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Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer (SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Aptamer-based protein sensing can be implemented using a variety of optical and non-optical detection methods. In this report, we present protein-induced fluorescence enhancement (PIFE) based detection of DNA aptamer binding to thrombin. We demonstrate that PIFE reports on direct binding of thrombin to the aptamer strand carrying the fluorophore and hence is unaffected by salt based stabilization of the aptamer conformations as observed in fluorescence resonance energy transfer (FRET) assays. PIFE based thrombin detection displayed excellent linearity in the range of 0.25 pM-25 nM with a detection limit of 8.9 pM. In an alternate scheme, PIFE was demonstrated by placing the fluorophore on a connector strand thus bypassing the requirement to label the aptamer directly. This strategy allowed us to examine thrombin binding by variously modified thrombin aptamers and can serve as a platform for aptamer screening. We finally show that PIFE based thrombin aptamer assay displays high specificity even in the presence of `natural' background such as blood plasma. We hence propose PIFE based aptamer sensing of protein ligands as a facile yet sensitive and general strategy for biosensing. (C) 2018 Elsevier B.V. All rights reserved.

Smartphone-based fluorescence detection is a promising avenue for biosensing that can aid on-site analysis. However, quantitative detection with fluorescence in the field has been limited due to challenges with robust excitation and calibration requirements. Here, we show that ratiometric analysis with Förster resonance energy transfer (FRET) between dye pairs on DNA aptamers can enable rapid and sensitive kanamycin detection. Since our detection scheme relies on ligand binding-induced changes in the aptamer tertiary structure, it is limited only by the kinetics of ligand binding to the aptamer. Our FRET-based kanamycin binding aptamer (KBA) sensor displays two linear ranges of 0.05-5 nM (detection limit of 0.18 nM) and 50-900 nM of kanamycin. The aptamer displays high specificity even in the presence of the 'natural' background from milk. By immobilizing the aptamer in the flow cell, our KBA sensor design is also suitable for repeated kanamycin detection. Finally, we show that the ratiometric FRET-based analysis can be implemented on a cheap custom-built smartphone setup. This smartphone-based FRET aptamer scheme detects kanamycin in a linear range of 50-500 nM with a limit of detection (LOD) of 28 nM. © 2019 The Royal Society of Chemistry.