The Soft Nanodots as Fluorescent Probes for Cell Imaging: Analysis of Cell and Spheroid Penetration Behavior of Single Chain Polymer Dots.

This study describes the formation, size control, and penetration behavior of polymer nanodots (Pdots) consisting of single or few chain polythiophene-based conjugated polyelectrolytes (CPEs) via nanophase separation between good solvent and poor solvent of CPE. Though the chain singularity may be associated with dilution nanophase separation suggests that molecules of a good solvent create a thermodynamically driven solvation layer surrounding the CPEs and thereby separating the single chains even in their poor solvents. This statement is therefore corroborated with emission intensity/lifetime, particle size, and scattering intensity of polyelectrolyte in good and poor solvents. Regarding the augmented features, Pdots are implemented into cell imaging studies to understand the nuclear penetration and to differentiate the invasive characteristics of breast cancer cells. The python based red, green, blue (RGB) color analysis   depicts that Pdots have more nuclear penetration ability in triple negative breast cancer cells due to the different nuclear morphology in shape and composition and Pdots have penetrated cell membrane as well as extracellular matrix in spheroid models. The current Pdot protocol and its utilization in cancer cell imaging are holding great promise for gene/drug delivery to target cancer cells by explicitly achieving the very first priority of nuclear intake.

PCR-Free Methodology for Detection of Single-Nucleotide Polymorphism with a Cationic Polythiophene Reporter.

This study presents a nonamplification-based nucleic acid assay for the detection of single-nucleotide polymorphism (SNP) associated with familial Mediterranean fever (FMF) besides polymerase chain reaction (PCR)-based methodologies. The major objective is to show the potential of the proposed assay for rapid screening of FMF in a Mediterranean region of 400 million population. The assay relies on binding difference of specially designed wild and mutant primers to the target genomic DNA, followed by determination of unbound primers by quick titration of a cationic polythiophene reporter. The fluorescent reporter exhibits signal transition from 525 to 580 nm in the presence of unbound primers, and it correlates the binding affinity of label-free primers to the homozygous wild and mutant genomes. As a proof of concept, 26 real samples are studied relying on the ON and OFF fluorescence signals of the cationic polythiophene reporter. The results are analyzed by principal component analysis (PCA), which provides clear separation of healthy and patient individuals. The further analysis by support vector machine (SVM) classification has revealed that our assay converges to 96% overall accuracy. These results support that the PCR-free nucleic acid assay has a significant potential for rapid and cost-effective screening of familial Mediterranean fever.

Pixelated colorimetric nucleic acid assay.

Conjugated polyelectrolytes (CPEs) have been widely used as reporters in colorimetric assays targeting nucleic acids. CPEs provide naked eye detection possibility by their superior optical properties however, as concentration of target analytes decrease, trace amounts of nucleic acid typically yield colorimetric responses that are not readily perceivable by naked eye. Herein, we report a pixelated analysis approach for correlating colorimetric responses of CPE with nucleic acid concentrations down to 1 nM, in plasma samples, utilizing a smart phone with an algorithm that can perform analytical testing and data processing. The detection strategy employed relies on conformational transitions between single stranded nucleic acid-cationic CPE duplexes and double stranded nucleic acid-CPE triplexes that yield distinct colorimetric responses for enabling naked eye detection of nucleic acids. Cationic poly[N,N,N-triethyl-3-((4-methylthiophen-3-yl)oxy)propan-1-aminium bromide] is utilized as the CPE reporter deposited on a polyvinylidene fluoride (PVDF) membrane for nucleic acid assay. A smart phone application is developed to capture and digitize the colorimetric response of the individual pixels of the digital images of CPE on the PVDF membrane, followed by an analysis using the algorithm. The proposed pixelated approach enables precise quantification of nucleic acid assay concentrations, thereby eliminating the margin of error involved in conventional methodologies adopted for interpretation of colorimetric responses, for instance, RGB analysis. The obtained results illustrate that a ubiquitous smart phone could be utilized for point of care colorimetric nucleic acids assays in complex matrices without requiring sophisticated software or instrumentation.

Single Chain Cationic Polymer Dot as a Fluorescent Probe for Cell Imaging and Selective Determination of Hepatocellular Carcinoma Cells.

This letter describes formation of single chain cationic polymer dots (Pdots) made of poly[1,4-dimethyl-1-(3-((2,4,5-trimethylthiophen-3-yl)oxy)propyl)piperazin-1-ium bromide] conjugated polyelectrolyte (CPE). The single chain Pdot formation relies on a simple process which is a rapid nanophase separation between CPE solution of ethylene glycol and water. Pdots show narrow monodisperse size distribution with a 3.6 nm in diameter exhibiting high brightness and excellent colloidal and optical stability. It has been demonstrated that photoluminescent Pdots provide selective nuclear translocation to hepatocellular carcinoma cells as compared to healthy liver cells. The Pdot labeling effectively discriminates cancer cells in the coculture media. Pdots hold great promise as a luminescent probe to diagnose cancer cells in histology and may guide surgeons during operations to precisely separate out cancerous tissue due to augmented fluorescence brightness.

Hand-Held Volatilome Analyzer Based on Elastically Deformable Nanofibers.

This study reports on a hand-held volatilome analyzer for selective determination of clinically relevant biomarkers in exhaled breath. The sensing platform is based on electrospun polymer nanofiber-multiwalled carbon nanotube (MWCNT) sensing microchannels. Polymer nanofibers of poly(vinylidene fluoride) (PVDF), polystyrene (PS), and poly(methyl methacrylate) (PMMA) incorporated with MWCNT exhibits a stable response to interferences of humidity and CO2 and provides selective deformations upon exposure of exhaled breath target volatilomes acetone and toluene, exhibiting correlation to diabetes and lung cancer, respectively. The sensing microchannels "P1" (PVDF-MWCNT), "P2" (PS-MWCNT), and "P3" (PMMA-MWCNT) are integrated with a microfluidic cartridge (µ-card) that facilitates collection and concentration of exhaled breath. The volatilome analyzer consists of a conductivity monitoring unit, signal conditioning circuitries and a low energy display module. A combinatorial operation algorithm was developed for analyzing normalized resistivity changes of the sensing microchannels upon exposure to breath in the concentration ranges between 35 ppb and 3.0 ppm for acetone and 1 ppb and 10 ppm for toluene. Subsequently, responses of volatilomes from individuals in the different risk groups of diabetes were evaluated for validation of the proposed methodology. We foresee that proposed methodology provides an avenue for rapid detection of volatilomes thereby enabling point of care diagnosis in high-risk group individuals.