Article pubs.acs.org/Biomac
Ethidium Bromide-Adsorbed Graphene Templates as a Platform for Preferential Sensing of DNA Sudipta Nandi, Parimal Routh, Rama K. Layek, and Arun K. Nandi* Indian Association for the Cultivation of Science, Jadavpur, Kolkata-700 032, India S Supporting Information *
ABSTRACT: Sulfonated graphene (SG) and graphene oxide (GO) are used as an ethidium bromide (EtBr, E) binding platform, to preferentially sense DNA (D) among the other biomolecules such as RNA (R), bovine serum albumin (BSA, P) and glucose (G) using spectroscopic techniques. EtBr loses its intrinsic fluorescence property after binding with SG. DNA can “turn on” the quenched fluorescence of an SG−EtBr hybrid to a greater extent compared to the RNA, BSA, and glucose. UV−vis absorption spectra and circular dichroism (CD) spectra also support the higher ability of DNA to release adsorbed EtBr from the SG surface in comparison to the above-mentioned biomolecules. Compared to GO−EtBr, the SG−EtBr hybrid is superior to preferentially sense DNA, as the enhancement of fluorescence intensity is 16 times in the later but it is 4.5 times in the former from their respective complexes. An analysis of Raman spectral data indicates that the interaction of EtBr in its adsorbed state on an SG template is greater with DNA than with RNA.
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which gets enhanced after binding with nucleic acids.22 It is generally agreed that strong fluorescence enhancement accompanies intercalation22−26 of the dye into the helix of the nucleic acids, but there is also evidence for additional nonintercalative, less fluorescence-enhanced sites that are presumed to involve electrostatic binding.27,28 This very common dye is universally used in fluorescence staining of chromosomes29 and it is also widely used for staining nucleic acid during electrophoretic determination of nucleic acid.30 In this article, we have compared the properties of SG/EtBr and GO/EtBr templates in the preferential sensing of DNA than other biomolecules such as RNA, bovine serum albumin (BSA, a protein) and glucose (a sugar), which are usually present in a living cell.
INTRODUCTION Graphene, a single layered hexagonally arrayed sp2 bonded carbon atomic sheet,1 has attracted a great deal of research attention for its unique optical,2,3 electrical,3,4 and mechanical5,6 properties extending its potential application in various fields, such as electronics,3,4 supercapacitors,7−9 sensors,3,4,10−12 and composite materials.3,4,13−15 The difficulty of thin-layer graphene preparation in a large scale from graphite enhances the importance of graphene oxide (GO; Scheme 1) for the above purpose.16 The high specific surface area,2,3 excellent water solubility,17 and the intrinsic fluorescence property of GO in the visible and near-infrared (NIR) range18 make it attractive for several biological applications such as drug delivery,18,19 live cell imaging,18 biosensors,11 nitro aromatic-sensor,20 and so on. GO can be considered to be a weak acid cation exchange resin because of the ionizable carboxylic acid groups, and this excellent property of GO may be used to preferentially sense DNA using the dye ethidium bromide (EtBr; Scheme 1) by fluorescence spectroscopy. A better approach of it is to use sulfonated graphene (SG; Scheme 1) as it possesses better water solubility21 than GO due to the presence of charged SO3− units, which prevent the graphitic sheets from aggregation in solution.21 In our experiment, SG is used as an adsorbing platform for the water-soluble EtBr (a cationic dye), that acts as a fluorescent probe due to its excellent fluorescence property, © 2012 American Chemical Society
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EXPERIMENTAL SECTION Sample. Calf thymus DNA (Type 1; sodium salt, molecular weight = 8.6 × 106 Da),31 RNA (diethyl amino ethanol salt, type IX from Torula Yeast, molecular weight, determined from agarose gel electrophoresis, is 4 × 104 Da), BSA (P), graphite powder, sodium borohydride, and sulfanilic acid were Received: June 23, 2012 Revised: September 6, 2012 Published: September 17, 2012 3181
dx.doi.org/10.1021/bm3009632 | Biomacromolecules 2012, 13, 3181−3188
Biomacromolecules
Article
Scheme 1. Structure of EtBr and Different Graphene Derivatives Used in This Work
Table 1. Fluorescence Lifetime Data of EtBr, SGE, DE, RE, SGED, and SGER Samples Measured on Excitation at 440 nm systems
τ1 (ns)
relative amplitude (a1)
EtBr SG + EtBr (SGE) DNA + EtBr (DE) RNA + EtBr (RE) SGE + DNA (SGED) SGE + RNA (SGER)
1.58 1.59 5.63 6.14 13.31 8.36
100.00 100.00 −1.36 35.18 −18.44 44.85
τ2 (ns)
relative amplitude (a2)
20.6 14.78 54.14 19.33
101.36 43.49 3.98 47.16
τ3 (ns)
1.63 19.31 1.47
relative amplitude (a3)
av. lifetime (ns)
21.33 114.46 8.00
1.58 1.59 20.80 8.94 21.80 12.98
100 °C. Finally, it was washed with water thoroughly and was dried in vacuum at 60 °C. Estimation of Immobilized Functional Groups on GO and SG Samples. In order to quantitatively evaluate immobilized functional groups such as −COOH, −OH, and −SO3H on a graphene template, the Boehm titration method33 and elemental analysis were used. In the titration method, to quantify the amount of total acidic groups on GO and SG, first 10 mg of material was sonicated for 20 min in 10 mL of 0.1 (M) NaOH solution. After sonication, the resulting mixture was left to equilibrate for 1 h under nitrogen flow and stirring to remove dissolved carbon dioxide. Then the excess NaOH solution was collected by centrifuging in a Beckman Coulter (Allegra Model 64R) centrifuge. Ten milliliters of 0.1(M) HCl was added to the excess NaOH solution and was kept stirring under nitrogen gas flow. Finally, the excess HCl was determined by titration with 0.1(N) NaOH. In order to determine the amount of −COOH group on GO and the amount of (−COOH + −SO3H) groups on SG, the above procedure was followed except that 10 mL of 0.1(M) NaHCO3 solution was used in place of NaOH before sonication. To obtain the amount of −SO3H group present in SG, sulfur analysis was performed using a “vario Macro CHNS analyzer”, Elementar GmbH, Germany. Sample Preparation. DNA, RNA, BSA, glucose solutions (0.02% w/v), and 10−3 (M) EtBr solution were prepared by dissolving the required amount of above-mentioned biomolecules, sugar, and dye in triple distilled water (pH 6.9). Aqueous solutions of SG and GO (0.005% w/v) were made by sonicating in an ultrasonic bath (60 W, model AVIOC, Eyela) for half an hour. First, 0.2 mL of 10−3 (M) EtBr solution
purchased from Aldrich Chemical Co., USA. Sodium nitrate, potassium permanganate, 35% hydrochloric acid (G.R. grade), hydrazine hydrate solution (99%, synthetic grade), glucose (G) and EtBr (E) were purchased from E-Merck (Mumbai). All the reagents were used as received. Preparation of GO and SG. First, GO was prepared from graphite powder by oxidizing with a KMnO4/NaNO3 mixture in concentrated H2SO4 medium using Hummers method.32 To prepare SG,21 75 mg of GO was dispersed in 75 mL of water (0.1% w/v) and was sonicated for 1 h in an ultrasonic bath (60 W, frequency 28 kHz, model AVIOC, Eyela) when a clear brownish dispersion of GO was formed. The synthesis of SG from GO consisted of three steps: (1) prereduction of GO with sodium borohydride; (2) sulfonation with the aryl diazonium salt of sulfanilic acid; and (3) postreduction with hydrazine to remove epoxy (>O) functionality completely . The pH of GO dispersion was maintained between pH 9−10 with 5% (w/v) sodium carbonate solution. Then 15 mL of sodium borohydride solution (4%, w/v) in water was mixed with the GO dispersion and was kept under constant stirring at 80 °C for 1 h. The product was washed with water until its pH became 7, and it was redispersed in water for diazonium coupling. For this purpose, 46 mg of sulphanilic acid and 10 mg of sodium nitrite were dissolved in 10 mL water with the addition of 1.15 mL 12 N HCl under ice cold conditions. The mixture was then added to the dispersion at 0 °C and was kept for 2 h with stirring. It was centrifuged and washed repeatedly with water until the pH became 7. The product was then redispersed in 100 mL water and was reduced with 2 mL of hydrazine hydrate solution under refluxed condition for 24 h at 3182
dx.doi.org/10.1021/bm3009632 | Biomacromolecules 2012, 13, 3181−3188
Biomacromolecules
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Figure 1. TEM images of (a) GO and (b) SG.
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RESULTS AND DISCUSSION Characterization of Graphene Templates. The transmission electron microscopy (TEM) micrographs of GO and SG (Figure 1) indicate that graphene sheets are exfoliated in both the samples due to the presence of −COOH, epoxy, and −OH groups in the former and −SO3H groups along with −COOH and −OH groups in the latter (Scheme 1). The presence of the above functional groups in the respective graphene derivatives is evident from their Fourier transform infrared (FTIR) spectra.21 The FTIR spectrum of GO (SIFigure 1a) shows the characteristic peaks at 1060 cm−1 and 1235 cm−1 for the C−O and C−OH stretching, respectively. The 1390 cm −1 peak arises from −OH deformation, 1621 cm−1 for skeletal vibrations of unoxidized graphitic domains, and 1724 cm−1 peak is attributed to the C O stretching vibration of carboxylic acid and carbonyl moieties.21 The FTIR spectrum of SG (Supporting Information (SI) Figure 1b) exhibits peaks at 1175 cm−1 and 1126 cm−1 corresponding to the S−O bond vibrations, and the 1036 cm−1 peak is attributed to the S-phenyl vibration confirming the attachment of sulfonic acid group with the graphene rings. The peaks at 1009 cm−1 and 830 cm−1 characterize the C−H inplane bending and out-of-plane wagging vibrations of the pdisubstituted phenyl group, respectively.21 The amounts of −COOH and −OH groups on GO and the amount of −COOH, −OH, and −SO3H groups on SG are calculated from acid−base titration. The total amount of acidic sites (−COOH and −OH groups) in GO is determined to be 3.2 mmol/g, and the amount of −COOH group is measured to be 1.06 mmol/g. Consequently, the amount of −OH group is calculated to be (3.2−1.06) = 2.14 mmol/g. For SG, the amount of sulfur from elemental analysis is 1.745 wt %, and hence the amount of −SO3H group is calculated to be 0.5453 mmol/g. The total acid (−SO3H, −COOH, −OH group) content is measured to be 1.28 mmol/g, and the amounts of −COOH and −SO3H groups are found to be 0.8478 mmol/g. Hence the amount of −COOH group is calculated to be (0.8478−0.5453) = 0.3025 mmol/g, and the amount of −OH group is (1.28−0.8478) = 0.4322 mmol/g. Thus a decrease in the amount of −OH and −COOH groups in SG than that of GO is observed, and it may be attributed to the two-step reduction processes used to prepare SG from GO. To these
was added to 5 mL of 0.005% SG/GO solution and was allowed to mix for 30 min. To the SG-EtBr (SGE) or GO-EtBr (GOE) solution, 3.75 mL of the above biomolecule solutions were added and homogenized. For spectroscopic studies, these solutions were used. The sample designations used in the work are presented in Table 1. Characterization. Microscopy. The TEM micrographs of the samples were made by casting a drop of dispersed aqueous solutions of GO and SG on the carbon-coated copper grids and drying at 30 °C in air and finally in vacuum. The micrographs are taken from a high-resolution transmission electron microscope (JEOL, 2010EX) operated at an accelerated voltage of 200 KV fitted with a CCD Camera. Spectral Characterization. FT-IR spectra of the samples were obtained from thin films cast from aqueous solutions (2% w/v) by spreading over the silicon wafer using a Perkin-Elmer FT-IR instrument (spectrum100). The circular dichroism (CD) spectra of the aqueous solutions were performed using a spectro-polarimeter (JASCO, Model J-815) in a 1-cm quartz cuvette at 30 °C. The ultraviolet−visible (UV−vis) absorption spectra of the aqueous solution were taken from a UV−vis spectro photometer (Hewlett−Packard, Model 8453) in a quartz cell of thickness 1 cm in the wavelength range 190−1100 nm at 30 °C. Photoluminescence (PL) study of the solutions was performed using a quartz cell of 1 cm path length with a Horiba Jobin Yvon Fluoromax-3 instrument. The solutions were excited at 480 nm, and the emission scans were taken from 500 to 850 nm using a slit width of 2 nm with an increment of 1 nm wavelength having an integration time of 0.1 s. Fluorescence lifetime values were measured using a timecorrelated single photon counting fluoremeter (Fluorocube, Horiba Jobin Yvon). The system was excited with a 440 nm nano LED of Horiba Jobin Yvon having λmax at 439 nm with pulse duration of
