Paper-Based Solid-Phase Multiplexed Nucleic Acid Hybridization Assay with Tunable Dynamic Range Using Immobilized Quantum Dots As Donors in Fluorescence Resonance Energy Transfer

Abstract

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.

Ratiometric fluorescence transduction by hybridization after isothermal amplification for determination of zeptomole quantities of oligonucleotide biomarkers with a paper-based platform and camera-based detection

“…In addition, hybridization reactions done with uidA(93) FC TGT that was fortified with 86% (v/v) goat serum in BBS showed the same response as the hybridization reactions done in BBS only and retained excellent resistance to non-specific adsorption of oligonucleotides (see Figure S5 for details). These results are consistent with our previous studies that have also shown that the GSH-QDs exhibit excellent resistance to non-specific adsorption of oligonucleotides for the development of paper-based QD-FRET nucleic acid hybridization assays [34,35].…”

“…For these experiments, the GSH-QDs were modified with SMN1 probes. The optimization of stringency conditions for SNP discrimination was based on our previously published results that involved a concurrent adjustment of the ionic strength and formamide concentration of the solution [34,35]. This method for SNP discrimination is advantageous as it allows mismatch discrimination at room temperature conditions.…”

“…Previous single-color (one donor-one acceptor) solid-phase QD-FRET nucleic acid hybridization assays have been reported using optical fibers [36,40], glass beads [41], polystyrene well plates [42], paper substrates [29,34,35] and within microfluidic channels [43,44]. These assays were conducted using one density of immobilized QDs for a given solid substrate.…”

“…FRET offers the possibility of a ratiometric quantification approach, which is practically advantageous as the ratiometric detection is less prone to signal variations that are caused by fluctuations in the intensity of excitation source or the detector sensitivity response [33]. A paper-based solid-phase nucleic acid hybridization assay that employs immobilized QDs as donors in FRET-based transduction method offers additional advantages of improved assay sensitivity, containment of QDs, and elimination of the requirement of maintaining the colloidal stability of QDs [34,35]. We have recently reported a ratiometric solid-phase QD-FRET nucleic acid hybridization assay on a paper-based platform that used a low-cost iPad camera as a readout detector, rendering the assay format more suitable for field portable and point-of-care applications [29].…”

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

“…[1] QDs are popular RET donors due to their high quantum yield and tuneable photoluminescence (PL).The surface area of QDs allows the immobilization of multiple recognition elements, where a single QD can participate in multiple RET events [1]. Our group has paired green-emitting QDs (gQDs) and red-emitting QDs (rQDs) with various molecular dyes as RET acceptors in nucleic acid hybridization assays [2,3].While QDs are popular RET donors, their use as RET acceptors has been limited to time gated measurements [4], chemiluminescence resonance energy transfer (CRET) [5] and bioluminescence resonance energy transfer (BRET) [6]. The broad absorbance band of QDs that stretches into the UV region of the spectrum makes them susceptible to direct excitation in the process of exciting the RET donor.…”