CN110478943A - 磷酸化丝氨酸印迹聚合物整体微柱的制备方法 - Google Patents
磷酸化丝氨酸印迹聚合物整体微柱的制备方法 Download PDFInfo
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Abstract
本发明涉及整体柱制备技术领域,尤其涉及一种磷酸化丝氨酸印迹聚合物整体微柱的制备方法。磷酸化丝氨酸印迹聚合物整体微柱的制备方法,包括步骤:(a)将磷酸化丝氨酸和四丁基氢氧化铵以及甲醇和水混合,反应得到产物;(b)在密封的反应容器内以该反应产物为模板化合物,加入甲醇、功能单体、交联剂和引发剂进行水相原位聚合反应,反应完成后洗涤,得到磷酸化丝氨酸印迹聚合物整体微柱。得到的磷酸化丝氨酸印迹聚合物整体微柱能够实现对磷酸化丝氨酸的特异性吸附。
Description
技术领域
本发明涉及整体柱制备技术领域,尤其涉及一种磷酸化丝氨酸印迹聚合物整体微柱的制备方法。
背景技术
氨基酸的磷酸化在许多生物化学过程中起着关键性的调节作用。磷酸化丝氨酸(P-Ser)来源于磷酸化蛋白质或细胞磷脂的分解,其含量的增加与疾病的严重程度有关,虽然,目前无法量化P-Ser和疾病的关系,但研究表明,P-Ser与肝肾功能不全、癌症等疾病相关,P-Ser水平异常通常预示着疾病的存在,所以P-Ser的分析检测有着重要的医学意义。质谱(MS)化学信息丰富、可以快速和多组分分析,是生物样品分析的有效技术,但是生物样品基质复杂,高丰度非磷酸化氨基酸的干扰大,P-Ser含量低,而且P-Ser质谱分析时的电离效率很低。所以,建立复杂生物样品中P-Ser有效分离和富集的样品前处理方法是质谱分析的关键步骤。
发明内容
为了解决以上问题,本发明的目的是提供一种磷酸化丝氨酸印迹聚合物整体微柱的制备方法,能够对磷酸化丝氨酸的特异性吸附,从而,实现对磷酸化丝氨酸的有效分离富集。
以下磷酸化丝氨酸印迹聚合物整体微柱检测猪肝样品中磷酸化丝氨酸含量的方法简称为SPME-LC-MS/MS;液相色谱-串联质谱简称HPLC-MS/MS;固相微萃取简称SPME。
为实现上述目的,本发明所设计的磷酸化丝氨酸印迹聚合物整体微柱的制备方法,包括步骤:
(a)将磷酸化丝氨酸和四丁基氢氧化铵的甲醇溶液以及甲醇和水混合反应得到反应产物;
(b)以反应产物为模板化合物,加入甲醇、功能单体、交联剂和引发剂进行水相原位聚合反应,经在密封的反应容器内聚合、洗涤后得到磷酸化丝氨酸印迹聚合物整体微柱。
与现有的分子印迹聚合物整体微柱的制备方法相比,本发明首先利用四丁基氢氧化铵(TBAOH)增加P-Ser在有机溶剂中的溶解性,然后与功能单体进行水相原位聚合反应,经洗涤后得到对P-Ser具有特异性吸附功能的磷酸化丝氨酸印迹聚合物整体微柱。因为水相聚合制备整体微柱,使得该材料应用于SPME中的水溶液上样时,不易溶胀,使用寿命长。
作为优选方案,所述功能单体为丙烯酰胺;所述交联剂为乙二醇二甲基丙烯酸酯;所述引发剂为偶氮二异丁腈。
作为优选方案,所述步骤(a)的具体过程为,取1.0~1.2mmol的磷酸化丝氨酸和0.5~1.0mol/L的四丁基氢氧化铵的甲醇溶液2~3ml混合均匀后,加入到4~6ml的甲醇中,搅拌均匀后再滴加1~2ml水得到反应产物。
作为优选方案,所述步骤(b)中,模板分子200~300μL,甲醇200~300μL,丙烯酰胺8~9mg,乙二醇二甲基丙烯酸酯100~110μL,偶氮二异丁腈4~6mg。
作为优选方案,所述反应容器为移液枪枪头。即在移液枪小枪头中制备整体微柱。
一种利用磷酸化丝氨酸印迹聚合物整体微柱检测样品中磷酸化丝氨酸含量的方法,包括步骤:将样品溶液注入磷酸化丝氨酸印迹聚合物整体微柱中,磷酸化丝氨酸被选择性吸附于磷酸化丝氨酸印迹聚合物整体微柱后,从整体微柱中洗脱磷酸化丝氨酸,得到洗脱液,最后利用HPLC-MS/MS检测分析洗脱液中磷酸化丝氨酸的含量。
与传统HPLC检测P-Ser技术相比,本发明首先利用磷酸化丝氨酸印迹聚合物整体微柱在复杂的基质中选择性吸附P-Ser,然后利用洗脱剂将P-Ser洗脱下来得到洗脱液,洗脱液的组成较原样品组成简单,干扰小,而且增加了P-Ser的浓度,最后利用HPLC-MS/MS分析检测洗脱液中P-Ser的含量,进而获得P-Ser在样品中的含量,磷酸化丝氨酸印迹聚合物整体微柱对P-Ser的选择性吸附而提高了分析选择性,利用磷酸化丝氨酸印迹聚合物整体微柱对P-Ser的富集作用,以及采用了串联质谱(MS/MS)技术,从而有效提高了检测的灵敏度。
作为优选方案,将含有磷酸化丝氨酸印迹聚合物整体微柱的移液枪枪头卡扣在注射器上,将样品溶液置于注射器内,在微量注射泵的推动下,注射器内样品溶液流过磷酸化丝氨酸印迹聚合物整体微柱。
作为优选方案,所述样品为猪肝。
附图说明
图1为磷酸化丝氨酸印迹聚合物整体微柱的合成流程图;
图2为磷酸化丝氨酸印迹聚合物整体微柱和丙烯酰胺的红外光谱图;
图3为磷酸化丝氨酸印迹聚合物整体微柱的电镜扫描图;
图4为磷酸化丝氨酸印迹聚合物整体微柱的热重图;
图5为磷酸化丝氨酸和四丁基氢氧化铵反应产物结构图;
图6磷酸化丝氨酸印迹聚合物整体微柱对P-Ser的吸附曲线;
图7空白猪肝样品(a)、加标500ng g-1P-Ser(b)、加标500ng g-1P-Ser后SPME(c)的LC-MS/MS图。
具体实施方式
为更好地理解本发明,以下将结合附图和具体实例对发明进行详细的说明。
为解决现有P-Ser的质谱检测技术中存在受生物样品基质的干扰大、P-Ser含量低的问题,本发明提供一种磷酸化丝氨酸印迹聚合物整体微柱的制备方法,本发明首先利用四丁基氢氧化铵(TBAOH)增加P-Ser在有机溶剂中的溶解性,然后与功能单体进行水相原位聚合反应,经洗涤后得到磷酸化丝氨酸印迹聚合物整体微柱。本发明的磷酸化丝氨酸印迹聚合物整体微柱能够实现对P-Ser特异性吸附。并且通过采用水相聚合法,使得样品水溶液上样时,整体微柱不易水溶胀,增加了整体微柱的使用寿命。以下将通过具体的实施例来对本发明的磷酸化丝氨酸印迹聚合物整体微柱的制备方法的优选方式进行详细地说明。
实施例1
结合图1所示的本发明制备磷酸化丝氨酸印迹聚合物整体微柱的流程原理图,磷酸化丝氨酸印迹聚合物整体微柱的制备方法具体包括步骤:
(a)取P-Ser(1.1mmol,0.2g)和四丁基氢氧化铵的甲醇溶液(0.8mol/L,2.75ml)混合均匀后,加入到5ml的甲醇中,搅拌均匀后再滴加1.55ml水得到反应产物。
(b)取240μL反应产物,加入250μL甲醇、8.1mg丙烯酰胺、超声1h后加入108μLEGDMA和5mg AIBN超声10min得到预聚物,移取100μL预聚物到移液枪枪头中,移液枪枪头的一端利用甲醇封口,移液枪枪头另一端用硅胶封口,预聚物在密封的移液枪枪头内进行水相原位聚合反应,60℃下反应24h,去除两端的封口,用50%甲醇的水溶液清洗干净,即得磷酸化丝氨酸印迹聚合物整体微柱。
将本实施例得到的磷酸化丝氨酸印迹聚合物整体微柱以及丙烯酰胺进行红外光谱分析,结果如图2所示,其中(a)为磷酸化丝氨酸印迹聚合物整体微柱的红外光谱图,(b)为丙烯酰胺的红外光谱图;图2(b)中3364、1671和1611cm-1分别是丙烯酰胺的N-H、C=O和C=C的特征吸收峰,在图2(a)中,1161cm-1处C=C的吸收峰消失,但保留了图2(b)中3364cm-1处N-H和1671cm-1处C=O的吸收峰,说明AM已参加聚合反应;EGDMA位于1732cm-1的C=O伸缩振动峰仍存在于图2(a)中,说明磷酸化丝氨酸印迹聚合物通过二甲基丙烯酸乙二醇酯(EGDMA)聚合而成,图2表明磷酸化丝氨酸印迹聚合物整体微柱成功合成。
再将本实施例得到的磷酸化丝氨酸印迹聚合物整体微柱进行电镜扫描和热重测试,结果如图3和图4所示,从图3中可以看出磷酸化丝氨酸印迹聚合物整体微柱具有骨架和孔的双连续结构,骨架中的大、中和小孔提供良好的溶液渗透性和大比表面积。从图4中可以看出,磷酸化丝氨酸印迹聚合物整体微柱从250℃以后开始分解,说明磷酸化丝氨酸印迹聚合物整体微柱的热稳定性良好,适用于常温下固相微萃取。
图5是P-Ser与季铵碱反应的产物结构,以这个反应产物为模板进行分子印迹聚合物整体柱合成,主要是为了解决P-Ser、交联剂EGDMA、水和有机溶剂不能相互混合溶解的问题,聚合反应前,P-Ser与氢氧化四丁基铵(TBAOH)反应,这个反应产物极性小,增加了P-Ser在有机溶剂中的溶解性,使预聚物有更好的均一性,确保了聚合物内部结构的完整性和印迹位点的均匀性。
该反应产物作为模板进行分子印迹,其中弱极性的季铵盐部分的丁基不参与分子印迹作用,实际上还是该产物中的P-Ser部分的磷酸基团、羧基和氨基在印迹过程中在聚合物中形成印迹位点。所以,该分子印迹整体柱在吸附过程中,是印迹位点与P-Ser的磷酸基团、羧基、和氨基作用,从而特异性吸附P-Ser。
实施例2
利用实施例得到的磷酸化丝氨酸印迹聚合物整体微柱分离富集磷酸化丝氨酸,并建立了猪肝样品中磷酸化丝氨酸高灵敏度高选择性的HPLC-MS/MS分离分析的方法,包括步骤:将猪肝样品溶液注入磷酸化丝氨酸印迹聚合物整体微柱中,磷酸化丝氨酸被选择性吸附在磷酸化丝氨酸印迹聚合物整体微柱后,以洗脱剂洗脱磷酸化丝氨酸印迹聚合物整体微柱中的磷酸化丝氨酸,得到洗脱液,最后利用HPLC-MS/MS技术分析洗脱液中磷酸化丝氨酸的含量。将猪肝样品溶液注入磷酸化丝氨酸印迹聚合物整体微柱中的具体过程为,将含有磷酸化丝氨酸印迹聚合物整体微柱的移液枪枪头卡扣在注射器上,将猪肝样品溶液置于注射器内,在微量注射泵的推动下,注射器内猪肝样品溶液流过磷酸化丝氨酸印迹聚合物整体微柱。
图6是磷酸化丝氨酸印迹聚合物整体微柱对P-Ser吸附容量实验结果,磷酸化丝氨酸印迹聚合物整体微柱对P-Ser的吸附容量达到16mg g-1,说明本发明的磷酸化丝氨酸印迹聚合物整体微柱对P-Ser具有良好的吸附性能。
图7是空白猪肝样品(a)、空白样品加标500ng g-1(b)、空白样品加标500ng g-1经过磷酸化丝氨酸印迹聚合物整体微柱固相微萃取后(c)的HPLC-MS/MS色谱图。可以看出,经过磷酸化丝氨酸印迹聚合物整体微柱固相微萃取后,色谱峰明显增大,说明利用磷酸化丝氨酸印迹聚合物整体微柱对猪肝样品中P-Ser富集效果明显。
由于猪肝中存在的丝氨酸(Ser)和甘氨酸(Gly)组分与P-Ser具有相似的结构,对P-Ser的检测产生影响,因此将Ser和Gly用作干扰物测试磷酸化丝氨酸印迹聚合物整体微柱对P-Ser的选择性。具体实验过程为:取相同浓度Ser、Gly、P-Ser的混合溶液上样,用醋酸-醋酸钠缓冲液洗脱,洗脱液进行HPLC检测。富集因子(EF)和印迹因子(IF)用来评估磷酸化丝氨酸印迹聚合物整体微柱对P-Ser的选择性吸附。
非印迹聚合物整体微柱:其合成方法除不加P-Ser外,其余步骤与实施例1的相同。
其中,EFMIM和EFNIM分别表示相同萃取条件下磷酸化丝氨酸印迹聚合物整体微柱(MIM)和非印迹聚合物整体微柱(NIM)的富集因子(EF),ce和co分别是洗脱液和上样溶液中P-Ser的浓度。根据实验结果,P-Ser-MIM的IF为2.14,表明P-Ser-MIM对P-Ser的吸附性能优于NIM,表明P-Ser-MIM的印迹效果较好;P-Ser-MIM对P-Ser的EF(49.10)远大于Ser(2.14)和Gly(2.23),表明该微柱分子印迹位点的形成及对P-Ser有优异的选择性和富集能力。
本发明的SPME-HPLC-MS/MS方法的线性范围、检出限和定量限,见表。P-Ser在12.5-20000ng g-1范围内线性良好,线性方程为Y=0.0248X-0.4642,R2=0.9974,LOD(S/N=3)为2.5ng g-1,本方法能够满足于肝脏样品中P-Ser的灵敏检测。
表1线性范围、检出限和定量限
为评价该SPME-LC-MS/MS方法检测肝脏样品中P-Ser的准确度和可靠性,以低、中、高三个浓度水平的P-Ser溶液进行SPME-LC-MS/MS分析,考察方法的回收率和精密度,对同一浓度在同一天内和四天中平行检测四次,计算日内日间相对标准偏差(RSD),结果见表2。可见,日内RSD在2.2-4.4%范围内,日间RSD在0.48-3.3%范围内,回收率在95.5-100.4%范围内。因此,该方法重现性好、回收率高、灵敏度高,满足猪肝样品中P-Ser的分析检测。
表2精密度和回收率
用该SPME-LC-MS/MS方法对超市购买的猪肝样品进行P-Ser检测,实验结果表明,猪肝中检测到P-Ser含量为15.1μg g-1,与文献报道肝脏中P-Ser水平相当,说明利用SPME-LC-MS/MS方法的检出结果可靠。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (8)
1.一种磷酸化丝氨酸印迹聚合物整体微柱的制备方法,其特征在于,包括步骤:
(a)将磷酸化丝氨酸和四丁基氢氧化铵的甲醇溶液以及甲醇和水混合反应得到反应产物;
(b)以反应产物为模板化合物,加入甲醇、功能单体、交联剂和引发剂进行水相原位聚合反应,经在密封的反应容器内聚合、洗涤后得到磷酸化丝氨酸印迹聚合物整体微柱。
2.根据权利要求1所述的磷酸化丝氨酸印迹聚合物整体微柱的制备方法,其特征在于,所述步骤(a)的具体过程为,取1.0~1.2mmol的磷酸化丝氨酸和0.5~1.0mol/L的四丁基氢氧化铵的甲醇溶液2~3ml混合均匀后,加入到4~6ml的甲醇中,搅拌均匀后再滴加1~2ml水得到反应产物。
3.根据权利要求2所述的磷酸化丝氨酸印迹聚合物整体微柱的制备方法,其特征在于,所述功能单体为丙烯酰胺;所述交联剂为乙二醇二甲基丙烯酸酯;所述引发剂为偶氮二异丁腈。
4.根据权利要求3所述的磷酸化丝氨酸印迹聚合物整体微柱的制备方法,其特征在于,所述步骤(b)中,模板分子200~300μL,甲醇200~300μL,丙烯酰胺8~9mg,乙二醇二甲基丙烯酸酯100~110μL,偶氮二异丁腈4~6mg。
5.根据权利要求1~4中任一项所述的磷酸化丝氨酸印迹聚合物整体微柱的制备方法,其特征在于,所述反应容器为移液枪枪头。
6.一种利用权利要求1所述的磷酸化丝氨酸印迹聚合物整体微柱检测样品中磷酸化丝氨酸含量的方法,包括步骤:将样品溶液注入磷酸化丝氨酸印迹聚合物整体微柱中,磷酸化丝氨酸被选择性吸附在磷酸化丝氨酸印迹聚合物整体微柱后,用洗脱剂洗脱磷酸化丝氨酸印迹聚合物整体微柱中的磷酸化丝氨酸,得到洗脱液,最后利用液相色谱质谱/质谱联用技术分析洗脱液中磷酸化丝氨酸的含量。
7.根据权利要求6所述的利用磷酸化丝氨酸印迹聚合物整体微柱检测样品中磷酸化丝氨酸含量的方法,其特征在于,将样品注入磷酸化丝氨酸印迹聚合物整体微柱中的具体过程为,将含有磷酸化丝氨酸印迹聚合物整体微柱的移液枪枪头卡扣在注射器上,将样品溶液置于注射器内,在微量注射泵的推动下,注射器内样品溶液流过磷酸化丝氨酸印迹聚合物整体微柱。
8.根据权利要求6所述的磷酸化丝氨酸印迹聚合物整体微柱检测样品中磷酸化丝氨酸含量的方法,其特征在于,所述样品为猪肝。
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Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050070676A1 (en) * | 2003-09-29 | 2005-03-31 | Council Of Scientific And Industrial Research | Cobalt imprinted polymer composition for selective removal of cobalt, process for preparation thereof, and process for removal of cobalt |
JP2010100708A (ja) * | 2008-10-22 | 2010-05-06 | Mukogawa Gakuin | 分子インプリントポリマーおよびその製造方法 |
CN101775103A (zh) * | 2009-12-29 | 2010-07-14 | 湖北工业大学 | 一种蛋白质分子印迹薄膜的制备方法 |
US20120225962A1 (en) * | 2009-08-05 | 2012-09-06 | Monash University | Molecularly imprinted polymers, methods for their production and uses thereof |
CN104356308A (zh) * | 2014-11-14 | 2015-02-18 | 中北大学 | 一种牛血清白蛋白分子表面印迹聚合物微球的制备方法 |
CN106009012A (zh) * | 2016-06-07 | 2016-10-12 | 吉首大学 | 纳米二氧化钛负载l-丝氨酸印迹聚合物薄层板及其制法 |
CN106512963A (zh) * | 2015-11-11 | 2017-03-22 | 大连出入境检验检疫局检验检疫技术中心 | 一种河豚毒素分子印迹整体柱的制备方法及整体柱和应用 |
CN106582066A (zh) * | 2016-11-24 | 2017-04-26 | 上海交通大学 | 肉桂酸酯类紫外防晒剂分子印迹固相萃取柱及制备与应用 |
-
2019
- 2019-07-25 CN CN201910676296.1A patent/CN110478943B/zh active Active
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050070676A1 (en) * | 2003-09-29 | 2005-03-31 | Council Of Scientific And Industrial Research | Cobalt imprinted polymer composition for selective removal of cobalt, process for preparation thereof, and process for removal of cobalt |
JP2010100708A (ja) * | 2008-10-22 | 2010-05-06 | Mukogawa Gakuin | 分子インプリントポリマーおよびその製造方法 |
US20120225962A1 (en) * | 2009-08-05 | 2012-09-06 | Monash University | Molecularly imprinted polymers, methods for their production and uses thereof |
CN101775103A (zh) * | 2009-12-29 | 2010-07-14 | 湖北工业大学 | 一种蛋白质分子印迹薄膜的制备方法 |
CN104356308A (zh) * | 2014-11-14 | 2015-02-18 | 中北大学 | 一种牛血清白蛋白分子表面印迹聚合物微球的制备方法 |
CN106512963A (zh) * | 2015-11-11 | 2017-03-22 | 大连出入境检验检疫局检验检疫技术中心 | 一种河豚毒素分子印迹整体柱的制备方法及整体柱和应用 |
CN106009012A (zh) * | 2016-06-07 | 2016-10-12 | 吉首大学 | 纳米二氧化钛负载l-丝氨酸印迹聚合物薄层板及其制法 |
CN106582066A (zh) * | 2016-11-24 | 2017-04-26 | 上海交通大学 | 肉桂酸酯类紫外防晒剂分子印迹固相萃取柱及制备与应用 |
Non-Patent Citations (2)
Title |
---|
何新秀、汪源成: "《色谱分析》", 30 September 1994, 成都科技大学出版社 * |
郑平等: "《分子印迹固相萃取技术及其在食品安全分析中的应用》", 30 November 2011, 合肥工业大学出版社 * |
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