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
ATP（腺苷三磷酸，ATP）、r-凝血酶（r-thrombin）；样品基质：缓冲液、1%预处理人血浆（pretreated human plasma）
检测原理
该传感器采用无标记阻抗换能。Au/PCS-MBA/MCE界面带负电，排斥[Fe(CN)6]4-/3-，使电子转移电阻Ret较高。ATP与MBA中的ABA结合后，MBA从PCS上脱离，界面负电荷密度降低，对探针排斥减弱，Ret下降，呈signal-off响应；TBA串联在ABA上延长识别链，增强空间位阻与信号变化，提高灵敏度。r-凝血酶与TBA结合后，大体积蛋白复合物保留在电极表面，形成疏水阻断面层，阻碍电子转移，Ret上升，呈signal-on响应。EIS在5 mV、10 kHz–0.1 Hz下读取Ret，CV辅助验证。ATP检测后可用PCS再杂交再生；r-凝血酶检测后可用高浓度ATP置换MBA再杂交再生。
检测灵敏度
ATP: LOD: 0.01 µM (1 × 10-8 M)；线性范围: 0.01–100 µM；R^2 = 0.9946；检测范围可延伸至 10-4 M。r-thrombin: LOD: 0.01 nM (1 × 10-11 M)；线性范围: 0.01–76 nM；R^2 = 0.9983；检测范围: 1 × 10-11–1 × 10-7 M。
效应效果
选择性方面，0.1 mM GTP/CTP、100 nM BSA、20 nM α-凝血酶和20 nM γ-凝血酶均无明显EIS响应，错配适配体MAS也无响应。重现性方面，ATP平均RSD为0.01609，r-凝血酶平均RSD为0.03096。稳定性方面，传感器在纯水中4 °C保存15天后EIS信号与使用前一致。可再生性方面，界面可重复使用3次以上，超过3次后MBA难以被ATP完全置换，仅能识别而难以定量。实际样品中，在1%预处理人血浆里，r-凝血酶20–80 nM、ATP 20–120 µM有显著响应，但灵敏度低于缓冲液。作者认为其无标记、可再生、多功能，适合小分子与蛋白检测。
传感器的构成
- 基底/换能器电极：金电极（Au electrode，直径1.2 mm）作为工作电极，配合Ag/AgCl参比电极和Pt对电极，用于EIS/CV信号读出
- 自组装固定层：5'-巯基部分互补链（PCS，5′HS-(CH2)6-TACAGACCTTCC 3′）通过Au-S键自组装固定于Au表面，提供MBA杂交位点
- 识别元件：混合适配体链（MBA，5′ GGTTGGTGTGGTTGGACCTGGGGGAGTATTGCGGAGGAAGGTCTGTA 3′），内含ATP结合适配体（ABA）和r-凝血酶结合适配体（TBA），与PCS杂交后伸出识别区
- 封闭/规整层：2-巯基乙醇（MCE，10 mM Tris-HAc，pH 7.41）处理界面，使DNA阵列更规整并降低非特异吸附
- 氧化还原探针：[Fe(CN)6]4-/3-（5 mM K4[Fe(CN)6]/K3[Fe(CN)6]，67 mM PBS，pH 7.0）作为无标记EIS/CV电子转移指示剂
- 被测物：ATP（腺苷三磷酸）和r-凝血酶（r-thrombin），分别与ABA和TBA特异性结合
- 再生试剂：PCS再杂交用于ATP检测后再生；高浓度ATP置换MBA后与PCS再杂交用于r-凝血酶检测后再生
中文摘要
本文报道了一种基于整合适配体的多功能、可再生、无标记电化学生物传感器，用于平行检测腺苷三磷酸（ATP）和r-凝血酶（r-thrombin）。该传感器以金电极为传感表面，固定5'-巯基部分互补链（PCS），并与含ATP结合适配体（ABA）和r-凝血酶结合适配体（TBA）的混合适配体链（MBA）杂交形成部分双链，再用2-巯基乙醇（MCE）规整封闭。检测采用电化学阻抗谱（EIS）和循环伏安法（CV），以[Fe(CN)6]4-/3-为氧化还原探针。ATP结合ABA后使MBA脱离电极表面，降低界面负电荷，电子转移电阻下降；r-凝血酶结合TBA后在界面形成大体积蛋白层，阻碍电子转移，电阻上升。ATP检出限为1×10^-8 M，范围可至10^-4 M；r-凝血酶检出限为1×10^-11 M，范围为1×10^-11–10^-7 M。传感器可通过再杂交或高浓度ATP置换再生，并在1%预处理人血浆中表现出检测能力，具有无标记、高灵敏、可再生和多功能识别优势。
英文摘要
Aptamers, which are in vitro selected functional oligonucleotides, have been employed to design novel biosensors (i.e., aptasensors) due to their inherent selectivity, affinity, and their multifarious advantages over traditional recognition elements. In this work, we reported a multifunctional reusable label-free electrochemical biosensor based on an integrated aptamer for parallel detection of adenosine triphosphate (ATP) and alpha-thrombin, by using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). A Au electrode as the sensing surface was modified with a part DNA duplex which contained a 5'-thiolated partly complementary strand (PCS) and a mixed aptamer (MBA). The unimolecular MBA contained small-molecule ATP binding aptamer (ABA) and also protein alpha-thrombin binding aptamer (TBA). Thus, the aptasensor could be used for detection of ATP and alpha-thrombin both. The detection limit of ATP was 1 x 10(-8) M, and its detection range could extend up to 10(-4) M, whereas the detection limit of alpha-thrombin was 1 x 10(-11) M, and its detection range was from 1 x 10(-11) to 1 x 10(-7) M. Meanwhile, after detecting alpha-thrombin, the sensing interface could be used for ATP recognition as well. The aptasensor regeneration could be realized by rehybridizing of the MBA strand with the partly complementary strand immobilized on the Au surface after ATP detection or by treating with a large amount of ATP and then rehybridizing the MBA strand with the partly complementary strand immobilized on the Au surface after alpha-thrombin detection. The aptasensor fabricated exhibited several advantages such as label-free detection, high sensitivity, regeneration, and multifunctional recognition. It also showed the detectability in biological fluid. Therein it held promising potential for integration of the sensing ability such as the simultaneous detection for multianalysis in the future.