A label-free DNA reduced graphene oxide-based fluorescent sensor for highly sensitive and selective detection of hemin Yan Shi, Wei Tao Huang, Hong Qun Luo and Nian Bing Li* Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People s Republic of China 1. Synthesis of Reduced Graphene Oxide Graphene oxides (GO) was synthesized by oxidation of natural graphite powder (325 mesh, spectral pure, Sinopharm Chemical Reagent Co., Ltd., China) according to the Hummers method. 1 Reduced graphene oxide (rgo) was prepared by the chemical reduction of GO with hydrazine. 2,3 The concentration of rgo was determined by drying rgo dispersion over phosphorus pentoxide in a vacuum desiccator for a week and was found to be 0.14 mg L 1. 2. Reagents Acridine orange (AO, Fluka) and Dimethyl sulfoxide (DMSO, HPLC grade) were purchased from Shanghai Chemical Co., China. Tris-HCl buffer A solution (ph 7.4) contains 20 mm Tris. Tris-HCl buffer B solution (ph 7.4) contains 20 mm Tris, 100 mm NaCl, 5 mm KCl, and 5 mm MgCl 2. The stock solution of hemin (5 mm) was prepared in DMSO and stored in the dark at -20 C. The required concentration of hemin was diluted with the 20 mm Tris-HCl buffer B. Oligonucleotide sequence (PS2.M, hemin aptamer, 5 -GTGGGTAGGGCGGGT- TGG-3 ) and another oligonucleotide sequence (ADNA, 5 -CACACACACACACAC- ACACAC-3 ) for contrast were synthesized by Shanghai Sangon Biotechnology Co. S1
Ltd. (Shanghai, China). The PS2.M (100 µm) solution was prepared in the Tris-HCl buffer B solution and heated at 88 C for 10 min to dissociate any intermolecular interaction, and then gradually cooled to room temperature. Subsequently, this solution was put in refrigerator at 4 C for 24 h and allowed to form G-quadruplex structures. The PS2.M concentration was accurately quantified using UV-vis absorption spectroscopy with the following extinction coefficients (ε 260 nm, expressed in units of M 1 cm 1 ) for each nucleotide: A = 15400, G = 11500, C = 7400, T = 8700. The control experiments were conducted by preparing the PS2.M (100 µm) solution in the Tris-HCl buffer A solution. ADNA was treated with a similar proceduce as PS2.M to prepare a 100 µm solution. All other chemicals not mentioned here were of analytical reagent grade and were used as received. Double distilled water was used throughout. 3. Construction of Fluorescent Sensor The AO-rGO complex was prepared by titrating with rgo to AO solution till the fluorescence of AO was just quenched completely. The concentration of the AO-rGO complex was calculated by the concentration of AO. After that, a suitable amount of PS2.M solution was added to the AO-rGO complex solution and kept for 20 min. Successively, the mixture was incubated with hemin for 30 min and then used for the determination of fluorescence. 4. Characterizations Fluorescence emission spectra were recorded on a Hitachi F-4500 spectrofluorophotometer (Hitachi Ltd., Tokyo, Japan). The XPS measurements were carried out with an ESCALAB 250 high performance photoelectron spectrometer (Thermo, USA) with an Al Kα (1486.6 ev) radiation. All obtained spectra were calibrated to a C 1s peak at 284.6 ev. Fig. S1 shows the C1s high-resolution XPS spectra of GO and rgo and there is a significant decrease of the signals of oxygen-containing functional groups, indicating an efficient reduction of GO to rgo by hydrazine. But rgo is not as perfect as pristine graphene. S2
It still has some residual oxygen-containing functional groups, such as epoxy, hydroxyl, and carboxylic groups, which make rgo in aqueous dispersion being negatively charged, so rgo is easier to combine with other molecules than pristine graphene. Fig. S1 High-resolution C1s XPS spectra of (a) unreduced GO and (b) rgo. Atomic force microscopic (AFM) images were taken using a NanoScope Quadrex Veeco Multimode atomic force microscopy (Veeco Instruments, USA) in tapping mode with a TESP7 Veeco tapping tip. As shown in Fig. S2, the average thickness of a single-layer rgo and AO-rGO complex were measured to be approximately 1.0 nm and 1.7 nm, respectively, which are consistent with the results reported previously. 4 The fact that the single-layer of AO-rGO complex is thicker than that of rgo also indicates that AO has strong electrostatic and π-π stacking interactions with rgo. a b Fig. S2 Tapping mode AFM images of (a) rgo and (b) AO-rGO complex on mica. S3
5. Fitting of the fluorescence titration data of AO by G-quadruplex. 5.1 The binding-stoichiometry ratio We investigated the binding of AO and PS2.M by titrating increasing amount of AO to PS2.M. In Fig. S3, by subtracting the titration curve of AO from that of AO-PS2.M, curve c that corresponds to the contribution of PS2.M on the increased fluorescence of AO is obtained. When the concentration of PS2.M is fixed at 2.5 μm, the concentration of AO at the intersection point on the titration curve is 2.5 μm also. So, we deduce that AO molecule may be bound at 1 : 1 molar ratio to PS2.M. Fig. S3 Fluorescence titrations of AO to (a) 2.5 μm PS2.M in 20 mm Tris-HCl buffer B solution and (b) 20 mm Tris-HCl buffer B solution. (c) is obtained by subtracting (b) from (a). 5.2 Determination of the binding affinity (K d ) of G-quadruplex with AO According to previous methods 5,6 and based on the binding-stoichiometry ratio of 1 : 1, Equation (1) can be obtained: F Fmin F Fmin [ PS 2. M ] T = Kd + [ AO] T (1) F F F F max max min S4
Where, F corresponds to the fluorescence of AO. Subscript min and max denotes the value when all of AO is in free form and in complex form, respectively. [AO] T and [PS2.M] T correspond to the total concentrations of added AO and PS2.M, respectively. K d refers to the dissociation constant. Curve b in Fig. 3 can be well fitted by Equation (1) using the fitting ability of Origin software to afford a submicromolar affinity (K d 85 nm) of AO with G-quadruplex. 6. The possible binding models of PS2.M and hemin Fig. S4 shows the possible binding models proposed by Dipankar Sen and co-workers. 7,8 It can be seen that two similar but topologically distinct folded models were postulated, both of which feature two guanine quartets and slightly different loop topologies. Fig. S4 Two possible models for the potassium-folded PS2.M proposed by Dipankar Sen and co-workers. The postulated binding sites for hemin are shown by the arrows. The hydrogen bonds in the two guanine quartets (G4, G8, G14, and G17, and G3, G9, G13, and G18) are shown by the dashed lines. 7. The effect of hemin on the fluorescence of AO The effect of hemin on the fluorescence of AO has been investigated and the results are shown in Fig. S5. There is no spectroscopic overlap between hemin and AO, and the addition of increasing amounts of hemin to AO shows little effect on the fluorescence intensity of AO. S5
Fig. S5 (a) The fluorescence spectra of hemin and AO, [AO] = [hemin] = 2.5 μm; (b) The fluorescent titration curve by adding increasing amount of hemin to 2.5 um AO. 8. The role of rgo The addition of hemin to AO-PS2.M sensor will also lead to the diminution of the fluorescent signal. The role of rgo in AO-PS2.M/rGO sensor is that it can amplify the fluorescence signal to some degree. In Fig. 3b, the fluorescence intensities of AO are increased by G-quadruplex about six times the intensity of free AO. So, the addition of hemin to AO-PS2.M sensor will doom to bring down the intensity at most six-fold. But with the aid of rgo which has an excellent quenching ability, the addition of hemin to AO-PS2.M/rGO sensor brings down the fluorescence intensity about eighteen-fold (as shown in Fig. 4). So, we think that the role of rgo is to amplify the fluorescence signal and it is important for the sensor. Supplementary References 1 W. S. Hummers Jr and R. E. Offeman, J. Am. Chem. Soc., 1958, 80, 1339 1339. 2 D. Li, M. B.Muller, S. Gilje, R. B. Kaner and G. G. Wallace, Nat. Nanotechnol., 2008, 3, 101 105. 3 Y. Xu, L. Zhao, H. Bai, W. Hong, C. Li and G. Shi, J. Am. Chem. Soc., 2009, 131, 13490 13497. 4 S. Park and R. S. Ruo, Nat. Nanotechnol., 2009, 4, 217 224. S6
5 T. Li, E. Wang and S. Dong, Chem. Eur. J., 2009, 15, 2059 2063. 6 D. M. Kong, Y. E. Ma, J. Wu and H. X. Shen, Chem. Eur. J., 2009, 15, 901 909. 7 P. Travascio, D. Sen and A. J. Bennet, Can. J. Chem., 2006, 84, 613 619. 8 H. W. Lee, D. J. F. Chinnapen and D. Sen, Pure Appl. Chem., 2004, 76, 1537 1545. S7