Use of Semiconductor Nanocrystals to Encode Microbeads for Multiplexed Analysis of Biological Samples
In recent years, interest in optically encoded microbeads has been growing due to their potential applications in medical diagnostics . Microbeads can be encoded with either organic fluorophores or semiconductor quantum dots (QDs) [2-6]. Quantum dots constitute a new class of fluorophores that have a number of advantages over routinely used fluorescent organic dyes, such as a high brightness, resistance to photobleaching, a wide excitation spectrum, and a narrow emission one [8-9]. The unique optical characteristics of QDs make it possible to use them in multiplexed analyses, as well as in multicolor imaging , because QDs of different colors can be simultaneously excited using a single radiation source, whereas their fluorescence peaks can be effectively separated and detected in different channels of a flow cytometer. Therefore, multiplexed analysis by means of flow cytometry using QD-encoded microbeads is easy to perform and does not require sophisticated equipment.
To obtain a population of microbeads encoded with QDs with different fluorescence peaks and fluorescence intensities, we used the method of layer-by-layer deposition : the surface of microbeads was successively coated with layers of differently charged polyelectrolytes and negatively charged QDs. The number of QDs adsorbed on each microbead 4.08 μm in diameter was 1.8–2 ∙ 106. The fluorescence lifetime and the degree of fluorescence quenching were measured to analyze Förster resonance energy transfer (FRET) for QDs in the solution and QDs adsorbed on the encoded microbeads. The fluorescence of the adsorbed QDs was quenched more strongly than that of suspended QDs. However, we cannot conclude that FRET occurred between the absorbed QDs, because the QD fluorescence lifetime was the same for microbeads encoded with one and several layers of QDs.
Figure 1. Fluorescence lifetime of QDs 508 nm in a solution and adsorbed on microbeads.
Figure. 2. Typical fluorescence microscopy images of microbeads of different sizes encoded with QDs 508 nm (A) or QDs 590 nm (B).
Populations of QD-encoded microbeads have been studied in detail using immunofluorescence. The technique of optical encoding of microbeads with QDs developed in this study makes it possible to obtain microbead populations for multiplexed analysis of biological markers using flow cytometry.