Quantum-dot-based suspension microarray for multiplex detection of lung cancer markers: preclinical validation and comparison with the Luminex xMAP® system

Abstract

A novel suspension multiplex immunoassay for the simultaneous specific detection of lung cancer markers in bronchoalveolar lavage fluid (BALF) clinical samples based on fluorescent microspheres having different size and spectrally encoded with quantum dots (QDEM) was developed. The designed suspension immunoassay was validated for the quantitative detection of three lung cancer markers in BALF samples from 42 lung cancer patients and 10 control subjects. Tumor markers were detected through simultaneous formation of specific immune complexes consisting of a capture molecule, the target antigen, and biotinylated recognition molecule on the surface of the different QDEM in a mixture. The immune complexes were visualized by fluorescently labeled streptavidin and simultaneously analyzed using a flow cytometer. Preclinical validation of the immunoassay was performed and results were compared with those obtained using an alternative 3-plex immunoassay based on Luminex xMAP® technology, developed on classical organic fluorophores. The comparison showed that the QDEM and xMAP® assays yielded almost identical results, with clear discrimination between control and clinical samples. Thus, developed QDEM technology can become a good alternative to xMAP® assays permitting analysis of multiple protein biomarkers using conventional flow cytometers.

Tempo-spectral multiplexing in flow cytometry with lifetime detection using QD-encoded polymer beads

“…These beads were used as templates for QD-encoding employing a polyelectrolyte-based layer-by-layer (LbL) deposition technology with oppositely charged poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrensulfonate) (PSS) polymers (Sigma-Aldrich) and electrostatic immobilization of water-soluble negatively charged QDs presented elsewhere (see also Fig. S2 of the Electronic Supplementary Information (ESI)) 35 . We used CdSe(core)/ ZnS(shell) QD500 or CdSe(core)/CdS/ZnS(multilayer shell) QD645 core/shell QDs with fluorescence maxima at 500 nm and 645 nm, respectively.…”

“…Ideal colour barcodes are semiconductor quantum dots (QDs) due to their versatile excitability with virtually all wavelengths below the optical bandgap in conjunction with the relatively narrow and symmetrically shaped emission bands. Even though there are concerns about the environmental impact of the QDs, their very attractive optical-spectroscopic properties seem to outweigh this drawback for certain applications [34][35][36] . In the last decades, numerous examples for their use as optical reporters in spectrally encoding or multiplexing approaches in the life sciences have been reported, ranging from luminescence assays over immunohistochemistry to the flow cytometric detection of biomarkers in body liquids 35,[37][38][39] .…”

“…Even though there are concerns about the environmental impact of the QDs, their very attractive optical-spectroscopic properties seem to outweigh this drawback for certain applications [34][35][36] . In the last decades, numerous examples for their use as optical reporters in spectrally encoding or multiplexing approaches in the life sciences have been reported, ranging from luminescence assays over immunohistochemistry to the flow cytometric detection of biomarkers in body liquids 35,[37][38][39] . Their luminescence lifetimes have been, however, rarely exploited as encoding parameter 21,36 , although they cover a time range which is not accessible with other luminophores.…”

Immunoassay Techniques Highlighting Biomarkers in Immunogenetic Diseases

“…Old traditional ELISA technique was developed in 1971 by Engvall and Perlmann [16] and Van Weemen and Schuurs [17] and continues to be nowadays widely used as a routine diagnostic method allowing quantitation of a large variety of proteins [18]. The single-plex ELISA, the most utilized assay method performed in 96-or 384-well plates, has played a prominent role in the quantitative and qualitative identification of analytes.…”

Biofunctionalized Polyelectrolyte Microcapsules Encoded with Fluorescent Semiconductor Nanocrystals for Highly Specific Targeting and Imaging of Cancer Cells

“…Micro-and nanosized carriers can be fluorescently labeled or optically encoded through the incorporation of QDs into them [7,8]. In this case, the shell of the carrier serves as a barrier restricting the interaction of QDs with biological structures, which increases their general biocompatibility.…”