组成图示
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传感器类型
表面等离子共振(SPR)生物传感器
检测对象
RsaI内切酶（RsaI endonuclease）；样品基质：Tris–HCl缓冲液（pH 8.0，含MgCl2）
检测原理
巯基化Probe DNA1自组装于Au膜并经MCH封闭后，与Au-NPs标记的cDNA杂交形成含GTAC位点的双链DNA。加入RsaI内切酶后，酶识别GTAC并切割双链，使Au-NPs标记片段从表面脱离，同时残余Probe DNA1由线性结构折叠为发夹结构。Au-NPs脱离导致表面质量、折射率下降，并削弱Au膜与Au-NPs局域表面等离子体之间的电磁耦合；发夹构象变化进一步改变界面折射率分布。二者共同引起SPR谷偏移增大。RsaI浓度越高，切割越多，SPR谷偏移越大，从而实现实时定量检测。
检测灵敏度
LOD: 5 × 10−10 M；检测水平: 5 × 10−8 M；剂量依赖范围: 5 × 10−8 M–1 × 10−5 M
效应效果
该方法可实时监测DNA切割过程，RsaI检测水平为5×10−8 M，LOD为5×10−10 M，低于电化学和荧光方法。XPS显示Au-NPs非特异吸附约68%，但因SPR记录Au-NPs被切割脱离的过程，非特异吸附不影响结果。阴性对照EcoRI内切酶不产生SPR信号变化，说明特异性良好。Au-NPs放大使信号约为无Au-NPs时的2倍，发夹探针灵敏度高于线性探针。重复测量RSD均小于10%，单次样品量仅50 μL，对应RsaI约2.5×10−12 mol，适合限制性内切酶活性分析。
传感器的构成
- 基底/换能器：BK7玻璃片溅射2 nm Cr和50 nm Au膜，作为SPR换能基底
- 封闭层：6-巯基-1-己醇(MCH)修饰Au膜，降低非特异吸附
- 识别探针：巯基化探针DNA1(Probe DNA1)通过硫醇自组装固定于Au膜，含可形成发夹的茎区
- 杂交识别层：Probe DNA1与Au-NPs标记cDNA杂交形成双链DNA，含GTAC识别位点
- 信号标记物：13 nm金纳米粒子(Au-NPs)偶联巯基化cDNA，提供SPR质量与电磁耦合放大
- 构象放大元件：切割后残余Probe DNA1形成发夹结构，引起SPR谷偏移变化
- 读出系统：自制FI-SPR仪器配双元光电探测器，记录SPR角位移/谷偏移
中文摘要
金纳米粒子（Au-NPs）常用于放大表面等离子共振（SPR）信号，但严重非特异吸附限制了其实际应用。本文开发了一种不受非特异吸附影响的Au-NPs增强SPR生物传感器：利用RsaI内切酶将Au-NPs从传感器表面切割下来。为进一步提高灵敏度，作者对探针DNA进行了特殊设计。切割反应后，残余探针DNA形成发夹结构，引起SPR谷偏移的显著变化。在Au-NPs与探针DNA构象变化的共同作用下，SPR信号被显著放大。该方法可实时监测DNA切割过程，检测水平达5×10−8 M。X射线光电子能谱（XPS）结果证实存在较大非特异吸附，但由于SPR记录的是Au-NPs被切割脱离的过程，严重非特异吸附并未影响实验结果。
英文摘要
Gold nanoparticles (Au-NPs) are usually used to amplify surface plasmon resonance (SPR) signals, however, the serious nonspecific adsorption has largely limited their practical applications. Here, we developed a novel Au-NPs enhanced biosensor without the effect of nonspecific adsorption: cutting Au-NPs off from the biosensor surface by RsaI endonuclease. In order to further improve the sensitivity, the probe DNA was designed specially. After the cleavage reaction, the residual probe DNA formed hairpin structure, which also resulted in a great change in SPR dip shift. Then, with the coaction of Au-NPs and conformation change of probe DNA, the SPR signal was amplified greatly. Using this method, we monitored the process of DNA cleavage in real-time and achieved a detection level of 5×10(-8) M. Moreover, the result of X-ray photoelectron spectroscopy (XPS) experiment further confirmed that large nonspecific adsorption existed. However, because SPR recorded a process in which the Au-NPs were cut off, the serious nonspecific adsorption did not affect the experimental result.