Abstract
Single-molecule techniques are being increasingly applied to biomedical investigation, notwithstanding the numerous challenges they pose in terms of signal-to-noise ratio issues. Non-specific binding of probes to glass substrates, in particular, can produce experimental artifacts due to spurious molecules on glass, which can be particularly deleterious in live-cell tracking experiments. In order to resolve the issue of non-specific probe binding to substrates, we performed systematic testing of a range of available surface coatings, using three different proteins, and then extended our assessment to the ability of these coatings to foster cell growth and retain non-adhesive properties. Linear PEG, a passivating agent commonly used both in immobilized-molecule single-molecule techniques and in tissue engineering, is able to both successfully repel non-specific adhesion of fluorescent probes and to foster cell growth when functionalized with appropriate adhesive peptides. Linear PEG treatment results in a significant reduction of tracking artifacts in EGFR tracking with Affibody ligands on a cell line expressing EGFR-eGFP. The findings reported herein could be beneficial to a large number of experimental situations where single-molecule or single-particle precision is required.
Highlights
Since they were first described around 20 years ago, there has been a near-exponential growth in the use of singlemolecule techniques for the investigation of biological systems and processes [1]
For non-cell experiments we investigated the non-specific binding of three proteins: Human epidermal growth factor (EGF), antihuman epidermal growth factor receptor 2 (HER2) Affibody [14,15], and hen egg white lysozyme (HEWL), each labelled with three different fluorescent probes
We studied the effect of surface treatments on non-specific binding of anti-human epidermal growth factor receptor (EGFR) Affibody labelled with two different fluorescent probes
Summary
Since they were first described around 20 years ago, there has been a near-exponential growth in the use of singlemolecule techniques for the investigation of biological systems and processes [1]. This is expected to continue as singlemolecule methods move out of specialist physics laboratories and find more applications in the biomedical sciences. Fluorescence-based methods can be divided into measurements on fluorescent molecules in solution, those on immobilized fluorescent molecules, and measurements on fluorescent molecules in cultured cells These measurements can be used to investigate stoichiometry, inter- and intramolecular interactions, and molecular conformation. The principle of single-molecule localization lies behind sub-diffraction limit imaging techniques such as stochastic optical reconstruction microscopy (STORM) [7] and photoactivation localization microscopy (PALM) [8]
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