Biosensors and Bioelectronics 65 (2015) 226–231
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Surface assembly of nano-metal organic framework on amine functionalized indium tin oxide substrate for impedimetric sensing of parathion Akash Deep a,b,n, Sanjeev Kumar Bhardwaj a,b, A.K. Paul a,b, Ki-Hyun Kim c, Pawan Kumar a,b,1 a
Central Scientific Instruments Organisation (CSIR-CSIO), Sector 30C, Chandigarh 160030, India Academy of Scientific and Innovative Research, CSIR-CSIO, Sector 30C, Chandigarh 160030, India c Department of Civil & Environmental Engineering, Hanyang University, 222 Wangsimni-Ro, Seoul 133-791, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 14 July 2014 Received in revised form 8 October 2014 Accepted 9 October 2014 Available online 22 October 2014
The present paper reports the assembly and pesticide sensing application of a nanometal organic framework [Cd(atc)(H2O)2]n (‘atc’ ¼2-aminoterephthalic acid). The assembly of the NMOF film has been achieved by sequential dipping of a 2-aminobenzylamine (2-ABA) modified indium tin oxide (ITO) slide in organic linker ‘atc’ and metal ion ‘Cd2 þ ’ solutions. The different structural and morphological characteristics of the NMOF thin film have been characterized. The availability of pendent –COOH functional groups on the assembled NMOF film is exploited to synthesize a pesticide immunosensor by conjugating the NMOF film with anti-parathion antibody. This immunosensor has been explored for the electrochemical impedance spectroscopy (EIS) based analysis of parathion in the concentration range of 0.1– 20 ng/mL. The proposed detection is specific with respect to other organophosphate compounds, e.g. malathion, paraoxon, fenitrothion, monochrotophos and dichlorovos. The proposed sensor shows the detection limit of 0.1 ng/mL and it is applicable for analysis of parathion in a rice sample. The sensor's performance is validated by comparting the obtained results with gas chromatographic data. & Elsevier B.V. All rights reserved.
Keywords: Metal organic framework Sensing Impedance Parathion Self-assembly
1. Introduction Nanocrystal metal organic frameworks (NMOFs) have been projected useful in application areas such as, catalysis, nonlinear optics, electroluminescent devices, biomedical imaging and small molecules sensing (Krische and Lehn, 2000; Tanabe and Cohen, 2011; Cui et al., 2011; Della Rocca et al., 2011; Kumar et al., 2014a). NMOFs are characterized with better dispersive nature than the micsron scale MOFs and they can easily sense small molecules with no loss in their intrigue framework architectures and topologies (Wang et al., 2011a, 2011b). NMOFs with Eu(III), Zn(II) and Cd (II) as metal ion units have been reported for the detection of nitro aromatic compounds, small molecules and explosives (Kumar et al., 2014b; Wang et al., 2011a, 2011b; Li et al., 2008; Pramanik et al., 2011). Availability of pendent functional groups on some NMOF structures may be utilized to tag them with biomolecules n Corresponding author at: Central Scientific Instruments Organisation (CSIR-CSIO), Sector 30C, Chandigarh 160030, India. Fax: þ91 0172 2657287. E-mail addresses: [email protected], [email protected] (A. Deep). 1 Present address: Department of Civil & Environmental Engineering, Hanyang University, 222 Wangsimni-Ro, Seoul 133-791, Korea.
http://dx.doi.org/10.1016/j.bios.2014.10.045 0956-5663/& Elsevier B.V. All rights reserved.
such as nucleic acid, protein, antibody and enzymes (DeKrafft et al., 2009; McKinlay et al., 2010). A Cd ion based NMOF ‘[Cd(atc) (H2O)2]n’ (atc¼2-aminoterephthalic acid) has recently been proposed for the fluorescence quenching based detection of nitro aromatic compounds, including explosives and pesticides (Wang et al., 2012; Kumar et al., 2014a). Aqueous dispersibility and availability of pendent –COOH groups on this NMOF structure (Fig. S1) makes it an interesting choice for exploring in molecular sensing applications. Development of impedimetric immunosensors is an exciting field of research as this technique provide the realization of labon-chip kind of devices which can be integrated with microfluidic sample chambers and are easy to calibrate. Modern impedimetric immunosensors rely on the use of nanosized electrode materials such as carbon nanotubes (CNTs) and graphene because of their large surface areas (Zhang et al., 2014; Wu et al., 2013; Musameh et al., 2013; Yan et al., 2013). The NMOFs are also another class of nanomaterials which are known for their extremely large surface areas and availability of functional pendent groups. Due to hierarchical chemical assembly, the use of NMOF nanomaterials may offer the development of well-defined and ordered sensor geometry, which, in turn, is advantageous in the impedimetric
A. Deep et al. / Biosensors and Bioelectronics 65 (2015) 226–231
227
Fig. 1. FE-SEM imaging of (A): Bare ITO, (B): 2-ABA/ITO, (C): NMOF grown on 2-ABA/ITO surface, and (D): high resolution image for NMOF grown on 2-ABA/ITO surface.
immunosensor applications mainly in terms of sensor stability and sensitivity. The ready availability of functional groups on NMOFs is another useful property as the additional step of chemical oxidation (like in the case of CNTs and graphene) can be avoided. Though NMOFs have been investigated for optical sensing of some analytes, such as pesticides and explosives, their exploitation in electrochemical sensor technology is still an area to be explored. The MOF thin films are seen with significant potential in the future development of luminescent devices, magnetic films, redox-active films, and proton/electron-conducting films (Allendorf et al., 2011; Sadakiyo et al., 2009). Surface assembly technique offers one of the most robust routes for synthesizing the MOF thin films. In this work, we report the assembly of NMOF on a 2-aminobenzylamine (2-ABA) modified indium tin oxide (ITO) electrode. 2-ABA is a derivative of aniline, and its electrochemical deposition on ITO leaves behind free –NH2 moiety which is reacted with the carboxylate groups of the organic ligand atc. The morphological appearance of 2-ABA/ITO surface is shown in Fig. 1. The grafting of the organic ligand is then followed by sequential assembly of the metal center (Cd) and organic ligand. Five numbers of such steps provided the growth of rod like NMOF structure on the 2-ABA modified ITO. The pendent –COOH groups on thus assembled NMOF were then exploited for tagging it with anti-parathion antibody. The whole strategy has been depicted as schematic in Fig. S2. It is pertinent to mention here that parathion is one of the most toxic and widely used insecticides. High practical relevence of developing parathion biosensing devices has motivated the researchers to articulate on different strategies (Mulchandani and Rajesh, 2011). Amperometric and electrochemical techniques have been suggested for rapid and low cost detections of organophosphates (Zhang et al., 2014, Yoon et al., 2014; Li et al., 2014; Yan et al., 2013; Wu et al., 2013; Musameh et al., 2013; Funari et al.,
2013; Gong et al., 2013; Zhao et al., 2013; Mulchandani and Rajesh, 2011; Du et al., 2010; Lei et al., 2004; Mulchandani et al., 2001). Nafion-coated glassy carbon electrode has been proposed for the detection of as low as 50 nM of parathion (Funari et al., 2013). Organophosphate hydrolase functionalized electrodes have also been proposed (Thakur et al., 2013;Du et al., 2010; Lei et al., 2007). A limit of detection of 20 nM for both methyl-parathion and paraoxon has been reported by Mulchandani et al. (2001).
2. Materials and methods 2.1. Reagents and chemicals Indium tin oxide (ITO) substrates, cadmium nitrate, 2-aminoterephthalic acid (atc), EDC [ethyl(dimethylaminopropyl) carbodiimide, 99%], 2-aminobenzylamine (2-ABA), and MES buffer (2ethanesulfonic acid) were high purity chemical purchased from Sigma-Aldrich. Anti-parathion antibody (polyclonal IgG, rabbit anti-parathion) was purchased from Abcam. All other chemicals (including pesticides) and solvents were also AR/GR grade products from Sigma-Aldrich/Merck. All the experiments were carried out at room temperature (25 72 °C). 2.2. Preparation of sodium salt of 2-aminoterephthalic acid NaOH solution (2.2 g in 10 mL DI water) was mixed with atc solution (5 g in 20 mL DI water) under stirring, which resulted into formation of white precipitate. After 10 min of stirring at room temperature, the precipitate was collected by centrifugation and washed with water). This salt was dried at 100 °C.
228
A. Deep et al. / Biosensors and Bioelectronics 65 (2015) 226–231
2.3. Electrochemical grafting of 2-ABA on ITO
2.7. Characterizations
Chronoamperometry technique was employed to assemble 2-ABA on ITO substrate. 10 mM solution of 2-ABA was taken (phosphate buffer, pH 7) in a three-electrode configured electrochemical cell. ITO, platinum and Ag/AgCl were selected as working, counter and reference electrodes, respectively. A potential of 0.8 V was applied for 120 s to electro-deposit the 2-ABA film. The above value of the potential was selected based on a separate cyclic voltammetry experiment (Fig. S3). In this study, the cyclic voltammetry assisted electrodeposition of 10 mM 2-ABA solution was investigated, which revealed the appearance of cathodic peak at around 0.75–0.80 V. The duration of the electrodeposition experiment was optimized so as to attain smooth coverage (as investigated by FE-SEM) of 2-ABA on ITO slides. The electrodeposition cycles of 120 seconds offered smooth coverage of 2-ABA molecules. After electrodeposition, the film was washed and dried in vacuum. The adhesion of organic compound 2-ABA on the metallic ITO surface occurs at the metal/organic coating interface when interatomic bonds take place. These bonds can be primary (covalent or ionic bonds), secondary (dispersion forces, dipole interactions, and van der Waals forces) or hydrogen bridge type. Metal/polymer interfacial bonds are generally secondary or hydrogen bridge type.
Characterization were done with different instrumental techniques such as Fourier transform infrared spectroscopy (Nicolet, iS10), field-emission scanning electron microscopy (FE-SEM, 4300/ SE, Hitachi), X-ray diffractometry (XPert3 Powder, Panalytical), and electrochemistry workstation (Autolab, Metrohm). Gas chromatography experiments were carried out on GC (DANI Master) coupled with the electron capture detector.
2.4. Assembly of NMOF on 2-ABA/ITO Separate solutions of 0.05 M Cd(NO3)2 and 0.5 M sodium salt of atc were prepared. 2-ABA/ITO was first left in contact (dipping method) with the ligand solution for 10 min. Afterwards, the 2-ABA/ITO slide was washed (by dipping) with water and then introduced into the metal ion solution for 10 min. The said incubation time was optimized by investigating the assembly of metal–organic components for different time intervals (2, 4, 6, 8, and 10 min). Based on the desired product appearance (nano-rod like structure) on FE-SEM, it was found that minimum of 8 min of incubation time was required for the growth of the desired product. Further increase in the incubation time did not influence the formation of the desired product. In all the other experiments a uniform incubation time of 10 min was provided. Sequential dipping of the slide in the ligand and metal ion solutions were done for 5 times. NMOF were nucleated and grown to the surface in a controlled step by step procedure leading to the formation of its layers, which was visually apparent as a whitish film. Finally, the NMOF/2-ABA/ITO substrate was washed and then bioconjugated with anti-parathion antibody 2.5. Conjugation of anti-parathion antibody on NMOF/2-ABA/ITO For activation of the pendent –COOH functional groups on the synthesized NMOF, the slide was first left in contact with 5 mL of 0.1 M MES buffer (pH 5.0). 100 mg of EDC was added and the sample was incubated for 30 minutes. 1 mg mL 1 aqueous solution of anti-parathion was introduced and the sample was further incubated for 2 h at room temperature. The slide was carefully washed and thus prepared anti-parathion/NMOF/2-ABA/ITO sensing platform was stored in vacuum desiccator. 2.6. Electrochemical impedance immunoassay Impedance spectrum was recorded at the formal potential of redox couple (ferricyanide/ferrocyanide) i.e. 0.172 V (vs. screen printed Ag/AgCl) at an amplitude of 5 mV within the frequency range of 1 Hz–100 kHz.
2.8. Analysis of parathion in rice sample The origin of rice sample was in Punjab, India. Recovery of pesticide content from the sample was done according to the literature (Barkar, 2006). Dehusked rice sample was grinded and dissolved in water. The mixture was centrifuged at 5000 rpm for 15 min to collect the supernatant. The obtained solution was then passed through solid phase extraction (SPE) C18 column. It was followed by the elution step with methanol. The eluted sample was left to dry overnight in an oven. The dried mass was re-solubilized in water (for analysis by anti-parathion/NMOF/2-ABA/ITO sensor) or n-hexane (for analysis by GC). The sample thus obtained was analyzed by anti-parathion/NMOF/2-ABA/ITO sensor and gas chromatography (GC). Appropriate dilutions of the sample were prepared in order to fit the EIS signal within the calibration range of the sensor. For GC experiments, the oven temperature was first kept at 40 °C for 5 min and incremented to 250 °C with step rate of 5 °C/ min. A temperature of 250 °C was maintained for 5 min. The flow rate of carrier gas (N2) was maintained at 1 mL/minute. The injector port temperature was programmed at 250 °C. Electron capture detector (ECD) was kept at 320 °C and 30 mL/min flow of N2 was maintained for the detection of organophosphates. 2 mL of the sample was injected each time for the sample analysis.
3. Results and discussion 3.1. Synthesis and characterization of NMOF on 2-ABA/ITO The structural morphology of bare ITO slide, the electrodeposited 2-ABA particles (on ITO slide), and NMOF grown onto the 2-ABA/ITO slide is presented in Fig. 1. It is fairly apparent that the 2-ABA particles have modified the ITO surface. The presence of 2-ABA is characterized with the appearance of globular nanosized particles. In comparison, the crystals of NMOF on 2-ABA/ITO were formed in nanorod shape (diameter 100–250 nm). There is an apparent modification of the 2-ABA/ITO surface after the assembly of NMOF. Five sequential dipping cycles of the 2-ABA/ITO slide in the organic linker solution and metal ion solution provided a smooth coverage of the NMOF on the 2-ABA/ITO surface without any apparent lack of continuity. Lesser numbers of cycles left voids on the electrode surface. Similar nanorod like morphology of the NMOF has been reported when bulk synthesis method was used (Wang et al., 2012; Chen et al., 2007). The formed layer of the NMOF was fairly uniform as suggested by the line profile analysis of the scanned atomic force microscopy (AFM) image (Fig. S4). The roughness of the film was limited in the range of 7 5 nm. The binding of the NMOF on the amine terminated 2-ABA/ITO surface is chemically achieved through the amide linkage between the – NH2 groups (of 2-ABA) and free –COOH pendent groups (available on the NMOF surface). Powder XRD data of the synthesized NMOF film (Fig. 2) matched with the previously reported findings (Wang et al., 2012; Chen et al., 2007). NMOF has monoclinic crystal structure with space group C2/c in which Cd(II) ions of equatorial sites are occupied by two bidentate carboxylate oxygen atoms, one
A. Deep et al. / Biosensors and Bioelectronics 65 (2015) 226–231
229
Fig. 2. PXRD data of bulk NMOF and NMOF grown on 2-ABA/ITO.
carboxylate oxygen atom from the second H2atc ligand, one oxygen atom from the water and one nitrogen atom of the amino group from the third H2atc ligand (Chen et al., 2007). FTIR spectrum of NMOF is shown in Fig. 3. The appearance of vibration bands at 1610, 1507, 1570, 1457 and 1358 cm 1 in NMOF may be assigned to the stretching vibrations of higher energy and bending vibrations of lower energy of C–O in carboxylate groups pertaining to the presence of organic ligand in structure (Rai et al., 2013 and Savonnet et al., 2012). Another band due to C–N appears at 1285 cm 1. The appearance of bands in the lower frequency region (500–750 cm 1) may be assigned to the involved Cd–O linkages. 3.2. Development of sensor Incubation of NMOF/2-ABA/ITO substrate with anti-parathion antibody has resulted into the formation of a novel immunosensing platform of parathion. Successful biofunctionalization was confirmed by FTIR analysis (Fig. 3). Characteristic C ¼O (CONH2) stretching was observed at 1637 cm 1 due to the attachment of protein through amide bond formation. The presence of two weak bands at 2825 and 2930 cm 1 are also suggestive of the successful grafting of protein molecules with NMOF. 3.3. Electrochemical detection of parathion Fig. 4 shows the Nyquist plots for electrochemical impedance spectroscopy (EIS) experiments done on ITO, 2-ABA/ITO, NMOF/2-
Fig. 3. FTIR of NMOF/2-ABA/ITO, anti-parthion antibody, and anti-parathion/ NMOF/2-ABA/ITO sensor.
Fig. 4. Nyquist plots for ITO, 2-ABA/ITO, NMOF/2-ABA/ITO and anti-parathion/ NMOF/2-ABA/ITO.
ABA/ITO and anti-parathion/NMOF/2-ABA/ITO platforms. In Nyquist plot, the imaginary impedance component (Z″, out-of-phase) is plotted against the real impedance component (Z′, in-phase) at each excitation frequency. In the case of the proposed immunosensor, a non-insulating dielectric antibody layer covers the surface of the electrode, which is able to catalyze the redox probe in the measuring solution. The measured parameter ‘charge transfer resistance’ is actually the real component of impedance. The antibody–antigen interactions cause an increase in its value as the faradaic reaction becomes increasingly hindered, which is clear from the non-existence of typical straight lines after the semi-circle shape. The immunosensor configuration resembles a capacitor in its ability to store charge. An event of antibody–antigen interaction leads to a decrease in the total capacitance of the system. Total capacitance ‘Ct’ of the whole system can be described according to the following equation:
1/Ct = (1/Cdl) + (1/Cbm)
(1)
where Cdl denotes the capacitance due to dielectric layer and Cbm denotes the capacitance due to outer biomolecule layer. Since the current must pass through the uncompensated resistance of the electrolyte solution, its value is also taken into account while evaluating the sensor response. The impedance data have been fitted with software Zview. Randle's equivalent circuit was chosen for determining the values of charge transfer resistance. This circuit is often used to model the interfacial phenomena and it takes into account the uncompensated resistance of the electrolyte in series with the capacitance of the dielectric layer (Cdl) and the charge transfer resistance (Rct). An additional component, the Warburg impedance (Zw) accounts for the diffusion of ions from bulk electrolyte to the electrode interface. Compared to 2-ABA/ITO, the NMOF/2-ABA/ITO shows increase in charge transfer resistance confirming the growth of NMOF on 2-ABA/ITO. Observed electrical conductivity of the NMOF/2-ABA/ ITO could be exploited for various biosensing applications. The attachment of protein further increases the charge transfer resistance, once again confirming the successful immobilization of protein. The increase in the value of charge transfer resistance at this step may be attributed to the hindrance in the diffusion of the ferricyanide (redox probe) toward the electrode surface as a
230
A. Deep et al. / Biosensors and Bioelectronics 65 (2015) 226–231
Table 1 Analysis of parathion in rice samples. Sample no.
GC analysis (mg/L)
Anti-parathion/NMOF/2-ABA/ITO analysis
1 2 3 4
2.57 2.65 2.50 2.49
2.48 mg/L 2.54 mg/L 2.37 mg/L 2.47 mg/L
(s.d. ¼ 0.060) (s.d. ¼0.061) (s.d. ¼ 0.058) (s.d. ¼0.065)
The applicability and validity of the sensor were demonstrated by detecting the parathion concentration in rice sample (Table 1). The analyzed concentration of parathion (2.57 ppm) in the sample agreed well with the results from gas chromatography.
4. Conclusions Fig. 5. EIS response of the anti-parathion/NMOF/2-ABA/ITO platform toward varying concentrations (0.1–20 ng/mL) of parathion.
results of protein layering. Fig. 5 shows the EIS response of the anti-parathion/NMOF/2-ABA/ITO platform towards varying concentrations (0.1–20 ng/mL) of parathion. The charge transfer resistance (Rct) increases as the concentration of the analyte increases. At concentrations lower than 0.1 ng/mL, the signal was unstable. The increase in the charge transfer resistance with the increasing parathion concentration is caused by the increased binding of the antigen molecules to the immobilized antibodies which acts as a definite kinetic barrier for the electron transfer. The increase in the charge transfer resistance values after the antibody–antigen binding may also be correlated with the fact that increasing antigen–antibody binding events eventually block the free spaces on the bioelectrode. Calibration of the measurements is also shown in Fig. 5. Clearly the detection is possible over a wide linear range of analyte concentration. The limit of detection was 0.1 ng/mL under the given experimental conditions, which is better than most of the earlier studies (Table S1). Specificity of the parathion detection was analyzed with respect to some other organophosphate pesticides, namely malathion, paraoxon, fenitrothion, monochrotophos and dichlorovos. For these experiments the response of the anti-parathion/NMOF/ 2-ABA/ITO sensor was recorded for 0.1 ng/mL parathion in the presence of three different concentrations (0.1/10/50 ng/mL) of the above possible interferants. The response of the sensor toward parathion was unchanged (variation in Rct ¼ 73.5%) thereby highlighting superior specificity of the proposed immunosensing system. In a separate experiment, the sensor response was also investigated against a fixed concentration (50 ng/mL) of the malathion, paraoxon, fenitrothion, monochrotophos and dichlorovos. The EIS response (estimated Rct) of the anti-parathion/NMOF/2ABA/ITO sensor in these cases was observed to be 5990 710 thus again suggesting that the proposed sensor was responsive to the target analyte (parathion) only. The reproducibility of the sensor was investigated through intra- and interassay precision. For intraassay precision, the sensor's response was periodically checked with respect to 0.1 ng/mL parathion. The sensor was stable (7 3% variation in the values of Rct) over a period of 10 days. The stability of the sensor's EIS response as a function of storage time was also evaluated. It was observed that the proposed sensor could retain its EIS characteristics (7 5% variation in the values of Rct) even after 25 days of storage (storage conditions: medium 10 mM PBS, pH 7.4, T¼ 4 °C). Similarly, interassay precision was estimated by measuring 0.1 ng/mL of parathion concentration with four separately prepared sensors. In this case, the variation in the sensors' response was limited to 74%.
In summary, the present work first time reports the use of NMOF based immunosensing platform for sensitive and highly specific impedimetric detection of an organophosphate pesticide. The self-assembly of NMOF onto a 2-ABA modified ITO may offer the development of many such immunosenors in future. The use of antibody has provided the achievement of the desired specificity. The sensitivity of the proposed sensor is better or comparable to the existing systems (Table S1). Being a hierarchical structure, NMOF assembles in a well-defined geometry which may also help in achieving more stable results than other random platforms such as nafion based electrodes. The proposed use of antibody offers a direct advantage in terms of the specificity over organophosphate hydrolase enzyme based sensors. Moreover, such sensor systems can also be explored as host–guest interaction based direct chemical sensing.
Acknowledgments Authors gratefully acknowledge the financial grant from CSIR India through project OMEGA/PSC0202/2.2.5. We are thankful to the Director, CSIR-CSIO, Chandigarh, India. The fourth author acknowledges partial financial support from the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (No. 2009-0093848).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.10.045
References Allendorf, M.D., Schwartzberg, A., Stavila, V., Talin, A.A., 2011. Chem. Eur. J 17, 11372–11388. Barkar, S.A., 2006. Use of matrix solid-phase dispersion for determining pesticides in fish and food. In: Vidal, J.L.M., Vidal, J.L.M. (Eds.), Pesticide Protocols: Methods in Biotechnology. Humana Press, NewJersy, pp. 285–296. Chen, B., Yang, Y., Zapata, F., Lin, G., Qian, G., Lobkovsky, E.B., 2007. Adv. Mater. 19, 1693–1696. Cui, Y., Yue, Y., Qian, G., Chen, B., 2011. Chem. Rev. 112, 1126–1162. DeKrafft, K.E., Xie, Z., Cao, G., Tran, S., Ma, L., Zhou, O.Z., Lin, W., 2009. Angew. Chem. Int. Ed. 48, 9901–9904. Della Rocca, J., Liu, D., Lin, W., 2011. Acc. Chem. Res 44, 957–968. Du, D., Chen, W., Zhang, W., Liu, D., Li, H., Lin, Y., 2010. Biosens. Bioelectron. 25, 1370–1375. Funari, R., Della Ventura, B., Schiavo, L., Esposito, R., Altucci, C., Velotta, R., 2013. Anal. Chem. 85, 6392–6397. Gong, J., Guan, Z., Song, D., 2013. Biosens. Bioelectron. 39 (1), 320–323.
A. Deep et al. / Biosensors and Bioelectronics 65 (2015) 226–231
Krische, M., Lehn, J.-M., 2000. The utilization of persistent h-bonding motifs in the self-assembly of supramolecular architectures. In: Fuiita, M. (Ed.), Molecular Self-Assembly Organic Versus Inorganic Approaches. Springer, Berlin Heidelberg, pp. 3–29. Kumar, P., Kumar, P., Bharadwaj, L.M., Paul, A.K., Deep, A., 2014a. Inorg. Chem. Commun. 43, 114–117. Kumar, P., Paul, A.K., Deep, A., 2014b. Anal. Method 6, 4095–4101. Lei, C., Valenta, M.M., Saripalli, K.P., Ackerman, E.J., 2007. J. Environ. Qual. 36, 233–238. Lei, Y., Mulchandani, P., Chen, W., Wang, J., Mulchandani, A., 2004. Biotechnol. Bioeng. 85, 706–713. Li, Y., Xu, M., Peipei, L., Dong, J., Ai, S., 2014. Anal. Methods 6 (7), 2157–2162. Li, Z.-Q., Qiu, L.-G., Wang, W., Xu, T., Wu, Y., Jiang, X., 2008. Inorg. Chem. Commun. 11, 1375–1377. McKinlay, A.C., Morris, R.E., Horcajada, P., Férey, G., Gref, R., Couvreur, P., Serre, C., 2010. Angew. Chem. Int. Ed. 49, 6260–6266. Mulchandani, A., Rajesh, 2011. Appl. Biochem. Biotechnol. 165, 687–699. Mulchandani, P., Chen, W., Mulchandani, A., 2001. Environ. Sci. Technol. 35 (12), 2562–2565. Musameh, M., Notivoli, M.R., Hickey, M., Huynh, C.P., Hawkins, S.C., Yousef, J.M., Kyratzis, I.L., 2013. Electrochim. Acta. 101, 209–215. Pramanik, S., Zheng, C., Zhang, X., Emge, T.J., Li, J., 2011. J. Am. Chem. Soc. 133, 4153–4155.
231
Rai, S., Bajpai, A., Lokhandwala, S., 2013. J. Polym. , http://dx.doi.org/10.1155/2013/ 278576 (Article ID 278576, 6 pages). Sadakiyo, M., Yamada, T., Kitagawa, H., 2009. J. Am. Chem. Soc. 131, 9906–9907. Savonnet, M., Canivet, J., Gambarelli, S., Dubois, L., Bazer-Bachi, D., Lecocq, V., Bats, N., Farrusseng, D., 2012. Cryst. Eng. Commun 14 (12), 4105–4108. Tanabe, K.K., Cohen, S.M., 2011. Chem. Soc. Rev. 40, 498–519. Thakur, S., Kumar, P., Reddy, M.V., Siddavattam, D., Paul, A.K., 2013. Sens. Actuators B Chem. 178, 458–464. Wang, C., Xie, Z., deKrafft, K.E., Lin, W., 2011a. J. Am. Chem. Soc. 133, 13445–13454. Wang, F., Ke, X., Zhao, J., Deng, K., Leng, X., Tian, Z., Wen, L., Li, D., 2011b. Dalton Trans. 40, 11856–11865. Wang, Y.-P., Wang, F., Luo, D.-F., Zhou, L., Wen, L.-L., 2012. A luminescent nanocrystal metal–organic framework for sensing of nitroaromatic compounds. Inorg. Chem. Commun. 19, 43–46. Wu, S., Huang, H., Lan, X., Wang, X., Wang, J., Meng, C., 2013. Sens. Actuators B Chem 177, 724–729. Yan, J., Guan, H., Yu, J., Chi, D., 2013. Pest. Biochem. Physiol. 105 (3), 197–202. Yoon, Y.-J., Li, K.H.H., Low, Y.Z., Yoon, J., Ng, S.H., 2014. Sens. Actuators B Chem. 198, 233–238. Zhang, T., Zeng, L., Han, L., Li, T., Zheng, C., Wei, M., Liu, A., 2014. Anal. Chim. Acta 822, 23–29. Zhao, L., Zhao, F., Zeng, B., 2013. Sens. Actuators B. Chem. 176, 818–824.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Surface assembly of nano-metal organic framework on amine functionalized indium tin oxide substrate for impedimetric sensing of parathion Akash Deep a,b,n, Sanjeev Kumar Bhardwaj a,b, A.K. Paul a,b, Ki-Hyun Kim c, Pawan Kumar a,b,1 a
Central Scientific Instruments Organisation (CSIR-CSIO), Sector 30C, Chandigarh 160030, India Academy of Scientific and Innovative Research, CSIR-CSIO, Sector 30C, Chandigarh 160030, India c Department of Civil & Environmental Engineering, Hanyang University, 222 Wangsimni-Ro, Seoul 133-791, Republic of Korea b
art ic l e i nf o
a b s t r a c t
Article history: Received 14 July 2014 Received in revised form 8 October 2014 Accepted 9 October 2014 Available online 22 October 2014
The present paper reports the assembly and pesticide sensing application of a nanometal organic framework [Cd(atc)(H2O)2]n (‘atc’ ¼2-aminoterephthalic acid). The assembly of the NMOF film has been achieved by sequential dipping of a 2-aminobenzylamine (2-ABA) modified indium tin oxide (ITO) slide in organic linker ‘atc’ and metal ion ‘Cd2 þ ’ solutions. The different structural and morphological characteristics of the NMOF thin film have been characterized. The availability of pendent –COOH functional groups on the assembled NMOF film is exploited to synthesize a pesticide immunosensor by conjugating the NMOF film with anti-parathion antibody. This immunosensor has been explored for the electrochemical impedance spectroscopy (EIS) based analysis of parathion in the concentration range of 0.1– 20 ng/mL. The proposed detection is specific with respect to other organophosphate compounds, e.g. malathion, paraoxon, fenitrothion, monochrotophos and dichlorovos. The proposed sensor shows the detection limit of 0.1 ng/mL and it is applicable for analysis of parathion in a rice sample. The sensor's performance is validated by comparting the obtained results with gas chromatographic data. & Elsevier B.V. All rights reserved.
Keywords: Metal organic framework Sensing Impedance Parathion Self-assembly
1. Introduction Nanocrystal metal organic frameworks (NMOFs) have been projected useful in application areas such as, catalysis, nonlinear optics, electroluminescent devices, biomedical imaging and small molecules sensing (Krische and Lehn, 2000; Tanabe and Cohen, 2011; Cui et al., 2011; Della Rocca et al., 2011; Kumar et al., 2014a). NMOFs are characterized with better dispersive nature than the micsron scale MOFs and they can easily sense small molecules with no loss in their intrigue framework architectures and topologies (Wang et al., 2011a, 2011b). NMOFs with Eu(III), Zn(II) and Cd (II) as metal ion units have been reported for the detection of nitro aromatic compounds, small molecules and explosives (Kumar et al., 2014b; Wang et al., 2011a, 2011b; Li et al., 2008; Pramanik et al., 2011). Availability of pendent functional groups on some NMOF structures may be utilized to tag them with biomolecules n Corresponding author at: Central Scientific Instruments Organisation (CSIR-CSIO), Sector 30C, Chandigarh 160030, India. Fax: þ91 0172 2657287. E-mail addresses: [email protected], [email protected] (A. Deep). 1 Present address: Department of Civil & Environmental Engineering, Hanyang University, 222 Wangsimni-Ro, Seoul 133-791, Korea.
http://dx.doi.org/10.1016/j.bios.2014.10.045 0956-5663/& Elsevier B.V. All rights reserved.
such as nucleic acid, protein, antibody and enzymes (DeKrafft et al., 2009; McKinlay et al., 2010). A Cd ion based NMOF ‘[Cd(atc) (H2O)2]n’ (atc¼2-aminoterephthalic acid) has recently been proposed for the fluorescence quenching based detection of nitro aromatic compounds, including explosives and pesticides (Wang et al., 2012; Kumar et al., 2014a). Aqueous dispersibility and availability of pendent –COOH groups on this NMOF structure (Fig. S1) makes it an interesting choice for exploring in molecular sensing applications. Development of impedimetric immunosensors is an exciting field of research as this technique provide the realization of labon-chip kind of devices which can be integrated with microfluidic sample chambers and are easy to calibrate. Modern impedimetric immunosensors rely on the use of nanosized electrode materials such as carbon nanotubes (CNTs) and graphene because of their large surface areas (Zhang et al., 2014; Wu et al., 2013; Musameh et al., 2013; Yan et al., 2013). The NMOFs are also another class of nanomaterials which are known for their extremely large surface areas and availability of functional pendent groups. Due to hierarchical chemical assembly, the use of NMOF nanomaterials may offer the development of well-defined and ordered sensor geometry, which, in turn, is advantageous in the impedimetric
A. Deep et al. / Biosensors and Bioelectronics 65 (2015) 226–231
227
Fig. 1. FE-SEM imaging of (A): Bare ITO, (B): 2-ABA/ITO, (C): NMOF grown on 2-ABA/ITO surface, and (D): high resolution image for NMOF grown on 2-ABA/ITO surface.
immunosensor applications mainly in terms of sensor stability and sensitivity. The ready availability of functional groups on NMOFs is another useful property as the additional step of chemical oxidation (like in the case of CNTs and graphene) can be avoided. Though NMOFs have been investigated for optical sensing of some analytes, such as pesticides and explosives, their exploitation in electrochemical sensor technology is still an area to be explored. The MOF thin films are seen with significant potential in the future development of luminescent devices, magnetic films, redox-active films, and proton/electron-conducting films (Allendorf et al., 2011; Sadakiyo et al., 2009). Surface assembly technique offers one of the most robust routes for synthesizing the MOF thin films. In this work, we report the assembly of NMOF on a 2-aminobenzylamine (2-ABA) modified indium tin oxide (ITO) electrode. 2-ABA is a derivative of aniline, and its electrochemical deposition on ITO leaves behind free –NH2 moiety which is reacted with the carboxylate groups of the organic ligand atc. The morphological appearance of 2-ABA/ITO surface is shown in Fig. 1. The grafting of the organic ligand is then followed by sequential assembly of the metal center (Cd) and organic ligand. Five numbers of such steps provided the growth of rod like NMOF structure on the 2-ABA modified ITO. The pendent –COOH groups on thus assembled NMOF were then exploited for tagging it with anti-parathion antibody. The whole strategy has been depicted as schematic in Fig. S2. It is pertinent to mention here that parathion is one of the most toxic and widely used insecticides. High practical relevence of developing parathion biosensing devices has motivated the researchers to articulate on different strategies (Mulchandani and Rajesh, 2011). Amperometric and electrochemical techniques have been suggested for rapid and low cost detections of organophosphates (Zhang et al., 2014, Yoon et al., 2014; Li et al., 2014; Yan et al., 2013; Wu et al., 2013; Musameh et al., 2013; Funari et al.,
2013; Gong et al., 2013; Zhao et al., 2013; Mulchandani and Rajesh, 2011; Du et al., 2010; Lei et al., 2004; Mulchandani et al., 2001). Nafion-coated glassy carbon electrode has been proposed for the detection of as low as 50 nM of parathion (Funari et al., 2013). Organophosphate hydrolase functionalized electrodes have also been proposed (Thakur et al., 2013;Du et al., 2010; Lei et al., 2007). A limit of detection of 20 nM for both methyl-parathion and paraoxon has been reported by Mulchandani et al. (2001).
2. Materials and methods 2.1. Reagents and chemicals Indium tin oxide (ITO) substrates, cadmium nitrate, 2-aminoterephthalic acid (atc), EDC [ethyl(dimethylaminopropyl) carbodiimide, 99%], 2-aminobenzylamine (2-ABA), and MES buffer (2ethanesulfonic acid) were high purity chemical purchased from Sigma-Aldrich. Anti-parathion antibody (polyclonal IgG, rabbit anti-parathion) was purchased from Abcam. All other chemicals (including pesticides) and solvents were also AR/GR grade products from Sigma-Aldrich/Merck. All the experiments were carried out at room temperature (25 72 °C). 2.2. Preparation of sodium salt of 2-aminoterephthalic acid NaOH solution (2.2 g in 10 mL DI water) was mixed with atc solution (5 g in 20 mL DI water) under stirring, which resulted into formation of white precipitate. After 10 min of stirring at room temperature, the precipitate was collected by centrifugation and washed with water). This salt was dried at 100 °C.
228
A. Deep et al. / Biosensors and Bioelectronics 65 (2015) 226–231
2.3. Electrochemical grafting of 2-ABA on ITO
2.7. Characterizations
Chronoamperometry technique was employed to assemble 2-ABA on ITO substrate. 10 mM solution of 2-ABA was taken (phosphate buffer, pH 7) in a three-electrode configured electrochemical cell. ITO, platinum and Ag/AgCl were selected as working, counter and reference electrodes, respectively. A potential of 0.8 V was applied for 120 s to electro-deposit the 2-ABA film. The above value of the potential was selected based on a separate cyclic voltammetry experiment (Fig. S3). In this study, the cyclic voltammetry assisted electrodeposition of 10 mM 2-ABA solution was investigated, which revealed the appearance of cathodic peak at around 0.75–0.80 V. The duration of the electrodeposition experiment was optimized so as to attain smooth coverage (as investigated by FE-SEM) of 2-ABA on ITO slides. The electrodeposition cycles of 120 seconds offered smooth coverage of 2-ABA molecules. After electrodeposition, the film was washed and dried in vacuum. The adhesion of organic compound 2-ABA on the metallic ITO surface occurs at the metal/organic coating interface when interatomic bonds take place. These bonds can be primary (covalent or ionic bonds), secondary (dispersion forces, dipole interactions, and van der Waals forces) or hydrogen bridge type. Metal/polymer interfacial bonds are generally secondary or hydrogen bridge type.
Characterization were done with different instrumental techniques such as Fourier transform infrared spectroscopy (Nicolet, iS10), field-emission scanning electron microscopy (FE-SEM, 4300/ SE, Hitachi), X-ray diffractometry (XPert3 Powder, Panalytical), and electrochemistry workstation (Autolab, Metrohm). Gas chromatography experiments were carried out on GC (DANI Master) coupled with the electron capture detector.
2.4. Assembly of NMOF on 2-ABA/ITO Separate solutions of 0.05 M Cd(NO3)2 and 0.5 M sodium salt of atc were prepared. 2-ABA/ITO was first left in contact (dipping method) with the ligand solution for 10 min. Afterwards, the 2-ABA/ITO slide was washed (by dipping) with water and then introduced into the metal ion solution for 10 min. The said incubation time was optimized by investigating the assembly of metal–organic components for different time intervals (2, 4, 6, 8, and 10 min). Based on the desired product appearance (nano-rod like structure) on FE-SEM, it was found that minimum of 8 min of incubation time was required for the growth of the desired product. Further increase in the incubation time did not influence the formation of the desired product. In all the other experiments a uniform incubation time of 10 min was provided. Sequential dipping of the slide in the ligand and metal ion solutions were done for 5 times. NMOF were nucleated and grown to the surface in a controlled step by step procedure leading to the formation of its layers, which was visually apparent as a whitish film. Finally, the NMOF/2-ABA/ITO substrate was washed and then bioconjugated with anti-parathion antibody 2.5. Conjugation of anti-parathion antibody on NMOF/2-ABA/ITO For activation of the pendent –COOH functional groups on the synthesized NMOF, the slide was first left in contact with 5 mL of 0.1 M MES buffer (pH 5.0). 100 mg of EDC was added and the sample was incubated for 30 minutes. 1 mg mL 1 aqueous solution of anti-parathion was introduced and the sample was further incubated for 2 h at room temperature. The slide was carefully washed and thus prepared anti-parathion/NMOF/2-ABA/ITO sensing platform was stored in vacuum desiccator. 2.6. Electrochemical impedance immunoassay Impedance spectrum was recorded at the formal potential of redox couple (ferricyanide/ferrocyanide) i.e. 0.172 V (vs. screen printed Ag/AgCl) at an amplitude of 5 mV within the frequency range of 1 Hz–100 kHz.
2.8. Analysis of parathion in rice sample The origin of rice sample was in Punjab, India. Recovery of pesticide content from the sample was done according to the literature (Barkar, 2006). Dehusked rice sample was grinded and dissolved in water. The mixture was centrifuged at 5000 rpm for 15 min to collect the supernatant. The obtained solution was then passed through solid phase extraction (SPE) C18 column. It was followed by the elution step with methanol. The eluted sample was left to dry overnight in an oven. The dried mass was re-solubilized in water (for analysis by anti-parathion/NMOF/2-ABA/ITO sensor) or n-hexane (for analysis by GC). The sample thus obtained was analyzed by anti-parathion/NMOF/2-ABA/ITO sensor and gas chromatography (GC). Appropriate dilutions of the sample were prepared in order to fit the EIS signal within the calibration range of the sensor. For GC experiments, the oven temperature was first kept at 40 °C for 5 min and incremented to 250 °C with step rate of 5 °C/ min. A temperature of 250 °C was maintained for 5 min. The flow rate of carrier gas (N2) was maintained at 1 mL/minute. The injector port temperature was programmed at 250 °C. Electron capture detector (ECD) was kept at 320 °C and 30 mL/min flow of N2 was maintained for the detection of organophosphates. 2 mL of the sample was injected each time for the sample analysis.
3. Results and discussion 3.1. Synthesis and characterization of NMOF on 2-ABA/ITO The structural morphology of bare ITO slide, the electrodeposited 2-ABA particles (on ITO slide), and NMOF grown onto the 2-ABA/ITO slide is presented in Fig. 1. It is fairly apparent that the 2-ABA particles have modified the ITO surface. The presence of 2-ABA is characterized with the appearance of globular nanosized particles. In comparison, the crystals of NMOF on 2-ABA/ITO were formed in nanorod shape (diameter 100–250 nm). There is an apparent modification of the 2-ABA/ITO surface after the assembly of NMOF. Five sequential dipping cycles of the 2-ABA/ITO slide in the organic linker solution and metal ion solution provided a smooth coverage of the NMOF on the 2-ABA/ITO surface without any apparent lack of continuity. Lesser numbers of cycles left voids on the electrode surface. Similar nanorod like morphology of the NMOF has been reported when bulk synthesis method was used (Wang et al., 2012; Chen et al., 2007). The formed layer of the NMOF was fairly uniform as suggested by the line profile analysis of the scanned atomic force microscopy (AFM) image (Fig. S4). The roughness of the film was limited in the range of 7 5 nm. The binding of the NMOF on the amine terminated 2-ABA/ITO surface is chemically achieved through the amide linkage between the – NH2 groups (of 2-ABA) and free –COOH pendent groups (available on the NMOF surface). Powder XRD data of the synthesized NMOF film (Fig. 2) matched with the previously reported findings (Wang et al., 2012; Chen et al., 2007). NMOF has monoclinic crystal structure with space group C2/c in which Cd(II) ions of equatorial sites are occupied by two bidentate carboxylate oxygen atoms, one
A. Deep et al. / Biosensors and Bioelectronics 65 (2015) 226–231
229
Fig. 2. PXRD data of bulk NMOF and NMOF grown on 2-ABA/ITO.
carboxylate oxygen atom from the second H2atc ligand, one oxygen atom from the water and one nitrogen atom of the amino group from the third H2atc ligand (Chen et al., 2007). FTIR spectrum of NMOF is shown in Fig. 3. The appearance of vibration bands at 1610, 1507, 1570, 1457 and 1358 cm 1 in NMOF may be assigned to the stretching vibrations of higher energy and bending vibrations of lower energy of C–O in carboxylate groups pertaining to the presence of organic ligand in structure (Rai et al., 2013 and Savonnet et al., 2012). Another band due to C–N appears at 1285 cm 1. The appearance of bands in the lower frequency region (500–750 cm 1) may be assigned to the involved Cd–O linkages. 3.2. Development of sensor Incubation of NMOF/2-ABA/ITO substrate with anti-parathion antibody has resulted into the formation of a novel immunosensing platform of parathion. Successful biofunctionalization was confirmed by FTIR analysis (Fig. 3). Characteristic C ¼O (CONH2) stretching was observed at 1637 cm 1 due to the attachment of protein through amide bond formation. The presence of two weak bands at 2825 and 2930 cm 1 are also suggestive of the successful grafting of protein molecules with NMOF. 3.3. Electrochemical detection of parathion Fig. 4 shows the Nyquist plots for electrochemical impedance spectroscopy (EIS) experiments done on ITO, 2-ABA/ITO, NMOF/2-
Fig. 3. FTIR of NMOF/2-ABA/ITO, anti-parthion antibody, and anti-parathion/ NMOF/2-ABA/ITO sensor.
Fig. 4. Nyquist plots for ITO, 2-ABA/ITO, NMOF/2-ABA/ITO and anti-parathion/ NMOF/2-ABA/ITO.
ABA/ITO and anti-parathion/NMOF/2-ABA/ITO platforms. In Nyquist plot, the imaginary impedance component (Z″, out-of-phase) is plotted against the real impedance component (Z′, in-phase) at each excitation frequency. In the case of the proposed immunosensor, a non-insulating dielectric antibody layer covers the surface of the electrode, which is able to catalyze the redox probe in the measuring solution. The measured parameter ‘charge transfer resistance’ is actually the real component of impedance. The antibody–antigen interactions cause an increase in its value as the faradaic reaction becomes increasingly hindered, which is clear from the non-existence of typical straight lines after the semi-circle shape. The immunosensor configuration resembles a capacitor in its ability to store charge. An event of antibody–antigen interaction leads to a decrease in the total capacitance of the system. Total capacitance ‘Ct’ of the whole system can be described according to the following equation:
1/Ct = (1/Cdl) + (1/Cbm)
(1)
where Cdl denotes the capacitance due to dielectric layer and Cbm denotes the capacitance due to outer biomolecule layer. Since the current must pass through the uncompensated resistance of the electrolyte solution, its value is also taken into account while evaluating the sensor response. The impedance data have been fitted with software Zview. Randle's equivalent circuit was chosen for determining the values of charge transfer resistance. This circuit is often used to model the interfacial phenomena and it takes into account the uncompensated resistance of the electrolyte in series with the capacitance of the dielectric layer (Cdl) and the charge transfer resistance (Rct). An additional component, the Warburg impedance (Zw) accounts for the diffusion of ions from bulk electrolyte to the electrode interface. Compared to 2-ABA/ITO, the NMOF/2-ABA/ITO shows increase in charge transfer resistance confirming the growth of NMOF on 2-ABA/ITO. Observed electrical conductivity of the NMOF/2-ABA/ ITO could be exploited for various biosensing applications. The attachment of protein further increases the charge transfer resistance, once again confirming the successful immobilization of protein. The increase in the value of charge transfer resistance at this step may be attributed to the hindrance in the diffusion of the ferricyanide (redox probe) toward the electrode surface as a
230
A. Deep et al. / Biosensors and Bioelectronics 65 (2015) 226–231
Table 1 Analysis of parathion in rice samples. Sample no.
GC analysis (mg/L)
Anti-parathion/NMOF/2-ABA/ITO analysis
1 2 3 4
2.57 2.65 2.50 2.49
2.48 mg/L 2.54 mg/L 2.37 mg/L 2.47 mg/L
(s.d. ¼ 0.060) (s.d. ¼0.061) (s.d. ¼ 0.058) (s.d. ¼0.065)
The applicability and validity of the sensor were demonstrated by detecting the parathion concentration in rice sample (Table 1). The analyzed concentration of parathion (2.57 ppm) in the sample agreed well with the results from gas chromatography.
4. Conclusions Fig. 5. EIS response of the anti-parathion/NMOF/2-ABA/ITO platform toward varying concentrations (0.1–20 ng/mL) of parathion.
results of protein layering. Fig. 5 shows the EIS response of the anti-parathion/NMOF/2-ABA/ITO platform towards varying concentrations (0.1–20 ng/mL) of parathion. The charge transfer resistance (Rct) increases as the concentration of the analyte increases. At concentrations lower than 0.1 ng/mL, the signal was unstable. The increase in the charge transfer resistance with the increasing parathion concentration is caused by the increased binding of the antigen molecules to the immobilized antibodies which acts as a definite kinetic barrier for the electron transfer. The increase in the charge transfer resistance values after the antibody–antigen binding may also be correlated with the fact that increasing antigen–antibody binding events eventually block the free spaces on the bioelectrode. Calibration of the measurements is also shown in Fig. 5. Clearly the detection is possible over a wide linear range of analyte concentration. The limit of detection was 0.1 ng/mL under the given experimental conditions, which is better than most of the earlier studies (Table S1). Specificity of the parathion detection was analyzed with respect to some other organophosphate pesticides, namely malathion, paraoxon, fenitrothion, monochrotophos and dichlorovos. For these experiments the response of the anti-parathion/NMOF/ 2-ABA/ITO sensor was recorded for 0.1 ng/mL parathion in the presence of three different concentrations (0.1/10/50 ng/mL) of the above possible interferants. The response of the sensor toward parathion was unchanged (variation in Rct ¼ 73.5%) thereby highlighting superior specificity of the proposed immunosensing system. In a separate experiment, the sensor response was also investigated against a fixed concentration (50 ng/mL) of the malathion, paraoxon, fenitrothion, monochrotophos and dichlorovos. The EIS response (estimated Rct) of the anti-parathion/NMOF/2ABA/ITO sensor in these cases was observed to be 5990 710 thus again suggesting that the proposed sensor was responsive to the target analyte (parathion) only. The reproducibility of the sensor was investigated through intra- and interassay precision. For intraassay precision, the sensor's response was periodically checked with respect to 0.1 ng/mL parathion. The sensor was stable (7 3% variation in the values of Rct) over a period of 10 days. The stability of the sensor's EIS response as a function of storage time was also evaluated. It was observed that the proposed sensor could retain its EIS characteristics (7 5% variation in the values of Rct) even after 25 days of storage (storage conditions: medium 10 mM PBS, pH 7.4, T¼ 4 °C). Similarly, interassay precision was estimated by measuring 0.1 ng/mL of parathion concentration with four separately prepared sensors. In this case, the variation in the sensors' response was limited to 74%.
In summary, the present work first time reports the use of NMOF based immunosensing platform for sensitive and highly specific impedimetric detection of an organophosphate pesticide. The self-assembly of NMOF onto a 2-ABA modified ITO may offer the development of many such immunosenors in future. The use of antibody has provided the achievement of the desired specificity. The sensitivity of the proposed sensor is better or comparable to the existing systems (Table S1). Being a hierarchical structure, NMOF assembles in a well-defined geometry which may also help in achieving more stable results than other random platforms such as nafion based electrodes. The proposed use of antibody offers a direct advantage in terms of the specificity over organophosphate hydrolase enzyme based sensors. Moreover, such sensor systems can also be explored as host–guest interaction based direct chemical sensing.
Acknowledgments Authors gratefully acknowledge the financial grant from CSIR India through project OMEGA/PSC0202/2.2.5. We are thankful to the Director, CSIR-CSIO, Chandigarh, India. The fourth author acknowledges partial financial support from the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST) (No. 2009-0093848).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.10.045
References Allendorf, M.D., Schwartzberg, A., Stavila, V., Talin, A.A., 2011. Chem. Eur. J 17, 11372–11388. Barkar, S.A., 2006. Use of matrix solid-phase dispersion for determining pesticides in fish and food. In: Vidal, J.L.M., Vidal, J.L.M. (Eds.), Pesticide Protocols: Methods in Biotechnology. Humana Press, NewJersy, pp. 285–296. Chen, B., Yang, Y., Zapata, F., Lin, G., Qian, G., Lobkovsky, E.B., 2007. Adv. Mater. 19, 1693–1696. Cui, Y., Yue, Y., Qian, G., Chen, B., 2011. Chem. Rev. 112, 1126–1162. DeKrafft, K.E., Xie, Z., Cao, G., Tran, S., Ma, L., Zhou, O.Z., Lin, W., 2009. Angew. Chem. Int. Ed. 48, 9901–9904. Della Rocca, J., Liu, D., Lin, W., 2011. Acc. Chem. Res 44, 957–968. Du, D., Chen, W., Zhang, W., Liu, D., Li, H., Lin, Y., 2010. Biosens. Bioelectron. 25, 1370–1375. Funari, R., Della Ventura, B., Schiavo, L., Esposito, R., Altucci, C., Velotta, R., 2013. Anal. Chem. 85, 6392–6397. Gong, J., Guan, Z., Song, D., 2013. Biosens. Bioelectron. 39 (1), 320–323.
A. Deep et al. / Biosensors and Bioelectronics 65 (2015) 226–231
Krische, M., Lehn, J.-M., 2000. The utilization of persistent h-bonding motifs in the self-assembly of supramolecular architectures. In: Fuiita, M. (Ed.), Molecular Self-Assembly Organic Versus Inorganic Approaches. Springer, Berlin Heidelberg, pp. 3–29. Kumar, P., Kumar, P., Bharadwaj, L.M., Paul, A.K., Deep, A., 2014a. Inorg. Chem. Commun. 43, 114–117. Kumar, P., Paul, A.K., Deep, A., 2014b. Anal. Method 6, 4095–4101. Lei, C., Valenta, M.M., Saripalli, K.P., Ackerman, E.J., 2007. J. Environ. Qual. 36, 233–238. Lei, Y., Mulchandani, P., Chen, W., Wang, J., Mulchandani, A., 2004. Biotechnol. Bioeng. 85, 706–713. Li, Y., Xu, M., Peipei, L., Dong, J., Ai, S., 2014. Anal. Methods 6 (7), 2157–2162. Li, Z.-Q., Qiu, L.-G., Wang, W., Xu, T., Wu, Y., Jiang, X., 2008. Inorg. Chem. Commun. 11, 1375–1377. McKinlay, A.C., Morris, R.E., Horcajada, P., Férey, G., Gref, R., Couvreur, P., Serre, C., 2010. Angew. Chem. Int. Ed. 49, 6260–6266. Mulchandani, A., Rajesh, 2011. Appl. Biochem. Biotechnol. 165, 687–699. Mulchandani, P., Chen, W., Mulchandani, A., 2001. Environ. Sci. Technol. 35 (12), 2562–2565. Musameh, M., Notivoli, M.R., Hickey, M., Huynh, C.P., Hawkins, S.C., Yousef, J.M., Kyratzis, I.L., 2013. Electrochim. Acta. 101, 209–215. Pramanik, S., Zheng, C., Zhang, X., Emge, T.J., Li, J., 2011. J. Am. Chem. Soc. 133, 4153–4155.
231
Rai, S., Bajpai, A., Lokhandwala, S., 2013. J. Polym. , http://dx.doi.org/10.1155/2013/ 278576 (Article ID 278576, 6 pages). Sadakiyo, M., Yamada, T., Kitagawa, H., 2009. J. Am. Chem. Soc. 131, 9906–9907. Savonnet, M., Canivet, J., Gambarelli, S., Dubois, L., Bazer-Bachi, D., Lecocq, V., Bats, N., Farrusseng, D., 2012. Cryst. Eng. Commun 14 (12), 4105–4108. Tanabe, K.K., Cohen, S.M., 2011. Chem. Soc. Rev. 40, 498–519. Thakur, S., Kumar, P., Reddy, M.V., Siddavattam, D., Paul, A.K., 2013. Sens. Actuators B Chem. 178, 458–464. Wang, C., Xie, Z., deKrafft, K.E., Lin, W., 2011a. J. Am. Chem. Soc. 133, 13445–13454. Wang, F., Ke, X., Zhao, J., Deng, K., Leng, X., Tian, Z., Wen, L., Li, D., 2011b. Dalton Trans. 40, 11856–11865. Wang, Y.-P., Wang, F., Luo, D.-F., Zhou, L., Wen, L.-L., 2012. A luminescent nanocrystal metal–organic framework for sensing of nitroaromatic compounds. Inorg. Chem. Commun. 19, 43–46. Wu, S., Huang, H., Lan, X., Wang, X., Wang, J., Meng, C., 2013. Sens. Actuators B Chem 177, 724–729. Yan, J., Guan, H., Yu, J., Chi, D., 2013. Pest. Biochem. Physiol. 105 (3), 197–202. Yoon, Y.-J., Li, K.H.H., Low, Y.Z., Yoon, J., Ng, S.H., 2014. Sens. Actuators B Chem. 198, 233–238. Zhang, T., Zeng, L., Han, L., Li, T., Zheng, C., Wei, M., Liu, A., 2014. Anal. Chim. Acta 822, 23–29. Zhao, L., Zhao, F., Zeng, B., 2013. Sens. Actuators B. Chem. 176, 818–824.
