Samer Doughan, Uvaraj Uddayasankar and Ulrich J. Krull*

A paper-based resonance energy transfer nucleic acid hybridization assay using upconversion nanoparticles as donors and quantum dots as acceptors

Samer Doughan, Uvaraj Uddayasankar and Ulrich J. Krull*

Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON, L5L 1C6, Canada

*Author to whom correspondence should be addressed:

Abstract:

Monodisperse aqueous upconverting nanoparticles (UCNPs) were covalently immobilized on aldehyde modified cellulose paper via reduction amination to develop a luminescence resonance energy transfer (LRET)-based nucleic acid hybridization assay. This first account of covalent immobilization of UCNPs on paper for a bioassay reports an optically responsive method that is sensitive, reproducible and robust. The immobilized UCNPs were decorated with oligonucleotide probes to capture HPRT1 housekeeping gene fragments, which in turn brought reporter conjugated quantum dots (QDs) in close proximity to the UCNPs for LRET. This sandwich assay could detect unlabeled oligonucleotide target, and had a limit of detection of 13 fmol and a dynamic range spanning nearly 3 orders of magnitude. The use of QDs, which are excellent LRET acceptors, demonstrated improved sensitivity, limit of detection, dynamic range and selectivity compared to similar assays that have used molecular fluorophores as acceptors. The selectivity of the assay was attributed to the decoration of the QDs with polyethylene glycol to eliminate non-specific adsorption. The kinetics of hybridization were determined to be diffusion limited and full signal development occurred within 3 minutes.

Keywords: Upconversion Nanoparticles, Quantum Dots, Luminescence Resonance Energy Transfer, Nucleic Acid Hybridization, Bioassay, Paper

1. Introduction:

Lanthanide doped upconverting nanoparticles (UCNPs) have attracted much attention for applications in bioanalysis due to their unique optical properties. Upconversion is based on the sequential absorption of two or more photons in the IR region of the electromagnetic spectrum followed by the narrow band emission of radiation of higher energy within the UV to NIR region. IR excitation minimizes autofluorescence from biological material, and reduces optical background that is commonly associated with an excitation source that operates in the UV or visible wavelength range. In addition, lanthanide doped UCNPs emit multiple narrow and well-defined emission peaks suitable for multiplexed optical analysis[1]. UCNPs have been used in bioassays as passive labels, and as energy donors in LRET for the detection of nucleic acids[2-4], proteins[5-7] and metal ions[8-10]. While UCNP LRET-based assays offer access to a ratiometric approach that provides for good precision, they suffer from LRET efficiencies that are generally well below 50%. Improving the LRET efficiency provides higher sensitivity and lower detection limits of bioassays. Reported strategies to improve LRET efficiency include surface decoration of UCNPs[11], adjustment of donor and acceptor distance[12] and optimization of LRET acceptor properties[13]. We have previously demonstrated the use of a sandwich-based assay format for the detection of thrombin where a dense monolayer of UCNPs deposited onto a glass surface allows a single LRET acceptor to interact with multiple donors[5]. The multiple donor - acceptor interactions at the surface provided about 4 fold enhancement of the LRET ratio.

To further improve sensitivity and dynamic range, in this work UCNPs were immobilized on paper to make use of the large surface area associated with the three dimensional matrix. Paper based assays have attracted much attention due to their low cost, fluid transport via capillary action, and easy modification and patterning[14]. More importantly, the three dimensional nature of paper was reported by Noor et al. to be capable of providing more than a 10 fold enhancement in fluorescence resonance energy transfer (FRET) ratio for immobilized quantum dots (QDs) and dye acceptors in a label free nucleic acid hybridization assay[15, 16]. The enhancement was attributed to the large available surface area, in combination with the contraction of wet paper upon drying that brought neighboring donors and acceptors into closer proximity[16]. Zhou et al. adsorbed UCNPs on paper for the detection of dye labeled oligonucleotides, however, the assay was limited in sensitivity and selectivity[17, 18]. Herein, we report a novel design for the sensitive and selective detection of unlabeled target oligonucleotide on paper using covalently immobilized UCNPs as donors and QDs as acceptors. QDs are more photostable, offer higher extinction coefficients and wider absorption spectra than molecular dyes, and are known as excellent LRET acceptors[19].However, QDs have broad spectral absorption profiles and this has limited their use as acceptors. This is primarily because the wavelengths that are used to excite donors will often concurrently directly excite QDs, making it impossible to excite the QDs only by resonance energy transfer from the donor[19]. Use of QDs as acceptors is typically limited to chemiluminescence energy transfer (CRET)[20] and bioluminescence energy transfer (BRET)[21] where no excitation source is used. QDs have also been used as acceptors with lanthanide complexes as donors in time gated measurements[22]. Herein, QDs are effectively used as LRET acceptors without the need for time gated measurements. An epifluorescence microscope equipped with a continuous 980 nm laser provides for photoluminescence from UCNPs that in turn can excite QDs, where the intensity of acceptor emission is measured using a photomultiplier tube in conjunction with appropriate band pass filters. The narrow and well defined emission peaks of both donor and acceptor makes it possible to collect LRET sensitized QD photoluminescence in the absence of any donor background.

This work presents the first account of use of covalently immobilized UCNPs on paper as LRET donors for the optical detection of unlabeled nucleic acid targets (Figure 1). Oligonucleotide probes decorating the UCNPs capture HPRT1 housekeeping gene fragments. An unhybridized segment of the HPRT1 target in turn hybridizes with an oligonucleotide reporter that is conjugated to QDs. This results in localization of QDs in close proximity to the UCNPs for LRET. The kinetics of hybridization are optimized, and non-specific adsorption by QDs is eliminated to build a hybridization assay that offers speed and high selectivity[17].

Figure 1: The strategy for the detection of target DNA. Probe oligonucleotide is conjugated to an UCNP, and the UCNP is immobilized on a paper substrate. Target oligonucleotide serves to bridge probe oligonucleotide on the UCNP and reporter oligonucleotide on the QD. Excitation of the UCNP at 980 nm provides for luminescence that excites the QD. The paper substrate is prepared to localize 3 reaction spots that are defined by wax printing. The orange, black and green nucleic acid strands represent the probe, target and reporter strands, respectively.

2. Experimental

A full list of Materials and Instrumentation can be found in the supporting information.

2.1 Synthesis of NaYF4: 0.5% Tm3+, 30% Yb3+/NaYF4 core/shell UCNPs

Core NaYF4: 0.5% Tm3+, 30% Yb3+ UCNPs were synthesized according to previous reports[23]. In short, 0.4562, 0.2534, and 0.0042 g of Y(CH3CO2)3.xH2O, Yb(CH3CO2)3.4H2O and Tm(CH3CO2)3.xH2O were stirred gently in 30 mL octadecene and 12 mL oleic acid (OA) under vacuum at 115 °C for 30 min. The mixture was then cooled to 50 °C under argon before a 20 mL methanol solution containing 0.20 g NaOH and 0.30 g NH4F was added. The mixture was stirred for 30 min and was then heated to 75 °C to evaporate the methanol. The temperature was rapidly increased to 300 °C and maintained for one hour. The solution was allowed to cool to room temperature before the core UCNPs were precipitated using ethanol and centrifugation at 4500 rpm. The UCNPs were resuspended in hexanes and recaptured with ethanol and centrifugation. Core UCNPs were stored in hexanes at 4 °C.

The core UCNPs were subsequently capped with a NaYF4 shell. In a three neck round bottom flask, 0.5738 g of Y(CH3CO2)3.xH2O were stirred gently in 30 mL octadecene and 12 mL OA under vacuum at 115 °C for 30 min. The temperature was lowered to 80 °C with the mixture under argon, and core UCNPs in hexanes were added. The reaction temperature was maintained until the hexane was evaporated after which the reaction was cooled to 50 °C. A volume of 20 mL methanol solution containing 0.14 g NaOH and 0.26 g NH4F was added and stirred for 30 min. The temperature was increased to 75 °C to evaporate the methanol, and then the temperature was rapidly increased to 300 °C and maintained for one hour. Core/shell UCNPs were precipitated using ethanol and centrifugation at 4500 rpm. The UCNPs were resuspended in hexanes and recaptured with ethanol and centrifugation three times. The core/shell UCNPs were stored in hexanes at 4 °C.

2.2 Preparation of water soluble UCNPs

Oleic acid capped core/shell UCNPs were made water soluble by ligand exchange with o-phosphorylethanolamine (PEA). In a typical reaction, 100 mg of OA-UCNPs in 2 mL hexanes were mixed with 400 mg of PEA and 1 mL of tetramethylammonium hydroxide (TMAH) in 10 mL of ethanol[5]. The reaction was allowed to stir vigorously overnight in a capped glass vial at 70 °C. The PEA coated UCNPs were recovered by centrifugation at 3500 rpm. The UCNPs were washed three times by sonication in ethanol for 5 min before the addition of hexanes and centrifugation at 4500 rpm. The washed PEA-UCNPs were suspended in 10 mL of water before passing through a 0.2 µm poly(ether sulfone) (PES) syringe filter to remove any aggregates. The aqueous UCNPs were stored at 4 °C in excess PEA.

2.3 Modification of paper and immobilization of PEA-UCNPs

Reaction zones in the form of spots with an inner diameter of 0.3 cm were defined on paper by wax printing. Hydroxyl groups of cellulose in the defined spots were oxidized using an aqueous solution containing 30 mg mL-1 lithium chloride and 10 mg mL-1 sodium periodate. Each spot was treated with 10 µL of the oxidizing solution and was allowed to incubate at 50 °C until dryness. The papers were washed with purified water in a 50 mL tube for 5 min and were dried in a desiccator.

PEA-UCNPs were immobilized on aldehyde functionalized paper via reduction amination. Excess PEA from the stock PEA-UCNP solution was first removed using a 100 kDa centrifugal filter. A 1.5 mg mL-1 PEA-UCNP solution was then prepared in HEPES buffer (100mM, pH 7.2) containing 1 mM sodium cyanoborohydride. Into each spot, 5 µL of the UCNP solution was pipetted and the spots were allowed to incubate for 10 minutes before they were washed with a 0.1% v/v aqueous Tween® 20 solution for 5 minutes. The papers were then washed with purified water for 2 minutes before they were dried in a desiccator.

2.4 Preparation of hexahistidine functionalized oligonucleotides and mPEG thiol

Modifications were based on previous reports[24, 25]. The thiol modified reporter nucleic acids (Table 1), diluted in 1 x PBS, were incubated with 500 molar equivalents of dithiothreitol (DTT) for 1 hour at room temperature to reduce the disulfides into sulfhydryl moieties. The nucleic acids were then isolated from excess DTT by extracting the aqueous solution with ethyl acetate four times. To functionalize the nucleic acid with the peptide, 10 molar equivalents of the maleimide-functionalized peptide (dissolved in dimethyl sulfoxide; (6-Maleimidohexanoic acid) – G(Aib)GHHHHHH) was added, and the solution was allowed to shake for 12 hours at room temperature. Excess peptide was then removed by passing the nucleic acid solution through a NAP-5 desalting column, as per the manufacturer’s instructions. The purified nucleic acid was quantified using UV-vis spectroscopy and then stored at -20 0C.

The thiol functionalized poly(ethylene glycol) (PEG) methyl ether (Mn = 6000 g mol-1) was incubated with 10 molar equivalents of tris (2-carboxyethyl) phosphine (TCEP) for 2 hours to reduce any disulfides. Excess TCEP was removed using a NAP-5 desalting column, and the thiol mPEG was then incubated with 10 equivalents of maleimide-functionalized peptide for 12 hours. Un-reacted peptide was subsequently removed using a NAP-5 desalting column.

2.5 Preparation of QD-reporter conjugates

Alkyl 575nm CdSxSe1-x/ZnS core/shell QDs were obtained from Cytodiagnostics Inc. (Burlington, ON, Canada) and were made water soluble via ligand exchange with reduced L-glutathione (GSH)[15]. In one glass vial, 75 µL of 10 µM alkyl QDs was suspended in 2mL chloroform, and in another glass vial, 0.2 g of GSH was dissolved in 600 µL of TMAH. The solution containing QDs was then added drop-wise to the GSH solution while swirling. The mixture was allowed to sit overnight and the QDs were collected by centrifugation. The GSH QDs were washed three times by resuspension in pH 9.25 borate buffer (50 mM, 100 mM NaCl) and collection with ethanol and centrifugation. The QDs were then stored in pH 9.25 borate buffer (50 mM, 100 mM NaCl) at 4 °C.

The hexahistidine modified oligonucleotides were incubated with GSH QDs at the desired QD:DNA ratio for one hour in pH 9.25 borate buffer (50 mM, 100 mM NaCl). The solution was then treated with hexahistidine modified PEG at 15x excess the amount of QDs for one hour. The solution was washed three times using a 100 kDa centrifugal filter. The PEG stabilized DNA conjugated QDs were collected and stored in pH 9.25 borate buffer (50 mM, 100 mM NaCl) at 4 °C.

2.6 Quantification of the average number of oligonucleotides per QD

After the QDs were incubated with the desired amount of hexahistidine modified DNA, the average number of oligonucleotides conjugated per QD was determined using a well-established method[20]. The QD conjugates were purified as previously described and their absorbance spectra was obtained. The contribution of the oligonucleotides to the absorbance at 260nm was obtained by subtracting the contribution provided by the presence of the QDs. The latter was obtained from a normalized absorbance spectrum of the QDs in the absence of DNA. The average number of oligonucleotides per QD was then determined by the ratio of the concentrations of oligonucleotides to QDs.

2.7 Hybridization and detection on paper substrates

Spots containing immobilized PEA-UCNPs were treated with 3 µL aliquots of a 2 mM aqueous solution of NHS-PEG4-biotin. The spots were allowed to dry, and the paper was washed for 2 min in purified water before it was dried in a desiccator. Subsequently, 5 µL aliquots of a 20 µM avidin solution in HEPES buffer (100 mM, pH 7.2) was pipetted onto the spots. The paper was allowed to dry before it was washed for 2 minutes with borate buffer (50 mM, pH 9.25) and dried again. Each spot was then treated with a 5 µL of solution containing 10 µM biotinylated oligonucleotide probe and 10 µM biotinylated oligonucleotide of non-complementary sequence but of the same length to ensure dispersion of the probe on the surface. The surface of the paper was then blocked by treatment with a 20 µM solution of unlabelled oligonucleotide to minimize non-specific adsorption and the paper was washed for 2 minutes in pH 9.25 borate buffer (50 mM, 100 mM NaCl). After the paper was dried, 3 µL solutions with the desired DNA target concentration ranging from 5 nM to 2 µM were introduced. The paper was allowed to dry before the addition of a 3 µL solution containing reporters consisting of PEG stabilized DNA conjugated to GSH-QDs. The solution was allowed to incubate for 5 minutes and was washed with pH 9.25 borate buffer (50 mM, 100 mM NaCl) containing 0.1% v/v Tween for 2 minutes. The paper was allowed to dry in a desiccator before it was imaged using an epi-fluorescence microscope.