Unraveling the Interaction of Silver Nanoparticles with Mammalian and

Article pubs.acs.org/JPCB

Unraveling the Interaction of Silver Nanoparticles with Mammalian and Bacterial DNA Srikrishna Pramanik,†,§ Sabyasachi Chatterjee,‡,§ Arindam Saha,† Parukuttyamma Sujatha Devi,*,† and Gopinatha Suresh Kumar‡ †

Sensor and Actuator Division, CSIR-Central Glass and Ceramic Research Institute, Kolkata 700 032, India Biophysical Chemistry Laboratory, Organic and Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, Kolkata 700 032, India

‡

S Supporting Information *

ABSTRACT: The focus of this study was to understand and unravel the interaction of silver nanoparticles (AgNPs) with different types of Deoxyribonucleic acid (DNA), mammalian and bacterial, having different base pair compositions. Binding of spherical silver nanoparticles (AgNPs) to Calf thymus (CT) DNA, Escherichia coli (EC) DNA and Micrococcus lysodeikticus (ML) DNA has been studied to gain insights into their mode of interaction and specificity. Interaction of AgNPs with synthetic DNA has also been carried out. On the basis of absorption, thermal melting, isothermal calorimetry and viscosity studies, we could establish the mode of binding and specificity of the synthesized silver nanoparticles with mammalian and bacterial DNA. Thermal melting (Tm) studies indicated a decrease in the Tm of all the DNAs, confirming the destabilization of DNA stacks on interaction with AgNPs. Comparative interaction studies with single stranded (ss) and double stranded (ds) DNAs further confirmed the specificity of the particles toward ds DNA. On the basis of the results we could confirm that the synthesized AgNPs could be used for selective detection of DNA through their DNA binding mechanism. In addition, the AgNPs−DNA complexes exhibited distinct differences in the SERS spectra making it an interesting SERS platform for identifying ds DNA. The optical and physical properties of AgNPs help in differentiating the DNAs of different base pair compositions through their binding affinity and specificity.

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INTRODUCTION Study on the interaction of nanostructured materials with biomolecules leading to biological applications has been an active area in recent years.1,2 In particular, nucleic acid sensing through interaction with nanoparticles can be used for the devolvement of novel biosensor for clinical diagnosis,3−5 pathogenic bacteria detection,6,7 imaging,8 and drug delivery applications.9−11 Deoxyribonucleic acid (DNA), one of the most well studied macromolecules, has a double helical structure with sugar phosphate backbone and nucleobase paired inside. Among the four base pairs present in DNA, adenine pairs up with thymine and guanine pairs with cytosine through hydrogen bonding.12 In recent years, several materials have been identified that selectively interact and detect biomolecules like DNA; the most important ones are noble metal nanoparticles of Au and Ag.13−21 Such interactions have been reported to critically depend on the size, shape and surface chemistry of the nanostructured materials under investigation. For example, Rahban et.al., reported the interaction of AgNPs (particles size 5−10 nm) with calf thymus DNA exhibiting intercalation of the nanoparticles with DNA.14 Goodman et al. demonstrated the effect of hydrophobic group functionalized AuNPs in DNA binding efficiency.17 Park et al. observed the shape-dependent © XXXX American Chemical Society

reversible binding affinity and self-assembly nature of Ag nanocube and DNA conjugates.18Tunable optoelectronic properties of plasmonic Au and Ag nanoparticles have been utilized as a superior sensing platform to detect DNA. Most of the groups reported fluorophore dye tagged DNA and used fluorescence probe to detect double and single stranded DNAs. Recently Fan et al. reported fluorescent FAM dye tagged DNA and its interaction with AuNPs by monitoring the fluorescence quenching.19 Zhang et al. conjugated thiolated oligonucleotides with AgNPs at lower pH to detect complementary linker DNA.20 There are also reports on thiolated DNA linkage to noble nanomaterials for the detection through salt induced agglomeration of Au colloidal solution and electrostatic interaction.21 Apart from fluorophore dye tagging and electrochemical detection,22,23 Surface enhanced Raman spectroscopy24−27 has also been used for successful recognition of DNA by nanoparticles. Though there are many reports on the interaction of metal nanoparticles with CT DNA, there are no comparative studies to understand the interaction of silver nanoparticles with Received: February 16, 2016 Revised: May 22, 2016

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DOI: 10.1021/acs.jpcb.6b01586 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B different DNA samples, viz. mammalian DNA and bacterial DNA.14−18 AgNPs can also hamper the bacteria and its DNA replication2 and is considered as a less studied plasmonic material for DNA recognition compared to Au. In this study, we have investigated the interaction of silver nanoparticles with a mammalian DNA, calf thymus (CT) DNA, a Gram-negative bacterial DNA, Escherichia coli (EC) DNA and a Gram-positive bacterial DNA, Micrococcus lysodeikticus (ML) DNA. The selected double stranded (ds) DNAs have different base pair compositions; CT DNA consists of 42% GuanineCytosine (GC) pairs and 58% adenine = thymine (AT) pairs and EC DNA contains 50% of GC and 50% AT compositions whereas ML DNA has 72% GC and 28% AT pairs. Therefore, it would be interesting to study the interaction of silver nanoparticles with different DNA samples consisting of different base pair composition.

N=

πρD3 6M

(1)

N is the number of atoms per nanoparticle, π = 3.14, ρ represents the density of face-cantered cubic silver (10.5 g/cm3), D is the average diameter of nanoparticles, M is the atomic mass of silver (107.868 g). Finally, the concentration (C) of AgNPs is calculated using following formulaC=

NTotal NVNA

(2)

Here NTotal represents the total amount of Ag atom used, V is the volume of the solution in liters, and NA is the Avogadro number. Methods. All the spectroscopic measurements were carried out at 25 ± 0.5 °C from 200 to 800 nm on a Shimadzu UV-3600 UV−vis−NIR spectrophotometer (Shimadzu Corporation, Kyoto, Japan) in 10 mm path length quartz cuvettes. Absorbance titration was carried out by the addition of 0.02 to 6.5 μM CT DNA, 0.02 to 6.13 μM EC DNA and 0.02 to 6 μM ML DNA into the as prepared Ag nanoparticles solution (AgNPs). Absorbance values were recorded after 1 min of each successive addition of DNA solution to obtain the equilibrium condition. The association constants (KBH) of AgNPs−DNA were calculated by employing Benesi−Hildebrand32,33 plot obtained from the following equation:

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MATERIALS AND METHODS Materials. Silver nitrate (SD. Fine-Chemicals Ltd., India) and Trisodium citrate (Sigma-Aldrich Corporation, St. Louis, MO) were used for the experiments. Calf thymus (CT) DNA (sodium salt, type XI, 42 mol % GC content) and E. coli (EC) DNA (Type I, 50 mol % GC content) and M. lysodeikticus (ML) DNA (Type XI, 72 mol % GC content) were also obtained from SigmaAldrich. Concentration of the DNA samples were determined spectroscopically by measuring the absorbance at 260 nm using molar absorption coefficient values of 13 200, 13 000, and 13,800 M−1 cm−1, respectively, for CT DNA, EC DNA, and ML DNAs.28,29 Poly(dA-dT)·poly(dA-dT), poly(dG-dC)·poly(dGdC) and Hoechst 33258 were purchased from Sigma-Aldrich and their concentrations were measured similarly using molar absorption coefficients of 13 200 M−1 cm−1 at 260 nm and 16 800 M−1 cm−1 at 255 nm, respectively, for poly(dA-dT)· poly(dA-dT) and poly(dG-dC)·poly(dG-dC).29 All experiments were conducted in citrate-phosphate (CP) buffer of pH 7.4, containing 10 mM Na2HPO4, the pH was adjusted with citric acid. The pH of the solutions was measured using a Sartorious PB-11 high precision bench pH meter (Sartorious GmBH, Germany) with an accuracy of >±0.01 units. The DNA samples were sonicated in a labsonic sonicator (B. Brown, Germany) to molecular weights of approximately (1−2) × 105 Da and were dialyzed under sterile conditions at 5 °C into the experimental buffer. The ratio of the A260/280 values for the DNA samples varied between 1.8 and 1.9 indicating the samples to be free from protein contamination. The buffer solution was filtered through Millipore filters (Millipore, India Pvt. Ltd., Bangalore, India) of 0.22 μm pore size. Synthesis. Citrate-capped silver nanoparticles (AgNPs) were synthesized by a modified technique.30 In this synthesis, 0.5 mL of 2.5 × 10 −3 M AgNO3 solution was mixed with 0.5 mL of 2.5 × 10 −3 M trisodium citrate and diluted with18 mL of water followed by the addition of 0.5 mL of 10−2 (M) NaBH4 to the solution. The mixture was stirred in the dark for 2 min followed by sonication for 10 min in an ultrasonication bath to get stable yellow colored solution. The as prepared AgNPs were characterized by UV−vis spectroscopy and TEM to confirm the formation of spherical AgNPs. The concentration of the prepared solution was 9.87 × 10−7 M/L. Concentration of the spherical AgNPs has been calculated using the following principle31 assuming 100% conversion. At first, the average number (N) of Ag atoms per nanoparticle was calculated using the formula

1 1 1 1 = + × ΔA ΔA max (ΔA max )kBH [M]

(3)

ΔA is the difference in absorbance and [M] is the DNA concentration. KBH is obtained from intercept to the slope ratio of the linear plot. The ζ-potential measurement was performed on a Horiba (SZ-100) analyzer. Surface charge variation of the AgNPs in the presence of DNA has been measured as above. All the measurements were carried out in the standard capillary electrophoresis cell. Tecnai G2 30ST (FEI) high-resolution transmission electron microscope operating at 300 kV and JEOL JEM 2100 HR with EELS 200 kV were used to evaluate the morphology. For the TEM study, 40 μL of Ag nanoparticles solution was drop casted on the carbon coated copper grid and dried at room temperature before checking the morphology. Average particle size was calculated using image j software. The viscosity of the DNA-AgNPs complexes was determined from the time dependent flow through a Cannon-Manning semi micro size 75 capillary viscometer (Cannon Instruments Company, State College, PA, USA) that was submerged in a thermostated bath (25 ± 1 °C). Flow time was recorded with an electronic stopwatch Casio Model HS-30W (Casio Computer Co. Ltd., Tokyo, Japan) and each data was taken as an average of three measurements. Relative viscosities of CT DNA in the presence and absence of AgNPs were calculated using the following equation. η′sp /ηsp = {(t − t0)/t0}/{(tcontrol − t0)/t0

(4)

where, η′sp and ηsp represents specific viscosity of DNA−AgNPs complex and DNA, respectively, t, t0, and tcontrol are the average time taken by the complex, DNA, and buffer to flow through the capillary. The relative viscosity in the presence and absence of AgNps was obtained from the following equation.

(η /η0)1/3 = 1 + βr B

(5) DOI: 10.1021/acs.jpcb.6b01586 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

Figure 1. (a) Absorbance spectrum of AgNPs. (b) TEM image of AgNPs at a scale of 50 nm (inset shows the magnified image). (c) Bright field image of individual AgNPs (scale bar =50 nm). (d) HRTEM image of AgNPs. (e) Average particle size of the synthesized AgNPs. (f) ζ-Potential of AgNPs dispersion in H2O.

where η and ηo are the corresponding values of intrinsic viscosity (reduced viscosity η = ηsp/C, where C is the concentration of DNA and β is the slope of the plot). AgNPs to DNA ratio was fixed in the range of 0.01 to 1 and concentration of the DNA was 50 μM. The minor groove binder Hoechst 33258 displacement assay was carried out by monitoring the emission spectra of different concentrations of AgNPs (0.2−15 μM) and DNA (10 μM), and Hoechst (12.25 μM) at 25 ± 1 °C after excitation at 341 nm.29,33 Circular dichroism (CD) spectra of DNA’s were measured on a J-815 Jasco unit (Jasco International Co. Ltd., Hachioji, Japan) equipped with a temperature controller (model PFD 425L/15) at 20 ± 0.5 °C. The CD experiments were conducted in a 10 mm path length rectangular quartz cuvette and measured in the range 200−400 nm. CD titrations were carried out by successive addition of AgNPs to 60 μM of the DNA samples placed in the cuvette until CD spectra of DNA showed no further change. Each spectrum was taken as an average of five successive scans and corrected after baseline subtraction. Melting study of DNA and DNA-AgNPs complexes was done on a Shimadzu Pharmaspec 1700 unit equipped with the Peltier controlled TMSPC-8 model accessory (Shimadzu Corporation). In this experiment AgNPs of different concentrations were added to the 5 μM DNA solutions and mixed well in an eight partitioned micro optical cuvette of 10 mm path length. The absorbance change for DNA at 260 nm was monitored on increasing the temperature at a heating rate 0.5 °C/min. The melting temperature (Tm) was detected as the midpoint of the melting transition from the differential of the absorbance versus temperature plots. All isothermal micro calorimetric titration experiments were performed in a VP-ITC unit (MicroCal, Inc., Northampton, MA, USA) at 25 °C. In the experiment degassed AgNP solution was injected from a rotating syringe (354 rpm) into the isothermal sample chamber containing the DNA solution (1.4235 mL).

Immediately, a corresponding control experiment to determine the heat of dilution of AgNP was performed by injecting identical volumes of the same concentration of the AgNP solution into the buffer solution. The area under each heat burst curve was determined by integration using the Origin 7.0 software to give the measure of the heat associated with the injections. The heat associated with each AgNPs−buffer mixing was subtracted from the corresponding heat of the AgNPs−DNA reaction to give the final heat of AgNPs−DNA binding. The heat of dilution of buffer−DNA mixing was observed to be negligible. The resulting corrected injection heats were plotted as a function of the AgNPs/DNA ratio and fit with a model for one set of binding sites and analyzed using Origin 7.0 software to provide the binding affinity (Ka), the binding stoichiometry (N) and the enthalpy of binding (ΔHo). The binding Gibbs energy change (ΔG°) and the entropic contribution to the binding (TΔS°) were then calculated from standard thermodynamic equation, ΔG° = ΔH ° − T ΔS°

(6)

SERS experiment was performed on an Agiltron R3000 Raman spectrophotometer with 785 nm excitation laser having 5 mW laser power and 15s integration time. All the measurements were performed in liquid phase. At first, SERS using AgNPs and DNA mixture was measured. We also measured the SERS in the presence of 0.01 M NaCl. For this, three different concentrations [2 × 10−7 M, 6 × 10 −6 M, and 50 × 10−9 M] of calf thymus DNA, E. coli DNA, and M. lysodeikticus DNA were added to 2.5 mL of AgNPs (98 μM). After incubating, 0.01 M NaCl was mixed with it and the reaction again incubated for 10 min before the spectra were taken. SERS enhancement factor (EF) was calculated using the formula,34 EF = (ISERS/IRaman) × (CRaman/CSERS), where ISERS and IRaman are intensities of the selected bands in SERS spectrum and normal Raman spectrum, respectively. CSERS and CRaman are the concentrations of DNA in the SERS spectrum and normal Raman spectrum, respectively. C


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Figure 2. (a−c) Absorption spectra of AgNPs in the presence of different concentrations of CT, EC, and ML DNA, respectively. (d−f) Linear plot obtained after plotting the difference in absorbance with DNA concentrations for CT (0.02−5.5 μM), EC(0.02−4 μM), and ML DNA(0.02−3 μM), respectively.

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RESULTS AND DISCUSSION Characterization of Silver Nanoparticles. AgNPs are well-known for their strong and sharp absorbance band in the visible region due to Localized Surface Plasmon Resonance (LSPR). The synthesized citrate capped AgNPs were characterized by UV−vis absorbance spectroscopy which shows a sharp band around 400 nm (Figure. 1a), the characteristic (LSPR) of AgNPs in colloidal solution. During reduction, color of the solution changed to yellow. Bright field image (Figure 1b and c) obtained from transmission emission microscopy represents spherical nature of AgNPs, which exists in close proximity due to citrate capping as evident from the microstructure shown in Figure 1. HRTEM image of a single particle confirms its single crystalline nature (Figure 1d). The measured spacing between adjacent lattice fringes of 0.203 nm is compatible to the 200 reflection of face centered cubic silver structure. The X-ray diffraction (XRD) analysis (Figure S1) also confirms the crystalline nature of the metal particles. The (111) and (200) plane of AgNPs appeared at 38.2° and 44.6° (JCPDS 03−0921) confirming the formation of phase pure silver nanoparticles. The silver nanoparticles in water with an average particle size of 16 ± 2 nm (Figure 1e) exhibited a negative surface charge of −41.2 mV as is evident from the ζ-potential value (Figure 1f). Spectroscopic Analysis. The main objective of this work was to understand the nature of binding of AgNPs to mammalian and bacterial DNA samples of different base pair compositions (Table S1). For this purpose, spectroscopic titration of a solution of the AgNPs with DNA was performed and the changes observed in the SPR band of the AgNPs in the presence of CT, EC and ML DNA was carefully monitored. Parts a−c of Figure 2 represent the variation in the absorbance spectra of AgNPs on addition of increasing concentration of CT (0- 6.5 μM), EC (0− 6.1 μM) and ML (0−6 μM) DNA, respectively. The spectroscopic titration was carried out until saturation was achieved (Figure 2). In all cases, a very strong hypochromism effect leading to static quenching in the SPR band of silver

nanoparticles around 400 nm has been observed as shown in Figure 2a−c. Additionally, in the presence of CT DNA, the SPR peak exhibited more red shift (bathochromic shift) indicative of enhanced agglomeration/aggregation of AgNPs due to the interaction with DNA. The characteristic absorption band of the DNA around 260 nm did not vary much for CT DNA and EC DNA. However, though minute the change in the absorbance was more pronounced for the ML and EC DNA compared to CT DNA. But in the case of ML DNA, an isosbestic point at 306 nm (shown by an arrow mark in Figure 2c) was observed indicating a strong interaction of AgNPs with the GC rich ML DNA. A recent work by Rahban et al. on the spectroscopic studies of silver nanoparticles with CT DNA, on the other hand, reported only hyperchromic effect (increase in absorption) in the DNA absorption band at 260 nm.14 Rahban et al. reported the interaction of AgNPs (particle size 5−10 nm) with only calf thymus DNA, where the DNA concentration was fixed and monitored the change in absorbance of DNA around 260 nm with the addition of silver nanoparticles resulting in a hyperchromic effect (increase in absorption) in the DNA absorption band at 260 nm. In our study in Figure 2a−c, we have fixed the silver nanoparticle concentration and monitored the change in the plasmonic absorption of Ag around 400 nm with the addition of DNA. Our result exhibited hypochromic effect (reduction in intensity) of the plasmonic absorption around 400 nm. Additionally, the characteristic DNA absorption band at 260 nm showed only a marginal change as discussed earlier. This is a clear indication that the size and shape of the particles play a major role in defining the mode and specificity of interaction with DNA. In order to further understand the nature of interaction in detail, we also monitored the change in the absorbance of DNA at 260 nm by the addition of increasing amount of silver nanoparticles to a fixed concentration of DNA, similar to what has been reported by Rahban et al.14 In this case saturation was observed at 30 μM addition of Ag nanoparticles (Figure S3a). Further, to understand the specificity between double stranded and single stranded DNAs, we have also studied the interaction D


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Figure 3. (a−c) Plots of A0 − A) versus different concentrations of CT, EC DNA, and ML DNA, respectively, and (d) plot of binding constant versus % GC content in DNAs with standard error.

Figure 4. (a and b) Absorbance spectra of AgNPs in the presence of different concentration of poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dGdC). (c and d) Shifting in the SPR peak in the presence AT polynucleotide and GC polynucleotide and (e and f) plots of 1/(A0 − A) versus 1/poly(dAdT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC), respectively.

of ss EC DNA and AgNPs. Absorbance data of ds DNA (Figure S3a) in the presence of AgNPs showed little enhancement whereas the absorbance spectra of ss DNA remained unchanged in the presence of AgNPs (Figure S3b). Generally absorbance value increases during unwinding of ds DNA to form ss DNA. These results depict strong interaction of ds DNA with AgNPs compared to ss DNA. Furthermore, the change in the SPR band

of AgNPs was not that significant in the presence of EC ss DNA compared to EC ds DNA (Figure 2b) as demonstrated in Figure S3. To determine the limit of detection (LOD) a plot of A0 − A versus concentration of DNAs was drawn to yield a linear calibration curve (Figure 2, parts d, e, and f). Here A0 is the initial absorbance of AgNPs and A is the absorbance of AgNPs in the E


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Figure 5. (a−c) ζ-Potential variation of AgNPs in the presence of different concentration of CT, EC DNA, and ML DNA, respectively. (d) TEM image of AgNPs in the presence of CT DNA(scale bar =20 nm), where the inset in part d shows a bright field image at a a different magnification (scale bar =100 nm). (e) TEM image of AgNPs in the presence of EC DNA (scale bar = 20 nm), where the inset in part e shows a bright field image at a different magnification (scale bar = 10 nm) indicating the linearly assembled particles.

AgNPs in the presence of AT pairs. The apparent binding constants to AT and GC polynucleotides were evaluated to be (1.81 ± 0.3) × 104 (Figure 4e) and (6.5 ± 0.2) × 104 M−1 (Figure 4f), respectively. This investigation revealed 3.5 times stronger affinity of AgNPs to poly(dG-dC)·poly(dG-dC) compared to poly(dA-dT)·poly(dA-dT). Although the binding constant of poly(dG-dC)·poly(dG-dC) was less than EC and ML DNA, but still these results verify the GC specific binding of AgNPs. In an earlier study, Sastry et al. have shown by ITC measurements that the binding strength of AgNPs to nucleobases change in the order C > G > A > T.36 Effect on the Properties of Silver Nanoparticles. The CT DNA induced aggregation or loss in stability was also proved by measuring the ζ-potential of the AgNPs in the presence of the same concentration of DNAs. The as-prepared AgNPs showed good colloidal stability with a ζ-potential of −41.2 mV. But in the presence of various concentrations of CT (Figure. 5a), EC (Figure. 5b), and ML DNAs (Figure 5c), the ζ-potential value decreased linearly up to the saturation concentration. A lower ζpotential value represents lower stability as well as a decrease in the charge particles velocity or electrophoretic mobility when electric field is applied. These data confirm better stabilization of EC DNA with AgNPs than CT DNA. TEM studies were performed to monitor any change in morphology, that could have occurred during the interaction of AgNPs with DNA. From Figure 5d, the enhanced agglomeration of AgNPs in the presence of CT DNA was evident corroborating the bathochromic shift observed in the absorbance data. But in case of EC DNA (Figure 5e), a distinct layer formation occurred around the AgNPs indicating the formation of a complex with less agglomeration. However, in the case of ML DNA the change in ζ-potential was higher compared to the EC DNA. The lower bathochromic shift in the absorbance data ascertained less agglomeration of AgNPs. Therefore, it can be ascertained that a decrease in zeta value could be due to the stronger interaction with the GC rich ML

presence of DNA. The LOD was calculated using the relation 3σ/S, where σ is the standard deviation of the blank (obtained from 10 blank measurements) and S is the slope obtained from the linear fitting plot.35 In the case of CT DNA titration, the obtained LOD was 6.3 nM whereas for EC DNA and ML DNA it was 5.4 nM and 5 nM, respectively. To compare the strength of the interaction, AgNPs−DNA association constants were calculated from the Benesi−Hildebrand plot using the UV−vis data. The apparent binding constants (KBH) were 5.048 ± 0.2 × 104 M−1, 1.158 ± 0.12 × 105 M−1, and 4.54 ± 0.06 × 105, respectively for CT (Figure 3a), EC (Figure 3b), and ML DNAAg complexes (Figure 3c). Apparent binding constant data confirm the stronger interaction of ML ds DNA having higher GC pairs leading to higher binding constant (Figure 3d). For kinetic studies, we have added same amount DNAs to the AgNPs solution to monitor the change in absorbance with time. In Figure S4a−c, a concurrent shift and hypochromic effect in the SPR band of AgNPs with time in the presence of CT, EC and ML DNAs is presented. It can be seen from Figure S5a−c that in the presence of CT DNA, the SPR band shift was 8 nm compared to 3 and 2 nm, in the presence of EC DNA and ML DNA, respectively. These bathochromic shifts indicate the CT DNA induced agglomerations of AgNPs without any salt addition. From the above result we can conclude that the interaction of GC rich bacterial DNA with synthesized AgNPs is stronger and the shift in the λ max could be used as a tool to identify the nature of DNAs. In order to verify the GC specificity of the interaction, we have studied the interaction of AgNPs with synthetic 100% GC and no GC (100% AT) compositions such as poly(dG-dC)· poly(dG-dC) and poly(dA-dT)·poly(dA-dT). Spectroscopic titration data ascertained similar kind of trend as the natural DNA as evident form Figure 4a and b). The SPR band shift is 7 nm (Figure 4c) in the presence of poly(dA-dT)·poly(dA-dT) and 4 nm in the presence of poly(dG-dC)·poly(dG-dC) (Figure 4d). A higher shift in the SPR band confirms more aggregation of F


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Figure 6. (a) Viscosity changes of DNA in the presence of AgNPs and (b) emission spectra of the Hoechst−DNA complex in the presence of an increase in concentration of AgNPs (0−15 μM).

Figure 7. CD spectra of (a) CT DNA, (b) EC DNA, and (c) ML DNA in the presence of AgNPs and (d−f) observed change in ellipticity (error bar is given and represents five successive runs for each band).

bind on the Au and Ag surface via the N7 atom.39 Similarly thymine via C4 keto oxygen40 and the N1 atom of cytosine along with the CO group have been shown to bind the Ag surface.40 Very recently, Quintela et al. reported that Ag2 clusters can bind covalently with DNA base pairs and Ag3 clusters can intercalate between the base pairs to cause changes in the structural property of the DNA.41 The lower ζ-potential value in the presence of DNAs observed have confirmed the minimization of negative charge on AgNPs surface due to interaction with nucleobases.

DNA and not due to decreased stability. DNA is a polynucleotide with negatively charged phosphate backbone. AgNPs bearing negative charge could cause electrostatic repulsion on approaching DNA. The mode of interaction therefore could be through DNA bases which may bind or interact to the negatively charged AgNPs. Earlier reports suggested different binding modes of nanoparticles surface to DNA bases like guanine binding via keto tautomer of nitrogen (N)9H37 and N7 atom and CO group adsorbed via lone pair on the gold surface.38 Adenine can also G


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The Journal of Physical Chemistry B Interaction of small molecules with DNA occurs mainly by intercalation and groove binding. In such interactions stacking to the base pairs and hydrogen bonding interactions play major role. The size of the AgNPs used in this study is little higher to act as a DNA intercalator. In order to understand whether the binding occurs by intercalation or groove binding, we have carried out viscosity measurements using CT DNA and AgNPs. Generally intercalation leads to lengthening of the DNA, resulting from an increased viscosity of DNA solution whereas groove binders do not influence the DNA strand and hence the viscosity of the DNA solution remains unchanged in the presence of groove binders.42 It can be seen from Figure 6a that the viscosity of the CT DNA does not change in the presence of increasing concentrations of AgNPs and this confirms surface binding of AgNPs on DNA in the DNA grooves. So, it can be assumed that interaction of DNA-AgNPs is mainly boosted by surface binding of the nucleosides either from major groove side or minor groove side. To further gain insight into the DNA binding mode of silver nanoparticles we have utilized Hoechst 33258, 2-(4-hydroxyphenyl)-5-[5-(4-methylpiperazine-1-yl) benzimidazo-2-yl] bezimidazole, a synthetic N-methylpiperazine and classical minor groove binder for competitive binding study. There is a strong increase in the fluorescence intensity of Hoechst upon binding with CT DNA. Upon addition of different concentrations of AgNPs to the Hoechst−DNA complex, a clear decrease in the fluorescence intensity was observed (Figure 6b). This result proved that AgNPs competes with the Hoechst for binding in the minor groove of DNA leading to displacement of Hoechst and confirm minor groove binding. Effect on the DNA Conformation. To investigate if there was any conformational change taking place in the presence of AgNPs, CD spectrum has been recorded. CD spectrum of CT DNA consists of a positive band around 275 nm due to base stacking and a negative band around 245 nm corresponding to right handed helicity of B-form of DNA (Figure. 7a), the CD spectrum of EC DNA exhibited these peaks at 277 and 247 nm, respectively, (Figure. 7b) whereas for ML DNA these bands appeared at 272 and 242 nm, respectively (Figure 7c). Both these bands are very sensitive to the mode by which other molecules/ particles interact with DNA. AgNPs are optically inactive and have no CD. With the addition of increasing concentration of AgNPs, there was a gradual decrease in the intensity of both negative and positive CD bands of CT, EC and ML DNA (Figure 7a−c). The observed changes in the CD spectra in the presence of AgNPs may be due to the deformity in base stacking and the helical structure of DNA.43 Nevertheless the observed intrinsic B-DNA CD spectra does not specify any major structural transformation of B-form to other forms. From the changes in ellipticity shown in Figure 7d−f, we can conclude that changes in both right-handed ellipticity and base stacking induced by AgNPs are more pronounced in CT DNA compared to EC DNA and ML DNA. The CD data also revealed that the ellipticity change is almost double in CT DNA compared to those in EC and ML DNAs. This finding proves the strong influence of GC content on the CD spectra of DNA in the presence of AgNPs. We also measured the melting temperature (Tm) of the DNA in the absence and in the presence of AgNPs. In this thermal denaturation experiment hyperchromicity effect in absorbance of DNA was observed with increase in temperature. The results show that AgNPs decreased the Tm of CT DNA (Figure. 8a) from 67.64 to 64.83 °C and that of EC DNA (Figure. 8b) from 69.05 to 66.54 °C and that of ML DNA from 89.6 to 87.01 °C

Figure 8. (a) Melting profile of free CT DNA and in the presence of AgNPs. (b) Melting temperature profile of free EC DNA and in the presence of AgNPs. (c) Melting profile of free ML DNA and in the presence of AgNPs.

(Figure 8c). The decrease in Tm indicates destabilization of DNA by disturbing base stacking interaction and interferes with their base pairs to trigger the unwinding process of DNA base pairs with rise in temperature. These results further confirmed that binding of AgNPs to DNA occurred through the grooves. A previous study by Rahban et al. on the interaction of AgNPs with CT DNA reported that synthetic AgNPs of 5−10 nm can act as a intercalator, disrupting the base stacking and thereby increasing the Tm.14 Jang et al., on the other hand reported the dissociation of ds DNA on interaction with Au through melting temperature measurement.44 In our case, the synthesized AgNPs of size 16 ± H


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Figure 9. (a−c) ITC profiles of CT DNA, EC DNA, and ML DNA in the presence of AgNPs. The top panels represent the raw data obtained after sequential injection of AgNPs into DNA, and the bottom part depict the integrated data points fitted to a one-site model against the molar ratio of AgNPs/DNA.

Table 1. ITC Data for AgNPs and DNA at 25 °C N CT DNA EC DNA ML DNA

1.88 2.07 1.33

Ka 6.32 × 10 ± 2.44 × 10 1.76 × 105 ± 5.03 × 102 2.37 × 105 ± 2.88 × 102 4

2

ΔHo (kcal/mol)

TΔSo (kcal/mol)

ΔGo (kcal/mol)

−0.105 ± 0.029 −0.051 ± 0.014 −0.192 ± 0.018

6.3 6.9 6.52

−6.40 −6.95 −6.712

DNAs compared to CT DNA, due to the higher GC pairs present in EC and ML DNAs.On the basis of the Tm, UV−vis and ITC studies we could confirm that the synthesized AgNPs could be used for selective identification of ML, EC and CT DNA, through their DNA binding mechanism. Surface Enhanced Raman Spectroscopy (SERS). In order to further establish the potential of the synthesized AgNPs for detecting DNA, SERS have been employed as presented below. SERS have been explored as a modern tool to detect small biomolecules using SERS active substrates like Au and AgNPs.45−48 Most of the reports on SERS analysis of DNA have been carried out by complex labeling technique but direct label free detection of double-stranded DNA has not been studied much compared to single-stranded DNA. There are very few reports that show nonthiolated or label free double stranded DNA detection using SERS. Usually thiol conjugated DNA has been utilized to strongly interact with Au nanoparticles to achieved good Raman signal.24 Previous reports have found better SERS result with AgNPs compared to AuNPs for unmodified single stranded and double stranded DNA recognition with a lower limit of detection of 10−8 M for Au.48 Here, we have studied the direct interaction of dsDNA of different base pair compositions with plasmonic silver nanoparticles using SERS technique up to nanomolar concentration. In the absence of any salt, SERS spectrum of CT DNA (Figure S6a) and AgNPs colloidal mixture showed very weak bands with a single intense band around 750 cm−1 which is attributed to the ring breathing mode of adenine.24 This band showed shift in the band position compared to normal CT DNA, implying a probable interaction of CT DNA with Ag NPs.24 In the case of EC DNA (Figure S6b) no strong detectable bands were observed on interaction with Ag NPs. The two weak bands around 405 and 342 cm−1 exhibited by CT, EC, and ML DNA are mainly due to

2 nm are not acting as intercalator but interact through base pairs by groove binding as confirmed from melting studies. Thermodynamic Characterization of the Binding. Isothermal titration calorimetry is an effective tool to thermodynamically characterize the binding of nanoparticle to nucleic acid structures and can provide key insights into the complex formation driven by the molecular forces. The advantage of ITC is that it can give a complete thermodynamic profile of the binding such as Gibbs energy change (ΔGo), enthalpy change (ΔHo) and entropy change (ΔSo) together with stoichiometry (N) and the affinity constant (Ka), and these are independent of the spectroscopic changes that occur during the reactions. The representative ITC profiles for the binding of AgNPs to all the three DNAs are presented in Figure 9. The binding is exothermic in nature. Each of the heat burst curves in Figure 9 corresponds to a single injection of AgNPs into the nucleic acid solution that was corrected by the corresponding dilution heats derived from a titration of identical amounts of AgNPs solution into the buffer. The resulting corrected heat plotted as a function of the molar ratio of AgNPs/DNA, is depicted in the lower panels of the figure. The data points represent experimental injection heats while the solid lines reflect the calculated fits of the data to the model. The thermodynamic parameters obtained for the binding of AgNP to the DNAs are depicted in Table 1. The ITC data yielded an association constant of 6.32 × 104 ± 2.44 × 102 M−1 for CT DNA, 1.76 × 105 ± 5.03 × 102 M−1 for EC DNA, and 2.37 × 105 ± 2.88 × 102 M−1 for ML DNA. It is observed that the binding affinity values obtained from ITC experiments are in agreement with those obtained from the spectroscopic study. Negative Gibbs energy suggests a thermodynamically favorable interaction occurred between AgNPs and DNA. The stoichiometry of the binding was similar in all the cases. The AgNPs binds strongly with bacterial I


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observed due to the stretching mode of adenine. In case of ML DNA strong band at 1336 cm−1 represents stretching mode of guanine. The broad band at 802 cm−1 mainly arise from the ring breathing mode of cytosine is also visible in all the DNAs. In addition, SERS spectra of DNAs showed a sharp peak at 1004 cm−1, which could be assigned to the nitrogen and sugar stretching (N−C) mode of nucleobases. Most intense Raman bands appeared at 1294 cm−1 due to the stretching mode of PO2− backbone. These features depict the strong adsorption of DNAs on AgNPs surface in the presence of salt. The 1477/1480 cm−1 band is affiliated to the adenine, thymine and cytosine bases. The strong band around 405 cm−1 and at 1576 cm−1 have been assigned to the bending mode of external nitrogen atom bonded to the guanine ring, out of plane ring bending mode and carbonyl stretching mode of guanosine, respectively. CT DNA and EC DNA display cytosine band at 1272 and 1214 cm−1, respectively. A new band around 1780 cm−1 is present in both EC and ML DNA which can be assigned to the CO stretching. Although some of the Raman bands exhibited shift in their position due to strong interaction in the liquid phase, most of the above observed Raman bands resemble the previously reported studies on DNA.24,38,40,45−50 Similar building blocks of all the three DNAs make it difficult to differentiate them in terms of Raman band. Therefore, we compared the relative intensities of base specific Raman bands as shown in Figure S7a,b. Therefore, we have tried to differentiate them in terms of relative band intensities as CT, EC and ML DNA have different GC content. For this purpose, we considered the bending vibration mode of guanine at 405 cm−1 and ring breathing modes of cytosine at 802/804 cm−1 which are common in all the three DNAs. The new data has been presented in the Supporting Information as Figure S7a,b. It can be observed from Figure S7a, that intensity of both peaks increases from CT DNA to ML DNA. The SERS enhancement factor also showed similar trend (Figure 7b). In addition, the ring breathing mode of adenine is reduced in ML DNA and EC DNA compared to CT DNA. Therefore, by utilizing the SERS phenomenon we were able to detect all the nucleoside bases as well as phosphate backbone of double stranded bacterial DNAs (ML DNA, EC DNA) and mammalian DNA (CT DNA) without any modification contrast to most previous results which were dominated by adenine band.

the ring bending vibration of guanine and thymine. To achieve good Raman signal, we have added 0.01 (M) NaCl to AgNPs− DNA mixtures. Addition of Na+Cl− salt reduces the repulsive charge barrier between the negatively charged AgNPs and negatively charged phosphate backbone of DNA and trigger the interaction through adsorption on Ag surface resulting in AgNPs aggregation. It can be seen from Figure 10 that SERS spectra of CT, EC, and ML DNA display several peaks upon salt addition. The assignments of all the Raman peaks are listed in Table S2. Adenine ring breathing mode has been observed around 751 cm−1 with a slight shift toward a higher wavenumber due to adsorption on silver surface. In addition two more bands around 1330 cm−1 (CTDNA) and 1430 cm−1 (EC DNA) can be

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CONCLUSIONS In this work, binding of spherical silver nanoparticles (AgNPs) to calf thymus (CT), E. coli (EC), and M. lysodeikticus (ML) DNA was studied to gain insights into their mode and specificity of interaction. On the basis of the absorption, thermal melting, isothermal calorimetry and viscosity studies, we could establish the mode of binding and specificity of the synthesized silver nanoparticles with mammalian and bacterial DNA. The viscosity measurement and competitive binding study with classical groove binder could unequivocally establish the mode of binding of the synthesized particles to be in the minor groove of the DNA. In order to verify the GC specificity of the interaction, we studied the interaction of AgNPs with synthetic DNAs poly(dGdC)·poly(dG-dC) and poly(dA-dT)·poly(dA-dT) having 100% GC and 0% GC base pair compositions. The apparent binding constants to GC and AT polynucleotides were evaluated to be (6.5 ± 0.2) × 104 M−1 and (1.81 ± 0.3) × 104, respectively. A 3.5 times stronger affinity of AgNPs to poly(dG-dC)·poly(dG-dC) compared to poly(dA-dT)·poly(dA-dT) observed further confirms the GC specificity of the nanoparticles. The isothermal calorimetric studies revealed a thermodynamically favorable

Figure 10. (a) (i) Raman spectra of CT DNA, (ii) SERS spectra of 50 × 10−9 (M) CT DNA, (iii) SERS spectra of 2 × 10−7 (M) CT DNA, and (iv) SERS spectra of 6 × 10−6 (M) CT DNA. (b) (i) Raman spectra of EC DNA, (ii) SERS spectra of 50 × 10−9 (M) EC DNA, (iii) SERS spectra of 2 × 10−7 (M) EC DNA, and (iv) SERS spectra of 6 × 10−6 (M). EC DNA. (c) (i) Raman spectra of ML DNA, (ii) SERS spectra of 50 × 10−9 (M) ML DNA, (iii) SERS spectra of 2 × 10−7 (M) ML DNA, and (iv) SERS spectra of 6 × 10−6 (M) ML DNA (all spectra were plotted with the same intensity scale). J


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(6) Jaganathan, H.; Ivanisevic, A. Gold−Iron Oxide Nanoparticle Chains Scaffolded on DNA as Potential Magnetic Resonance Imaging Agents. J. Mater. Chem. 2011, 21, 939−943. (7) Chung, H. J.; Castro, C. M.; Im, H.; Lee, H.; Weissleder, R. A Magneto-DNA Nanoparticle System for Rapid Detection and Phenotyping of Bacteria. Nat. Nanotechnol. 2013, 8, 369−375. (8) Kuan, G. C.; Sheng, L. P.; Rijiravanich, P.; Marimuthu, K.; Ravichandran, M.; Yin, L. S.; Lertanantawong, B.; Surareungchai, W. Gold-Nanoparticle Based Electrochemical DNA Sensor for the Detection of Fish Pathogen Phanomyces Invadans. Talanta 2013, 117, 312−317. (9) Hekmat, A.; Saboury, A. A.; Divsalar, A. The Effects of Silver Nanoparticles and Doxorubicin Combination on DNA Structure and Its Antiproliferative Effect against T47D and MCF7 Cell Lines. J. Biomed. Nanotechnol. 2012, 8, 968−982. (10) Khiati, S.; Luvino, D.; Oumzil, K.; Chauffert, B.; Camplo, M.; Barthelemy, P. Nucleoside-Lipid-Based Nanoparticles for Cisplatin Delivery. ACS Nano 2011, 5, 8649−8655. (11) Ruiz-Hernandez, E.; Baeza, A.; Vallet-Regí, M. Smart Druǵ Delivery through DNA/Magnetic Nanoparticle Gates. ACS Nano 2011, 5, 1259−1266. (12) Watson, J. D.; Crick, F. Molecular Structure of Nucleic Acid. Nature 1953, 171, 737−738. (13) Fong, K. E.; Yung, L-Y. L. Localized Surface Plasmon Resonance: A Unique Property of Plasmonic Nanoparticles for Nucleic Acid Detection. Nanoscale 2013, 5, 12043−12071. (14) Rahban, M.; Divsalar, A.; Saboury, A. A.; Golestani, A. Nanotoxicity and Spectroscopy Studies of Silver Nanoparticle: Calf Thymus DNA and K562 as Targets. J. Phys. Chem. C 2010, 114, 5798− 5803. (15) Polavarapu, L.; Perez-Juste, J.; Xu, Q.; Liz-Marzan, L. M. Optical Sensing of Biological, Chemical and Ionic Species through Aggregation of Plasmonic Nanoparticles. J. Mater. Chem. C 2014, 2, 7460−7476. (16) Deng, H.; Xu, Y.; Liu, Y.; Che, Z.; Guo, H.; Shan, S.; Sun, Y.; Liu, X.; Huang, K.; Ma, X.; et al. Gold Nanoparticles with Asymmetric Polymerase Chain Reaction for Colorimetric Detection of DNA Sequence. Anal. Chem. 2012, 84, 1253−1258. (17) Goodman, C. M.; Chari, N. S.; Han, G.; Hong, R.; Ghosh, P.; Rotello, V. M. DNA-Binding by Functionalized Gold Nanoparticles: Mechanism and Structural Requirements. Chem. Biol. Drug Des. 2006, 67, 297−304. (18) Park, H. G.; Joo, J. H.; Kim, H. G.; Lee, J. S. Shape-Dependent Reversible Assembly Properties of Polyvalent DNA-Silver Nanocube Conjugates. J. Phys. Chem. C 2012, 116, 2278−2284. (19) Li, F.; Pei, H.; Wang, L.; Lu, J.; Gao, J.; Jiang, B.; Zhao, X.; Fan, C. Nanomaterial-Based Fluorescent DNA Analysis: A Comparative Study of the Quenching Effects of Graphene Oxide, Carbon Nanotubes, and Gold Nanoparticles. Adv. Funct. Mater. 2013, 23, 4140−4148. (20) Zhang, X.; Servos, M. R.; Liu, J. Fast pH-Assisted Functionalization of Silver Nanoparticles with Monothiolated DNA. Chem. Commun. 2012, 48, 10114−10116. (21) Li, H.; Rothberg, L. Colorimetric Detection of DNA Sequences Based on Electrostatic Interactions with Unmodified Gold Nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 14036−14039. (22) Zhang, J.; Ting, B. P.; Jana, N. R.; Gao, Z.; Ying, J. Y. Ultrasensitive Electrochemical DNA Biosensors Based on the Detection of a Highly Characteristic Solid-State Process. Small 2009, 5, 1414−1417. (23) Hsieh, K.; Patterson, A. S.; Ferguson, B. S.; Plaxco, K. W.; Soh, H. T. Rapid, Sensitive, and Quantitative Detection of Pathogenic DNA at the Point of Care through Microfluidic Electrochemical Quantitative Loop-Mediated Isothermal Amplification. Angew. Chem., Int. Ed. 2012, 51, 4896−4900. (24) Barhoumi, A.; Zhang, D.; Tam, F.; Halas, N. J. Surface-Enhanced Raman Spectroscopy of DNA. J. Am. Chem. Soc. 2008, 130, 5523−5529. (25) Brabec, V.; Niki, K. Raman Scattering from Nuceic Acids Adsorbed at a Silver Electrode. Biophys. Chem. 1985, 23, 63−70. (26) Lin, T. W.; Wu, H.-Y.; Tasi, T. T.; Lai, Y.-H.; Shen, H.-H. SurfaceEnhanced Raman Spectroscopy for DNA Detection by the Self Assembly of Silver Nanoparticles onto Ag Nanoparticle-Graphene

binding of AgNPs to double stranded bacterial DNA. Comparative interaction studies with ss and ds DNAs further confirmed the specificity of the particles toward ds DNA. On the basis of these studies, we confirmed that the AgNPs could be used for selective detection of different Gram-negative and Gram-positive bacterial DNAs and CT DNA, where the ML and EC DNAs exhibited stronger interaction resulting in destabilization of the structure. Above all, we were able to detect all the nucleoside bases as well as phosphate backbone of double stranded DNA using the synthesized unmodified silver nanoparticles by the SERS spectra. Overall, the results present new insights into the base pair composition dependent specificity and interaction of silver nanoparticles that could be used for selective DNA detection.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b01586. Base pair composition table of all the DNAs, XRD of AgNPs, saturation plot for the UV−vis titration, time dependent absorbance spectra, concentration dependent SPR shift plot, absorbance spectra of ss EC DNA and ds EC DNA in the presence of AgNPs, absorbance spectra of AgNPs in the presence of ss EC DNA, Raman spectra of CT and EC DNA, assignments of Raman bands for dsDNAs, SERS intensity, and enhancement factor vs different DNAs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(P.S.D.) Telephone: +91 33-2422-3487. E-mail: psujathadevi@ cgcri.res.in; [email protected]. Author Contributions §

S.P. and S.C. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS S.P. and S.C. acknowledge Council of Scientific and Industrial Research (CSIR) and University Grant Commission (UGC), Government of India, respectively, for awarding junior research fellowships. P.S.D. acknowledges the CSIR for support through the CSIR network program on MULTIFUN CSC0101.Work at CSIR-IICB was supported by funds from the project Gen Code (BSC 0123).

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Unraveling the Interaction of Silver Nanoparticles with Mammalian and

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