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Multicolor fluorescent biosensor for multiplexed detection of DNA.

ABSTRACT: Development of efficient methods for highly sensitive and rapid screening of specific oligonucleotide sequences is essential to the early diagnosis of serious diseases. In this work, an aggregated cationic perylene diimide (PDI) derivative was found to efficiently quench the fluorescence emission of a variety of anionic oligonucleotide-labeled fluorophores that emit at wavelengths from the visible to NIR region. This broad-spectrum quencher was then adopted to develop a multicolor biosensor via a label-free approach for multiplexed fluorescent detection of DNA. The aggregated perylene derivative exhibits a very high quenching efficiency on all ssDNA-labeled dyes associated with biosensor detection, having efficiency values of 98.3 ± 0.9%, 97 ± 1.1%, and 98.2 ± 0.6% for FAM, TAMRA, and Cy5, respectively. An exonuclease-assisted autocatalytic target recycling amplification was also integrated into the sensing system. High quenching efficiency combined with autocatalytic target recycling amplification afforded the biosensor with high sensitivity toward target DNA, resulting in a detection limit of 20 pM, which is about 50-fold lower than that of traditional unamplified homogeneous fluorescent assay methods. The quencher did not interfere with the catalytic activity of nuclease, and the biosensor could be manipulated in either preaddition or postaddition manner with similar sensitivity. Moreover, the proposed sensing system allows for simultaneous and multicolor analysis of several oligonucleotides in homogeneous solution, demonstrating its potential application in the rapid screening of multiple biotargets.

SUBMITTER: Hu R

PROVIDER: S-EPMC4033642 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Multicolor fluorescent biosensor for multiplexed detection of DNA.

Hu Rong R Liu Tao T Zhang Xiao-Bing XB Huan Shuang-Yan SY Wu Cuichen C Fu Ting T Tan Weihong W

Analytical chemistry 20140429 10

Development of efficient methods for highly sensitive and rapid screening of specific oligonucleotide sequences is essential to the early diagnosis of serious diseases. In this work, an aggregated cationic perylene diimide (PDI) derivative was found to efficiently quench the fluorescence emission of a variety of anionic oligonucleotide-labeled fluorophores that emit at wavelengths from the visible to NIR region. This broad-spectrum quencher was then adopted to develop a multicolor biosensor via ...[more]

PMID: 24731194

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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.

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Multicolor fluorescent biosensor for multiplexed detection of DNA.

Publications

Multicolor fluorescent biosensor for multiplexed detection of DNA.

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