CN116656662A - 一种集成酶水凝胶复合材料、其制备方法及其在制备双模式检测葡萄糖试剂盒中的应用 - Google Patents
一种集成酶水凝胶复合材料、其制备方法及其在制备双模式检测葡萄糖试剂盒中的应用 Download PDFInfo
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- CN116656662A CN116656662A CN202310688939.0A CN202310688939A CN116656662A CN 116656662 A CN116656662 A CN 116656662A CN 202310688939 A CN202310688939 A CN 202310688939A CN 116656662 A CN116656662 A CN 116656662A
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- C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
本发明涉及生物传感技术领域，具体涉及一种集成酶水凝胶复合材料、其制备方法及其在制备双模式检测葡萄糖试剂盒中的应用。本发明的集成酶水凝胶复合材料，以水凝胶、具有类葡萄糖氧化酶性质的纳米酶、辣根过氧化物酶以及荧光染料为原料制备得到。本发明可将荧光染料包埋在琼脂糖水凝胶中，利用集成酶对目标物的级联催化产生的活性氧介导荧光信号变化，可得葡萄糖浓度与荧光信号变化的定量关系。利用集成酶对目标物级联催化产生的活性氧还可将常用的比色显色底物3,3’,5,5’‑四甲基联苯胺氧化为有色物质，通过手机摄像获得照片并进行分析，可得葡萄糖浓度与颜色变化的定量关系，实现灵敏、简便、经济的葡萄糖可视化检测。
Description
技术领域
本发明涉及生物传感技术领域，具体涉及一种集成酶水凝胶复合材料、其制备方法及其在制备双模式检测葡萄糖试剂盒中的应用，所述的应用基于集成酶水凝胶复合材料荧光/比色双模式检测传感器的构建，进而实现对葡萄糖的快速检测。
背景领域
葡萄糖是生物体内新陈代谢不可缺少的营养物质，是活细胞的能量来源和新陈代谢中间产物，被广泛用于发酵、食品、化工、生物医学领域中。然而，如果血液中的葡萄糖长期保持过高或过低的水平，则可能导致组织损伤、中风、肾衰竭、失明和心脏病等。由于血糖控制不良，每年有100多万人丧生，严重威胁人类健康和生活。此外，由于患者每天都需要进行血糖监测，对个人以及政府带来巨大的经济负担。因此，能够快速且准确地定量检测出葡萄糖浓度对于人体健康和工业生产都具有重要意义。
当前商业用血糖监测仪主要分为电化学法和光反射技术两大类，该技术不仅检测体系复杂，所需仪器昂贵，而且试纸的抗干扰能力差，准确性不足。实验室中比较常用的葡萄糖检测方法包括电化学检测法、高效液相色谱法、紫外分光光度法、荧光法、气相色谱法、比色法等。其中，荧光法检测葡萄糖具有较高的灵敏度，但也受限于生物分子背景信号高，以及分辨率不足等问题。基于酶活模拟酶的催化比色法是近年来发展起来的一种新型葡萄糖检测方法。该法具有操作简便、不依赖于特定的仪器设备等优势，但也面临检测灵敏度低等缺点。由于单模式检测策略都具有各自的局限性，双模式检测结果更加可靠，且节约时间、劳动力和试剂。在多种输出信号中，比色信号和荧光信号是应用最广泛的两种信号，也是发展双模式传感技术最常用的转导通道。将比色检测与荧光检测相结合，有利于提高葡萄糖的检测灵敏度和可靠性，并降低检测时间与成本(Biosensors and Bioelectronics,2021,190,113386)。值得注意的是，双模式检测探针大多是将两个不同的信号单元集成到一个传感平台上制备的。信号单元的复杂合成和不灵活的集成可能导致批次之间的鲁棒性和可重复性差(Sensors and actuators B,2018,310-317)。因此，开发在合成、功能修饰和某些单元的组装方面具有良好可控性的技术是未来制备双模式纳米探针的重要途径。
纳米酶是一类既有纳米材料的独特性能，又有催化功能的模拟酶。2006年，Rossi等人首次证明“裸”金纳米颗粒(AuNPs)具有类葡萄糖氧化酶的特性，可以在O2存在下催化氧化葡萄糖，产生葡萄糖酸和H2O2。与天然葡萄糖氧化酶相比，AuNPs纳米酶具有更大的比表面积和更高的稳定性。辣根过氧化物酶(HRP)是一种可催化双氧水分解成活性氧的蛋白酶，将葡萄糖氧化酶与过氧化物酶整合构筑具有级联催化性质的复合材料已被证明可以高效地检测葡萄糖(Analytical Chemistry,2022,94,14385)。然而，天然酶的许多固有缺陷比如稳定性差，难以回收利用等，阻碍了它们在实际中的应用。近年来发展的酶固定技术，利用固相载体稳定酶，是解决以上问题的有效策略。一些多孔材料，如介孔二氧化硅、金属有机框架、金属氧化物等已被用作酶载体。然而，这些材料存在制备过程复杂，材料本身的颜色干扰，酸性条件可能分解的问题。相比之下，水凝胶作为一种具有三维网状结构的聚合物，是用于酶固定的良好载体。当前已发展一些DNAzyme水凝胶(CN201711256475.7)、气凝胶(CN202010165964.7)等作为天然酶和纳米酶的固定载体用于葡萄糖检测。然而，这些凝胶在制备的过程中需要发生化学聚合反应，容易导致天然酶失活，还可能影响纳米酶的性质。
发明内容
针对现有技术存在的问题，本发明提供了一种集成酶水凝胶复合材料以实现荧光/比色双模式检测葡萄糖的新方法。本发明利用琼脂糖水凝胶低温自发成胶的特性，以及其透明、廉价、多孔的优势，实现多天然酶和纳米酶的整合，以及对荧光和比色指示剂的富集效应，不仅操作简便，准确度高还具有较高的稳定性，便于长期储存。此外，琼脂糖水凝胶还适用于血液中葡萄糖的检测，无需离心便可过滤掉血细胞，适用于复杂样品中的葡萄糖检测。
在本发明的第一方面，提供了一种集成酶水凝胶复合材料。
本发明提供了一种集成酶水凝胶复合材料，以水凝胶、具有类葡萄糖氧化酶性质的纳米酶、辣根过氧化物酶以及荧光染料为原料制备得到；水凝胶包裹具有类葡萄糖氧化酶性质的纳米酶、辣根过氧化物酶以及荧光染料；
所述水凝胶为琼脂糖水凝胶；
所述纳米酶是以聚乙烯吡咯烷酮(PVP)为保护剂，采用化学还原法制备水分散性良好的AuNPs。所述AuNPs可以替换为具有葡萄糖氧化酶活性的其他类型的纳米材料。
所述的荧光染料为荧光染料2',7'二氯氢化荧光素(H2DCF)；
所述水凝胶为浓度范围在0.1％wt-1％wt的琼脂糖水溶液；所述具有类葡萄糖性质的AuNPs纳米酶的浓度范围为10-80μg/mL；所述辣根过氧化物酶浓度为5-40μg/mL；所述荧光染料的终浓度为0.5mM。
本发明中，提供的集成酶水凝胶复合材料包含三个部分，琼脂糖水凝胶、酶以及荧光染料。琼脂糖水凝胶具有多孔结构且廉价易得，不仅可以将荧光和比色指示剂富集起来，还适用于血液中葡萄糖的检测，无需离心便可过滤掉血细胞。具有类葡萄糖氧化酶性质的AuNPs和辣根过氧化物酶形成级联酶反应体系，可以产生活性氧氧化荧光指示剂和比色指示剂以发生荧光信号变化以及比色颜色变化。
在本发明的第二方面，提供了如第一方面所述的集成酶水凝胶复合材料的制备方法，包括以下步骤：将金纳米颗粒AuNPs、辣根过氧化物酶HRP和荧光染料2',7'二氯氢化荧光素H2DCF混合于超纯水中，加入琼脂糖并搅拌均匀，将混合溶液加热至沸腾后冷却至室温，即可形成AuNPs-HRP-H2DCF-Agarose水凝胶复合材料。
进一步地，所述的制备方法中，金纳米颗粒AuNPs是以PVP为保护剂，采用化学还原法还原金属离子前驱体，制备水分散性良好的AuNPs；所制备的金纳米颗粒在中性或弱碱性条件下才能表现出葡萄糖氧化酶性质，在酸性条件下为类过氧化物酶性质。因此本实验所用体系为pH7.4的PBS缓冲溶液。所述的化学还原法中，化学还原剂是硼氢化钠，所述金属离子前驱体为氯金酸水溶液，浓度为100mM。
进一步地，所述的制备方法中，2',7'二氯氢化荧光素H2DCF制备方法为：将2',7'-二氯二氢荧光素二乙酸酯(H2DCF-DA)染料溶于氢氧化钠(NaOH)溶液中，制备出2',7'二氯氢化荧光素(H2DCF)水溶液。所述NaOH溶液为0.01M。
进一步地，所述的制备方法中，琼脂糖水溶液的浓度为0.1％wt-1％wt，更优选0.33％wt。所述加热方式为微波炉中高火(700W)加热。
在本发明的第三方面，提供了将如第一方面所述的集成酶水凝胶复合材料用于非诊断目的的双模式检测葡萄糖的方法。
利用水凝胶的双亲性质和多孔性质，本发明提供了一种双模式检测葡萄糖的新方法。采用上述集成酶水凝胶复合材料收集生物样本中的葡萄糖，然后将纳米酶和天然酶组合起来级联催化葡萄糖产生活性氧，活性氧氧化指示剂产生荧光信号和比色信号，进行葡萄糖的定量检测。天然酶和纳米酶的级联催化，兼具高选择性和强催化活性，保证葡萄糖到活性氧的转化过程。采用荧光/比色双模式检测，不仅能保证准确定量，而且具有成本低、灵敏度高、操作简便的优点。
本发明的非诊断目的的双模式检测葡萄糖的方法，是利用上述集成酶水凝胶，加入葡萄糖水溶液，孵育一段时间后，用酶标仪读取荧光强度，建立荧光强度与葡萄糖浓度的线性关系。再用智能手机对凝胶拍照，用软件分析图像的RGB值，建立R值与葡萄糖浓度的线性关系。
具体包括以下步骤：
1)将集成酶水凝胶与葡萄糖样品接触，并加入显色液3,3’,5,5’-四甲基联苯胺(TMB)溶液进行显色反应，孵育一段时间后，采用荧光光谱仪测定溶液的荧光强度，以荧光强度为纵坐标，葡萄糖浓度为横坐标，绘制出浓度-发射强度谱图，实现葡萄糖的荧光检测；
2)用智能手机对凝胶拍照，用软件分析图像的加成色(RGB)值，建立R值与葡萄糖浓度的线性关系，实现葡萄糖的比色检测。
进一步地，所述显色反应时间范围为5-30min，更优选地为10min。
进一步地，所述步骤1)中所得的荧光准曲线回归方程为Y＝13.78+1.78X,R2＝0.9989；其中，Y为荧光发射强度，X为葡萄糖浓度。
进一步地，所述步骤2)中所得的比色法准曲线回归方程为Y＝1.034+0.182*(1-e-0.01x),R2＝0.9888；其中，Y为R/G的比值，X为葡萄糖浓度。
本发明采用集成酶水凝胶复合材料检测葡萄糖的的流程图如图1所示。
本发明采用纳米酶作为级联反应的第一步是为了利用纳米酶的高稳定性和对目标物的高选择性。天然酶与纳米酶组合进行级联催化，可以保证将葡萄糖快速分解成过氧化氢，再利用辣根过氧化物酶将过氧化氢氧化成活性氧，将信号快速准确地转化为荧光信号和比色信号，方便检测。
在本发明的第四方面，提供了如第一方面所述的集成酶水凝胶复合材料在制备荧光/比色双模式检测葡萄糖试剂盒中的应用。
本发明具有如下技术效果：
1)为提高葡萄糖的检测灵敏度和可靠性，并降低检测时间与成本，本发明构建了荧光/比色双模式检测策略，将荧光染料和纳米酶以及天然酶集成在水凝胶的三维网络中，不仅设计简单，可操作性强，而且廉价易得。
2)本发明合成了具有葡萄糖氧化酶性质的金纳米颗粒纳米酶，与天然酶相比，纳米酶具有更高的稳定性，易于储存和运输。
3)相比于当前用于葡萄糖检测的气凝胶、DNAzyme水凝胶、聚丙烯酰胺水凝胶等，本发明构建的琼脂糖水凝胶的成胶过程是物理胶连而非化学交联，不会影响天然酶和纳米酶的活性。
4)本发明提供的集成酶水凝胶复合材料制备简单，无需复杂的仪器和苛刻的实验条件。本发明制备的金纳米材料具有较好的类葡萄糖氧化酶性质和稳定性。本发明制备的纳米酶功能划水凝胶具有大的孔状结构，可自动过滤掉血细胞，无需离心去除血细胞，在护理点葡萄糖的检测应用中具有很大潜力。本发明构建荧光/比色双模式探针，不仅具有较高的灵敏度和选择性，而且具有可视化、检出限低、线性范围宽、检测时间短等优点，为发展其他类型的生物传感器提供了新的思路。本发明可将荧光染料包埋在琼脂糖水凝胶中，利用集成酶对目标物的级联催化产生的活性氧介导荧光信号变化，可得葡萄糖浓度与荧光信号变化的定量关系。利用集成酶对目标物级联催化产生的活性氧还可将常用的比色显色底物3,3’,5,5’-四甲基联苯胺氧化为有色物质，通过手机摄像获得照片并进行分析，可得葡萄糖浓度与颜色变化的定量关系，实现灵敏、简便、经济的葡萄糖可视化检测。
5)从本发明具体实施方式的图5a所示，在荧光模式下，AHHA凝胶对葡萄糖检测的LOD约为1.9μM。拟合曲线显示AHHA凝胶对葡萄糖检测在0-0.1mM的浓度范围内具有良好的线性。如图5b所示，在比色模式下，AHHA凝胶对葡萄糖检测的LOD为5.2μM，线性范围为0.01mM-0.1mM。从具体实施方式的图6所示，当干扰物浓度为葡萄糖浓度2倍且其它条件相同时，葡萄糖样品的荧光强度与干扰物有显著差异，表明AHHA凝胶对于葡萄糖具有良好的选择性，有望用于复杂生理环境中的葡萄糖检测。从本发明具体实施方式的表1可以看出，本发明的集成酶水凝胶复合材料实现了比色/荧光双模式葡萄糖检测，提高了检测灵敏度，并降低检测时间与成本。
附图说明
图1为AHHA凝胶检测体系的构建及其对葡萄糖的荧光/比色双模式检测的原理示意图；
图2为AuNPs的电镜照片；其中，(a)为AuNPs的透射电子显微镜照片，(b)为AuNPs的高分辨率电镜照片。
图3为AuNPs的类葡萄糖氧化酶活性检测体系的紫外-可见近红外吸收光谱图。
图4为琼脂糖凝胶与AHHA凝胶的照片。其中，(a)为琼脂糖凝胶的照片，(b)为AHHA凝胶的照片。
图5为AHHA凝胶体系检测葡萄糖的灵敏性。其中，(a)AHHA凝胶的荧光强度随葡萄糖浓度的变化关系；(b)葡萄糖浓度对R/B的关系图。
图6为AHHA凝胶体系对葡萄糖检测的选择性。
具体实施方式
为了使本发明的目的、技术方案及优点更加清晰明了，以下结合附图及实施例，对本发明进行进一步详细说明，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。
其中，氯金酸(HAuCl4，≧99％)、葡萄糖氧化酶(GOx，160kDa，99％)、2',7'-二氯二氢荧光素二乙酸酯(H2DCF-DA，≧97％)、辣根过氧化物酶(HRP，≥250units/mg)、聚乙烯吡咯烷酮(PVP，30kDa)、四甲基联苯胺盐酸盐(TMB，99％)、氢氧化钠(NaOH，片状，≧98％)、半乳糖(Gal，≥99％)、果糖(Fru，≥99％)、D-蔗糖(Suc，99％)、甘露糖(Man，≥99％)、盐酸多巴胺盐酸盐(DA)、抗坏血酸(AA，99％)、尿素(UA)均购于Sigma-Aldrich(美国)。二甲亚砜(DMSO，99％)、硼氢化钠(NaBH4，96％)购于国药化学试剂集团。
实施例1AuNPs的制备
称取20mg PVP于50mL烧杯中，加入45mL超纯水，搅拌溶解。用注射器吸取1mLHAuCl4水溶液(100mM)，使用针式过滤器(0.22μm)过滤。随后，称取19mgNaBH4于试管中，加入5mL纯水溶解，得到浓度为0.1M的NaBH4水溶液，将其置于冰水浴中冷却。取0.5mL过滤后的HAuCl4溶液加入盛有PVP溶液的烧杯中，搅拌5min。取5mL冰水浴过的NaBH4溶液(0.1M)，快速加入上述反应体系中，搅拌后溶液呈变为黑褐色。反应2h后，将溶液离心(8000rpm，10min)，取上清液并丢弃沉淀，此步骤可得到AuNPs。
由图2可知，AuNPs呈球状，平均尺寸约为3.76nm。金纳米颗粒具有清晰的晶格条纹，晶面间距为0.132nm，归属于(110)晶面。
对制备的AuNPs进行类葡萄糖氧化酶性质测定，结果如图3所示，Au+Glu+HRP+TMB组和Au+Glu+TMB组在652nm处存在显著的吸收峰，而其它组吸收较弱，表明H2O2的产生是基于AuNPs的催化作用，即小尺寸AuNPs具有类葡萄糖氧化酶活性。
实施例2AuNPs-H2DCF-HRP凝胶体系的制备
本实施例的凝胶体系为AuNPs-H2DCF-HRP，以琼脂糖水凝胶、具有类葡萄糖氧化酶性质的纳米酶AuNPs(实施例1制备)、辣根过氧化物酶HRP，以及荧光染料H2DCF为原料制备得到。
具体制备步骤如下：
步骤1)2',7'二氯氢化荧光素(H2DCF)的制备
称取9.8mg H2DCF-DA并溶解在2mL DMSO中(0.01M)。将20mg NaOH加入50mL水中，配制浓度为0.01M的NaOH水溶液。向H2DCF-DA溶液中加入四倍体积的0.01M NaOH，室温下避光搅拌30min，此步骤可得H2DCF。
步骤2)AuNPs-HRP-H2DCF-Agarose(AHHA)凝胶的制备
在避光的条件下，按顺序分别向96孔板中加入3μLAuNPs溶液(2mg/mL)、1.5μL HRP溶液(2mg/mL)、37.5μL H2DCF溶液(2mM)，震荡形成AuNPs-H2DCF-HRP混合液。将0.05g琼脂糖加入10mL超纯水中，搅拌至无大颗粒沉淀，在微波炉中高火(700W)加热至沸腾，琼脂糖水溶液(0.5％wt)，取出搅拌并在室温下冷却。当琼脂糖溶液温度降低至45℃时，取100μL琼脂糖溶液加入含有50μLAuNPs-H2DCF-HRP混合液的96孔板中，快速搅拌混合均匀，冷却至室温，此步骤可得AuNPs-H2DCF-HRP-Agarose(AHHA)复合水凝胶。
得到复合水凝胶中，琼脂糖浓度为0.33％wt；具有类葡萄糖性质的AuNPs纳米酶的浓度为40μg/mL；辣根过氧化物酶浓度为20μg/mL；所述荧光染料的终浓度为0.5mM。
对制备的AuNPs-H2DCF-HRP凝胶进行形貌表征，结果如图4所示，当Agarose溶液和AuNPs-H2DCF-HRP-Agarose溶液温度高于45℃时为均一且透明溶液。静置冷却至室温后，液体凝固不流动，证明凝胶制备成功。
实施例3利用实施例2制备的AHHA凝胶体系检测葡萄糖的检测限和线性范围
设置浓度分别为0、5、10、20、40、80、100、200μM的葡萄糖水溶液，在黑暗的环境中，加入AHHA复合水凝胶表面，再加入40μL TMB水溶液(2mM)，在37℃下孵育10min。随后用酶标仪读取其荧光强度，建立荧光强度与葡萄糖浓度的线性关系。通过手机对凝胶拍照，用软件分析图像RGB值，建立R值与浓度的线性关系。按照LOD＝3S/N计算其检测限，其中，S为空白标样的标准偏差，N为斜率。
如图5a所示，在荧光模式下，所得的荧光准曲线回归方程为Y＝13.78+1.78X,R2＝0.9989；其中，Y为荧光发射强度，X为葡萄糖浓度。经计算，AHHA凝胶对葡萄糖检测的LOD约为1.9μM。拟合曲线显示AHHA凝胶对葡萄糖检测在0-0.1mM的浓度范围内具有良好的线性。如图5b所示，在比色模式下，所得的比色法准曲线回归方程为Y＝1.034+0.182*(1-e-0.01x),R2＝0.9888；其中，Y为R/G的比值，X为葡萄糖浓度。AHHA凝胶对葡萄糖检测的LOD为5.2μM，线性范围为0.01mM-0.1mM。由此可知，AHHA凝胶体系与其它类似的葡萄糖传感体系相比，具有较高的灵敏度和较宽的线性范围，为葡萄糖检测提供了一种荧光/比色双模式的检测平台。由表1可知，相比于其他凝胶传感器，AHHA凝胶传感器具有更低的检测限。
实施例4AHHA凝胶体系检测葡萄糖选择性
取40μL TMB水溶液(2mM)、3μLAuNPs水溶液(2mg/mL)、1.5μLHRP水溶液(2mg/mL)于96孔板中，吹打混匀，加入100μL浓度为0.5％wt的琼脂糖水溶液(温度为45℃)，冷却后形成AHHA复合凝胶。分别配制浓度为2mM(pH 7.4)的葡萄糖、果糖、半乳糖、甘露糖、蔗糖、尿酸、抗坏血酸、多巴胺溶液。黑暗条件下，分别取40μL以上溶液加入凝胶中，置于37℃下的摇床孵育10min，随后用酶标仪读取其荧光强度。每组实验重复三次，以确定探针的选择性。
发明人探究了潜在干扰物质如半乳糖、果糖、D-蔗糖、壳聚糖、L-半胱氨酸、甘露糖、谷胱甘肽、尿素等对AHHA传感体系的影响。如图6所示，当干扰物浓度为葡萄糖浓度2倍且其它条件相同时，葡萄糖样品的荧光强度与干扰物有显著差异，表明AHHA凝胶对于葡萄糖具有良好的选择性，有望用于复杂生理环境中的葡萄糖检测。
实施例5
将本发明制备的AHHA凝胶与现有技术中的催化凝胶检测体系的比较，结果如表1所示。
表1AHHA凝胶与其它催化凝胶检测体系的比较
由表1可知，相比于其他凝胶体系，本发明使用的琼脂糖水凝胶体系不仅可实现葡萄糖的荧光/比色双模式检测，而且具有更低的检测限，可实现葡萄糖的高灵敏检测。
参考文献
[1]Y.Zhu,J.Zhang,J.Song,J.Yang,Z.Du,W.Zhao,H.Guo,C.Wen,Q.Li,X.Sui,L.Zhang,A Multifunctional Pro-Healing Zwitterionic Hydrogel for SimultaneousOptical Monitoring of pH and Glucose in Diabetic Wound Treatment,AdvancedFunctional Materials 30(2019).
[2]C.Chen,Z.Q.Dong,J.H.Shen,H.W.Chen,Y.H.Zhu,Z.G.Zhu,2DPhotonicCrystal Hydrogel Sensor for Tear Glucose Monitoring,ACS Omega 3(2018)3211-3217.
[3]X.Huang,Y.Zhou,C.Liu,R.Zhang,L.Zhang,S.Du,B.Liu,M.Y.Han,Z.Zhang,Asingle dual-emissive nanofluorophore test paper for highly sensitivecolorimetry-based quantification of blood glucose,Biosens Bioelectron 86(2016)530-535.
[4]C.B.Ma,Y.Zhang,Q.Liu,Y.Du,E.Wang,Enhanced Stability of EnzymeImmobilized in Rationally Designed Amphiphilic Aerogel and Its Applicationfor Sensitive Glucose Detection,Anal Chem 92(2020)5319-5328.
由上述实施例可知，本发明提供了一种双模式检测葡萄糖的检测体系，通过级联反应将葡萄糖检测转变为荧光信合和颜色信号，操作简便。
以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。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，还可以对本发明进行若干改进和修饰，这些改进和修饰也要落入有本发明权利要求的保护范围内。
Claims (10)
1.一种集成酶水凝胶复合材料，其特征在于，以水凝胶、具有类葡萄糖氧化酶性质的纳米酶、辣根过氧化物酶以及荧光染料为原料制备得到；水凝胶包裹具有类葡萄糖氧化酶性质的纳米酶、辣根过氧化物酶以及荧光染料；
所述水凝胶为琼脂糖水凝胶；
所述纳米酶是以聚乙烯吡咯烷酮PVP为保护剂，采用化学还原法制备水分散性良好的AuNPs；
所述的荧光染料为荧光染料2',7'二氯氢化荧光素H2DCF；
所述水凝胶为浓度范围在0.1％wt-1％wt的琼脂糖水溶液；所述具有类葡萄糖性质的AuNPs纳米酶的浓度范围为10-80μg/mL；所述辣根过氧化物酶浓度为5-40μg/mL；所述荧光染料的终浓度为0.5mM。
2.如权利要求1所述的集成酶水凝胶复合材料的制备方法，其特征在于，包括以下步骤：将金纳米颗粒AuNPs、辣根过氧化物酶HRP和荧光染料2',7'二氯氢化荧光素H2DCF混合于超纯水中，加入琼脂糖并搅拌均匀，将混合溶液加热至沸腾后冷却至室温，即可形成AuNPs-HRP-H2DCF-Agarose水凝胶复合材料。
3.根据权利要求2所述的制备方法，其特征在于，所述金纳米颗粒AuNPs是以PVP为保护剂，采用化学还原法还原金属离子前驱体，制备水分散性良好的AuNPs。
4.根据权利要求3所述的制备方法，其特征在于，所述的化学还原法的反应体系为pH7.4的PBS缓冲溶液，化学还原剂是硼氢化钠，所述金属离子前驱体为氯金酸水溶液，浓度为100mM。
5.根据权利要求2所述的制备方法，其特征在于，所述2',7'二氯氢化荧光素H2DCF制备方法为：将2',7'-二氯二氢荧光素二乙酸酯H2DCF-DA染料溶于氢氧化钠溶液中，制备出2',7'二氯氢化荧光素H2DCF水溶液。
6.根据权利要求2所述的制备方法，其特征在于，所述琼脂糖水溶液浓度为0.1％wt-1％wt，所述加热方式为微波炉中高火700W加热。
7.利用如权利要求1所述的集成酶水凝胶复合材料进行非诊断目的的双模式检测葡萄糖的方法，其特征在于，包括以下步骤：
1)将集成酶水凝胶与葡萄糖样品接触，并加入显色液3,3’,5,5’-四甲基联苯胺TMB溶液进行显色反应，孵育一段时间后，采用荧光光谱仪测定溶液的荧光强度，以荧光强度为纵坐标，葡萄糖浓度为横坐标，绘制出浓度-发射强度谱图，实现葡萄糖的荧光检测；
2)用智能手机对凝胶拍照，用软件分析图像的加成色RGB值，建立R值与葡萄糖浓度的线性关系，实现葡萄糖的比色检测。
8.根据权利要求7的方法，其特征在于，所述步骤1)中显色反应时间范围为5-30min。
9.根据权利要求7的方法，其特征在于，
所述步骤1)中所得的荧光准曲线回归方程为Y＝13.78+1.78X,R2＝0.9989；其中，Y为荧光发射强度，X为葡萄糖浓度；
所述步骤2)中所得的比色法准曲线回归方程为Y＝1.034+0.182*(1-e-0.01x),R2＝0.9888；其中，Y为R/G的比值，X为葡萄糖浓度。
10.如权利要求1所述的集成酶水凝胶复合材料在制备荧光/比色双模式检测葡萄糖试剂盒中的应用。
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