Single‐Molecule Measurement of Carbonic Anhydrase in Cation Coordinated Environment Using MspA Nanopore^†

Abstract

Comprehensive Summary Carbonic anhydrase accounts for catalytic reaction of CO2/HCO3– transformation, thus resulting in neutralization and acidification of the cellular environment, thereby favoring tumor development. Hence, it is a classical protein model of greatly biocatalytic significance as well as a highly expressed biomarker with renal tumor. We herein proposed a single‐molecule measurement on carbonic anhydrase using MspA nanopore, in [BMIM+] and asymmetric K+/Ca2+ cationic coordinated environment, instead of usual symmetric KCl/NaCl electrolyte. Significantly, our empirical analysis showed that asymmetric K+/Ca2+ cationic environment contributes to distinguishable current modulations, thus yielding better resolution for carbonic anhydrase measurement, which is independent of applied voltage and more importantly, is stable enough at varied pH conditions and for very low concentration test in urine sample. Our results provide a classical model for nanopore protein analysis, and may also permit biocatalytic measurement at single‐molecule level.

Determination of Acetylamantadine by γ-Cyclodextrin-Assisted α-HL Nanopore for Potential Cancer Prediagnosis

“…Nanopore technology is a convenient single-molecule technique with major advantages of high throughput, high sensitivity, and easy operation, which has been used in detecting biomolecules, such as DNA or RNA, − peptides, − and proteins. − In decades, the adaptor-assistant nanopore method has been developed to sense smaller analytes, such as metal ions and organic molecules. − For example, β-cyclodextrin (β-CD), as the most commonly used adaptor, can reside in the α-hemolysin (α-HL) nanopore and reversibly host the small molecules to generate detectable signals to recognize drug molecules and toxic molecules. − The amantadine and its acetylation product, acetylamantadine, both cause secondary signals due to the strong host–guest interactions of β-CD, making it hard to detect the desired analyte with excessive the other . So, the β-CD-assistant nanopore is not suitable for the quantification of acetylamantadine in cancer patient urine samples due to the existence of excess amantadine caused by its incomplete transformation .…”