A Spectrometric Setup for Synchronous Total Internal Reflection Fluorescence Measurement at the Solid/Liquid Interface

A spectrometric setup to perform total internal reflection fluorescence (TIRF) and synchronous TIRF measurements at solid/liquid interfaces is presented. The combination of TIRF and synchronous fluorescence was proposed to analyze simultaneously different components at interfaces. The TIRF excitation, emission and synchronous spectra of a watersoluble porphyrin were obtained from water/glass interface using this setup without the existence of a surfactant.

One-step detection of T4 polynucleotide kinase activity based on single particle-confined enzyme reaction and digital particle counting

T4 polynucleotide kinase(T4 PNK) is a pivotal enzyme for DNA replication, recombination, and DNA damage repair. Herein, a robust single particle counting-based assay has been developed for the high-sensitive determination of T4 PNK activity through only a simple one-step reaction. Taking benefit of the exceptional space-confined enzymatic property of T4 PNK towards DNA substrates on a single nanoparticle,the T4 PNK activity can be precisely determined by counting the fluorescence-positive nanoparticles in a digital manner with a total internal reflection fluorescent microscope(TIRFM). Due to the featured spatial-confined enzymatic property of T4 PNK and the single particle counting-based signal readout, T4PNK can be effectively differentiated from other interfering enzymes. This facile strategy has been also successfully applied to screen T4 PNK inhibitor and accurately determine T4 PNK activity in complex biological samples, paving a potential avenue for the digital analysis of biomarkers.

Using single-molecule approach to visualize the nucleosome assembly in yeast nucleoplasmic extracts

In eukaryotic cells,the smallest subunit of chromatin is the nucleosome,which consists of a segment of DNA wound on histone protein cores. Despite many years of effort,the process of nucleosome assembly and disassembly is still not very clear. Here,we present a convenient method to investigate the process of nucleosome assembly at the single molecule level. We invented a novel system derived from the yeast nucleoplasmic extracts(YNPE),and demonstrated that the YNPE supports the nucleosome assembly under physiological condition. By combining the total internal reflection fluorescence microscopy with microfluidic flow-cell technique,the dynamic process of nucleosome assembly in YNPE was visualized at single-molecule level. Our system provides a novel in vitro single-molecule tool to investigate the dynamics of nucleosome assembly under physiological conditions.

Target Extension-Activated DNA Walker on Nanoparticles for Digital Counting-Based Analysis of MicroRNA

MicroRNAs(miRNAs),especially exosomal miRNAs,are promising noninvasive biomarkers in early-stage cancer diagnosis and disease treatment monitoring.However,their precise and sensitive quantification remains challenging due to their small size and low abundance.Herein,we have developed a nanoparticle-confined DNA walker strategy for the specific detection of miRNA.In the existence of the target miRNA;the on-particle DNA walking reaction will be initiated,providing a fluorescence-positive nanoparticle.Otherwise,the nanoparticle would be fluorescence-negative.Utilizing the total internal reflection fluorescent microscope(TIRFM)to digitally count the fluorescence-positive nanoparticles,the proposed method possesses a detection limit of 0.2 pmol/L miRNA and can accurately distinguish the single-base mismatched target.This design combines the merits of the DNA walker for signal amplification and the TIRFM for highly sensitive detection,paving a new way for the digital counting-based analysis of exosomal miRNAs.