Comprehensive characterization of molecular interactions based on nanomechanics

Molecular interaction is a key concept in our understanding of the biological mechanisms of life. Two physical properties change when one molecular partner binds to another. Firstly, the masses combine and secondly, the structure of at least one binding partner is altered, mechanically transducing the binding into subsequent biological reactions. Here we present a nanomechanical micro-array technique for bio-medical research, which not only monitors the binding of effector molecules to their target but also the subsequent effect on a biological system in vitro. This label-free and real-time method directly and simultaneously tracks mass and nanomechanical changes at the sensor interface using micro-cantilever technology. To prove the concept we measured lipid vesicle (approximately 748*10(6) Da) adsorption on the sensor interface followed by subsequent binding of the bee venom peptide melittin (2840 Da) to the vesicles. The results show the high dynamic range of the instrument and that measuring the mass and structural changes simultaneously allow a comprehensive discussion of molecular interactions

Quantitative time-resolved measurement of membrane protein–ligand interactions using microcantilever array sensors

Membrane proteins are central to many biological processes, and the interactions between transmembrane protein receptors and their ligands are of fundamental importance in medical research. However, measuring and characterizing these interactions is challenging. Here we report that sensors based on arrays of resonating microcantilevers can measure such interactions under physiological conditions. A protein receptor — the FhuA receptor of Escherichia coli — is crystallized in liposomes, and the proteoliposomes then immobilized on the chemically activated gold-coated surface of the sensor by ink-jet spotting in a humid environment, thus keeping the receptors functional. Quantitative mass-binding measurements of the bacterial virus T5 at subpicomolar concentrations are performed. These experiments demonstrate the potential of resonating microcantilevers for the specific, label-free and time-resolved detection of membrane protein–ligand interactions in a micro-array format

Surface-stress sensors for rapid and ultrasensitive detection of active free drugs in human serum

here is a growing appreciation that mechanical signals can be as important as chemical and electrical signals in biology. To include such signals in a systems biology description for understanding pathobiology and developing therapies, quantitative experiments on how solution-phase and surface chemistry together produce biologically relevant mechanical signals are needed. Because of the appearance of drug-resistant hospital ‘superbugs’, there is currently great interest in the destruction of bacteria by bound drug–target complexes that stress bacterial cell membranes. Here, we use nanomechanical cantilevers as surface-stress sensors, together with equilibrium theory, to describe quantitatively the mechanical response of a surface receptor to different antibiotics in the presence of competing ligands in solution. The antibiotics examined are the standard, Food and Drug Administration-approved drug of last resort, vancomycin, and the yet-to-be approved oritavancin, which shows promise for controlling vancomycin-resistant infections. The work reveals variations among strong and weak competing ligands, such as proteins in human serum, that determine dosages in drug therapies. The findings further enhance our understanding of the biophysical mode of action of the antibiotics and will help develop better treatments, including choice of drugs as well as dosages, against pathogens