CN104155248A - 用于检测葡萄糖的纳米材料的制备方法 - Google Patents
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Abstract
本发明公开了一种用于检测葡萄糖的纳米材料的制备方法,该方法包括如下步骤:(1)配置浓度为100-200mM的金属盐水溶液;(2)将葡萄糖氧化酶与过氧化物酶混合配置缓冲溶液;(3)将步骤(1)中的溶液与步骤(2)得到的溶液按体积比1∶150混合;(4)混匀步骤(3)得到的混合溶液,静置得到沉淀物既为所述纳米材料。本发明的方法可以简单高效地合成具有高催化活性的杂合纳米花,简化了实验操作步骤,大幅度提高了葡萄糖比色检测体系对葡萄糖的响应信号。
Description
技术领域
本发明涉及纳米材料制备领域,尤其涉及一种多蛋白质/磷酸盐的用于检测葡萄糖的纳米材料的制备方法。
背景技术
糖尿病是目前最常见的非传染性疾病之一,它被认为是世界性的第三大危险疾病,严重威胁着人类的健康。一般地来讲,经典的比色型葡萄糖传感器是基于葡萄糖氧化酶(GOx)催化氧化葡萄糖产生过氧化氢,而后者在辣根过氧化物酶的催化下氧化一些生色底物如3,3',5,5'-四甲基联苯胺(TMB)而产生颜色变化,实现葡萄糖检测。其检测步骤一般分两步:首先葡萄糖和葡萄糖氧化酶以及氧气在37℃下反应,葡萄糖产生葡萄糖酸和过氧化氢;然后把前一步产生的含有过氧化氢的反应液与含有辣根过氧化物酶以及生色底物(如TMB)的溶液混合,在37℃反应,产生颜色变化。但是这种传统的葡萄糖传感器存在一些缺陷,首先,葡萄糖氧化酶和辣根过氧化物酶都属于生物酶系,它们在室温下不稳定,容易失活和降解,因此这种传感器无法长期储存;第二,这种方法实质上是使用过氧化氢做中介体,产生颜色变化的反应,但是过氧化氢不稳定,在环境中很容易分解。所以这种方法的信号强度(变色程度)和灵敏度都不高。
发明内容
本发明所要解决的技术问题是提供一种用于检测葡萄糖的纳米材料的制备方法,该方法可以简单高效地合成具有很高催化活性的杂合纳米花,由于在这种纳米花上同时固定有葡萄糖氧化酶和辣根过氧化物酶,因而可以同时催化葡萄糖氧化生成过氧化氢反应和过氧化氢氧化TMB变色的反应,简化了传统葡萄糖检测过程中需要两步酶促才能完成的反应,简化了实验操作步骤,大幅度提高了葡萄糖比色检测体系对葡萄糖的响应信号。
本发明采用的技术方案是提供一种用于检测葡萄糖的纳米材料的制备方法,该方法包括如下步骤:
(1)配置浓度为100-200mM的金属盐水溶液;
(2)将葡萄糖氧化酶与过氧化物酶按质量比1:1-1:50混合后加入到浓度范围为5mM-0.5M的磷酸盐缓冲溶液中;
(3)将步骤(1)中的溶液与步骤(2)得到的溶液按体积比1:50-1:200混合;
(4)混匀步骤(3)得到的混合溶液,静置得到沉淀物即为所述纳米材料。
优选地,步骤(1)所述金属盐选自氯化钙、硫酸铜、氯化铁、氯化亚铁、氯化钴、硝酸银、氯化铯、硝酸铅或氯化铬。
优选地,步骤(2)所述磷酸盐缓冲溶液为磷酸钠、磷酸钾、磷酸二氢钠、磷酸氢二钠、磷酸二氢钾或磷酸氢二钾的磷酸缓冲溶液。
优选地,步骤(2)所述葡萄糖氧化酶选自黑曲霉的葡萄糖氧化酶或青霉的葡萄糖氧化酶。
优选地,步骤(2)所述过氧化物酶选自辣根的过氧化物酶、大豆的过氧化物酶或黑管菌的过氧化物酶。
优选地,步骤(2)所述葡萄糖氧化物酶与过氧化物酶按质量比为1:5的比例混合,
优选地,步骤(3)所述体积比为1:150。
优选地,步骤(4)所述静置时间12-120小时,静置温度为5-50℃。
本发明的效果是可以简单高效地合成具有很高催化活性的纳米材料即杂合纳米花,简化了实验操作步骤,大幅度提高了葡萄糖比色检测体系对葡萄糖的响应信号。
本发明使用葡萄糖氧化酶和辣根过氧化物酶共同作为蛋白组分,与水合磷酸金属盐制成了一种纳米花。这种纳米花具有十分规则的形貌,呈多层结构。传统的两步检测法可以在这种纳米花上一步完成。在这个体系中,葡萄糖和氧气在纳米花上的葡萄糖氧化酶催化下反应生成过氧化氢,过氧化氢可以立即被在同一纳米花表面上相邻的辣根过氧化物酶催化氧化TMB完成变色反应。这种双酶杂合的纳米花结构避免了过氧化氢的扩散和分解,因而大大提高了反应的信号强度和检测灵敏度。
附图说明
图1是本发明实例1中制备的纳米花材料粉末衍射图;
图2是本发明实例1中制备的纳米花的低分辨扫描电子显微镜图(SEM);
图3是本发明实例1中制备的纳米花的高分辨扫描电子显微镜图(SEM);
图4是本发明实施例1与对比实验的吸收光谱图。
具体实施方式
下面结合附图及实施例对本发明进一步加以说明。
实施例1
(1)使用分析天平和容量瓶配制120mM的硫酸铜水溶液。
(2)秤取1mg来源于黑曲霉的葡萄糖氧化酶(GOx)和1mg来源于辣根的过氧化物酶(HRP),加入到150mM磷酸盐缓冲溶液(PBS,pH=7)中,使用容量瓶定容到10mL,得到含0.5mg/mL GOx和0.1mg/mLHRP的PBS溶液。
(3)取20μL120mM的硫酸铜溶液加3mL含0.5mg/mL GOx和0.1mg/mL HRP的PBS溶液中,充分混匀该溶液,并在室温下放置3天。得到的沉淀物质即为GOx&HRP–Cu3(PO4)2·3H2O纳米花。
实施例2
(1)使用分析天平和容量瓶配制100mM的氯化铁水溶液。
(2)秤取5mg来源于黑曲霉的葡萄糖氧化酶(GOx)和1mg来源于大豆的过氧化物酶(HRP),加入到200mM磷酸盐缓冲溶液(PBS,pH=7)中,使用容量瓶定容到10mL,得到含0.5mg/mL GOx和0.1mg/mL HRP的PBS溶液。
(3)取15μL120mM的硫酸铜溶液加入3mL含0.5mg/mLGOx和0.1mg/mLHRP的PBS溶液中,充分混匀该溶液,并在室温下放置3天。得到的沉淀物质即为纳米花。
实施例3
(1)使用分析天平和容量瓶配制150mM的氯化钙水溶液。
(2)秤取1mg来源于青霉的葡萄糖氧化酶(GOx)和2mg来源于黑管菌的过氧化物酶(HRP),加入到250mM磷酸盐缓冲溶液(PBS,pH=7)中,使用容量瓶定容到10mL,得到含0.5mg/mL GOx和0.1mg/mL HRP的PBS溶液。
(3)取60μL120mM的硫酸铜溶液加入3mL含0.5mg/mL GOx和0.1mg/mLHRP的PBS溶液中。充分混匀该溶液,并在室温下放置3天。得到的沉淀物质即为纳米花。
应用例:
(1)在做葡萄糖测试时,将乙酸-乙酸钠缓冲溶液、3,3',5,5'-四甲基联苯胺(TMB)溶液、葡萄糖标准溶液混合配成悬浊液;
(2)该悬浊液在37℃下避光反应30min。使用离心机离心上清,测吸收光谱。
图4表示的是用实施例1中制备的纳米花催化氧化30μM葡萄糖产生过氧化氢,后者氧化TMB变色后测得的吸收光谱,与游离的葡萄糖氧化酶和过氧化物酶催化该反应后的变色TMB吸收光谱的对比。从吸收光谱可以看出,纳米花催化氧化葡萄糖并导致TMB变色的反应效率比游离的葡萄糖氧化酶和过氧化物酶高3倍。
Claims (8)
1.一种用于检测葡萄糖的纳米材料的制备方法,其特征在于,该方法包括如下步骤:
(1)配置浓度为100-200mM的金属盐水溶液;
(2)将葡萄糖氧化酶与过氧化物酶按质量比1:1-1:50混合后加入到浓度为5mM-0.5M的磷酸盐缓冲溶液中。
(3)将步骤(1)中的溶液与步骤(2)得到的溶液按体积比1:50-1:200混合;
(4)混匀步骤(3)得到的混合溶液,静置得到沉淀物即为所述纳米材料。
2.根据权利要求1所述的纳米材料的制备方法,其特征在于:步骤(1)所述金属盐选自氯化钙、硫酸铜、氯化铁、氯化亚铁、氯化钴、硝酸银、氯化铯、硝酸铅或氯化铬。
3.根据权利要求1所述的纳米材料的制备方法,其特征在于:步骤(2)所述磷酸盐缓冲溶液为磷酸钠、磷酸钾、磷酸二氢钠、磷酸氢二钠、磷酸二氢钾或磷酸氢二钾的磷酸缓冲溶液。
4.根据权利要求1所述的纳米材料的制备方法,其特征在于:步骤(2)所述葡萄糖氧化酶选自黑曲霉的葡萄糖氧化酶或青霉的葡萄糖氧化酶。
5.根据权利要求1所述的纳米材料的制备方法,其特征在于:步骤(2)所述过氧化物酶选自辣根的过氧化物酶、大豆的过氧化物酶或黑管菌的过氧化物酶。
6.根据权利要求1所述的纳米材料的制备方法,其特征在于:步骤(2)所述葡萄糖氧化物酶与过氧化物酶按质量比为1:1-1:10的比例混合。
7.根据权利要求1所述的纳米材料的制备方法,其特征在于:步骤(3)所述体积比为1:150。
8.根据权利要求1所述的纳米材料的制备方法,其特征在于:步骤(4)所述静置时间为12-120小时,静置温度为5-50℃。
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