CN111624187A - 一种基于葡萄糖氧化酶和类手枪脱氧核酶构成的荧光型葡萄糖传感器 - Google Patents
一种基于葡萄糖氧化酶和类手枪脱氧核酶构成的荧光型葡萄糖传感器 Download PDFInfo
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Abstract
本发明公开一种基于葡萄糖氧化酶和类手枪脱氧核酶构成的荧光型葡萄糖传感器，属于生物化学检测领域。在类手枪脱氧核酶5’端化学修饰荧光淬灭分子，在其DNA底物5’和3’端分别化学修饰荧光淬灭分子和荧光分子。当检测液中含有葡萄糖时，葡萄糖氧化酶催化葡萄糖生成过氧化氢和葡萄糖酸，过氧化氢与铜离子作用促使类手枪脱氧核酶催化底物断裂，同时产生荧光信号。该传感器检测葡萄糖下限为50纳摩尔/升，葡萄糖浓度和荧光信号之间曲线拟合符合Extreme方程，对葡萄糖检测显示出专一性。使用本发明可以实现对唾液中葡萄糖含量检测。
Description
技术领域
本发明属于生物化学检测领域，涉及一种葡萄糖传感器，具体涉及一种基于葡萄糖氧化酶和类手枪脱氧核酶的检测唾液中葡萄糖含量的荧光型传感器。
背景技术
在世界范围内，糖尿病已成为威胁人类健康的重要慢性疾病。在中国其发病率正不断上升，患者人数已占世界首位。为治疗或预防糖尿病，糖尿病患者及易感人群在日常生活中需不断监测体内葡萄糖含量。检测方式为血液检测，过程需针刺取血，易造成患者疼痛不适。为解决这一问题，糖尿病检测正朝非创伤性检测方向快速发展，检测对象由血液逐渐转向唾液、泪液以及汗液等身体代谢产物。唾液/泪液/汗液检测属近年来新兴发展的非创伤性检测技术(non-invasive technology)，可在不引起机体损伤或疼痛情况下获得所需检测数据，对于那些需频繁检测的糖尿病患者而言具有特别重要的现实意义。
发明内容
本发明的目的是提供一种基于葡萄糖氧化酶和类手枪脱氧核酶的检测唾液中葡萄糖含量的荧光型传感器。
为实现上述目的，本发明通过以下技术来实现：
荧光型葡萄糖传感器的检测原理：
荧光型葡萄糖传感器主要包括葡萄糖氧化酶、类手枪脱氧核酶及其DNA底物。
类手枪脱氧核酶3’端标记淬灭分子，底物5’和3’端分别淬灭分子和荧光分子。
当脱氧核酶与其底物识别结合时，荧光分子和淬灭分子接近，发生荧光淬灭现象，无荧光信号发射。
在检测过程中，葡萄糖作为一级信号分子，葡萄糖氧化酶催化葡萄糖生成葡萄糖酸和过氧化氢，过氧化氢作为二级信号分子被类手枪脱氧核酶识别并发生催化底物断裂反应。
底物脱离类手枪脱氧核酶，同时底物标记的荧光分子也与酶标记淬灭分子相互分离，分离后荧光分子发射荧光信号，其荧光强度与过氧化氢浓度正相关，进而与葡萄糖浓度存在数量关系(见图1)。
荧光型葡萄糖传感器检测条件：
葡萄糖氧化酶浓度浓度为0.01单位/微升，类手枪脱氧核酶浓度及其DNA底物都为50纳摩尔/升，氯化钠浓度为800毫摩尔/升，50毫摩尔/升MES缓冲液(pH 6.0)，检测液总体积为100微升，其中含唾液样品50微升，检测温度为23℃。
荧光型葡萄糖传感器检测范围：
传感器的检测铜离子浓度下限为50纳摩尔/升，在50纳摩尔/升至1毫摩尔/升范围内葡萄糖浓度与传感器荧光信号强度拟合曲线符合Extreme方程(见图2)。
荧光型葡萄糖传感器检测专一性：
选择麦芽糖、半乳糖、甘露糖、海藻糖、古洛糖、果糖、蔗糖、乳糖和葡萄糖进行实验。结果显示麦芽糖、半乳糖、甘露糖、蔗糖有信号输出，但是与同浓度的葡萄糖信号相比这4种糖的干扰信号弱很多(见图3)。
由于在口腔分泌的唾液中不存在麦芽糖、半乳糖、甘露糖、蔗糖，因此不会干扰葡萄糖荧光型传感器对唾液中葡萄糖含量检测。
荧光型葡萄糖传感器对唾液样品检测：
选择10个唾液样本，样品为早饭后约2小时收集。利用葡萄糖传感器对饭后唾液进行葡萄糖含量检测。样本葡萄糖最高浓度为149.4±12.9微摩尔/升，最低为26.9±8.5微摩尔/升。
附图说明
图1为荧光型葡萄糖传感器原理图。字母表示关键DNA序列。F表示荧光分子，Q表示荧光淬灭分子，PLQ表示类手枪脱氧核酶，SQF表示DNA底物。
图2为荧光型葡萄糖传感器检测范围和荧光信号关系图。
图3为荧光型葡萄糖传感器检测专一性图。
具体实施方式
本发明实验所使用仪器为Tecan Infinite F200多功能酶标仪。
所用葡萄糖氧化酶从Sigma公司购买，类手枪脱氧核酶及其DNA底物从生工生物工程 (上海)股份有限公司购买。
下面通过具体的实施方式对本发明荧光型葡萄糖传感器作进一步说明。
荧光型葡萄糖传感器对唾液样品检测：
检测体系组成包括50微升唾液样品，0.01单位/微升葡萄糖氧化酶，50纳摩尔/升类手枪脱氧核酶，50纳摩尔/升类手枪脱氧核酶底物，800毫摩尔/升氯化钠，50毫摩尔/升MES缓冲液(pH 6.0)，总体积为100微升。
23℃水浴中反应15分钟后，利用微量移液器将检测液转移到96孔荧光检测板中，使用 Tecan Infinite F200多功能酶标仪检测荧光信号，然后将荧光值代入公式中计算葡萄糖浓度。
序列表
<110> 吉林大学
<120> 一种基于葡萄糖氧化酶和类手枪脱氧核酶构成的荧光型葡萄糖传感器
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ctgggcc 7
<210> 2
<211> 6
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aagacg 6
Claims (10)
1.一种基于葡萄糖氧化酶和类手枪脱氧核酶构成的荧光型葡萄糖传感器，其特征在于：传感器由葡萄糖氧化酶、类手枪脱氧核酶、类手枪脱氧核酶DNA底物、铜离子、氯化钠以及pH缓冲体系组成。
2.如权利要求1所述一种基于葡萄糖氧化酶和类手枪脱氧核酶构成的荧光型葡萄糖传感器，其特征在于：类手枪脱氧核酶含有DNA序列为5’-ctgggcc-3’。
3.如权利要求1所述一种基于葡萄糖氧化酶和类手枪脱氧核酶构成的荧光型葡萄糖传感器，其特征在于：类手枪脱氧核酶5’端化学修饰的淬灭分子为黑洞淬灭分子1(BlackHole Quencher-1)。
4.如权利要求1所述一种基于葡萄糖氧化酶和类手枪脱氧核酶构成的荧光型葡萄糖传感器，其特征在于：类手枪脱氧核酶DNA底物含有序列为5’-aagacg-3’。
5.如权利要求1所述一种基于葡萄糖氧化酶和类手枪脱氧核酶构成的荧光型葡萄糖传感器，其特征在于：DNA底物5’端化学修饰的淬灭分子为黑洞淬灭分子1(Black HoleQuencher-1)，3’端化学修饰的荧光分子为羧基荧光素(Carboxyfluorescein)。
6.如权利要求1所述一种基于葡萄糖氧化酶和类手枪脱氧核酶构成的荧光型葡萄糖传感器，其特征在于：葡萄糖氧化酶浓度为0.01单位/微升。
7.如权利要求1所述一种基于葡萄糖氧化酶和类手枪脱氧核酶构成的荧光型葡萄糖传感器，其特征在于：铜离子使用浓度为1微摩尔/升。
8.如权利要求1所述一种基于葡萄糖氧化酶和类手枪脱氧核酶构成的荧光型葡萄糖传感器，其特征在于：类手枪脱氧核酶与DNA底物使用浓度同为50纳摩尔/升。
9.如权利要求1所述一种基于葡萄糖氧化酶和类手枪脱氧核酶构成的荧光型葡萄糖传感器，其特征在于：氯化钠浓度为800毫摩尔/升。
10.如权利要求1所述一种基于葡萄糖氧化酶和类手枪脱氧核酶构成的荧光型葡萄糖传感器，其特征在于：缓冲体系为50毫摩尔/升MES缓冲溶液(pH为6.0)。
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