Luminescence Resonance Energy Transfer-Based Nucleic Acid

Feb 7, 2014 - North, Mississauga, Ontario L5L 1C6, Canada. •S Supporting Information. ABSTRACT: A bioassay based on DNA hybridization on cellulose ...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/ac

Luminescence Resonance Energy Transfer-Based Nucleic Acid Hybridization Assay on Cellulose Paper with Upconverting Phosphor as Donors Feng Zhou, M. Omair Noor, and Ulrich J. Krull* Chemical Sensors Group, Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, Ontario L5L 1C6, Canada S Supporting Information *

ABSTRACT: A bioassay based on DNA hybridization on cellulose paper is a promising format for gene fragment detection that may be suited for in-field and rapid diagnostic applications. We demonstrate for the first time that luminescence resonance energy transfer (LRET) associated with upconverting phosphors (UCPs) can be used to develop a paper-based DNA hybridization assay with high sensitivity, selectivity and fast response. UCPs with strong green emission were synthesized and subsequently functionalized with streptavidin (UCP-strep). UCP-strep particles were immobilized on cellulose paper, and then biotinylated single-stranded oligonucleotide probes were conjugated onto the UCPs via streptavidin−biotin linkage. The UCPs served as donors that were LRET-paired with Cy3-labeled target DNA. Selective DNA hybridization enabled the proximity required for LRET-sensitized emission from Cy3, which was used as the detection signal. Hybridization was complete within 2 min, and the limit of detection of the method was 34 fmol, which is a significant improvement in comparison to an analogous fluorescence resonance energy transfer (FRET) assay based on quantum dots. The assay exhibited excellent resistance to nonspecific adsorption of noncomplementary short/long DNA and protein. The selectivity of the assay was further evaluated by one base pair mismatched (1BPM) DNA detection, where a maximum signal ratio of 3.1:1 was achieved between fully complementary and 1BPM samples. This work represents a preliminary but significant step for the development of paper-based UCP-LRET nucleic acid hybridization assays, which offer potential for lowering the limit of detection of luminescent hybridization assays due to the negligible background signal associated with optical excitation by near-infrared (NIR) light.

A

UCPs as direct labels in a sandwich format enzyme-linked immunosorbent assay (ELISA) and achieved superior sensitivity due to the low fluorescence background signal; Yliharsila et al.6 developed an array in microwell format with UCPs as direct signal reporters; Niedbala et al.7 developed a competitive assay on paper strips with UCPs as signal reporter; and Rantanen et al.8 developed a homogeneous multiplexing LRETbased sandwich assay with UCPs as LRET donors in which excitation was done with a 980 nm NIR source. These assays were conducted either in homogeneous solution or on traditional solid substrates (i.e., microwell plate) with UCPs

pplication of upconverting phosphors (UCPs) in the development of innovative sensors continues to garner attention due to the capability of converting low-energy infrared excitation radiation to UV−visible luminescence via sequential photon absorption and energy-transfer processes. Of significance is the ability of UCPs to produce strong anti-Stokes photoluminescence (PL) of narrow bandwidth when irradiated with near-infrared radiation (NIR).1,2 Such long excitation wavelengths (typically a 980 nm laser is used) have little interaction with biomolecules. Therefore, autofluorescence and scattered incident light can be virtually eliminated in luminescent assays, resulting in increased sensitivity by means of background reduction.3 Kuningas et al.4 developed a homogeneous competitive immunoassay based on luminescence resonance energy transfer (LRET) signals with UCPs as LRET donor and organic dye as acceptor; Pakkila et al.5 used © 2014 American Chemical Society

Received: December 19, 2013 Accepted: February 6, 2014 Published: February 7, 2014 2719

dx.doi.org/10.1021/ac404129t | Anal. Chem. 2014, 86, 2719−2726

Analytical Chemistry

Article

Scheme 1. Paper-Based LRET Assay Using UCPs as Donors

nanomaterials and paper-based analytical platforms to prepare cost-effective, sensitive, and stable assays that can include the capability of multiplexed detection.21−23 Most of these assays have utilized gold nanoparticles (GNPs). Detection can be achieved by making use of the significant color change that occurs due to plasmonic coupling upon particle aggregation or by methods such as induction of silver ion precipitation for signal amplification.24−26 Quantum dots (QDs) are well-suited for applications in fluorescence resonance energy transfer (FRET)-based detection and have been implemented in paperbased detection strategies.27,28 Noor et al.21,22 recently reported a FRET-based ratiometric quantitative DNA hybridization assay using QD-probe conjugates that were immobilized on cellulose paper substrates. Petryayeva and Algar 29 subsequently developed a proteolytic assay on paper that was based on QD-FRET with use of a cell phone camera as a detector. These methods using QDs typically rely on ultraviolet excitation, which can be limiting in terms of background fluorescence and light scatter from a sample matrix. Herein we report the first LRET-based hybridization assay on cellulose paper with UCP as a donor. The assay is presented in Scheme1. The SMN1 probe-target sequence was selected for study, being a representative example of a mixed-base oligonucleotide system that has clinical relevance in diagnostics of the neuromuscular disease known as spinal muscular atrophy. Synthesized oleate-coated UCPs were first converted into ligand-free nanoparticles and then were subsequently coated with sodium citrate. Streptavidin was subsequently conjugated onto the UCPs, and the UCP-strep nanoparticles were spotted onto cellulose paper. Biotinylated SMN1 DNA probe was then immobilized onto the UCP-strep. After hybridization with Cy3-labeled oligonucleotide targets, LRET-sensitized Cy3 emission upon excitation of UCP-probe conjugates at 980 nm was collected via an epifluorescence microscope. The hybridization process was complete in minutes and demonstrated high sensitivity and selectivity. The intention of this work was an initial exploration of the capability and advantages offered by UCPs in development of paper-based LRET assays.

as donors in LRET or as labels to directly report the signal of binding interactions. Paper-based analytical devices (PADs) hold great promise to revolutionize point-of-care diagnosis, especially in underdeveloped regions where laboratory resources are scarce.9,10 A number of features combine to make paper-based assays desirable. The three-dimensional microstructures of paper enable transport of fluid via capillary wicking, which obviates the need for an external power supply to achieve sample transport. The movement of sample through pores of micrometer dimensions ameliorates the limitations imposed by diffusion for interfacial reactions, resulting in substantial reduction of assay time.11 Concurrently, the porosity of paper can serve as a filtering medium to separate particles and aggregated materials from a reaction zone. Many welldeveloped methods can be used to modify the surface chemistry of paper.12,13 It is possible to introduce a variety of functional groups that can be further conjugated for immobilization of various biomolecules and nanomaterials with very small volumes of reagents and samples (typically a few microliters to nanoliters). Paper is also notably inexpensive, easily obtained, and portable in comparison with other substrates that are commonly used in assay development (microplates, glass slides, etc.)9,14 Therefore, sensors based on paper are considered as inexpensive and effective alternative substrates that are well-suited for some types of medical diagnosis, environmental monitoring, and food quality control.15−17 Recently, Allen et al.18 developed a nonenzymatic nucleic acid amplification circuit on a paper-based platform, implementing a catalytic hairpin assembly in conjunction with strand exchange reactions. In association with loop-mediated isothermal amplification (LAMP), as few as 1200 nucleic acid templates could be detected with this approach. Crooks and coworkers19 developed a competitive DNA assay on origamibased paper, where hybridization-induced fluorescence provided a limit of detection as low as 5 nM. Wang et al.20 reported a sensitive microfluidic photoelectrochemical (PEC) system for detection of DNA on a paper-based platform. A photocurrent generated by charging of the supercapacitor (PS) was used as the detection signal, and it was demonstrated that a single base pair mismatch could be discerned by this method. Moreover, there have been numerous reports of the combination of

■

EXPERIMENTAL SECTION Synthesis of β-NaYF 4:2% Er 3+ , 18% Yb3+ Core Upconverting Phosphors. β-NaYF4:2% Er3+, 18% Yb3+ 2720

dx.doi.org/10.1021/ac404129t | Anal. Chem. 2014, 86, 2719−2726

Analytical Chemistry

Article

Coating the Ligand-Free Upconverting Phosphors with Sodium Citrate. Sodium citrate coated ligand-free UCPs were prepared by using a previously reported method.32 Sodium citrate was added into a 1 mg/mL aqueous solution of ligand-free UCP, making the final concentration of sodium citrate 5 mg/mL. Then the solution was sonicated for 60 min, and the sodium citrate-coated UCPs (UCP-SC) were obtained. Conjugation of Streptavidin onto UCP-SC. To eliminate the free sodium citrate in the UCP-SC solution, 0.3 mL of solution was filter-centrifuged (cutoff size 100 KDa) at 6000 rpm for 5 min and then washed with 2-(N-morpholino)ethanesulfonic acid buffer (MES, pH 6.0). The UCP-SC was resuspended in 300 μL of MES buffer. A volume of 100 μL of “activation solution” [2 mg of 1-ethyl-3-(3dimethylaminoisopropyl)carbodiimide (EDC) and 1 mg of N-hydroxysulfosuccinimide (sulfo-NHS) in 1 mL of MES buffer, freshly prepared] was added to the UCP-SC solution, followed by 15 min of stirring. Then 50 μL of streptavidin solution (5 mg/mL) was added and the reaction was allowed to proceed at room temperature for 2 h with continuous stirring. The solution was then filtered and centrifuged to eliminate the unconjugated streptavidin. The washing was done twice with borate buffer (50 mM, pH 9.25). Finally, the UCP-strep nanoparticles were redispersed in 0.3 mL of borate buffer. Immobilization of UCP-Strep and Biotin−Probe Conjugation. UCP-strep solution (1.5 μL, 1 mg/mL) was pipetted into spots that were defined by printing a wax ring on cellulose paper (inner diameter 3 mm), followed by drying under vacuum. The paper was then soaked in borate buffer and rinsed thoroughly for 10 min to eliminate any loosely adsorbed UCP-strep. Subsequently, the treated paper was wicked on absorbent paper to assist drying. For biotin−probe conjugation, 1.5 μL of biotin−probe (10 μM) in borate buffer was pipetted onto the spots that contained UCP-strept. The paper was allowed to stand at room temperature for 1 h. Finally, the paper was rinsed in borate buffer to remove any probes that had not been covalently conjugated. Hybridization Assay. The oligonucelotide sequences used for hybridization assays are listed in Table 1. All hybridization

core UCPs were synthesized via a reported procedure with minor modifications.30 Briefly, Y(CH3CO2)3 hydrate (99.9%, 0.535 g, 1.6 mmol), Yb(CH3CO2)3 hydrate (99.9%, 0.155 g, 0.360 mmol), and Er(CH3CO2)3 hydrate (99.9%, 0.018 g, 0.04 mmol) were added to a 100 mL three-neck round-bottom flask containing octadecene (30 mL, 90%) and oleic acid (12 mL, 90%). The solution was heated to 160 °C under argon protection for 30 min with vigorous stirring. After formation of the lanthanide oleate complexes, the temperature was lowered to 50 °C. Subsequently, 20 mL of methanol solution containing ammonium fluoride (0.2964 g, 8 mmol) and sodium hydroxide (0.2 g, 5 mmol) was slowly injected into the above reaction, and the cloudy mixture was stirred for 30 min. The reaction temperature was then slowly increased to 75 °C to evaporate the methanol. Subsequently the reaction temperature was increased to 300 °C (20−30 min) and maintained for 60 min. A clear solution that was slightly yellow was obtained. After the solution was cooled to room temperature, the UCPs were precipitated by addition of ethanol and acetone and then centrifuged at 8000 rpm for 10 min. The resulting pellets were dispersed in hexane and were then precipitated again with excess ethanol/acetone. This purification process was repeated three times. The purified pellets were redispersed in hexanes (10−15 mL) for the subsequent shell growth procedure. Synthesis of β-NaYF4:2% Er3+, 18% Yb3+/β-NaYF4 Core/Shell Upconverting Phosphors. Y(CH3CO2)3 hydrate (99.9%, 0.5741 g, 1.75 mmol) was added to a 100 mL three-neck round-bottom flask containing octadecene (30 mL) and oleic acid (12 mL). Similar to the process of synthesizing the core, the solutions were first heated slowly to 160 °C under vacuum with magnetic stirring for 30 min, and then the temperature was lowered to 80 °C under an argon atmosphere. The hexane solution containing core UCPs (10−15 mL) was injected, and the resulting solution was maintained at 80 °C until all the hexanes were completely evaporated. The reaction mixture was then cooled to 50 °C, and a solution of ammonium fluoride (0.2593 g, 7 mmol) and sodium hydroxide (0.1400 g, 3.5 mmol) in methanol (20 mL) was injected. The resulting cloudy mixture was stirred for 30 min, and then the methanol was completely evaporated at 75 °C. The reaction temperature was then increased to 300 °C (20−30 min), and this temperature was maintained for another 60 min. Finally, the mixture was allowed to cool to room temperature. The subsequent washing process was identical to that used to prepare the core. The purified core/shell UCPs were kept in hexane at room temperature. Synthesis of Ligand-Free Water-Soluble Upconverting Phosphors. Ligand-free UCPs were prepared according to a reported process.31 A volume of the stock hexane solution containing 10 mg of UCPs was transferred to 2 mL of water, followed by vigorous shaking for dispersion. The pH of the solution was adjusted to 4 by addition of 0.1 M HCl solution, followed by stirring for 2−3 h to allow phase transfer of the UCPs to proceed. During this process, the carboxylate groups of the oleate ligand were protonated, and the ligand was released from the UCP surface. The aqueous solution was then shaken with diethyl ether to remove the oleic acid generated in solution. This was done for a number of cycles until the solution was completely clear. The ligand-free UCPs in the water fraction were collected and washed by centrifugation after precipitation with acetone. The water-soluble nanoparticles could be readily dispersed in water.

Table 1. Oligonucleotide Sequences Used in Hybridization Assaysa SMN1 probe SMN1 FC target SMN1 1 BPM target SMN1 NC target polyT (T30)

biotin−5′-ATT TTG TCT GAA ACC CTG T-3′ Cy3−3′-TAA AAC AGA CTT TGG GAC A-5′ Cy3−3′-TAA AAC ACA CTT TGG GAC A-5′ Cy3−3′-TGT CCC AAA GTC TGT TTT A-5′ 3′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT5′

a

FC = fully complementary, 1BPM = 1 base pair mismatch, NC = noncomplementary. The mismatched base in 1BPM sequence is underlined. Melting temperatures (Tm) of SMN1 probe with its FC and 1BPM targets are 65 ± 1 and 57.4 ± 1 °C, respectively.

assays were done with oligonucleotide target solutions in borate buffers (50 mM borate, pH 9.25, and 100 mM NaCl) unless otherwise stated. Hybridization assays were done by pipetting a 1.5 μL aliquot of oligonucleotide target solutions of various concentrations (0.025−25.6 μM) onto the spots on the paper. After hybridization, the paper was rinsed in borate buffer and dried in a vacuum desiccator. Subsequently, the paper was scanned with an epifluorescence microscope equipped with a NIR laser for excitation (980 nm, 800 mW). To investigate single base pair mismatched (1BPM) target discrimination, the 2721

dx.doi.org/10.1021/ac404129t | Anal. Chem. 2014, 86, 2719−2726

Analytical Chemistry

Article

Figure 1. (a) TEM image of UCP-oleate, (b) HRTEM image of UCP-oleate, and TEM images of (c) UCP-SC and (d) UCP-strep.

size of the UCP-SCs remained unchanged (Figure 1c), and DLS analysis indicated that 98% of the UCP-SCs had a hydrodynamic diameter and size distribution of 25.2 ± 3.7 nm (Figure S2b, Supporting Information). Any changes induced by conjugation of streptavidin could not be observed in TEM images, and the size and morphology of UCPs remained unchanged (Figure 1d). The successful conjugation of streptavidin was confirmed by ninhydrin reaction and by fluorescence from the conjugation of Cy3-labeled biotinylated SMN1 probe (data not shown). The emission spectra of UCPoleate, ligand-free UCPs, UCP-SC, and UCP-strep are shown in Figure 2. The UCPs in water exhibited strong emission at 525 and 542 nm. The upconversion luminescence spectra were similar to those for the samples in hexane, with a slight decrease in intensity due to a surface quenching effect of water molecules.33 These results reinforce that the characteristic upconversion property of the nanoparticles was unaffected by the surface modification process. Moreover, the normalized emission spectrum of UCPs and the absorbance/emission spectrum of Cy3 overlapped well with the absorbance region (450−550 nm) of Cy3 (Figure S3, Supporting Information). The UCPs were then implemented as LRET donors for Cy3. Characterization and Stability of UCP-Strep on Cellulose Paper. The stability of UCP-strep on cellulose paper is the prerequisite for any subsequent assay development. Based on results from the environmental scanning electron micrograph (ESEM) shown in Figure S4 (Supporting Information), it can be clearly seen that, even after extensive washing with buffer, the UCPs formed stable layers on the fibrous cellulose matrix. According to the UCP-strep emission intensity, there were no significant changes induced by extensive rinsing (Figure S5, Supporting Information). This is consistent with previous reports that have also suggested that

paper was exposed to fully complementary (FC) and 1BPM target solutions and subsequently treated for 10 min with formamide solution. Formamide concentrations of 10% and 20% were used to explore the impact on stringency. For assays involving more complicated matrices that were selected to impose potential for nonspecific adsorption, 2.3 × 10−6 mol/L FC target in borate buffer was spiked with either salmon sperm DNA (long sequences, ∼2.2 × 10−6 mol/L) solution, polyT (short sequences, 2.2 × 10−6 mol/L) solution, or bovine serum albumin (BSA) solution (3.8 × 10−4 mol/L). For all the quantitative analyses, four replicates were measured (n = 4) unless otherwise stated.

■

RESULTS AND DISCUSSION Synthesis and Characterization of β-NaYF4:2% Er3+, 18% Yb3+/β-NaYF4 Core/Shell Upconverting Phosphors. UCPs with strong green emission were synthesized via a protocol reported elsewhere.30 Figure 1 shows a representative transmission electron microscopy (TEM) image of the UCPs that were synthesized. The UCPs had an average diameter of 22.2 ± 3.2 nm, were roughly spherical, and dispersed well in hexane (Figure 1a). The high-resolution transmission electron microscopy (HRTEM) image of UCP-oleate (Figure 1b) shows a distance between lattice fringes of 0.52 nm, which can be ascribed to the (100) plane of the hexagonal phase. Furthermore, as shown in Figure S1 (Supporting Information), the X-ray diffraction (XRD) patterns of the samples were consistent with a pure hexagonal phase of NaYF4 nanocrystals. The hydrodynamic diameter and size distribution of the UCPs measured by dynamic light scattering (DLS) showed that 100% of the UCPs were 20.3 ± 2.9 nm (Figure S2a, Supporting Information). After removal of the oleate coating and subsequent coating with sodium citrate, the morphology and 2722

dx.doi.org/10.1021/ac404129t | Anal. Chem. 2014, 86, 2719−2726

Analytical Chemistry

Article

Performance and Optimization of Hybridization Assay. It has been previously reported that paper-based DNA hybridization assays are advantageous due to the speed of nucleic acid hybridization, with the hybridization signal profiles being reported to reach a plateau within a few minutes.21 The fast kinetics of hybridization was postulated to be due to the capillary wicking effect, which could overcome diffusion-limited kinetics. The potential of the UCPs to change or block flow through paper necessitated a similar study of the rate of signal development for cellulose paper that had been treated with UCPs. According to the results indicated by Figure S6 (Supporting Information), the LRET ratio reached a plateau within 2 min at room temperature and 40 °C. In comparison, most of the UCP-LRET nucleic acid hybridization assays for solution-phase work have reported anywhere from 30 min or more to achieve equilibration.6 Therefore, this paper-based hybridization assay using UCPs is notably advantageous in terms of short assay time and is competitive with other paperbased hybridization assays. A ratiometric approach based on fluorescence intensities of the UCPs and Cy3 was used to quantify the amount of target oligonucleotide. A ratiometric approach for quantification has been previously reported for QD-FRET-based assays on paper.21,22 This approach offers superior accuracy and precision in comparison to those that use only the luminescence from

Figure 2. Emission spectra of 1 mg/mL UCPs with different coatings. UCP-oleate was dispersed in hexane, and the other water-soluble samples were dispersed in deionized H2O.

interactions between proteins and cellulose paper can be utilized to immobilize nanoparticles.34

Figure 3. Quantitative determination of Cy3-labeled fully complementary (FC) target by UCP-mediated LRET-sensitized Cy3 emission. (a) Pseudocolor epifluorescence images collected via green filter channel, Cy3 filter channel, and the composite of green and Cy3 imaging channels corresponding to (a) 0, (b) 0.0375, (c) 0.075, (d) 0.15, (e) 0.3, (f) 0.6, (g) 1.2, (h) 2.4, (i) 4.8, (j) 9.6, (k) 19.2, and and (l) 38.4 pmol of Cy3labeled FC target respectively. (b) Calibration curve corresponding to the LRET ratio responses was collected by ImageJ analysis of the two imaging channels. (Inset) Linear fitting results of added Cy3-labeled FC target from the detection limit to 1.2 pmol (n = 4). 2723

dx.doi.org/10.1021/ac404129t | Anal. Chem. 2014, 86, 2719−2726

Analytical Chemistry

Article

UCPs as the signal. The response of the assay to increasing amounts of SMN1 FC target is shown in Figure 3. A linear increase in response for target amounts ranging from the detection limit to above 1.2 pmol was obtained. The limit of detection (LOD) of the assay was calculated to be 34 fmol, based for a signal that was 3 standard deviations higher than the background LRET ratio. The LOD was almost 1 order of magnitude lower than the 300 fmol LOD reported for analogous paper-based FRET assays with QDs as donors and a similar probe-target combination with Cy3 dye as acceptor.21 Definitive quantitative comparison of LOD is not appropriate given the different methods of nanoparticle immobilization and laser source powers used for the earlier QD-FRET work and the new UCP-LRET work herein. However, the data suggest that the UCP-LRET system is competitive in terms of detection limit. 1BPM Discrimination Assay. In nucleic acid assays, it is crucial for a detection system to discriminate 1BPM DNA effectively. A combination of ionic strength and formamide concentration was used to enhance the stringency of nucleic acid hybridization for 1BPM oligonucleotide discrimination. Lowering the ionic strength promotes the destabilization of a DNA duplex, while formamide helps to lower the melting temperature of a DNA duplex by acting as a hydrogen-bond disrupter. This approach was reported to be effective in the discrimination of 1BPM at room temperature, which obviates the need for external heating.35 Therefore, effects of formamide and salt concentration were used to control the stringency of nucleic acid hybridization in this UCP-based LRET assay. The results presented in Figure S7 (Supporting Information) indicate that when the concentration of NaCl reached 150 mM, the LRET signal was significantly higher than when 25 or 50 mM NaCl was used and was slightly higher than that observed for 100 mM NaCl. Given that even higher concentrations of NaCl might decrease the selectivity in DNA hybridization, 100 mM salt was chosen in the following hybridization assays. It can be seen in Figure 4 that, after the rinse with borate buffer, no significant LRET signal differences could be observed between the signal from FC and 1BPM targets. However, after treatment with increasing concentrations of formamide solution, signal differentiation became more pronounced. The signal ratio between FC and 1BPM target signal increased from 1.2 (rinsed with borate buffer) to 1.9 (10% formamide treatment) and 3.1 (20% formamide treatment), while the signal from nonspecific hybridization of NC remained suppressed at a low level. Hybridization Assay in a High Background of Potential Interferents. The resistance of this paper-based hybridization assay to nonspecific adsorption of oligonucleotides and protein was evaluated. Hybridization assays were conducted with a background interferent in mixture with the target oligonucleotide. The three permutations that were investigated included ∼2.2 × 10−6 mol/L salmon sperm DNA (2000 bp fragment), 2.2 × 10−6 mol/L polyT (T30) solution, and 3.8 × 10−4 mol/L BSA. The SMN1 FC target was spiked into the above solutions to a final concentration of 2.3 × 10−6 mol/L. The data shown in Figure 5 indicate that no statistically significant differences can be observed among the LRET signals obtained from assays done in buffer and those with different potential interferents. These results demonstrate that solutions with a large quantity of unrelated short/long sequences of DNA or protein did not significantly affect the

Figure 4. Examination of selectivity of the paper-based hybridization assay for FC and 1BPM targets. (a) Pseudocolor epifluorescence images after hybridization with NC, 1 BPM, and FC targets (from right to left) after treatment with 20% (v/v) formamide (F) solution for 10 min, scanned by use of green signal channel and Cy3 signal channel as indicated. (b) LRET ratios calculated from the fluorescent images analyzed by ImageJ at different formamide concentrations. Note: numbers on the top of each group of bars indicate the signal ratio between FC and 1BPM targets (n = 4).

selective response of this paper-based assay. This is an attractive result for applications where efforts to purify target are to be minimized.

■

CONCLUSIONS DNA hybridization assay on cellulose paper with UCPs as donor and Cy3-labeled DNA as acceptor was realized for the first time. Hybridization enabled the proximity required for LRET-sensitized Cy3 emission, and the dye emission was used as the analytical signal. This new format for nucleic acid hybridization assay reached complete signal development within 2 min after introduction of Cy3-labeled target DNA. The dynamic range of the assay spanned almost 2 orders of magnitude, which compares favorably with the 1 order of magnitude range reported for solid-phase QD-FRET systems, and a LOD of 34 fmol was achieved without the use of any signal amplification method, which is competitive with previous 2724

dx.doi.org/10.1021/ac404129t | Anal. Chem. 2014, 86, 2719−2726

Analytical Chemistry

Article

this research. M.O.N. is grateful to Ontario Ministry of Training Colleges and Universities for provision of an Ontario Graduate Scholarship.

■

(1) Gnach, A.; Bednarkiewicz, A. Nano Today 2012, 7, 532−563. (2) Haase, M.; Schafer, H. Angew. Chem., Int. Ed. 2011, 50, 5808− 5829. (3) Hao, S. W.; Chen, G. Y.; Yang, C. H. Theranostics 2013, 3, 331− 345. (4) Kuningas, K.; Ukonaho, T.; Pakkila, H.; Rantanen, T.; Rosenberg, J.; Lovgren, T.; Soukka, T. Anal. Chem. 2006, 78, 4690−4696. (5) Pakkila, H.; Yliharsila, M.; Lahtinen, S.; Hattara, L.; Salminen, N.; Arppe, R.; Lastusaari, M.; Saviranta, P.; Soukka, T. Anal. Chem. 2012, 84, 8628−8634. (6) Yliharsila, M.; Valta, T.; Karp, M.; Hattara, L.; Harju, E.; Holsa, J.; Saviranta, P.; Waris, M.; Soukka, T. Anal. Chem. 2011, 83, 1456−1461. (7) Niedbala, R. S.; Feindt, H.; Kardos, K.; Vail, T.; Burton, J.; Bielska, B.; Li, S.; Milunic, D.; Bourdelle, P.; Vallejo, R. Anal. Biochem. 2001, 293, 22−30. (8) Rantanen, T.; Jarvenpaa, M. L.; Vuojola, J.; Arppe, R.; Kuningas, K.; Soukka, T. Analyst 2009, 134, 1713−1716. (9) Martinez, A. W.; Phillips, S. T.; Whitesides, G. M.; Carrilho, E. Anal. Chem. 2010, 82, 3−10. (10) Martinez, A. W.; Phillips, S. T.; Carrilho, E.; Thomas, S. W.; Sindi, H.; Whitesides, G. M. Anal. Chem. 2008, 80, 3699−3707. (11) Araujo, A. C.; Song, Y. J.; Lundeberg, J.; Stahl, P. L.; Brumer, H. Anal. Chem. 2012, 84, 3311−3317. (12) Missoum, K.; Belgacem, M. N.; Bras, J. Materials 2013, 6, 1745−1766. (13) Kalia, S.; Dufresne, A.; Cherian, B. M.; Kaith, B. S.; Averous, L.; Njuguna, J.; Nassiopoulos, E. Int. J. Polym. Sci. 2011, 1−35. (14) Martinez, A. W.; Phillips, S. T.; Butte, M. J.; Whitesides, G. M. Angew. Chem., Int. Ed. 2007, 46, 1318−1320. (15) Zhang, Y. F.; Rochefort, D. Anal. Chim. Acta 2013, 800, 87−94. (16) Kong, F.; Ni, Y. Water Sci. Technol. 2009, 60, 3083−3089. (17) Hamzah, H. H.; Yusof, N. A.; Salleh, A.; Abu Bakar, F. Sensors 2011, 11, 7302−7313. (18) Allen, P. B.; Arshad, S. A.; Li, B. L.; Chen, X.; Ellington, A. D. Lab Chip 2012, 12, 2951−2958. (19) Scida, K.; Li, B. L.; Ellington, A. D.; Crooks, R. M. Anal. Chem. 2013, 85, 9713−9720. (20) Wang, Y.; Ge, L.; Wang, P.; Yan, M.; Ge, S.; Li, N.; Yu, J.; Huang, J. Lab Chip 2013, 13, 3945−3955. (21) Noor, M. O.; Shahmuradyan, A.; Krull, U. J. Anal. Chem. 2013, 85, 1860−1867. (22) Noor, M. O.; Krull, U. J. Anal. Chem. 2013, 85, 7502−7511. (23) Yetisen, A. K.; Akram, M. S.; Lowe, C. R. Lab Chip 2013, 13, 2210−2251. (24) Zhao, W. A.; Ali, M. M.; Aguirre, S. D.; Brook, M. A.; Li, Y. F. Anal. Chem. 2008, 80, 8431−8437. (25) Luckham, R. E.; Brennan, J. D. Analyst 2010, 135, 2028−2035. (26) Rastogi, S. K.; Gibson, C. M.; Branen, J. R.; Aston, D. E.; Branen, A. L.; Hrdlicka, P. J. Chem. Commun. 2012, 48, 7714−7716. (27) Algar, W. R.; Tavares, A. J.; Krull, U. J. Anal. Chim. Acta 2010, 673, 1−25. (28) Vannoy, C. H.; Tavares, A. J.; Noor, M. O.; Uddayasankar, U.; Krull, U. J. Sensors 2011, 11, 9732−9763. (29) Petryayeva, E.; Algar, W. R. Anal. Chem. 2013, 85, 8817−8825. (30) Carling, C. J.; Nourmohammadian, F.; Boyer, J. C.; Branda, N. R. Angew. Chem., Int. Ed. 2010, 49, 3782−3785. (31) Bogdan, N.; Vetrone, F.; Ozin, G. A.; Capobianco, J. A. Nano Lett. 2011, 11, 835−840. (32) Peng, J.; Sun, Y.; Zhao, L.; Wu, Y.; Feng, W.; Gao, Y.; Li, F. Biomaterials 2013, 34, 9535−9544. (33) Wang, F.; Wang, J. A.; Liu, X. G. Angew. Chem., Int. Ed. 2010, 49, 7456−7460. (34) Chiu, W. C.; Huang, C. C. Anal. Chem. 2013, 85, 6922−6929.

Figure 5. Hybridization with FC target in solutions containing different interfering DNA and proteins (n = 4).

analogous work that used QDs as FRET donors.21 The combination of paper substrate and streptavidin coating on UCPs enabled the assay to be resistant to nonspecific adsorption of high concentrations of potential DNA and protein interferents. A 1BPM target was effectively discriminated by controlling concentrations of salt and formamide solution, and a maximum signal ratio of 3.1:1 was achieved in comparison of FC to 1BPM hybridization. A Cy3-labeled oligonucleotide was used as target in this preliminary study to offer control of location of the acceptor dye for LRET. Other formats of detection without labeling of the target are available.36−38 Since UCPs with different emission bands can be synthesized, the platform we have demonstrated herein can be extended to applications that are multiplexed, by use of different LRET pairs of UCPs and corresponding fluorescent dyes. We speculate that, in future developments to move such technology forward for in-field applications, cell phone cameras may be suitable as alternative portable imaging technology that is capable of quantitative spectroscopy. In-field applications can also be limited by the need for washing steps to remove background, but once hybridization has taken place the paper can be washed. This can be achieved by providing a crossstream of a wash solution that is wicked across a sensing pad by directing the flow via wax borders printed on paper.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional text with description of reagents, instrumentation, and equation used in data analysis; and seven figures showing characterization of LRET pairs and additional results. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We are grateful to the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support of 2725

dx.doi.org/10.1021/ac404129t | Anal. Chem. 2014, 86, 2719−2726

Analytical Chemistry (35) (36) (37) (38) 2013,

Article

Noor, M. O.; Krull, U. J. Anal. Chim. Acta 2011, 708, 1−10. Algar, W. R.; Krull, U. J. Langmuir 2010, 26, 6041−6047. Algar, W. R.; Krull, U. J. Anal. Chem. 2010, 82, 400−405. Vannoy, C. H.; Chong, L.; Le, C.; Krull, U. J. Anal. Chim. Acta 759, 92−99.

2726

dx.doi.org/10.1021/ac404129t | Anal. Chem. 2014, 86, 2719−2726