CN114740137A - 一种基于GOx@GA@MIL-101快速简便识别葡萄糖的方法 - Google Patents
一种基于GOx@GA@MIL-101快速简便识别葡萄糖的方法 Download PDFInfo
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Abstract
本发明公开了一种基于GOx@GA@MIL‑101快速简便识别葡萄糖的方法，以交联法将GOx固定于MIL‑101（Fe）表面，然后将模拟类过氧化物酶MIL‑101（Fe）与天然葡萄糖氧化酶（GOx）进行串联催化协同氧化，从而实现过氧化氢和葡萄糖的共识别。上述方案中，将模拟类过氧化物酶MIL‑101（Fe）与天然葡萄糖氧化酶（GOx）结合，将二者进行串联催化协同氧化，从而达到对过氧化氢和葡萄糖共识别的目的；与其他铁基催化剂相比，具有更小的Km值，对底物有更高的亲和力，从而可以实现底物的快速识别；通过交联葡萄糖氧化酶对葡萄糖达到专一识别，不受其他小分子干扰。
Description
技术领域
本发明涉及葡萄糖识别技术领域，具体涉及一种基于GOx@GA@MIL-101快速简便识别葡萄糖的方法。
背景技术
目前，传统的葡萄糖检测的方法主要包括DNS比色，菲林滴定和高效液相色谱等，均需要花费大量的时间才能完成检测，同时一个样品往往需要多次重复操作，能够进行的实验组数十分有限，并且具有灵敏度差，专一性不强的特点，容易造成实验数据误差导致实验重复性差。
发明内容
为解决上述问题，本发明提供了一种基于GOx@GA@MIL-101快速简便识别葡萄糖的方法，样品数量远超于传统方法，同时其检测样品不需要预处理，比以往的传统方法提高了检测效率，且人体内其他小分子对其检测并不产生影响，对葡萄糖的检测具有专一性。
为实现上述目的，本发明采取的技术方案为：
一种基于GOx@GA@MIL-101快速简便识别葡萄糖的方法，以交联法将GOx固定于MIL-101（Fe）表面，然后将模拟类过氧化物酶MIL-101（Fe）与天然葡萄糖氧化酶（GOx）进行串联催化协同氧化，从而实现过氧化氢和葡萄糖的共识别；具体的：包括如下步骤：
S1、制备MIL-101：
取0.675gFeCl3·6H2O和0.206g对苯二甲酸置于烧杯中，经15mLN，N-2甲基甲酰胺溶解，在室温下超声搅拌15min,转移至25mL聚四氟乙烯反应釜中，110℃下反应20h后，将反应釜降至室温，将得到的产品以乙醇离心反复洗涤3-4次，60℃烘干6h，以玛瑙研钵研磨至粉末状备用；
S2、将10mg葡萄糖氧化酶（GOx）分散在10mL磷酸盐缓冲液中，室温下在磁力搅拌器中分散30min后，加入2.5wt%的戊二醛水溶液（GA），交联，得交联酶；
S3、取50mg制备好的MIL-101，分散到10mL配置好的磷酸盐缓冲液中，室温磁力搅拌分散30min，使之充分分散后，加入所得的交联酶，室温下与MIL-101交联1h后,以PBS洗涤未交联上的载体或酶，离心收集，转移至预先制冷的冷冻干燥机内干燥6h，得到GOx@GA@MIL-101，置于-20℃冰箱内冷藏备用；
S4、取30mg制备好的MIL-101分散于10mL水中，体系中加入1mLPBS缓冲液，850μLTMB，50μL不同浓度的H2O2，50μL浓HCl，在45℃条件下水浴加热，反应30s，将得到的变色样品置于452nm条件下检测得到吸光度值，以H2O2浓度为横坐标，吸光度值为纵坐标，进行二次拟合，得到浓度在1.125x10-6-8.8x10-4mol/L的H2O2检测范围；
S5、取30mg制备好的GOx@GA@MIL-101分散于10mLPBS中，体系中加入1mLPBS缓冲液，850μLTMB，50μL不同浓度的葡萄糖（Glu），50μL浓HCl，在45℃条件下水浴加热，反应30s，将得到的变色样品置于452nm条件下检测得到吸光度值，以葡萄糖浓度为横坐标，吸光度值为纵坐标，进行线性拟合，得到浓度在1-10 mM/L的Glu检测范围。
上述方案中，将模拟类过氧化物酶MIL-101（Fe）与天然葡萄糖氧化酶（GOx）结合，将二者进行串联催化协同氧化，从而达到对识别过氧化氢和葡萄糖共识别的目的；与其他铁基催化剂相比，具有更小的Km值，对底物有更高的亲和力，从而可以实现底物的快速识别；通过交联葡萄糖氧化酶对葡萄糖达到专一识别，不受其他小分子干扰。
附图说明
图1为交联葡萄糖氧化酶的实验操作及原理。
图2为MIL-101对不同浓度过氧化氢的检测结果。
图3为GOx@GA@MIL-101对不同浓度葡萄糖的检测结果。
图4为不同浓度葡萄糖与吸光度之间的线性关系。
图5为GOx@GA@MIL-101对葡萄糖的识别专一性结果。
图6为实验流程及原理示意图。
图7为比色原理化学反应方程式。
具体实施方式
为了使本发明的目的及优点更加清楚明白，以下结合实施例对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
实施例
S1、制备MIL-101：
取0.675gFeCl3·6H2O和0.206g对苯二甲酸置于烧杯中，经15mLN，N-2甲基甲酰胺溶解，在室温下超声搅拌15min,转移至25mL聚四氟乙烯反应釜中，110℃下反应20h，待反应结束后，将反应釜降至室温，将得到的产品以乙醇离心反复洗涤3-4次，60℃烘干6h，以玛瑙研钵研磨备用；
S2、将10mg葡萄糖氧化酶（GOx）分散在10mL磷酸盐缓冲液中，室温下在磁力搅拌器中分散30min后，向其中加入2.5wt%的戊二醛水溶液（GA），开始交联，示意图1所示。
S3、取50mg制备好的MIL-101，分散到10mL配置好的磷酸盐缓冲液中，室温磁力搅拌分散30min，使之充分分散后，将制备好的交联酶加入，室温下与MIL-101交联1h后,以PBS洗涤未交联上的载体或酶，离心收集，转移至预先制冷的冷冻干燥机内干燥6h，得到GOx@GA@MIL-101，置于-20℃冰箱内冷藏备用。
S4、取S2制备好的MIL-101取30mg分散于10mL水中。体系中加入1mLPBS缓冲液，850μLTMB，50μL不同浓度的H2O2，50μL浓HCl，在45℃条件下水浴加热，反应30s，将得到的变色样品置于452nm条件下检测得到吸光度值。以H2O2浓度为横坐标，吸光度值为纵坐标，进行二次拟合，得到浓度在1.125x10-6~8.8x10-4mol/L的H2O2检测范围，如图2所示：
S5、取S3制备好的GOx@GA@MIL-101 30mg分散于10mLPBS中。体系中加入1mLPBS缓冲液，850μLTMB，50μL不同浓度的葡萄糖（Glu），50μL浓HCl，在45℃条件下水浴加热，反应30s，将得到的变色样品置于452nm条件下检测得到吸光度值。以葡萄糖浓度为横坐标，吸光度值为纵坐标，进行线性拟合，得到浓度在1-10 mM/L的Glu检测范围，如图3和图4所示：
经计算得到上述制备材料对H2O2和TMB的Km值为5.047×10-4、0.0196，比其他的Fe基催化剂和天然过氧化物酶对底物有着更高的亲和力，加快了反应进程。同时，对该材料进行专一性考察，模拟了人的体液环境，分别对尿酸（UA）、多巴胺（DA）、抗坏血酸（AA）、氯化钠（NaCl）进行检测，得到结果如图5所示。
本发明对H2O2和Glu两种底物的检测机理解释如图6所示；GOx@GA@MIL-101在O2的参与下，以交联的GOx为第一反应催化剂，氧化Glu，同时产生H2O2，为第二步的显色反应提供了底物；在H2O2的氧化及类过氧化物酶MIL-101的催化下，将无色的TMB底物氧化为黄色氧化态TMB。通过这一级联串联反应，鉴别低浓度葡萄糖的存在最终以TMB的吸光度值展现出来。具体反应式如图7所示。
综上所述，所制备的GOx@GA@MIL-101对H2O2和葡萄糖有着更灵敏的检测，以裸眼辩色即可达到检测低浓度葡萄糖的目的。此项方法的研究为纳米材料在医学领域的应用奠定了一定基础。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以作出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (2)
1.一种基于GOx@GA@MIL-101快速简便识别葡萄糖的方法，其特征在于：以交联法将GOx固定于MIL-101（Fe）表面，然后将模拟类过氧化物酶MIL-101（Fe）与天然葡萄糖氧化酶（GOx）进行串联催化协同氧化，从而实现过氧化氢和葡萄糖的共识别。
2.如权利要求1所述的一种基于GOx@GA@MIL-101快速简便识别葡萄糖的方法，其特征在于：包括如下步骤：
S1、制备MIL-101：
取0.675gFeCl3·6H2O和0.206g对苯二甲酸置于烧杯中，经15mLN，N-2甲基甲酰胺溶解，在室温下超声搅拌15min,转移至25mL聚四氟乙烯反应釜中，110℃下反应20h后，将反应釜降至室温，将得到的产品以乙醇离心反复洗涤3-4次，60℃烘干6h，以玛瑙研钵研磨至粉末状，备用；
S2、将10mg葡萄糖氧化酶（GOx）分散在10mL磷酸盐缓冲液中，室温下在磁力搅拌器中分散30min后，加入2.5wt%的戊二醛水溶液（GA），交联，得交联酶；
S3、取50mg制备好的MIL-101，分散到10mL配置好的磷酸盐缓冲液中，室温磁力搅拌分散30min，使之充分分散后，加入所得的交联酶，室温下与MIL-101交联1h后,以PBS洗涤未交联上的载体或酶，离心收集，转移至预先制冷的冷冻干燥机内干燥6h，得到GOx@GA@MIL-101，置于-20℃冰箱内冷藏备用；
S4、取30mg制备好的MIL-101分散于10mL水中，体系中加入1mLPBS缓冲液，850μLTMB，50μL不同浓度的H2O2，50μL浓HCl，在45℃条件下水浴加热，反应30s，将得到的变色样品置于452nm条件下检测得到吸光度值，以H2O2浓度为横坐标，吸光度值为纵坐标，进行二次拟合，得到浓度在1.125x10-6-8.8x10-4mol/L的H2O2检测范围；
S5、取30mg制备好的GOx@GA@MIL-101分散于10mLPBS中，体系中加入1mLPBS缓冲液，850μLTMB，50μL不同浓度的葡萄糖（Glu），50μL浓HCl，在45℃条件下水浴加热，反应30s，将得到的变色样品置于452nm条件下检测得到吸光度值，以葡萄糖浓度为横坐标，吸光度值为纵坐标，进行线性拟合，得到浓度在1-10 mM/L的Glu检测范围。
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