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摘要:INTRODUCTION Endotoxin,a liposaccharide,is one of the analytes that requires a highly sensitive detection for medical safety. The Limulus amebocyte lysate(LAL) assay is the most commonly used method for endotoxin detection utilizing a cascade reaction of three zymogens induced by endotoxin. In conventional LAL assay,the activation of the proclotting enzyme at the last step of LAL reaction induced by endotoxin is detected by optical methods. We have developed electrochemical LAL assay methods for highly sensitive and easy-to-use endotoxin sensor using p-aminophenol(pAP)-conjugated peptide(Boc-Leu-Gly-Arg-pAP:LGR-pAP) with a small chip device1,2. In the present study,we developed a nanogap electrode device for highly sensitive endotoxin detection by amplifying the current signal by redox cycling(Fig. 1). METHODS The nanogap electrode device was fabricated using photolithography and sputtering. Ti/Pt bottom ring electrode(Fig. 2 a),a 190 nm thick Cr sacrificial layer(Fig. 2 b),and Pt top ring electrode(Fig. 2 c) was deposited onto a glass slide with lift-off process. An insulation layer was fabricated on the device except for sensor area with SU-8 negative photoresist(Fig. 2 d). After removal of Cr sacrificial layer by wet etching process,a device with a pair of ring electrodes facing each other across a 190 nm gap was obtained(Figs. 2 e and f). Characterization of the device was performed with cyclic voltammetry(CV) using 500 μM ferrocenemethanol(FMA) dissolved in 0.1 M KCl introduced into the device. The potential of the top electrode was swept at a scan rate of 20 mV/s while the bottom electrode was biased at 0 V. We also simulated the CV of non-redox cycling condition using COMSOL Multiphysics with the device model without bottom electrode. Finally,we performed endotoxin assay using 0.5 mM LGR-pAP(Watanabe Chemical Industries) as a substrate of commercially available LAL reagent(Endospecy,Seikagaku Corp). After 60 or 30 min LAL reaction,the solution was introduced to the nanogap electrode device and then current of the bottom electrode biased at-0.2 V was monitored while the potential applied to the top electrode was stepped from-0.2 to 0.5 V at 10 s. RESULTS From the results of CV of FMA with redox-cycling condition,the cycling efficiency(current from the bottom electrode/current from the top electrode) of the fabricated device was revealed to be 98%. This result indicates almost all of oxidant generated at the top electrode reached the bottom electrode. Comparing the CV with redox-cycling condition and non-redox cycling condition,the current was amplified by 1660 times with redox cycling. These results show our nanogap electrode device can be used for a highly sensitive sensor platform. From the amperograms of endotoxin assay,the detection limit was 0.2 and 0.5 EU/L with 60 and 30 min of LAL reaction time,respectively. These results is more sensitive and rapid than conventional method. CONCLUSIONS In this study,we developed a highly sensitive
会议名称:

中国化学会第十二届全国微全分析系统学术会议、第七届全国微纳尺度生物分离分析学术会议、第七届国际微流控学学术论坛、The 11th International Symposium on Microchemistry and Microsystems

会议时间:

2019-05-17

会议地点:

中国陕西西安

  • 专辑:

    工程科技Ⅰ辑; 信息科技

  • 专题:

    材料科学; 自动化技术

  • DOI:

    10.26914/c.cnkihy.2019.085661

  • 分类号:

    TP212;TB383.1

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