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
L-乳酸（l-lactate/L-lactic acid），样品基质为磷酸盐缓冲液（PBS, pH 7.0）溶液
检测原理
该传感器以LOx为识别元件，L-乳酸为底物。LOx催化L-乳酸氧化为丙酮酸，同时酶被还原；溶液中的HMF氧化态接受电子，使LOx再生并生成HMF还原态。HMF还原态在3DOM金电极表面发生氧化，电子进入金膜并传递至三电极体系，产生阳极催化电流。3DOM金的高比表面积增加LOx负载量，三维互连金属网络降低电子传递阻力，DTSP共价固定提高酶层稳定性。随着L-乳酸浓度升高，催化电流增大，响应呈Michaelis–Menten型，低浓度范围内近似线性，因此可用阳极峰电流定量乳酸。
检测灵敏度
Au3DOM-DTSP-LOx: LOD: 3.93 μM；线性范围: 最高1.3 mM；灵敏度: 1.63 μA mM−1；KM: 1.02 mM；IMAX: 150.0 μA cm−2；定量限: 13.08 μM
效应效果
该传感器对酒石酸、柠檬酸、葡萄糖和果糖无明显干扰，仅抗坏血酸产生干扰。重复测量RSD为0.3%，不同电极重现性RSD为3.5%；30天稳定性保留100%，优于无DTSP的89%和粗糙金DTSP传感器的85%。与粗糙金DTSP-LOx相比，LOD由21.50 μM降至3.93 μM，定量限由71.81 μM降至13.08 μM，灵敏度由1.49 μA mM−1提高至1.63 μA mM−1。作者认为3DOM金电极的高比表面积、良好生物相容微环境和导电网络可提高酶负载、电子传递和稳定性，是通用生物分析平台，适用于医学和食品工业乳酸检测。
传感器的构成
- 基底/换能器电极：玻璃载片上热蒸镀10 nm Cr粘附层和200 nm Au，形成Au/Cr/glass工作电极基底
- 大孔金修饰层：以500 nm聚苯乙烯（PS）胶体球为模板电化学沉积金（ECF 60镀金液，10 g/L Au），去除PS后形成3DOM金膜（Au3DOM），提供高比表面积和导电网络
- 功能化自组装单层：DTSP（3,3'-dithiodipropionic acid di(N-succinimidyl ester)）自组装单层，提供琥珀酰亚胺酯活性基团用于酶共价固定
- 识别元件：乳酸氧化酶（LOx），催化L-乳酸氧化为丙酮酸
- 信号介质：羟甲基环戊二烯铁（HMF，hydroxymethylferrocene）在溶液中作为氧化还原介质传递电子
- 检测读出：三电极体系，循环伏安（CV）和电化学阻抗谱（EIS）读取阳极催化电流
中文摘要
本文报道了一种基于三维有序大孔金（3DOM）金膜修饰电极的通用生物分析平台，并以乳酸氧化酶（LOx）为模型构建乳酸生物传感器。该电极采用倒置opal模板法电化学制备，使3DOM金电极的比表面积最高可达裸平金电极的18倍。作者利用场发射扫描电子显微镜（FE-SEM）、原子力显微镜（AFM）和X射线衍射（XRD）对新换能器进行形貌与结构表征。生物传感器通过在DTSP自组装单层修饰的3DOM金电极上固定LOx制备，并用电化学阻抗谱（EIS）表征各修饰步骤。3DOM金电极不仅提供良好生物相容微环境，还提高导电性和稳定性，因此所得LOx生物分析平台表现出比多晶金换能器更高的介导生物电催化活性。在溶液中加入羟甲基环戊二烯铁（HMF）作为氧化还原介质时，可获得对不同乳酸浓度的响应；与无DTSP相比，DTSP共价结合酶的响应得到改善。
英文摘要
A general bioanalytical platform for biosensor applications was developed based on three-dimensional ordered macroporous (3DOM) gold film modified electrodes using lactate oxidase (LOx) as a case study, within the framework of developing approaches of broad applicability. The electrode was electrochemically fabricated with an inverted opal template, making the surface area of the 3DOM gold electrode up to 18 times higher than that of bare flat gold electrodes. These new electrochemical transducers were characterized by using Field Emission Scanning Electron Microscopy (FE-SEM), Atomic Force Microscopy (AFM) and the X-ray diffraction (XRD). The biosensor was developed by immobilization of lactate oxidase (LOx), on a 3DOM gold electrode modified with a self-assembled monolayer of dithiobis-N-succinimidyl propionate (DTSP). The resulting lactate oxidase biosensor was characterized by electrochemical impedance spectroscopy (EIS). The 3DOM gold electrode not only provides a good biocompatible microenvironment but also promotes the increase of conductivity and stability. Thus, the developed lactate oxidase bioanalytical platforms showed higher mediated bioelectrocatalytic activity compared to others previously described based on polycrystalline gold transducers. The response to varying lactate concentrations has been obtained in the presence of hydroxymethylferrocene as redox mediator in solution. Under these conditions, the bioanalytical platform response for DTSP covalently bound enzyme was improved with respect to that obtained in absence of DTSP.