Abstract
Bioassays based on cellulose paper substrates are gaining increasing popularity for the development of field portable and low-cost diagnostic applications. Herein, we report a paper-based nucleic acid hybridization assay using immobilized upconversion nanoparticles (UCNPs) as donors in luminescence resonance energy transfer (LRET). UCNPs with intense green emission served as donors with Cy3 dye as the acceptor. The avidin functionalized UCNPs were immobilized on cellulose paper and subsequently bioconjugated to biotinylated oligonucleotide probes. Introduction of unlabeled oligonucleotide targets resulted in a formation of probe-target duplexes. A subsequent hybridization of Cy3 labeled reporter with the remaining single stranded portion of target brought the Cy3 dye in close proximity to the UCNPs to trigger a LRET-sensitized emission from the acceptor dye. The hybridization assays provided a limit of detection (LOD) of 146.0 fmol and exhibited selectivity for one base pair mismatch discrimination. The assay was functional even in undiluted serum samples. This work embodies important progress in developing DNA hybridization assays on paper. Detection of unlabeled targets is achieved using UCNPs as LRET donors, with minimization of background signal from paper substrates owing to the implementation of low energy near-infrared (NIR) excitation.
Highlights
Upconversion nanoparticles (UCNPs) are a category of luminescent materials with intriguing properties for development of bioanalytical assays [1]
Sandwich format DNA assay on paper using upconversion nanoparticles (UCNPs) as energy donors in luminescence resonance energy transfer (LRET)
LRET-based sandwich format hybridization assays have been developed for determination of unlabeled oligonucleotide target on cellulose paper using UCNPs as energy donors and Cy3-labeled oligonucleotide reporters as energy acceptors
Summary
Upconversion nanoparticles (UCNPs) are a category of luminescent materials with intriguing properties for development of bioanalytical assays [1]. Superior sensitivity and detection limit were achieved in comparison to an analogous DNA hybridization assay using quantum dots (QDs) as energy donors due to reduction of background signal by use of 980 nm laser excitation We further extended this method to a two-plex DNA hybridization assay using two different dye-labeled targets that interacted with two different wavelengths of emission from one form of UCNP [16]. Labeling of target DNA with molecular dyes in a real samples of complex composition can be challenging, and adds time and cost to analysis One solution to this issue is implementation of sandwich assays where a reporter oligonucleotide associates with a probe-target hybrid [20]. The ratiometric method allowed for quantification of the un-labeled target oligonucleotide with a competitive limit of detection (LOD), and selectivity for one base pair mismatch (1BPM) target discrimination, using samples containing various interferents, and even samples prepared using undiluted serum
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
