通过在金纳米颗粒和葫芦的聚集体中形成精确的等离子体纳米结来定量检测尿酸
Summary
在水溶液中形成葫芦[7]尿酸和尿酸的宿主 – 客体复合物，然后向Au NP溶液中加入少量，以使用模块化光谱仪进行定量表面增强拉曼光谱（SERS）传感。
Abstract
这项工作描述了一种快速且高度灵敏的方法，用于通过表面增强拉曼光谱（SERS）定量检测重要的生物标志物尿酸（UA），使用模块化光谱仪对指纹区域中的多个特征峰具有约0.2μM的低检测限。这种生物传感方案由大环，葫芦[7]uril（CB7）和UA之间的宿主 – 客体络合以及随后在自组装的Au NP：CB7纳米聚集体内形成精确的等离子体纳米结介导。基于经典的柠檬酸盐还原方法，还进行了SERS底物所需尺寸的简单Au NP合成，并可以选择使用实验室制造的自动合成器来促进。该方案可以很容易地扩展到体液中生物标志物的多重检测，用于临床应用。
Introduction
尿酸是嘌呤核苷酸代谢的最终产物，是血清和尿液中诊断痛风，先兆子痫，肾脏疾病，高血压，心血管疾病和糖尿病等疾病的重要生物标志物1，2，3，4，5。目前用于尿酸检测的方法包括比色酶法、高效液相色谱法和毛细管电泳，这些方法既耗时又昂贵，并且需要复杂的样品制备6，7，8，9。
表面增强拉曼光谱是一种用于常规床旁诊断的有前途的技术，因为它允许通过其振动指纹选择性检测生物分子，并提供许多优点，如高灵敏度，快速响应，易用性以及无或最少的样品制备。基于贵金属纳米颗粒（例如，Au NPs）的SERS衬底可以通过表面等离子体共振引起的强电磁增强10将分析物分子的拉曼信号增强4至10个数量级10。与耗时的复杂金属纳米复合材料12相比，定制尺寸的Au NPs可以很容易地合成，因此由于其优越的性能13，14，15，16，因此被广泛用于生物医学应用。将大环分子，葫芦[n]尿素（CBn，其中n = 5-8，10）附着在Au NPs的表面上可以进一步增强分析物分子的SERS信号，因为高度对称和刚性的CB分子可以控制Au NPs之间的精确间距，并通过形成宿主 – 客体复合物将分析物分子定位在中心或靠近等离子体热点（图1）17，18，19，20.以前使用Au NP的SERS研究示例：CBn纳米聚集物包括硝基爆破剂，多环芳烃，二氨基二苯乙烯，神经递质和肌酐21，22，23，24，25，SERS测量在比色皿中进行或通过将小液滴加载到定制的样品架上。该检测方案对于快速定量具有高再现性的复杂基质中的生物标志物特别有用。
本文使用模块化光谱仪展示了一种简单的方法来形成CB7的宿主 – 客体复合物和重要的生物标志物UA，并通过CB7介导的Au NPs在水介质中的聚集物以0.2μM的检测限量化UA，这在诊断和临床应用中是有希望的。
Protocol
1. 金核素的合成 通过传统的图尔克维奇方法合成金种子26 通过溶解98.5mg HAuCl4 ， 制备10 mL 25 mM HAuCl4溶液3H2O前驱体与10mL去离子水在玻璃小瓶中。注意：将少量HAuCl4 前驱体转移到称量船中，并使用塑料刮刀代替金属刮刀称量晶体，因为HAuCl4 前驱体会腐蚀金属实验室器具。称量步骤应尽可能快地进行，因为HAuCl4 具有…
Representative Results
在所提出的Au NP合成中，紫外 – 可见光谱显示LSPR峰在10个生长步骤后从521 nm移动到529 nm（图4A，B），而DLS数据显示随着Au NPs的尺寸从25.9 nm增加到42.8 nm而窄尺寸分布（图4C，D）。从TEM图像（图4E）测量的G0，G5和G10的平均尺寸分别为20.1±2.1nm，32.5±2.3nm和40.0±2.2nm，每种情况下计数200个颗粒。这些结果表明，?…
Discussion
协议中描述的自动合成方法允许可重复地合成尺寸增加的Au NPs。虽然有一些元素仍然需要手动进行，例如在种子合成过程中快速添加柠檬酸钠并定期检查以确保PEEK管是安全的，但这种方法允许大尺寸（高达40nm）的Au NPs，这通常需要多次手动注射HAuCl4 和柠檬酸钠， 通过长时间的连续加法合成。
可以进行进一步的表征以阐明CB配合物的基本性质。例如，宿主 – 客体复合…
Disclosures
The authors have nothing to disclose.
Acknowledgements
TCL感谢英国皇家学会研究补助金2016 R1（RG150551）和UCL BEAMS未来领袖奖的支持，该奖项由EPSRC的机构赞助奖（EP/P511262/1）资助。WIKC，TCL和IPP感谢A * STAR-UCL研究实习计划通过EPSRC M3S CDT（EP / L015862 / 1）资助的学生奖学金。GD和TJ要感谢EPSRC M3S CDT（EP / L015862 / 1）赞助他们的学生奖学金。TJ和TCL感谢Camtech Innovations对TJ学生的贡献。所有作者都感谢伦敦大学学院开放获取基金。
Materials
| 40 nm gold nanoparticles | NanoComposix | AUCN40-100M | NanoXact, 0.05 mg/ mL, bare (citrate) |
| Centrifuge tube | Corning Falcon | 14-432-22 | 50 mL volume |
| Cucurbit[7]uril | Lab-made | see ref. 19 | |
| Gold(III) chloride trihydrate | Sigma aldrich | 520918 | ≥99.9% trace metals basis |
| Luer lock disposable syringe | Cole-Parmer | WZ-07945-15 | 3 mL volume |
| Luer-to-MicroTight adapter | LuerTight | P-662 | 360 μm outer diameter Tubing to Luer Syringe |
| PEEK tubing | IDEX | 1572 | 360 μm outer diameter, 150 μm inner diameter |
| PEEK tubing cutter | IDEX | WZ-02013-30 | Capillary Polymer Chromatography Tubing Cutter For 360 µm to 1/32" OD tubing |
| Raman spectrometer | Ocean Optics | QE pro | |
| Sodium citrate tribasic dihydrate | Sigma aldrich | S4641 | ACS reagent, ≥99.0% |
| Sonicator | |||
| Standard Probe | Digi-Sense | WZ-08516-55 | Type-K |
| Syringe pump | Aladdin | ALADDIN2-220 | 2 syringes, maximum syringe volume 60 mL |
| Thermocouple thermometer | Digi-Sense | WZ-20250-91 | Single-Input Thermocouple Thermometer with NIST-Traceable Calibration |
| ThermoMixer | Eppendorf | 5382000031 | With an Eppendorf SmartBlock for 50 mL tubes |
| Uric acid | Sigma aldrich | U2625 | ≥99%, crystalline |
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Cite This Article
Chio, W. K., Davison, G., Jones, T., Liu, J., Parkin, I. P., Lee, T. Quantitative SERS Detection of Uric Acid via Formation of Precise Plasmonic Nanojunctions within Aggregates of Gold Nanoparticles and Cucurbit[n]uril. J. Vis. Exp. (164), e61682, doi:10.3791/61682 (2020).