Ultrasensitive Electrochemical DNA Assay Based on Counting of Single Magnetic Nanobeads by a Combination of DNA Amplification and Enzyme Amplification

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• 作者：Xiaoli Zhang ; Linlin Li ; Lu Li ; Jia Chen ; Guizheng Zou ; Zhikun Si ; Wenrui Jin*
• 刊名：Analytical Chemistry
• 出版年：2009
• 出版时间：March 1, 2009
• 年：2009
• 卷：81
• 期：5
• 页码：1826-1832
• 全文大小：198K
• 年卷期：v.81,no.5(March 1, 2009)
• ISSN：1520-6882

文摘

An ultrasensitive electrochemical method for determination of DNA is developed based on counting of single magnetic nanobeads (MNBs) corresponding to single DNA sequences combined with a double amplification (DNA amplification and enzyme amplification). In this method, target DNA (t-DNA) is captured on a streptavidin-coated substrate via biotinylated capture DNA. Then, MNBs functionalized with first-probe DNAs (p1-DNA−MNBs) are conjugated to t-DNA sequences with a ratio of 1:1. Subsequently, the p1-DNA−MNBs are released from the substrate via dehybridization. The released p1-DNA−MNBs are labeled with alkaline phosphatase (AP) using biotinylated second-probe DNAs (p2-DNAs) and streptavidin−AP conjugates. The resultant AP−p2-DNA−p1-DNA−MNBs with enzyme substrate disodium phenyl phosphate (DPP) are continuously introduced through a capillary as the microsampler and microreactor at 40 °C. AP on the AP−p2-DNA−p1-DNA−MNBs converts a huge number of DPP into its product phenol, and phenol zones are produced around each moving AP−p2-DNA−p1-DNA−MNB. The phenol zones are continuously delivered to the capillary outlet and detected by a carbon fiber disk bundle electrode at 1.05 V. An elution curve with peaks is obtained. Each peak is corresponding to a phenol zone relative to single t-DNA sequence. The peaks on the elution curve are counted for quantification. The number of the peaks is proportional to the concentration of t-DNA in a range of 5.0 × 10⁻¹⁶ to 1.0 × 10⁻¹³ mol/L.