16 research outputs found
Detection of cystic fibrosis transmembrane conductance regulator Δf508 gene mutation using a paper-based nucleic acid hybridization assay and a smartphone camera
Diagnostic technology that makes use of paper platforms in conjunction with the ubiquitous availability of digital cameras in cellular telephones and personal assistive devices offers opportunities for development of bioassays that are cost effective and widely distributed. Assays that operate effectively in aqueous solution require further development for implementation in paper substrates, overcoming issues associated with surface interactions on a matrix that offers a large surface-to-volume ratio and constraints on convective mixing. This report presents and compares two related methods for determination of oligonucleotides that serve as indicators of cystic fibrosis, differentiating between the normal wild-type sequence, and a mutant-type sequence that has a 3-base replacement. The transduction strategy operates by selective hybridization of oligonucleotide probes that are conjugated to fluorescent quantum dots, where hybridization of target sequences causes a molecular fluorophore to approach the quantum dot and become emissive through fluorescence resonance energy transfer. Detection can rely on hybridization of a target that is labelled with Cy3 fluorophore, or in the presence of an unlabelled target when a sandwich assay format is implemented with a labelled reporter oligonucleotide. Selectivity to determine the presence of mismatched sequences involves appropriate selection of nucleotide sequences to set melt temperatures, in conjunction with control of stringency conditions using formamide as a chaotrope. It was determined that both direct and sandwich assays on paper substrates are able to distinguish between wild-type and mutant-type samples.Natural Sciences and Engineering Research Council of Canad
A Paper-Based Sandwich Format Hybridization Assay for Unlabeled Nucleic Acid Detection Using Upconversion Nanoparticles as Energy Donors in Luminescence Resonance Energy Transfer
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
Paper-based biodetection using luminescent nanoparticles
Point-of-care and in-field technologies for rapid, sensitive and selective detection of molecular biomarkers have attracted much interest. Rugged bioassay technology capable of fast detection of markers for pathogens and genetic diseases would in particular impact the quality of health care in the developing world, but would also make possible more extensive screening in developed countries to tackle problems such as associated with water and food quality, and tracking of infectious organisms in hospitals and clinics. Literature trends indicate an increasing interest in the use of nanomaterials, and in particular luminescent nanoparticles, for assay development. These materials may offer attributes for development of assays and sensors that could achieve improvements in analytical figures of merit, and provide practical advantages in sensitivity and stability. There is opportunity for cost-efficiency and technical simplicity by implementation of luminescent nanomaterials as the basis for transduction technology, when combined with the use of paper substrates, and the ubiquitous availability of cell phone cameras and associated infrastructure for optical detection and transmission of results. Luminescent nanoparticles have been described for a broad range of bioanalytical targets including small molecules, oligonucleotides, peptides, proteins, saccharides and whole cells (e.g., cancer diagnostics). The luminescent nanomaterials that are described herein for paper-based bioassays include metal nanoparticles, quantum dots and lanthanide-doped nanocrystals. These nanomaterials often have broad and strong absorption and narrow emission bands that improve opportunity for multiplexed analysis, and can be designed to provide emission at wavelengths that are efficiently processed by conventional digital cameras. Luminescent nanoparticles can be embedded in paper substrates that are designed to direct fluid flow, and the resulting combination of technologies can offer competitive analytical performance at relatively low cost.J.Q., M.O.N and U.J.K. gratefully acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support of this research. This work is also supported by NSFC (Nos. 61505077 and 21501089), and the Natural Science Foundation of Jiangsu Province, China (BK20150939, and BK20150936). M.O.N is also grateful to the Ontario Centres of Excellence (OCE) for provision of a TalentEdge postdoctoral fellowship
Paper-Based Solid-Phase Multiplexed Nucleic Acid Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As Donors in Fluorescence Resonance Energy Transfer
A multiplexed
solid-phase nucleic acid hybridization assay on a paper-based platform
is presented using multicolor immobilized quantum dots (QDs) as donors
in fluorescence resonance energy transfer (FRET). The surface of paper
was modified with imidazole groups to immobilize two types of QD-probe
oligonucleotide conjugates that were assembled in solution. Green-emitting
QDs (gQDs) and red-emitting QDs (rQDs) served as donors with Cy3 and
Alexa Fluor 647 (A647) acceptors. The gQD/Cy3 FRET pair served as
an internal standard, while the rQD/A647 FRET pair served as a detection
channel, combining the control and analytical test zones in one physical
location. Hybridization of dye-labeled oligonucleotide targets provided
the proximity for FRET sensitized emission from the acceptor dyes,
which served as an analytical signal. Hybridization assays in the
multicolor format provided a limit of detection of 90 fmol and an
upper limit of dynamic range of 3.5 pmol. The use of an array of detection
zones was designed to provide improved analytical figures of merit
compared to that which could be achieved on one type of array design
in terms of relative concentration of multicolor QDs. The hybridization
assays showed excellent resistance to nonspecific adsorption of oligonucleotides.
Selectivity of the two-plex hybridization assay was demonstrated by
single nucleotide polymorphism (SNP) detection at a contrast ratio
of 50:1. Additionally, it is shown that the use of preformed QD-probe
oligonucleotide conjugates and consideration of the relative number
density of the two types of QD-probe conjugates in the two-color assay
format is advantageous to maximize assay sensitivity and the upper
limit of dynamic range
Camera-Based Ratiometric Fluorescence Transduction of Nucleic Acid Hybridization with Reagentless Signal Amplification on a Paper-Based Platform Using Immobilized Quantum Dots as Donors
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
Paper-Based Solid-Phase Nucleic Acid Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence Resonance Energy Transfer
A paper-based solid-phase assay is presented for transduction
of
nucleic acid hybridization using immobilized quantum dots (QDs) as
donors in fluorescence resonance energy transfer (FRET). The surface
of paper was modified with imidazole groups to immobilize QD–probe
oligonucleotide conjugates that were assembled in solution. Green-emitting
QDs (gQDs) were FRET-paired with Cy3 acceptor. Hybridization of Cy3-labeled
oligonucleotide targets provided the proximity required for FRET-sensitized
emission from Cy3, which served as an analytical signal. The assay
exhibited rapid transduction of nucleic acid hybridization within
minutes. Without any amplification steps, the limit of detection of
the assay was found to be 300 fmol with the upper limit of the dynamic
range at 5 pmol. The implementation of glutathione-coated QDs for
the development of nucleic acid hybridization assay integrated on
a paper-based platform exhibited excellent resistance to nonspecific
adsorption of oligonucleotides and showed no reduction in the performance
of the assay in the presence of large quantities of noncomplementary
DNA. The selectivity of nucleic acid hybridization was demonstrated
by single-nucleotide polymorphism (SNP) detection at a contrast ratio
of 19 to 1. The reuse of paper over multiple cycles of hybridization
and dehybridization was possible, with less than 20% reduction in
the performance of the assay in five cycles. This work provides an
important framework for the development of paper-based solid-phase
QD–FRET nucleic acid hybridization assays that make use of
a ratiometric approach for detection and analysis
Inorganic Nanoparticles as Donors in Resonance Energy Transfer for Solid-Phase Bioassays and Biosensors
Bioassays for the rapid detection and quantification of specific nucleic acids, proteins and peptides are fundamental tools in many clinical settings. Traditional optical emission methods have focused on the use of molecular dyes as labels to track selective binding interactions, and as probes that are sensitive to environmental changes. Such dyes can offer good detection limits based on brightness, but typically have broad emission bands and suffer from time-dependent photobleaching. Inorganic nanoparticles such as quantum dots and upconversion nanoparticles are photo-stable over prolonged exposure to excitation radiation and tend to offer narrow emission bands, providing greater opportunity for multi-wavelength multiplexing. Importantly, in contrast to molecular dyes, nanoparticles offer substantial surface area and can serve as platforms to carry a large number of conjugated molecules. The surface chemistry of inorganic nanoparticles offers both challenges and opportunities for control of solubility and functionality for selective molecular interactions by assembly of coatings through coordination chemistry. This report reviews advances in the compositional design and methods of conjugation of inorganic quantum dots and upconversion nanoparticles, and the assembly of combinations of nanoparticles to achieve energy exchange. The interest is exploration of configurations where the modified nanoparticles can be immobilized to solid substrates for the development of bioassays and biosensors that operate by resonance energy transfer (RET).This work was sponsored by the Natural Sciences and Engineering Council of Canad
Biosensing with Quantum Dots: A Microfluidic Approach
Semiconductor quantum dots (QDs) have served as the basis for signal development in a variety of biosensing technologies and in applications using bioprobes. The use of QDs as physical platforms to develop biosensors and bioprobes has attracted considerable interest. This is largely due to the unique optical properties of QDs that make them excellent choices as donors in fluorescence resonance energy transfer (FRET) and well suited for optical multiplexing. The large majority of QD-based bioprobe and biosensing technologies that have been described operate in bulk solution environments, where selective binding events at the surface of QDs are often associated with relatively long periods to reach a steady-state signal. An alternative approach to the design of biosensor architectures may be provided by a microfluidic system (MFS). A MFS is able to integrate chemical and biological processes into a single platform and allows for manipulation of flow conditions to achieve, by sample transport and mixing, reaction rates that are not entirely diffusion controlled. Integrating assays in a MFS provides numerous additional advantages, which include the use of very small amounts of reagents and samples, possible sample processing before detection, ultra-high sensitivity, high throughput, short analysis time, and in situ monitoring. Herein, a comprehensive review is provided that addresses the key concepts and applications of QD-based microfluidic biosensors with an added emphasis on how this combination of technologies provides for innovations in bioassay designs. Examples from the literature are used to highlight the many advantages of biosensing in a MFS and illustrate the versatility that such a platform offers in the design strategy