Camera-Based Ratiometric Fluorescence Transduction of Nucleic Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform Using Immobilized Quantum Dots as Donors

Abstract

Paper-based diagnostic assays are gaining increasing popularity for their potential application in resource-limited settings and for point-of-care screening. Achievement of high sensitivity with precision and accuracy can be challenging when using paper substrates. Herein, we implement the red-green-blue color palette of a digital camera for quantitative ratiometric transduction of nucleic acid hybridization on a paper-based platform using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). A nonenzymatic and reagentless means of signal enhancement for QD-FRET assays on paper substrates is based on the use of dry paper substrates for data acquisition. This approach offered at least a 10-fold higher assay sensitivity and at least a 10-fold lower limit of detection (LOD) as compared to hydrated paper substrates. The surface of paper was modified with imidazole groups to assemble a transduction interface that consisted of immobilized QD-probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as an acceptor. A hybridization event that brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs was responsible for a FRET-sensitized emission from the acceptor dye, which served as an analytical signal. A hand-held UV lamp was used as an excitation source and ratiometric analysis using an iPad camera was possible by a relative intensity analysis of the red (Cy3 photoluminescence (PL)) and green (gQD PL) color channels of the digital camera. For digital imaging using an iPad camera, the LOD of the assay in a sandwich format was 450 fmol with a dynamic range spanning 2 orders of magnitude, while an epifluorescence microscope detection platform offered a LOD of 30 fmol and a dynamic range spanning 3 orders of magnitude. The selectivity of the hybridization assay was demonstrated by detection of a single nucleotide polymorphism at a contrast ratio of 60:1. This work provides an important framework for the integration of QD-FRET methods with digital imaging for a ratiometric transduction of nucleic acid hybridization on a paper-based platform.

Cellphone-based diagnostic devices

“…Noor and Krull reported on a DNA diagnostic platform ( Figure 14) using a paper-based fluorescence resonance energy transfer (FRET) sensor and an iPhone or iPad for fluorescence detection [85]. …”

Resonance Energy Transfer-Based Nucleic Acid Hybridization Assays on Paper-Based Platforms Using Emissive Nanoparticles as Donors

“…Salt aging is used to increase the loading capacity of oligonucleotide probes to the surface of QDs by screening the electrostatic repulsion associated with the negative charge of the sugar-phosphate backbone of oligonucleotides [21]. We have previously reported that without the salt-aging step, an average of 17 oligonucleotide probes can be self-assembled to the surface of similar GSH-QDs when incubated with 40 times molar excess of the oligonucleotide probes [21]. However, with the salt-aging step, an average of 35-40 oligonucleotide probes can be self-assembled to the surface of the GSH-QDs for the aforementioned preparation [21].…”

“…We have previously reported that without the salt-aging step, an average of 17 oligonucleotide probes can be self-assembled to the surface of similar GSH-QDs when incubated with 40 times molar excess of the oligonucleotide probes [21]. However, with the salt-aging step, an average of 35-40 oligonucleotide probes can be self-assembled to the surface of the GSH-QDs for the aforementioned preparation [21].…”

“…We have previously reported that data collection from dry paper substrates offers at least an order of magnitude higher assay sensitivity and at least an order of magnitude lower limit of detection as compared to the corresponding hydrated paper substrates [21].…”

Nano-Technological Approaches to Improve the Efficiency of Bio-Assays

“…The state of the art personal healthcare industry has put significant emphasis on the development of easy to use portable diagnostic platforms like Integrated quantum dot barcode smartphone optical device for wireless multiplexed diagnosis [17], camera-based ratio metric fluorescence transduction on a paper-based platform using immobilized quantum dots as FRET donors [18]. Algar et al, showed QDs as simultaneous acceptors and donors in time gated FRET relay events and their application in biosensing [19].…”