Graphene Enhanced Electron Transfer at Aptamer Modified Electrode

Jul 26, 2012 - Ultrasensitive Label-Free Resonance Rayleigh Scattering Aptasensor for Hg ... Graphene-based label-free electrochemical aptasensor for ...
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Graphene Enhanced Electron Transfer at Aptamer Modified Electrode and Its Application in Biosensing Li Wang, Miao Xu, Lei Han, Ming Zhou, Chengzhou Zhu, and Shaojun Dong* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, China S Supporting Information *

ABSTRACT: Graphene (GN), a two-dimensional and one-atom thick carbon sheet, is showing exciting applications because of its unique morphology and properties. In this work, a new electrochemical biosensing platform by taking advantage of the ultrahigh electron transfer ability of GN and its unique GN/ssDNA interaction was reported. Adenosine triphosphate binding aptamer (ABA) immobilized on Au electrode could strongly adsorb GN due to the strong π−π interaction and resulted in a large decrease of the charge transfer resistance (Rct) of the electrode. However, the binding reaction between ABA and its target adenosine triphosphate (ATP) inhibited the adsorption of GN, and Rct could not be decreased. On the basis of this, we developed a new GN-based biosensing platform for the detection of small molecule ATP. The experimental results confirmed that the electrochemical aptasensor we developed possessed a good sensitivity and high selectivity for ATP. The detection range for ATP was from 15 × 10−9 to 4 × 10−3 M. The method here was label-free and sensitive and did not require sophisticated fabrication. Furthermore, we can generalize this strategy to detect Hg2+ using a thymine (T)-rich, mercury-specific oligonucleotide. Therefore, we expected that this method may offer a promising approach for designing high-performance electrochemical aptasensors for the sensitive and selective detection of a spectrum of targets.

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tions including gene diagnosis, protein analysis, and intracellular tracking.25−28 However, exploration of the unique GN/ ssDNA interaction in electrochemical biosensing is still at an early stage. Considering the high density of edge-plane-like defects present in GN sheets which allow high electron transfer29 and the unique GN/ssDNA interaction, it is highly possible to design an excellent electrochemical aptasensor using GN. In this work, a new electrochemical aptasensor by taking advantage of the ultrahigh electron transfer ability of GN and its unique GN/ssDNA interaction was reported. As shown in Figure 1, adenosine triphosphate binding aptamer (ABA) was first immobilized on Au electrode via the Au−S chemistry. The aptamer can strongly adsorb GN due to the strong π−π interaction between GN and ABA and resulted in a large decrease of the charge transfer resistance (Rct). However, the binding reaction between aptamer and its target adenosine triphosphate (ATP) would inhibit the GN’s adsorption on Au electrode; as a result, Rct could not be decreased. On the basis of this, we fabricated an electrochemical aptasensor for the sensitive detection of ATP using ATP binding aptamer modified Au (ABA/Au) electrode and GN, and it is fairly easy to generalize this strategy to detect Hg2+ using a thymine (T)-rich, mercury-specific oligonucleotide (MSO) probe.

ptamers are in vitro selected functional DNA or RNA structures from random-sequence nucleic acid libraries and can be chemically synthesized.1−3 Recently, aptamers have offered a new generation of biosensing platforms because of their high binding affinity and specificity to a broad range of targets from small inorganic and organic substances to proteins and cells.4−10 In the electrochemical field, it is very facile to convert aptamer−target recognition events into detectable electrochemical signals; aptamer modified films are widely used to fabricate electrochemical biosensors, and they play an important role in this field due to their significant advantages such as simplicity, low cost, and high stability.11−15 At the same time, advances in nanotechnology have generated nanomaterials with unique optical, electrical, mechanical, and catalytic properties, and the coupling of nanomaterials with aptamers will lead to novel molecular diagnostic tools with more promising performances such as improving the sensitivity of the sensors.16−18 Graphene (GN) is a single-atom-thick and two-dimensional carbon material, and it has attracted great attention due to its excellent properties such as high thermal conductivity, strong mechanical strength, excellent electronic transport properties, and high surface area.19−22 Particularly, GN could adsorb dye labeled ssDNA probe due to the strong π−π stacking between nucleotide and GN, and the fluorescence of the dye was quenched due to the high fluorescence quenching efficiency of GN, while the fluorescence was recovered when the probe bound the specific target to form a double helix.23,24 The unique GN/ssDNA interaction has shown fascinating applica© 2012 American Chemical Society

Received: February 21, 2012 Accepted: July 26, 2012 Published: July 26, 2012 7301

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electrochemical measurements (CV, EIS) were performed in the solution of 5 mM K4[Fe(CN)6]/K3[Fe(CN)6] (in 67 mM PBS buffer, pH = 7.0) at room temperature. Surface plasmon resonance (SPR) measurements were carried out for characterizing the fabrication of the sensing interface using an Autolab SPR instrument (Eco Chemie BV, The Netherlands). This instrument worked with a laser diode fixed at a wavelength of 670 nm, using a vibrating mirror to modulate the angle of incidence of the p-polarized light beam on the SPR gold substrate. Atomic force microscopy (AFM) was performed on a SPI3800N microscope instrument (Seiko Instruments, Inc., Japan) in tapping-mode in air at room temperature. X-ray photoelectron spectroscopy (XPS) measurement was performed on an ESCALAB-MKII spectrometer (UK) with Al KR radiation (1486.6 eV) as the X-ray source for excitation. Raman spectra were recorded at ambient temperature on a Renishaw Raman system model 1000 spectrometer with a 200 mW argon-ion laser at an excitation wavelength of 514.5 nm. XPS and Raman spectra of GN were showed in Figure S1 (Supporting Information). Electrode Cleaning, Pretreatment, and Fabrication of the Sensing Interface. Au electrode (3 mm in diameter) was polished with 1.0 μm and 0.3 μm α-Al2O3 and then washed ultrasonically with pure water for three times successively, followed by electrochemically cleaning in 0.1 M H2SO4 by potential scanning between −0.2 and 1.6 V until a reproducible cyclic voltammogram was obtained. Then, it was rinsed with a copious amount of pure water and finally blown dry with nitrogen before modification. Au electrode was first immersed into the 10 μM ABA solution in order to assemble the monolayer of aptamer through the Au−S bond between DNA and gold film. The assembly was kept for 18 h at room temperature, followed by rinsing with Tris−HCl buffer and pure water for several times. Then, the electrode was dried in a nitrogen stream, after which the interface was covered with 5 μL of 50 μM MCE and kept at room temperature for 1 h, followed by rinsing with Tris−HCl buffer and pure water. The sensing interface (ABA/Au) electrode was obtained after dried with nitrogen. Electrochemical Detection of ATP. We have optimized the concentration of ABA employed during sensor fabrication. As shown in Figure S2 (Supporting Information), maximum detection performance was observed at the ABA/Au film prepared by 10 μM ABA. Therefore, 10 μM ABA was used to fabricate the sensing interface. As shown in Figure 1, the new as-prepared sensing interface was immersed in different concentrations of ATP and reacted for 1 h, followed by washing with the buffer and water. Then, the electrode was immersed in 0.3 mg/mL GN solution for 1 h. Control experiments were also carried out; the detection processes were the same as those used previously except using TTP, GTP, and CTP instead of ATP. Electrochemical Detection of Hg2+ Based on MSO and GN. We have optimized the concentration of MSO to fabricate the Hg2+ sensor. As shown in Figure S3 (Supporting Information), maximum detection performance was observed at MSO/Au film prepared by 10 μM MSO. Therefore, 10 μM MSO was used to fabricate the sensing interface. The pretreatment of Au electrode (3 mm in diameter) was the same as that previously described. Cleaned Au electrode was first immersed into 10 μM MSO in order to assemble the monolayer of MSO through the Au−S bond between DNA and gold film. The assembly was kept for 18 h at room temperature,

Figure 1. Electrochemical sensing strategy for the detection of ATP: (A) GN was adsorbed on ABA/Au electrode due to the strong π−π interaction, and a large Rct decrease was obtained. (B) In the presence of ATP, ATP binding aptamer (ABA) bound ATP and formed duplex which could not adsorb GN and resulted in a small Rct decrease. Electrochemical sensing strategy for the detection of Hg2+ was the same way as that of ATP except using MSO/Au electrode instead of ABA/Au electrode. For the detailed information, please see the Experimental Section.

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EXPERIMENTAL SECTION Reagents. Well dispersed GN was prepared by the chemical reduction of graphene oxide with hydrazine according to the literature.30 Procedures were as follows: 144 μL of ammonia solution (25 wt %) and 8.7 μL of hydrazine hydrate (50 wt %) were added to 0.3 mg/mL GO (20 mL). After being vigorously stirred for a few minutes, the vial was put in a water bath (95 °C); after being continually stirred at 95 °C for 1 h, dispersed GN was obtained. ATP binding aptamer sequences (ABA) (5′ SH-ACCTGGGGGAGTATTGCGGAGGAAGGT-3′), and thymine (T)-rich, mercury-specific oligonucleotide (MSO) sequences (5′SH-ATTCTTTCTTCCCCCCGGTTGTTTGTTT-3′) were synthesized by Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). DNA oligonucleotide stock solutions were prepared with Tris−HCl buffer (25 mM Tris−HCl, 150 mM NaCl, pH = 8.0) and kept frozen. ATP, cytidine triphosphate (CTP), thymidine triphosphate (TTP), and guanosine (GTP) were prepared in the Tris−HCl buffer (25 mM Tris−HCl, 150 mM NaCl, pH = 8.0). The metal salts (Hg(Ac)2, Pb(Ac)2, CdCl2, FeCl3, FeSO4, Mg(Ac)2, Ca(Ac)2, Al(Ac)3, and Cu(Ac)2) used were purchased from Sinopharm Group Chemical Reagent Co., Ltd. (Shanghai, China). 2Mercaptoethanol (MCE) and 2,6-pyridinedicarboxylic acid (PDCA) was purchased from Aldrich. All other chemicals not mentioned here were of analytical reagent grade and used as received. Double distilled water was used throughout. Instrumentation. Cyclic voltammetry (CV) experiments were performed with a model CH Instrument 832C electrochemical workstation (Shanghai Chenhua Equipments, China). Electrochemical impedance spectroscopy (EIS) experiments were performed on an Autolab PGSTAT30 (Utrecht, The Netherlands, controlled by GPES4 and Fra software). A conventional three-electrode system, with a Au electrode (3 mm in diameter) as working electrode, a Ag−AgCl reference electrode, and a platinum wire as counter electrode, was used. The cell was housed in a homemade Faraday cage to reduce stray electrical noise. All the measurements with the Autolab were carried out at room temperature. EIS was performed under an oscillation potential of 5 mV over the frequency range of 10 kHz to 0.01 Hz. The amplitude of the alternate voltage is 5 mV. We carried out simulations using the equivalent circuit on the commercial Autolab FRA software (supplied by Eco Chemie, The Netherlands) to obtain the Rct values. All the 7302

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Figure 2. (A) AFM image of GN on mica substrates. (B) AFM images of ABA/Au electrode and (C) GN adsorbed on ABA/Au electrode. (D) ABA/Au electrode first reacted with 2 mM ATP, and then incubated with GN.

due to the π-stacking interaction between aptamers and GN, and we could distinguish the edges of individual sheets, including kinked areas. However, GN was almost not adsorbed on the ABA/Au electrode which first reacted with 2 mM ATP to form duplex (as shown in Figure 2D). SPR spectroscopy is one of the most exciting surface-sensitive techniques for detecting analytes adsorbed on a metal surface33 and can be used in situ to characterize the process of the adsorption of GN on ABA/Au electrode with high sensitivity. Therefore, these processes were then characterized by SPR technique. The angle-resolved SPR curves (Figure S4, Supporting Information) and angle-time SPR response (Figure 3) showed that, in the absence of ATP, ABA/Au electrode could adsorb a large amount of GN due to the strong noncovalent binding of GN with nucleobases of ssDNA and resulted in a large SPR angle shift (about 280 mDeg). However, the ABA/Au electrode was

followed by rinsing with Tris−HCl buffer and pure water for several times. Then, the electrode was dried in a nitrogen stream, after which the interface was covered with 5 μL of 50 μM MCE and kept at room temperature for 1 h, followed by rinsing with Tris−HCl buffer and pure water. The sensing interface (MSO/Au) electrode was obtained after being dried with nitrogen. For the detection of Hg2+, the MSO/Au electrode was immersed in different concentrations of Hg2+ and reacted for 1 h, followed by washing. Last, the electrode was immersed in 0.3 mg/mL GN solution for 1 h. Control experiments were also carried out; the detection processes were the same as those used previously except using other metal ions instead of Hg2+.

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RESULTS AND DISCUSSION Preparation of GN. GN was prepared by the chemical reduction of graphene oxide with hydrazine.30 As shown in Figure 2A, the lateral size of GN was 200 nm to 1.2 μm. The thickness of GN was ∼0.96 nm. This value matched well with the reported apparent thickness of GN, suggesting the single sheet nature of GN obtained in this work.31 The final oxygen and nitrogen contents of GN were calculated from the XPS spectrum (Figure S1A, Supporting Information) to be 14.6% and 3.5%, respectively. The Raman spectrum (Figure S1B, Supporting Information) of GN exhibited the presence of D and G bands, and the ratio of the D and G lines (ID/IG) of GN was ∼1.16, indicating there were significant edge-plane-like defective sites existing on the surface of GN.32 Study of the Adsorption of GN on ABA/Au Electrode by Atomic Force Microscopy (AFM) and Surface Plasmon Resonance (SPR) Spectroscopy. AFM was performed first to characterize the adsorption of GN on ABA/Au electrode. Figure 2B exhibited an AFM image of the ABA/Au electrode surface. After adsorbing GN on the ABA/Au electrode surface, the Au electrode surface showed a significant change of the surface morphology (Figure 2C). The image clearly indicated GN was adsorbed on the ABA/Au electrode

Figure 3. SPR angle−time curves for the ABA/Au sensing interface with 0.3 mg/mL GN for 1 h (a). ABA/Au first reacted with 2 mM ATP and then incubated with 0.3 mg/mL GN for 1 h (b). 7303

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observed because ABA/Au first reacted with ATP to form stable DNA duplexes which resulted in very little adsorption of GN. CV responses in different conditions were also investigated; as shown in Figure 4B, the ABA/Au electrode showed remarkable blocking of the redox probes from effective charge transfer at the interface (curve a of Figure 4B). In the absence of ATP, the CV response was much enhanced after the adsorption of GN on the ABA/Au electrode (curve b of Figure 4B). In the presence of 2 mM ATP, the CV response could almost not be enhanced due to almost no GN being adsorbed on the ABA/Au electrode (curve c of Figure 4B). It was worthy to note that the formed GN/ABA/Au electrode in the absence of ATP was found to be very stable, which was illustrated by the almost unchanged redox peak currents in the presence of [Fe(CN)6]3−/4−. The redox peak currents were essentially unchanged after continuously cycling the electrodes for 100 cycles (not shown). This substantially suggests that the π-stacking interaction between GN and ABA essentially makes it possible to stably adsorb GN onto the Au electrode in a controllable manner. In addition to their potential uses in other research fields, such as GN-based nanodevices, herein, the controllable adsorption of GN onto the ABA/Au electrode is used for the sensitive detection of ATP. In the absence of ATP, GN was adsorbed on ABA/Au electrode and small Rct value was obtained. When ATP was present, less GN was adsorbed and the Rct value was large. Figure 5A shows the EIS response of different concentrations of ATP; when the concentration of ATP was increased, the Rct gradually increased. We were able to reliably detect 15 nM ATP, indicating a detection limit of 15 nM. There was a linearity relationship between Rct and the logarithm of the concentrations of ATP, and ATP could be quantified over a concentration range from 15 nM to 4 mM (R2 = 0.991) (as shown in Figure 5B), which was more sensitive than the most electrochemical aptasensors that existed for ATP detection.34,35 Control experiments were carried out to detect the selectivity of the aptasensor. As shown in Figure 5C, there was no remarkable Rct shift for CTP, TTP, and GTP, all of which small molecules belong to the nucleoside family. Generality of the Platform for the Detection of Hg2+. Hg2+ is a highly toxic heavy metal ion and is known as a hazardous pollutant with recognized accumulative character in the environment.36 Thus, as a further step, we attempted to fabricate another sensing strategy for Hg2+ using a thymine (T)-rich, mercury-specific oligonucleotide (MSO) probe to testify the generality of the integrated sensing platform. MSO probe could strongly and selectively bind Hg2+ due to the T− Hg2+−T coordination, and successful sensors for Hg2+ based on the unique T−Hg2+−T coordination have been developed.37 As shown in Figure 1, instead of the ABA, the MSO probe was immobilized on Au electrode through the formation of Au−S bond between MSO and Au electrode to capture Hg2+ in aqueous solution. After 2-mercaptoethanol (MCE) assembly, the sensing interface of MSO/Au electrode was obtained and could be used to detect Hg2+. MSO/Au electrode was immersed in different concentrations of Hg2+ and reacted for 1 h; then, the electrode was immersed in 0.3 mg/mL GN solution for 1 h, and then EIS spectra were recorded. In the absence of Hg2+, MSO/Au electrode could adsorb a large amount of GN due to the strong noncovalent binding of GN with nucleobases of ssDNA and resulted in the small Rct, as shown in Figure 6A. However, when MSO/Au electrode was

first reacted with 2 mM ATP and then was incubated with GN, and small SPR angle shift (about 20 mDeg) was obtained because ABA was reacted with ATP to form DNA duplexes, which could almost not adsorb GN. AFM and SPR studies both indicated that GN can be adsorbed on the ABA/Au electrode, while almost not on the ABA/Au electrode which had reacted with ATP. Effect of GN on the Electron Transfer of ABA/Au Electrode and the Sensitive Electrochemical Detection of ATP. To understand the enhancement of GN on the electron transfer of the ABA/Au electrode, electrochemical behaviors of ABA/Au electrode in different conditions were then studied by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) in the presence of [Fe(CN)6]3−/4−. The circuit (Figure S5, Supporting Information) was used to fit the EIS data. As shown in Figure 4A, ABA was

Figure 4. EIS (A) and CV (B) response during the different steps (ABA/Au film (a), ABA/Au film incubation with the 0.3 mg/mL GN for 1 h (b), ABA/Au first reacted with 2 mM ATP and then incubated with 0.3 mg/mL GN for 1 h (c)).

first immobilized on Au electrode with its ss-DNA structure and showed a large charge transfer resistance (Rct) (curve a of Figure 4A). The negative charges of the assembled TBA repelled the negatively charged redox probes, thus leading to the large Rct value. The steric hindrance introduced with the formation of the TBA on the electrode surface also contributed to the large Rct. In the absence of ATP, a large amount of GN was adsorbed due to the strong noncovalent binding of GN with nucleobases. The adsorbed GN could highly enhance the electron transfer; therefore, a large Rct decrease was observed. However, when ABA/Au was first reacted with 2 mM ATP and then treated with 0.3 mg/mL GN, a small Rct decrease was 7304

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Figure 5. (A) EIS response for the detection of different concentrations of ATP. (a) 4 mM; (b) 2 mM; (c) 500 μM; (d) 40 μM; (e) 10 μM; (f) 1 μM; (g) 0.1 μM. ABA/Au electrode was first reacted with ATP for 1 h and then incubated with 0.3 mg/mL GN for 1 h; finally, EIS spectra were recorded. (B) The relationship between Rct and the concentration of ATP. The error bars represent the standard deviation of three measurements. (C) Selectivity for the detection of ATP. ATP and other tested molecules were all used at 30 μM. The error bars represent the standard deviation of three measurements.

Figure 6. (A) EIS response during the different steps (MSO/Au film (a), MSO/Au film incubated with GN (b), MSO/Au first reacted with 800 nM Hg2+ and then incubated with GN (c)). (B) The relationship between the EIS response and the concentration of Hg2+. The error bars represent the standard deviation of three measurements. (C) Selectivity for the detection of Hg2+. Hg2+ and other tested molecules were all used at 100 nM. The error bars represent the standard deviation of three measurements.

0.995) (shown in Figure 6B). To test the specificity of sensing Hg2+, the interferences of other metal ions for detecting Hg2+ were performed. The severe Pb2+ and Cd2+ interference with Hg2+ detection had been reported previously, whereas it could be overcome by addition of 2,6-pyridinedicarboxylic acid (PDCA).38 Figure 6C showed that, in the presence of PDCA, no interference from other metal ions was found, and PDCA had no obvious influence on the target ion Hg2+, which was consistent with the previous report.38 The result for detection

reacted with 800 nM Hg2+ and then incubated with GN, a large Rct was obtained because MSO first reacted with Hg2+ to form stable DNA duplexes, which could almost not adsorb GN. We were able to reliably detect 0.5 nM Hg2+, indicating a detection limit of 0.5 nM for analyzing aqueous Hg2+. By analyzing the Rct value with the concentrations of Hg2+, we obtained a linear relationship between the Rct value and the logarithm of the concentrations of Hg2+ over a range of 0.5−500 nM (R2 = 7305

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of Hg2+ was comparable favorably to that of electrochemical and optical sensors.39,40 The U.S. Environmental Protection Agency permits the maximum level of mercury in drinking water to be 10 nM. In blood, the mercury concentrations were 1.25−67 nM. In our work, we obtained a linear relationship between the Rct value and the logarithm of the concentration of Hg2+ over a range of 0.5−500 nM (R2 = 0.995); therefore, our sensor might show the potential in real clinical detection of Hg2+.

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CONCLUSIONS In conclusion, we have developed an efficient, label-free electrochemical aptasensor by taking advantage of the ultrahigh electron transfer ability of GN, the unique GN/aptamer interaction, and the specific aptamer−target recognition. GN could be preferentially adsorbed on the aptamer modified Au electrode and highly enhanced the electron transfer; while for the aptamer modified Au electrode which had reacted with its target, GN could not. On the basis of this, we have successfully detected ATP down to a detection limit of 15 nM with a detection range of 15 × 10−9 to 4 × 10−3 M. Meanwhile, a GNbased electrochemical sensor for the sensitive detection of Hg2+ was also developed on the basis of the same strategy using the Hg2+-specific oligonucleotide, and the detection limit of Hg2+ was 0.5 nM. The GN-based electrochemical biosensing platform has obvious advantages over the conventional method. First, by combing of the high electron transfer of GN and the unique GN/aptamer interaction, label-free detection is realized which makes the sensing process quite simple and convenient. Second, the GN-based method is cost-effective. GN can be prepared from graphite available at very low cost. More significantly, it would be easy to generalize this strategy to detect a spectrum of targets using GN and different functional DNA or RNA structures.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.”

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 431 85262101. Fax: +86 431 85689711. E-mail: [email protected]. 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 (200935003, 21075116) and 973 projects (2011CB911002, 2010CB933603).

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REFERENCES

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