Biosensors based on intrinsic detection methods have attracted growing interest. The use of Fourier transform infra-red (FTIR) spectroscopy with the attenuated internal total reflection (ATR) mode, in the biodetection context, requires appropriate surface functionalization of the ATR optical element. Here, we report the direct grafting of a thin organic layer (about 20 Å depth) on the surface of a germanium crystal. This covering, constructed with novel amphiphilic molecules 2b (namely, 2,5,8,11,14,17,20-heptaoxadocosan-22-yl-3-(triethoxysilyl) propylcarbamate), is stable for several hours under phosphate buffered saline (PBS) flux and features protein-repulsive properties. Photografting of molecule 5 (namely, O-succinimidyl 4-(p-azidophenyl)butanoate) affords the activated ATR element, ready for the covalent fixation of receptors, penicillin recognizing proteins BlaR-CTD for instance. The different steps of the previous construction have been monitored by water contact angle (θ w ) measurements, spectroscopic ellipsometry (covering depth), X-ray photoelectron spectroscopy (XPS) by using a fluorinated tag for the control of surface reactivity, and FTIR-ATR spectroscopy for the structural analysis of grafted molecules. Indeed, contrarily to silicon device, germanium device offers a broad spectral window (1000-4000 cm −1 ) and thus amide I and II absorption bands can be recorded. This work lays the foundations for the construction of novel FTIR biosensors.
Biosensors are composite devices suitable for the investigation of receptor–ligand interactions. In this paper we present the specific application to a membrane embedded protein of a new sensor device, so-called BIA–ATR, based on Attenuated Total Reflection–Fourier Transform Infrared (ATR–FTIR) spectroscopy. It consists in a functionalised ATR germanium crystal whose surface has been covalently modified to adsorb a biomembrane. Detection of the ligand–receptor interaction is achieved using FTIR spectroscopy. We report the specific detection of the phosphorylation/dephosphorylation of the H+/K+gastric ATPase. The H+, K+-ATPase is a particularly large protein entity. This glycosylated protein contains more than 1300 residues and is embedded in a lipid membrane. Yet we demonstrate that the BIA–ATR sensor is capable of monitoring the binding of a single phosphate on such a large protein entity. Furthermore, we also demonstrate the potential of the approach to monitor the kinetics of binding and dissociation of the ligand.
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