Aptamer-Based Cantilever Array Sensors for Oxytetracycline Detection

Letter pubs.acs.org/ac

Aptamer-Based Cantilever Array Sensors for Oxytetracycline Detection Hui Hou,†,□,§ Xiaojing Bai,†,§ Chunyan Xing,†,□ Ningyu Gu,‡ Bailin Zhang,*,† and Jilin Tang*,† †

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China ‡ Department of Chemistry, Nanchang University, Nanchang 330031, P. R. China □ University of Chinese Academy of Sciences, Beijing 100049, People's Republic of China ABSTRACT: We present a new method for specific detection of oxytetracycline (OTC) at nanomolar concentrations based on a microfabricated cantilever array. The sensing cantilevers in the array are functionalized with self-assembled monolayers (SAMs) of OTC-specific aptamer, which acts as a recognition molecule for OTC. While the reference cantilevers in the array are functionalized with 6-mercapto-1-hexanol SAMs to eliminate the influence of environmental disturbances. The cantilever sensor shows a good linear relationship between the deflection amplitude and the OTC concentration in the range of 1.0−100 nM. The detection limit of the cantilever array sensor is as low as 0.2 nM, which is comparable to some traditional methods. Other antibiotics such as doxycycline and tetracycline do not cause significant deflection of the cantilevers. It is demonstrated that the cantilever array sensors can be used as a powerful tool to detect drugs with high sensitivity and selectivity.

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extensively used as a veterinary antibiotic and an animal growth promoter. For this reason, they always accumulate in our food products, such as meat,3,4 milk,5 and eggs,6 and cause serious side effects to human health. Among the above TCs, OTC is the most frequently used antibiotic because of its effective antimicrobial properties. It is urgently needed to provide a simple and effective approach to detect the OTC in contaminated food products and pharmaceutical preparations, but due to the high structural similarity of TCs derivatives, the existing conventional methods such as dipstick colorimetric method,7 amperometric detection method using multiwall carbon nanotubes,8 and antibody-based colorimetric methods9 are usually lacking specificity and sensitivity.7−10 A more simple and effective approach to detect OTC is important to aid in its utilizations and the deeper understanding of action mechanisms. Recently, aptamer-based biosensor has attracted a growing interest in developing sensitive methods for the detection of OTC.10,11 Aptamers are single-stranded oligonucleotides which can recognize and bind to their respective targets through formation of unique tertiary structures.12 As nucleic acid backbones of aptamers are much flexible and smaller than their protein counterparts, the binding can result in large structural changes of aptamers.13 Moreover, aptamers also have many other advantages including high binding affinity, simplicity of synthesis, ease of labeling, and excellent stability.14 With these

etracyclines (TCs) are a group of broad-spectrum polyketide antibiotics, which have a naphthalene ring and three acidic groups: tricarbonyl, phenolic β-diketone, and dimethylamine (Figure 1).1,2 The TCs especially oxytetracycline (OTC), doxycycline (DOX), and tetracycline (TET) are

Figure 1. Structure of tetracyclines (TCs). The X1 and X2 are variable functional groups involved in their structural analogs; R represents the alkyl group. © 2013 American Chemical Society

Received: December 24, 2012 Accepted: January 28, 2013 Published: January 28, 2013 2010

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characteristics, aptamers have been widely employed as recognition elements for biosensing applications, such as colorimetry,10 surface plasmon resonance,15 and quartz crystal microbalance (QCM).16 In 2007, Javed et al. selected a DNA aptamer which can be used to specifically detect OTC.17 The aptamer was used for OTC detection through electrochemical and colorimetric biosensor.10,11 In this study, we demonstrate another method which can specifically and sensitively detect OTC based on the cantilever sensor system. The cantilever sensors have attracted significant attention in recent decades as a label-free, real-time, and highly sensitive approach to detect biomolecules.18 Compared with more conventional sensors, the cantilever sensor offers improved dynamic response, greatly reduced size, increased reliability, and high precision. Based on the cantilever sensor, molecular recognition can be directly and specifically transduced into nanomechanical responses. It can be achieved by immobilizing receptor molecules on one side of the cantilevers. When target molecules bind to the receptors, the biochemical interaction generates a difference in surface stress between the functionalized and the nonfunctionalized sides of the cantilever, which bends the cantilever. Cantilever deflections were measured by monitoring the position of a laser beam reflected from the cantilever onto a four-quadrant position sensitive detector (PSD).Thus, receptor−ligand interaction could be detected.19 It has been demonstrated that the cantilever sensor has been wildly applied for the labelfree detection approach, such as heavy metal ion detection,20 DNA hybridization,21 detection of single-nucleotide mismatches in DNA,22 protein−ligand binding,23 and protein− DNA interaction.12 The basic principle of OTC detection using the cantilever array sensor is illustrated in Figure 2. Briefly, the sensing

Letter

EXPERIMENTAL SECTION

Reagents. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was bought from Alfa Aesar (USA). MCH was purchased from Sigma-Aldrich (St. Louis, MO., USA). OTC and TET were purchased from Aladdin Chemistry Co. Ltd. (Shanghai, China). DOX and OTC aptamer with sequences of 5′-GGA ATT CGC TAG CAC GTT GAC GCT GGT GCC CGG TTG TGG TGC GAG TGT TGT GTG GAT CCG AGC TCC ACG TG-(CH2)6-SH-3′ were bought from Sangon Biotech Co., Ltd. (Shanghai, China). All other chemicals were of analytical reagent grade and used as received without further purification. Pure water (18.2 MΩ cm) used in the experiment was obtained with a Milli-Q system (Millipore). The running buffer (pH 7.6) contained 20 mM Tris-HCl, 100 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 1 mM CaCl2. The thiol-modified aptamer was dissolved in Tris buffer (20 mM Tris-HCl, 100 mM NaCl, 1 mM TCEP, pH 7.4) and stored at −20 °C until use. The TCEP was used to allow a free-sulfhydryl group of aptamer to remain in reduced form. Cantilever Array Functionalization. The cantilever arrays used here consist of eight identical silicon cantilevers (500 μm × 100 μm × 1 μm) with a 20 nm layer of gold coated on the top side (Concentris GmbH, Switzerland). Before being used, the cantilever array was cleaned with ethanol and pure water for 3 times, respectively, and then treated in a UV−ozone cleaning cycle for 20 min. The aptamer immobilization was performed by inserting four of the cantilevers into an array of microcapillaries that filled with 1 μM aptamer for 3 h. Then, the cantilever array was incubated with 2 mM MCH ethanol solution for 1 h to remove nonspecific DNA adsorption on the four sensing cantilevers and form MCH SAMs on the four reference cantilevers. Finally, the cantilever array was washed consecutively with ethanol and pure water for three times, dried under a stream of nitrogen gas, and stored dry. Measurements and Apparatus. The deflection measurements were carried out on the commercial Cantisens sensor platform (Concentris GmbH, Switzerland) equipped with a measurement cell of 5 μL, an automated liquid handling system, and an integrated temperature control with sample preheating stage. Temperature of the experiments was controlled at 25.0 ± 0.01 °C. The cantilever array was initially placed into the measurement cell and equilibrated in the running buffer at a constant buffer flow of 0.42 μL s−1 until a stable baseline was obtained. Then, 250 μL of OTC sample solution was injected via a six-way valve that allowed for switching of different solutions. Following the measurement, the cantilever array was treated with 2 M NaCl for 10 min to regenerate the sensor cantilever and then washed with running buffer. After each regeneration step, the deflection signal was evaluated to confirm the efficiency of the regeneration method. The resulting nanomechanical deflection of each cantilever was measured in real time by monitoring the position of a laser beam reflected off the apex of the cantilever onto a fourquadrant photodiode. The bending of cantilever toward or outward the gold side was defined as the positive or the negative deflection; the surface stresses were tensile or compressive, respectively.

Figure 2. Illustration of the procedure for label-free biodetection of OTC. Sensing cantilevers 1, 3, 5, and 7 (blue) are coated with aptamer SAMs to detect OTC, while cantilevers 2, 4, 6, and 8 (yellow) are coated with MCH SAMs and used as reference cantilevers. The injection of OTC generates a compressive surface stress, which causes the sensing cantilevers to bend downward compared with the reference cantilevers.

cantilevers (blue) were functionalized with aptamer selfassembled monolayers (SAMs) to detect OTC, while the reference cantilevers (yellow) were functionalized with 6mercapto-1-hexanol (MCH) SAMs to eliminate the influence of environmental disturbances, such as temperature and nonspecific adsorption. The cantilever array sensor for OTC detection relies on resolving surface stress changes induced by the formation of OTC−aptamer complexes. The interactions between OTC and the aptamer can be transmitted instantaneously to deflections of the cantilever. On the basis of the cantilever array sensor, OTC could be detected with high sensitivity and specificity.

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RESULTS AND DISCUSSION Cantilever Deflection Caused by OTC Binding. The interaction between OTC and the aptamer was investigated using the modified cantilever array. The sensing and reference 2011

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assume the charge−charge repulsion between neighboring OTC−aptamer complexes can also be an important element for the compress stress under our experimental condition. After the injection of OTC, the liquid cell was rinsed with running buffer only. As shown in Figure 3B, the deflection signal reaches a differential equilibrium value of 60 nm at about 1000 s, indicating that the OTC has a very low dissociation rate attributed to the high affinity of OTC to aptamer. Relationship between Deflection Amplitudes and the Concentrations of OTC. Figure 4 shows the cantilever

cantilevers in the array have been coated with aptamer SAMs and MCH SAMs, respectively. Before any measurements were attempted, the cantilever array was exposed to the running buffer and equilibrated until a stable baseline was obtained. Then, the OTC was introduced into the measurement cell at a concentration of 50 nM (shaded area, Figure 3). Throughout

Figure 4. Differential deflection response of the cantilever sensor to different concentrations of OTC as a function of time. Figure 3. Detection of OTC with aptamer SAMs modified cantilever arrays. (A) Averaged deflections (Avrg.Defl) response of aptamercoated cantilevers (shown in dark yellow) and MCH-coated cantilevers (shown in black) against time. Upon injection of OTC, the deflection of the cantilevers were measured in situ. The shaded area corresponds to the injection of 50 nM OTC solution. (B) Differential deflections (Diff.Defl) reveal the specific biomolecular interactions between aptamer and OTC derived by subtracting the MCH reference.

deflection as a function of time for different concentrations of OTC. The concentrations of OTC in the solution are varied from a low concentration of 1 nM to a high concentration of 100 nM. For each concentration, the experiments were repeated at least three times to assess the reproducibility of the experimental measurement. Sensing experiments on different cantilever arrays presented similar deflection signals with a standard deviation of 5%. In the concentration range of 1−100 nM, the deflection increases with the increasing concentrations of OTC. At concentration of 100 nM, the signal rapidly reached a 110 nm differential deflection at about 1000 s. The magnitude of the differential deflection did not change at OTC concentration higher than 100 nM. Figure 5 illustrates the calibration curve for the cantilever array sensor. Each point represents the average for three determinations, and

the process, the absolute deflection of individual cantilever is recorded in real-time, and the differential signal can be extracted simultaneously. The average deflection in Figure 3A represents the average measurements for the identically functionalized cantilevers within an array. Corresponding differential deflection between the sensing and reference cantilevers is plotted in Figure 3B to provide a true molecular recognition signal between OTC and the aptamer. As can be seen in Figure 3A, upon the injection of OTC (at about 170 s), the averaged deflection signals of all cantilevers in the array exhibited an obvious negative deflection, but this deflection could not be observed in the differential signal (Figure 3B), which indicates the effect can be ascribed to the nonspecific interactions and adsorption of OTC. At about 220 s, the sensing cantilevers started to bend downward with larger amplitude, but no significant change could be observed for the reference cantilevers. We can infer that the bending of the sensing cantilevers is based on the specific interaction between aptamer and OTC. It is known that DNA aptamer is the long nucleotide chain with loop region, where OTC can bind to.16 The formation of OTC−aptamer complexes may drive physical steric crowding due to the high density of aptamers that immobilized on the surface of the cantilevers. As a result, the OTC−aptamer complexes were trying to expand and finally led to an increased repulsion between the neighboring chains. Besides, as the OTC molecules are charged,1 it is reasonable to

Figure 5. Calibration curve of the cantilever sensor for OTC. Inset shows the linear responses at low OTC concentrations. The error bars illustrate the relative standard deviation (RSD) for three replicates. 2012

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recognition molecule, was immobilized on sensing cantilever surface through Au−S bond binding. When OTC interacted with the aptamer, a mechanical bending of the cantilever was generated due to the change in surface stress. The cantilever biosensor is able to detect OTC with the lowest detectable concentration down to 0.2 nM, which is comparable to other methods.9,10 These results provide evidence that aptamercoated cantilevers can be applied successfully to detect trace amounts of OTC in solution. At present, experiments are under way to develop new sensors for other drugs using cantilevers modified by different molecular recognition materials. We anticipate that this technology will become an ideal platform for the specific and simultaneous detection of drugs in pharmaceutical preparations and food products.

the error bars represent the standard deviation of data. The linear response range of the cantilever biosensor to OTC concentration was from 1.0 to 100 nM, with a correlation coefficient of 0.993. The linear progress equation is Y = 29.65 + 0.57X, where Y is the differential deflection (nm) and X is the concentration of OTC (nM). The limit of detection (LOD) of the OTC was calculated as three times the standard deviation for the average measurements of blank samples (LOD = 3 × RSD/slope). The LOD was determined to be as low as 0.2 nM. Specificity of the Cantilever Array Sensor. To learn the specificity of the cantilever array biosensors to OTC over other antibiotics, the modified cantilever array was exposed to three structurally similar tetracycline group antibiotics, such as OTC, DOX, and TET, with the same concentration (50 nM), respectively. The above antibiotics differ in minor functional groups like −H or −OH on fifth and/or sixth carbon atoms on the tetracycline nucleus (B and C rings) of OTC, DOX, and TET (Figure 1). As shown in Figure 6, none of these antibiotics

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +86-431-85262734 (J.T.); +86-431-85262430 (B.Z.). E-mail: [email protected] (J.T.); [email protected] (B.Z.). Author Contributions §

These authors contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Basic Research Program of China (973 Program; No. 2011CB935800) and the National Natural Science Foundation of China (Nos. 21075121, 21275140).

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Figure 6. Differential deflection response of the cantilever sensor to OTC, DOX, and TET at the same concentration (50 nM).

REFERENCES

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induces obvious deflection of cantilever. It is possible to distinguish the responses obtained for OTC from responses with structurally similar antibiotics (DOX and TET). The results indicate that the cantilever array sensor could be a powerful tool to detect OTC with high specificity. Regeneration of the Cantilever Array Sensor. The reusability is particularly important for a practical biosensor application. To verify the effective regeneration of cantilever array sensor, the cantilever array was treated with 2 M NaCl after OTC binding. Then, OTC with the same concentration was injected again after each regeneration step. The signals induced by a given concentration were about 90% of the original signal. It is because the hydrogen bonds and electrostatic interactions responsible for the aptamer−target association can be disrupted, owing to the high concentration of NaCl; subsequently, the bound OTC is released from aptamer.24 The results show that the regeneration step can successfully reestablish the initial conditions of the cantilever array and enabled the same cantilever array to be recycled for at least 10 successive experiments, which indicates the high reproduction of the cantilever array sensor.

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CONCLUSION In summary, a simple and highly sensitive method for the detection of OTC based on a cantilever biosensor has been successfully developed. The DNA aptamer, which acted as a 2013

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(16) Liss, M.; Petersen, B.; Wolf, H.; Prohaska, E. Anal. Chem. 2002, 74, 4488−4495. (17) Niazi, J. H.; Lee, S. J.; Kim, Y. S.; Gu, M. B. Biorg. Med. Chem. 2008, 16, 1254−1261. (18) Goeders, K. M.; Colton, J. S.; Bottomley, L. A. Chem. Rev. 2008, 108, 522−542. (19) Fritz, J.; Baller, M. K.; Lang, H. P.; Rothuizen, H.; Vettiger, P.; Meyer, E.; Guntherodt, H. J.; Gerber, C.; Gimzewski, J. K. Science 2000, 288, 316−318. (20) Cherian, S.; Gupta, R. K.; Mullin, B. C.; Thundat, T. Biosens. Bioelectron. 2003, 19, 411−416. (21) McKendry, R.; Zhang, J. Y.; Arntz, Y.; Strunz, T.; Hegner, M.; Lang, H. P.; Baller, M. K.; Certa, U.; Meyer, E.; Guntherodt, H. J.; Gerber, C. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 9783−9788. (22) Hansen, K. M.; Ji, H. F.; Wu, G. H.; Datar, R.; Cote, R.; Majumdar, A.; Thundat, T. Anal. Chem. 2001, 73, 1567−1571. (23) Wu, G. H.; Ji, H. F.; Hansen, K.; Thundat, T.; Datar, R.; Cote, R.; Hagan, M. F.; Chakraborty, A. K.; Majumdar, A. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 1560−1564. (24) Radi, A. E.; Sanchez, J. L. A.; Baldrich, E.; O’Sullivan, C. K. Anal. Chem. 2005, 77, 6320−6323.

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