Available online at www.sciencedirect.com ScienceDirect Procedia Engineering 168 (2016) 448 – 451 30th Eurosensors Conference, EUROSENSORS 2016 Molecularly imprinted polymer based sensor to detect isoborneol in aqueous samples G.S Bragaa,b*, P.A Lieberzeitb, F.J Fonsecaa b a University of São Paulo, Polytechnic School, Av Prof Luciano Gualberto, travessa 3, 158, São Paulo, 05508-010, Brazil University of Vienna, Faculty for Chemistry, Department of Physical Chemistry, Währinger Strasse, 42, Vienna, 1090, Austria Abstract Herein we report the development of a selective chemical sensor based on molecularly imprinted polymers (MIP) and quartz crystal microbalance (QCM) to detect isoborneol (ISO) in aqueous samples Their response scales linearly with isoborneol concentration and exhibits sensitivity of -16.23 Hz mM-1 cm-2 at the active area, with a gradient of -25 Hz mM-1 Moreover, they respond rapidly, reversibly and reproducibly Sensor layers are stable within a period of four weeks during use as compared to a few days for natural receptors Moreover, ISO-MIP sensor calculated theoretical detection limit is 0.58 mM © Authors Published by Elsevier Ltd This ©2016 2016The The Authors Published by Elsevier Ltd.is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference Keywords: molecularly imprinted polymer; quartz crystal microbalance; isoborneol; aqueous samples; methacrylic acid Introduction Sensors based on molecularly imprinted polymers (MIP) are usually highly selective and often called “biomimetic sensors” because their interaction type is very similar to the ones found in biological materials, such as enzyme-substrate and antibody-antigen MIPs exhibit some advantages over biological materials, such as increased shelf time due to the strength and resistance of the polymer and higher resistance to extremes environmental conditions of temperature and humidity [1] * Corresponding author Tel.: +55 11 3091-0631; fax: +55 11 3091-5300 E-mail address: gbraga@lme.usp.br 1877-7058 © 2016 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference doi:10.1016/j.proeng.2016.11.542 G.S Braga et al / Procedia Engineering 168 (2016) 448 – 451 During MIP synthesis, polymerization reactions occur in the presence of the template After the matrix has hardened, template is removed from it with a suitable solvent The rebinding effect is based on reversible (selective) interactions between polymer matrix and template and is detected using quartz crystal microbalance (QCM) among others QCM is a sensitive acoustic device that changes its fundamental frequency depending on mass bound to its surface [2] Within this work, we describe the development of MIP based sensor to detect isoborneol (ISO) in aqueous samples ISO is a component of several essential plant oils and is used as antiviral for herpes [3], antibacterial [4], for cardiovascular diseases treatment [5], and as a natural repellent [6,7] It is structurally related to 2-methylisoborneol, an important pollutant in water To the best of our knowledge, this is the first ISO MIP based sensor developed Experimental 2.1 Synthesis of ISO imprinted polymer 130 Pmol of ISO and 802 Pmol of methacrylic acid (MAA) were dissolved in 200 Pml of hexane by sonication for 10 minutes Later, they were incubated for hours to form ISO-MAA complex [8] 1,060 Pmol ethylene glycol dimethacrylate (EGDMA) and 30.4 Pmol 2,2-azobis(isobutyronitrile) (AIBN) were added to the solution and sonicated for another 10 minutes After purging with nitrogen, this solution was pre-polymerized in hot water bath (60o C) for about one hour Polymerization is stopped prior to the gel point After one hour, the translucent solution turned opaque, indicating successful pre-polymerization [9], which enhances film stability and adhesion [10] The control, non-imprinted polymer, was prepared in the same way in the absence of the template 2.2 Molecularly imprinted sensor fabrication Sensor device consisted of gold electrodes screen printed (brilliant gold paste 10%) onto AT-cut crystal sheets of 10 MHz (14 mm in diameter and 168 Pm in thickness) coated with ISO-imprinted polymer (ISO-MIP) solution Polymer layers were deposited onto transducer surfaces by spin coating PL of the pre-polymerized ISO-imprinted solution onto the electrodes (10 seconds at 500 RPM), followed by UV polymerization (360 nm, 210W) for about 48 hours After polymerization, sensors were placed in ethanol (15 minutes) for template removal A reference sensor was prepared in the same way with the non-imprinted polymer (NIP) solution 2.3 Sensor measurements QCM based sensors were placed within a dedicated measuring cell which was connected to a homemade oscillator circuit The oscillator resonance frequency as function of time was recorded by a frequency counter (AGILENT 53131A) Sensor signals were transferred to a computer through a homemade platform using LabVIEW 2.4 Analytical procedure The frequency response of the ISO-MIP based sensor was monitored by first immersing the sensor into deionized water After achieving stable base line (reference value) ISO solution was added to the measuring cell After reaching the equilibrium and noting sensor response time, deionized water was flushed into the cell to remove bound ISO molecules The aforementioned procedure consisted a measuring cycle and was repeated for every measurement at least three times Frequency shift was calculated subtracting the frequency response obtained for ISO solution from the reference value Results and discussion The frequency responses of the sensors (ISO-MIP and NIP), shown in Fig 1, decrease linearly as ISO concentration increases Such decrease was expected, since the more concentrated the solution, the more molecules are available to bind to the imprinted cavities present at polymer matrix [2] Also, sensor signal, i.e the difference between 449 450 G.S Braga et al / Procedia Engineering 168 (2016) 448 – 451 MIP and NIP for each concentration, respectively, decreases linearly with good correlation (R2 = 0.982) as ISO concentration increases from mM to mM They exhibited a sensitivity of -16.23 Hz mM-1 cm-2, with an area of 1.54 cm2 and a gradient of -25 Hz mM-1 Moreover, sensor signal was used to calculate a theoretical detection limit (LoD): 0.58 mM ISO-MIP based sensors achieved equilibrium after about 90 seconds following addition of mM ISO solution and circa of 60 seconds after adding more diluted ISO solution (2.5 mM) as shown at Fig These results demonstrate direct relationship between the response time of the sensor and solution concentration, i.e., the more concentrated the solution is, the longer it takes for the signal to get stable One hypothesis for this behavior is that template binds preferably with the cavities at the polymeric layer’s surface (faster binding) and, as the concentration increases, ISO molecules have to permeate further into the polymeric matrix for rebinding (lower mobility), increasing the time to achieve a stable signal Isoborneol concentration (mM) Frequency shift (Hz) y = -10.953x + 10.451 R² = 0.9564 -50 y = -24.862x + 14.374 R² = 0.9823 -100 y = -35.815x + 24.825 R² = 0.9992 -150 -200 MIP NIP Sensor signal Fig MIP (circle), NIP (triangle) and sensor signal (square) calibration curves and its respective fitting equations Frequency (Hz) 600 mM 500 2.5 mM 2.5 mM 594 s 370 s 400 2.5 mM 756 s 124 s 300 634 s 200 818 s 448 s 100 216 s 0 ISO-MIP SO-MIP 100 200 300 400 500 600 700 800 900 Time (s) Fig ISO-MIP sensor frequency response in function of time towards 2.5 mM and mM isoborneol solutions (indicated in light blue) Sensor responses turned out appreciably repeatable after 10 consecutive measuring cycles (deionized water – mM ISO solution), indicated by the low change in frequency shift value (-249.93 Hz ± 12.45 %) and signal loss of 3.26% (Fig 3a) However long term experiments reveal some signal loss of circa 11% measured in a four-week period, even though it remained quite stable during the first two weeks (Fig 3b) This can be expected to be overcome by improving adhesion between polymer and gold electrode Sensor signal loss exhibited linear behavior as can be noted by the respective linear fit with R-squared value of 0.979 451 G.S Braga et al / Procedia Engineering 168 (2016) 448 – 451 Measuring cycles A) B) -50 -100 -150 -200 -250 -300 ISO-MIP Frequency shift(Hz) Frequency shift (Hz) -350 Number of weeks 10 -50 ISO-MIP -100 -150 -200 y = 9.6516x - 272.37 R² = 0.9795 -250 -300 Fig 3: ISO-MIP sensor response repeatability in function of ten consecutive measuring cycles (A) and for a period of four weeks (B) Conclusion The developed MIP sensors exhibited a sensitivity of -16.23 Hz mM-1 cm-2 towards isoborneol, with a sensor signal about three times higher than the one obtained with the non-imprinted polymer (NIP) based sensors Sensor response scales linearly with isoborneol concentration in the range of to mM, which is basically the solubility limit of isoborneol in water Moreover, they possess fast response time of about 90 seconds for high concentrated ISO solutions (5 mM) and even faster response time for less concentrated solutions (average of 60 seconds) and appreciable repeatability on sensor response with a signal loss of circa 3% after 10 measuring cycles However long term experiments reveal some signal loss of circa 11% measured in a four-week period Overall, the calculated theoretical detection limit is 0.58 mM As a first step towards real-life sensing, the experimental results obtained so far are very encouraging Acknowledgements Authors wish to thanks FAPESP (2015/05359-2 and 2013/19421-6) for its financial support References [1] M Hussain, J Wackerlig, P.A Lieberzeit; Biomimetic strategies for sensing biological species; Biosensors (2013) 89-107 [2] S.Z Bajwa, Template polymer nanostructures-chemical sensors towards CU(II) ions and TiO2 nanoparticles, PhD Thesis, Universität Wien, Austria, 2012, 122 pages [3] M Armaka, et al.: Antiviral properties of isoborneol, a potent inhibitor of herpes simplex virus type Antiviral Research 43 (1999) 79-92 [4] N Chaftar, et al.: Activity of six essential oils extracted from tunisian plants against Legionella pneumophila Chem & Biodiversity 12 (2015), 1565-1574 [5] X Wu, et al.: Gas chromatography-mass spectrometry and high-performance liquid chromatography analysis of the drug absorption characteristics in the buccal mucosa via a circulating device Biomedical Reports (2015) 51-54 [6] V-U Bläske, et al.: Repellent effects of isoborneol on subterranean termites (isopteran: rhinotermitidae) in soils of different composition J of Economic Entomology 96 (2003) 1267-1274 [7] H Sun, et al.: Effects of aphid herbivory on volatile organic compounds of Artemisia annua and Chrysanthemum morifolium Biochem Sys And Ecol.60 (2015) 225-233 [8] W Dong; et al.; Effects of solvents on the adsorption selectivity of molecularly imprinted polymers: molecular simulation and experimental validation; Separation and Purification Technology 53 (2007) 183-188 [9] N.V.H Phan, H.F Sussitz, P.A Lieberzeit; Polymerization parameters influencing the QCM response characteristics of BSA MIP; Biosensors (2014) 161-171 [10] S.Z Bajwa, R Dumler, P.A Lieberzeit, Molecularly imprinted polymers for conductance sensing Cu+2 in aqueous solutions; Sensors and Actuators B 192 (2014) 522-528 ... mM-1 cm-2 towards isoborneol, with a sensor signal about three times higher than the one obtained with the non -imprinted polymer (NIP) based sensors Sensor response scales linearly with isoborneol. .. first immersing the sensor into deionized water After achieving stable base line (reference value) ISO solution was added to the measuring cell After reaching the equilibrium and noting sensor response... fundamental frequency depending on mass bound to its surface [2] Within this work, we describe the development of MIP based sensor to detect isoborneol (ISO) in aqueous samples ISO is a component