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Nanobiosensors exploiting specific interactions between an enzyme and herbicides in atomic force spectroscopy (jnn)

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Transcripts - Nanobiosensors exploiting specific interactions between an enzyme and herbicides in atomic force spectroscopy (jnn)

  • 1. Delivered by Publishing Technology to: UNIVERSIDADE SAO PAULO IF IP: 143.107.252.60 On: Tue, 25 Mar 2014 22:19:28 Copyright: American Scientific Publishers Copyright © 2014 American Scientific Publishers All rights reserved Printed in the United States of America Article Journal of Nanoscience and Nanotechnology Vol. 14, 6678–6684, 2014 www.aspbs.com/jnn Nanobiosensors Exploiting Specific Interactions Between an Enzyme and Herbicides in Atomic Force Spectroscopy Aline C. N. da Silva1 , Daiana K. Deda1 ∗ , Carolina C. Bueno1 , Ariana S. Moraes1 , Alessandra L. Da Roz1 , Fabio M. Yamaji1 , Rogilene A. Prado2 , Vadim Viviani2 , Osvaldo N. Oliveira, Jr3 , and Fábio L. Leite1 ∗ 1 Department of Physics, Chemistry and Mathematics, Nanoneurobiophysics Research Group, Federal University of São Carlos, P.O. Box 3031, 18052-780, Sorocaba, SP, Brazil 2 Laboratory of Biochemistry and Biotechnology of Bioluminescence, Department of Physics, Chemistry and Mathematics, Federal University of São Carlos, P.O. Box 3031, 18052-780, Sorocaba, SP, Brazil 3 São Carlos Institute of Physics, University of São Paulo, São Carlos, P.O.Box 369, 13560-970, SP, Brazil The development of sensitive methodologies for detecting agrochemicals has become important in recent years due to the increasingly indiscriminate use of these substances. In this context, nanosensors based on atomic force microscopy (AFM) tips are useful because they provide higher sensitivity with operation at the nanometer scale. In this paper we exploit specific interactions between AFM tips functionalized with the enzyme acetolactate synthase (ALS) to detect the ALS- inhibitor herbicides metsulfuron-methyl and imazaquin. Using atomic force spectroscopy (AFS) we could measure the adhesion force between tip and substrate, which was considerably higher when the ALS-functionalized tip (nanobiosensor) was employed. The increase was approximately 250% and 160% for metsulfuron-methyl and imazaquin, respectively, in comparison to unfunctionalized probes. We estimated the specific enzyme-herbicide force by assuming that the measured force comprises an adhesion force according to the Johnson–Kendall–Roberts (JKR) model, the capillary force and the specific force. We show that the specific, biorecognition force plays a crucial role in the higher sensitivity of the nanobiosensor, thus opening the way for the design of similarly engineered tips for detecting herbicides and other analytes. Keywords: Enzymes, Herbicides, Nanobiosensors, Atomic Force Microscopy, Atomic Force Spectroscopy, Chemical Force Microscopy. 1. INTRODUCTION The growing global demand for food has led to the use of pesticides in ever increasing quantities,1 2 of which only an estimated 0.1% reach their targeted pests.3 4 The remaining 99.9% translocate to other environmental areas, causing direct damage to flora, fauna and human health due to their highly cytotoxic and genotoxic effects.3–8 Detecting agro- chemicals with greater efficiency, speed, and sensitivity9–14 than traditional chromatographic methods15–17 has there- fore become important. Sensors and biosensors based on chemically modified cantilevers may, in this context, be a promising alternative to detection18–24 due to their excel- lent performance in detecting analytes,25–29 including agro- chemicals. Specific interactions, such as the “lock and key” or “host-guest” mechanisms, for they are selective ∗ Authors to whom correspondence should be addressed. with the binding of analytes to sensing molecules.30 31 These devices utilize a combination of biomolecule recep- tors and a physicochemical detector, which together enable the recognition of a specific analyte in a medium.32 33 An essential requirement is then a well-controlled immobiliza- tion of functional biomolecules on surfaces or nanoma- terials, which has indeed been used in clinical diagnosis, investigation of biomolecular interactions, environmental monitoring, and quality control of food.20 30 34–41 Sensors based on specific interactions may employ atomic force microscopy (AFM) as a force appara- tus,19 42–46 where cantilevers are functionalized with sen- sitive materials such as polymers, enzymes or antibodies. With the interaction with target molecules that selectively adsorb or bind by chemical affinity onto the cantilever, selective, sensitive sensors can be produced.20 47 This is the case of a nanobiosensor designed with microcantilevers 6678 J. Nanosci. Nanotechnol. 2014, Vol. 14, No. 9 1533-4880/2014/14/6678/007 doi:10.1166/jnn.2014.9360
  • 2. Delivered by Publishing Technology to: UNIVERSIDADE SAO PAULO IF IP: 143.107.252.60 On: Tue, 25 Mar 2014 22:19:28 Copyright: American Scientific Publishers Silva et al. Nanobiosensors Exploiting Specific Interactions Between an Enzyme and Herbicides in Atomic Force Spectroscopy functionalized with the enzyme acetolactate synthase (ALS) for detecting metsulfuron-methyl.48 Even though there are sensors that use the technique of covalent func- tionalization of silicon surfaces,49 as well as the enzyme immobilization for enhancing the recognition (as sense element),50 the technology described in this work is the first report about using the synergetic effect of the covalent character of the Si C bond combined to mimetic mech- anism of enzymatic inhibition by herbicides. This unique structure provided by AFS is a favorable microenviron- ment to maintain the bioactivity of an enzyme, which led to a rapid recognition response through force curves. In this paper, we extend the previous work to detect another herbicide, imazaquin, and estimate the specific, biorecog- nition force between the enzyme and the herbicide. This is performed with a series of atomic force spectroscopy (AFS) measurements, whose data are evaluated using the- oretical models to calculate the adhesion and the capillary forces. We shall show that the specific interaction is essen- tial for the high sensitivity of the nanobiosensors. 2. METHODOLOGY 2.1. Expression of Recombinant ALS Recombinant ALS was kindly provided by Dr. Tsutomu Shimizu from Life Science Research Institute, Shizuwoka, Japan. The cDNA of the ALS gene (from Oryza sativa) was incorporated into Eco RI sites of the pGEX 2T vector and used to transform the E. coli BL21-DE3 strain. The colonies were grown in 500–1000 mL of LB medium con- taining ampicillin at a temperature of 37 C until reaching an OD600 of 0.4, then induced at 22 C for 3–4 h. The cell suspension was centrifuged at 2,500 g for 15 min and resuspended in 1X PBS buffer containing complete pro- tease inhibitors (Roche), then freeze-thawed three times in dry ice and centrifuged at 15,000 g for 15 min at 4 C. The supernatant (crude extract) was used for cantilever functionalization. 2.2. Chemical Functionalization of Tips and Substrates The nanobiosensor was fabricated according to the method reported in Ref. [48]. The functionalization procedure for the Si3N4 tips, cantilevers and substrates (muscovite mica) was adapted from the method described by Wang and collaborators.51 The tips and substrates were cleaned by irradiation in a UV chamber (240 nm; Procleaner, UV.PC.220, BIOForce Nanosciences, Ames, IA, USA).52 The functionalization was initiated by gaseous evapora- tion of 3-aminopropyl triethoxysilane (APTES) in the presence of triethylamine (both as commercial solutions), followed by the addition of a small aliquot of a glutaralde- hyde solution (1×10−3 M). Subsequently, 100 L of the ALS enzyme extract (0.200 mg/mL) were added to the probe tips, and 200 L of the ALS-inhibiting herbicides metsulfuron-methyl and imazaquin were added to the sub- strates (1×10−3 M). All reagents used, with the exception of the ALS (Section 2.2), were purchased from Sigma. 2.3. Atomic Force Spectroscopy (AFS) Force spectroscopy experiments were performed at 25 C and a relative humidity of approximately 35%. The force curves were obtained using an Atomic Force Micro- scope Multimode-VS System with the PicoForce pack- age (dedicated to force spectroscopy). The Si3N4 AFM tips (V-shaped, model NP-10 by Veeco) employed in the measurement of force curves possessed a nominal spring constant of 0.12 N/m. Considerable variations can occur between the nominal and real value of the spring constant; each AFM tip was therefore calibrated using the thermal noise method.53 The detection of herbicides was confirmed by examining the force curves obtained using two types of tips: (i) tips functionalized with the ALS enzyme and (ii) unfunctional- ized tips. To evaluate the efficiency of the nanobiosensor, adhesion force values were obtained on various substrates, at different points on each substrate and using different tips. The adhesion force values reported represent the aver- age of 30 force curves obtained at the same point on the substrate. 2.4. Contact Angle and Surface Energy Contact angle analysis was performed to determine the surface energies for calculating the work of adhesion and the theoretical adhesion force between the AFM tips and the substrates contaminated with herbicides. Con- tact angle measurements were performed at 25 C using CAM200 equipment by KSV. Due to the small size of the tip, the system was reproduced on the macroscopic scale using a functionalized silicon plate. Addition- ally, measurements were performed on mica/metsulfuron- methyl and mica/imazaquin substrates. The measurements employed water, formamide and diiodomethane as liquids, whose surface tensions are 72.2, 58.3 and 50.8 mJ/m2 , respectively. Surface energies were calculated using the Owens– Wendt theory54 described by Eq. (1): L 1+cos 2 d L = d S + p L d L p S (1) where p S and d S are the polar and dispersive surface energies of the solid, respectively, and p L and d L are the polar and dispersive surface energies of the liquid, respec- tively. L represents the total surface energy ( p L + d L . The surface energy ( p L, d L employed, in nN/m, was (51.0, 21.8), (18.0, 39.0) and (0, 50.8) for water, formamide and diiodomethane, respectively.55 The data were plotted for using Eq. (1), in which the linear coefficient is d S and the angular coefficient is p S , from which the surface energy of the solid material can be determined.56 J. Nanosci. Nanotechnol. 14, 6678–6684, 2014 6679
  • 3. Delivered by Publishing Technology to: UNIVERSIDADE SAO PAULO IF IP: 143.107.252.60 On: Tue, 25 Mar 2014 22:19:28 Copyright: American Scientific Publishers Nanobiosensors Exploiting Specific Interactions Between an Enzyme and Herbicides in Atomic Force Spectroscopy Silva et al. 2.5. Determination of the Specific Force The specific force resulting from the interaction between the ALS enzyme and the herbicides was determined from the difference between the theoretical and experimental adhesion force. The theoretical adhesion force was deter- mined from the sum of the capillary force (determined from contact angle measurements) and theoretical adhe- sion force values determined using the Johnson–Kendall– Roberts (JKR) model.57 All calculations performed, values and equations employed are described in Section 3.2.3. 3. RESULTS AND DISCUSSION 3.1. Nanobiosensor Characterization A representative surface topography of a silicon nitride substrate (simulating the AFM tips) outlining the function- alization steps is depicted in Figures 1(a)–(d). The depo- sition of APTES (Fig. 1(b)) did not significantly affect the surface roughness compared to the unmodified surface (Fig. 1(a)). In both cases, a roughness of approximately 0.4 nm for a surface area of 400 m2 was observed. Fol- lowing glutaraldehyde modification (Fig. 1(c)), the rough- ness of the substrate increased to 1.9 nm. Enzyme coating (Fig. 1(d)) resulted in uniform surface coverage and an increase in roughness to 5.4 nm, indicating that the func- tionalization was successful even after the washing steps. 3.2. Application of the Nanobiosensor to Herbicide Detection The nanobiosensor design and construction were based on the biomimicry of the natural process of host-guest Figure 1. Surface topography of the silicon nitride surface (a) uncoated and coated with (b) APTES, (c) APTES followed by coating with glu- taraldehyde and (d) following functionalization with the ALS enzyme. interactions; i.e., the nanobiosensor harnessed the specific binding interactions of the herbicides metsulfuron-methyl and imazaquin with the enzyme ALS. As described by Chipman,58 these agrochemicals bind to the ALS enzyme to inhibit its action inside the plant cell. Atomic force spectroscopy (AFS) was used to quantify the interactions between the AFM tip and the herbicide samples by measuring the corresponding adhesion force. In AFS, force versus distance curves (force curves) are used to identify recognition events that can be used for detection,48 59 60 and to obtain insights into ways to modify the tip with immobilization of target analytes and sens- ing molecules.61–63 Figure 2 displays typical force curves for the interaction between unfunctionalized and ALS- functionalized tips and metsulfuron-methyl- or imazaquin- contaminated substrates. Increases in the adhesion force of approximately 250% and 160% were observed when the nanobiosensor interacted with the herbicides metsulfuron- methyl and imazaquin, respectively, compared to the use of an unfunctionalized tip (16 nN). In the black curve depicted in Figure 2, only small adhesive forces occasion- ally appear, indicating the absence of any strong interac- tion between the unfunctionalized tip and the herbicide. The black curve was obtained using a substrate modified with metsulfuron-methyl, but similar values were obtained for imazaquin (Table I). In contrast, when the nanobiosen- sor was employed, adhesion forces of ca. 42 and 56 nN were observed for imazaquin (blue line) and metsulfuron- methyl (red line), respectively. The considerable differ- ences in adhesion force were due to the specific binding between the ALS enzyme and the herbicides, analogously to previous studies in which cantilevers functionalized with specific antibodies were used to detect herbicides.52–55 Control experiments, in which the biorecognition process was inhibited, were used to confirm the specificity of the detected specific recognition events. This control was achieved by saturating the tip with the complementary blocking agent (i.e., anti-ALS antibody). The adhesion Figure 2. Force curves for an unfunctionalized tip interacting with metsulfuron-methyl (black line) and with a tip functionalized with the ALS enzyme to detect the herbicides metsulfuron-methyl (red line) and imazaquin (blue line). 6680 J. Nanosci. Nanotechnol. 14, 6678–6684, 2014
  • 4. Delivered by Publishing Technology to: UNIVERSIDADE SAO PAULO IF IP: 143.107.252.60 On: Tue, 25 Mar 2014 22:19:28 Copyright: American Scientific Publishers Silva et al. Nanobiosensors Exploiting Specific Interactions Between an Enzyme and Herbicides in Atomic Force Spectroscopy Table I. Average values of the adhesion force and the coefficients of variation using unfunctionalized and functionalized AFM tips on sub- strates contaminated with imazaquin and metsulfuron-methyl.48 The val- ues were obtained from force curves collected at either three different spots on the same substrate (spots 1 to 3), at a single spot on three dif- ferent substrates (Substrates 1 to 3), or using three different tips (tips 1 to 3). Coefficients of variation (%) Unfunctionalized Functionalized Herbicide Variant tip tip Imazaquin Spot 1 1.4 2.0 Spot 2 1.3 2.2 Spot 3 1.3 1.8 ¯F spot adh (nN) 16 0±1 0 42 0±4 0 Substrate 1 1.3 1.3 Substrate 2 4.2 2.2 Substrate 3 1.4 2.7 ¯F subs adh (nN) 18 0±2 0 40 0±4 0 Tip 1 0.9 1.3 Tip 2 1.4 4.8 Tip 3 1.2 3.3 ¯F Tip adh (nN) 13 9±0 8 44 0±5 0 Total average ¯F Exp adh (nN) 15 9±2 4 42 0±7 5 Metsulfuron Spot 1 1.1 1.4 methyl Spot 2 3.7 3.5 Spot 3 0.7 1.5 ¯F Spot adh (nN) 16 0±2 0 57 0±4 0 Substrate 1 0.8 1.5 Substrate 2 0.9 0.9 Substrate 3 1.0 0.7 ¯F Subs adh (nN) 13 0±2 0 58 0±7 0 Tip 1 1.1 0.7 Tip 2 1.4 1.9 Tip 3 4.5 1.7 ¯F Tip adh (nN) 14 6±0 5 66 0±3 0 Total average ¯F Exp adh (nN) 14 5±2 9 60 3±8 6 values were insignificant and below the value of 16 nN for unfunctionalizated tips (see Fig. 2, in green). The pur- pose of such experiments was to exclude any possibility of incorrect functionalization of the tip and, consequently, a possible interaction between herbicide with the amino or aldehyde groups. The distributions of adhesion forces in the analysis of 2000 force curves, collected from three different spots on a substrate modified with imazaquin or metsulfuron-methyl and using either unfunctionalized tips or the nanobiosen- sor, are displayed in Figure 3. Small deviations were observed in the measurements collected from the three points on the substrate as can be observed from the aver- age values of adhesion force presented in black, blue and pink. Because the standard deviation is a measure of the dispersion relative to an average value, and since the measured points in our study exhibited different arith- metic averages, the standard deviation is not a suit- able means of comparison. Therefore, the coefficient of variation (Eq. (2))64 was used, expressed as a percentage and calculated for each evaluated condition on both the imazaquin and metsulfuron-methyl substrates. Coefficient of variation = Standard deviation Arithmetic mean ×100 (2) The variability in adhesion force in Table I on the sub- strate modified with imazaquin was ≤1.4% and ≤2.2% for unfunctionalized tips and tips functionalized with the ALS enzyme, respectively. For the substrate containing the herbicide metsulfuron-methyl, variations in the adhe- sion force were ≤3.7% and ≤3.5% for unfunctional- ized tips and tips functionalized with the ALS enzyme, respectively. Minor changes were also observed between the force curves collected from three different substrates (using the same tip) or using three different tips on the same substrate. The average values of adhesion force for each variable (spot, substrate and tip) and the correspond- ing coefficients of variation are summarized in Table I. The small changes in adhesion force demonstrate the repro- ducibility of the tip and substrate functionalization method and the reliability of the results. We could therefore deter- mine the experimental adhesion force ( ¯F Exp adh ) from the average among the adhesion force obtained on different spots ( ¯F spot adh ), different substrates ( ¯F Subs adh ) and using differ- ent tips ( ¯F Tip adh ). These values are also presented in Table I and were employed to determine the specific force, as described in Section 3.2.3. 3.3. Contact Angle Measurements and Surface Energy Calculations Table II displays the measured contact angles and the polar ( p), dispersive ( d) and total surface energies of the solid ( s), which were calculated as specified in Section 2.4. These values are necessary to calculate the work of adhe- sion and the theoretical adhesion force between the AFM tips and the substrates, as described in Section 3.2.3. 3.4. Calculation of the Specific Force One expects the efficiency of the nanobiosensor to derive primarily from the specific interaction between the ALS enzyme and the herbicides. Therefore this interaction should have an important contribution to the adhesion force obtained experimentally. But, the adhesion force determined from the force curves has other contribut- ing components, including the capillary force that occurs under ambient conditions owing to the formation of a thin film of water on the surface under analysis. This attractive capillary force (Fcap) is described by Eq. (3) in Table III, where 1 and 2 are the contact angles between the water and the plane and the water and the sphere, respectively.65 The increased capillary force for the functionalized tip in Table III is due to the hydrophilicity of the ALS enzyme, as indicated by the contact angle and the higher surface energy compared to the unfunctionalized tip (Table II). J. Nanosci. Nanotechnol. 14, 6678–6684, 2014 6681
  • 5. Delivered by Publishing Technology to: UNIVERSIDADE SAO PAULO IF IP: 143.107.252.60 On: Tue, 25 Mar 2014 22:19:28 Copyright: American Scientific Publishers Nanobiosensors Exploiting Specific Interactions Between an Enzyme and Herbicides in Atomic Force Spectroscopy Silva et al. Figure 3. The histograms (fitted to Gaussian functions, continuous lines) associated with the force curves collected on three different spots of a same substrate using unfunctionalized (a) and (b) and functionalized (c) and (d) tips and obtained from the herbicides metsulfuron-methyl (above) and imazaquin (below). The values presented in black, blue and pink represent the average adhesion force determined from 2000 force curves obtained in each spot analyzed. The results for metsulfuron-methyl were reproduced from Ref. [48], and are included here as a comparison with imazaquin. The theoretical adhesion force was calculated using the Johnson–Kendall–Roberts (JKR) model (F JKR adh ), which is adapted to treat adhesive interactions. JKR theory includes an adhesive force inside the contact area and is considered more suitable for soft samples with a low elastic modulus and a large tip radius, as used in AFM experiments.66–68 JKR theory is described by Eq. (5), where Rt is the radius of the tip and Wikj is the work of adhesion between two surfaces i and j in a medium k. Radii of curvature of 20 nm and 30 nm were used for the unfunctionalized and functionalized tips, respectively (values determined by SEM images). The work of adhesion (Wikj) was calculated Table II. Measurements of the contact angle and surface energies (total, s; polar, p; dispersive, d) obtained for the tips and the substrate. Contact angle Surface energy (mN/m) Surface Water Formamide Diiodomethane p d s Imazaquin 68.15 30.43 47.37 14.80 29.70 44.60 Metsulfuron- 58.55 30.78 39.14 17.20 30.80 47.90 methyl Silicon 51.11 39.84 41.31 24.10 25.20 49.20 ALS 25.26 22.73 35.18 40.70 23.40 64.10 using Eq. (4) from the ratio between the polar ( p ) and dispersive ( d ) surface energies of the material under study (determined by measurements of the contact angle), in which the tip is represented by (i) and the substrate by (j). The results in Table III point to a higher work of adhesion between the nanobiosensor and the herbicide-contaminated substrates than for the unfunctionalized tip. The theoreti- cal value for the total adhesion force (F Total adh ) between the AFM tips and the substrates was obtained with Eq. (6), which represents the sum of Fcap and F JKR adh .65 We take the difference between the experimentally measured force of adhesion and the predicted theoret- ical value as being the contribution from the specific interaction between the nanobiosensor and the herbicides metsulfuron-methyl and imazaquin (Eq. (7)). More specif- ically, we assume that the total force comprises the adhe- sion force, predicted theoretically with the JKR theory, the capillary force and the specific interaction, where the latter is not accounted for in the total theoretical force. Figure 4 displays the values of F JKR adh , ¯F Exp adh and Fspec. The specific force plays a critical role in the interaction between the nanobiosensor and the metsulfuron-methyl-contaminated 6682 J. Nanosci. Nanotechnol. 14, 6678–6684, 2014
  • 6. Delivered by Publishing Technology to: UNIVERSIDADE SAO PAULO IF IP: 143.107.252.60 On: Tue, 25 Mar 2014 22:19:28 Copyright: American Scientific Publishers Silva et al. Nanobiosensors Exploiting Specific Interactions Between an Enzyme and Herbicides in Atomic Force Spectroscopy Table III. Values for the work of adhesion (Wij), adhesion force (F JKR adh ), capillary force (Fcap), total theoretical adhesion force (F Total adh ), experimental adhesion force ( ¯F Exp adh ) and specific force (Fspec) as calculated using JKR theory. MET = metsulfuron-methyl; IMA = imazaquin. Nanobiosensor Unfunctionalized tip Parameter MET IMA MET IMA Equations Fcap (nN) 19.6 18.5 10.5 9.8 Fcap = 2 R L cos 1 +cos 2 (3) Wij (mN/m) 101.6 95.8 95.6 70.5 Wij = 4 d i d j d i + d j + 4 p i p j p i + p j (4) F JKR adh (nN) 14.4 13.5 9.0 6.6 F JKR adh = 3 2 RtWikj (5) F Total adh (nN) 34.0 32.0 19.5 16.5 F Total adh = Fcap +F JKR adh (6) F Exp adh (nN) 60 3±8 6 42 0±7 5 14 5±2 9 15 9±2 4 See Table I Fspec (nN) 26 3±8 6 10 0±7 5 −5 0±2 9 −0 6±2 4 Fspec = ¯F Exp adh −F Total adh (7) Figure 4. Theoretical (F JKR adh ) and experimental adhesion force (F Exp adh ) and the specific force (Fspec) for the interaction between the nanobiosensor and the herbicides metsulfuron-methyl and imazaquin. substrate, contributing with 44% and 24% of the exper- imentally obtained value for metsulfuron-methyl and imazaquin, respectively. This confirms that the sensitiv- ity of the nanobiosensor is primarily due to the presence of specific interactions between ALS and the herbicides. In contrast, the contribution of the specific force to the tip-herbicide interaction for the unfunctionalized tip was negligible, as indicated by the negative values in Table III, ascribed to the absence of specific interactions. The higher contribution for metsulfuron-methyl is explained by the stronger inhibition of the ALS enzyme by herbicides belonging to the sulfonylurea group.69 Metsulfuron-methyl and imazaquin are only two com- pounds among a larger group of herbicides considered to be ALS enzyme inhibitors. Our future work aims to extend these studies to other herbicides from this group (and other groups using other nanobiosensors) and capitalize on the resulting variations in specific force that can be expected. 4. CONCLUSIONS The use of cantilevers chemically modified with the enzyme ALS proved promising for detecting the herbicides metsulfuron-methyl and imazaquin. The adhesion force measured with the nanobiosensor was considerably higher than those obtained with unfunctionalized tips, with increases of approximately 250% and 160% for metsulfuron-methyl and imazaquin, respectively. The cAl- culation of the total theoretical adhesion force corroborated our experimental results and allowed the calculation of the specific force for the interaction between the ALS enzyme and the herbicides. The results indicated that the specific interaction was the primary source of the greater adhe- sion force when using the nanobiosensor, especially for the interaction of ALS with metsulfuron-methyl. This illus- trates the importance of surface chemical modifications in promoting molecular recognition and, consequently, the specific interactions which enable detecting substances with high selectivity. Acknowledgments: The authors acknowledge FAPESP (Proc. 2007/05089-9, Proc. 2010/00463-2, Proc. 2010/04599-6 and 2009/09120-3), CAPES (Proc. 23038006985201116 and Proc. 02880/09-1), CNPq (Proc. 483303/2011-9) and nBioNet network for the financial support. References and Notes 1. K. Arrow, P. Dasgupta, L. Goulder, G. Daily, P. Ehrlich, G. Heal, S. Levin, K. G. Maler, S. Schneider, D. Starrett, and B. Walker, J. Econ. Perspect. 18, 147 (2004). 2. V. Seidel and W. Lindner, Anal. Chem. 65, 3677 (1993). 3. N. V. Gonzalez, S. Soloneski, and M. L. Larramendy, Mutat. Res. Genet. Toxicol. Environ. Mutagen. 634, 60 (2007). 4. V. M. Kale, S. R. Miranda, M. S. Wilbanks, and S. A. Meyer, J. Biochem. Mol. Toxicol. 22, 41 (2008). 5. A. Martinez, I. Reyes, and N. Reyes, Biomedica 27, 594 (2007). 6. N. Nikoloff, S. Soloneski, and M. L. Larramendy, Toxicol. Vitro 26, 157 (2012). 7. S. Soloneski, N. V. Gonzalez, M. A. Reigosa, and M. L. Larramendy, Cell Biol. Int. 31, 1316 (2007). 8. S. Soloneski and M. L. Larramendy, J. Hazard. Mater. 174, 410 (2010). 9. J. Masojidek, P. Soucek, J. Machova, J. Frolik, K. Klem, and J. Maly, Ecotox. Environ. Safe. 74, 117 (2011). 10. R. Liu, G. Guan, S. Wang, and Z. Zhang, Analyst 136, 184 (2011). J. Nanosci. Nanotechnol. 14, 6678–6684, 2014 6683
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