Abstract
One of the most important goals in proteomics is to detect the real-time kinetics of diverse biomolecular interactions. Fluorescence, which requires extrinsic tags, is a commonly and widely used method because of its high convenience and sensitivity. However, in order to maintain the conformational and functional integrality of biomolecules, label-free detection methods are highly under demand. We have developed the oblique-incidence reflectivity difference (OI-RD) technique for label-free, kinetic measurements of protein-biomolecule interactions. Incorporating the total internal refection geometry into the OI-RD technique, we are able to detect as low as 0.1% of a protein monolayer, and this sensitivity is comparable with other label-free techniques such as surface plasmon resonance (SPR). The unique advantage of OI-RD over SPR is no need for dielectric layers. Moreover, using a photodiode array as the detector enables multi-channel detection and also eliminates the over-time signal drift. In this paper, we demonstrate the applicability and feasibility of the OI-RD technique by measuring the kinetics of protein-protein and protein-small molecule interactions in sandwich assays.
Highlights
In modern proteomics, it is important and necessary to be able to analyze the interactions between proteins and other biomolecules
The kinetics of antigens binding to solid substrates has been an important area in immunodiagnostics for many years [33]
The oblique-incidence reflectivity difference (OI-RD) technique is applicable in label-free kinetic measurements of protein-biomolecule interactions
Summary
It is important and necessary to be able to analyze the interactions between proteins and other biomolecules. Quantitative analysis of binding kinetics helps biologists to better understand the structures and functions of these proteins Traditional methods such as chromatography and immunoprecipitation are widely used in detecting protein-protein interactions, but they are limited to qualitative measurements. Determines the changes in thickness and refractive index of a thin deposited layer by measuring the interference of light source caused by partial reflection at the interface [15,16,17,18] All these methods are applicable in kinetic analysis of biomolecular interactions and are of very high sensitivity. The OI-RD microscopy, a particular and the most sensitive form of optical elliposometry, has a comparable sensitivity (few pg protein/mm2) to all above-mentioned methods This technique is suitable for both real-time and end-point measurements, and, comparing to SPR or RM, no dielectric layers is required. Being able to study the heterogeneity of proteins and detect small molecular protein ligands, the feasibility of the OI-RD technique, in combination with microarrays, can definitely benefit the development of proteomics, or the large-scale studies of proteins, their structures and functions
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