Nanopore-Based Electrical and Label-Free Sensing of Enzyme Activity in Blood Serum

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.

Label-Free Nanopore Single-Molecule Measurement of Trypsin Activity

“…It should be noted that, one of the challenges to analyze clinical samples using the protein nanopore sensor is that the amphiphilic proteins in serum samples might disturb the lipid bilayer 34 using the vertical design used in this work, although it has been successfully used in other horizontal conducting chambers. 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 .…”

Identification of single amino acid differences in uniformly charged homopolymeric peptides with aerolysin nanopore

“…Pioneering studies were devoted to the dynamics of peptides transport 9 , 10 , 13 , 14 , 16 , 30 , and then to the dynamics of unfolded proteins transport 18 , 31 , 48 through the nanopore. Others studies focused on protein-antibodies complex 32 , 41 , 42 , on intrinsically disordered proteins 49 , on enzymatic reactions 15 , 17 , 23 , 60 , 61 and on the aggregation of amyloidogenic peptides 36 , 47 , 55 . The unfolding of proteins was investigated, either in the presence of an unfolding agent 12 , 31 , 44 , or by varying temperature 24 , or with an electrical force 50 , 62 , or with a molecular motor such as an unfoldase 27 .…”

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.…”