Paper-Based Solid-Phase Nucleic Acid Hybridization Assay Using

Dec 29, 2012 - Paper-based analytical devices (PADs) have promise to revolutionize point-of-care diagnosis in the developing nations and for point-of-...
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Paper-Based Solid-Phase Nucleic Acid Hybridization Assay Using Immobilized Quantum Dots as Donors in Fluorescence Resonance Energy Transfer M. Omair Noor, Anna Shahmuradyan, 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 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.

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same device. As a result, another format that has been reported with PADs is based on spatially resolved microzones to conduct parallel analyses. For example, a paper-based 96-microzone plate for conducting enzyme-linked immunosorbent assay (ELISA) has been reported with faster analysis time and reduced sample volumes as compared to the conventional ELISA done on a traditional 96-microwell plate.9 Despite the progress reported in the literature for the incorporation of various assays on PADs, little work has appeared about the integration of nanomaterials with PADs for assay development. From the standpoint of nucleic acid detection, most studies that focus on PADs have reported the use of gold nanoparticles (AuNPs) as labels to aid visual detection.5,7,10 However, this strategy is limited in terms of offering a ratiometric approach for signal processing, which is desirable for quantitative analysis. Numerous ratiometric methods have been reported using semiconductor quantum dots (QDs) as integrated nanomaterials within biosensors and bioassays.11−15 For assay development, QDs offer a number of

aper-based analytical devices (PADs) have promise to revolutionize point-of-care diagnosis in the developing nations and for point-of-care screening due to low cost and ease of use. Paper has a number of attractive features that render this substrate advantageous for conducting chemical and biological analysis. These features include passive transport of fluid via capillary wicking, compatibility with biological samples, wellestablished methods for modifying the surface with a wide variety of functional groups to immobilize proteins, DNA, and other molecules, ease of safe disposal by incineration, and the commercial availability of materials that offer a wide variety of physical properties such as pore size and flow rate.1 A format commonly used for PADs is based on a lateral flow device, and this consists of a test zone and a control zone.2 A test zone is used to interrogate the sample solution for the analyte of interest, while a control zone is used to evaluate and ensure the proper functioning of the selective chemistry. PADs that are based on a lateral flow format have been reported for the detection of pesticides,3 nucleic acids,4−6 metal ions,7 and antigen−antibody interactions.8 While the lateral flow format remains the most commonly used format for conducting analyses with PADs due to its simplicity, it is inherently limited with respect to development of multiplexed analysis on the © 2012 American Chemical Society

Received: November 7, 2012 Accepted: December 29, 2012 Published: December 29, 2012 1860

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Figure 1. (a) Synthetic steps involved in the surface modification of paper (cellulose) with the imidazole ligands for the immobilization of QDs and QD−probe oligonucleotide conjugates on the surface of paper. (b) Design of the paper-based solid-phase nucleic acid hybridization assay using immobilized QDs as donors in FRET.

reported solid-phase QD−FRET nucleic acid hybridization assays.20,23 A ratiometric approach that was based on a FRET ratio was used for a quantitative determination of target oligonucleotides. The hybridization assays were functional in the presence of a large background of noncomplementary matrix. Single-nucleotide polymorphism (SNP) detection is demonstrated with an excellent contrast ratio. A further intriguing aspect of this work is the improved regeneration capability as compared to the previously reported solid-phase QD−FRET nucleic acid hybridization assays, where regeneration has been reported to be a challenge.

unique optoelectronic properties that render them useful for chemical and biosensing purposes. These properties include broad absorption spectra, narrow, symmetrical, and size-tunable emission spectra, long photoluminescence lifetimes (>20 ns), and greater photostability and brightness than organic fluorophores.16−18 These properties are well-suited for applications that use QDs in optical multiplexing and where QDs can serve as donors in fluorescence resonance energy transfer (FRET) based applications.19 Solid-phase assays that utilize QDs as integral components of bioassays and biosensors are also advantageous from the standpoint of improved reusability,20 sensitivity,21 and in reduction of environmental contamination by containment of the QDs on a solid matrix. Very few studies have been published about the integration of QDs with PADs for signal transduction. One example by Yuan et al. reported the use of polymer QD−enzyme hybrid films on PADs to quantitatively probe the presence of a substrate for the corresponding enzyme.22 Two different polymer QD−enzyme hybrid films based on use of tyrosinase or glucose oxidase were reported. In the presence of a corresponding substrate for the enzyme, the conversion of substrates into products quenched the photoluminescence (PL) of QDs. The quenching of PL served as an analytical signal and could be used to determine substrate concentration or enzyme activity. The signal transduction was based on absolute PL measurements of QDs. Such an approach is less precise than a ratiometric approach that can be implemented when using QDs in a FRET based transduction scheme.14 No work has appeared in the literature describing the integration of QD−FRET assays on PADs. We report herein a paper-based solid-phase assay for selective transduction of nucleic acid hybridization using immobilized QDs as donors in FRET. A schematic of the assay design is shown in Figure 1. The surface of a paper was chemically modified with imidazole ligands (Figure 1a) to immobilize QD−probe conjugates that had been assembled in solution. Addition and subsequent hybridization of Cy3-labeled oligonucleotide targets provided FRET-sensitized Cy3 emission upon excitation of QD−probe conjugates (Figure 1b). Rapid hybridization kinetics allowed equilibration within minutes, compared to the hours of incubation required with previously

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EXPERIMENTAL SECTION

A detailed description of the materials and reagents, experimental procedures, buffers, and instrumentation used in these experiments can be found in the Supporting Information. Preparation of GSH-Coated QDs. Oleic acid capped CdSxSe1−x/ZnS (core/shell) green-emitting QDs (gQDs, peak PL = 525 nm) in toluene were made water-soluble by a ligandexchange reaction with glutathione (GSH). Approximately 0.2 g of GSH dissolved in 600 μL of tetramethylammonium hydroxide solution (TMAH) was added to 0.35 μM solution of organic QDs dissolved in 2 mL of chloroform. The solution was agitated on a vortex mixer and allowed to stand at room temperature overnight. The GSH-modified QDs (GSH−QDs) were then extracted with 200 μL of 50 mM borate buffer (BB, pH 9.25) containing 250 mM NaCl (top layer), and the organic layer (bottom layer) was discarded. Ethanol was then added to the aqueous layer containing QDs until the solution became turbid. The mixture was centrifuged at 7500 rpm for 10 min to obtain a pellet of QDs. The supernatant was discarded, and the pellet was redissolved in 200 μL of BB buffer containing 250 mM NaCl. Two addition ethanol−buffer precipitations were done, and the resulting QD pellet was finally dissolved in 200 μL of 50 mM BB buffer (pH 9.25, no NaCl). UV−vis absorption spectroscopy was used to determine the concentration of QDs (molar extinction coefficient at 504 nm = 411 400 M−1 cm−1). GSH-capped gQDs were stored at 4 °C. Preparation of QD−Probe Oligonucleotide Conjugates. The oligonucleotide probes were terminated with a dithiol moiety and were conjugated to the surface of GSH− 1861

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2 μL aliquot of oligonucleotide target solutions of various concentrations (0.25−7 μM) at the pads where QD−probe conjugates were immobilized. The hybridization experiments were done in 50 mM BB buffer saline (BBS, pH 9.25, 100 mM NaCl), and the target solution was allowed to sit for 30 min at room temperature prior to image scans and measuring PL spectra without any washing of the paper. In case of SNP discrimination experiments, the paper was initially exposed to FC and 1 BPM targets in BBS buffer and subsequently exposed to increasing formamide concentration in BB buffer for 10 min. For experiments involving a more complex matrix, the 2 μM solution of FC TGT in BBS buffer was fortified with either 0.88 mg/mL of salmon sperm DNA or 50 mg/mL of BSA with 0.1% SDS.

QDs by self-assembly. For a typical preparation, GSH−QDs were incubated with 20 times molar excess of probe oligonucleotides in the presence of 500 times molar excess of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) dissolved in 100 mM Tris−borate buffer (TB, pH 7.4, 20 mM NaCl). The mixture was then agitated overnight on a vortex mixer. The solution containing QD−probe conjugates was used without further purification and stored at 4 °C. Chemical Modification of Paper with Imidazole. The protocol for surface modification of paper with an aldehyde functionality was based on the periodate oxidation of cellulose as reported elsewhere.24 Briefly, ca. 0.5 g of chromatography paper (Whatman cellulose chromatography papers, grade 1) cut to a dimensions of 25 mm by 60 mm was immersed in 75 mL of Milli-Q water containing sodium (meta)periodate (NaIO4) and lithium chloride (LiCl) at 26 mM (0.15 g) and 47 mM (0.42 g) concentrations, respectively. The temperature of the solution was adjusted to 55 °C, and the reaction was allowed to proceed for 1 h. These conditions provide 86% yield in terms of aldehyde modification of cellulose as reported elsewhere.24 The paper was then rinsed twice with Milli-Q water and dried in an oven at 40 °C. Aldehyde-functionalized paper was stored in a desiccator and was subsequently modified with imidazole in an array format (array size = 3 × 6; spot size (diameter) = 5 mm) using imine formation. The imine was reduced in situ to secondary amines using sodium cyanoborohydride (NaCNBH3). The location of each spot was marked by highlighting its circumference with a graphite pencil. For each spot, a 2 μL aliquot of solution containing 1-(3aminopropyl)imidazole (API) at 160 mM concentration and NaCNBH3 at 200 mM concentration dissolved in 100 mM 4(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, pH 8.0) buffer was pipetted on the paper. The spotted paper was incubated for 1 h at room temperature and then washed for 10 min in BB buffer containing 0.1% sodium dodecyl sulfate (SDS). Immobilization of QD−Probe Oligonucleotide Conjugates. Prior to the immobilization of QD−probe conjugates on imidazole-modified paper, the surface of the paper was blocked with 0.1% bovine serum albumin (BSA) in TB buffer for 30 min. The paper was then washed with BB buffer for 10 min. For each spot, a 2 μL aliquot of a solution of QD−probe conjugates at 400 nM concentration was pipetted onto the paper. The spotted paper was stored in the dark at room temperature for 2 h. The paper was then washed with BB buffer for 10 min. Hybridization Assays and Selectivity. The oligonucleotide target sequences used for the hybridization assays are given in Table 1. Hybridization experiments were done by pipetting a

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RESULTS AND DISCUSSION Immobilization of QD−Probe Conjugates on Imidazole-Modified Paper. In the previously reported QD−FRET solid-phase assays for oligonucleotides, the immobilization of QDs and oligonucleotide probes for the assembly of a biorecognition interface proceeded as a two-step sequential process. The QDs were first immobilized on the surface of a solid support, and then oligonucleotide probes were conjugated to the surface of immobilized QDs.20,21,23,25−28 In the work presented herein, an alternative approach was taken for the immobilization of QD−probe conjugates on paper. Oligonucleotide probes were immobilized onto QDs in solution, and then these bioconjugates were immobilized onto the surface of paper that had been modified with a coating of imidazole groups. The assembly of oligonucleotide probes to the surface of GSH−QDs in solution was driven by a dithiol moiety appended to the 5′ terminus of the oligonucleotide probes. The oligonucleotides probes conjugated to the surface of the ZnS shell of GSH−QDs by self-assembly via a ligand-exchange reaction, as reported elsewhere.28 The conjugation was confirmed by gel electrophoresis and solution-phase hybridization experiments (see Figures S2 and S3 in the Supporting Information). Data confirming the immobilization of GSH−QDs and QD− probe conjugates onto paper that had been modified with imidazole ligands is shown in Figure 2. In the absence of imidazole ligands (aldehyde-modified paper), no immobilization of either QDs or QD−probe conjugates was observed in epifluorescence images and the corresponding PL spectra. The presence of imidazole ligands correlated with fluorescence that was consistent with the retention of QDs on the surface of paper. The average PL contrast ratio for GSH−QDs immobilized on paper modified with and without imidazole ligands was 100 to 1, respectively. The immobilization of QD− probe conjugates provided a PL contrast ratio of 90 to 1 for imidazole-modified and unmodified paper, respectively. Hypsochromic and bathochromic shifts were not observed for the QD PL spectra of GSH−QDs and QD−probe conjugates when comparing solution-phase spectra with spectra collected from these materials that had been immobilized on the paper. Additionally, no increase in the full width at-half-maximum of the QD PL spectra was observed after immobilization, suggesting that the optical properties of the QDs were not compromised upon immobilization. The use of multidendate imidazole ligands for bioconjugation of oligonucleotides,29 peptides,13 and proteins,30 and for the immobilization of QDs and gold nanoparticles on the surface of optical fibers and glass

Table 1. Oligonucleotide Sequences Used in the Hybridization Assays name SMN1 SMN1 SMN1 SMN1

probe FC TGT 1 BPM TGT NC TGT

sequencea DTPA−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′

a

TGT = target, FC = fully complementary, 1 BPM = 1 base pair mismatch, NC = noncomplementary, DTPA = dithiol phosphoramidite. The mismatched base in 1 BPM TGT is bolded and underlined. 1862

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Figure 2. Pseudocolor epifluorescence images and PL spectra showing immobilization of (a) GSH−gQDs and (b) gQD−probe conjugates in the (i) presence and (ii) absence of imidazole functionalized paper. The paper was functionalized with imidazole in an array format, and representative data from only one element of an array is shown in the figure. White dash circles in the figure identify the area on the paper where GSH−QDs and QD− probe conjugate solutions were dispensed for immobilization.

beads27 has been previously reported. Compared to other thiolbased ligands,25 the use of imidazole ligands for the immobilization of QDs has been reported to be advantageous from the standpoint of retention of optical properties of QDs such as quantum yield.27 However, the immobilization of QD− probe conjugates on the surface of a solid support via imidazole ligands has not been previously demonstrated. It is also important to note that, as compared to other solid-phase QD− FRET nucleic acid hybridization assays that required days to chemically modify solid surfaces t o immobilize QDs,20,21,23,25,26,28 it was possible to chemically modify paper within hours for the immobilization of QD−probe conjugates. Quantitative Hybridization Assays. In this work, a ratiometric approach based on a FRET ratio (see eqs S4 and S5 in the Supporting Information) was used to quantify the signal that was associated with the amount of target DNA added. Use of a FRET ratio accommodates any donor PL quenching and sensitization of acceptor PL. This approach to quantification avoids reliance on the magnitude of donor PL quenching (FRET efficiency). A ratiometric approach for quantification has been previously reported for the QD−FRET based assays.12,13,23 Such an approach is less susceptible to signal variations caused by changes in instrumental response or variations imposed by sample preparation.14,15,31 The response of the assay to increasing amounts of SMN1 FC TGT is shown in Figure 3. A linear increase in response for target amounts ranging from 300 fmol to 5 pmol corresponds to a dynamic range of 1 order of magnitude. A similar dynamic range has previously been reported for solid-phase QD−FRET nucleic acid hybridization assays using optical fibers,20,23,26 glass beads,28 and microfluidics.21 The limit of detection (LOD) of the assay without any amplification steps was found to be 300 fmol and is based on a FRET ratio signal that was 3 standard deviations higher than the background FRET ratio signal. The LOD of 300 fmol for the paper-based solid-phase assay presented herein is approximately 3-fold better than the 1 pmol LOD reported previously for solid-phase QD−FRET nucleic acid hybridization assays done using optical fibers.23 The paper-based assay exhibited rapid transduction of nucleic acid hybridization, with the FRET ratio response reaching a

steady state within 2 min (see Figure S4 in the Supporting Information). It is hypothesized that the fluid transport mediated by a capillary wicking in paper serves as an active delivery method to move oligonucleotide targets to the biorecognition elements. Fast hybridization kinetics are achieved by overcoming diffusion-limited kinetics, as reported previously.32 In comparison, other solid-phase QD−FRET nucleic acid hybridization assays done using optical fibers and glass beads have reported hybridization times of 30 min to 4 h in order to reach steady-state signals.20,23,28 Dry storage of the paper coated with QD−probe conjugates at 4 °C maintained the activity of the assay without any reduction in the assay performance for at least 2 weeks (see Figure S5 in the Supporting Information). Nonspecific Adsorption and SNP Detection. Nonspecific adsorption can occur on both the QDs and the solid matrix that is used to immobilize the QDs. The extent of nonspecific adsorption of oligonucleotides on immobilized QD−probe conjugates was investigated using SMN1 NC TGT. In the previous QD−FRET based solid-phase assays for nucleic acid detection, the nonspecific adsorption of oligonucleotides on the surface of mercaptopropionic acid (MPA)-coated QDs has been a challenge, requiring blocking agents such as BSA or NeutrAvidin to achieve selectivity.20 The nonspecific adsorption of oligonucleotides on MPA-coated QDs was found to be pH and ionic strength dependent33 and was driven by hydrogen bonding between nucleobases and neutral acid groups associated with the ligands on the surface of MPA-capped QDs.34 In this work, the surface capping ligand used to render QDs water-soluble was GSH. GSH contains two carboxylic acid groups and one primary amine, in addition to a thiol that was used to assemble GSH on the ZnS shell of QDs. Examples of signals obtained for the exposure of immobilized QD−probe conjugates on the surface of imidazole-functionalized paper to SMN1 FC and SMN1 NC targets are given in Figure 4. It can be seen that GSH−QDs conjugated with oligonucleotide probes provided excellent resistance to nonspecific adsorption of oligonucleotides, to the extent that no blocking step was required to prevent nonspecific adsorption of oligonucleotides. The FRET ratio response for the exposure of 1863

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Figure 4. Selectivity of the nucleic acid hybridization assay and SNP discrimination. (a) Pseudocolor epifluorescence images for the hybridization of NC target in BBS and FC and 1 BPM targets after exposure to 10% (v/v) formamide (F) solution in BB buffer. The spots were sequentially scanned under the gQD and Cy3 signal channels. (b) FRET ratios acquired from the PL spectra for the hybridization of NC, 1 BPM, and FC targets under various stringency conditions. Note: a white dashed circle in panel a highlights the location of the spot, and the FRET ratio corresponding to NC target in panel b is indicated by a black arrow.

Previous studies have shown that the nonspecific adsorption of oligonucleotides is more pronounced at lower pH on QDs capped with ligands that are terminated with carboxylic acid groups.33 Solution-phase hybridization experiments with the same targets yielded a contrast ratio of ca. 70 to 1 for FC and NC TGTs, respectively (Figure S3 in the Supporting Information). An improvement in the contrast ratio seen in case of a solid-phase assay when compared with a solutionphase assay can be attributed to the enhancement of FRET efficiency previously reported for solid-phase QD−FRET assays due to a greater number of energy-transfer pathways.16,21 In the case of solution-phase QD−FRET assays, the ensemble average construct is composed of a single QD donor surrounded by multiple acceptors. The presence of multiple acceptors can offer some enhancement of FRET efficiency when interacting with a single QD donor.16,19 In the solid-phase configuration, the physical arrangement consists of a layer of QDs in close proximity as donors and a layer of acceptors. As a result, multiple donors can potentially interact with a single acceptor, which gives rise to additional energy-transfer pathways and enhancement of FRET efficiency that ultimately can improve analytical sensitivity. To further explore the resistance of the paper-based solidphase assay to nonspecific adsorption of oligonucleotides and protein, hybridization assays were done using a large excess of background material relative to the target oligonucleotide. One set of samples contained sheared salmon sperm DNA (587 and 831 base pair fragments) at 0.88 mg/mL concentration, and the other contained a large background of BSA at 50 mg/mL concentration in the presence of 0.1% (w/v) SDS. The target, SMN1 FC TGT, was present at 14 μg/mL concentration. The assays yielded a FRET ratio response of 0.75 ± 0.05 in the presence of the salmon sperm DNA, and the samples

Figure 3. Quantification of labeled SMN1 oligonucleotide targets using gQDs mediated FRET-sensitized Cy3 emission. (a) Pseudocolor epifluorescence images collected using the gQD imaging channel, the Cy3 imaging channel, and a merger of the gQDs and Cy3 imaging channels corresponding to (i) 0, (ii) 1, (iii) 3, and (iv) 6 pmol of Cy3labeled SMN1 targets. (b) PL spectra corresponding to the aforementioned amounts of target oligonucleotides. (c) Calibration curve showing the FRET ratio response obtained from the two imaging channels for various amounts of Cy3-labeled oligonucleotide targets. Note: the white dashed circles highlight the location of the spots that are not otherwise clearly visible.

immobilized QD−probe conjugates to FC TGT was 0.80 ± 0.05, while NC TGT yielded a FRET ratio response of 0.002 ± 0.002. This corresponds to a contrast ratio of 335 to 1 for FC and NC targets, respectively. A plausible reason for the resistance of nonspecific adsorption of oligonucleotides on GSH−QDs could be the partial zwitterionic character associated with GSH due to the presence of amine and carboxylic acid functional groups on the same molecule.27 Additionally, since the hybridization experiments were done in BBS buffer at pH 9.25, the carboxylic groups are expected to be ionized under these conditions.33 As a result, the electrostatic repulsion between the carboxylate groups and the negatively charged phosphate backbone of DNA should be more pronounced at this pH, thus minimizing nonspecific adsorption. 1864

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containing BSA yielded a FRET ratio response of 0.98 ± 0.05. Assays done in buffer containing SMN1 FC TGT target at 14 μg/mL concentration yielded a FRET ratio response of 0.61 ± 0.03. These results demonstrate that the presence of a large background of protein or DNA based complex matrix did not negatively affect the performance of the assay. In the presence of both the salmon sperm and BSA background, the FRET ratios exhibited an enhanced response when compared to that from the target in buffer; 1.23 and 1.59 fold higher, respectively. Effects of nonspecific adsorption of oligonucleotides on the surface of QD−probe conjugates that were immobilized on the paper were not observed, which is noteworthy since no washing steps were done following an application of the target solution to a pad with immobilized QD−probe conjugates (Figure 4). Some of the target DNA could occupy vacant sites elsewhere on the paper in locations where QD−probe conjugates were not present. The nonspecific adsorption of oligonucleotides elsewhere on the paper was confirmed by using labeled noncomplementary oligonucleotide sequence (data not shown). However, since the transduction mechanism was based on FRET, any adsorption of oligonucleotides that is not within the close proximity of QDs did not contribute to the FRET signal; this highlights another advantage of the FRET based transduction. In the presence of a large background of adsorbing material, the adsorption sites are more likely to be occupied by a background material than the target of interest, hence yielding a higher assay response. The experimental results confirmed that the analytical performance of the assay was not degraded in the presence of a significant background of noncomplementary protein or DNA. For many nucleic acid based applications, it may be necessary to resolve an FC target from one containing a single base pair mismatch (1 BPM). Such SNP discrimination might, for example, be indicative of a genetically related disease that is caused by a mutation at a single base pair level. One example of such a disease is a neuromuscular dysfunction associated with spinal muscular atrophy. The survival motor neuron sequence, SMN1, can be used in the diagnosis of spinal muscular atrophy and requires SNP discrimination to determine the extent of disease.35 Selection of temperature is commonly used method to control the stringency of nucleic acid hybridization for SNP detection.36 However, this is a less desirable approach owing to the temperature dependence of the quantum yields of QDs and molecular dyes.37 As a result, in this study a combination of ionic strength and formamide were used to tune the stringency of hybridization to achieve selectivity for SNP detection at room temperature. Formamide serves as a hydrogen-bond disrupter and lowers the melt temperature of a DNA duplex.38 Lowering the ionic strength destabilizes a DNA duplex by preventing the charge screening that is required to stabilize the two strands of a hybrid due to the electrostatic repulsion between the negatively charged sugar−phosphate backbone of DNA.39 An advantage of this approach to tune the stringency of nucleic acid hybridization for SNP discrimination is that external heaters are not required. Furthermore, precise control of the temperature is not required to achieve selectivity for quantitative analysis. The results for SNP discrimination at various experimental conditions are shown in Figure 4, parts a and b. Hybridization assays done with FC and 1 BPM targets in the presence of BBS buffer (50 mM borate, 100 mM NaCl) yielded no contrast ratio between a FC TGT and 1 BPM TGT. Incubating the paper in

BB buffer (50 mM borate, 0 mM NaCl) yielded a contrast ratio of 1.9 to 1 for FC and 1 BPM targets, respectively. Formamide was used to further improve the contrast ratio for SNP discrimination. When the paper was incubated in BB buffer containing 5% (v/v) formamide, the contrast ratio for SNP detection improved to 3.2 to 1. Increasing the formamide concentration to 10% (v/v) in BB buffer yielded the maximum contrast ratio of 19 to 1 for SNP discrimination that was observed in this work. Further increasing the formamide concentration to 15% (v/v) in BB buffer had an overall effect of lowering the contrast ratio to 5.6 to 1 for FC and 1 BPM targets, respectively. As compared to previous solid-phase QD−FRET nucleic acid hybridization assays that have been reported using the same probe and target sequences, considerably lower formamide concentration (10%) was required for SNP discrimination in this work (cf. 25% formamide for SNP detection in the work by Algar and Krull23,28). A possible reason for this effect is that, in the previous studies, ionic strength was not explored as a factor to optimize the stringency of nucleic acid hybridization for SNP discrimination, and the optimization of the SNP detection was done solely on the basis of controlling the concentration of formamide and/or temperature. Regeneration. An important attribute of biosensors is the regeneration of a biorecognition element. Given the success of the paper-based assay in providing excellent selectivity for SNP discrimination, regeneration of the biorecognition element was attempted by subjecting the immobilized QD−probe conjugates to multiple cycles of hybridization and dehybridization using FC target. As shown in Figure 5, incubating the paper for

Figure 5. Regeneration and reuse of paper-based solid-phase QD− FRET assay for multiple cycles of hybridization and dehybridization. Regeneration was based on incubating the paper in BB buffer containing 25% (v/v) formamide for 10 min. Abbreviations: X, initial sample exposure; S, sample exposure; R, regeneration cycle; NC, noncomplementary target.

10 min in 25% (v/v) formamide in BB buffer yielded the same baseline signal (FRET ratio = 0.021 ± 0.005) as in the presence of a NC target (FRET ratio = 0.027 ± 0.004), suggesting a probe regeneration (melting of probe and target hybrids) efficiency of 100% (within the precision of experimental measurement). It should be noted that, within the precision of the experimental measurement, the NC target yielded no signal above the background FRET ratio signal, i.e., in the absence of targets. In terms of the signal regeneration (signal recovery), each successive cycle of hybridization yielded a FRET ratio signal that was within 90−97% of the previous hybridization cycle suggesting that there was little deleterious effect of the 1865

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presence of a large background of noncomplementary nucleic acid sequences. Using a stringency control provided by a combination of ionic strength and formamide concentration, SNP discrimination was possible with a contrast ratio of 19 to 1. Regeneration of the biorecognition interface was also possible for at least five cycles with less than 20% decrease in the assay response. As a proof of concept, this work used labeled oligonucleotide targets for detection, but certainly other configurations can be used to eliminate the need to directly label target oligonucleotides.40 The methods presented herein can be extended to high-density arrays on a paper-based platform for parallel and high-throughput detection of multiple nucleic acid hybridization reactions using a fluorescence plate reader,41 and use of such a detection system would also be amenable to multiplexing owing to the unique optical properties of QDs.

regeneration conditions on the integrity of the chemistry that was used to assemble the biorecognition interface. In five cycles of regeneration, less than 20% decrease in the FRET ratio signal was observed to occur. The regeneration of previously reported solid-phase QD− FRET nucleic acid hybridization assays done on the surface of glass beads and optical fibers has been reported to be a challenging issue. A primary factor that has limited the reusability of such assays was the stability of the interfacial chemistry that was used to assemble oligonucleotide probes on the surface of immobilized QDs upon exposure to the regeneration conditions. For assays that have relied on the physisorption of NeutrAvidin on the surface of immobilized QDs to immobilize biotinylated oligonucleotide probes, each regeneration cycle resulted in reduced regeneration efficiency and nonspecific adsorption of oligonucleotides.23 This was attributed to the transient destabilization of the physisorbed protein layer of the interfacial chemistry upon exposure to regeneration conditions that were based on either the use of 70% (v/v) formamide or 40% (v/v) formamide at 45 °C for the same oligonucleotide probe and target sequences used in this work.20,23 The use of protein-free interfacial chemistry for the immobilization of oligonucleotide probes has often relied on the self-assembly of disulfide-terminated oligonucleotides to the ZnS shell of QDs27,28 (the same bioconjugation chemistry used in this work). Attempts at regeneration offered rather limited reusability when done using with 50% formamide at 25 °C, due to the deleterious effects of formamide on the QD PL.28 A possible explanation for the improvement of the regeneration efficiency achieved with the paper-based solid-phase assay presented in this work is that considerably milder regeneration condition (cf. 25% formamide in this work compared with 50− 80% formamide in the other assays) was used to achieve efficient regeneration. It was possible to use lower concentrations of formamide to achieve efficient regeneration because stringency control could be achieved by lowering the ionic strength of the solution (from 100 mM NaCl to no NaCl). Additionally, these results suggest that imidazole ligands serve as a robust platform for the immobilization of QDs for assay development. One potential disadvantage of the paper-based assay is the limited number of regeneration cycles that can be afforded with this solid-phase matrix due to the limited structural integrity of the paper over multiple washing cycles.

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ASSOCIATED CONTENT

* Supporting Information S

Detailed experimental procedures, description of instrumentation used, characterization of the FRET pair and equations used in the data analysis, solution-phase hybridization experiments, results for the time-based hybridization experiments, and stability results. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) for financial support of this research. M.O.N. is also grateful to the Ontario Ministry of Training, Colleges and Universities (MTCU) for provision of an Ontario Graduate Scholarship (OGS).

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REFERENCES

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CONCLUSIONS The integration of a solid-phase QD−FRET nucleic acid hybridization assay on a paper-based platform was investigated. The surface of paper was modified with imidazole ligands in a two-step synthetic protocol to immobilize QD−probe conjugates that were assembled in solution. Rapid transduction of nucleic acid hybridization (within 2 min) was possible upon introduction of Cy3-labeled oligonucleotide targets, where the hybridization event provided the proximity necessary for FRET-sensitized Cy3 emission that served as an analytical signal. FRET based transduction eliminated the need for extra washing steps required to remove nonhybridized oligonucleotide sequences, while also facilitating time-based monitoring of the hybridization reaction. Using a ratiometric approach for analysis (FRET ratio), the LOD of the assay was found to be 300 fmol without using any amplification steps. The use of GSH−QDs for the development of QD−FRET hybridization assay also showed excellent resistance to nonspecific adsorption of oligonucleotides, enabling hybridization assays in the 1866

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NOTE ADDED AFTER ASAP PUBLICATION This paper was published ASAP on the Web on January 9, 2013. Corrections were made to the Supporting Information, and the corrected version was reposted on January 10, 2013.

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