College of Chemistry & Chemical Engineering, Shanghai Jiaotong University, 800 Dongchuan Road, Shanghai 200240, PR China.
Analyst. 2012 Aug 21;137(16):3659-66. doi: 10.1039/c2an35503e. Epub 2012 Jun 28.
In this paper, we report a new strategy for highly sensitive determination of hydrogen peroxide, glucose and uric acid based on fluorescence resonance energy transfer (FRET) using gold nanoparticles (AuNPs) as energy acceptors. The principle is based on highly sensitive reaction of tetramethyl rhodamine (TMR) labeled tyramide and hydrogen peroxide catalysed by horseradish peroxidase (HRP), and the fluorescence spectrum of TMR (EX(max) 575 nm) partially overlaps with the visible absorption bands of AuNPs. We demonstrated an efficient FRET between tyramide labeled TMR (as energy donors) and HRP (BSA) conjugated AuNPs (as energy acceptors) due to the formation of TMR-labeled HRP-AuNPs or TMR-labeled BSA-AuNPs in the presence of H(2)O(2). We observed that the quenching of the fluorescence signal depended linearly on the H(2)O(2) concentration within a range of concentrations from 25 to 400 nM and the detection limit of this assay was 10 nM. Based on the principle for determination of H(2)O(2), we developed a new strategy for assay of glucose and uric acid by coupling with glucose oxidase (GOx)-mediated and uricase-mediated reaction. The established methods were successfully used for determination of glucose and uric acid levels in human sera, and the results obtained are in good agreement with commercially available methods. Our methods are at least 1 order of magnitude more sensitive than the commercially available methods. More importantly, our method described here can be extended to other assay designs using different oxidase enzymes, energy donors and energy acceptors, such as fluorescent quantum dots, near-infrared (NIR)-to-visible upconversion nanoparticles and even other metallic nanoparticles.
在本文中，我们报告了一种新的策略，基于金纳米粒子（AuNPs）作为能量受体的荧光共振能量转移（FRET），用于高度敏感地测定过氧化氢、葡萄糖和尿酸。该原理基于辣根过氧化物酶（HRP）催化的TMR 标记的酪胺与过氧化氢的高度灵敏反应，并且 TMR 的荧光光谱（EX（max）575nm）与 AuNPs 的可见吸收带部分重叠。我们证明了由于在 H2O2 的存在下形成了 TMR 标记的 HRP-AuNPs 或 TMR 标记的 BSA-AuNPs，TMR 标记的酪胺（作为能量供体）和 HRP（BSA）共轭的 AuNPs（作为能量受体）之间存在有效的 FRET。我们观察到，荧光信号的猝灭与 H2O2 浓度在 25 到 400 nM 的浓度范围内呈线性关系，该测定的检测限为 10 nM。基于测定 H2O2 的原理，我们通过与葡萄糖氧化酶（GOx）介导和尿酸酶介导的反应偶联，开发了一种测定葡萄糖和尿酸的新策略。所建立的方法成功地用于测定人血清中的葡萄糖和尿酸水平，并且获得的结果与市售方法非常吻合。我们的方法比市售方法至少灵敏 1 个数量级。更重要的是，我们在这里描述的方法可以扩展到使用不同的氧化酶、能量供体和能量受体的其他测定设计，例如荧光量子点、近红外（NIR）到可见上转换纳米粒子，甚至其他金属纳米粒子。
