New technologies getting informations on repartition, orientation and identification of biomolecules on SPR chip
Résumé
We present herein an original approach, consisting to lead in parallel a global analysis (SPR) and nanoscale characterizations (AFM, mass spectrometry, TOF-SIMS) of bio-molecules on the biochip surface. The questions we want to answer concern especially the organization, orientation and identification of immobilized and captured biomolecules. In order to characterize the sensing layer in terms of molecular structure and orientation, we engaged a SPR-TOF SIMS first set of experiments (collaboration: Pr Aoyagi, Japan). Our results demonstrated that analysis of orientation of the immobilized cytochrome b5 is possible (ref2&3) and help to optimize and develop our sensing layer. We also recently developed an innovative concept to identify directly « on the chip » (without any elution) the biomolecules retained by mass spectrometry. We first validated the concept on a simple model (monolayer of albumin) with sensitivity for MS identification less than 1 fmol/mm2 (ref1). We further continued by developing immunosensors to catch specifically, in complex biological fluids like sera, low concentrated biomarkers (lag3 protein). Controlling the surface coverage and repartition of molecules by AFM, mass spectrometry “on the chip” allowed to identify without ambiguity both antibody and biomarker. Our objective is now to increase the throughput of analysis. For that, we are currently working on micro-arrays. Our expertise in molecule grafting and nanocharacterization will make our clinical proteomic research relevant, thanks to deeply controlled steps of biosensor development and use. References (1) Lucchi G., Rouleau A., Boireau W., Ducoroy P. SMAP 2007 (Congress on Mass Spectrometry and Proteomics Analysis), Pau (France). (2) S. Aoyagi, W. Boireau, C. Elie-Caille, A. Rouleau, M. Dohi, N. Kato, M. Kudo. 45th Annual Meeting of the Biophysical Society of Japan (Dec 2007), Pacifico Yokohama. (3) S. Aoyagi, A. Rouleau, W. Boireau. Applied Surface Science, (in press).