Fluorescent Conjugated Polyelectrolyte as an Indicator for Convenient Detection of DNA Methylation

Abstract

A convenient, sensitive, and label-free method to determine the DNA methylation status of CpG sites of plasmid and human colon cancer cell has been developed. The system relies on highly selective single base extension reaction and significant optical amplification of cationic conjugated polyelectrolytes (CCP-1). The higher fluorescence resonance energy transfer efficiency between CCP-1 and fluorescein-labeled dGTP (dGTP-Fl) is correlated to the incorporation of dGTP-Fl into the probe DNA by single base extension reaction when the target/probe pair is complementary at the methylation site. As low as 1% methylation status can be determined by this new assay method. Because of the optical amplification property of CCP-1, the method exhibited high sensitivity with a concentration of analyte DNA at the picomolar level. The CCP-1 can form a complex with negatively charged DNA through electrostatic interactions, avoiding labeling the DNA target and probe by covalent linking. The isolation steps employed in other typical assays were avoided to simplify operations and increase repeatability. These features make the system promising for future use for early cancer diagnosis.

Bimolecular Excited-State Electron Transfer with Surprisingly Long-Lived Radical Ions

“…6 By virtue of their light-harvesting properties, CPEs have also been utilized as photosensitizers in solar cells [7][8] and fluorescent sensors. [9][10][11] The basis of the successful utilization of CPEs in optoelectronic applications is their semiconducting properties, including their large optical densities and high emission intensities for fluorescence resonance energy transfer. 12 In particular, key features of CPEs for solar cell applications are their flexibility and their simple, large-scale, and low-cost fabrication.…”

eMethylsorb: rapid quantification of DNA methylation in cancer cells on screen-printed gold electrodes

“…While many assays have been developed for the early detection of DNA methylation [6][7][8][9][10][11][12][13] on bisulte treated samples, the majority of these methylation detection techniques usually involve complex surface chemistries, chemical labels, long experimental time or relatively tedious experimental procedures. These limitations restrict their use to research settings rather than for clinical usage.…”

Fluorescence‐Amplifying Assay for Irradiated DNA Lesions Using Water‐Soluble Conjugated Polymers

“…Upon adding the CCP, the strong electrostatic interactions between the DNA duplex and CCP keep them in close proximity, which allows for efficient FRET from CCP to fluorescein. [24][25][26] If the DNA duplex is irradiated under UV light to induce the formation of CPD and PPD lesions (see Scheme 1b), the primer extension is blocked and the dUTP-Fl remains in the solution. In this case, the relatively weak electrostatic interactions between fluorescein and CCP leave the fluorescein far removed from the CCP and an inefficient FRET between them is observed.…”

“…[17][18][19][20][21][22][23][24][25][26] The signal amplification property of CPs by a collective optical response imparts high sensitivity to the sensor and, therefore, offers a key advantage over sensors based on small molecules. [27,28] In this paper, we develop a new homogeneous platform for DNA lesion detection that interfaces a single base extension (SBE) reaction [24,25] with the light harvesting property of CPs without requiring any isolation and washing steps. The DNA consumption in our new assay is greatly reduced as compared to the UV methodology, and the total treatment procedures are completed within just 30 min to one hour.…”