OPTICAL DETECTION OF LIGAND-RECEPTOR INTERACTIONS

 

Life is based on soft interactions that enable all the molecules of key biological importance to associate and ultimately form living beings. Their softness ensures reversibility and enables crucial functions, such as cell duplication, and, above all, allows the wide spectrum of inter and intramolecular configurations essential for evolvability.

Despite their importance, soft molecular interactions are hard to measure and thus a great effort in contemporary research is devoted to try new schemes to access interaction strength or visualize interaction events. Our group has recently proposed a method to detect interactions based on the use of nanoscopic dispersed colloids having refractive index equal to the one of water. The basic concept is that the scattering cross section of nearly index-matched nanoparticles significantly changes as molecules accumulate on their surface. This fact enables measuring the intermolecular interactions via a two steps method: first a surfactant/receptor monolayer is adsorbed on the particles; second, ligand molecules are added to the dispersion. If the added molecules interact with the previously adsorbed monolayer, the scattering cross section increases. The quantitative study of the adsorbed mass on the particles vs. added molecules enables determining the ligand-receptor binding strength.

The resulting method is simple, accurate and particularly suitable to study binding phenomena at the solid-liquid interface, mimicking recognition events occurring at the level of living cell wall. The method has sensitivity comparable to that of the best available techniques for detecting molecular interactions, such as Surface Plasmon Resonance and quartz crystal microbalance.

 

The base of our method is the use of nanoparticles – here shown in a freeze-fracture TEM image – having refractive index matched to the one of water. As ligand molecules (green) stick on the head groups (blue) of previously adsorbed receptors, the intensity of light scattered by the particles (red arrows) increases, thus revealing the occurrence of the molecular interactions.

 

We have tested this method by adsorbing on the particles amphiphilic molecules having their hydrophilic group terminating with a tripeptide typical of bacterial cell walls. Addition of the antibiotic Vancomycin results in an increase of the molecular mass adhering to the particles. The analysis of the scattered intensity vs. added Vancomycin enables extracting the binding strength between the antibiotic and the bacteria cell wall.

 

The scattered intensity strongly depends on the total molecular mass on the particles surfaces. First we adsorb by hydrophobic action peptidic receptor-bearing amphiphilic molecules (red dots) and later Vancomycin (green), which sticks on the receptors until they are saturated. Data analysis (orange line) enables determining the binding coefficient for the interaction.

 

Our group is actively studying, with this method, other biologically relevant interactions such as avidine-biotine, enzymatic activity, DNA recognition. The group is also investigating the efficiency of similar experimental schemes with important changes in the optics, materials and particles shape and mutual interactions.