组成图示
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传感器类型
荧光生物传感器
检测对象
硫酸化胆汁酸（sulfated bile acids, SBAs；以甘氨胆酸硫酸酯 GLCA-S 为代表）；样品基质：尿液
检测原理
该传感器以固定化酶催化作为识别事件：SBAs在BSS催化下脱硫酸生成3β-羟基胆汁酸；3β-HSD进一步催化3β-羟基胆汁酸氧化为3-酮类固醇，同时NAD+被还原为NADH。NADH作为电子供体将1-MPMS还原为1-MPMSH2，并再生NAD+，形成循环传递；1-MPMSH2再将resazurin还原为强荧光产物resorufin。resorufin在540 nm激发、580 nm发射，荧光差值ΔF随SBAs浓度升高而增大。酶促级联与NAD+/1-MPMS循环构成信号放大，使微量SBAs产生可测荧光。固定化酶可提高灵敏度并便于重复使用。
检测灵敏度
LOD: 0.16 μmol L−1；线性范围: 0.5–5.0 μmol L−1；灵敏度斜率: 71.286；相关系数: r = 0.9991
效应效果
该传感器抗干扰能力较好，尿素（50–1000 mmol/L）、抗坏血酸（0.25–2.0 mmol/L）、尿酸（0.25–2.5 mmol/L）、葡萄糖（50–200 mg/L）和NaCl（100–500 mg/L）均不干扰；尿液预反应10 min可消除未知干扰。稳定性方面，同一传感器每周测3次、连续1个月，荧光均值165.4±2.4，RSD<1.4%（n=12），4 ℃保存至少6个月。重现性RSD为2.2%–3.3%。尿液加标回收率为95.5%–106%。与商用UBASTEC试剂盒相比结果无明显差异；固定化传感器斜率71.286高于液相酶法38.055。单次反应体积仅18 μL，试剂用量约为传统荧光法的1/400，适合临床尿液SBAs检测。
传感器的构成
- 基底/反应腔：医用毛细管（medical capillary，内径0.9 mm），作为反应容器和荧光检测窗口
- 表面修饰层：3-氨基丙基三乙氧基硅烷（APTES），共价结合于毛细管内壁羟基，提供氨基
- 交联固定层：戊二醛（glutaraldehyde），与APTES氨基反应形成醛基交联层，用于固定酶
- 催化识别元件：胆汁酸硫酸酯酶（BSS，EC 2.8.2.14）和3β-羟基类固醇脱氢酶（3β-HSD，EC 1.1.1.51），催化SBAs脱硫酸及3β-羟基胆汁酸氧化
- 封闭层：0.5 mmol/L Tris缓冲液，封闭残余醛基
- 信号放大试剂：NAD+、1-MPMS和resazurin，构成NADH/1-MPMS循环并将resazurin还原为resorufin
- 读出装置：F-4500荧光分光光度计与自制毛细管夹持器，540 nm激发、580 nm发射检测resorufin荧光
中文摘要
本文提出一种固定化酶荧光毛细管生物传感器（SBAs-IE-FCBS），用于尿液中硫酸化胆汁酸（SBAs）的测定。该传感器将胆汁酸硫酸酯酶（BSS）和3β-羟基类固醇脱氢酶（3β-HSD）固定于医用毛细管内壁。在BSS催化下，SBAs脱硫酸生成3β-羟基胆汁酸；在3β-HSD催化下，3β-羟基胆汁酸与NAD+反应生成3-酮类固醇和NADH。NADH与1-甲氧基-5-甲基吩嗪甲基硫酸盐（1-MPMS）循环反应生成NAD+和1-MPMSH2，1-MPMSH2进一步将resazurin还原为resorufin（λex/λem：540/580 nm），通过resorufin荧光强度定量SBAs。优化条件下，BSS和3β-HSD固定化浓度均为5 kU/L，1-MPMS和resazurin均为25 μmol/L，Tris-HCl缓冲液和NAD+分别为100和400 μmol/L，反应总体积18 μL，37 ℃反应15 min。SBAs浓度在0.5–5.0 μmol/L范围内与荧光强度呈线性关系，RSD小于3.4%，检出限为0.16 μmol/L，回收率为95.5%–106%。该传感器可用于尿液SBAs定量，辅助诊断肝胆疾病。
英文摘要
The authors have proposed an immobilized enzymatic fluorescence capillary biosensor (SBAs-IE-FCBS) for the determination of sulfated bile acids (SBAs). The reaction principle of the biosensor is that under the catalysis of the bile acid sulfate sulfatase (BSS) and beta-hydroxysteroid dehydrogenase (beta-HSD) immobilized on inner surface of a medical capillary, SBAs desulfates to 3beta-hydroxyl bile acids, then the latter reacts with nicotinamide adenine dinucleotide (NAD(+)), and is converted into 3-ketosteroid; meanwhile, NAD(+) is converted to reduced nicotinamide adenine dinucleotide (NADH). NADH continuously reacts with 1-methoxy-5-methylphenazinium methyl sulfate (1-MPMS) and is converted into NAD(+) circularly and 1-MPMSH(2). Finally resazurin is reduced into resorufin by 1-MPMSH(2), the formed resorufin (lambda(ex)/lambda(em): 540 nm/580 nm) is used for quantifying the concentration of SBAs. Optimized conditions being suitable with the biosensor are as follows: the concentrations of BSS and beta-HSD used for the immobilization all are 5 kUL(-1); the concentrations of 1-MPMS and resazurin all are 25 micromolL(-1); the concentrations of Tris-HCl buffer and NAD(+) are 100 and 400 micromolL(-1), respectively; total volume of the enzyme, reagent and sample is only 18 microL per time for determining; the reaction temperature is 37 degrees C; the reaction time is 15min. The concentration of SBAs is directly proportional to the fluorescence intensity of the biosensor measured from 0.5 to 5.0 micromolL(-1). The relative standard deviation is less than 3.4%, and the detection limit was 0.16 micromolL(-1). The recoveries are in the range 95.5-106%. This SBA-IE-FCBS can be used for quantifying SBAs in urine to diagnose and judge hepatobiliary diseases, etc.