Real-time label-free measurement of HIV-1 protease activity by nanopore analysis

Abstract

A label-free method for the measurement of the activity of HIV-1 protease is developed by real-time monitoring of the cleavage of a peptide substrate by HIV-1 protease in a nanopore. The method is rapid and sensitive: picomolar concentrations of HIV-1 protease could be detected in ~10 minutes. Simulated clinical samples are analyzed, and the activity of HIV-1 protease could be accurately detected. Our developed nanopore sensor design strategy should find useful application in the development of stochastic sensors for other proteases of medical, pharmaceutical, and biological importance.

Label-Free Nanopore Single-Molecule Measurement of Trypsin Activity

“…24 One feasible approach to overcome this difficulty is to use spin-columns to remove interfering blood serum components 46 . In our previous work, we found that by incubating mock serum samples with peptide substrates, and then analyzing the mixture solution using the protein nanopore sensor, we could sensitively determine the protease activity in the serum within minutes, thus overcoming the lipid bilayer’s short life-time limitation 31 . This same approach was utilized to analyze the mock trypsin serum sample in this work.…”

“…It should be noted that nanopore stochastic sensors have been utilized to detect enzymatic reactions 31–34 . By using a substrate (tethered or untethered) for a particular enzyme/protease and monitoring its translocation in the nanopore, we can readily identify whether the sample contains the target enzyme/protease or not.…”

Nanopore Sensing

“…Indirect detection of proteins via α-HL pores has been reported previously. 38, 41, 45, 142 In one approach, the activity of a protein enzyme is measured by monitoring the ion current modulations that occur due to protease–substrate reactions. In the absence of protease, translocation of intact peptide substrate molecules through a nanopore will produce one type of event.…”

Pattern recognition of adipokines in whole blood samples using stochastic sensing

“…The signature (t off value) of the analytes is the identification tools in the diagrams obtained, while the t on values are used for all quantitative measurements. Natural and engineered pores are proposed for stochastic sensing (Stefan-van Staden et al 2014;Bayley et al 2000;Wang et al 2014) with good results, although it is believed that engineered pores/channels are well-defined, and uniform, and should be preferred to the natural one.…”

“…The role of adipose tissue is not only to store excess Stochastic sensors are promising tools for biomedical analysis (Stefan-van Staden et al 2014;Bayley et al 2000;Wang et al 2014), due to their capacity of recognizing the analytes (qualitative analysis) in complex matrices, and also to reliable quantify them. The signature (t off value) of the analytes is the identification tools in the diagrams obtained, while the t on values are used for all quantitative measurements.…”