Unveiling the Interaction of Duplex DNA with Graphene Oxide in the

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Unveiling the Interaction of Duplex DNA with Graphene Oxide in Presence of Two Diverse Binders: A Detailed Photophysical Study Sangita Kundu, Arghajit Pyne, Rupam Dutta, and Nilmoni Sarkar J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b10752 • Publication Date (Web): 27 Feb 2018 Downloaded from http://pubs.acs.org on February 27, 2018

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The Journal of Physical Chemistry

Unveiling the Interaction of Duplex DNA with Graphene Oxide in Presence of Two Diverse Binders: A Detailed Photophysical Study Sangita Kundu, Arghajit Pyne, Rupam Dutta, and Nilmoni Sarkar* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India E-mail: [email protected] Fax: 91-3222-255303 Abstract Coupling of biomolecules with nanomaterials has drawn immense attraction due to the improved synergistic properties, functions and biocompatible nature. Thus this process manifests its important role and fascinating potential in various nanobiotechnogical, biomedical, biosensing and imaging applications. In this work, fundamental understanding of the interfacial properties and the interaction of double-stranded DNA (dsDNA) with graphene oxide (GO) has been systematically investigated employing two different DNA binding probes. Our results suggest that the unusual adsorption of duplex DNA onto GO surface has been facilitated due to the partial deformation of the helical structure of DNA as evident from the circular dichroism (CD) spectroscopy. Depending on the location of the probes inside DNA helix, the photophysical properties of the dye bound DNA in presence of GO has been changed. Interestingly, the translational diffusion and rotational motion of the minor groove binding probe, DAPI (4'-6diamidino-2-phenylindole) bound DNA has been significantly altered with the addition of GO. In contrast, efficient electron transfer may occur from the DNA-intercalated EB (ethidium bromide) to GO with a time constant ~300 fs as evident from the ultrafast time-resolved measurement. Conclusively, basic understanding of the interaction mechanism and dynamics of two different probes inside DNA and at GO interface pave us for the future development of various nano/bio applications. 1. Introduction The integration between nanomaterials and biocomponents is an emerging area of research in material sciences, molecular biotechnology, biomedical imaging and so forth.1-6 The carbonaceous nanostructures such as carbon nanotubes (CNT), graphene, water-soluble C60 derivatives have been extensively used in biosensing, bioimaging, drug delivery and gene 1 ACS Paragon Plus Environment

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therapy.7-14 Graphene, a single-layered two-dimensional sheet of sp2 carbon atoms having extended honeycomb network has drawn much attention because of its superior thermal, mechanical, electrical and optical properties.15, 16 The limited solubility of graphene enhances the significance of graphene oxide (GO) for the application purposes. The most facile route to obtain GO is the oxidation followed by the exfoliation of graphite using strong oxidizing agents.17 In GO, the contiguous aromatic lattice structure is interrupted by the multiple reactive oxygen functionalities which incorporate themselves in such a way that hydroxyl and epoxy groups are on the basal planes whereas, the carboxylic acid and carbonyl groups are at the periphery of the graphitic platelets.18 High specific surface area enriched with several functional groups, excellent aqueous stability, physiological stability of GO19, 20 have attracted to employ it in drug delivery and biosensing after the covalent or noncovalent functionalization with drug molecules or aromatic molecules.12,

21, 22

Furthermore, GO can be used as the substrate material for the

biomolecules including proteins, nucleic acids, cells which lead to the intriguing applications in the field of cell imaging,23 molecular recognition,24 enzyme assays etc.25 The biomolecules can be adsorbed onto the graphene surface via hydrogen bonding, hydrophobic interaction, π-π stacking or electrostatic forces.26-28 Varghese et al.29 have reported that the four DNA nucleobases and nucleosides bind with graphene through van der Waals interaction. The relative strength of interaction with graphene sheet follows the order guanine (G), adenine (A), cytosine (C) and thymine (T) in aqueous solution suggesting the importance of solvation energy. Furthermore, the interaction and binding mechanism of biomolecules especially nucleic acids with GO has been extensively investigated to study the exact interaction, structural changes of the biomolecules.30-32 Both, experimental and theoretical results demonstrate that the singlestranded DNA (ssDNA) or RNA can be preferentially adsorbed onto the graphene surface through the hydrophobic and π-stacking interaction between the ring atoms of the nucleobases and the carbon rings of the graphene surface. Whereas, the affinity of DNA duplex towards graphene is comparatively lower due to the efficient shielding of the end base pairs within the negatively charged sugar-phosphate backbone of double-stranded DNA (dsDNA).33-35 Additionally, GO can quench the fluorescence of probes conjugated to ssDNA due to extensive quenching capability. However, in presence of a target the dye labeled DNA is desorbed from the surface of GO due to the hybridization of the ssDNA resulting in the enhancement of the fluorescence signal.30, 36, 37 Several groups have reported that the adsorption and desorption of 2 ACS Paragon Plus Environment

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oligonucleotides onto graphene vary as a function of DNA length, temperature, ionic strength and pH.35, 38, 39 Based on these basic understandings, Lu et al.40 have reported GO as a promising DNA protecting agent and transporter during cellular delivery as GO has the stronger binding affinity to ssDNA. On the other hand, Lie et al.41 have shown that the binding ability of dsDNA onto the graphene surface is facilitated in presence of metal ions and divalent cations are more effective compared to monovalent cations for increasing the adsorption efficiency of dsDNA onto GO. Zhao et al.42 have performed molecular dynamics (MD) simulations to explore the interaction between DNA duplex and graphene in aqueous environment. They have found that the dsDNA segments can self-assemble onto the surface of graphene forming hybrid nanostructure. This type of structure is stabilized via the π-stacking interaction between the end base pairs of DNA that are already broken due to such type of attachment and the graphitic carbon atoms. More interestingly, AT base pairs of DNA are more likely to be deformed than GC end and initiate hydrophobic π type interaction with the carbon surface. Li and his coworkers43 have shown experimentally that the interaction of duplex DNA with graphene is possibly facilitated due to the partial deformation of the helical structure of the DNA and the efficiency of interaction may be increased via hydrogen bond formation between the oxygen functional groups of graphene and end base pairs while the primary governing force is the π-π stacking interaction. In another report, Liu et al.38 have proposed that dsDNA can be trapped between two stacked graphene layers at high salt concentration. They have demonstrated that the presence of salt decreases the electrostatic repulsion between phosphate backbone of dsDNA and graphene to form the stable structure via van der Waals and aromatic interaction. It is well established that the fluorescence of the biomolecules or fluorophores are quenched due to the adsorption of the biomolecules on the surface of GO. This quenching may be due to the fluorescence resonance energy transfer (FRET), electron transfer or nonradiative dipole- dipole interaction.24, 44, 45 Based on these phenomenon, graphene and graphene-based complexes with organic dyes have been employed to construct DNA biosensors.46-50 Herein, our main aim is to unveil the restricted interaction of duplex DNA with GO in presence of two fluorescent DNA binding dye molecules. One is DNA intercalating dye, ethidium bromide (EB) and another is minor groove binder, 4'-6-diamidino-2-phenylindole (DAPI) and they have been employed to examine the interaction mechanism with the help of several microscopic and spectroscopic techniques. The excited state behavior of the probe molecules in presence of GO are quite 3 ACS Paragon Plus Environment


different due to their respective binding nature with DNA. Static quenching is observed in DAPIDNA in presence of GO. In contrary, very fast electron transfer from EB-DNA to GO has been exhibited from ultrafast spectroscopy. Furthermore, the organization and diffusion properties of dye inside DNA and in presence of GO have been analyzed using fluorescence correlation spectroscopy (FCS) in a single molecular level. This study may facilitate for the development of the graphene-DNA nano-biointerface in the field of nanobiotechnology, biomedical, biosensing. 2. Experimental Section 2.1. Materials and Sample Preparation Ethidium bromide (EB), 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were obtained from Sigma-Aldrich. Sodium salt of deoxyribonucleic acid (Calf Thymus DNA, ctDNA) and graphite flakes were bought from Sisco Research Chemical Laboratory (SRL), India. The solution of ctDNA was prepared in 10 mM phosphate buffer solution (pH7) and the solution was kept at 4°C for 2 days with occasional shaking. The absorbance ratio of the ctDNA solution at 260 nm and 280 nm was 1.83 suggesting the absence of protein contamination in the sample. The concentration of the stock DNA solution was determined by measuring the absorbance at 260 nm using molar extinction coefficient (εDNA) of 6600 M−1 cm−1. The structures of the GO and the fluorophores are shown in Scheme 1. 2.2. Synthesis of Graphene Oxide Graphene oxide (GO) was synthesized using the modified Hummer’s method reported earlier.17 Briefly, 1 g of graphite powder was mixed to the 0.5 g of NaNO3 and then 50 mL of concentrated H2SO4 was added to the mixture and it was cooled down to 0 °C. Then 3 g KMnO4 was added with constant stirring keeping the mixture in an ice bath. Then it was stirred for half an hour at room temperature followed by dilution with water and temperature was increased to 90°C and kept for 1h. Then 30% H2O2 was added slowly until the effervescence was stopped indicating the complete oxidation of the graphite powder. The supernatant was decanted off and the brown precipitate was centrifuged and washed with 1 N HCl followed by water for several times. The product was redispersed in water by ultrasonication for 4 h followed by centrifuging at 4000 rpm for an hour. The supernatant was again centrifuged at 13,500 rpm and vaccum–dried for overnight. This solid GO was dispersed in water and sonicated for 1h to prepare 1 mg/ml single layered GO solution. This solution was used as the stock solution in the experiment.

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Scheme 1. Chemical structures of GO, Ethidium Bromide (EB), DAPI 2.3. Instrumentation 2.3.1. Spectroscopic Measurements Steady-state absorption and fluorescence spectra were recorded in Shimadzu (model no UV2450) and Hitachi (model no F-7000) spectrofluorimeter respectively. For all the spectroscopic experiments, the DNA solution was added to the respective dyes followed by the addition of GO solution. The EB was excited at 440 nm and DAPI was excited at 375 nm. The anisotropy measurement was performed using a 1 cm quartz cell in Fluorolog-3. Steady-state anisotropy can be defined as,



=

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− ∙

(1) + 2 ∙

where, G is the correction factor. The emission intensity of the sample when both excitation

polarizer and emission polarizer oriented vertically is represented as , and when excitation polarizer is oriented vertically and the emission polarizer is horizontally oriented, it is represented as . Time-resolved lifetimes were measured by time-correlated single photon counting (TCSPC) technique at two different excitation wavelengths of 375 nm and 440 nm. The signals were detected at a magic angle (54.7°) using Hamamatsu microchannel plate photomultiplier tube (3809U). The instrument response function (IRF) of TCSPC set up is ∼170 ps and ∼130 ps

depending on the excitation source of 440 nm and 375 nm respectively. The decays were fitted using IBH DAS-6 decay analysis software. Time-resolved anisotropy was performed in the same instrument and for our set up, G factor is 0.6. The femtosecond (fs) time-resolved experiments were performed on an up-conversion setup (Fluoromax, IB Photonics) where solid state Tisapphire laser (Mai Tai HP, Spectra Physics) was used as an excitation source.51 In our experiment, we used 880 nm pulsed laser with duration 100 fs and 80 MHz repetition rate. The fundamental 880 nm beam frequency was doubled to 440 nm in a nonlinear barium Borate (BBO) crystal. The fluorescence collected from the sample was upconverted on another thin BBO type I crystal using gate pulse of the fundamental beam (880 nm). The sum frequency of the emission wavelength (609 nm) and gate pulse was upconverted using BBO type I I crystal and the signal was detected as a function of time delay between gate and excitation pulses. The signal was collected into the double monochromator and detected by a Hamamatsu photomultiplier tube keeping the angle of polarization of 54.7°. All samples were excited at 12 mW average power of pump laser taken in a rotating sample holder. The instrument response function (IRF) is found around ~100 fs. The femtosecond fluorescence decays were collected at the respective emission maxima and deconvoluted using a Gaussian-shaped IRF by Labview Software using a multi-exponential function,

I ( λ ,t ) = ∑ ai ( λ ,t ) exp ( −t τ i ( λ ) ) i

contribution of corresponding τi(λ) decay times. 2.3.2. Fluorescence Correlation Spectroscopy (FCS)


, where ai(λ) is the

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FCS measurements were performed using DCS 120 Confocal Laser Scanning Microscope (CLSM) system (Becker & Hickl DCS-120) with inverted microscope of Zeiss. The FCS traces were collected using picosecond diode lasers with excitation source connected to the inverted microscopes of Zeiss, equipped with a 40× water-immersion objective (NA 1.2). For FCS measurements, the fluorophores containing DNA solution in presence of GO solution were placed onto the glass coverslip and excited by the picosecond diode laser of 488 nm. The autocorrelation function G(τ), which was obtained due to the temporal fluctuation of the fluorescence intensity can be defined as52 (τ) =

〈()( + τ)〉 (2) 〈()〉

where τ denotes delay and ⟨F(t)⟩ is the average fluorescence intensity.

Where δF(t) and δF(t + τ) are the amounts of fluctuation in intensity around at time t and t + τ

() = () − 〈()〉 (3) ( + τ) = ( + τ) − 〈()〉 (4)

3D diffusion model has been used to fit the autocorrelation traces. For n fraction of dyes which are diffused within the system with distinct diffusion coefficients, the correlation function G(τ) can be defined as,

() =

1 % $ !"

1

. %

*+ - $

1

.

*+ -

' 1 ' 1+& ) 1+ & ) / '( , '( , # #

"⁄

(5)

Where, N denotes the number of fluorescent particles within the observation volume, is the

fractional weighting factor for the ith contribution to the autocorrelation curve and '( is the

diffusion time of the fluorophores and ' is the delay or lag time. The structure parameter / is related to ω = l/r where l is the longitudinal radii and r is the transverse radii. The transverse radii is related to the diffusion coefficient by the following equation,



23 = (6) 4τ( Using a sample having known diffusion coefficient [Rhodamine 6G (R6G) in water, Dt = 426 µm2 s−1],53 the structural parameter (ω) of the excitation volume was determined using the given equation

"

1 423 τ 7" 423 τ 7 (τ) = 51 + 6 51 + 6 (7) ω

In the fitting analysis, r and ω were kept as linked global parameters. ω value is obtained as 5 after fitting correlation trace of R6G in water and observation volume (Veff) is calculated from the following equation

= 6Γ * (9)

The value of α in equation5 and equation9 denotes the extent of deviation from normal diffusion (α =1). For α >1, the process is termed as superdiffusion and for α

Unveiling the Interaction of Duplex DNA with Graphene Oxide in the

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