Bead-based Padlock Rolling Circle Amplification under Molecular Crowding Conditions: The Effects of Crowder Charge and Size

Abstract

Bead-based padlock rolling circle amplification under molecular crowding conditions, which we have developed for ultrasensitive detection of DNA, is examined to improve the detection efficiency and sensitivity of the method as well as to gain insight into the mechanism of the method. Both non-magnetic and magnetic sepharose microbeads were employed. Biotinylated DNA had to be pre-immobilized onto the microbeads in order to obtain products on the magnetic beads. The optimal concentration of biotinylated DNA was found to be about 5 μM, above which the number of products decreased. The effect of the crowder charge was examined, and neutral polymers were found to be effective on ligation and the hybridization step, while charged polymers were only effective on the hybridization step and inhibited the ligation and primer extension. The effect of the molecular weight of neutral dextran on the number of products was investigated, and the number of products was found to be increased with an increase in the molecular weight of dextran.

Capping Gold Nanoparticles to Achieve a Protein-like Surface for Loop-Mediated Isothermal Amplification Acceleration and Ultrasensitive DNA Detection

“…PEG and PVP are two common polymers, and they had a weak effect on LAMP acceleration in the absence of AuNPs (accelerated for ∼5 to 10 min, Figure A, C, and D). PEG was reported to increase the activity of polymerase, which could also be helpful for the isothermal nucleic acid amplification. , When the AuNPs were added to PEG and PVP, similarly boosted LAMP amplifications were obtained, and they showed ∼10 min acceleration compared to using the AuNPs alone (Figure D). Therefore, the effect of these polymers and AuNPs appeared to be additive.…”

J-aggregation of 5, 10, 15, 20-tetraphenyl-21H, 23H-porphinetetrasulfonic acid in a molecular crowding environment simulated using dextran

“…For example, Actis et al demonstrated a 1000-fold increase in the molecular count of proteins and sixfold increase in the peak current for DNA by the molecular crowding in the nanopore sensing system [32]. Sasaki et al reported a 5.5-fold increase in the sensitivity of DNA padlock rolling circle amplification in the molecular crowding environment [33]. Although the molecular crowding effect is useful for the increase in the sensitivity of the sensing system, the detailed mechanism remains unclear.…”