Development of a microscopic platform for real-time monitoring of biomolecular interactions

Abstract

We developed a new microscopic platform for the real-time analysis of molecular interactions by combining microbead-tagging techniques with total internal reflection fluorescent microscopy (TIRFM). The optical manipulation of probe microbeads, followed by photo immobilization on a solid surface, enabled us to generate arrays with extremely high density (>100 microbeads in a 25 µm × 25 µm area), and TIRFM made it possible to monitor the binding reactions of fluorescently labeled targets onto probe microbeads without removal of free targets. We demonstrated the high performance of this platform through analyses of interactions between antigen and antibody and between small compounds and proteins. Then, recombinant protein levels in total cellular lysates of Escherichia coli were quantified from the association kinetics using antibody-immobilized microbead arrays, which served as a model for a protein-profiling array. Furthermore, in combination with in vitro synthesis-coupled protein labeling, we could kinematically analyze the interaction of nuclear factor B (p50) with DNA. These results demonstrated that this platform enabled us to: (1) monitor binding processes of fluorescently labeled targets to multiple probes in real-time without removal of free targets, (2) determine concentrations of free targets only from the association kinetics at an early phase, and (3) greatly reduce the required volume of the target solution, in principle to subnanoliter, for molecular interaction analysis. The unique features of this microbead-based microarray system open the way to explore molecular interactions with a wide range of affinities in extremely small volumes of target solutions, such as extracts from single cells.

The trip of the tip: understanding the growth cone machinery

“…Combinations of these microscopy methods with new cross correlation speckle tracking algorithms for quantitatively measuring the details of cytoskeletal dynamics have provided further insights into the roles that actin and MTs have during normal vehicular function of the growth cone36,120, including the finding that exploratory MTs might have a role in early signalling from guidance cues, whereas actin dynamics guide and control more stable MTs to fix the direction of new growth. The same types of quantitative methodology will need to be applied to understand protein-protein interactions and catalytic activations of signalling molecules, through advanced fluorescent sensors and tags121-123. This is an exciting time for studying growth cone dynamics, with new techniques and microscopy tools that are now providing even more extensive opportunities to explore the outstanding questions of the growth cone vehicle and its navigation.…”

A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

“…Recent studies have shown the development of TIRF-based systems for microarray studies using microscopic beads [30]. Other new developments include the design of fiber optic microarrays [5] and the combination of microarrays with micro-fluidics [31].…”

“…New developments include the use of molecular beacons [ 28 ] and the design of protein microarray technology (for a review see [ 29 ]). Recent studies have shown the development of TIRF-based systems for microarray studies using microscopic beads [ 30 ]. Other new developments include the design of fiber optic microarrays [ 5 ] and the combination of microarrays with micro-fluidics [ 31 ].…”

Laser direct-write of single microbeads into spatially-ordered patterns

“…The precise placement of physiologically relevant microbead subunits into spatially-ordered patterns has broad implications for biomaterial processing and biomedical applications. Microbeads possess versatile applicability as probes for biomolecule screening [1], vehicles for targeted drug delivery [2,3], microtools for cell manipulation [4] and microbioreactors for scalable cell culture systems [5,6]. For example, positioning of cell-laden or protein-conjugated beads from blueprinted patterns can greatly enhance spatial detection in bioMEMS sensor technology [7][8][9], and the arrangement of drug-loaded microbeads allows for the formation of spatially defined concentration gradients [10].…”