Biosensing using antibody-modulated motility of actin filaments on myosin-coated surfaces.

Motor proteins, such as myosin and kinesin, are biological molecular motors involved in force generation and intracellular transport within living cells. The characteristics of molecular motors, i.e., their motility over long distances, their capacity of transporting cargoes, and their very efficient energy consumption, recommend them as potential operational elements of a new class of dynamic nano-devices, with potential applications in biosensing, analyte concentrators, and biocomputation. A possible design of a biosensor based on protein molecular motor comprises a surface with immobilized motors propelling cytoskeletal filaments, which are decorated with antibodies, presented as side-branches. Upon biomolecular recognition of these branches by secondary antibodies, the 'extensions' on the cytoskeletal filaments can achieve considerable lengths (longer than several diameters of the cytoskeletal filament carrier), thus geometrically impairing or halting motility. Because the filaments are several micrometers long, this sensing mechanism converts an event in the nanometer range, i.e., antibody-antigen sizes, into an event in the micrometer range: the visualization of the halting of motility of microns-long cytoskeletal filaments. Here we demonstrate the proof of concept of a sensing system comprising heavy-mero-myosin immobilized on surfaces propelling actin filaments decorated with actin antibodies, whose movement is halted upon the recognition with secondary anti-actin antibodies. Because antibodies to the actin-myosin system are involved in several rare diseases, the first possible application for such a device may be their prognosis and diagnosis. The results also provide insights into guidelines for designing highly sensitive and very fast biosensors powered by motor proteins.

Spatially Addressable Multiplex Biodetection by Calibrated Micro/Nanostructured Surfaces.

A challenge of any biosensing technology is the detection of very low concentrations of analytes. The fluorescence interference contrast (FLIC) technique improves the fluorescence-based sensitivity by selectively amplifying, or suppressing, the emission of a fluorophore-labeled biomolecule immobilized on a transparent layer placed on top of a mirror basal surface. The standing wave of the reflected emission light means that the height of the transparent layer operates as a surface-embedded optical filter for the fluorescence signal. FLIC extreme sensitivity to wavelength is also its main problem: small, e.g., 10 nm range, variations of the vertical position of the fluorophore can translate in unwanted suppression of the detection signal. Herein, we introduce the concept of quasi-circular lenticular microstructured domes operating as continuous-mode optical filters, generating fluorescent concentric rings, with diameters determined by the wavelengths of the fluorescence light, in turn modulated by FLIC. The critical component of the lenticular structures was the shallow sloping side wall, which allowed the simultaneous separation of fluorescent patterns for virtually any fluorophore wavelength. Purposefully designed microstructures with either stepwise or continuous-slope dome geometries were fabricated to modulate the intensity and the lateral position of a fluorescence signal. The simulation of FLIC effects induced by the lenticular microstructures was confirmed by the measurement of the fluorescence profile for three fluorescent dyes, as well as high-resolution fluorescence scanning using stimulated emission depletion (STED) microscopy. The high sensitivity of the spatially addressable FLIC technology was further validated on a diagnostically important target, i.e., the receptor-binding domain (RBD) of the SARS-Cov2 via the detection of RBD:anti-S1-antibody.

Polymer surface properties control the function of heavy meromyosin in dynamic nanodevices.

The actin-myosin system, responsible for muscle contraction, is also the force-generating element in dynamic nanodevices operating with surface-immobilized motor proteins. These devices require materials that are amenable to micro- and nano-fabrication, but also preserve the bioactivity of molecular motors. The complexity of the protein-surface systems is greatly amplified by those of the polymer-fluid interface; and of the structure and function of molecular motors, making the study of these interactions critical to the success of molecular motor-based nanodevices. We measured the density of the adsorbed motor protein (heavy meromyosin, HMM) using quartz crystal microbalance; and motor bioactivity with ATPase assay, on a set of model surfaces, i.e., nitrocellulose, polystyrene, poly(methyl methacrylate), and poly(butyl methacrylate), poly(tert-butyl methacrylate). A higher hydrophobicity of the adsorbing material translates in a higher total number of HMM molecules per unit area, but also in a lower uptake of water, and a lower ratio of active per total HMM molecules per unit area. We also measured the motility characteristics of actin filaments on the model surfaces, i.e., velocity, smoothness and deflection of movement, determined via in vitro motility assays. The filament velocities were found to be controlled by the relative number of active HMM per total motors, rather than their absolute surface density. The study allowed the formulation of the general engineering principles for the selection of polymeric materials for the manufacturing of dynamic nanodevices using protein molecular motors.

Protein immobilisation on micro/nanostructures fabricated by laser microablation.

The performance of biomedical microdevices requires the accurate control of the biomolecule concentration on the surface, as well as the preservation of their bioactivity. This desideratum is even more critical for proteins, which present a significant propensity for surface-induced denaturation, and for microarrays, which require high multiplexing. We have previously proposed a method for protein immobilisation on micro/nanostructures fabricated via laser ablation of a thin metal layer deposited on a transparent polymer. This study investigates the relationship between the properties of the micro/nanostructured surface, i.e., topography and physico-chemistry, and protein immobilisation, for five, molecularly different proteins, i.e., lysozyme, myoglobin, α-chymotrypsin, human serum albumin, and human immunoglobulin. Protein immobilisation on microstructures has been characterised using quantitative fluorescence measurements and atomic force microscopy. It has been found that the sub-micrometer-level, combinatorial nature of the microstructure translates in a 3-10-fold amplification of protein adsorption, as compared to flat, chemically homogenous polymeric surfaces. This amplification is more pronounced for smaller proteins, as they can capitalize better on the newly created surface and variability of the nano-environments.