Quantifying the Degree of Aggregation from Fluorescent Dye-Conjugated DNA Probe by Single Molecule Photobleaching Technology for the Ultrasensitive Detection of Adenosine.

In this work, we demonstrated a single molecule photobleaching-based strategy for the ultrasensitive detection of adenosine. A modified split aptamer was designed to specifically recognize individual adenosine molecules in solution. The specific binding of dye-labeled short strand DNA probes onto the elongated aptamer strand in the presence of adenosine resulted in a concentration-dependent self-aggregation process. The degree-of-aggregation (DOA) of the short DNA probes on the elongated aptamer strand could then be accurately determined based on the single molecule photobleaching measurement. Through statistically analyzing the DOA under different target concentrations, a well-defined curvilinear relationship between the DOA and target molecule concentration (e.g., adenosine) was established. The limit-of-detection (LOD) is down to 44.5 pM, which is lower than those recently reported results with fluorescence-based analysis. Owing to the high sensitivity and excellent selectivity, the sensing strategy described herein would find broad applications in biomolecule analysis under complicated surroundings.

Background-Free Imaging of a Viral Capsid Proteins Coated Anisotropic Nanoparticle on a Living Cell Membrane with Dark-Field Optical Microscopy.

Exploring the diffusion dynamics of a viral capsid proteins (VCP)-functionalized nanocarrier on a living cell membrane could provide much kinetic information for the better understanding of their biological functionality. Gold nanoparticles are an excellent core material of nanocarriers because of the good biocompatibility as well as versatile surface chemistry. However, due to the strong scattering background from subcellular organelles, it is a grand challenge to selectively image an individual nanocarrier on a living cell membrane. In this work, we demonstrated a convenient strategy to effectively screen the scattering background from living cells for single-particle imaging with a polarization-resolved dual-channel imaging module. By taking advantage of the polarization of anisotropic gold nanoparticles (gold nanorods, GNRs), the signals from cell components could be counteracted after subtracting the sequential images one by one, while those transiently rotating GNRs on the cell membrane still exist in the processed image. In contrast to the previously reported methods, this method does not require a complicated optical setup alignment and sophisticated digital image analysis process. According to the single-particle imaging results, the majority of VCP-GNRs were anchoring on the cell membrane with confined diffusion. Interestingly, on further inspection of the diffusion trajectories, the particles displayed anomalous confined diffusion with randomly distributed large walking steps during the whole track. Non-Gaussian step distribution was noted, indicating heterogeneous binding and desorption processes on the cell membrane. As a consequence of the robust background screening capability, this approach would find broad applications for single-particle imaging under a noisy environment, e.g., living cells.