Electrochemical microfluidic biosensor for the detection of nucleic acid sequences
Electrochemical microfluidic biosensor for the detection of nucleic acid sequences
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DOI：
10.1039/b513239h
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发表时间：
2006-03-01
期刊：
影响因子：
6.1
通讯作者：
Baeumner, AJ
中科院分区：
文献类型：
--
作者：
Goral, VN;Zaytseva, NV;Baeumner, AJ
开发了一种具有电化学检测功能的微流体生物传感器，用于核酸序列的定量。与大多数基于荧光产生信号的微生物传感器相比，它利用了电流分析和比色检测系统提供的简单性和高灵敏度。在玻璃芯片中制造叉指超微电极阵列（IDUA），并直接与聚（二甲基硅氧烷）（PDMS）制成的微通道集成。该组件被封装在 Plexiglas(R) 外壳中，提供流体和电气连接。 IDUA 通过电流分析和循环伏安法对可逆氧化还原对 - 六氰基铁酸钾 (II)/六氰基铁酸 (III)（铁/亚铁氰化物）的存在的静态和动态响应进行表征。 0.5 μM 的铁/铁氰化物组合浓度被确定为动态范围为 5 个数量级的检测下限。背景信号可以忽略不计，并且 IDUA 以高度可逆的方式对不同体积和不同浓度的电化学标记物的注射做出反应。为了检测核酸序列，用DNA探针标记包埋电化学标记物的脂质体，并用第二个DNA探针包被超顺磁珠。单链 DNA 靶序列与两个探针杂交。夹层结构通过位于外壳中的磁铁捕获在 IDUA 上游的微流体通道中。使用去污剂裂解脂质体，并在通过 IDUA 时对释放的亚铁/铁氰化物的量进行定量。研究了磁体相对于 IDUA 的最佳位置，研究了硫酸葡聚糖对杂交反应的影响，并优化了测定中使用的磁珠量。使用不同浓度的靶 DNA 分子绘制的剂量响应曲线显示了 1 fmol 测定 (-1) 的检测限以及 1 至 50 fmol 之间的动态范围。整个测定需要 6 分钟才能完成，再加上 15 - 20 分钟的预孵育，并且只需要一个简单的恒电位仪来记录和解释信号。
A microfluidic biosensor with electrochemical detection for the quantification of nucleic acid sequences was developed. In contrast to most microbiosensors that are based on fluorescence for signal generation, it takes advantage of the simplicity and high sensitivity provided by an amperometric and coulorimetric detection system. An interdigitated ultramicroelectrode array (IDUA) was fabricated in a glass chip and integrated directly with microchannels made of poly( dimethylsiloxane) ( PDMS). The assembly was packaged into a Plexiglas(R) housing providing fluid and electrical connections. IDUAs were characterized amperometrically and using cyclic voltammetry with respect to static and dynamic responses for the presence of a reversible redox couple - potassium hexacyanoferrate (II)/hexacyanoferrate(III) (ferri/ferrocyanide). A combined concentration of 0.5 mu M of ferro/ferricyanide was determined as lower limit of detection with a dynamic range of 5 orders of magnitude. Background signals were negligible and the IDUA responded in a highly reversible manner to the injection of various volumes and various concentrations of the electrochemical marker. For the detection of nucleic acid sequences, liposomes entrapping the electrochemical marker were tagged with a DNA probe, and superparamagnetic beads were coated with a second DNA probe. A single stranded DNA target sequence hybridized with both probes. The sandwich was captured in the microfluidic channel just upstream of the IDUA via a magnet located in the outside housing. Liposomes were lysed using a detergent and the amount of released ferro/ferricyanide was quantified while passing by the IDUA. Optimal location of the magnet with respect to the IDUA was investigated, the effect of dextran sulfate on the hybridization reaction was studied and the amount of magnetic beads used in the assay was optimized. A dose response curve using varying concentrations of target DNA molecules was carried out demonstrating a limit of detection at 1 fmol assay(-1) and a dynamic range between 1 and 50 fmol. The overall assay took 6 min to complete, plus 15 - 20 min of preincubation and required only a simple potentiostat for signal recording and interpretation.