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Ternary surface monolayers for ultrasensitive (zeptomole) amperometric detection of nucleic acid hybridization without signal amplification.

ABSTRACT: A ternary surface monolayer, consisting of coassembled thiolated capture probe, mercaptohexanol and dithiothreitol, is shown to offer dramatic improvements in the signal-to-noise characteristics of electrochemical DNA hybridization biosensors based on common self-assembled monolayers. Remarkably low detection limits down to 40 zmol (in 4 ?L samples) as well as only 1 CFU Escherichia coli per sensor are thus obtained without any additional amplification step in connection to the commonly used horseradish peroxidase/3,3',5,5'-tetramethylbenzidine system. Such dramatic improvements in the detection limits (compared to those of common binary alkanethiol interfaces and to those of most electrochemical DNA sensing strategies without target or signal amplification) are attributed primarily to the remarkably higher resistance to nonspecific adsorption. This reflects the highly compact layer (with lower pinhole density) produced by the coupling of the cyclic- and linear-configuration "backfillers" that leads to a remarkably low background noise even in the presence of complex sample matrixes. A wide range of surface compositions have been investigated, and the ternary mixed monolayer has been systematically optimized. Detailed impedance spectroscopy and cyclic voltammetric studies shed useful insights into the surface coverage. The impressive sensitivity and high specificity of the simple developed methodology indicate great promise for a wide range of nucleic acid testing, including clinical diagnostics, biothreat detection, food safety, and forensic analysis.

SUBMITTER: Wu J

PROVIDER: S-EPMC3038188 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Ternary surface monolayers for ultrasensitive (zeptomole) amperometric detection of nucleic acid hybridization without signal amplification.

Wu Jie J Campuzano Susana S Halford Colin C Haake David A DA Wang Joseph J

Analytical chemistry 20100930 21

A ternary surface monolayer, consisting of coassembled thiolated capture probe, mercaptohexanol and dithiothreitol, is shown to offer dramatic improvements in the signal-to-noise characteristics of electrochemical DNA hybridization biosensors based on common self-assembled monolayers. Remarkably low detection limits down to 40 zmol (in 4 μL samples) as well as only 1 CFU Escherichia coli per sensor are thus obtained without any additional amplification step in connection to the commonly used hor ...[more]

PMID: 20883023

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Project description:Biomolecular self-assembly has spurred substantial research efforts for the development of low-cost point-of-care diagnostics. Herein, we introduce an isothermal enzyme-free concatenated hybridization chain reaction (C-HCR), in which the output of the upstream hybridization chain reaction (HCR-1) layer acts as an intermediate input to activate the downstream hybridization chain reaction (HCR-2) layer. The initiator motivates HCR-1 through the autonomous cross-opening of two functional DNA hairpins, yielding polymeric dsDNA nanowires composed of numerous tandem triggers T as output of the primary sensing event. The reconstituted amplicon T then initiates HCR-2 and transduces the analyte recognition into an amplified readout, originating from the synergistic effect between HCR-1 and HCR-2 layers. Native gel electrophoresis, atom force microscopy (AFM) and fluorescence spectra revealed that C-HCR mediated the formation of frond-like branched dsDNA nanowires and the generation of an amplified FRET signal. As a versatile and robust amplification strategy, the unpreceded C-HCR can discriminate DNA analyte from its mutants with high accuracy and specificity. By incorporating an auxiliary sensing module, the integrated C-HCR amplifier was further adapted for highly sensitive and selective detection of microRNA (miRNA), as a result of the hierarchical and sequential hybridization chain reactions, in human serum and even living cells through an easy-to-integrate "plug-and-play" procedure. In addition, the C-HCR amplifier was successfully implemented for intracellular miRNA imaging by acquiring an accurate and precise signal localization inside living cells, which was especially suitable for the ex situ and in situ amplified detection of trace amounts of analyte. The C-HCR amplification provides a comprehensive and smart toolbox for highly sensitive detection of various biomarkers and thus should hold great promise in clinical diagnosis and assessment. The infinite layer of multilayered C-HCR is anticipated to further strengthen the amplification capacity and reliability (anti-invasion performance) of intracellular imaging approach, which is of great significance for its bioanalytical applications.

Dataset Information

Ternary surface monolayers for ultrasensitive (zeptomole) amperometric detection of nucleic acid hybridization without signal amplification.

Publications

Ternary surface monolayers for ultrasensitive (zeptomole) amperometric detection of nucleic acid hybridization without signal amplification.

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