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
晚期糖基化终末产物（AGEs）、Nε-(羧甲基)赖氨酸（CML）；样品基质：Zucker糖尿病肥胖大鼠（ZDF）和Zucker瘦大鼠（ZL）血清，另测尿液
检测原理
金芯片经半胱氨酸标记蛋白G固定抗AGEs或抗CML抗体，并用BSA封闭。血清点样后，AGEs/CML与表面抗体特异性结合，结合物质量增加改变金膜表面等离子体共振条件，使反射光强度或图像斑点强度发生变化。棱镜耦合光照射芯片，CCD相机采集反射图像，SPRI软件对斑点强度进行定量。信号随被测物浓度升高而增强，在0.001–1000 µg/ml范围内呈线性，LOD为10 ng/ml。该方法为无标记直接检测，不依赖荧光、酶或化学放大。
检测灵敏度
LOD: 10 ng/ml；线性范围: 0.001–1000 µg/ml；R^2 = 0.958
效应效果
SPRI蛋白芯片对PBS和BSA阴性对照几乎无信号，显示良好选择性。20周龄ZDF大鼠血清AGEs和CML水平均显著高于ZL大鼠；15%稀释血清中ZDF与ZL的相对差异为19.12%（85%和30%稀释分别为7.21%和11.34%）。竞争性ELISA测得ZDF血清AGEs为1.384±0.148 mg/ml，ZL为0.6748±0.095 mg/ml，与SPRI趋势一致。系统无需荧光或酶标记，可高通量同时检测数百样品，节省时间、资源和人力。尿液AGEs未检出显著差异，作者认为因尿中AGEs低于血清且样品量少，需超滤预浓缩。
传感器的构成
- 基底/换能器：裸金芯片（bare gold chip, K-MAC, Korea），经Piranha溶液清洗，作为SPR换能基底
- 连接层：半胱氨酸标记重组蛋白G（cysteine-tagged recombinant protein G, 100 µg/ml PBS），通过金-硫键固定于金表面，用于捕获抗体Fc段
- 封闭层：牛血清白蛋白（BSA, 1 mg/ml PBS），防止非特异性结合
- 识别元件：抗AGEs单克隆抗体6D12（Wako）或抗CML抗体（Abcam），固定于蛋白G修饰金表面，特异性结合血清中AGEs/CML
- 样品层：ZDF/ZL大鼠血清或尿液，稀释于含15%甘油的PBS后点样于芯片
- 读出层：棱镜耦合器（prism coupler）、CCD相机（Sony）和SPRI系统（Spot analysis program multi-gauge V3.1），无标记直接检测结合引起的反射图像变化
中文摘要
晚期糖基化终末产物（AGEs）与糖尿病并发症密切相关。为测定Zucker糖尿病肥胖大鼠（ZDF）和Zucker瘦大鼠（ZL）血清中AGEs，尤其是Nε-(羧甲基)赖氨酸（CML）的水平，作者采用蛋白芯片与表面等离子共振成像（SPRI）新方法。20周龄雄性ZDF和ZL大鼠血清样品中，抗AGEs或抗CML抗体被固定于经半胱氨酸标记蛋白G构建体修饰的金表面。点样血清的金芯片与棱镜耦合器光学耦合，用CCD相机获取反射图像。SPRI直接分析糖化蛋白及终产物显示，ZDF血清中AGEs和CML水平均高于ZL血清。AGEs最低检测限为10 ng/ml，工作范围覆盖生理水平。结果表明，蛋白芯片与SPRI系统适合测定血清中糖化蛋白和终产物，无需荧光或其他标记，具有高灵敏度，并节省时间、资源和人力，可作为糖尿病并发症诊断工具。
英文摘要
Advanced glycation end products (AGEs) have been implicated in diabetic complications. To measure AGEs, especially N(epsilon)-(carboxymethyl)lysine (CML), in sera from Zucker diabetic fatty rats (ZDF) and Zucker lean rats (ZL), we used a novel method of protein chip and surface plasmon resonance imaging (SPRI). Serum samples were obtained from male ZDF and ZL rats at 20 weeks of age. Antibodies to AGEs or CML were immobilized on a gold surface, which was modified by cysteine-tagged, protein-G constructs. The gold chip upon which the serum was spotted was optically coupled with a prism coupler. The reflected images from the gold chip were obtained using a charge-coupled device (CCD) camera. The direct analysis of the glycated proteins and products using SPRI showed that AGEs and CML levels were elevated in ZDF serum, compared with ZL serum. The lowest detection limit of AGEs was 10 ng/ml, with a working range covering the physiological range. These results indicate that the protein chip and SPRI system is very suitable for the measurement of glycated proteins and end products in serum samples. This system offers high sensitivity without any fluorescent or other labeling of the components and saves a substantial amount of time, resources, and labor. Our results suggest that SPRI systems can be used as a tool to diagnose diabetic complications.