Letter pubs.acs.org/ac
Photoelectrochemical Aptasensing of Kanamycin Using Visible LightActivated Carbon Nitride and Graphene Oxide Nanocomposites Ruizhen Li,†,‡ Yong Liu,† Ling Cheng,† Changzhu Yang,‡ and Jingdong Zhang*,† †
School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China School of Environmental Science and Engineering, Huazhong University of Science and Technology, Wuhan 430074, China
‡
S Supporting Information *
ABSTRACT: Photoactive material and recognition element are two crucial factors which determine the sensitivity and selectivity of the photoelectrochemical (PEC) sensor. Herein we developed a novel PEC aptamer sensor for the specific detection of kanamycin using water-dispersible graphite-like carbon nitride (w-g-C3N4) as visible light-active material and aptamer as the biorecognition element. While a suitable amount of graphene oxide (GO) was doped in w-g-C3N4, the visible light photocurrent response was enhanced, which was beneficial to the construction of PEC sensor. On the other hand, the large specific surface area and π-conjugated structure of GO/w-g-C3N4 provided an excellent platform for immobilizing the kanamycinbinding DNA aptamer on the surface of the sensor via π−π stacking interaction. On such a sensor, the capture of kanamycin molecules by aptamer resulted in increased photocurrent. The PEC response of the sensor was found to be linearly proportional to the concentration of kanamycin in the range from 1 nM to 230 nM with a detection limit (3S/N) of 0.2 nM. Moreover, the proposed sensor displayed high selectivity, good reproducibility, and high stability, demonstrating the successful combination of GO/w-g-C3N4 with aptamer in fabricating high performance PEC sensors.
A
been made in the fabrication of various aptamer-based biosensors including colorimetric sensor,20 electrochemical sensor,21 cantilever sensor,22 PEC sensor,23 and fluorometric sensor.24 However, almost all these biosensors needed timeconsuming and costly surface confinement of aptamers via covalent bonding,25 avidin−biotin interaction,26 or DNA− compound interactions.27 Herein, we reported the first kanamycin PEC aptamer sensor using low-cost, unmodified, and label-free DNA aptamer in cooperation with graphene oxide (GO) and water-dispersible graphite-like C3N4 (w-g-C3N4) nanocomposites. Because of the π-conjugated structure and large specific surface area of GO and w-g-C3N4, the according kanamycin-binding DNA aptamer (5′-TGGGG GTTGA GGCTA AGCCG A-3′) could be facilely immobilized on the surface of GO/w-g-C3N4 nanocomposites through π−π stacking interaction between the nucleobases and the GO/w-g-C3N4 nanocomposites.28−31 Kanamycin, a kind of aminoglycoside antibiotic widely used to treat various infections, was selected as the detection target. Because kanamycin has some side effects such as loss of hearing, toxicity to kidneys, and excessive residual antibiotics in the environment can cause antibiotic resistance,32 it is of great
s a newly developed technique for the sensing platform, photoelectrochemical (PEC) sensors have attracted considerable attention owing to their high sensitivity, rapid measurement speed, and inexpensive instrumentation.1−3 Such PEC sensors combine the advantages of optical methods and electrochemical sensors, and thus show great promise for analytical applications.4 To fabricate PEC sensors, various semiconductors especially visible light-active materials such as CdS, BiOI, and CdSe have been used to convert light illumination to electronic response.5−8 Recently a new type of metal-free photoactive material, namely, graphite-like carbon nitride (g-C3N4) composed of earth abundant, inexpensive, and nontoxic elements has been intensively studied in photocatalysis.9−13 Owing to its narrow band gap (2.7 eV), g-C3N4 shows high photocatalytic activity under visible light illumination and exhibits unique electronic and optical properties.14 On the basis of C3N4 and AgX hybrid materials, a PEC sensor for the detection of Cu(II) ion has been designed.15 Aptamers, single-stranded oligonucleotides with specific sequences, have been used as a strong competitor to antibodies in analytical applications due to their unique advantages of lowcost and easy in vitro synthesis, less immunogenic response, high stability, and inherent binding affinity.16,17 Aptamers can be selected via the systematic evolution of ligands by exponential enrichment (SELEX) technique.18,19 Also they have the ability to recognize and bind to various specific targets such as small molecules, proteins, and cells. Great progress has © 2014 American Chemical Society
Received: July 15, 2014 Accepted: September 14, 2014 Published: September 15, 2014 9372
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Scheme 1. Schematic Illustration of PEC Aptasensing Principle Using the GO/w-g-C3N4/FTO Electrode
Figure 1. (A) SEM images of w-g-C3N4 (a) and GO/w-g-C3N4 (b) modified electrodes. (B) Transient photocurrent responses of w-g-C3N4 (a) and GO/w-g-C3N4 (b) modified electrodes recorded in 0.1 M Na2SO4 at a bias potential of +0.8 V (vs SCE).
uniform and compact film was obtained on the surface of FTO, indicating that GO improved the dispersion and film-forming ability of w-g-C3N4. This result is consistent with Fourier transform-infrared (FT-IR) spectra (see Figure S1 in the Supporting Information). As shown in Figure S1 in the Supporting Information, the representative absorption peaks for −COO− at 1386 and 1575 cm−1 were found in both the samples of w-g-C3N4 and GO/w-g-C3N4.35,36 The −COO− formed during the synthetic process of w-g-C3N4 is very beneficial to the water-dispersibility.37 Moreover, the broad peak at about 3160 cm−1 which might correspond to the primary, secondary amines, and their intermolecular hydrogen bonding as well as O−H stretching38 were also observed in both w-g-C3N4 and GO/w-g-C3N4. Nevertheless, the peak intensity of the latter is stronger than that of the former, due to the contribution of GO. In order to evaluate the PEC performance of the samples, the transient photocurrent responses of w-g-C3N4 and GO/w-gC3N4 modified electrodes were recorded in 0.1 M Na2SO4 at a bias potential of +0.8 V (vs SCE) under visible light irradiation (λ > 420 nm). As illustrated in Figure 1B, w-g-C3N4/FTO electrode exhibits sensitive photocurrent response to light illumination, indicating the photocatalytic activity of w-g-C3N4. It is believed that photogenerated holes may oxidize water at the surface of photoanode while photogenerated electrons may be transferred to the counter electrode to reduce water or dissolved O2 in electrolyte, leading to the generation of photocurrent.39,40 Similarly, the photocurrent is also generated
significance to develop highly sensitive and selective strategy to detect kanamycin. To fabricate the sensor, the GO/w-g-C3N4 nanocomposites were prepared to modify F-doped SnO2 (FTO) conducting glass substrate via the drop-coating method (for experimental details, see the Supporting Information). After aptamer was assembled on the surface of GO/w-g-C3N4/FTO electrode, the generated photocurrent was very low due to decreased interfacial electron transfer on the GO/w-g-C3N4/FTO electrode by the aptamer. While kanamycin molecules were present in the solution, the aptamer immobilized on GO/w-gC3N4/FTO electrode could specifically and sensitively capture kanamycin molecules on the surface of sensor. Then, an amplified photocurrent was obtained, as illustrated in Scheme 1. Since antibiotics are readily oxidized on various photocatalysts,33 the tentative mechanism for the generation of high photocurrent for kanamycin on the PEC sensor is that the captured kanamycin molecules on the sensor surface are quickly oxidized by the photogenerated holes and the recombination of photogenerated electrons and holes is inhibited.34 Thus, the concentration of kanamycin can be determined by recording the change of photocurrent response. The morphology of w-g-C3N4 and GO/w-g-C3N4 modified FTO electrodes were observed by scanning electron microscopy (SEM). It can be seen from Figure 1A that w-g-C3N4 with stacked lamellar texture on the surface of FTO aggregates into particles with a size range from tens of nanometers to hundreds of nanometers. After forming GO/w-g-C3N4 nanocomposites, a 9373
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on the GO/w-g-C3N4/FTO electrode; nevertheless, the photocurrent of GO/w-g-C3N4/FTO is nearly 2 times higher than that of w-g-C3N4/FTO. This result implies that GO may act as a light absorber and electron acceptor41,42 to enhance the light absorption and reduce the recombination of electrons and holes, which can be confirmed by UV−visible diffuse reflectance spectroscopic (DRS) measurements. As shown in Figure S2 in the Supporting Information, the introduction of GO leads to redshift of the absorption edge of GO/w-g-C3N4, meaning that the band gap of w-g-C3N4 is narrowed and the absorption of w-g-C3N4 in the visible light region is enhanced. The influence of the content of GO on the PEC performance of GO/w-g-C3N4 nanocomposites was investigated (Figure S3 in the Supporting Information). The transient photocurrent response of GO/w-g-C3N4/FTO electrode increased significantly with increasing the weight ratio of GO from 1% to 5%. Nevertheless, when the weight ratio of GO exceeded 5%, the photocurrent response decreased. The reason for this decrease may be due to the fact that excessive GO in the nanocomposites can act as a charge recombination center rather than electron acceptor.43 On the other hand, excessive GO can reduce the visible light absorption of w-g-C3N4. Therefore, the weight ratio of GO introduced in GO/w-g-C3N4 in the present work is 5% unless otherwise specified. The aptamer concentration showed an obvious effect on the PEC response to kanamycin. The PEC response of aptamer/ GO/w-g-C3N4/FTO electrode to kanamycin was evaluated by the difference of photocurrent before and after incubation with kanamycin (ΔPI). As shown in Figure S4 in the Supporting Information, the PEC response increased with increasing the aptamer concentration up to 1 μM. While the aptamer concentration was further increased to more than 1 μM, the PEC response declined. It is known that optimum aptamer concentration on the surface of electrode can capture kanamycin molecules sensitively; however, excessive aptamer hinders the kanamycin-capture process due to steric hindrance.44 Thus, 1 μM aptamer was used in the following experiments. The interfacial behavior of each sensor fabrication step was probed by recording the electrochemical impedance spectra (EIS) of [Fe(CN)6]3‑/4‑ on various modified electrodes (Figure S5 in the Supporting Information). The semicircle diameter of Nyquist plot representing the interfacial charge-transfer resistance (Rct) of FTO electrode is observed to be increased after coating with w-g-C3N4, owing to the low conductivity of w-g-C3N4. For GO/w-g-C3N4/FTO electrode, the Rct value is much smaller than that for w-g-C3N4/FTO, indicating that the introduction of GO facilitates the electron transfer due to the higher conductivity of GO. The dramatically increased Rct value of aptamer/GO/w-g-C3N4/FTO means that the negatively charged aptamer is successfully immobilized on the electrode surface which induces electrostatic repulsion between the electrode surface and negatively charged redox species of [Fe(CN)6]3‑/4‑.45 The developed PEC aptamer sensor was applied to the detection of kanamycin. Figure 2 shows the PEC responses of aptamer/GO/w-g-C3N4/FTO electrodes to kanamycin at different concentrations. The photocurrent response increased with increasing the concentration of kanamycin, showing that more captured kanamycin molecules participated in the PEC process. Moreover, the steady-state photocurrent in the presence of kanamycin might imply the binding/dissociation of the analyte to sustain the current. The PEC response of the
Figure 2. Photocurrent responses of aptamer/GO/w-g-C3N4/FTO electrode in 0.1 M Na2SO4 at a bias potential of +0.8 V (vs SCE) before and after incubation with different concentrations of kanamycin: (a) 0, (b) 1, (c) 30, (d) 60, (e) 100, (f) 150, (g) 200, (h) 230 nM. Insert: linear relationship between PEC response of sensor and kanamycin concentration. The error bars are the standard error of the mean.
sensor was found to be linearly proportional to the kanamycin concentration in the range of 1 nM to 230 nM with a correlation coefficient of 0.996. The detection limits (3S/N) was estimated to be 0.2 nM. Compared with the previously reported kanamycin sensors, this PEC aptamer sensor shows lower detection limit (see Table S1 in the Supporting Information). The selectivity of this PEC aptamer sensor was evaluated by analyzing the PEC response of aptamer/GO/w-g-C3N4/FTO toward various antibiotics such as chloramphenicol, erythromycin, doxycycline, chlorotetracycline, ciprofloxacin, and ofloxacin (Figure 3). It can be seen that all these antibiotics
Figure 3. PEC response of aptamer/GO/w-g-C3N4/FTO electrode in 0.1 M Na2SO4 at a bias potential of +0.8 V (vs SCE) before and after incubation with 200 nM kanamycin, chloramphenicol, erythromycin, doxycycline, chlorotetracycline, ciprofloxacin, and ofloxacin, respectively. The error bars are the standard error of the mean. 9374
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show negligible response on the PEC aptamer sensor as compared with kanamycin, revealing the high selectivity of this sensor for kanamycin detection, attributed to the specific interaction of aptamer and target kanamycin molecules. In addition, the reproducibility of PEC aptamer sensor was studied by checking the responses of five independently prepared aptamer/GO/w-g-C3N4/FTO electrodes toward 200 nM kanamycin. A relative standard deviation value of 2.7% was obtained, showing a good reproducibility. At the same time, no obvious change was observed for the response of an aptamer/ GO/w-g-C3N4/FTO electrode stored at 4 °C after 10 days, indicating the high stability of the sensor. In summary, this work developed a novel visible light PEC aptamer sensor based on metal-free GO/w-g-C3N4 nanocomposites for kanamycin detection. The doping of GO improved the dispersion and film-forming ability of w-g-C3N4 as well as the PEC performance of w-g-C3N4 modified electrode. Because of π−π stacking interaction, the aptamer without any modification can be effectively anchored on GO/ w-g-C3N4. The results demonstrate that the PEC aptamer sensor with high sensitivity and selectivity not only broadens the application of w-g-C3N4 to the field of aptamer sensor but also provides a novel strategy to the fabrication of various aptamer sensors for other target molecules.
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ASSOCIATED CONTENT
S Supporting Information *
Additional experimental details including materials and methods, synthesis procedures, fabrication procedure of the PEC sensor, and the analytical procedure. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +86-27-87792154. Fax: +86-27-87543632. 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 (Grant No. 61172005). We also thank the Analytical and Testing Center of HUST for the help in the characterization of synthesized materials.
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REFERENCES
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