CN115121225B - 一种用于富集thc的磁性脂质体、制备方法及其应用 - Google Patents
一种用于富集thc的磁性脂质体、制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种用于富集THC的磁性脂质体、制备方法及其应用。本发明所述磁性脂质体包括Fe3O4磁珠、包覆在所述Fe3O4磁珠上的脂质体以及含有特异性吸附受体的细胞膜,所述特异性吸附受体为1型大麻素受体,所述脂质体包括质量比为15~20:2~5:0.5~1.5的大豆磷脂、胆固醇以及维生素E。本发明制备的磁性脂质体用于富集或者检测THC简便快捷,灵敏度高,易实现高通量检测,专属性高。
Description
技术领域
本发明涉及富集THC检测技术,特别涉及到一种用于富集THC的磁性脂质体、制备方法及其应用。
背景技术
四氢大麻酚(THC)是一种流行的精神活性药物,属于大麻不同部位的大麻素群,结构式如下:
四氢大麻酚口服或吸烟进入人体后主要以四氢大麻酚酸(THCA)的形式存在。THCA通过加热会使四氢大麻酚脱羧,进一步代谢为11-羟基-四氢大麻酚(11-OH-THC),最后生成主要代谢物11-羧基-四氢大麻酚(THCA)。吸食大麻初期有欣快感,同时会发生短程记忆受损,视、听、触或味觉变得更加敏锐等现象,但是随着吸食的量的增加会引起懒散、意识混乱、无方向感、时空扭曲,动作协调性差等不良反应。尽管市场上新的精神活性物质正在与日俱增,但大麻仍然是毒驾最为相关的精神活性物质,血液中四氢大麻酚及其两种代谢物的浓度取决于使用方式、使用量及代谢时间。处理警察交通管制时采集的样本,其中THC和THCA血药浓度分别低于20ng/mL和200ng/mL。THC和其代谢物的定量可通过气相色谱-质谱(GC-MS)、柱前衍生化和液相色谱串联质谱(LC-MS/MS)等方法进行检测。前处理步骤对最终检测的结果起着至关重要的作用,目前主要通过使用固相萃取(SPE)、液-液萃取(LLE)等方法对样品进行前处理,以得到更快的分析时间,更低的检测限。环境及污水中四氢大麻酚及其代谢物的测定为毒品溯源和打击毒品犯罪活动提供重要依据,但THC易氧化,且易吸附于环境介质中不易被检测到,同时一般磁固相萃取(MSPE)是基于理化性质进行富集只具备一定的特异性,THC在人体内的主要受体是1型大麻素受体(CB1R),这是一种G蛋白偶联受体,CB1R中存在Arg2143.50和Asp3386.30,拮抗剂/反向激动剂化合物通过TMH1和TMH7之间的间隙进入结合袋,配体结合袋主要是通过与细胞外环2(ICL2)、TMH3、TMH5、TMH6和TMH7上残基的疏水相互作用形成的。
发明内容
发明目的:本发明提供了一种用于富集THC的磁性脂质体、制备方法及其应用,本发明的脂质体基于CB1R与THC的特异性结合,可实现对THC富集和检测。
技术方案:本发明所述的用于富集THC的磁性脂质体,所述磁性脂质体包括Fe3O4磁珠、包覆在所述Fe3O4磁珠上的脂质体以及含有特异性吸附受体的细胞膜,所述特异性吸附受体为1型大麻素受体(CB1R),所述脂质体包括质量比为15~20:2~5:0.5~1.5的大豆磷脂、胆固醇以及维生素E。
作为本发明的一种优选实施方式,所述Fe3O4磁珠与所述维生素E的质量比为0.8~1:0.8~1。
作为本发明的一种优选实施方式,所述细胞膜与所述Fe3O4磁珠的质量比为0.15~0.2:0.8~1。
作为本发明的一种优选实施方式,所述Fe3O4磁珠、脂质体以及细胞膜的质量比为5:130:1。
本发明所述的用于富集THC的磁性脂质体的制备方法,包括以下步骤:
(1)用薄膜分散法制备脂质体包覆的Fe3O4磁珠,得到脂质体包覆的Fe3O4磁珠Lip@Fe3O4;
(2)将步骤(1)得到的脂质体包覆的Fe3O4磁珠与含有特异性吸附受体的细胞膜混合后分散均匀,过滤,得到磁性脂质体CM@Lip@Fe3O4。
作为本发明的一种优选实施方式,步骤(1)中,所述薄膜分散法包括以下步骤:大豆磷脂、胆固醇以及维生素E加入三氯甲烷中,使其完全溶解,在45~50℃的水浴加热下去除三氯甲烷,形成一层淡黄色均匀薄膜,加入分散于PBS溶液中的Fe3O4磁珠,继续使用旋转蒸发仪于25~30℃的水浴中进行水化,将黑色磁性产物用磁铁分离收集,用去离子水和PBS溶液分别清洗后,溶解于PBS溶液中,使用细胞破碎仪破碎,得到Lip@Fe3O4。
作为本发明的一种优选实施方式,步骤(2)中,含有特异性吸附受体的细胞膜通过以下方法制备:将导入有1型大麻素受体序列的HEK 293F细胞,加入蛋白酶抑制剂,并且在细胞破碎仪中破碎,破碎完毕后将其置于冷冻离心机中,在6000~8000rmp转速下离心,取上清液,将收集的上清液使用超速冷冻离心机以30000rpm的转速离心,弃去上清液取沉淀,得到含有特异性吸附受体的细胞膜。
本发明还提供了上述的用于富集THC的磁性脂质体在富集THC中的应用。
作为本发明的一种优选实施方式,所述应用方法为:在待富集样品中加入质量浓度为50~100mg/L的磁性脂质体,调整溶液pH为6~7,磁性脂质体对THC进行富集。
作为本发明的一种优选实施方式,吸附有THC的磁性脂质体的洗脱条件为:6000~8000r/min离心20~30min,洗脱液为:70~80%甲醇水溶液。
本发明提供一种使用CM@Lip@Fe3O4富集污水中THC的方法,包括以下步骤:
(1)获取镶嵌着CB1R的细胞膜CM:将载入CB1R序列的HEK 293F细胞进行培养48小时,加入通用型蛋白酶抑制剂在细胞破碎仪中破碎,通过高速离心和超速离心获得细胞膜,通过冻干置于-20℃保存;
(2)使用水热法制备Fe3O4磁珠,再使用薄膜分散法制备脂质体的水合阶段加入Fe3O4磁珠获得脂质体包覆的Fe3O4磁珠Lip@Fe3O4;
(3)取1mg冻干的细胞膜溶解于1mLPBS溶液中,将其与Lip@Fe3O4混匀,使用脂质体挤出器进行挤压获得CM@Lip@Fe3O4。
(4)将制备好的CM@Lip@Fe3O4加入到添加到THC污水样品进行磁固相萃取,使用HPLC-MS/MS进行检测,以甲氧那明作为内标物使用内标法进行计算,以THC浓度为横坐标,THC的响应强度除内标物的响应强度为纵坐标,绘制标准曲线;
(5)将制备好的CM@Lip@Fe3O4添加到相同体积的THC的污水样品,混匀,进行磁固相萃取,根据响应强度计算样品中THC的浓度。
本发明制备的CM@Lip@Fe3O4吸附THC最佳条件的进行探索,包括pH,材料用量。确定了最佳条件为pH=7,材料用量为75mg/L,以80%的甲醇水作为洗脱液,8000r/min离心30min为洗脱条件。本发明的专属性良好,本方法灵敏度高。
有益效果:本发明CM@Lip@Fe3O4可检测实际污水样品中的THC,定量下限低,方法灵敏度高,专属性高,通过对实际污水样品中THC的定量检测,从而实现对区域内吸食大麻情况的鉴定。
附图说明
图1为CM@Lip@Fe3O4制备过程及样品前处理过程,其中,A图为CM@Lip@Fe3O4制备流程,B图为CM@Lip@Fe3O4对污水中THC的富集过程;
图2为本发明实施例1制备的CM@Lip@Fe3O4的透射电镜图;
图3为CB1R的序列以及WB图,其中,A图为CB1R的序列,B图为模拟的CB1R的空间结构,C图为CB1R的WB图,1、2、3组为未载入CB1R序列组,4、5、6组为载入CB1R序列组;
图4为CM@Lip@Fe3O4富集THC的最优条件,其中,A图为pH对THC吸附率的影响,B图为吸附材料添加浓度对THC吸附率的影响,C图为在离心条件下洗脱液中甲醇浓度对回收率的影响,D图为在离心条件下pH对回收率的影响;
图5为CM@Lip@Fe3O4在不同浓度下的线性曲线;
图6为CM@Lip@Fe3O4对不同浓度的THC的回收率;
图7为CM@Lip@Fe3O4对六种不同毒品的回收率。
具体实施方式
实施例1:CM@Lip@Fe3O4磁性纳米粒子的构建
(1)Fe3O4磁珠的构建
通过溶剂热反应进行制备Fe3O4磁珠,具体步骤如下:室温下,称取4.30g FeCl3·6H2O、2.35g柠檬酸三钠和8.75g醋酸钠(NaAc)置于250mL干燥圆底烧瓶中,加入150mL乙二醇并通过机械搅拌使其溶解。将得到的黄色溶液转移并密封在聚四氟乙烯内衬不锈钢高压釜中高压灭菌器中,在200℃下加热10h,然后冷却至室温。将黑色磁性产物用磁铁分离收集,分别用去离子水和乙醇清洗3次后,用磁铁吸附产物于烧杯底部,上清液倒出,之后在60℃真空干燥箱中干燥12h,密封备用。
(2)Lip@Fe3O4磁性纳米粒子的构建
用薄膜分散法进行制备脂质体包覆的Fe3O4磁珠,具体操作方法如下:室温下,称取100mg大豆磷脂(生物技术级,来自于麦克林CAS:8002-43-5,Lot#:C12061184),25mg胆固醇,5mg维生素E置于250mL烧瓶底部,加入10mL三氯甲烷并通过水浴超声使其完全溶解。在50℃的水浴加热下使用旋转蒸发仪去除三氯甲烷,最终在瓶底形成一层淡黄色均匀薄膜。室温下称取5mg的Fe3O4,将其分散于10mLPBS溶液中并通过水浴超声使其完全分散。将该溶液加入挥干后的烧瓶中,继续使用旋转蒸发仪于30℃的水浴中进行1小时的旋转水化。将黑色磁性产物用磁铁分离收集,用去离子水和PBS分别清洗3次后,将其溶解于10mL PBS溶液中,使用细胞破碎仪以10%的功率破碎8min,每开启2s暂停1s,得到Lip@Fe3O4。
(3)镶嵌CB1R的细胞膜的提取
取15mL导入CB1R序列(载有CB1R序列的质粒来自于金斯瑞生物科技有限公司,CB1R的氨基酸序列如图3中A图所示,其三维空间折叠结构如图3中B图所示)的HEK 293F细胞中加入150μL的通用型蛋白酶抑制剂在细胞破碎仪中以20%的功率破碎10min,每开启3s暂停2s,破碎完毕后将其置于冷冻离心机以8000rpm的转速离心3次每次10分钟,每次离心后取上清液,将收集的上清液使用超速冷冻离心机以30000rpm的转速离心30分钟,弃去上清液取沉淀,得到镶嵌CB1R的细胞膜,将其冻干12小时后置于-20℃保存。
(4)CB1R的WB(Western blotting)表征
取1mg上述细胞膜,溶解于0.8mL PBS溶液中,加入200μL loading buffer(5×),涡旋后煮沸10min,作为实验组放置在冰上冷却保温,同法处理未导入CB1R序列的细胞膜作为对照组。将Marker Protein,实验组,对照组加到SDS-PAGE胶的加样孔中,在电泳槽中加入电泳缓冲液,在70V的电压下进行30分钟电泳,样品loading buffer跑至分离胶后将电压增至110V,电泳60分钟后终止电泳。海绵垫放在转膜使用的夹子上,取出SDS-PAGE胶,将PVDF膜(0.45μm)放在黑色夹子的海绵垫上,SDS-PAGE胶放在红色夹子的海绵垫上,夹紧夹子后将其放入转移槽中,加入适量电转液后将转移槽放入冰水中,在90V的电压下进行120分钟的转膜。将转膜后的PVDF膜放置在摇床上用TBST洗3遍,每次10分钟。将清洗完毕的PVDF膜使用封闭液进行2h的封闭过程,封闭结束后加入His标签的1抗溶液,在4℃的条件下孵育12h,孵育结束后回收1抗溶液,将PVDF膜放置在摇床上用TBST洗3遍,每次10分钟。将2抗溶液加入到清洗完毕的PVDF膜上在室温下进行2h的孵育,将PVDF膜放置在摇床上用TBST洗3遍,每次10分钟。将清洗完毕的PVDF膜放置在显影仪台面上,取适量显影液于PVDF膜上,将PVDF膜用镊子夹起来正反面充分和显影液接触,使用显影仪进行曝光拍摄,如图3中C图所示,实验组的条带清晰,且分子量位于50-62kD之间,与预测的分子量相符,这说明了CB1R成功得在表达在了细胞膜上。
(5)CM@Lip@Fe3O4磁性脂质体的构建
取10mL上述Lip@Fe3O4溶液,以及1mg细胞膜,将细胞膜加入溶液中涡旋15s分散均匀,再使用水浴超声使其完全溶解,将溶液使用脂质体挤出器进行挤压,依次通过1000nm,800nm,400nm,200nm的滤膜,取滤液,用磁铁将黑色磁性物质吸附至瓶底,用PBS冲洗三次,得到CM@Lip@Fe3O4磁性脂质体,其透射电镜图如图2所示,将其置于10mL PBS溶液中4℃保存。
实施例2:磁性脂质体CM@Lip@Fe3O4吸附THC的条件筛选
(1)对本发明的CM@Lip@Fe3O4吸附THC的条件包括pH、材料用量进行筛选,确定了最佳条件为pH=7,材料用量为75mg/L。同时对于洗脱过程的条件进行考察,包括洗脱液乙腈含量、甲醇含量以及离心步骤,确定了最终以80%的甲醇水作为洗脱液,8000r/min离心30min为洗脱条件。
(2)吸附率的测定:在100mL超纯水中,加入50μL的THC(1.0μg/mL)使其浓度为500ng/L,放置1小时后加入7.5mg CM@Lip@Fe3O4,涡旋混匀后水浴超声30s,震荡20min后使用强磁铁将CM@Lip@Fe3O4吸附到杯壁上,吸取0.8mL上清液至2mL EP管中,加入0.2mL 1μg/mL的内标物甲氧那明混匀后通过0.2μm的有机滤膜,得到样品组。将50μL的THC(1.0μg/mL)直接加到100mL超纯水中,震荡均匀后吸取0.8mL上清液至2mL EP管中,加入0.2mL 1μg/mL的内标物甲氧那明,作为对照组。
检测THC的方法为:
液相色谱条件:色谱柱:Ecllpse Plus C18(100mm×2.1mm×5μm);柱温:40℃;流速:1mL/min;进样体积:5μL;流动相:A为0.1%(体积分数)甲酸缓冲水溶液,B为乙腈;梯度洗脱程序:0~5min,95%B相。
质谱条件:离子源:ESI,正离子模式;监测模式:d-MRM模式;雾化器流速:4L/min;干燥器流速:10L/min;加热气流速:20L/min;接口电压:5000V;接口温度:300℃。
将制备好的样品和对照品按照检测THC的方法进行HPLC-MS/MS测定,记录各组分的峰面积,经内标法计算方法得出其浓度,计算吸附率。
(3)对本发明的CM@Lip@Fe3O4吸附THC的条件pH进行筛选,取十二份超纯水各100mL,将超纯水分为俩组,一组为样品组一组为对照组,俩组的六份超纯水通过加入HCl(1M)和NaOH(1M)调节溶液的pH,使其pH分别为2,3,5,7,9,10,按吸附率的测定方法测定各pH下THC的吸附率,结果如图4中A图与D图所示,结果表明pH为7时吸附率及回收率最高,之后的实验的pH均为7。
(4)对本发明的CM@Lip@Fe3O4吸附THC的条件材料用量进行筛选,取七份超纯水各100mL,标为1-7号,将7号作为对照组,按吸附率的测定方法测定不同材料用量下的THC的吸附率,其中1-6号样品中分别添加2.5mg,5mg,7.5mg,10mg,15mg,22.5mg的吸附材料CM@Lip@Fe3O4,结果如图4中B图所示,结果表明添加7.5mg的吸附材料CM@Lip@Fe3O4即可将THC完全吸附,之后实验中材料用量按照75mg/L的浓度添加。
(5)回收率的测定:取100mL污水,调整其pH为7,加入50μL的THC(1.0μg/mL)使其浓度为500ng/L,放置1小时后加入7.5mg CM@Lip@Fe3O4,涡旋混匀后水浴超声30s,震荡5min后使用强磁铁将CM@Lip@Fe3O4吸附到杯壁上,吸取所有液体后添加0.8mL洗脱液,混匀后水浴超声30s,使用强磁铁将CM@Lip@Fe3O4吸附到杯壁上,吸取洗脱液至2mL EP管中,加入0.2mL1μg/mL的内标物甲氧那明混匀后通过0.2μm的有机滤膜,得到样品组。
(6)将50μL的THC(1.0μg/mL)直接加到0.8mL洗脱液中,震荡均匀后加入0.2mL1μg/mL的内标物甲氧那明,作为对照组。
将制备好的样品和对照品按照检测THC的方法进行HPLC-MS/MS测定,记录各组分的峰面积,经内标法计算方法得出其浓度,计算回收率。
(7)对本发明的CM@Lip@Fe3O4吸附THC的条件洗脱液成分进行筛选,取5份污水各100mL,按回收率的测定计算各组回收率,其中洗脱液成分分别为20%甲醇水溶液,40%甲醇水溶液,60%甲醇水溶液,80%甲醇水溶液,甲醇溶液,且在添加洗脱液后增加30min8000rpm/min的离心步骤。结果如图4所示,图4中C图为甲醇溶液结合离心处理对于回收率的影响结果,结果表明回收率基本上随着溶剂浓度增加有显著提升,且80%甲醇水溶液回收率最高,后续实验以80%甲醇水溶液作为洗脱液。
实施例3:HPLC-MS/MS检测THC标准曲线的建立
取六份污水各100mL,加入不同体积的1μg/mL的THC使其终浓度分别为100ng/L,200ng/L,300ng/L,400ng/L,500ng/L,1000ng/L,分别加入不同体积的污水使总体积一致,放置1小时后加入相同体积的CM@Lip@Fe3O4使其终浓度为75mg/L,调整其pH为7,涡旋混匀后水浴超声30s,震荡5min。使用强磁铁将CM@Lip@Fe3O4吸附到杯壁上,吸取剩余液体后加入0.8mL 80%甲醇水溶液将杯壁上的CM@Lip@Fe3O4冲洗下来,将收集到的溶液转移至2mL的EP管中,以8000rpm/min的转速离心30min,使用磁铁将CM@Lip@Fe3O4吸附到EP管的管壁上,将0.8mL的洗脱液转移至新的2mL EP管内,加入0.2mL 1μg/mL的内标物甲氧那明混匀后通过0.2μm的有机滤膜,将制备好的样品进行HPLC-MS/MS检测,THC的定量离子对是314.2/193.2,内标物甲氧那明的定量离子对为181.0/121.0,以甲氧那明作为内标物使用内标法进行计算,以THC浓度为横坐标,THC的响应强度除内标物的响应强度为纵坐标,绘制标准曲线,如图5所示。绘制标准曲线后得到线性方程y=0.04864x+4.995,R2=0.9836。添加CM@Lip@Fe3O4最佳浓度为75mg/L,检测THC的线性范围为1-2000ng/L。
实施例4:CM@Lip@Fe3O4磁性脂质体检测污水样品中的THC的准确度
(1)污水样品的前处理:检测前将污水样品静置,使用0.4μm的滤膜进行抽滤,取滤液作为实验使用的基质。
(2)方法准确度考察:取8份100mL抽滤后的污水样品,分为2组每组4份污水样品标为1-4号样品,加入不同体积的1μg/mL的THC使其终浓度分别为含有50ng/L,100ng/L,500ng/L,1000ng/L,分别加入不同体积的污水使总体积一致,混匀后放置一小时,三组样品分别添加CM@Lip@Fe3O4使其终浓度分别为75mg/L,150mg/L,调整其pH为7,涡旋混匀后水浴超声30s,震荡5min。使用强磁铁将CM@Lip@Fe3O4吸附到杯壁上,吸取剩余液体后加入0.8mL80%甲醇水溶液将杯壁上的CM@Lip@Fe3O4冲洗下来,将收集到的溶液转移至2mL的EP管中,以8000rpm/min的转速离心30min,使用磁铁将CM@Lip@Fe3O4吸附到EP管的管壁上,将0.8mL的洗脱液转移至新的2mL EP管内,加入0.2mL 1μg/mL的内标物甲氧那明混匀后通过0.2μm的有机滤膜,将制备好的样品进行HPLC-MS/MS检测,THC的定量离子对是314.2/193.2,内标物甲氧那明的定量离子对为181.0/121.0,与标准品进行对比,如图6所示回收率在98.25%-109.28%,证明了该发明的准确性。
实施例5:CM@Lip@Fe3O4磁性脂质体检测污水样品中的THC的特异性
取6份100mL污水,标为1-6号样品,分别加入1μg/mL的四氢大麻酚(THC),四氢大麻酚酸,海洛因,芬太尼,甲卡西酮,甲基苯丙胺使其终浓度为500ng/L,分别添加CM@Lip@Fe3O4使其终浓度分别为75mg/L,调整其pH为7,涡旋混匀后水浴超声30s,震荡5min。使用强磁铁将CM@Lip@Fe3O4吸附到杯壁上,吸取剩余液体后加入0.8mL 80%甲醇水溶液将杯壁上的CM@Lip@Fe3O4冲洗下来,将收集到的溶液转移至2mL的EP管中,以8000rpm/min的转速离心30min,使用磁铁将CM@Lip@Fe3O4吸附到EP管的管壁上,将0.8mL的洗脱液转移至新的2mL EP管内,加入0.2mL 1μg/mL的内标物甲氧那明混匀后通过0.2μm的有机滤膜,将制备好的样品进行HPLC-MS/MS检测THC的定量离子对是314.2/193.2,内标物甲氧那明的定量离子对为181.0/121.0,四氢大麻酚酸的定量离子对是357/215.1,海洛因的定量离子对是370.2/165.3,芬太尼的定量离子对是337/202.16,甲卡西酮的定量离子对是164.1/105.1,甲基苯丙胺的定量离子对是150.1/118.9,使用标准品进行对比,计算回收率,结果显示专属性良好。
从以上实验可以看出,本发明构建的材料可以有效吸附污水中的THC,有很好的富集效果,与HPLC-MS/MS联用可实现对污水环境中的THC的检测,同时具有方便、快速的萃取程序,灵敏度高,定量下限低,专属性高,灵敏度高和环境友好性等。
Claims (7)
1.一种用于富集THC的磁性脂质体,其特征在于,所述磁性脂质体包括Fe3O4磁珠、包覆在所述Fe3O4磁珠上的脂质体以及含有特异性吸附受体的细胞膜,所述特异性吸附受体为1型大麻素受体,所述脂质体包括质量比为15~20:2~5:0.5~1.5的大豆磷脂、胆固醇以及维生素E,所述Fe3O4磁珠与所述维生素E的质量比为0.8~1:0.8~1,所述 Fe3O4磁珠、脂质体以及细胞膜的质量比为5:130:1,所述磁性脂质体的制备方法包括以下步骤:用薄膜分散法制备脂质体包覆的Fe3O4磁珠,得到脂质体包覆的Fe3O4磁珠Lip@Fe3O4;将脂质体包覆的Fe3O4磁珠与含有特异性吸附受体的细胞膜混合后分散均匀,过滤,得到磁性脂质体CM@Lip@Fe3O4。
2.一种权利要求1所述的用于富集THC的磁性脂质体的制备方法,其特征在于,包括以下步骤:
(1)用薄膜分散法制备脂质体包覆的Fe3O4磁珠,得到脂质体包覆的Fe3O4磁珠Lip@Fe3O4;
(2)将步骤(1)得到的脂质体包覆的Fe3O4磁珠与含有特异性吸附受体的细胞膜混合后分散均匀,过滤,得到磁性脂质体CM@Lip@Fe3O4。
3.根据权利要求2所述的用于富集THC的磁性脂质体的制备方法,其特征在于,步骤(1)中,所述薄膜分散法包括以下步骤:大豆磷脂、胆固醇以及维生素E加入三氯甲烷中,使其完全溶解,在45~50 ℃的水浴加热下去除三氯甲烷,形成一层淡黄色均匀薄膜,加入分散于PBS溶液中的Fe3O4磁珠,继续使用旋转蒸发仪于25~30℃的水浴中进行水化,将黑色磁性产物用磁铁分离收集,用去离子水和PBS溶液分别清洗后,溶解于PBS溶液中,使用细胞破碎仪破碎 ,得到Lip@Fe3O4。
4.根据权利要求2所述的用于富集THC的磁性脂质体的制备方法,其特征在于,步骤(2)中,含有特异性吸附受体的细胞膜通过以下方法制备:将导入有1型大麻素受体序列的HEK293F细胞,加入蛋白酶抑制剂,并且在细胞破碎仪中破碎,破碎完毕后将其置于冷冻离心机中,在6000~8000 rpm转速下离心,取上清液,将收集的上清液使用超速冷冻离心机以30000rpm的转速离心,弃去上清液取沉淀,得到含有特异性吸附受体的细胞膜。
5.一种权利要求1所述的用于富集THC的磁性脂质体在富集THC中的应用。
6.根据权利要求5所述的应用,其特征在于,所述应用方法为:在待富集样品中加入质量浓度为50~100mg/L的磁性脂质体,调整溶液pH为6~7,磁性脂质体对THC进行富集。
7.根据权利要求6所述的应用,其特征在于,吸附有THC的磁性脂质体的洗脱条件为:6000~8000 r/min离心20~30min,洗脱液为:70~80%甲醇水溶液。
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