组成图示
示意图生成中
传感器类型
荧光生物传感器
检测对象
雌酮（estrone, E1，主要验证对象）、17α-炔雌醇（17α-ethinylestradiol）、17β-雌二醇（17β-estradiol）、己烯雌酚（diethylstilbestrol）、雌三醇（estriol）；样品基质：Milli-Q 水加标、自来水。
检测原理
该传感器采用竞争抑制免疫分析。样品中的雌酮与荧光标记的 anti-total-estrogen (a-ES) 抗体孵育并达到平衡；分析物浓度越高，越多抗体被分析物占据，因而能结合到芯片表面雌酮衍生物 E13CME 的抗体越少。表面结合的荧光抗体被 635 nm 激光通过全内反射产生的倏逝场激发，发射长波长荧光，经聚合物光纤、700 nm 截止滤光片、光电二极管和锁相放大器读出。荧光信号与表面结合抗体量成正比，随雌酮浓度升高呈 logistic 下降，IC50 对应信号下降 50% 的浓度。该方法无需样品前处理和富集。
检测灵敏度
LOD: 0.19 ng L−1；LOQ: 1.39 ng L−1；IC50: 0.062 ± 0.004 µg L−1；SNR: 47；calibration range: 0–9 µg L−1（nine steps）。摘要另报告 LOD below 0.20 ng L−1、LOQ below 1.40 ng L−1。
效应效果
与 GC–MS/HPLC–MS 相比，该传感器无需前处理和富集，适合快速低成本筛查。最终雌酮校准各浓度步误差 0.79–4.76%（S.D.），空白误差 0.67%；长期稳定性测试显示芯片可完成超过 400 次测量并再生。自来水加标雌酮（0.27、0.9、2.7 ng/L）中，两个水平回收率在 70–120% 内，符合 AOAC；最接近 LOD 的 0.27 ng/L 回收率高于 120%，属典型高估。抗体可交叉识别多种雌激素，但交叉反应仍是同时检测限制。作者认为其可用于饮用水和环境水内分泌干扰物监测与预警。
传感器的构成
- 光波导基底：bulk optical glass slide（Schott 光学玻璃片），作为全内反射光波导与倏逝场换能器。
- 表面活化层：(3-glycidyloxypropyl)trimethoxysilane (GOPTS)，硅烷化玻璃表面并提供环氧基团用于共价固定氨基葡聚糖。
- 氨基葡聚糖修饰层：aminodextran Amdex（40 K/170 K Da），共价固定于 GOPTS 表面，提供氨基结合位点并降低非特异结合。
- 捕获/识别元件：estrone derivative E13CME（雌酮羧基间隔基衍生物），共价固定于氨基葡聚糖，作为表面抗原衍生物捕获未结合分析物的荧光抗体。
- 溶液相识别元件：anti-total-estrogen (a-ES) 多克隆 IgG 抗体，与样品中雌酮等雌激素结合，形成竞争抑制免疫分析。
- 信号标记物：荧光染料标记的 a-ES 抗体（CyDye Cy5.5 或 Alexa Fluor 680），被 635 nm 激光激发并发射长波长荧光。
- 封闭/背景抑制层：非偶联 aminodextran Amdex，覆盖非检测区域，减少非特异抗体吸附。
- 自动进样与读出系统：CTC Analytics HTC PAL 自动进样器、聚合物光纤、700 nm 截止吸收滤光片、光电二极管 (PD)、锁相放大器与 PC，完成稀释、孵育、进样、再生和荧光信号采集。
中文摘要
地表水中痕量污染物可显著影响水生生物内分泌系统，因此具有极低检出限（LOD）和定量限（LOQ）的免疫分析方法对环境和饮用水监测日益重要。抗生素、激素、内分泌干扰物和农药在真实水样中的监测对化学分析要求很高。生物传感器具有快速、灵敏、低成本等特性。本文以雌酮为例，描述全自动免疫分析优化过程，最终 LOD 低于 0.20 ng/L，LOQ 低于 1.40 ng/L。与 GC–MS 或 HPLC–MS 相比，该生物传感器无需样品前处理和富集。其高灵敏度来自高亲和抗体及连续优化；通过降低每样品抗体用量并改进芯片表面修饰，最终校准仅需 3.0 ng 抗体（样品体积 1.0 mL）。降低抗体用量可改善 LOD、LOQ 和 IC50，但也达到 River Analyser（RIANA）的设备相关限制。RIANA 可在低 ng/L 水平检测单一激素，是水生分析中补充常规分析方法的有力工具。
英文摘要
Certain contaminants at trace concentrations in surface waters can have dramatic effects on the hormonal system of organisms in the aquatic environment. Therefore, immunoanalytical methods at a very low limit of detection (LOD) and a low limit of quantification (LOQ) are becoming more and more important for environmental analysis and especially for monitoring drinking water quality. Environmental monitoring of antibiotics, hormones, endocrine disrupting chemicals, and pesticides in real water samples (e.g. surface, ground or drinking water) with difficult matrices places high demands on chemical analysis. Biosensors have suitable characteristics such as efficiency in allowing very fast, sensitive, and cost-effective detection. Here we describe an assay optimization process with a fully automated immunoassay for estrone which resulted in a LOD below 0.20ngL(-1) and a LOQ below 1.40ngL(-1). In contrast to common analytical methods such as GC-MS or HPLC-MS, the biosensor used requires no sample pre-treatment and pre-concentration. The very low validation parameters for estrone are the result of the continuous optimization of the immunoassay. The basis of our sensitive assay is the antibody with a high affinity constant towards estrone. During the optimization process, we reduced the amount of antibody per sample and improved the chip surface modification. Finally, this proceeding led to a calibration routine with an amount of antibody of only 3.0ng per sample (sample volume: 1.0mL). The reduction of the amount of antibody per sample results in better validation parameters (LOD, LOQ, and IC(50)), but this reduction leads to the current device-related limitation of the River Analyser (RIANA). For some endocrine disrupting compounds, no effect levels (NOELs) in the lower nanogram per liter range are reported. This defines the challenge, which analytical methods have to compete with and our RIANA instrument with its improved sensitivity for the detection of a single hormone in the lower nanogram per liter range is a powerful tool in aquatic analytics in addition to the common analytical methods.