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DNA Nanostructure-based Interfacial engineering for PCR-free ultrasensitive electrochemical analysis of microRNA.

ABSTRACT: MicroRNAs (miRNAs) have been identified as promising cancer biomarkers due to their stable presence in serum. As an alternative to PCR-based homogenous assays, surface-based electrochemical biosensors offer great opportunities for low-cost, point-of-care tests (POCTs) of disease-associated miRNAs. Nevertheless, the sensitivity of miRNA sensors is often limited by mass transport and crowding effects at the water-electrode interface. To address such challenges, we herein report a DNA nanostructure-based interfacial engineering approach to enhance binding recognition at the gold electrode surface and drastically improve the detection sensitivity. By employing this novel strategy, we can directly detect as few as attomolar (<1, 000 copies) miRNAs with high single-base discrimination ability. Given that this ultrasensitive electrochemical miRNA sensor (EMRS) is highly reproducible and essentially free of prior target labeling and PCR amplification, we also demonstrate its application by analyzing miRNA expression levels in clinical samples from esophageal squamous cell carcinoma (ESCC) patients.

SUBMITTER: Wen Y

PROVIDER: S-EPMC3499757 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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DNA Nanostructure-based Interfacial engineering for PCR-free ultrasensitive electrochemical analysis of microRNA.

Wen Yanli Y Pei Hao H Shen Ye Y Xi Junjie J Lin Meihua M Lu Na N Shen Xizhong X Li Jiong J Fan Chunhai C

Scientific reports 20121116

MicroRNAs (miRNAs) have been identified as promising cancer biomarkers due to their stable presence in serum. As an alternative to PCR-based homogenous assays, surface-based electrochemical biosensors offer great opportunities for low-cost, point-of-care tests (POCTs) of disease-associated miRNAs. Nevertheless, the sensitivity of miRNA sensors is often limited by mass transport and crowding effects at the water-electrode interface. To address such challenges, we herein report a DNA nanostructure ...[more]

PMID: 23162691

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Project description:BACKGROUND:DNA methylation detection is indispensable for the diagnosis and prognosis of various diseases including malignancies. Hence, it is crucial to develop a simple, sensitive, and specific detection strategy. METHODS:A novel fluorescent biosensor was developed based on a simple dual signal amplification strategy using functional dendritic DNA nanostructure and signal-enriching polystyrene microbeads in combination with ligase detection reaction (LDR). Dendritic DNA self-assembled from Y-DNA and X-DNA through enzyme-free DNA catalysis of a hairpin structure, which was prevented from unwinding at high temperature by adding psoralen. Then dendritic DNA polymer labeled with fluorescent dye Cy5 was ligated with reporter probe into a conjugate. Avidin-labeled polystyrene microbeads were specifically bound to biotin-labeled capture probe, and hybridized with target sequence and dendritic DNA. LDR was triggered by adding Taq ligase. When methylated cytosine existed, the capture probe and reporter probe labeled with fluorescent dye perfectly matched the target sequence, forming a stable duplex to generate a fluorescence signal. However, after bisulfite treatment, unmethylated cytosine was converted into uracil, resulting in a single base mismatch. No fluorescence signal was detected due to the absence of duplex. RESULTS:The obtained dendritic DNA polymer had a large volume. This method was time-saving and low-cost. Under the optimal experimental conditions using avidin-labeled polystyrene microbeads, the fluorescence signal was amplified more obviously, and DNA methylation was quantified ultrasensitively and selectively. The detection range of this sensor was 10-15 to 10-7 M, and the limit of detection reached as low as 0.4 fM. The constructed biosensor was also successfully used to analyze actual samples. CONCLUSION:This strategy has ultrasensitivity and high specificity for DNA methylation quantification, without requiring complex processes such as PCR and enzymatic digestion, which is thus of great value in tumor diagnosis and biomedical research.

Dataset Information

DNA Nanostructure-based Interfacial engineering for PCR-free ultrasensitive electrochemical analysis of microRNA.

Publications

DNA Nanostructure-based Interfacial engineering for PCR-free ultrasensitive electrochemical analysis of microRNA.

Similar Datasets

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