组成图示
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传感器类型
微流控生物传感器
检测对象
葡萄糖（glucose）、乙醇（ethanol）、酵母细胞密度（cell density）、溶解氧分压（pO2）；样品基质为酿酒酵母发酵液/培养液（Saccharomyces cerevisiae fermentation broth）
检测原理
双电化学生物传感器中，PyOx 催化葡萄糖氧化为 2-脱氢-D-葡萄糖，AOx 催化乙醇氧化为乙醛，两个反应均消耗溶解氧。在 -0.7 V vs Ag/AgCl 下，金电极监测氧还原电流变化；底物浓度越高，酶促氧消耗越强，电流变化越大，从而在 0.025–0.5 mM 内线性响应。采用负电位检测避免了 H2O2 在正电位下的扩散串扰，PAMAM 树状分子多分支共价固定提高酶稳定性。辅助通道中，光纤 pO2 传感器利用荧光猝灭/相移法将氧浓度转换为相移信号；原位显微镜以透射亮场成像，Canny 边缘检测识别细胞轮廓并计数，细胞密度与计数呈线性。FA 系统自动稀释 300 倍，使发酵液葡萄糖进入线性范围。
检测灵敏度
线性范围: 0.025–0.5 mM（乙醇和葡萄糖）
效应效果
双传感器混合标准无干扰，葡萄糖回收率 97.73–99.42%，乙醇 90.46–102.69%。0.5 mM 重复进样 RSD：葡萄糖 0.87%（n=5），乙醇 4.51%（n=5）。连续 11 h、85 次测量后，葡萄糖信号保持 98%，乙醇 94.1%，摘要称损失 6.0% 初始活性。1.0 mM H2O2 不干扰，>1.0 mM 反向；葡萄糖 >0.5 mM 时乙醇电极轻微串扰，经 FA 300 倍稀释消除。ISM 与 Neubauer 计数强线性相关，可校准测细胞密度；pO2 13 h 由 90% 降至 15% 且无漂白。系统用于酿酒酵母 H620 发酵 13 h 旁线监测，无需额外采样或 HPLC/比色离线方法。
传感器的构成
- 基底/换能器电极：金双工作电极（Au dual working electrode，3.0 mm 直径、1.0 mm 间隙）、Ag/AgCl 参比电极和 Pt 辅助电极，置于薄层流通池（flow-through cell）中，用于流通式电流检测。
- 修饰层：半胱胺自组装单分子层（cysteamine SAM）修饰金表面，提供连接位点并规整界面。
- 固定层：聚酰胺胺树状分子（PAMAM dendrimer，25% C12 Generation 4）接枝到 SAM 上，提供多分支共价固定位点。
- 识别元件：吡喃糖氧化酶（PyOx）和乙醇氧化酶（AOx）分别固定于两个金电极表面，通过戊二醛（glutaraldehyde）交联，分别识别葡萄糖和乙醇。
- 信号机制：酶催化底物氧化并消耗 O2，在 -0.7 V vs Ag/AgCl 下监测 O2 还原电流变化（ΔI, μA），无外加电子供体或荧光标记物。
- 流动分析模块：蠕动泵、硅管、稀释室和 50 mM 磷酸钠缓冲液（pH 7.0）载体流，实现自动进样、300 倍稀释和流通测量。
- 辅助传感模块：光纤 pO2 传感器（Presens GmbH，荧光猝灭/相移法）嵌入流通池；原位显微镜（ISM，III XTF，透射亮场，4×物镜，CCD，588 nm LED）改造为 4.5 mm3 流通池用于细胞密度。
- 读出模块：PalmSens 电位计/多路复用器和 PSlite 软件记录电流；Canny 边缘检测图像处理算法计数细胞；光纤相移法输出 pO2。
中文摘要
本文报道一种用于生物过程旁线（at-line）监测的多分析物传感装置，可同时监测葡萄糖、乙醇、溶解氧分压（pO2）和细胞密度。该装置将双电化学生物传感器、改造的原位显微镜和光纤 pO2 传感器集成到流动分析（FA）系统中。双生物传感器基于薄层流通池中的金双工作电极（串联构型），通过表面修饰实现无串扰检测。每个金表面先用半胱胺自组装单分子层（SAM）修饰，随后接枝聚酰胺胺（PAMAM）树状分子，并分别以戊二醛交联固定吡喃糖氧化酶（PyOx）和乙醇氧化酶（AOx）。葡萄糖和乙醇的响应在 0.5 mM 以下呈线性；连续运行 11 h 后仅损失 6.0% 初始活性。装置在酵母发酵过程中平行测定乙醇和葡萄糖，同时用原位显微镜监测细胞密度，并用光纤传感器监测 pO2，实现无需额外采样或耗时离线测量的多参数旁线监测。
英文摘要
A multi-analyte sensing device is described, for simultaneous at-line monitoring of glucose, ethanol, pO₂-value and cell density. It consists of a dual biosensor, a modified microscope and a fiber optical pO₂-sensor that are integrated into a flow analysis (FA) system. The biosensor is based on a conventional thin layer flow-through cell equipped with a gold (Au) dual electrode (serial configuration). The biosensors with no cross-talking were produced by modifying the electrochemical transducers. Each Au surface was initially modified by self-assembled monolayer (SAM) of cysteamine. Alcohol oxidase (AOx) and pyranose oxidase (PyOx) were immobilized each onto a gold surface by means of PAMAM (polyamidoamine) dendrimer via glutaraldehyde cross-linking. The responses for glucose and ethanol were linear up to 0.5 mM. The operational stability of the biosensors was very promising, after 11 h continuous operation, only 6.0% of the initial activity was lost. The potential of the described biosensor was demonstrated by parallel determination of ethanol and glucose in yeast fermentation process. Simultaneously the cell density of the culture was monitored with an in situ microscope (ISM), which was integrated into the FA system. Both the used in situ microscope and the image processing algorithm used for the analysis of the acquired image data are described. Furthermore the pO₂-value was monitored using a fiber optical sensor, which was embedded in a flow cell. The multi-sensor device allows the at-line monitoring of several process values without the need for further sampling or time consuming offline measurements.