RESUMO
The chelating reaction between glutathione peptides and divalent cadmium ion was used as a typical model for investigating the coordination chemistry of SH-containing peptides and heavy metal ions, which was essential to understand the mechanism of intracellular cadmium detoxification.Here, the mutant (CM113R)7 αHL protein nanopore equipped with a first synthesized per-6-quaternary ammonium-β-cyclodextrin (p-QABCD) was used as nanoreactor and detector to investigate the single-molecule reaction of GSH molecules and Cd2+ ions.Different reaction pathways, intermediates, and products could be recognized.Cd2+-GSH reaction was highly dependent on the solution pH.The Cd(GSH)2 was formed at pH 7.4, while the Cd(GSH)2 and Cd2(GSH)2 were formed at pH 9.0.Cd2(GSH)2 was formed by two possible pathways: (1) A Cd2+ ion primarily coordinated with the thiol group of two GSH molecules to form Cd(GSH)2, and then the second Cd2+ ion quickly incorporated with the deprotonated amino group of Cd(GSH)2 to form Cd2(GSH)2;(2) Two Cd2+ ions separately coordinated with the thiol and deprotonated amino group of one GSH molecule to form Cd2(GSH)1, and the second GSH molecule quickly bounded to Cd2+ ions to form Cd2(GSH)2.This work studied the chelating reaction between metal ions and bioactive glutathione at single-molecule level without labeling and chemical modification, and would be important to further understand intracellular mechanisms of detoxification of heavy metals and greatly expand the research field of nanopore single-molecule technique.
RESUMO
Nanopore technique is a low-cost, ultrafast method for single-molecule level analysis without labels. Nanopore technique was first proposed more than 20 years ago and exhibited an excellent potential in DNA sequencing. So far, the commercial development of nanopore strand-sequencing as a portable device has been realized. Meanwhile, a remarkable number of studies have demonstrated that nanopore represents versatile single-molecule sensors for a wide range of molecule. Therefore, in this article we mainly review the use of nanopore technique based on the interface interactions between biological pore and the analytes such as protein/peptide to obtain kinetic and thermodynamic information at single-molecule level. And a large number of biological molecules and metal ions are quantitatively detected by nanopore analysis, allowing its development for the future biotechnologies and medicine applications. Besides, electrochemical detection system is crucial to nanopore technique. Therefore, we focus on advancements in relative software and ultralow current instrumentations with high-bandwidth.
RESUMO
A novel amplifier system was proposed for low-noise recording of pico-ampere current in nanopore experiment (<100 pA). As an example, the amplifier system was applied in α-hemolysin based nanopore detection of DNA-PEG-DNA conjugate to record the signals of translocation and bumping events in buffer solution (1 mol/L KCl, 10 mmol/L Tris--HCl, 1 mmol/L EDTA and pH=8. 0). The amplified current signal was filtered by a 3 kHz Bessel filter and sampled by a 100 kHz analog-digital convertor. As a result, the presented amplifier system could lower the noise in recording the current. The current blockages (<10 pA) of single molecules with low amplitude were recovered due to the high signal-to-noise ratio.