Nanopore-based electrical and label-free sensing of enzyme activity in blood serum.

Mikiembo Kukwikila,Stefan Howorka

doi:10.1021/acs.analchem.5b01764

Abstract

A generic strategy to expand the analytical scope of electrical nanopore sensing is presented. We specifically and electrically detect the activity of a diagnostically relevant hydrolytic enzyme and remove the analytically harmful interference from the biochemically complex sample matrix of blood serum. Our strategy is demonstrated at the example of the renin protease which is involved in regulation of blood pressure. The analysis scheme exploits a new approach to reduce sample complexity while generating a specific read-out signal. Within a single spin-column (i), the protease cleaves a resin-tethered peptide substrate (ii) which is affinity-purified using the same multifunctional resin to remove interfering blood serum components, followed by (iii) detecting the peptide via electrical nanopore recordings. Our approach is beneficial in several ways. First, by eliminating serum components, we overcome limitations of nanopore sensing when challenging samples lead to membrane instability and a poor signal-to-noise ratio. Second, the label-free sensing avoids drawbacks of currently used radiolabel-immunoassays for renin. Finally, the strategy of simultaneous generation and purification of a signal peptide within a multifunctional resin can very likely be expanded to other hydrolytic enzymes dissolved in any analyte matrix and exploited for analytical read-out methods other than nanopore sensing.

Highlights

Nanopore analytics is an increasingly popular approach to detect molecules in a label-free fashion
The approach is generic and can be implemented with different types of nanopores. These include biological protein pores embedded into a lipid bilayer as well as with man-made solid-state pores drilled into thin membranes composed of SiN, glass, or organic polymers.[1]
Reflecting its generic principle, nanopore analytics can be exploited for the specific and label-free sensing of nucleic acid[4−8] including DNA sequencing,[9,10] the detection of proteins,[11−14] and enzymatic activity and peptides,[15−19] which are of relevance to the present report.[5]

Summary

Introduction

Nanopore analytics is an increasingly popular approach to detect molecules in a label-free fashion. Signal generation is achieved by adding renin-containing blood serum to microscale beads P displaying a substrate peptide (Figure 2A) derived from angiotensinogen.

Results

Conclusion