组成图示
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传感器类型
电化学生物传感器
检测对象
硫胺素（thiamine，维生素B1）；样品基质：维生素片剂水溶液（稀释液）及磷酸盐缓冲液标准液
检测原理
传感器以固定化丙酮酸氧化酶（POX）为生物识别/催化元件，以溶解氧（DO）探针为换能器。在含丙酮酸和磷酸盐的氧饱和缓冲液中，POX催化丙酮酸氧化脱羧生成乙酰磷酸、CO2和H2O2，同时消耗O2。硫胺素（维生素B1）作为POX的激活剂/辅因子，提高酶催化速率，使酶层与聚四氟乙烯膜间溶解氧下降更快。氧表记录加入丙酮酸前后达到稳态的DO浓度差（ΔDO）；在固定丙酮酸浓度下，ΔDO随硫胺素浓度增加而增加。测定基于无硫胺素与有硫胺素响应差值，实现硫胺素定量。
检测灵敏度
LOD: 0.025 μM；线性范围: 0.025–0.5 μM；灵敏度斜率: 0.1419（y = 0.1419x + 0.0152）；R^2 = 0.9979；响应时间: 2 min
效应效果
选择性：0.1 μM硫胺素使0.025 μM丙酮酸响应由0.11 mg/L增至0.13 mg/L（+18.2%），等浓度吡哆醇、核黄素、烟酸无干扰。重现性：0.2 μM硫胺素（n=7）SD ±0.006 μM，CV 3.07%。稳定性：原文称21天后保留约10%初始活性。维生素片剂测定：One a day标称1.50 mg/片，测得1.56±0.0132 mg/片，回收率104.0%；Bemiks标称10 mg/片，测得10.1±0.0890 mg/片，回收率101.0%；Men’s One a Day标称2.25 mg/片，测得2.28±0.0158 mg/片，回收率101.3%。作者认为可作硫胺素常规分析替代方法。
传感器的构成
- 换能器基底：YSI 5700溶解氧（DO）探针，作为氧敏感电化学换能器。
- 氧选择性膜：高灵敏度聚四氟乙烯（teflon）膜（0.0005 in），控制氧扩散并保护酶层。
- 表面预处理层：0.5%十二烷基硫酸钠（SDS）磷酸盐缓冲液处理，降低膜表面张力。
- 固定化基质层：牛皮肤明胶（gelatin，5.0 mg cm−2），形成生物活性层并承载酶。
- 识别催化元件：丙酮酸氧化酶（POX，2.5 U cm−2），催化丙酮酸氧化脱羧并消耗O2。
- 交联固定剂：戊二醛（glutaraldehyde，2.5%），与明胶和POX形成席夫碱共价键。
- 反应介质：50 mM磷酸盐缓冲液（pH 7.0）、丙酮酸（pyruvate）和溶解氧，提供酶促反应条件。
- 信号读出：YSI Model 58数字氧表，检测DO浓度差值。
中文摘要
本研究开发了一种基于丙酮酸氧化酶（POX）的生物传感器，用于研究硫胺素（维生素B1）分子对酶活性的影响。该传感器采用化学共价固定化方法，以明胶为基质、戊二醛为交联剂，将POX固定于溶解氧（DO）探针表面。POX在磷酸盐和氧气存在下催化丙酮酸氧化脱羧生成乙酰磷酸、CO2和H2O2，并消耗溶解氧。硫胺素是POX的激活剂，传感器响应随硫胺素存在而增加，且增加幅度与硫胺素浓度相关。测定方法基于氧表上无硫胺素与有硫胺素时传感器响应的差值。在0.025–0.5 μM范围内，传感器响应与硫胺素浓度呈线性关系，响应时间为2 min。优化结果表明，最适酶量为2.5 U cm−2，最适工作条件为50 mM磷酸盐缓冲液（pH 7.0）和35°C。此外，对传感器的激活剂效应、干扰效应和重现性等参数进行了表征。
英文摘要
A biosensor based on pyruvate oxidase (POX) enzyme was developed for the investigation of the effect of thiamine (vitamin B(1)) molecule on the activity of the enzyme. The biosensor was prepared with a chemical covalent immobilization method on the dissolved oxygen (DO) probe by using gelatin and cross-linking agent, glutaraldehyde. POX catalyzes the degradation of pyruvate to acetylphosphate, CO(2) and H(2)O(2) in the presence of phosphate and oxygen. Thiamine is an activator for POX enzyme and determination method of the biosensor was based on this effect of thiamine on the activity of the enzyme. The biosensor responses showed increases in the presence of thiamine. Increases in the biosensor responses were related to thiamine concentration. Thiamine determination is based on the assay of the differences on the biosensor responses on the oxygenmeter in the absence and the presence of thiamine. The biosensor response depend linearly on thiamine concentration between 0.025 and 0.5 microM with 2 min response time. In the optimization studies of the biosensor the most suitable enzyme amount was found as 2.5 U cm(-2) and also phosphate buffer (pH 7.0; 50 mM) and 35 degrees C were obtained as the optimum working conditions. In the characterization studies of the biosensor some parameters such as activator and interference effects of some substances on the biosensor response and reproducibility were carried out.