Precise Control of the Assembly of Dye-Coded Oligonucleotide Silver

Dec 18, 2009 - Strathclyde, Thomas Graham Building, 295 Cathedral St, Glasgow, U.K. G1 1XL. ReceiVed: October 8, 2009; ReVised Manuscript ReceiVed: ...
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Precise Control of the Assembly of Dye-Coded Oligonucleotide Silver Nanoparticle Conjugates with Single Base Mismatch Discrimination Using Surface Enhanced Resonance Raman Scattering† David G. Thompson, Karen Faulds, W. Ewen Smith, and Duncan Graham* Centre for Molecular Nanometrology, Dept. Pure and Applied Chemistry, WestCHEM, UniVersity of Strathclyde, Thomas Graham Building, 295 Cathedral St, Glasgow, U.K. G1 1XL ReceiVed: October 8, 2009; ReVised Manuscript ReceiVed: NoVember 15, 2009

The control of the assembly of nanostructures of dye-coded oligonucleotide-silver nanoparticle (DOSN) conjugates (silver nanoparticles cofunctionalized with thiol modified DNA and a Raman reporter molecule) in a sandwich assay format with single base discrimination in an unmodified target oligonucleotide is reported. Rational placement of a single base mismatch in the 18 base duplex formed between target and DOSN conjugates completely controls the hybridization event, preventing or allowing nanostructure formation. The assembly process was monitored using UV-vis spectrometry and surface-enhanced resonance Raman scattering (SERRS). These two supporting analysis techniques show that there is an initial formation of small, highly SERRS active assemblies followed by the formation of larger superaggregates. The rapid increase in SERRS intensity obtained from DOSN conjugate hybridization has been utilized for the near-immediate discrimination of a single base mismatch using SERRS. This report highlights the exquisite control and detection possibilities offered by coupling specific molecular recognition events, such as DNA hybridization, with the optical and SERRS responses possible by nanoparticle assembly. 1. Introduction Bottom-up organization of nanomaterials into submicroarchitectures is one of the major research drivers in the current climate of miniaturization. The ability to create ordered nanostructures will have a massive effect upon the fields of electronics, through synthesis of nanowires; power generation, by fabrication of new types of solar cells; and optics, by creating semiconductors with unique optical properties. The methodology behind the rational organization of nanobuilding blocks is one of the critical determinants with this work and one in which nature has already provided a suitable scaffold: deoxyribose nucleic acid (DNA). DNA is arguably the most studied chemical moiety of the past half century. The structure of DNA, and the formation of the double helix, is well understood and this indepth knowledge has allowed scientists to use the programmable nature of DNA to create nanostructures composed solely of DNA,1 with both two- and three-dimensional structures having been reported.2–4 While these nanomaterials composed solely of DNA are of considerable interest by themselves, they are also excellent starting points for the organization of other materials, namely metallic nanoparticles, on the nanoscale. Two methods are currently used for the organization of metallic nanoparticles by DNA; templating of metals onto the oligonucleotides via salt exchange5 or by conjugation of DNA to a metal nanoparticle.6 Oligonucleotide-gold nanoparticle (OGN) conjugates and oligonucleotide-silver nanoparticle (OSN) conjugates are now well established research tools consisting of synthetic oligonucleotides coupled to metal nanoparticles.7–9 These conjugates have been used to detect a massive variety of biological entities such as genomic DNA,10 bacterial DNA,11 duplex and triplex †

Part of the “Martin Moskovits Festschrift”. * Corresponding author. E-mail: [email protected].

binding sequences,12 and proteins,13 utilizing the change in surface plasmon caused by the reversible aggregation of the nanoparticles due to hybridization of the conjugated oligonucleotide. OGN and OSN conjugates are traditionally used in an oligonucleotide sandwich approach where two nanoparticles are functionalized with noncomplementary sequences followed by exposure and hybridization to a nonfunctionalized target oligonucleotide complementary to both. As the conjugates are densely functionalized with oligonucleotides and the target is present in excess, the aggregates formed are typically large and random. However, control is possible by altering the density of the conjugated oligonucleotides on the surface of the nanoparticle and the variation available in nanoparticle size. This has allowed for the creation of a wide variety of structures such as dimers14,15 and trimers14 between different sized nanoparticles, “halos” of small nanoparticles surrounding larger nanoparticles16 and controlled structures containing both silver and gold nanoparticles.15,17 All of these reported structures are organized either by manipulation of the sequence composition to force the nanoparticles into a particular geometry14,16 or by the addition of a secondary oligonucleotide that removes bound structures from a supporting microparticle15 using multiple base pairings. OGN and OSN conjugates have been used with a wide variety of analytical methods including optical,18,19 electrochemical,20 and vibrational21,22 techniques. These use the enhancement properties of the nanoparticle to facilitate detection but recently both OGN and OSN conjugates have been used to “turn on” detection via enhanced Raman scattering solely through oligonucleotide hybridization.22,23 When the nanoparticle surface is functionalized with both DNA and a Raman active molecule, hybridization to a target utilizing an oligonucleotide sandwich causes an increase in the surface enhanced resonance Raman scattering (SERRS) intensity achieved. With a large range of

10.1021/jp909639b  2010 American Chemical Society Published on Web 12/18/2009

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Figure 1. Composite diagram of the properties of DOSN conjugates featuring; (A) a schematic representation of before (i) and after (ii) exposure to a complementary target, (B) UV-vis spectra before (i) and after (ii) target addition showing the decrease and red-shift of the surface plasmon and, (C) SERRS spectra before (i) and after (ii) target addition showing the increase in the SERRS intensity. Inset graph is the increase in SERRS intensity monitored at the spectral maximum (1368 cm-1) monitored over 60 min. The UV-vis spectra were obtained from 10 pM DOSN conjugates exposed to 2.5 nM target. The SERRS spectra were obtained from 30 pM DOSN conjugates exposed to 7.5 nM target. Conditions: excitation wavelength 532 nm, 1 s scan time, 1 accumulation.

SERRS active molecules available,24–27 and the ability to detect in vivo,28 this combined technique shows great promise. This study fully expands on the single base mismatch discrimination concept to comprehensively analyze every possible mismatch at a set point critical for duplex formation using two mutually cooperative techniques: SERRS and UV-vis spectrometry. 2. Experimental Methodology 2.1. Materials. All chemicals, with the exception of those used in oligonucleotide synthesis, were purchased from SigmaAldrich, U.K. All DNA monomers, reagents and CPG columns were purchased from Link Technologies, Bellshill, U.K. Disposable cuvettes for SERRS experiments were purchased from VWR, U.K. All HPLC columns were purchased from GE Healthcare Life Sciences, U.K. 2.2. Instrumentation. All UV-vis analyses were performed using a Varian Cary 300 UV-visible spectrometer fitted with a Peltier temperature controller. All timed SERRS experiments were performed using an Avalon Instruments RamanStation R3 spectrometer fitted with a cuvette holder, excitation wavelength 532 nm. All near-immediate SERRS experiments were performed using a Renishaw InVia Microscope system fitted with a cuvette holder, excitation wavelength 514 nm. DNA synthesis was performed using a Bioautomation MerMade 6 DNA synthesizer, Plano, TX. DNA purification was performed using a Dionex P680 HPLC. 2.3. DOSN Conjugate Synthesis. DOSN conjugates were prepared by the following method. A 30 µL aliquot of 3,5-dimethoxy-4-(6′azobenzotriazolyl)phenol (stock concentration 1 × 10-3 M) was added to 3 mL of silver nanoparticles in a 5 mL glass vial. A 1 mL solution of 10 µM thiol modified DNA and 10 µM dithiothreitol was prepared in 60 mM phosphate buffer (pH 8.5). This was added directly to a solution of the modified silver nanoparticles via a HiTrap Sephadex superfine desalting column. The resulting solution was stored

in the dark for 24 h. A 0.333 mL sample of 60 mM phosphate buffer (pH 7) was then added, and the solution was left in the dark for a further 24 h. Then 123 µL of 2 M sodium chloride solution was added and the conjugate solution left again for 24 h. A 126 µL amount of 2 M sodium chloride was added, and the resulting solution was left for 24 h. The conjugate solution was then pipetted into four autoclaved 1.5 mL centrifuge tubes and centrifuged at 7000 rpm for 20 min. The supernatants were discarded and the oily pellets combined into a single centrifuge tubes with 1000 µL of 0.3 M phosphate buffered saline (PBS) (10 mM phosphate pH 7/0.3 M sodium chloride) added. This solution was centrifuged at 7000 rpm for 20 min. The supernatant was discarded and the pellet resuspended in 1000 µL of 0.3 M PBS pH 7 in an autoclaved centrifuge tubes. Silver nanoparticles (35 nm diameter) were synthesized by a citrate reduction method.29 3,5-Dimethoxy-4(6′-azobenzotriazoyl)phenol was synthesized by a previously published method.26 2.4. SERRS Analysis. For timed analysis the DOSN conjugates and target oligonucleotide were kept separate until dilution in the hybridization buffer. The final concentration of DOSN conjugate used in these experiments was 30 pM hybridizing to a final concentration of target oligonucleotide of 7.5 nM in a final volume of 750 µL of PBS. The full spectrum was then taken using a one second scan time, for one accumulation. For near-immediate analysis the DOSN conjugates and target oligonucleotide were premixed in a 100 µL centrifuge tubes tube followed by dilution to 150 µL in PBS. The full spectrum was then taken using one second scan time, one accumulation. 2.5. UV-vis Analysis. The UV-vis hybridization experiments were performed at room temperature using the “Kinetic” program set up to monitor the absorbance at 417 nm. The absorbance was measured every 15 s for 4 h. The final DOSN

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Figure 2. Schematic representation of the sequence composition of the two 9 base probes (P1 and P2) designed for hybridization only when the target is fully complementary. The diagram also illustrates their nanoparticle orientation (head to tail) and the position of the mismatch within the formed duplex (highlighted in yellow, bases denoted as X and Y). The recognition areas of the sequences are separated from the nanoparticle surface by a tether of 10 adenine bases.

conjugate concentration was 10 pM, hybridizing to a final concentration of target oligonucleotide of 2.5 nM in a final volume of 2 mL. The UV-vis melting experiments were performed using the “Thermal” program set up to monitor the absorbance at 417 nm. The temperature was initially set to 15 °C. The solutions were then heated to 45 °C and cooled back to 15 °C at a rate of 1 °C/min. The absorbance was measured every 0.1 °C. The final concentration of DOSN conjugates in the solution was 20 pM, hybridizing to a 5 nM final concentration of target oligonucleotide in a final volume of 2 mL.

Thompson et al. 3. Results and Discussion 3.1. DOSN Conjugate Assembly. DOSN conjugates consist of silver nanoparticles cofunctionalized with a Raman active species and a 5′ thiol functionalized oligonucleotide.22 With an oligonucleotide sandwich approach not only is there a decrease and red-shift in the surface plasmon of the silver nanoparticles but also there is an increase in the SERRS intensity received from the Raman active species. A combined diagram illustrating the attributes of the DOSN conjugate assembly is shown in Figure 1. This assembly process was performed at room temperature and can discriminate between a fully complementary target and noncomplementary control. 3.2. Oligonucleotide Probe Design. Typically, the oligonucleotide sequences conjugated to nanoparticles are greater than 12 bases long to ensure hybridization to the target of interest and conjugate stability. However, using probe sequences of this length means they hybridize not only to fully complementary targets but also to targets with a degree of complementarity such as those containing single base mismatches. A number of studies have elucidated the different melting temperatures obtained from target sequences containing noncomplementary bases at different positions,30–32 but no study has investigated the full effect of every exact match and mismatch at a fixed point within a sequence. Reducing the number of base pairs formed during hybridization lowers the melting temperature. Also, sequences with too few base pairs will not hybridize at room temperature. However, by careful control of the sequence and mismatch placement it is possible to design sequences that will hybridize at room temperature when fully complementary and will not when the target differs by a single base (Figure 2). These

Figure 3. UV-vis melting curves of DOSN conjugates monitored at their respective absorbance maximum (417 nm) for the four different probe sequences when exposed to the four different targets. (i) is the result of the DOSN conjugate with adenine as the probe base exposed to every possible base (A, C, G, and T). (ii) has cytosine as the probe base, (iii) guanine, and (iv) thymine. For ease, the mismatch base pairings are always written probe base:target base hence A:T is adenine probe:thymine target. Conditions: 20 pM DOSN conjugate concentration, 5 nM target concentration, and 2 mL final volume. The melting temperatures attained were (i) A:T 31.97 °C, (ii) C:G 34.02 °C, (iii) G:C 36.07 °C, and (iv) T:A 29.32 °C

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Figure 4. Decrease in the UV-vis absorbance of DOSN conjugates at 417 nm due to the hybridization of fully complementary targets to DOSN conjugates. Each graph contains the results of each probe sequence and every possible target base at the mismatch point. (i) is the result of the DOSN conjugate with adenine as the probe base exposed to every possible base (A, C, G, and T). (ii) has cytosine as the probe base, (iii) guanine, and (iv) thymine. All experiments were performed at room temperature. Conditions: 10 pM DOSN conjugate concentration, 2.5 nM target concentration, and 2 mL final volume.

sequences consist of two nine base probes (P1 and P2) that hybridize to an 18 base target. The mismatch point is at the center of P1. To fully investigate all possible mismatches, four different probe sequences were necessary, each containing a different base at the mismatch point as well as four different targets also containing a different base at the mismatch point. Having four probe sequences and four target sequences leads to sixteen separate experiments with each probe being exposed to its exact complement and its three possible mismatches. The viability of these probe and target sequences was investigated by analyzing the UV-vis melting characteristics of the DOSN conjugate sandwich (Figure 3). The melting curves and melting temperatures obtained show that the probe sequences were successfully designed to only allow hybridization, and hence aggregation, at room temperature to a target that is fully complementary. Any target containing a single base mismatch does not hybridize or aggregate. As expected, A:T and T:A have lower melting temperatures than C:G and G:C due to having fewer hydrogen bonds. However, rather than the same pairs resulting in the same melting temperature, there is a difference. This is due to the nearest neighbor effect of DNA base pairs, where the stability of a given base pair depends upon the identity of its neighbors.33 It should also be noted that some degree of duplex formation could be observed for a single base mismatch involving G:T or T:G when using the plasmon melting approach. 3.3. Analysis of Nanoparticle Assembly with Single Base Pair Discrimination. To analyze nanoassembly formation, two types of analysis were performed; UV-vis spectrometry (Figure 4) and SERRS (Figure 5). Figure 4 shows the decrease in UV-vis absorbance of DOSN conjugates solely due to the hybridization of a complementary target at room temperature.

Only a single mismatch (G:T, Figure 4(iii)) shows a decrease, and this is much smaller and slower than that of the complement. Probes with A, C, and G at the mismatch point take approximately 120 min to reach a stable plateau, while T probes have not plateaued by the end of the experimental time period (240 min). Figure 5 shows the increase in intensity at the spectral maximum of the DOSN conjugates due to their hybridization to a complementary target. None of the possible mismatches shows an increase in intensity above the initial baseline values, compared to the fully complementary sequences. Probes with A, C, and G at the mismatch point reached a plateau after 10 min while the T probe reached its plateau after 40 min. Comparing the SERRS and UV-vis results shows that the SERRS results reach completion a lot sooner than the UV-vis results and that the SERRS results achieved better discrimination, with only the complementary sequences showing an increase in intensity. This difference in rate indicates two distinct assembly processes. The initial hybridization of single DOSN conjugates to each other and hence the formation of small nanoassemblies such as dimers and trimers would only cause a minimal decrease in plasmon. This observation of smaller nanoassemblies not affecting the plasmon has been reported by other groups.34,35 However, the formation of smaller nanoassemblies can be monitored by SERRS as these dimers and small aggregates are highly SERRS active with large intensities observed.36,37 The hybridization of the small assemblies into larger aggregates causes the observed SERRS signal to level out due to the larger aggregates having a smaller SERRS intensity than the small assemblies.36 The formation of large aggregates and assemblies can be observed by the decrease in plasmon maximum in the UV-vis results.

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Figure 5. Increase in SERRS intensity at 1368 cm-1 due to the hybridization of fully complementary targets to DOSN conjugates. Each graph contains the results of each probe sequence and every possible target base at the mismatch point. (i) is the result of the DOSN conjugate with adenine as the probe base exposed to every possible base (A, C, G, and T). (ii) has cytosine as the probe base, (iii) guanine, and (iv) thymine. The Raman excitation wavelength was 532 nm with an accumulation time of 1 s. All experiments were performed at room temperature. Conditions: 30 pM DOSN conjugate concentration, 7.5 nM target concentration, and 750 µL final volume.

Figure 6. SERRS intensity of the most intense band after near-immediate single base mismatch discrimination. The complementary base pairs are highlighted and show a considerably higher spectral intensity at 1368 cm-1 than the mismatched targets. The intensities achieved for the complementary duplexes were A:T, 2040 counts, C:G, 2850 counts, G:C, 3140 counts, T:A, 1220 counts. The Raman excitation wavelength used was 514 nm.

3.4. Near-Immediate Single Base Mismatch Discrimination. To completely harness the rapid increase in SERRS intensity caused by hybridization, the preparation methodology was altered. For the previous SERRS experiments the DOSN conjugates were deliberately kept separate from the target prior

to dilution via careful pippetting. This allowed an accurate comparison to be performed of the single base mismatch targets. However, to fully take advantage of the fast increase in SERRS intensity, the DOSN conjugates and target were quickly premixed in their undiluted states followed by dilution to the

Assembly of Oligonucleotide Ag Nanoparticle Conjugates working volume of the analysis and immediate SERRS analysis. These results are shown in Figure 6. Figure 6 shows the immediate increase in SERRS intensity where the target is complementary to the DOSN conjugates. None of the targets containing a single base mismatch show any increase in intensity. This replicates the discrimination shown by the timed SERRS results (Figure 5) that also did not show any increase from the single base mismatch targets but is also significantly faster to reach a definitive result, especially with the least favorable probe:target configuration, T:A. Figure 5(iv) shows the intiensity increase for the complement is only significantly above the baseline after 20 min. The premixing of target and conjugate has shortened this drastically. As with previous results, the T:A duplex appears to be less stable than the others, having a significantly smaller intensity than the others. The maximum intensity data show excellent correlation to the melting temperatures previously attained (Figure 2). The duplex with the highest melting temperature also has the highest spectral intensity and this trend is replicated by the other results. DNA hybridization is commonly thought of as relatively slow, requiring incubation times in the minute or hour range. By showing these near-immediate results, we hope it could act as a cue to other researchers that simple method manipulation can drastically change the speed at which results are attained. 4. Conclusion This report details the control of DNA-nanoparticle assembly using the minimum amount of sequence manipulation possible. The hybridization, and hence formation, of nanoassemblies is solely controlled by the presence of a single base mismatch. Analysis using SERRS and UV-vis spectrometry has shown an initial formation of small, highly SERRS active nanoassemblies followed by the formation of larger aggregates that have a comparatively lower SERRS activity. The quick formation of the small, highly SERRS active nanoassemblies has been fully utilized by altering the methodology to facilitate nearimmediate discrimination of a single base mismatch in a target. This work illustrates the control possible in DNA-nanoparticle assembly and will hopefully lead to even more precise DNAmediated assembly becoming possible. Acknowledgment. We thank the Royal Society of Chemistry’s Analytical Chemistry Trust Fund for the award of their Analytical Grand Prix Fellowship for funding to D.T. and D.G. References and Notes (1) Seeman, N. C. Mol. Biotechnol. 2007, 37, 249–257. (2) Wang, Y.; Mueller, J. E.; Kemper, B.; Seeman, N. C. Biochemistry 1991, 30, 5667–5674. (3) Chen, J.; Seeman, N. C. Nature 1991, 350, 161–166. (4) Shih, W. M.; Quispe, J. D.; Joyce, G. F. Nature 2004, 427, 618– 621.

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