Introduction We have recently demonstrated an efficient

Introduction We have recently demonstrated an efficient methodology for the synthesis of self assembled monolayers (SAMs) based on thiolated long-chain alkanethiols (LCAT) incorporating oligoethyleneglycol (OEG) linkers of variable length [1]. LCAT-OEGs have been shown to be ideally suited for integrating immuno-receptors with a sensor platform [2]. Here, the LCAT component ensures spontaneous assembly of an ordered and densely packed monolayer onto coinage metal electrodes, typically gold, while the OEG component is efficient at minimising non-specific adsorption of peripheral molecules contained in a complex sample matrix, such as a clinical sample. Our solid-phase synthesis approach is not only efficient but also highly versatile and can be used to create bespoke LCAT-OEGs of variable length and derivatized with a wide range of functional groups that allow tuning of surface chemistry and conjugation of a range of molecular and biomolecular components. For example we have demonstrated an amine derivatized LCAT-OEG (named LCAT-OEG-NH2 and shown in Fig. 1(a)) to which we can covalently couple proteins and peptides, either pre- or post-monolayer assembly, via free amine or carboxylic Pyrrolidinedithiocarbamate ammonium Supplier groups exposed on a protein or peptide surface. We have exploited this monolayer to immobilize antibodies onto electrode surfaces and demonstrated that the biocompatibility and hydrophilicity of the OEG linker ensures antibody specificity and selectivity is preserved following immobilization [1]. Using the same methodology, we have also synthesized an innovative LCAT-OEG SAM derivatized with the redox active molecule methylene blue (named LCAT-OEG-MB and shown in Fig. 1) and that has been engineered specifically to act as a transduction element for monitoring molecular binding events electronically. Methylene blue (MB) is a heterocyclic aromatic chemical compound that is used widely as a dye, as a therapeutic for a range of medical conditions [5] and increasingly as a redox-active indicator for probing molecular interactions, particularly for the detection of DNA hybridisation [6] and for monitoring DNA aptamer binding [7]. The MB redox reaction is a two electron, one proton process described by the Nernst equation: E is the applied potential and and are the concentration of surface immobilized oxidised and reduced methylene blue, respectively. The formal potential, , is given by: Here, is the standard electrode potential, is the potential at the plane of electron transfer i.e. the plane in which the electroactive molecule lies, and is the solution potential (see Fig. 1(c)). From these equations it is clear that at a fixed ionic strength, i.e. constant , a change in local pH or in , for example due to binding of a charged protein at the plane of electron transfer, will lead to a shift in the formal potential, providing a mechanism to transduce molecular binding into a quantitative, electronic signal. In this manuscript, we demonstrate a mixed molecular monolayer that consists of LCAT-OEG-MB and LCAT-OEG-NH2 components where the NH2 component acts as a scaffold for the covalent immobilization of immuno-receptors, and the MB is an indirect probe of molecular interactions. Here, binding of a target ligand to the immobilised immuno-receptor leads to a change in the electrostatic environment local to the MB leading to a quantitative, electrochemical measurement of the molecular interaction. Unlike other similar proposals, Ho et al. [3,4] our approach based on LCAT-OEG SAMs provide a number of significant benefits, including; immobilisation and sensing in a single bio-compatible, stable and anti-fouling monolayer; access to a variety of surface chemistries for the integration of a wide range of small molecules/immuno-receptors; simple and precise control over the ratio of components assembled in the mixed monolayer, providing a facile approach for regulating the surface density of immuno-receptors; precise control of the length of both the LCAT and OEG components and thus the location of the MB sensing component relative to the immobilized immuno-receptors.