Biosensors and Bioelectronics 56 (2014) 112–116

Contents lists available at ScienceDirect

Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios

Short communication

Label-free detection of kanamycin using aptamer-based cantilever array sensor Xiaojing Bai a,b, Hui Hou a,b, Bailin Zhang a,n, Jilin Tang a,nn a b

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China University of Chinese Academy of Sciences, Beijing, 100049, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 22 September 2013 Received in revised form 14 December 2013 Accepted 31 December 2013 Available online 10 January 2014

A label-free detection method of kanamycin using aptamer-based cantilever array sensor was developed. The cantilever array was composed of sensing cantilevers and reference cantilevers. This configuration allowed direct detection of individual cantilever deflections and subsequent determination of differential deflection of sensing/reference cantilever pair. The sensing cantilevers were functionalized with kanamycin aptamer, which was used as receptor molecules while the reference cantilevers were modified with 6-mercapto-1-hexanol (MCH) to eliminate the influence of environmental disturbances. The kanamycin–aptamer interaction induced a change in cantilever surface stress, which caused a differential deflection between the sensing and reference cantilever pair. The surface stress change was linear with kanamycin concentration over the range of 100 μM–10 mM with a correlation coefficient of 0.995. A detection limit of 50 μM was obtained, at a signal-to-noise ratio of 3. The sensor also showed good selectivity against other antibiotics such as neomycin, ribostamycin and chloramphenicol. The facile method for kanamycin detection may have great potential for investigating more other molecules. & 2014 Elsevier B.V. All rights reserved.

Keywords: Cantilever array sensor Kanamycin Aptamer Surface stress change

1. Introduction Kanamycin is an aminoglycoside bacteriocidal antibiotic isolated from Streptomyces kanamyceticus (Garrod et al., 1981). It is widely used as a veterinary drug (Megoulas and Koupparis, 2005) and can be accumulated in animal body and transferred into food chain. Abuse of kanamycin can cause ototoxicity and nephrotoxicity (Song et al., 2011). The presence of kanamycin in animal derived foods is a potential serious threat to human health. It is desirable to develop techniques with simple procedures and quick response to detect the concentration of kanamycin in animal derived foods such as milk. Cantilevers are nanomechnical transducers, which convert molecular interactions into mechanical motion (Fritz et al., 2000). In recent years, the cantilever sensors have become an attractive and promising technology for biosensing applications (Arlett et al., 2011; Boisen et al., 2011; Buchapudi et al., 2011; Eom et al., 2011; Singamaneni et al., 2008; Zougagh and Rios, 2009). They can be operated in static mode (cantilever deflection is measured) or dynamic mode (resonant frequency shift is measured). In the static mode, binding of analytes and recognition molecules immobilized on one surface of the cantilever causes a change in cantilever surface stress and deflects the cantilever

n

Corresponding author. Tel./fax: þ 86 431 85262430. Corresponding author. Tel./fax: þ86 431 85262734.

nn

0956-5663/$ - see front matter & 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.12.068

(Biswal et al., 2006). In the dynamic mode, analyte–receptor binding adds an additional mass to the oscillating cantilever, decreasing its resonance frequency. Static mode works well in gas and liquid phases. Dynamic mode works efficiently in gas phase but in liquid environment it is hindered by viscous damping of the oscillation (Ziegler, 2004). Owing to the unique detection principles, the cantilever sensors can avoid pre-treatment labeling and the use of expensive reagents. Label-free detection methods for various molecules, including proteins such as cyanovirin (Gruber et al., 2011) and prostate specific antigen (Yue et al., 2008), antibiotic oxytetracycline (Hou et al., 2013), and pollutants like atrazine (Raman Suri et al., 2008), trimethylamine (Huang et al., 2011) and mycotoxin (Ricciardi et al., 2013), have been developed using the cantilever sensors. Besides being label-free, this technology is of high sensitivity due to the small size and high surface-to-volume ratio of the cantilevers (Buchapudi et al., 2011). It has been applied in investigating DNA hybridization and discrimination of single-nucleotide mismatches (Fritz et al., 2000; Zhang et al., 2012). In addition, the cantilevers can be fabricated into arrays, providing the internal reference cantilever sensor that can eliminate nonspecific signals and permitting detection of multiple species in a single step (Joo et al., 2012). Meanwhile, the cantilevers can be batch fabricated, leading to a decrease in production costs and the development of a portable system (Carrascosa et al., 2006). Compared with conventional analytical techniques, the cantilever sensors have several advantages, including simple procedures, quick response, high sensitivity, low sample consumption, and the potential ability

X. Bai et al. / Biosensors and Bioelectronics 56 (2014) 112–116

113

Fig. 1. Schematic representation of kanamycin detection. Sensing cantilevers (1, 3, 5, and 7, from left to right, blue) were functionalized with kanamycin aptamer while reference cantilevers (2, 4, 6, and 8, green) were coated with MCH. Upon injection of kanamycin solution, the interaction between kanamycin and aptamer causes a compressive surface stress, resulting in a downward bending of the sensing cantilevers compared with the reference cantilevers. Relying on resolving surface stress changes, kanamycin can be facilely detected. (For interpretation of the reference to color in this figure, the reader is referred to the web version of this article).

to monitor multiple species simultaneously (Buchapudi et al., 2011; Wu et al. 2001). Aptamers are artificially synthesized oligonucleotides selected by SELEX (systematic evolution of ligands by exponential enrichment) (Ellington and Szostak, 1990; Tuerk and Gold, 1990). They possess high recognition ability to specific targets ranging from small molecules to proteins and even cells (Famulok et al., 2000; Jayasena, 1999; Tombelli et al., 2005). Owning to their low cost, inherent selectivity, and high stability, aptamers are used as receptors in various detection technologies (Cheng et al., 2007; Hansen et al., 2006; Temur et al., 2012). The chemically modified aptamers can be easily immobilized on the surface of cantilevers and used as recognition molecules. By combining the advantages of cantilever sensors and aptamers, we could apply the aptamer modified cantilever sensor for detecting kanamycin with simple procedures, quick response and high selectivity. In this article, an aptamer functionalized cantilever array sensor working in static mode was utilized for the detection of kanamycin. The functionalized cantilever array was composed of sensing cantilevers modified with thiolated kanamycin aptamer and reference cantilevers modified with 6-mercapto-1-hexanol (MCH). The interaction between aptamer and kanamycin causes a change in cantilever surface stress, resulting in a differential deflection of the cantilevers (Fig. 1). Relying on resolving surface stress changes, the kanamycin–aptamer interaction could be screened in real time. The cantilever array sensor can be used to detect kanamycin facilely without any complicated labeling process.

2. Material and methods 2.1. Materials Kanamycin aptamer (50 -(SH)-(CH2)6-TGG GGG TTG AGG CTA AGC CGA C-30 ) was synthesized by Sangon (Shanghai, China). Kanamycin sulfate, neomycin sulfate hydrate, ribostamycin sulfate and chloramphenicol were purchased from Sangon. MCH was obtained from Sigma-Aldrich. Tris-(2-carboxyethyl) phosphine hydrochloride (TCEP) was bought from Alfa Aesar (Tianjin, China). Other reagents were of analytical grade or higher. All of the reagents were used as received without any further purification. The running buffer was 20 mM Tris–HCl containing 50 mM NaCl, 5 mM KCl, and 5 mM MgCl2, pH 7.2. Pure water (18.2 MΩ cm) was obtained with a Millipore water system and used throughout the experiment.

2.2. Cantilever array functionalization The cantilever array consisted of eight identical silicon cantilevers which were coated with a 20 nm layer of gold (Concentris GmbH, Switzerland) on one side. Prior to functionalization, the

cantilever array was thoroughly washed by pure water, ethanol and then incubated in UV–ozone environment for 30 min. The cantilevers (1, 3, 5, and 7, Fig. 1) within an array were functionalized in parallel by inserting separately into four microcapillaries filled with functionalization solution for 3 h at room temperature. The functionalization solution (pH 8.0) contained 10 mM Tris–HCl, 1 μM aptamer and 100 μM TCEP. After being thoroughly rinsed with water, the cantilever array was immersed in 1 mM MCH for 1 h. MCH was used to displace nonspecific interactions between aptamer and gold, rendering the aptamer to stand up and be more accessible to targets (Zheng et al., 2011). Meanwhile, reference cantilevers (2, 4, 6, and 8, Fig. 1) modified with MCH were obtained. The reference cantilevers were designed to eliminate environmental disturbances. Finally the array was thoroughly rinsed with ethanol and water.

2.3. Deflection measurement The deflection experiments were performed on the commercial Cantisens sensor platform (Concentris GmbH, Switzerland). The functionalized cantilever array was inserted into a cantilever array holder and mounted inside the measurement cell. The temperature in the measurement cell was kept constant at 25 1C with a stability of 0.1 1C. A constant flow rate of 0.42 μL/ s was maintained during the experiment. The cantilever array was equilibrated in running buffer until a stable baseline was obtained. For deflection measurements, 250 μL sample solution was injected into the sample loop and transported to the measurement cell at the desired time. This arrangement allows the cantilever array exposed to desired solutions continuously. Besides, owing to the small volume of the measurement cell (5 μL), a relatively fast replacement of the liquid can be achieved. The resulting nanomechanical deflection of each cantilever was detected in real time by the optical beam deflection system of the platform. We define bending toward silicon side as bending down (negative) and toward the gold side as bending up (positive). In general, the downward bending is caused by repulsion or expansion of molecules, which is so called compressive stress; vice versa, the upward bending is caused by attraction or contraction of molecules on the gold surface, which is called tensile surface stress (Alvarez and Lechuga, 2010). Using Stoney0 s formula (Stoney, 1909), the surface stress change (△s) can be written as ! ET 2 Δs¼ ΔZ ð1Þ 3ð1  νÞL2 where ΔZ is the observed deflection at the end of the cantilever, L is the length of the cantilever (500 nm), T is the thickness (1 μm), E is Young0 s modulus (156 GPa for silicon), and ν is the Poisson ratio of silicon (0.2152).

114

X. Bai et al. / Biosensors and Bioelectronics 56 (2014) 112–116

3. Results and discussion

3.2. Cantilever response to different concentrations of kanamycin

3.1. Deflection of cantilevers induced by kanamycin–aptamer interaction

To examine the relationship between cantilever response and kanamycin concentration, we measured the cantilever deflections induced by different concentrations of kanamycin. According to the Langmuir model, the surface coverage is related to the concentration of the ligand in the solution. For cantilever studies, the surface coverage determines the number of binding events which in turn determines the change of the surface stress and the cantilever deflection (Raorane et al., 2008). As illustrated in Fig. 3A, the bending speed of cantilevers increased with the increase of kanamycin concentration. Higher kanamycin concentrations resulted in larger differential deflections at the end of sample injection (about 775 s). This phenomenon can be ascribed to the formation of more kanamycin–aptamer complex on the cantilever sensing surface and the increase of the surface stress change under higher kanamycin concentration. Fig. 3B shows the differential cantilever deflection (obtained at about 775 s) as a function of kanamycin concentration in solution. The differential cantilever deflection increased with the increase of kanamycin concentration in the range of 100 μM–10 mM. For kanamycin with higher concentrations than 10 mM, the differential cantilever deflection did not change obviously. The concentration-dependent cantilever deflection is in agreement with the Langmuir adsorption isotherm model. The inset in Fig. 3B shows that the surface stress change increased linearly with kanamycin concentration over the range from 100 μM to 10 mM. The linear equation was calibrated as Δs¼  4.32 1.45nc (c, kanamycin concentration) with a correlation coefficient of 0.995. The detection limit was 50 μM, at a signal-to-noise ratio of 3. The error bars illustrate the relative standard deviation (RSD) of four identical cantilevers within an array. Compared with other cantilever sensors developed for the detection of small molecules (Table S-1), the detection limit obtained here is not satisfied. The difference of the detection limit between this work and others may lie in the difference of the target–probe binding interaction and the cantilever deflection mechanism. To improve the detection sensitivity, further efforts are required to fully understand the molecular origin of surface stress change and develop better surface modification methods for aptamers to generate larger surface stress change during molecular recognition and enhance the response of the cantilever.

A remarkable advantage of cantilever sensors is that deflection induced by the interaction between ligand and acceptor can be measured in real time. Taking advantage of this, we utilized the aptamer-based cantilever array sensor to investigate the interaction between kanamycin and aptamer. The sensing and reference cantilevers of the cantilever array were modified with kanamycin aptamer and MCH, respectively. The functionalized cantilever array was immersed in running buffer. After a stable baseline was obtained, 1 mM kanamycin was introduced into the measurement cell. Time-dependent cantilever deflection is shown in Fig. 2. The average deflection (Fig. 2A) is the mean value of four identically functionalized cantilevers within an array. As can be observed, a stable baseline was obtained for both sensing and reference cantilevers before sample injection. Upon injection of 1 mM kanamycin solution, the sensing cantilevers underwent a significant negative deflection (red, Fig. 2A) while a much weaker negative deflection can be seen for the reference cantilevers (black, Fig. 2A). The weak deflection of reference cantilevers was ascribed to the influence of environmental changes. A negative differential deflection was obtained, as illustrated in Fig. 2B. The differential deflection is obtained by subtracting the averaged signal of the four reference cantilevers from that of the four sensing cantilevers. A differential deflection of  25.8 nm was obtained at about 775 s after 250 mL sample was injected. The bending signal corresponded to a compressive surface stress (Δs) of  6.84 mN/m. The surface stress change was induced by specific interaction between kanamycin and aptamer. It was reported that kanamycin could stabilize the stem and loop structure of the aptamer (Song et al., 2011). Upon binding to kanamycin, the aptamer undergoes a conformation change from an unstructured single strand to a hairpin structure which gives rise to an intermolecular repulsion between neighboring molecules. A compressive surface stress is therefore produced and bends the cantilever downward to increase the available surface area. Exposure of 5 mM kanamycin to three different cantilever arrays prepared under the same conditions presented similar deflection amplitudes and the standard deviation was within 15%, indicating good reproducibility of the sensor.

Fig. 2. Average/differential cantilever deflection as a function of time for 1 mM kanamycin injection (shaded area). The shaded area corresponds to an injection of 1 mM kanamycin in buffer. (A) Average cantilever deflection of sensing cantilevers (sensing, shown in red) and reference cantilevers (reference, shown in black). (B) Differential deflection signal (calculated by subtracting the average deflection of reference cantilevers from that of sensing cantilevers). (For interpretation of the reference to color in this figure, the reader is referred to the web version of this article).

X. Bai et al. / Biosensors and Bioelectronics 56 (2014) 112–116

115

negligible or only slight responses compared with that of 5 mM kanamycin (Fig. 3C). The results reveal that the aptamer-based cantilever array sensor has a good selectivity for kanamycin.

4. Conclusions In summary, we have developed a facile and label-free detection method for kanamycin using an aptamer-based cantilever array sensor. The interaction between kanamycin and aptamer immobilized on cantilever surface induced a change in surface stress of the cantilever. The surface stress change was linearly dependent on kanamycin concentration over the range from 100 μM–10 mM with a correlation coefficient of 0.995. The sensor showed good selectivity to kanamycin and a detection limit of 50 μM was obtained. Compared with other techniques, the method developed here is not superior in detection limit (Table S-2) but it is rapid, facile, and reagentless. The future work will be addressed towards improving the detection sensitivity and achieving a more reliable integrated system, able to work with complex real-life samples. Our work indicates that the aptamerbased cantilever array sensors have great potential in investigating target–aptamer interactions and will be useful for the detection of more molecules.

Acknowledgments This work was supported by the National Basic Research Program of China (No. 2011CB935800), and the National Natural Science Foundation of China (No. 21375122).

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.12.068. References

Fig. 3. (A) Differential deflection as a function of time for different concentrations of kanamycin: 0 mM (Blank), 100 mM, 1 mM, 5 mM and 10 mM (from up to down). The shaded area corresponds to the injection of kanamycin. (B) Differential cantilever deflection (obtained at about 775 s) as a function of kanamycin concentration in solution. The inset shows that the cantilever surface stress change is linear with kanamycin concentration over the range from 100 μM to 10 mM. (C) Differential deflection as a function of time for 5 mM Na2SO4, glucose, neomycin, ribostamycin, chloramphenicol and kanamycin.

3.3. Selectivity In order to investigate the selectivity of the cantilever array sensor, we exposed the modified cantilever arrays to Na2SO4, glucose, neomycin, ribostamycin and chloramphenicol with the same concentration (5 mM). The cantilever array sensor had

Alvarez, M., Lechuga, L.M., 2010. Analyst 135 (5), 827. Arlett, J.L., Myers, E.B., Roukes, M.L., 2011. Nat. Nanotechnol. 6 (4), 203–215. Biswal, S.L., Raorane, D., Chaiken, A., Birecki, H., Majumdar, A., 2006. Anal. Chem. 78 (20), 7104–7109. Boisen, A., Dohn, S., Keller, S.S., Schmid, S., Tenje, M., 2011. Rep. Prog. Phys. 74, 3. Buchapudi, K.R., Huang, X., Yang, X., Ji, H.F., Thundat, T., 2011. Analyst 136 (8), 1539–1556. Carrascosa, L.G., Moreno, M., Alvarez, M., Lechuga, L.M., 2006. Trac-trend Anal. Chem. 25 (3), 196–206. Cheng, A.K.H., Ge, B., Yu, H.Z., 2007. Anal. Chem. 79 (14), 5158–5164. Ellington, A.D., Szostak, J.W., 1990. Nature 346 (6287), 818–822. Eom, K., Park, H.S., Yoon, D.S., Kwon, T., 2011. Phys. Rep. 503 (4-5), 115–163. Famulok, M., Mayer, G., Blind, M., 2000. Acc. Chem. Res. 33 (9), 591–599. Fritz, J., Baller, M.K., Lang, H.P., Rothuizen, H., Vettiger, P., Meyer, E., Guntherodt, H.J., Gerber, C., Gimzewski, J.K., 2000. Science 288 (5464), 316–318. Garrod, L.P., et al., 1981. Antibiotic and Chemotherapy. Churchill Livingstone, Edinburgh. Gruber, K., Horlacher, T., Castelli, R., Mader, A., Seeberger, P.H., Hermann, B.A., 2011. ACS Nano 5 (5), 3670–3678. Hansen, J.A., Wang, J., Kawde, A.N., Xiang, Y., Gothelf, K.V., Collins, G., 2006. J. Am. Chem. Soc. 128 (7), 2228–2229. Hou, H., Bai, X., Xing, C., Gu, N., Zhang, B., Tang, J., 2013. Anal. Chem. 85 (4), 2010–2014. Huang, X., Li, M., Xu, X., Chen, H., Ji, H.F., Zhu, S., 2011. Biosens. Bioelectron. 30 (1), 140–144. Jayasena, S.D., 1999. Clin. Chem. 45 (9), 1628–1650. Joo, J., Kwon, D., Yim, C., Jeon, S., 2012. ACS Nano 6 (5), 4375–4381. Megoulas, N.C., Koupparis, M.A., 2005. Anal. Chim. Acta 547 (1), 64–72. Raman Suri, C., Kaur, J., Gandhi, S., Shekhawat, G.S., 2008. Nanotechnology 19 (23) (235502-235502). Raorane, D.A., Lim, M.D., Chen, F.F., Craik, C.S., Majumdar, A., 2008. Nano Lett. 8 (9), 2968–2974.

116

X. Bai et al. / Biosensors and Bioelectronics 56 (2014) 112–116

Ricciardi, C., Castagna, R., Ferrante, I., Frascella, F., Marasso, S.L., Ricci, A., Canavese, G., Lore, A., Prelle, A., Gullino, M.L., Spadaro, D., 2013. Biosens. Bioelectron. 40 (1), 233–239. Singamaneni, S., LeMieux, M.C., Lang, H.P., Gerber, C., Lam, Y., Zauscher, S., Datskos, P.G., Lavrik, N.V., Jiang, H., Naik, R.R., Bunning, T.J., Tsukruk, V.V., 2008. Adv. Mater. 20 (4), 653–680. Song, K.M., Cho, M., Jo, H., Min, K., Jeon, S.H., Kim, T., Han, M.S., Ku, J.K., Ban, C., 2011. Anal. Biochem. 415 (2), 175–181. Stoney, G.G., 1909. Proc. R. Soc. London Ser. A-c 82 (553), 172–175. Temur, E., Zengin, A., Boyaci, I.H., Dudak, F.C., Torul, H., Tamer, U., 2012. Anal. Chem. 84 (24), 10600–10606.

Tombelli, S., Minunni, A., Mascini, A., 2005. Biosens. Bioelectron. 20 (12), 2424–2434. Tuerk, C., Gold, L., 1990. Science 249 (4968), 505–510. Yue, M., Stachowiak, J.C., Lin, H., Datar, R., Cote, R., Majumdar, A., 2008. Nano. Lett. 8 (2), 520–524. Zhang, J., Lang, H.P., Yoshikawa, G., Gerber, C., 2012. Langmuir 28 (15), 6494–6501. Zheng, S., Choi, J.H., Lee, S.M., Hwang, K.S., Kim, S.K., Kim, T.S., 2011. Lab Chip 11 (1), 63–69. Ziegler, C., 2004. Anal. Bioanal. Chem. 379 (7-8), 946–959. Zougagh, M., Rios, A., 2009. Analyst 134 (7), 1274–1290.

Label-free detection of kanamycin using aptamer-based cantilever array sensor.

A label-free detection method of kanamycin using aptamer-based cantilever array sensor was developed. The cantilever array was composed of sensing can...
1MB Sizes 0 Downloads 0 Views