Abstract
Quantum dot (QD) labeling combined with fluorescence lifetime imaging microscopy is proposed as a powerful transduction technique for the detection of DNA hybridization events. Fluorescence lifetime analysis of DNA microarray spots of hybridized QD labeled target indicated a characteristic lifetime value of 18.8 ns, compared to 13.3 ns obtained for spots of free QD solution, revealing that QD labels are sensitive to the spot microenvironment. Additionally, time gated detection was shown to improve the microarray image contrast ratio by 1.8, achieving femtomolar target sensitivity. Finally, lifetime multiplexing based on Qdot525 and Alexa430 was demonstrated using a single excitation-detection readout channel.
Highlights
DNA and protein microarrays are important tools for biomolecular detection with applications ranging from gene expression to clinical diagnostics [1,2,3]
Were constructed by spotting a matrix of twelve solubilized Qdot605-streptavidin-conjugates (1nM) on to epoxy silane coated glass slides and 2.) “Quantum dot (QD)-labeled DNA microarrays” were constructed by spotting twelve HCMV DNA probes on to epoxy coated glass slides and subsequently hybridizing with biotinylated complementary HCMV target labeled with Qdot605-streptavidin-conjugates
We observe that spots on the “QD microarray” had a distinctively shorter lifetime compared to spots on the “QD-labeled DNA microarray”
Summary
DNA and protein microarrays are important tools for biomolecular detection with applications ranging from gene expression to clinical diagnostics [1,2,3]. The multiplexing of labels is limited by the broad emission profiles associated with most organic dyes and requires a complex experimental setup, incorporating several excitation wavelength and detection channels In this manuscript, we explore an alternative transduction approach that combines quantum dot (QD) labeling and fluorescence lifetime imaging microscopy [10]. Quantum dots possess a number of advantages over traditional dyes, such as: a high quantum yield, long photostability, potential for multiplexing and a relatively long excited state lifetime [11] These properties have triggered a strong interest within the biosensing community [12,13,14,15,16] resulting in QD being used to label antigen and oligonucleotide microarrays [17,18,19]. Applications of QD labeling combined with fluorescence lifetime detection, both in terms of sensitivity and multiplexing capabilities
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