Using Scanning Electrochemical Atomic Force Microscopy to Image Redox Immunomarked Proteins on Viruses

Introduction

In Scanning Electrochemical Microscopy AFM (SECM-AFM), a metal-coated AFM tip, insulated except its very apex, is used as an electrode to generate/detect chemical species in an electrolyte solution at a desired location (the principle is shown in Fig.1). The main interest of this technique is that it combines the standard capabilities of an AFM (such as generating a 3D-profile of the surface or applying a controlled force to the sample) to the possibility of electrochemically generating/detecting species under a specific potential.

Thus the AFM probe can be used both as a force sensor and a microelectrode. Until recently, this technique had never been applied to characterize actual biological samples [1].

In the present application note, the tip has been brought into contact with redox immunomarked viral particles in order to electrochemically interrogate them. The aim was to achieve in situ mapping of specific viral proteins.

Viruses are natural nanomachines. In spite of their simple architecture, they can display complex activities and can infect both prokaryotic and eukaryotic cells. Over the last twenty years, they have also been used as nanovectors in nanomedicine [2], nano-vehicles for enzymatic catalysis [3] or scaffolds for complex constructs having structural and functional properties [4]. For nanotechnology applications, two types of viruses are often used: bacteriophages [5] and plant viruses [6], since they are harmless to humans. In this study, two filamentous plant viruses (Potyvirus gender), the lettuce mosaic virus (LMV) and the potato virus A (PVA), have been investigated by SECM-AFM. Until recently, the best resolution achieved in SECM was in the micrometer range [7], which is certainly enough to probe processes at a large scale (for instance on a living cell) but not to address nano-bio objects like viruses. This technical note describes a new technique called Mt / (Mediator-tethered) AFM-SECM in which a microelectrode probe is used to electrochemically contact redox-labeled molecules attached onto an electrode surface [8]. This type of construct has been proven to enable detection of the dynamics of PEGs [9] (Polyethylene-glycols) or DNA chains [10]. Mt/AFM-SECM can be combined with redox immunomarking (the use of antibodies grafted with redox PEG chains) to specifically locate ~100 nm size antigens attached to a synthetic surface [11]. Measuring the activity in situ of redox-labeled, immobilized active biostructures requires both a nanometer range spatial resolution and the ability to measure very low electrochemical current (down to the tens of femto Ampere range)