Macromolecular binding and kinetic analysis with optically sectioned planar format assays

Abstract

Real-time analysis of macromolecular interactions and competitive binding of ligands to receptors on surfaces are not typically performed using fluorescence intensity methods due to background interference from solution fluorescence. Separation-free optically sectioned planar format assays (OSPFAs) with confocal detection remove this problem. We report OSPFAs for indicator displacement and kinetic assessment of binding. A commercial androgen receptor binding domain indicator displacement assay adapted as an OSPFA yielded an IC 50 of 6.5 nM for testosterone with Z 0 ¼ 0.77. These measured IC 50 and Z 0 values are in ranges suitable for drug screening applications with Z 0 > 0.5 indicating good to excellent screenability. An OSPFA was applied to study the rate of antibody binding to a sandwich immunoassay on a planar surface. Langmuir fits provided forward rate constants in the range 2 Â 10 3 M À1 s À1 to 6 Â 10 4 M À1 s À1 and reverse constants 1 Â 10 À4 s À1 to 4 Â 10 À3 s À1 which cover a useful range for characterising probe-target interactions. This work demonstrates the suitability of OSPFAs for investigating kinetics and binding interactions using fluorescence under wash-free conditions. OSPFAs are an alternative to other separation-free methods such as acoustic, surface plasmon resonance, ellipsometry, fluorescence polarisation and other related methods. OSPFAs should make practical dynamic binding studies for small and large molecules including cases where the reaction under investigation results in no appreciable mass change on a surface.

Analysis of layered assays and volume microarrays in stratified media

“…33 In the last case, wash-free, optically sectioned planar format assays (OSPFAs) in a single layer have been presented previously in extended xy formats. 33,34 In these assays, fluorescent material concentrates in a thin layer due to binding to components attached to a substrate surface. This paper reports OSPFAs detected using quantum dot (QD) reporters arrayed along the optical z-axis on mica or glass substrates to form volume encoded arrays.…”

Multiphoton-Polymerized 3D Protein Assay

“…So far, only micro-bead assays, , a 2.5D polymer living graft polymerization assay, and stacked 2D FLISA microarrays ,, have been realized and introduced as 3D assays. On the one hand, because the stacked 2D FLISA consists of planar assays, the background signal is present in each section.…”

“…55−57 To improve the biocompatibility, the platforms could be fabricated out of unsensitized photoresists, avoiding the addition of potentially fluorescent or toxic photoinitiators. 58 So far, only micro-bead assays, 59,60 a 2.5D polymer living graft polymerization assay, 61 and stacked 2D FLISA microarrays 46,49,62 have been realized and introduced as 3D assays. On the one hand, because the stacked 2D FLISA consists of planar assays, 49 the background signal is present in each section.…”

Platform for Screening Abiotic/Biotic Interactions Using Indicator Displacement Assays

“…Adapting this approach into the field of abiotic/biotic interactions would represent an important advance and could enable comprehensive screening of interactions at the analyte/material interface. Screenable displacement assays in a planar format have been described for receptor ligand interaction studies and this approach can indeed be adapted to measure molecular interactions with abiotic/biotic surfaces, as we will prove in this paper.…”