pubs.acs.org/NanoLett
A DNA-Silver Nanocluster Probe That Fluoresces upon Hybridization Hsin-Chih Yeh, Jaswinder Sharma, Jason J. Han, Jennifer S. Martinez,* and James H. Werner* Center for Integrated Nanotechnologies, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 ABSTRACT DNA-templated silver nanoclusters (DNA/Ag NCs) are an emerging set of fluorophores that are smaller than semiconductor quantum dots and can have better photostability and brightness than commonly used organic dyes. Here we find the red fluorescence of DNA/Ag NCs can be enhanced 500-fold when placed in proximity to guanine-rich DNA sequences. On the basis of this new phenomenon, we have designed a DNA detection probe (NanoCluster Beacon, NCB) that “lights up” upon target binding. Since NCBs do not rely on Fo¨rster energy transfer for quenching, they can easily reach high (>100) signal-to-background ratios (S/B ratios) upon target binding. Here, in a separation-free assay, we demonstrate NCB detection of an influenza target with a S/B ratio of 175, a factor of 5 better than a conventional molecular beacon probe. Since the observed fluorescence enhancement is caused by intrinsic nucleobases, our detection technique is simple, inexpensive, and compatible with commercial DNA synthesizers. KEYWORDS Noble metal fluorescent nanoclusters, nucleic acid detection, separation-free probes, nanobiosensors
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problems associated with double labeling such as high cost, low yield, and singly labeled impurities. Here we show that DNA-templated silver nanoclusters can be used to detect specific nucleic acid targets in a manner that circumvents many of the shortcomings of conventional MBs. DNA/Ag NCs represent a new class of fluorophores16-21 that already have seen applications in cellular imaging22 and chemical/biological detection.16,23,24 While DNA/Ag NCs are promising as small and photostable fluorescent reporters, DNA/Ag NCs can sometimes change color,19,20,25,26 a conversion process neither well understood nor generally shared by organic dyes or photoluminescent nanocrystals. While these conversions can be viewed as a drawback, in the appropriate context, they can be used as new signal transduction mechanisms for molecular sensing. Often reversible, transformations among NC species having distinct emission wavelengths can depend upon a number of factors, including time, temperature, oxygen, and salt content.19,25,26 In this Letter, we report a new factor, guanine proximity, that can trigger reversible transformation of NCs between a dark species and a bright red-emitting species that we exploit for DNA target detection further below. As shown in Figure 1a, dark DNA-Ag NCs can be transformed into bright red-emitting clusters when placed in proximity to guanine bases. In these experiments, Ag NCs were formed on DNA Strand_1 as described in Supporting Information S2. Strand_1 has a 12-base NC-nucleation sequence represented by the red line in Figure 1a and a 30base hybridization sequence represented by the blue line, with these sequences listed in Table S1 (Supporting Information). Eighteen hours after the initiation of NC nucleation, the reaction solution had weak green fluorescence emission. Upon addition and hybridization of a complement having a guanine-rich tail (3′-G4(TG4)2TG3), a strong red fluorescence
he detection and quantification of specific biomolecules, ions, or metabolites are important for in vivo real-time monitoring of cellular processes1,2 and for in vitro biosensing and clinical diagnosis.3,4 Of the fluorescent probes used for these applications, those that enable detection without separation are more desirable, especially for intracellular studies, where removal of unbound probes is difficult. To this end, a number of detection strategies have been developed wherein reporters fluoresce upon probetarget binding, including split green fluorescent proteins,5 electron transfer-based probes,6,7 biarsenic organic dyes,8 triphenylmethane dyes,9 intercalating dyes,10 fluorescence resonance energy transfer (FRET)-based indicators,11 and molecular beacons (MBs).12 In particular, molecular beacons, hairpin-shaped nucleic acid probes that fluoresce upon hybridization with specific nucleic acid targets, have seen tremendous use since their introduction in 1996.12 While being one of the most successful separation-free probes,1-4 MBs have certain drawbacks. First, fluorescence enhancement of MBs is generally limited by background fluorescence, which comes from imperfect quenching of donors4 and conformational fluctuations of the hairpin structure.13 Well-designed MBs can achieve a signal-to-background ratio (S/B ratio),14 (Itarget Ibuffer)/(Ino target - Ibuffer), of more than 100-fold but often require special quenchers14 or sophisticated thermodynamic analysis for stem-loop sequence selection.15 Second, a MB needs to be labeled with two non-native moieties (i.e., a donor fluorophore and a quencher), thus suffering from
* To whom correspondence should be addressed,
[email protected] (J.H.W.) and
[email protected] (J.S.M.). Received for review: 05/19/2010 Published on Web: 07/19/2010 © 2010 American Chemical Society
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DOI: 10.1021/nl101773c | Nano Lett. 2010, 10, 3106–3110
FIGURE 1. Schematic and data showing the red fluorescence enhancement of DNA-templated silver nanoclusters (DNA/Ag NCs) caused by guanine proximity. (a) Schematic showing red fluorescence enhancement of DNA/Ag NCs through proximity with a G-rich overhang, 3′G4(TG4)2TG3, caused by DNA hybridization and photographs of the resulting emission under UV (366 nm) irradiation. (b) 3D- and 2D-countur plots of excitation/emission spectra of the Ag NCs before (left) and after (right) hybridizing NC-bearing Strand_1 with Strand_HC_15G (see Table S1). Inset: Integrated red fluorescence emission with the buffer fluorescence subtracted in arbitrary units. The excitation/emission peaks for aged NCs on Strand_1 before hybridization were at 460 nm/543 nm. The excitation/emission peaks changed to 580 nm/636 nm after hybridization. The integrated red fluorescence emission was enhanced ∼500-fold after duplex formation.
emission was observed from the solution, with a bulk enhancement ratio greater than 500 (Figure 1b). We performed a series of control experiments, including a gel shift assay, to ensure that the observed fluorescence enhancement was due to guanine proximity caused by DNA hybridization (Supporting Information S3 and S7). Measurements taken on a real-time PCR thermal cycler (Figure 2) provided additional verification that the red fluorescence emission came from a DNA duplex and demonstrated the reversibility of the fluorescence enhancement mechanism. In addition to testing fluorescence enhancement from guanine proximity, we explored possible enhancement from the proximity of other nucleosides. No red fluorescence enhancement was observed for adenine- and thymine-rich strands. However, cytosine-rich strands appeared to induce an irreversible cluster transfer, and thymine-rich strands produced a green emission (see Supporting Information S5). To further investigate the guanine proximity-induced fluorescenceenhancement,wehybridizedNC-bearingStrand_1 with a number of complements of varying guanine content on the overhang opposite to the NCs. The extent of red fluorescence enhancement was found to exponentially increase with an increasing number of guanine bases in proximity to the NCs (Figure 3). To better understand this systematic increase, we measured the per cluster brightness of the red Ag NCs using fluorescence correlation spectroscopy. The brightness of individual nanoclusters was essentially identical when different complements were used (Supporting Information Table S9). The observed increase in the bulk fluorescence for strands of increasing guanine © 2010 American Chemical Society
content (Figure 3) was caused by an increase in the number of Ag nanoclusters “turned on” due to guanine proximity. Approximately 6% of NC-bearing Strand 1 was transformed into bright red fluorophores when duplexed with Strand_ HC_15G. This relatively low conversion ratio already generated a more than 500-fold bulk fluorescence enhancement. Clearly, increasing the conversion percentage, possibly through an optimization of cluster formation conditions or a better selection of NC-nucleation and G-rich sequence pairs, can push the bulk red fluorescence enhancement further. At present, we do not fully understand the physical mechanism driving the increase in red fluorescence emission. It is possible that the G-rich sequences may form secondary structures (i.e., G-quadraplex) that favor formation of red-emitting NCs. A second possibility is that the conformational change of DNA due to cytosine-guanine base pairing may lead to dissociation of nonemissive NCs and formation of red-emitting NCs. However, these two possibilities are weakened by the finding that deoxyguanosine triphosphate (dGTP), once added to the NC-bearing Strand_1 solution, can slightly enhance the red fluorescence emission (Supporting Information S4). These possibilities also do not easily explain the trend shown in Figure 3, where the red fluorescence emission grows exponentially with guanine content. A third possibility is that the red fluorescence emission is due to electron transfer from guanines to the nanoclusters. Guanines have the lowest oxidation potential among the four nucleotides27 and can often alter the emission rates of organic dyes.7,27-32 Here, guanines may 3107
DOI: 10.1021/nl101773c | Nano Lett. 2010, 10, 3106-–3110
FIGURE 3. The red fluorescence enhancement of DNA/Ag NCs increases with the number of guanines in proximity. Integrated red fluorescence emission (left axis) and bulk fluorescence enhancement (right axis) from 9 samples. “No complement” represents a sample containing only NC-bearing Strand_1, whose emission is used as a baseline for the enhancement ratio calculation. The other eight samples contain NC-bearing Strand_1 and a complement. The complement’s overhang sequence is listed on the bottom axis; strands having overhangs 3′-G to 3′-G4(TG4)2TG3 correspond to strand numbers 7 to 13 in Table S1. The molar concentrations of NCbearing Strand_1 and complement were both at 15 µM. Hybridization was carried out in a 20 mM pH 6.6 sodium phosphate buffer. The mixture was first heated to 95 °C for 1 min, followed by cooling to room temperature for at least 50 min. “No overhang” represents a sample using Strand_HC as complement.
FIGURE 2. Competitive binding assays demonstrating red fluorescence enhancement is due to guanine proximity driven by DNA hybridization. (a) Representative fluorescence trace of DNA/Ag NCs during a thermal cycling process. The sample initially contained NCbearing Strand_1 and Strand_HC_15G (at 1:1 molar ratio). A 5-fold excess of naked Strand_1 was spiked into the solution after three thermal cycles, as marked by the arrow. Due to competitive binding, the fluorescence recovered to approximately 1/6 of its original value at 25 °C, indicating that the highly emissive NCs resulted from DNA hybridization. (b) Fluorescence intensity versus inhibitor/Strand_1 molar ratio. In this competitive binding experiment, we tested another inhibitor, Strand_HC, which also prevented NC-bearing Strand_1 from complexing with Strand_HC_15G. The resulting red emission had a 1/(1 + RI) relationship (solid line) with the amount of inhibitor used, where RI is the molar ratio between inhibitor and Strand_1. The samples were heated to 95 °C for 1 min and gradually cooled to room temperature before their fluorescence was measured. Markers (4, ×, O) represent three replicate measurements.
for separation-free detection of a specific DNA target. The NCB consists of two short linear DNA probes (a NC probe and a G-rich probe) that are brought into proximity through hybridization with target DNA (Figure 4a). With our initial NCB design (NCB_1), we observed a 76-fold increase in red emission in the presence of target DNA (a sequence from human Braf oncogene), as compared to a sample without target DNA or with a nonspecific target (Figure 4b). Complementary single NCB imaging on a total internal reflection fluorescence (TIRF) microscope showed a significant increase in the number of red emitting NCBs with target (Figure 4c), as compared to the sample without target (Figure 4d). These experiments demonstrate that NCBs can be used for both quantitative ensemble measurements and singlemolecule-based digital analysis of specific DNA targets. To directly compare NCB performance to a molecular beacon, we designed another NCB (NCB_2) to detect an influenza target (a sequence from influenza A virus (S-OIV) (H1N1)). We achieved a S/B ratio of 175 with the NCB_2 versus a S/B ratio of 32 for the molecular beacon on the same target (Figure 4e). The benefits of using NCBs lie in their simple, low cost “one-step” preparation process and potential to achieve an extraordinarily high S/B ratio. Conventional fluorescently labeled linear hybridization probes require only a single labeling step, but they can be used to detect specific targets only through binding with multiple fluorescent probes.34 FRET-based probes offer separation-free detection and a good S/B ratio, but they typically require dual labeling steps. Our method is unique not only because it requires only a
serve as electron donors, reducing oxidized-NC species (in this case, nonemissive NCs) into bright red-emitting NCs. The simple trend in Figure 3 (the red fluorescence intensity doubles approximately for every two guanines added) may imply that electron transfer is the mechanism responsible for the observed phenomenon. To further test this electrontransfer hypothesis, we performed an experiment where the five guanines of sequence Strand_HC_5G were substituted with five 7-deazaguanines, which is a stronger electron donor than guanine.33 We found that no red fluorescence emission resulted from hybridization of NC-bearing Strand_1 with the deazaguanine-substituted Strand_HC_5G (Supporting Information S10), weakening the electron transfer hypothesis. Another experimental result (Supporting Information S5) that weakens the electron-transfer hypothesis is that thymine-proximity produces a green fluorescence enhancement, with thymine being a worse electron donor than adenine (adenine proximity did not generate any measurable fluorescence enhancement). We are still investigating the underlying fluorescence enhancement mechanism. Taking advantage of this tremendous fluorescence enhancement upon guanine proximity, we designed a simple “light-up” probe, which we call a NanoCluster Beacon (NCB), © 2010 American Chemical Society
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FIGURE 4. Schematic and data of a NCB for specific DNA detection. (a) Schematic of NCB probe design. (b) Quantitative and specific detection of target DNA (a sequence from human Braf oncogene) from the initial NCB design (NCB_1). Kras sequence was used as a nonspecific control. “Braf 1:1” represents a sample with one to one target/probe molar ratio, in this case, 7.5 µM each. The average signal-to-background (S/B) ratios of three Braf samples are listed above each bar. This ratio scales linearly with the concentration of Braf target. Red emission from the sample without target and the Kras sample were similar. The NC and G-rich probes used here were purified by desalting during DNA synthesis. No purification was performed after NC formation. Bars (black, gray, white) represent three separate measurements. (c) Fluorescence image of individual NCBs from Braf 1:1 sample nonspecifically adsorbed to a coverslip taken with a total internal reflection fluorescence microscope. (d) Fluorescence image of individual NCBs from a sample without target. (e) The S/B ratio (red bar, left axis), target-specific signal (Itarget Ibuffer, light blue bar, right axis), and background fluorescence (Ino target - Ibuffer, dark blue bar, right axis) from the second NCB design (NCB_2) and a molecular beacon compared for influenza target detection. The probe and target concentrations were both at 5 µM.
single preparation step (i.e., NC formation on NC probes), but because there is no need to remove excess silver ions or borohydride ions from solution after NC formation is completed, as these are essentially nonfluorescent. Unlike conventional MBs, the fluorescence background of NCBs is not limited by Fo¨rster quenching, but rather by the existence of sparse NCBs that are red emitting in absence of target (Figure 4d). These background-fluorescence-generating NCBs are a small fraction (
