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

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.

Engineering of Optically Encoded Microbeads with FRET-Free Spatially Separated Quantum-Dot Layers for Multiplexed Assays.

Quantum dot (QD) encoded microbeads are emerging for multiplexed analysis of biological markers. The quantitative encoding of microbeads prepared with different concentrations of QDs of different colors suffers from resonance energy transfer from the QDs fluorescing at shorter wavelengths to the QDs fluorescing at longer wavelengths. Here, we used the layer-by-layer deposition technique to spatially separate QDs of different colors with several polymer layers so that the distance between them would be larger than the Förster energy transfer radius. We performed fluorescence lifetime measurements to investigate and determine the conditions excluding significant resonance energy transfer between QDs within QD-encoded microbeads. Additionally, the number of QDs adsorbed onto microbeads was systematically established and multilayer structures of the QD-encoded microbead shells were characterized by scanning probe nanotomography. Finally, we prepared eight populations of FRET-free microbeads encoded with QDs of three colors at two intensity levels and demonstrated that all the optical codes are excitable at a single wavelength and may be clearly identified in three channels of a flow cytometer. The developed approach for engineering QD-encoded microbeads that are free from optical artefacts related to inter-QD resonance energy transfer paves the way to quantitative QD-based multiplexed assays.

Quantum Dot-Based Nanotools for Bioimaging, Diagnostics, and Drug Delivery.

Quantum dots (QDs) are highly fluorescent nanocrystals with advanced photophysical and spectral properties: high brightness and stability against photobleaching accompanied by broad excitation and narrow emission spectra. Water-soluble QDs functionalized with biomolecules, such as proteins, peptides, antibodies, and drugs, are used for biomedical applications. The advantages of QD-based approaches to immuno-histochemical analysis, single-molecule tracking, and in vivo imaging (over traditional methods with organic dyes and fluorescent proteins) are explained. The unique spectral properties of QDs offer opportunities for designing systems for multiplexed analysis by multicolor imaging for the simultaneous detection of multiple targets. Conjugation of drug molecules with QDs or their incorporation into QD-based drug-delivery particles makes it possible to monitor real-time drug tracking and carry out image-guided therapy. Because of the tunability of their photophysical properties, QDs emitting in the near-infrared have become an attractive tool for deep-tissue mono- and multiphoton in vivo imaging. We review recent achievements in QD applications for bioimaging, targeting, and drug delivery, as well as challenges related to their toxicity and non-biodegradability. Key and perspectives for further development of advanced QD-based nanotools are addressed.

Two-photon-induced Förster resonance energy transfer in a hybrid material engineered from quantum dots and bacteriorhodopsin.

Energy transfer from nanostructures to biological supramolecular photosystems is an important fundamental issue related to the possible influence of nanoobjects on biological functions. We demonstrate here two-photon-induced Förster resonance energy transfer (FRET) from fluorescent CdSe/ZnS quantum dots (QDs) to the photosensitive protein bacteriorhodopsin (bR) in a QD-bR hybrid material. The two-photon absorption cross section of QDs has been found to be about two orders of magnitude larger than that of bR. Therefore, highly selective two-photon excitation of QDs in QD-bR complexes is possible. Moreover, the efficiency of FRET from QDs to bR is sufficient to initiate bR photoconversion through two-photon excitation of QDs in the infrared spectral region. The data demonstrate that the effective spectral range in which the bR biological function is excited can be extended beyond the band where the protein itself utilizes light energy, which could open new ways to use this promising biotechnological material.

Quantum dot-based lab-on-a-bead system for multiplexed detection of free and total prostate-specific antigens in clinical human serum samples.

An immunodiagnostic lab-on-a-bead suspension microarray based on microbeads encoded with quantum dots (QDs) has been developed and preclinically validated for multiplexed quantitative detection of prostate cancer markers in human serum samples. The sensitivity and specificity of the microarray are similar to those of "gold-standard" single-analyte ELISA. Moreover, the array has an improved immunoassay capacity, ensures quantitative detection of multiple cancer biomarkers and may be operational in a considerably wider dynamic range of concentrations. The array is characterized by reduced time and cost of analysis and is compatible with classical flow cytometers. Proof-of-concept preclinical tests ensured simultaneous quantitative determination of free and total prostate-specific antigens in human serum, with clear discrimination between the control and clinical samples. The proposed approach is flexible and paves the way to development of a wide variety of immunodiagnostic assays for multiplexed early diagnosis of various diseases. FROM THE CLINICAL EDITOR: Early diagnosis of cancer can result in better prognosis for patients. Thus, the use of specific tumor markers is widely employed in clinical practice. Traditional screening methods only employ single markers. The authors here developed a microarray system based on microbeads encoded with quantum dots (QDs), which can be used for multiplexed quantitative detection. The validated results on patient samples should lead to the development of a wider variety of assays for other diseases.

Quantum dot surface chemistry and functionalization for cell targeting and imaging.

Quantum dots (QDs) are highly fluorescent nanoscale crystals with size-dependent emission spectra. Due to their excellent photophysical properties, QDs are a promising alternative to organic fluorescent dyes and fluorescent proteins for cell targeting, imaging, and drug delivery. For biomedical applications, QDs should be chemically modified to be stable in aqueous solutions and tagged with the recognition molecules or drugs. Here, we review surface modification approaches to, and strategies for, conjugation of bioactive molecules with QDs. There are a variety of methods of QD surface modification and QD incorporation into larger delivery systems that yield fluorescent nanocarriers from 10 nm to several micrometers. Conjugates of QDs with peptides, proteins, antibodies, oligonucleotides, and small molecules have been used for fluorescent targeting, tracking, and imaging both in vitro and in vivo. Due to an extremely high stability to photobleaching, QDs were used for long-term visualization. QD applications pave the way for new generations of ultrasensitive detection, diagnostic systems, as well as drug delivery approaches, combining accurate targeting, delivery, and imaging in a single assay.