Light scattered by model phantom bacteria reveals molecular interactions at their surface - Analytical Methods & Tools for Lyophilization

Light scattered by model phantom bacteria reveals molecular interactions at their surface
Published online before print October 21, 2005,
approved September 12, 2005 (received for review July 13, 2005)
A. Ghetta *, D. Prosperi , , F. Mantegazza , L. Panza ?, S. Riva ||, and T. Bellini *
PNAS | November 1, 2005
*Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universit? di Milano, Via Fratelli Cervi 93, 20090 Segrate, Italy; Istituto di Scienze e Tecnologie Molecolari, Consiglio Nazionale delle Ricerche, Via Golgi 19, 20133 Milan, Italy; Dipartimento di Medicina Sperimentale, Ambientale e Biotecnologie Mediche, Universit? di Milano-Bicocca, Via Cadore 48, 20052 Monza, Italy; ?Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universit? del Piemonte Orientale, Via Bovio 6, 28100 Novara, Italy; and ||Istituto di Chimica del Riconoscimento Molecolare, Consiglio Nazionale delle Ricerche, Via Mario Bianco 9, 20131 Milan, Italy
Edited by Arnold L. Demain, Drew University, Madison, NJ
Abstract
Testing molecular interactions is an ubiquitous need in modern biology and molecular medicine. Here, we present a qualitative and quantitative method rooted in the basic properties of the scattering of light, enabling detailed measurement of ligand-receptor interactions occurring on the surface of colloids. The key factor is the use of receptor-coated nanospheres matched in refractive index with water and therefore optically undetectable ("phantom") when not involved in adhesion processes. At the occurrence of ligand binding at the receptor sites, optically unmatched material adsorbs on the nanoparticle surface, giving rise to an increment in their scattering cross section up to a maximum corresponding to saturated binding sites. The analysis of the scattering growth pattern enables extracting the binding affinity. This label-free method has been assessed through the determination of the binding constant of the antibiotic vancomycin with the tripeptide L-Lys-D-Ala-D-Ala and of the vancomycin dimerization constant. We shed light on the role of chelate effect and molecular hindrance in the activity of this glycopeptide.
binding affinity | nanoparticles | vancomycin | ligand-receptor recognition
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The formation of supramolecular complexes, involving interactions between biologically relevant macromolecules through noncovalent reversible bindings, is one of the most intensely investigated interdisciplinary topics today. Specifically, the evaluation and quantification of lock-and-key molecular interactions is of paramount importance in modern biology and molecular medicine. Traditional UV, mass and NMR spectroscopies, microcalorimetry, and several surface-sensitive techniques (e.g., evanescent wave spectroscopies, ellipsometry, and quartz crystal microbalance technique) allow the investigation of these complex and often cooperative binding events. As each of these techniques provides information on different aspects of the interactions, but not a complete description of the phenomena at play (refs. 1-3 and references therein), there is a continuous exploration for new methodologies capable of detecting and measuring binding affinities. Recent examples exploit the dispersion of colloids, either by detecting the adhesion of sedimented microspheres (4) or inducing interactions or spectral changes in suspensions of nanoparticles (ref. 5 and references therein). Here, we present a tool based on the high sensitivity offered by the measurement of scattered light intensity (I) when the binding occurs on the surface of index-matched colloids.
Although broadly used in a large variety of contexts, light scattering has never been applied to measure binding affinities in biomolecular interactions (6). As a matter of fact, binding of insulated ligands and receptors in dilute solutions produces a negligible increment of scattered light, and the use of mesoscopic particles hosting multiple receptors or ligands, including real bacteria, is typically of little help because particles generally scatter too much light compared with the contributions caused by molecular adhesion on their surface. We have found that this difficulty can be overcome by supporting the receptors on nanoscale latex spheres whose refractive index closely matches the one of water, a feature that makes them hard to detect with commercial light-scattering particle sizers. To this aim, we used highly hydrophobic nanocolloids, easily covered, by lipophilic interaction, with suitable ligands and/or receptors for the study of reversible interactions and adhesion processes. The dispersed phantom scatterer (DPS) technique we describe allows very sensitive quantitative measurements, so far accessible only with surface sensing techniques, such as the widespread technology based on surface plasmon resonance (SPR).
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