Base-Stacking-Determined Fluorescence Emission of DNA Abasic

Aug 11, 2012 - Job,s plot analysis shows that Ag2 NCs are the species responsible for the ..... emission intensities on the concentration ratios, good...
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Base-Stacking-Determined Fluorescence Emission of DNA Abasic Site-Templated Silver Nanoclusters Kun Ma, Yong Shao,* Qinghua Cui, Fei Wu, Shujuan Xu, and Guiying Liu Zhejiang Key Laboratory for Reactive Chemistry on Solid Surfaces, Institute of Physical Chemistry, Zhejiang Normal University, Jinhua 321004, Zhejiang, People’s Republic of China S Supporting Information *

ABSTRACT: DNA-templated silver nanoclusters (Ag NCs) are emerging sets of fluorophores that are widely applicable because of high brightness, good photostability, and visible to near-infrared emissions tunable using the DNA sequence and length to change the NC size. We find that fluorescent Ag NCs can be size-selectively grown at DNA abasic sites (AP site) using a constrained duplex environment opposed by a cytosine and flanked by two guanines. The size of the AP site-grown Ag NCs is not affected by the increasing Ag+ concentration. A Job’s plot analysis shows that Ag2 NCs are the species responsible for the observed emissions. Although varying the DNA sequence one base away from the AP site (i.e., the Ag NC growth site) does not alter the size of the fluorescent Ag NCs, the emissions of the formed Ag NCs are still gradually red shifted as the sequence changes from thymine (T) to cytosine (C), adenine (A), and guanine (G). Furthermore, this emission shift is strongly dependent on the base-stacking direction of the 3′side sequence of the 5′-G stack exactly flanking the AP site, which exhibits a larger emission alteration than altering the 5′-side sequence of the 3′-G stack flanking the AP site on the other side of the site. The excited-state lifetimes of the Ag NCs are inversely proportional to the singlet energies (ΔE0,0) of the Ag NCs relative to their ground state and of the vertical ionization potentials of the guanines directly flanking the AP site as determined by the base stacking. All of these results support the conclusion that the Ag NC excited state becomes more stable by interacting with a guanine base because of the larger electronic dipole moment that can be modified by the stacked sequences. Additionally, the size of the formed Ag NCs seems to be dependent on the consecutive AP site number. Thus, the AP site design in this work provides an easy way to shed light on the role of DNA base stacking in the optical properties of Ag NCs.

1. INTRODUCTION Noble metal nanoclusters with sizes comparable to the Fermi wavelength of an electron possess molecule-like fluorescent properties having discontinuous energy band structures. Such energy band gaps are much larger than the thermal motion energy of the electron.1−4 Among these nanoclusters, silver nanoclusters (Ag NCs) are seen as novel fluorophores for their special emission properties of brightness and photostability and have been used in the fields of sensing, catalysis, and surfaceenhanced Raman scattering (SERS).5−10 Many efforts have been made to constrain Ag NC growth to a useful size range by employing chitosan, ionic liquids, dendrimers, and polymers11−16 as capping templates. In contrast to peptides17−19 and proteins,20−22 nucleic acids (mainly DNA23) are widely accepted as novel biocompatible templates, being very convenient in DNA chemical synthesis for variable DNA sequences and lengths. Because of its flexibility in conformations, single-stranded DNA (ss-DNA) is widely employed as a useful template for creating Ag NC emitters with tunable emissions and alterable quantum yields.24−51 However, solvent conditions such as pure water26,28,31,37,50 and added buffer salt26−35,39,42,44−51 have a profound effect on Ag NC creation and subsequent optical properties. This sensitivity makes the © 2012 American Chemical Society

modeling of DNA sequence-dependent Ag NC emissions very intricate. However, a polycytosine bulge,52 mismatch,53 and abasic site (AP site)54 have been inserted into double-stranded DNA (ds-DNA) to accommodate fluorescent Ag NCs so as to sense, in situ, the DNA sequences that surround the Ag NCs. Many factors affect the Ag NC emission behavior. Previous studies have shown that, in addition to its determinative role in the Ag NC size, DNA can also control Ag NC emission by exactly following external stimuli-induced DNA conformation changes such as pH-induced i-motif formation,27,30 K+-induced G-quadruplex conversion,35 and even the protonation and deprotonation of cytosine.24,40 All of these observations can be easily explained by the fact that silver species favor specific binding to DNA’s heterocyclic bases rather than to its phosphate backbones.55,56 Therefore, interactions between the clusters and the surrounding DNA bases are the key criteria for modulating the Ag NC emissions. For example, Neidig et al. demonstrated the DNA template’s cooperative role in Ag NC emission by determining the size and simultaneously chelating Received: January 3, 2012 Revised: August 9, 2012 Published: August 11, 2012 15313

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to Ag atoms.57 In previous work, we found that DNA compactness around the NCs, which can be altered by neutralizing the negative charges of the DNA phosphate backbones in the presence of Mg2+ favoring the interaction between the DNA bases and silver, is also responsible for variations in Ag NC emissions.58 Fygenson36 also recognized that this factor is able to modulate Ag NC emission. However, there are other factors that could serve as effective Ag NC emission tuners. For example, Martinez and Werner29 reported that preformed Ag NCs changed their emission intensity and position upon hybridizing with, and thus being approached by, guanine-rich strands. We also investigated the selective growth of fluorescent Ag NCs at an AP site, and the results showed that guanines near the AP site play a crucial role in Ag NC emissions.54 In this article, we try to get more information about the sequence’s role in Ag NC emissions by employing ds-DNA with an embedded AP site as a promising binding site. The constrained space provided by the AP site should limit the growth of the Ag clusters to a defined size, thus offering a convenient tool for studying the role of nucleobases near the AP site. The sequences one base away from the AP site are systematically changed, and those bases in direct contact with the AP site are preserved to maintain intact the environment directly surrounding the Ag NCs (Scheme 1). We found that

is caused by the dipole moment of the 5′-guanine, which can stabilize the excited state of the Ag NCs to a much higher degree than the corresponding ground state.

2. EXPERIMENTAL SECTION DNA strands were synthesized by TaKaRa Biotechnology Co., Ltd. (Dalian, China), and all DNA samples were purified by HPLC. Other reagents were analytical grade and were used without further purification. Tetrahydrofuran residue was used as the chemically stable AP site for the replacement of naturally tautomeric deoxyribose structures in order to avoid its Tollens reaction with Ag+.59,60 As a proof of concept, the DNA sequences used in this article were derived from easily mutated fragments near codon 177 of cancer suppression gene p53. For the control experiments, we introduced minor modifications near the AP site (Scheme 1). Prior to hybridization, the DNA concentrations in the single-stranded format were measured at 260 nm in pure water on the basis of extinction coefficients calculated using nearest-neighbor analysis.61 To prepare the DNA duplex solutions, the two single-stranded DNAs (Scheme 1) were mixed in equimolar amounts and annealed in a thermocycler (starting at 92 °C and then slowly cooled to room temperature) in 20 mM phosphate buffer (pH 7.0) containing 1 mM Mg2+. AgNO3 (SigmaAldrich Ltd.) in a 20 mM phosphate buffer (pH 7.0) containing 1 mM Mg2+ was added to the DNA solution (5 μM DNA duplex solution if not stated) in an appropriate molar ratio. After being mixed, the solution was incubated for 15 min with gentle stirring and then reduced by the fast addition of freshly prepared NaBH4 to an appropriate quantity and allowed to react for up to 1 h at ambient temperature. A concentration ratio of 1:4 NaBH4/Ag+ was finally optimized to get a high emission signal. The resulting solutions were examined at room temperature within 1 h. Nanopure water (18.2 mΩ; Millpore Co., USA) was used in all experiments. Fluorescence spectra were acquired with a FLSP920 spectrofluorometer (Edinburgh Instruments Ltd., U.K.) at 20 ± 1 °C, equipped with a temperaturecontrolled circulator (Julabo, Germany). The Job’s plot analysis for the stoichiometric numbers of Ag+ binding to the duplex Y-MPs, tsX-YZs, and tcX-CC (Scheme 1) was carried out by keeping the total concentrations of the Ag+ and DNA duplex mixtures at 30 μM and successively changing the DNA/Ag+ concentration ratios. The absorbance resulting from the simultaneously formed large nanoparticles was corrected at the excitation wavelength for the corresponding Ag NCs. The singlet energy (ΔE0,0) relative to the ground state of the as-prepared Ag NCs was measured from the intersection point by normalizing the corresponding excitation and emission spectra. Fluorescence lifetimes were measured on a timecorrelated single-photon-counting FLSP920 system, with excitation at 350 nm and detection at the Ag NC emission maxima. The excitation source was an nF900 ns pulse hydrogen lamp, and a Ludox solution was used as the scatter for the instrument response. The data were fitted with a single-exponential decay, and χ2 was less than 1.2. The melting temperatures (Tm) were determined using a UV2550 spectrophotometer (Shimadzu Corp., Japan) equipped with a TMSPC-8 system accessory that can simultaneously control the chamber temperature and detect up to eight micromulticell samples with a low deviation in temperature between samples.

Scheme 1. Schematic Representation of the AP-SiteConstrained Formation of Fluorescent Ag NCsa

The AP site was flanked by two guanines and opposed by a base Y (where Y = C, A, G, or T). Sequences M/N and P/Q (M/N, P/Q = G/C, C/G, T/A, or A/T) one base away from the AP site were systematically changed in order to investigate their roles in the electronic properties of the guanines directly flanking the AP site. The AP site’s two flanking guanines were thus different in sequence direction. The guanine flanked by M was located on the 3′ side (3′-G), and the guanine flanked by P was located on the 5′ side (5′-G). The DNA sequences used follow the nomenclature of Y-MP. Control sequences containing two separated AP sites (tsX-YZ) were used to contrast Ag NC growth at the AP site. Additionally, a DNA containing two consecutive AP sites (tcX-CC) was employed to investigate the capability of synthesizing Ag NCs having a larger size than possible with DNAs having one AP site as the template. a

3. RESULTS AND DISCUSSION 3.1. Ag NC Growth at the AP Site to a Well-Defined Size. Variable-length, variable-sequence single-stranded DNA (ss-DNA) has been widely proven to be a promising candidate for creating fluorescent Ag NCs with emissions tunable between the visible and the near-infrared.24−51 Different emissions have been observed simply by varying the assembly manner of all of the bases while keeping the ss-DNA at the same length and base composition.26 In previous work, we observed preformed Ag NCs emitting at different wavelengths upon addition of electrolytes.58 It is difficult to achieve a clear

Ag NC emissions are strongly dependent on not only the sequence contexts but also on the sequence direction. The Ag NC band gaps are mainly determined by alterations in the basestacking-induced vertical ionization potentials (VIP) of the 5′guanine directly flanking the AP site in tandem with 3′-guanine also flanking the AP site (i.e., the lower the VIP of the 5′guanine, the narrower the band gap). We believe that this result 15314

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Figure 1. (A) Fluorescence excitation and emission spectra of Ag NCs with Y-GG (Y = C, A, G, or T) duplexes as the templates. The fully matched DNA (FM) with M/N = P/Q = X/Y = G/C was used as a control. (Inset) Photographs of the fluorescent Ag NCs generated using the Y-GGs and FM as templates under UV illumination. (B) Fluorescence excitation and emission spectra of Ag NCs templated by C-GG with increasing Ag+ concentration: [Ag+]/[C-GG] = 2:1, 5:1, 7:1, 10:1, 13:1, 15:1, 17:1, and 20:1. The Ag NCs were synthesized in a 20 mM phosphate buffer (pH 7.0) with 1 mM Mg2+. (Inset) Photographs of the fluorescent Ag NCs templated using C-GG with increasing Ag+ concentration (from left to right) under UV illumination.

Figure 2. Ag NC emission spectra dependency of the excitation wavelength for templates (A) C-GG and (B) C-GC.

GG), guanine (G-GG), or thymine (T-GG) opposite the AP site. This observed strongest emission for C-GG is in good agreement with the strong binding capacity of unpaired base C to Ag+.24,62 There are two possible locations for the formed Ag NCs on these ds-DNA's: an AP site or a groove. However, we did not observe any emission for the fully matched DNA being devoid of the AP site (FM, Scheme 1, X = G, Y = C, and Figure 1A), indicating that groove binding of the fluorescent Ag NCs is not possible. Previously, we proved that the Ag NC emission is strongly dependent on the AP site’s flanking bases, even through the base opposite the AP site was C (Figure S1, Supporting Information).54 According to these facts, it is certain that the formed Ag NC is located at the AP site. This selective growth of an Ag NC at the AP site can even be discriminated with the naked eye under UV illumination (inset of Figure 1A). However, similar excitation (596 ± 9 nm) and emission (685 ± 4 nm) maxima exist for all Y-GGs with different unpaired bases opposite the AP site, although the emission intensities are highly Y-dependent (Figure 1A). This suggests that the formed Ag NC emission wavelengths are less dependent on the unpaired bases, possibly by growing the Ag NCs to a similar size. Thus, the incubated Ag+ concentrations before reduction are not a final factor affecting Ag NC growth to a critical size at the AP site because of the finite void defined by the AP site. For example, the C-GG spectral profiles obtained for Ag+ to C-GG concentration ratios increasing from 2:1 to 20:1 are almost identical, with the invariable excitation and emission maxima located at about 588 and 670 nm, respectively (Figure 1B). The photographs of the fluorescent Ag NCs under UV illumination (inset in Figure 1B) show that the red color is visible even with an Ag+ to C-GG concentration ratio of as low as 5:1, and the color only deepens by further

picture of how a base’s electronic properties can affect Ag NC emission because of the fact that more than one emissive Ag species is simultaneously formed when using ss-DNA as the template (the excitation-energy-dependent Ag NC emissions prove the existence of multiple species).24 The flexible conformation and plurality of ss-DNA Ag binding sites make it much more difficult to determine the effect of the DNA bases’ electronic properties on Ag NC emissions correctly. To grow fluorescent Ag NCs to a well-defined size in a onepot synthesis, double-stranded DNA (ds-DNA) with a particular site to accommodate the Ag clusters serves as an efficient platform because of its rigid structure. Previously, we found only negligible emissions when the bases directly flanking the AP site were As, Ts, or Cs, a result independent of the base opposite the AP site.54 However, fluorescent Ag NCs can also be selectively formed at a DNA AP site that is directly flanked by guanines (Figure S1, Supporting Information). In this case, the unpaired base (Y) opposite the AP site does play a crucial role in the creation of the Ag NCs emitting between 600 and 800 nm.54 We first examine the effect of base Y on the emission of the AP-site-bound Ag NCs by maintaining the sequences (M/N and P/Q) one base away from the AP site as G/Cs (YGG, Scheme 1). In an optimized experiment, a 5 μM Y-GG duplex was mixed with AgNO3 solution in the desired concentration ratios. The resultant solution was incubated for 15 min under stirring, reduced by fast addition of freshly prepared NaBH4 to an optimized concentration ratio of 1:4 NaBH4/Ag+ (Figure S2, Supporting Information), and then left to react for 1 h to produce stable Ag NCs (Figure S3, Supporting Information). As shown in Figure 1A, the DNA with cytosine opposite the AP site (C-GG) produces the strongest emission in comparison to those with adenine (A15315

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Figure 3. Job’s plot analysis of the stoichiometric numbers of Ag+ binding to (A) C-GG and (B) C-GT in 20 mM phosphate buffer (pH 7.0) containing 1 mM Mg2+. The total Ag+ and DNA duplex concentrations for all mixtures were kept at 30 μM. The fluorescence intensities were measured at excitation/emission wavelengths of 588/670 and 467/548 nm for C-GG and C-GT, respectively, by successively changing the DNA/ Ag+ concentration ratio from 0.1 to 0.9. (Inset) Corresponding emission spectra.

within ±8 nm. Because a lower fluorescent intensity seems to be accompanied by a slightly higher variation in the Ag NC emission wavelength, it is reasonable to assume that these small variations are caused by large nanoparticles possessing surface plasmon resonance (SPR) optical properties (Figure S4, Supporting Information) formed independently of the DNAtemplated Ag NCs. Mass spectrometry (MS) is widely used to count the Ag atoms of Ag NCs encapsulated by ss-DNA and ds-DNA.23,38,53 However, this technique usually identifies many Ag species for a DNA molecule. For example, Ag2, Ag3, Ag4, Ag5, and Ag6 species are simultaneously observed by MS with a 12-mer ssDNA template,23 and Ag3, Ag4, Ag5, and Ag6 species are simultaneously observed for a 22-mer ds-DNA template containing a mismatch site, even though there is only one fluorescence emission peak.53 These facts indicate that some of the Ag species measured by MS should be nonfluorescent. Thus, arbitrarily attempting to attribute just one of the MSsized multiple Ag species (usually the most abundant one) as the size responsible for the Ag NC emission in a solution is a risky challenge. Furthermore, the incompatibility of MS with the buffer conditions used for DNA-templated Ag NCs and the MS-induced flexible DNA duplex structure26,52 must also be seriously considered. These drawbacks prevent us from using MS to size the Ag NCs created for this article accurately. Fortunately, the independence of the observed emission energy with respect to the DNA/Ag+ concentration ratio and the dependence of the emission intensities on that same ratio inspired us to develop a Job’s plot method to size the Ag NCs. Typical Job’s plots are shown in Figure 3, and the results are collected in Table 1. On the basis of the dependence of the

increasing the Ag+ concentration ratio. This result would not be possible with ss-DNA templates. For example, a 5-fold increase in Ag+ concentration produces a difference of up to 290 nm in the dominant emission wavelength of Ag NCs templated with ss-DNA,46 although the Ag+ concentration-sensitive shift in the emission is still sequence-dependent. Therefore, the AP site in duplex DNA is a promising platform for creating identically sized Ag NCs, which could provide the possibility of gaining insight into the effect of the DNA sequence on the optical properties of Ag NCs. Note that, because of the strong binding preference of Ag+ to C,24,62 sequences (C-MPs) with an unpaired C base opposite the AP site were employed for subsequent experiments. In contrast to our current results, we believe that the previously observed excitation-wavelength-dependent emission behavior for ssDNA-templated Ag NCs is due to multiply sized Ag species forming at the same time from the heterogeneous template. Assuming that there is constrained Ag NC growth to a well-defined size at the AP site, excitation at various wavelengths should result in identical emission wavelengths. As shown in Figure 2, the narrow variation in the emission maxima from 675 to 682 nm for C-GG is followed by excitation between 350 and 630 nm, strongly suggesting that only a single size of Ag NCs is formed. To investigate the effect of the bases’ electronic properties on Ag NC emissions in this work and to maintain a similar microenvironment for producing Ag NCs of a certain well-defined size at the AP site, the guanines directly flanking the AP site were kept intact while systematically changing the base pairs (M/N and P/Q) one base away from the AP site (C-MPs, named by these sequences, Scheme 1). Most importantly, these changes in the sequence do not affect the geometric constraints and binding sites provided by the AP site for accommodating Ag NCs and should not alter the expected growth size of the clusters. To verify this point, the P/ Q base pair in C-GG was changed to one of the other base pairs (e.g., C-GC), causing the induced emission to be located at a shorter wavelength. As can be seen in Figure 2B, the Ag NCs templated by C-GC emit at 565−578 nm upon excitation at 300−500 nm. The small variation in Ag NC emission at different excitation energies occurs for all of the other C-MPs as well, indicating that only a single Ag NC species is being formed using a C-MP template, unlike the simultaneous formation of multiple Ag species that occurs when using an ssDNA as the template. Unlike organic dyes, which exhibit constant emission energy at different excitation energies, this small variation is not caused by different sizes of Ag NCs (next paragraph). Note that the excitation-energy-dependent variation of the emission wavelength for all observed C-MPs is

Table 1. Binding Stoichiometric Numbers of C-MP to Ag+ Obtained from Job’s Plot Analysis C-MPs binding stoichiometric numbers of CMP to Ag+

C-GG

C-GT

C-GC

C-GA

C-AG

C-CG

C-TG

0.47

0.51

0.46

0.51

0.51

0.46

0.54

emission intensities on the concentration ratios, good Job’s plot behaviors are obtained for Ag NCs templated with various CMPs. For example, the DNA/Ag+ stoichiometric numbers for C-GG and C-GT are about 0.47 and 0.51 (Figure 3), respectively. From the obtained stoichiometric numbers listed in Table 1, we can imagine that this number is fixed at around 0.5 for all C-MPs. Therefore, we can reasonably conclude that the Ag2 dimer (more discussion in Section 3.2) is responsible 15316

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Figure 4. (A) Fluorescence emission spectra of Ag NCs with tsX-YZ duplexes (each 5 μM) as templates. A Ag+ concentration of 125 μM was used to saturate the binding at the AP site. (B) Job’s plot analysis for the stoichiometric numbers of Ag+ binding to tsX-CC in a 20 mM phosphate buffer (pH 7.0) containing 1 mM Mg2+. The total concentrations of Ag+ and the DNA duplex were kept at 30 μM. The fluorescence intensities were measured at excitation/emission wavelengths of 588/670 nm by successively changing the DNA/Ag+ concentration ratio from 0.1 to 0.9. (Inset) Corresponding emission spectra (the observed slight shifts in emission wavelengths are likely caused by the more flexible structure of the 23-mer DNA as compared to that of the 15-mer C-GG).

Note that we determined the stoichiometry according to the added Ag+ concentration and not directly from Ag0, which puts doubt on the feasibility of using a Job’s plot analysis to count the atom numbers for the Ag NCs. However, because the APsite-bound Ag+ is located within the DNA helix, we can reasonably say that the AP-site-bound Ag+ is less available to be promptly reduced by the added NaBH4 relative to the DNAbackbone-bound Ag+ and the Ag+ free in solution. The prompt appearance of the SPR band for the large nanoparticles and the relatively slow evolution of the absorption band for the fluorescent Ag NCs support this supposition (Figure S5, Supporting Information). Thus, we conclude that Ag NC fluorescence comes from the originally bound Ag+ at the AP site. Additionally, this also excludes the possibility that any reduced Ag0, other than at the AP site, re-entered the AP site to form a fluorescent Ag species because no fluorescence emission was observed from adding AgNO3 solution to any of the preincubated DNA-NaBH4 solutions. This means that the initial binding of Ag+ at the AP site is the starting point for the production of fluorescent Ag NCs. All of these statements support the fact that the atom number of the fluorescent Ag0 cluster is determined by the bound Ag+ at the AP site before reduction. Additionally, the bound Ag+ at the AP site is in equilibrium with the added Ag+. Thus, the stoichiometry can be obtained from the added Ag+ concentration by the Job’s plot method. DNA duplexes containing two separated AP sites (tsX-YZ, Scheme 1) were employed to further verify the formation of fluorescent Ag2 NCs at the cytosine-opposed AP site. The tsXYZ sequences were redesigned to avoid the disturbance of the possible DNA second structures so as to make an accurate determination of the single-stranded ingredient DNA concentration before hybridization that could be caused by doubly repeating the length of the C-MP sequences. As shown in Figure 4A, tsX-TT with two thymines opposite the two AP sites exhibits no fluorescence response, which is in agreement with the result for T-GG (Figure 1A) containing one AP site that is opposed by one thymine. However, the fluorescence intensity for tsX-CC with two cytosines opposite the two AP sites is about 2 times higher than that of tsX-CT with one cytosine and one thymine opposite the AP sites. Additionally, the stoichiometric number of Ag+ ions bound at these AP sites must be four so that each AP site in the tsX-CC duplex can be used to form Ag2 NC. This means that a turning point in the Job’s plot analysis should occur at 0.25. The measured value of

for the emissions and that any variations in the sequence one base away from the AP site in C-MPs do not affect Ag2 formation. This also indicates the crucial role that the AP site plays in constraining Ag NC growth. Although it is not clear at present why only the Ag2 dimer dominated in our case, the unusual stability of Ag2 clusters has been proven through its extraordinarily high oxidation potential. Thus, Ag2 is the most thermodynamically stable type of cluster for Agn nanoclusters where n < 7.63 However, only a few reports have clearly defined Ag2 NC emission, which is mainly at less than 506 nm in noble gas matrices.4,64 Recently, Scaiano et al. investigated the stability of fluorescent Ag2 nanoclusters in solution using a photogenerated ketyl radical as a reductant and pointed out that polar cyclohexylamine-protected Ag2 emitted at 540 nm.65,66 Thus, it is reasonable to expect the AP site-born Ag2 NCs observed in this work also to emit at longer wavelengths if we consider the stronger polarization interaction of Ag2 with the polar DNA bases compared to any interactions with the noble gas4,64 or cyclohexylamine matrices.65,66 This process induces a large loss in energy before emission because of excited-state relaxation. Note also that, among all of the previously reported fluorescent Ag NC species, Ag2 seems to be the simplest species for Ag−Ag bonding geometry, which would simplify the effects of different DNA binding sites67 on Ag NC optical properties. Soto-Verdugo et al.67 and Neidig et al.57 previously showed that the cytosine’s doubly bonded ring N3 and C2 carbonyl are the binding sites for Ag NCs. The Ag− Ag bond length in nanoclusters is less than 2.89 Å,57 so the diameter of Ag2 is not more than 5.78 Å. The width of the AP site for C-MPs is determined by the discarded guanine and is estimated to be about 7.56 Å. However, the AP site provides a height constraint of about 6.80 Å as a result of two B-helix steps. Therefore, the AP site can geometrically be expected to provide a pocket large enough to accommodate a Ag2 nanocluster, whereas previously used flexible ss-DNA templates24−51 are more suitable for accommodating Ag NCs with much larger sizes. Therefore, the limited space provided by the AP site and the extraordinary thermodynamic stability of Ag2 over that of the other nanoclusters support the preferable formation of Ag2 at the AP site. Because the size of the Ag NCs templated by C-MPs is identical within the constrained space provided by the AP site, it is very convenient to investigate the effect of DNA base stacking near the AP site on the fluorescence properties of the Ag NCs. 15317

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Werner observed a significant red shift in emission energy, accompanied by a 500-fold enhancement in the fluorescence intensity, when the preformed Ag NCs approached a guaninerich DNA strand.29 These authors also proved that an electron transfer between excited-state Ag NCs and guanine is impossible. Furthermore, Ag NC formation did not really affect the thermostability of any of the used DNA types, as evidenced by the small variations in their melting temperatures (Tm, Table 2). The Ag NC is expected to have a similar affinity

that turning point is 0.27 as shown in Figure 4B, which again strongly validates Ag2 NC formation at the AP site. From the above results, we can conclude that one AP site has space enough to grow a Ag2 NC. Thus, one would imagine that Ag NCs larger than Ag2 should be formed by this AP site design when the AP sites are placed at consecutive positions. To verify this point, DNA containing two consecutive AP sites (Scheme 1, tcX-CC) was used. The sequence of tcX-CC is similar to that of C-GG with the only exception of having two consecutive cytosines opposite the two consecutive AP sites. tcX-CC is stable enough to be in the duplex state at room temperature (the measured melting temperature being at 49.5 °C). As shown in Figure 5, the measured turning point is 0.28 via the

Table 2. Melting Temperatures of the C-MPs in the Absence and Presence of the Formed Ag NCsa C-MPs

C-GG

C-GA

C-GC

C-GT

C-AG

C-CG

C-TG

Tm (°C) for CMPs only Tm (°C) for CMPs + Ag NCs ΔTm (°C)

52.3

46.3

50.7

49.2

47.6

52.7

49.1

52.8

47.8

49.8

49.1

47.5

51.4

49.2

0.5

0.5

−0.9

−0.1

−0.1

−1.3

0.1

a

Absorbance as a function of the solution temperature at 260 nm was measured in 20 mM phosphate buffer (pH 7.0) containing 1 mM Mg2+. [DNA] = 5 μM, [Ag+] = 50 μM, and [NaBH4] = 12.5 μM.

for the single- and double-stranded DNAs, and thus only a weak effect on Tm was observed. Therefore, these small alterations in the DNA thermostability are not a crucial factor in determining Ag NC emission properties. Additionally, the small alterations also indicate that Ag NCs at the AP site interact only with the flanking bases through a weak interaction such as the dipole interaction. (See the following text.) Theoretically, if a fluorophore’s dipole moment in the excited state is larger than in the ground state, the excited state will be better stabilized relative to the ground state by its surrounded matrix dipole or the induced dipole. Increasing the matrix dipole causes the fluorophore’s emission band to red shift. Dickson et al.50 reported that Ag NCs in the excited state have a much larger dipole moment than when in the ground state because the free electrons of an Ag cluster exhibit a large polarizability through their free movement within the cluster.68 These highly polarizable electrons in the excited state must be stabilized before the photoejection of the high-energy electrons in order to dissipate the absorbed energy through irradiative transition. Additionally, Ag NC interaction with the surrounding matrix causes a shift in the emission energy.69 We expect that the dipole interaction between Ag2 NCs and any nearestneighbor guanines plays a dominant role in determining the Ag NC emission energy. Although the Ag2 NCs formed at the AP site for all of the DNAs studied in this work were invariably surrounded by an opposite cytosine and two flanking guanines, the Ag2 emission was largely affected by the flanking guanines because of the potentially direct communication of Ag2 with their π-electron system. Because the dipole moment of a base in a DNA environment is affected by stacking interactions with its neighboring bases, it is reasonable to assume that the Ag2 emission should also be controlled by the directly flanking guanines through variable dipole moments modulated according to M/N and P/Q sequences located one base away from the AP site. As shown in Figure 6, the Ag2 emissions indeed display a significant dependence on the M/N and P/Q sequences. If we maintain the 3′-side sequence of the AP site as GG stacking for C-GP, as noted in Scheme 1, changing the P/Q sequences in C-GP to T/A (C-GT), C/G (C-GC), A/T (C-GA), and G/C

Figure 5. Job’s plot analysis for the stoichiometric number of Ag+ ions binding to tcX-CC in a 20 mM phosphate buffer (pH = 7.0) containing 1 mM Mg2+. The fluorescence intensities were measured at excitation/emission wavelengths of 550/618 nm by successively changing the DNA/Ag+ concentration ratios while the total concentrations of Ag+ and DNA duplex were kept at 30 μM. (Inset) Corresponding emission spectra.

Job’s plot analysis. However, in comparison to C-GG, the excitation/emission maxima for tcX-CC are blue shifted to 550/618 nm. Thus, we expect the formation of Ag NCs with a size different from that of Ag2, most likely Ag4, because the same surrounding sequence environment is met for the C-GGtemplated Ag2 and tcX-CC-templated Ag4. This result suggests that our AP site strategy could also be capable of tuning the Ag NC size through expanding the consecutive AP site number. 3.2. Base-Stacking-Determined Emissions of Ag NCs Templated by C-MPs. All fluorescent Ag2 NCs are produced at the AP site, which means that they are directly and invariably surrounded by a cytosine opposite and two guanines flanking the site when C-MP is used as the template. However, Figures 2 and 3 show differences in the emission energy when varying the bases one base away from the AP site, indicating that factors other than the Ag NC size also control the emission energy. Dickson et al. hypothesized that electron transfer from excitedstate Ag NCs to the surrounding cytosine causes a blinking Ag NC emission.50 However, this electron transfer results only in a long-lived dark state and does not alter the emission energy. If we consider the oxidation potentials of the four DNA bases, guanine is the most oxidizable base. The possibility that the flanking guanines transfer electrons to the excited-state Ag NCs, and thus induce the creation of a radiation state, is excluded because further addition of NaBH4 (a stronger electron donator than guanine) to the preformed Ag NC solution causes only a decrease in emission intensity instead of an alteration in the emission energy. By contrast, Martinez and 15318

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Figure 6. Fluorescence emission spectra of Ag NCs with C-MP duplexes as templates. (Inset) Corresponding excitation/emission maxima.

Figure 7. Dependency of Ag2 NC singlet energies (ΔE0,0) on changes in the vertical ionization potential (ΔVIP) of the 5′-G and 3′-G sequences stacked with different bases. The VIP data are given relative to 5′-GG-3′ in order to eliminate any basic energy set differences arising from the different computational methods (symbols with the same shape and color all use the same method).70−73 Some of the raw IP data is derived from sequences with pyrimidine(s) located on the 5′ side and/or the 3′ side of the corresponding stacking. The curved lines are averages drawn only for convenience so as to distinguish the effects of 5′-side base variations versus 3′-side variations.

(C-GG) causes the Ag2 emission to shift to 546, 575, 628, and 670 nm, respectively. However, successively changing the M/N sequences in C-MG to T/A (C-TG), C/G (C-CG), A/T (CAG), and G/C (C-GG) while keeping the 5′-side sequences of the AP site intact as GG stacking for C-MG (Scheme 1) induces Ag2 emission shifts to 619, 661, 667, and 670 nm, respectively. Thus, although the red shift in the emissions consistently follows the order of T < C C-GA > C-GC > C-GT and C-GG > C-AG > C-CG > C-TG, which are inversely proportional to the corresponding ΔE0,0 values. In addition, AG NC lifetimes are much more affected by the 5′-G stacking sequences than by the 3′-G stacking sequences, a result that is in agreement with the results shown in Figure 7. Therefore, the optical properties of Ag NCs templated with C-MGs (M = T, C, A, and G) are mainly controlled by the 5′-GG-3′ stacking and are only slightly modified by the 5′-MG-3′ stacking. Because the available excitation wavelength of the excitation source was less than 400 nm, we did not excite the samples at 15319

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to indicate only Ag NC formations composed of multiple silver atoms, at least with DNA as the template. Thus, this linear relationship of our NC emissions to the emissions from previously reported DNA-templated Ag NCs and Ag2 NCs protected by cyclohexylamine65 and noble gas matrices4 again supports the conclusion that only the Ag2 dimer is responsible for the observed emissions in this work and excludes the possibility that two individually separated Ag atoms (i.e., Ag1 species) per DNA are formed. As shown in Figure 9, 150 data points (stars),23−54,58 mainly for the dominant emissions of Ag NCs, follow this linear preference with few exceptions.58 Emissions under high-energy UV excitation74 were not considered. Note that this linear relationship is also followed by differently sized Ag NCs synthesized with ss-DNA templates as well. However, the mystery behind this empirical linearity is still not solved. The linear dependence of the dominant emissions for different Ag NC excitation energies should illuminate the involvement of the electron interaction between the Ag NC emissive states and the DNA bases. We suggest that the electron interplay (likely a dipole or induced dipole interaction) between DNA bases and the Ag NCs plays a significant role in modulating Ag NC emission behavior, as observed for the Ag2 NCs investigated in this work (squares in Figure 9). We expect that this dipole interaction occurs much more easily between Ag NCs and ss-DNA templates because of their flexible structures. Therefore, the present method paves a new way to investigating Ag NC optical properties further as modulated by surrounding molecule dipoles.

Figure 8. Dependence of ΔE0,0 on the excited-state lifetime of Ag NCs templated with C-GP (circles) and C-MG (squares).

their corresponding excitation maxima. However, for steadystate emissions under 350 nm excitation, we observed only one emission peak occurring in almost the same wavelength region as occurs under excitation at the corresponding excitation maximum (e.g., Figure 2). Most importantly, the 350 nm highenergy excitation excites the Ag NCs to an excited state possessing a higher energy than the emissive state. However, the transition from this high-energy excited state to the emissive state is completed in tens of picoseconds.50 Thus, the measured nanosecond lifetimes correspond to the transition from the emissive state to the ground state, and the emissivestate lifetime of the Ag NC is not seriously affected by the highenergy excitation wavelength used in this work. This assertion is supported by the fact that the measured lifetimes for Ag2 NCs templated by C-MPs are indeed within the previously reported region (between 0.2 and 4.3 ns for DNA-encapsulated Ag NCs).4 Lastly, Ag2 NC optical properties, which are guanine-dipolemoment-dependent, also have an emission−excitation (Eem − Eex) energy (eV) relationship of Eem = 0.40 + 0.70Eex, with a relative standard deviation of 0.97. This relationship was previously proposed by us58 for Ag NCs encapsulated using various DNA sequences and lengths used by other groups in other studies (Figure 9). Note that cyclohexylamine-protected Ag2 in solution65 and one of the Ag2 NCs in a noble gas matrix4 also adhere to this energy relationship, but Ag1 does not.4 Therefore, this emission−excitation energy relationship seems

4. CONCLUSIONS A DNA AP site was used size-selectively to grow fluorescent Ag NCs, whose emission energy was independent of the DNA/Ag+ concentration ratios used in this work. Additionally, the excitation energy does not seriously affect the Ag NC emission energy, a result that directly contrasts with observations of Ag NCs previously templated with ss-DNA. These properties are very convenient for determining the Ag NC size through a Job’s plot analysis. We found that the sequences one base away from the AP site (which was also the Ag NC growth site) do not change the size of the fluorescent Ag NCs and that the Ag2 species is responsible for the emissions. The AP-site-constrained Ag2 NCs showed a clear emission dependence on the sequences one base away from the AP site, even though the sequences directly flanking the AP site were guanine-invariable. The Ag NC emission gradually red shifted as the sequences one base away from the AP site were changed in the order of T, C, A, and G. Furthermore, the 3′-side sequences of the 5′-G stack directly flanking the AP site induced larger emission shifts than the 5′-side sequences of the 3′-G stack. We propose that this sequence-dependent emission behavior is caused by basestacking-induced variations in the dipole moments of the guanines directly flanking the AP site. This work concludes that the electronic properties of DNA bases with different stacking environments significantly influence Ag NC emissions.

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Figure 9. Linear dependence of the emission energies on the corresponding excitation energies of fluorescent Ag NCs synthesized using DNA templates employed in previous studies (stars) and this work (squares). Our data (squares) also agree with this linear preference. Also shown are the data for previously reported Ag2 NCs protected by cyclohexylamine (triangles65) and noble gas matrices (diamonds,4 where only a clearly assigned excitation−emission of 407/ 485 nm was plotted).

ASSOCIATED CONTENT

S Supporting Information *

Effect of AP site’s flanking bases. Molar ratio experiments of NaBH4 to Ag+. Time evolution of fluorescence intensities. Absorption spectra. This material is available free of charge via the Internet at http://pubs.acs.org. 15320

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AUTHOR INFORMATION

Corresponding Author

*Fax: 86 579 82282595. Tel: 86 579 82282234. E-mail: yshao@ zjnu.cn. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (grant no. 21075112), the Zhejiang Provincial Natural Science Foundation of China (grant no. LR12B05001), the Foundation of State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry (grant no. SKLEAC2010001), and the Scientific Research Foundation for the Returning Overseas Chinese Scholars, State Education Ministry.

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