The Use of Quantum Dot Luminescent Probes for Western Blot Analysis

Abstract

Western blot analysis (1,2) is one of the most widely used methods for analyzing specific protein targets using colorimetric or chemiluminescent substrates (3). However, the reporting labels that are typically used for protein detection are unsuitable for simultaneous detection of multiple targets. In order to detect different proteins the blot is usually stripped and reprobed, an approach that is time- and labor-intensive, and often leads to loss of immobilized proteins from the blot. Detection of multiple antigens on a single protein blot without stripping off prior antibodies is possible, but requires sequential reactions (4). More recent approaches employ multiple fluorescent probes made from small organic dye molecules ((5) and references therein), but such probes have several limitations, described below. The use of QD luminescent labels has the potential to eliminate most of these problems.

QDs are semiconductor nanoparticles (e.g. CdSe, InP, InAs) with diameters in the range of 2 to 10 nm whose fundamental physical properties are influenced by quantum confinement effects (6). QDs exhibit absorption and emission peaks that progressively shift to longer wavelengths with increasing particle size. For applications as fluorescent markers, the relevant quantum confinement effect is the tuning of the semiconductor band gap and hence the color of the QD emission achieved simply by changing the size of the particle. In this way, the luminescence wavelength of QDs can be tuned from the infrared to the ultraviolet (7). Fig. 1 shows the photoluminescence of CdSe nanocrystals spanning the visible spectrum with particles ranging in diameter from 2 to 6 nm.