The mutation detections of KRAS and BRAF genes are of significant importance to predict the responses to anti-cancer therapy and develop new drugs. In this paper, we developed a multi-step fluorescence resonance energy transfer (FRET) assay for multiplex detection of KRAS and BRAF mutations using cationic conjugated polymers (CCP). The newly established detection system could detect as low as 2% mutant DNAs in DNA admixtures. By triggering the emission intensity change of CCP and the dyes labeled in the DNA, four possible statuses (three mutations and one wildtype) can be differentiated in one extension reaction. The detection efficiency of this new method in clinical molecular diagnosis was validated by determining KRAS and BRAF mutations of 51 formalin-fixed paraffin-embedded (FFPE) ovary tissue samples. Furthermore, the result of the CCP-based multi-step FRET assay can be directly visualized under UV light so that no expensive instruments and technical expertise are needed. Thus, the assay provides a sensitive, reliable, cost-effective and simple method for the detection of disease-related gene mutations.

A visual colorimetric method for detecting multiplex DNA mutations has been developed using multicolor fluorescent coding based on multistep FRET of cationic conjugated polymers. Expensive instruments and technical expertise are not required in this method. Also our visual system provides a quantitative detection by simply analyzing RGB values of images. Genomic DNAs extracted from 60 FFPE colon tissues can be sensitively determined by utilizing our visual assay with a high-throughput manner. Thus, it proves to be sensitive, reliable, cost-effective, simple, and high-throughput for mutation detection.Keywords: conjugated polymers; DNA mutations; FRET; multicolor fluorescent coding; sensors;

A new water-soluble conjugated polymer containing fluorene and boron-dipyrromethene repeat units in the backbones (PBF) that exhibits red emission was synthesized and characterized. Cationic PBF forms uniform nanoparticles with negatively charged disodium salt 3,3′-dithiodipropionic acid (SDPA) in aqueous solution through electrostatic interactions. The nanoparticles display absorption maximum at 550 nm and emission maximum at 590 nm. Upon photoexcitation with white light (400–800 nm) with 90 and 45 mW·cm–2 for bacteria and cancer cells killing respectively, PBF nanoparticles can sensitize the oxygen molecule to readily produce reactive oxygen species (ROS) for rapidly killing neighboring bacteria and cancer cells. Furthermore, PBF nanoparticles concurrently provide optical imaging capability. PBF nanoparticles are therefore a promising multifunctional material for treating cancers and bacteria infections, while concurrently providing optical monitoring capabilities.

Pathogen infections caused by Candida albicans (C. albicans) and Escherichia coli (E. coli) have led to extremely serious consequences. Cultures of blood samples, which are regarded as the diagnosis standard, often exhibit variable sensitivity and always take several days to detect certain pathogens. Here we demonstrate for the first time that a blend of two cationic conjugated polymers, between which fluorescence resonance energy transfer (FRET) can be induced, is used to detect and discriminate C. albicans and E. coli in a rapid, simple and visual way without the need for any complicated instrumentation. Coupled with general polymerase chain reaction (PCR) technology, the method can detect pathogens specifically independent of electrophoresis analyses and real-time PCR, which avoids utilizing detrimental nucleic dyes and expensive fluorophore-labeled primers. Our new cationic conjugated polymer blend has two unique features. Firstly, the assay is simple and rapid. Upon mixing fungi or bacteria with the polymer blend, one can trigger the signal response directly in solution using a fluorimeter, which may have a far-reaching impact on clinical diagnosis. Secondly, the most important characteristic of the assay is direct visualization under UV light, which makes it more convenient than other methods that require sophisticated instruments. The cationic conjugated polymer blend exhibits great potential as an ideal diagnostic agent for the identification of pathogens.

The L858R mutation of epidermal growth factor receptor (EGFR) in nonsmall cell lung cancer is associated with the increased sensitivity to EGFR tyrosine kinase inhibitors. In this paper, a simple and sensitive method for identification of L858R mutation in cell lines and tumor tissues was developed using cationic conjugated polymer-based fluorescence resonance energy transfer technology (CCP-based FRET). The new detection system can detect even as low as 4–8% mutation of the total DNA. Through the detection results for 48 DNA samples from tumor tissues, a sensitivity of 95.24% (20/21) and a specificity of 96.30% (26/27) were demonstrated. Further, the application of this method in clinical molecular diagnosis was validated by detecting T790 M in EGFR of 35 patients. In comparison with DNA sequencing and real-time PCR methods, our new protocol simplifies procedures by eliminating the need for primer labeling, cumbersome workups and sophisticated instruments and improves sensitivity by amplifying fluorescence signals. Our CCP-based FRET technology is particularly attractive because of its higher sensitivity, cost-effective, and simple characteristics. Particularly, this new method could confirm the suspected positive samples arisen by DNA sequencing and real-time PCR methods. Thus, the CCP-based FRET technology opens up an avenue for clinical therapy by guiding medication to lung cancer patients responsive to anti-EGFR therapy.Keywords: conjugated polymers; DNA mutation; EGFR; fluorescent sensor; FRET; mutation detection;

A new positively charged fluorescent probe poly(p-phenylene vinylene) derivative (PPV-1) has been developed for the detection of an important biological process, apoptosis, in a simple and fluorescent label-free way.

The development of methods for DNA detection is of importance in disease diagnosis, gene-targeted drug discovery and molecular biology field. In this paper, we synthesize a new cationic water-soluble CP containing fluorene moiety and flexible ethylenic moiety in the backbone (PFV) for label-free DNA detection. The conformational freedom of PFV provides stronger interactions with double-stranded DNA (dsDNA) and optimizes the orientation of transition moments between PFV and ethidium bromide (EB) intercalated in dsDNA. The efficient FRET from PFV (donor) to EB (acceptor) intercalated in dsDNA is observed and the emission of EB is amplified by the good light-harvesting ability of conjugated polymers. The interactions between PFV and DNA can also be probed by measuring the FRET ratio between PFV and EB intercalated in DNA. In comparison to other DNA detection assays based on FRET and conjugated polymers, synthesis of dye-labeled DNA probe is avoided in our method, which significantly reduces the cost and the synthetic complexity. The PFV/dsDNA/EB system provides promising applications on DNA detection with a simply, fast and label-free manner.

A new positively charged fluorescent probe poly(p-phenylene vinylene) derivative (PPV-1) has been developed for the detection of an important biological process, apoptosis, in a simple and fluorescent label-free way.

Pathogen infections caused by Candida albicans (C. albicans) and Escherichia coli (E. coli) have led to extremely serious consequences. Cultures of blood samples, which are regarded as the diagnosis standard, often exhibit variable sensitivity and always take several days to detect certain pathogens. Here we demonstrate for the first time that a blend of two cationic conjugated polymers, between which fluorescence resonance energy transfer (FRET) can be induced, is used to detect and discriminate C. albicans and E. coli in a rapid, simple and visual way without the need for any complicated instrumentation. Coupled with general polymerase chain reaction (PCR) technology, the method can detect pathogens specifically independent of electrophoresis analyses and real-time PCR, which avoids utilizing detrimental nucleic dyes and expensive fluorophore-labeled primers. Our new cationic conjugated polymer blend has two unique features. Firstly, the assay is simple and rapid. Upon mixing fungi or bacteria with the polymer blend, one can trigger the signal response directly in solution using a fluorimeter, which may have a far-reaching impact on clinical diagnosis. Secondly, the most important characteristic of the assay is direct visualization under UV light, which makes it more convenient than other methods that require sophisticated instruments. The cationic conjugated polymer blend exhibits great potential as an ideal diagnostic agent for the identification of pathogens.