imprinted nano-fluorescent material to saxitoxin.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Detailed description of the preferred embodiment
A preparation method of saxitoxin molecularly imprinted nano fluorescent material comprises the following steps: taking saxitoxin as a template molecule, adding a quantum dot fluorescent nano material, initiating polymerization in the presence of a cross-linking agent ethyl orthosilicate and a functional monomer, and removing the template molecule in the obtained polymer by adopting an ultrasonic-assisted extraction method to obtain the saxitoxin molecularly imprinted nano fluorescent material capable of specifically identifying saxitoxin. The method comprises the following specific steps:
(1) stirring 7.5mL of cyclohexane and 1.8mL of Triton X-100 for 15min, then adding 500 mu g of quantum dot fluorescent nano material, 50 mu L of tetraethyl orthosilicate TEOS and 100 mu L of ammonia water, stirring for 2h, finally adding 156 mu L of saxitoxin solution with the concentration of 1mg/mL and 22.8 mu L of functional monomer 3-Aminopropyltriethoxysilane (APTES) or methacryloxypropyl tris (trimethylsiloxy) silane (MPTES), and stirring for room temperature polymerization reaction for 12h to obtain a mixed solution;
(2) adding 10mL of acetone into the mixed solution obtained in the step (1), centrifuging to remove supernatant after precipitation, adding 6mL of double distilled water for dispersion, centrifuging to remove supernatant, adding 5mL of solution consisting of ethanol and acetonitrile in a volume ratio of 8:2, uniformly dispersing by ultrasonic waves, standing for 40min, and centrifuging;
(3) and (3) repeating the step (2) until the saxitoxin is completely removed, thus obtaining the saxitoxin molecularly imprinted nano fluorescent material.
The quantum dot fluorescent nano material is CdSe/ZnS, and the particle size is between 2.5nm and 6 nm.
Scanning electron microscope and transmission electron microscope images of the saxitoxin molecularly imprinted nano fluorescent material are shown in figure 1, which shows that the obtained MIP-QDs are highly dispersed and uniform in size.
Detailed description of the invention
A novel sample pretreatment mode QuEChERS is combined with a saxitoxin molecularly imprinted nano fluorescent material (STX-MIP-QDs) detection method, so that a rapid and high-sensitivity detection method for saxitoxin in shellfish samples is established, and the specific process is as follows:
1. shellfish sample pretreatment method based on QuEChERS
Accurately weighing 1.00g of shellfish meat, adding 2mL of acetonitrile water extract containing 0.1wt% of formic acid, performing vortex oscillation for 1min, performing ultrasonic extraction for 10min in an ice water state, centrifuging at 4500rpm below 15 ℃ for 10min, repeatedly performing ultrasonic extraction on the precipitate for 2 times by using acetonitrile water extract containing 0.1wt% of formic acid (the volume ratio of acetonitrile to water is 80: 20), combining the centrifuged supernatants, freezing for 1h at-20 ℃, taking out, rapidly discarding an upper organic phase within 1min, and freeze-drying a lower layer to be nearly dry; redissolving with 0, wt1% formic acid solution and fixing volume to 1mL, adding 50mg C18(octadecylsilane chemically bonded silica) and 50mg acidic alumina adsorbent, vortex oscillating for 1min, centrifuging at 4500rpm below 15 deg.C for 10min, and filtering the supernatant with 0.22 μm nylon filter membrane to obtain shellfish sample solution to be measured.
2. Saxitoxin detection based on molecularly imprinted nano fluorescent material
Dissolving saxitoxin molecularly imprinted nano fluorescent material in ethanol to prepare saxitoxin molecularly imprinted nano fluorescent material ethanol solution with concentration of 15 mug/mL, taking 500 mul of saxitoxin molecularly imprinted nano fluorescent material ethanol solution in a quartz cuvette, adding 500 mul of shellfish sample solution to be detected, and measuring fluorescence value F0Then, after 5min, the fluorescence value F was measured, and the fluorescence measurement conditions: the slit widths of the excitation wavelength and the emission wavelength are both 5.0nm, the excitation wavelength is set to 270nm, the emission wavelength is set to 618nm, the voltage is 700eV, and each experiment is carried out in parallel for three times to ensure the accuracy.
And finally, calculating the concentration of saxitoxin according to the following formula: [ Q ]]=（（F0/F)-1)/Ksv，[Q]The concentration of saxitoxin in the shellfish sample to be tested, F0The fluorescence value when the saxitoxin molecularly imprinted nano fluorescent material and the sample to be detected are just mixed, F is the fluorescence value after 5min of reaction, and Ksv is 0.56.
Detailed description of the preferred embodiment
1. Specificity of
The halichondric acid toxin molecularly imprinted-quantum dot polymer is synthesized by taking halichondric acid (OA) toxin as a template molecule, Saxitoxin (STX), actinomycin (ATX-a), Gonyautoxin (DTX) and Neosaxitoxin (NEO) are selected as structural analogues, the specificity of the obtained MIP-QDs is analyzed (a fluorescence quenching system is expressed by an equation,F 0 /F=1+Ksv[Q]，F 0 andFrespectively shows the initial fluorescence value of MIP-QDs and the fluorescence value after cypermethrin is added,Ksvis a constant parameter in the Stem-Volmer equation, [ Q ]]Is the concentration of the quencher. (F 0 -F) Showing fluorescence quenching values before and after addition of cypermethrin, ((ii))F 0 -F）/FThe imprinting efficiency of MIP-QDs. Of MIP-QDs and NIP-QDsKsvThe ratio of values indicates the imprinting factor: (IF) And is used to evaluate selectivity of MIP-QDs. ).
As can be seen from FIG. 2, in the experiments on toxins and analogs thereof, MIP-QDs have been shown to react with the template saxitoxinKsvIs far higher than the structural analogue, which indicates that the obtained MIP-QDs have specific fluorescence inhibition on saxitoxin.
2. High sensitivity
In the experiment of the fluorescence sensitivity of the toxin, we selected 5 concentrations of saxitoxin 20 μ g/L, 40 μ g/L, 60 μ g/L, 80 μ g/L and 100 μ g/L, and used them to perform the experiment by using the method of the second embodiment, and it can be seen from FIG. 3 that the fluorescence inhibition is reduced to different degrees with the increase of the toxin concentration. Suppression valueF 0 /FThe-1 is used as the ordinate, and saxitoxin is used as the abscissa to make a standard curve, so that the inhibition effect of MIP-QDs can be more visually seen. The standard curve equation is as follows: y =0.0014x + 0.0122, R = 0.9932, the limit of detection being the concentration corresponding to the standard deviation of the blank signal three times by fluorescence quenching. The results are shown in the following table 1,
TABLE 1 minimum detection limits for STX
Toxin sample Common mussel Oyster shell Clam
STX detection limit [ mu ] g/L 7.5 15 20
3. Recovery rate
The recovery rate is determined by adopting a matrix labeling experiment before extraction, adding STX toxin into a sample to make the final concentration of the STX toxin be 30.0, 40.0 and 50.0 mu g/kg, then separating and purifying, detecting by a MIP-QDs system, measuring the obtained concentration, and calculating the recovery rate. The results are shown in the following table 2,
TABLE 2 quantitation and detection limits of STX in 2 different matrices, μ g/kg
Figure DEST_PATH_IMAGE001
As can be seen from Table 2, the recovery rate was between 85.8% and 114.6% and the Relative Standard Deviation (RSD) was between 4.0% and 9.4%, indicating that the method has good accuracy and precision.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims (5)

1.一种贝类样品中石房蛤毒素含量的检测方法，其特征在于具体步骤如下：将石房蛤毒素分子印迹纳米荧光材料溶于乙醇中制得浓度为15μg/mL的石房蛤毒素分子印迹纳米荧光材料乙醇溶液，取500μL石房蛤毒素分子印迹纳米荧光材料乙醇溶液于石英比色皿中，再加入500μL待测贝类样品溶液，测量荧光值F0，然后5min后，测量荧光值F，荧光测量条件：激发波长和发射波长的狭缝宽度均为5.0nm，激发波长设定为270nm，发射波长设定为618nm，电压为700eV，最后按下列公式计算得到石房蛤毒素浓度：[Q]＝((F0/F)-1)/Ksv，[Q]为待测贝类样品中石房蛤毒素的浓度，F0刚混合石房蛤毒素分子印迹纳米荧光材料和待测样品时的荧光值，F为反应5min后的荧光值，Ksv为0.56，其中所述的石房蛤毒素分子印迹纳米荧光材料的制备方法包括以下步骤：以石房蛤毒素为模板分子，加入量子点荧光纳米材料，在交联剂正硅酸乙酯和功能单体3-氨丙基三乙氧基硅烷存在条件下，引发聚合后，采用超声辅助萃取法除去所得聚合物中模板分子，即获得可特异识别石房蛤毒素的石房蛤毒素分子印迹纳米荧光材料。1. a detection method for saxitoxin content in a shellfish sample, characterized in that the specific steps are as follows: dissolving the saxitoxin molecularly imprinted nano-fluorescent material in ethanol to obtain a saxitoxin molecule with a concentration of 15 μg/mL Imprinted nano-fluorescent material ethanol solution, take 500 μL of saxitoxin molecularly imprinted nano-fluorescent material ethanol solution in a quartz cuvette, add 500 μL of shellfish sample solution to be tested, measure the fluorescence value F 0 , and then measure the fluorescence value after 5 minutes F. Fluorescence measurement conditions: the slit width of excitation wavelength and emission wavelength are both 5.0nm, the excitation wavelength is set to 270nm, the emission wavelength is set to 618nm, the voltage is 700eV, and finally the saxitoxin concentration is calculated according to the following formula: [Q]=((F 0 /F)-1)/Ksv, [Q] is the concentration of saxitoxin in the shellfish sample to be tested, F 0 is just mixed with the saxitoxin molecularly imprinted nano-fluorescent material and the sample to be tested F is the fluorescence value after 5 min of reaction, Ksv is 0.56, wherein the preparation method of the saxitoxin molecularly imprinted nano-fluorescent material comprises the following steps: using saxitoxin as a template molecule, adding quantum dots Fluorescent nanomaterials, in the presence of crosslinking agent ethyl orthosilicate and functional monomer 3-aminopropyltriethoxysilane, after initiating polymerization, ultrasonic-assisted extraction method is used to remove template molecules in the obtained polymer, that is to obtain Saxitoxin molecularly imprinted nano-fluorescent material that can specifically recognize saxitoxin. 2.根据权利要求1所述的一种贝类样品中石房蛤毒素含量的检测方法，其特征在于所述的待测贝类样品溶液的制备方法具体步骤如下：精确称取1.00g贝肉，加入2mL含0.1wt％甲酸的乙腈水提取液，涡旋振荡1min后冰水状态下超声提取10min，于15℃以下，4500rpm离心10min，取沉淀用含0.1wt％甲酸的乙腈水提取液反复超声提取2次，将离心后得到的上清液合并，于-20℃条件下冷冻1h，取出后于1min内迅速弃去上层有机相，下层冷冻干燥至近干；用0.1wt％甲酸水溶液复溶并定容至1mL后，加入50mg十八烷基硅烷键合硅胶C18和50mg酸性氧化铝吸附剂，涡旋振荡1min，于15℃以下，4500rpm离心10min，取上层液过0.22μm尼龙滤膜，即得到待测贝类样品溶液。2. the detection method of saxitoxin content in a kind of shellfish sample according to claim 1, it is characterized in that the concrete steps of the preparation method of described shellfish sample solution to be tested are as follows: accurately weigh 1.00g shellfish meat, Add 2 mL of acetonitrile water extract containing 0.1 wt% formic acid, vortex for 1 min and then ultrasonically extract for 10 min in ice-water state, centrifuge at 4500 rpm for 10 min at below 15 °C, take the precipitate and use acetonitrile water extract containing 0.1 wt % formic acid to repeatedly ultrasonicate Extracted twice, the supernatants obtained after centrifugation were combined, frozen at -20 °C for 1 h, the upper organic phase was quickly discarded within 1 min after removal, and the lower layer was freeze-dried to near dryness; reconstituted with 0.1 wt% formic acid aqueous solution and After the volume was adjusted to 1 mL, 50 mg of octadecylsilane-bonded silica gel C 18 and 50 mg of acidic alumina adsorbent were added, vortexed for 1 min, centrifuged at 4500 rpm for 10 min at below 15 °C, and the upper layer was taken through a 0.22 μm nylon filter membrane. That is, the shellfish sample solution to be tested is obtained. 3.根据权利要求2所述的一种贝类样品中石房蛤毒素含量的检测方法，其特征在于：所述的乙腈水提取液中乙腈与水的体积比为80：20。3. the detection method of saxitoxin content in a kind of shellfish sample according to claim 2, is characterized in that: the volume ratio of acetonitrile and water in described acetonitrile water extract is 80:20. 4.根据权利要求1所述的一种贝类样品中石房蛤毒素含量的检测方法，其特征在于所述的石房蛤毒素分子印迹纳米荧光材料的制备方法具体步骤如下：4. the detection method of saxitoxin content in a kind of shellfish sample according to claim 1, it is characterized in that the concrete steps of the preparation method of described saxitoxin molecularly imprinted nano fluorescent material are as follows: (1)将7.5mL环己烷与1.8mL Triton X-100搅拌15min，然后加入量子点荧光纳米材料500μg、四乙基原硅酸盐50μL、氨水100μL，搅拌2h，最后加入浓度为1mg/mL的石房蛤毒素溶液156μL和功能单体3-氨丙基三乙氧基硅烷22.8μL，搅拌室温聚合反应12h，得到混合液；(1) Stir 7.5 mL of cyclohexane and 1.8 mL of Triton X-100 for 15 minutes, then add 500 μg of quantum dot fluorescent nanomaterials, 50 μL of tetraethylorthosilicate, and 100 μL of ammonia water, stir for 2 hours, and finally add a concentration of 1 mg/mL 156 μL of the saxitoxin solution and 22.8 μL of the functional monomer 3-aminopropyltriethoxysilane were stirred at room temperature for polymerization reaction for 12 h to obtain a mixed solution; (2)在步骤(1)得到的混合液中加入10mL丙酮待沉淀后离心弃上清液，加入6mL双蒸水分散后离心弃上清液，加入5mL由乙醇和乙腈按体积比8:2组成的溶液，超声分散均匀静置40min后离心；(2) 10 mL of acetone was added to the mixed solution obtained in step (1) and the supernatant was discarded by centrifugation after precipitation, 6 mL of double distilled water was added to disperse and the supernatant was discarded by centrifugation, and 5 mL of ethanol and acetonitrile were added in a volume ratio of 8:2 The formed solution was dispersed by ultrasonic evenly and left for 40 minutes, and then centrifuged; (3)重复步骤(2)直到完全除去石房蛤毒素为止，即获得石房蛤毒素分子印迹纳米荧光材料。(3) Step (2) is repeated until the saxitoxin is completely removed, that is, the saxitoxin molecularly imprinted nano-fluorescent material is obtained. 5.根据权利要求4所述的一种贝类样品中石房蛤毒素含量的检测方法，其特征在于：所述的量子点荧光纳米材料为CdSe/ZnS，粒径介于2.5nm-6nm之间。5. the detection method of saxitoxin content in a kind of shellfish sample according to claim 4, is characterized in that: described quantum dot fluorescent nanomaterial is CdSe/ZnS, and particle diameter is between 2.5nm-6nm .
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