Atomic Force Microscopy-Based Molecular Recognition - Formatex ...

Current Microscopy Contributions to Advances in Science and Technology (A. Méndez-Vilas, Ed.)

Atomic Force Microscopy-Based Molecular Recognition: A Promising Alternative to Environmental Contaminants Detection D. K. Deda*, C. C. Bueno, G. A. Ribeiro, A. S. Moraes, P. S. Garcia, B. Brito, F. L. Leite Universidade Federal de São Carlos, 18052-780, Sorocaba, São Paulo, Brazil Nowadays, there are major concerns about global environmental problems, resulting from high population growth and consequent increase in industrial production and contaminants generation. The high global demand for food, for example, is causing the excessive use of pesticides and fertilizers, used to increase and ensure the quality of production. However, most of these chemicals accumulate, causing soil and groundwater contamination. Within this context, the development of new methods, with greater sensitivity and selectivity, in order to detect these compounds even at extremely low concentrations, is essential for the prospection of contaminated areas. Regarding the sensitivity, the employment of nanosensors has proved to be extremely efficient for pesticide detection. When combined with biomolecules, those nanosystems become selective being able to detect only one type or class of pesticides. This chapter will present potential methods for agrochemicals detection, based on a combination of nanobiosensors with different operating modes of Atomic Force Microscopy, in particular, Chemical Force Microscopy. Keywords Atomic Force Microscopy; Chemical Force Microscopy; Atomic Force Spectroscopy; biosensor; herbicides

1.Introduction Agrochemicals (pesticides and fertilizers) are substances of different chemical classes, applied at various stages of cultivation, providing protection against weeds and pests. Furthermore, agrochemicals are also applied during postharvest storage for preserving the quality of the crop in many cases [1]. The use of pesticides and fertilizers has increased considerably in recent years due to the high global demand for food. As a result of these practices, there is also an increased exposure of ecosystems and humans to those highly toxic compounds [2]. Several studies have been carried out to assess the cytotoxic and genotoxic effects of these substances [3-8], which highlight the negative aspects of the use of agrochemicals. Thus, it becomes important to study and develop methods with greater sensitivity, to detect these substances. The classic methods that continue to be employed involve chromatographic techniques with UV [9, 10] or mass spectrometry [11] detection. However new methodologies for analysis, mainly of pesticide residues in environmental samples, have been developed in recent years. In this respect, immunochemical techniques, sensors and biosensors have been quite promising and are showing excellent results [12-17]. In recent years, studies have highlighted the use of chemically modified micro-cantilevers in development of sensors and biosensors for various applications. These advances have broadened the fields of application of the Atomic Force Microscopy, transforming it into a promising technique for the detection of different analytes [18-21], including agrochemicals.

2. Agrochemical detection Many agrochemicals have high water solubility and are readily absorbed by the target plants. These physicochemical properties facilitate the dispersion of agrochemicals, especially in aquatic environments where high concentrations of pesticides become highly toxic to microorganisms [22]. Prado and coworkers [23] investigated the effects caused by the paraquat herbicide in microalgae. The results indicated that this pesticide induces oxidative stress in cells, in addition to compromising the metabolic activity and integrity of the cytoplasmic membrane, and reducing the levels of intracellular proteins. These important physiological changes suggest that severe toxic effects are caused in cells exposed to this agrochemical. The entire population is continually exposed to agrochemicals as a number of crops including corn, soybeans, sugar cane and fruit, which comprise our daily diet, are treated with herbicides, as well as many types of vegetation existing in residential and public areas [24]. This is only one example that demonstrates the growing concern about the effects caused by the excessive use of agrochemicals, and several studies have shown that some pesticides in use are known to be highly toxic, mutagenic, carcinogenic or teratogenic [3-8]. As a result, the use of agrochemicals must be considered as a potential hazard to both aquatic ecosystems and the quality of potable water, since they can easily reach the groundwater once they are in the soil. In this respect, studies have detected high concentrations of the herbicide MCPANa (2-methyl-4-chlorophenoxyacetic acid) in the groundwater of agricultural areas and in rivers in various parts of the world [25]. This herbicide easily binds to proteins, resulting in conformational changes, and, once accumulated in the environment, this herbicide can produce neurotoxic effects, as well many drugs with a carboxylic acid structure [26].

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Due to their lipophilic character, slow hydrolysis and high absorption by organic matter, agrochemicals are considered to be potential contaminants of organisms of all trophic levels. Animal studies claim that exposure to herbicides, such as atrazine, results in changes on reproductive, development and immune systems. On investigating the effects of atrazine in Wistar rats, it was found that this substance induces oxidative stress and premature lipid peroxidation in organisms, as well as liver degeneration with the death of hepatocytes and micronucleus formation in cells, which are considered to be strong evidence of the cytotoxic and genotoxic potential of this herbicide [24]. Therefore, because of the highly toxic character combined with the long retention time of the herbicides in nature, there is a need for careful detection and evaluation of these substances, and because of that, more sensitive and selective methods are being investigated. Usually, the use of chromatography in different matrices is presented as a pertinent technique with regards to the detection of pesticides on the environment. Several studies have reported the use of chromatographic techniques coupled to a variety of detection methods. The analytes are separated by chromatographic techniques (gas chromatography, GC, or liquid chromatography, LC) and detected by methods such as selective electron-capture, and phosphorus-nitrogen flame photometric detection. However, analytes are more commonly detected by mass spectrometry detection, although all techniques provide information about the structure and weight of the agrochemical investigated [11, 27-29]. Nguyen and coworkers [30] identified 107 pesticides via gas chromatography with electron impact mass spectrometric detection in the selected ion monitoring mode (GC-MS-SIM). These pesticides, present in commercial cabbage and radish samples, were simultaneously analyzed after sample preparation, based on the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) sample preparation method, and the methodology employed proved to be very effective presenting low detection limits. A more recent study, using liquid chromatography with detection by mass spectrometry, was performed to detect different organophosphate (OP) and non-organophosphate (NOP) pesticides in grape and apple samples. A series of pesticides, including OPs, was detected at concentrations that were considered high due to in view of the considerable consumption of these fruit by the population. Chronic exposure to organophosphate pesticides may compromise neurophysiological processes, triggering diseases like Parkinson's [31]. As an alternative to sophisticated mass spectrometry techniques, the combination of chromatography with other detection methods has also been employed in the detection of agrochemicals. Seccia and coworkers [9] reported the simultaneous detection of eight sulfonylurea herbicides in bovine whole milk by employing high-performance liquid chromatography (HPLC) followed by the analysis of absorption in the ultraviolet region. This method allows the determination of herbicides, such that the sensitivity of the method was considered suitable for detection at concentrations below their respective maximum limits of residue determined by the responsible agencies. Fluorescence spectroscopy has also been used for the detection of agrochemicals [32, 33]. Phenylurea herbicides were detected in rice samples, using a technique employing separation by HPLC followed by UV treatment, resulting in the formation of primary amines that can be marked with o-phthalaldehyde to form fluorescent isoindole derivatives [34]. Capillary electrophoresis has also been shown to be a promising technique for the detection of agrochemicals [35, 36]. A new methodology, based on matrix solid phase dispersion-capillary electrophoresis with electrochemiluminescence detection (CE-ECL-MSPD) for the simultaneous determination of three kinds of phenylurea herbicides in green vegetable and rice, was reported by Wang and coworkers, showing excellent results [13]. More recently, sensitive analytical techniques and simpler methods of sample preparation proved to be necessary to detect and quantify herbicide residues. This is mainly due to the fact that many pesticides currently in use are not amenable to detection by chromatographic techniques, or at least not in concentrations low enough to satisfy the limits considered safe for fauna, flora and human health [27]. In this respect, methods based on the use of sensors and biosensors have been widely highlighted, especially when fluorescent detection methods are employed [12-14, 17]. An example of this is a fluoroimmunoassay, which uses a biosensor based on a specific antibody against the herbicide Diuron. This antibody is labeled with the fluorescent tag rhodamine isothiocyanate, which allows the detection of the herbicide with both high sensitivity and selectivity [16]. Recent studies have reported the use of chemically modified microcantilevers in the development of biosensors, expanding the area of employment of Atomic Force Microscopy [18-21]. These biosensors have been successfully applied in studies of DNA hybridization, immunoassays [37] and the detection of viruses, bacteria, cells [38] and parasites [39]. However, there are no reports of the use of nanosensors based on cantilevers in the detection of agrochemicals. Atomic Force Microscopy, and in particular Chemical Force Microscopy, has great potential as a technique for detecting such agents, even in small quantities. This has been a topic of research in our group in recent years; general aspects on the use of biosensors based on cantilevers and the capability of those for detection of herbicides are presented in this chapter.

3. Atomic Force Microscopy Atomic Force Microscopy (AFM) emerged in 1986 when Binnig, Quate and Gerber combined the principles of Scanning Tunneling Microscopy (STM) with a stylus profilometer, an instrument used to measure the roughness of surfaces [40]. With regards to STM [41], AFM has several advantages, including the fact that it allows the analysis of non-conductive samples, since the method does not use a tunneling current to produce images. Furthermore, it is able to

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acquire images in a liquid medium, which allows the analysis of biomolecules, whilst maintaining the characteristics of their natural environment and retaining their properties. Sample preparation is usually quite simple and the technique is non-destructive [42]. The fundamental principle of AFM is to obtain images of the surface by measuring the deflection of a tip of nanometric dimensions, mounted on a holder of 100-200 mm in length. These deflections are caused by attractive or repulsive forces between the tip and the sample, which depend on several factors, such as the materials that comprise the sample and the tip, the distance between them, the geometry of the tip and any kind of contamination present on the sample surface. In general, two modes of AFM operation have been found that are directly related to those factors: when the AFM operates in the region where the force between the tip and the sample is repulsive, is called contact mode; when the force is sometimes attractive and sometimes repulsive, the intermittent contact mode is characterized [40, 43]. The choice of appropriate operational mode depends on the type of sample and the type of information that will be extracted from the image. In its 25-years history, the applications of AFM have been rapidly expanding to several fields of science [44]. The many techniques derived from the AFM may generate a wide range of information, in addition to the visualization of the topography of different types of samples. Current-sensing Atomic Force Microscopy (C-AFM), Dynamic Force Microscopy (DFM), Kelvin Probe Force Microscopy (KPFM), Magnetic Force Microscopy (MFM) and Chemical Force Microscopy (CFM) are a few examples of AFM development that are of particular relevance [45]. Also in relation to the forces present in the tip-sample system, recent studies have addressed the analysis of these forces in order to quantify the interactions between the tip and the sample, especially when treating specific interactions. Therefore, it is necessary to chemically functionalize the AFM tips, developing so-called nanosensors. The use of chemically-functionalized tips for AFM imaging is called Chemical Force Microscopy [46-48]. CFM enhances a specific intermolecular interaction and suppresses other interfering interactions, thereby selectively probing surfacefunctional groups of interest in the range from microscale to nanoscale. In addition, CFM enables the measurement of a variety of interactions, including non-covalent chemical and biological forces, as a function of tip-sample distance [49, 50]. The use of those nanosensors in AFM and the quantification of the forces between the tip and the sample provide additional information to the topographic images, and it is known as Atomic Force Spectroscopy (discussed in detail below). 3.1

Chemical Force Microscopy

AFM is mostly used to obtain topographical images of samples. However, it cannot provide information about the physical and chemical properties of the material analyzed. Because of this limitation, other techniques derived from AFM have been developed in order to provide more tools for the characterization of materials [45]. In this respect, CFM has shown to be able to directly detect not only the interaction between specific functional groups using chemically modified AFM tips (cantilevers), but also detect and measure biological interactions. The chemical functionalization gives the tips selectivity and sensitivity to interactions at the molecular level. Moreover, it allows the analysis of the extent of these interactions, which have variable magnitudes for different molecular groups, and enable the detailed mapping of surface chemistry at a nanoscale resolution [48, 50]. Chemical modification of the cantilevers, allows the recognition of a specific substance or a group of substances, that results in powerful nanosensors that are able to detect a single molecule [51]. In many cases, the functionalization occurs via the use of an active biological component, which is able of specifically binding to certain substrates. This mimicry of biological systems, by the development of sensors that act through specific interactions similar to the antibody-antigen or key-lock scenario, consists of so-called biosensors or nanobiosensors [19, 20]. The biomolecule attached to the surface of a transducer converts the biological signal into an electrical signal, and provides information such as friction, adhesion and conformation [52]. The Enzyme-Linked Immunosorbent Assay (ELISA) method, based on an enzyme immunoassay which uses a detection system based on enzyme-labeled conjugates, is a prime example of nanobiosensors, and is widely used for the recognition of molecular interactions that comprises the diagnosis of various diseases [53]. Advances in AFM combined with the development of nanobiosensors (or functionalized cantilevers) have made CFM an important tool for material characterization, due to its fast response, selectivity and accuracy [52]. Functionalization of the AFM tip with organic monolayers, such as SAM’s (self-assembly monolayers) which have terminations with well-defined functional groups, makes an ideal tip-sample system, that describe the interactions between the functionalized tip and the sample analyzed through Van der Waals forces (~> pN) or strong covalent bonds (~ 0.1 μN). The resolution of the force measured by AFM is several orders of magnitude higher than that of weaker chemical bonds, which allows the measurement of individual molecular interactions [48]. However, it is important to mention that, at the nanoscale level, measurements of adhesion between surfaces are influenced by complex factors, such as surface roughness, contaminants, the radius of the curvature of the tip, and other interactions including capillary, electrostatic, hydrophobic and magnetic, which may affect the surface dynamic or structure of the image, and the scattering of results [47]. Therefore, it is necessary to obtain a large number of measurements for a good statistical analysis of the events of interaction force being studied.

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Several methodologies for the functionalization of AFM tips are described in the literature [19, 47, 54]. Generally, the tips consist of Si3N4 and are coated with a polycrystalline thin gold layer (50-100 nm). These are immersed in a solution containing the organic material to form an auto-mounted film on the surface of the gold. Generally, this modification consists of the formation of thiol (-SH) or organic silane monolayers, which allow the subsequent connection to other molecules that have terminations consisting of functional groups, such as CH3, COOH, CH2OH and NH2. These terminations allow the tips and the substrate to be covalently linked to different molecules and biomolecules, enabling the development of nanosensors and nanobiosensors for many different applications. 3.1.1 Chemical Force Microscopy Applications Studies related to the employment of CFM are motivated mainly due to the advantage of the promotion and management of molecular interactions of interest, whilst repressing other interactions. Furthermore, the technique allows the analysis of samples either in air, in solution or in vacuum, providing information about the sample surface under conditions approaching reality and with a much higher resolution than other microscopic techniques [55]. The molecular recognition and interactions that occur in biological systems can be studied by the CFM technique. These issues are widely discussed by scientists who seek to investigate chemical functional groups and their specific interactions, in order to contribute to clinical, biological, industrial and environmental areas [18]. CFM has been used for the detection and characterization of biomolecules and systems of biological importance, such as the streptavidin– biotin system, which is very important for biotechnological applications, since this interaction can serve as a linker for others biotinylated proteins [56, 57]. Another study involving a biomolecule shows that CFM may be a powerful ally for the characterization of protein conformations, if combined with other techniques. BSA (Bovine Serum Albumin), an important protein used in many scientific works, suffers important conformational changes in response to changes in the pH of a solution. Li and coworkers characterized these changes using CFM and the information provided by the smallangle neutron scattering (SANS) technique. Changes in pH significantly changed the strength of the intra- and intermolecular interactions, which have been easily detected by CFM [58]. The employment of enzymatic nanobiosensors has also been widely explored [59]. They consist of enzymes immobilized on the tip in AFM, which are able to interact with a molecule or a class of molecules of interest. Even more complex systems, such as tissues and cells, have also been studied by AFM. In recent years, studies have reported the use of CFM as a powerful technique for mapping cells, in their physiological conditions, with nanometer resolution [60, 61]. Dague and coworkers [62] reported the efficiency of CFM in mapping living cells, identifying hydrophobic surfaces that are important in many biological processes, such as protein folding, membrane fusion and cell adhesion. CFM also has been used in several areas of engineering and material science in order to characterize and evaluate different properties [63], to directly quantify the strength of intermolecular interactions [47, 64], and to monitor chemical reactions that occur in real-time on the surface of the tips [65]. An example of a direct measurement of the strength of intermolecular interactions was performed by Kado and coworkers [66], where the strength of hydrogen bonding between phenylurea groups on a probe tip and carboxyl groups in self-assembled monolayers was evaluated. With CFM it is also possible to investigate, at the nanoscale level, chemical properties on the sample surface, as its viscosity, elasticity [50] and chirality [67, 68]. McKendry and coworkers [69] have used CFM to investigate supramolecular interactions between chiral molecules linked to the probe tip and the substrate surface. It is also possible to determine the apparent pKa of functional groups, based on the fact that the processes of protonation and deprotonation of the surface functional groups present on the tip or on the substrate are fully dependent on the pH. The occurrence of those processes promotes changes in electrostatic and hydrophobic interactions, resulting in changes in the magnitude of interaction forces between the tip and the sample, which makes the analysis of chemical functional groups by CFM possible [50, 70]. In general terms, it can be said that CFM provides information that goes beyond the topographical characteristics of the sample. The use of nanosensors (modified cantilevers) can provide important information regarding the chemical composition and conformation of the molecules or biomolecules present in the substrate. This fact makes CFM a powerful analytical technique, with great potential to be applied not only as a tool for characterization, but also in detecting substances such as environmental pollutants, for example. The following section details the methodology of the measurement of these interactions. 3.2 Atomic Force Spectroscopy The term “spectroscopy” is normally used in physical and analytical chemistry for the identification of substances and the characterization of materials. Through the use of the atomic force microscope, Atomic Force Spectroscopy (AFS) is a dynamic analytical technique which allows the study of the mechanical properties and chemical bonds of molecules. AFS measures the behavior of a surface or a molecule under stretching and torsional forces, as well as its indentation response as a function of the interaction between the cantilever tip and the surface being studied [43, 71, 72]. Studies employing AFS involve the analysis of graphics, denominated force versus distance curves or force curves, where the approach (to the contact) and retraction (to the non contact) between the tip and the sample were represented,

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and used to measure the tip and sample adhesion. In addition, the material properties, such as elasticity and wettability can be investigated [72, 73]. There are many factors that can influence the behavior of force curves, such as the functionalization process of the surface and cantilever tip, the kind of sample being studied (hard or soft matter) and the medium of the measure (air, liquid or vacuum environment) [74, 75]. As the cantilever behaves like a spring in AFM analyses, knowing the spring constant of the cantilever is fundamental for the measurement of the quantitative interaction forces between the probe and the sample. Thus, the interaction force in an AFS study can be described by Hooke’s law presented in Eq. 1: (1) where F is the force, k is the spring constant and x is the cantilever deflection. A force curve is divided into three major regions: the contact line, the non-contact region, and line zero. In Fig. 1a, a force curve can be observed, showing where cantilever deflections occur [74].

Fig. 1 (a) Force-displacement curve illustrating the points where jump-to-contact (approach) and jump-off-contact (withdrawal) occur. (Adapted from [5]). (b) Adhesion force histogram.

In order to understand how AFS studies are proceeded, what is happening at each point of the force curve should be highlighted, as shown above. This resulting plot contains information about the magnitude and functional dependence of long-range attractive and adhesive forces, the point of tip-sample contact, the tip-sample contact area, and the elastic modulus and plasticity of thin and thick films [76]. Some details of these features are given hereafter:[77, 78] Curve Region A-B: The piezo extends to achieve contact with the AFM tip; Curve Region B-C: The tip is taken to point C due to attractive forces near the surface, and will jump to contact if there is enough force on the sample; Curve Region C-D: The cantilever shifts up because of the tip, which is pressed against the surface; Curve Region D-E: The piezo begins to retract and the cantilever begins to get in balance with surfaces forces; Curve Region E-F: The piezo continues to retract and the cantilever deflects down with the surface attraction; Curve Region F-G: At point F, the rupture moment between the tip and the surface can be observed. At point G, the cantilever returns to its original position and the piezo continues to retract. The vertical distance (Fadh) corresponds to the value of the adhesion force; Curve Region G-H: The tip and the sample do not have any further contact. The most important data that can be extracted from the force curve is the adhesion force (Fadh). The adhesion force corresponds to the vertical distance showed in Fig. 1a and it relates to the force that had to be made to separate the AFM tip from the sample’s surface. A range of the Fadh values can be made through several (n) measurements of force curves and its results presented as a pattern of frequency values. By plotting these values in accordance to the frequencies that they showed up, an adhesion force histogram can be created (Fig. 2b). Going back to the concepts briefly introduced in this topic, the importance of the adhesion force histogram relies on the fact that binding event implies on a pattern of a recognition process, i.e., by this histogram, a force pattern value can be recognized on a complex sample. In other words, when a nanobiosensor is used to detect agrochemicals in a water sample, for example, if a force pattern value is found, it means that the analyte of interest has been recognized.

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Additionally, the specific force-binding map or adhesion map can also be obtained as a supplementary analysis (Fig. 3). This adhesion map indicates whether the interest molecule are uniformly or no uniformly distributed onto the substrate surface and can give an important information about the occurrence of adhesion events in a portion of area expressed by the number of events/cm2 or mm2, for example [79]. In an adhesion map imaging, both topographic and interaction force data are generate across the surface. A schematic diagram of adhesion mapping performed over a multicomponent film using a functionalized tip is shown in Fig. 3. As an adhesion map is being made, force curves are obtained on the surface for every pixel of the scan producing a twodimensional array of force curves [5], and consequently, the strength of the tip-surface interaction in each pixel is used to generate an adhesion map of the entire surface [80].

Fig. 2 Schematic representation of an adhesion map. (a) A functionalized probe scanning a multicomponent layer, (b) the topographic image and the array of force curves across the surface, (c) an adhesion map can be generated by calculating the strength of adhesion from individual force curves. (Adapted from [80]).

By interpretating the force curves, the results can lead to the initiation of several types of nanomechanical studies, which can extend the applicability and development of force spectroscopy, as will be introduced the next section. 3.2.1 Atomic Force Spectroscopy Applications The AFS approach has been employed in several research areas, such as materials and science engineering, chemistry, biochemistry and biotechnology [43, 71, 72]. In recent years, AFS has allowed researchers to collect additional information about the mechanics of different substrates through the investigation of the forces that occur at the nanoscale [81]. In most cases, AFS has been applied to the understanding of the nanomechanical properties of a vast number of well-defined biological systems, such as proteins [73], unfolding polyproteins [81, 82] and cells and their behavior at the nanoscale, with additional importance in areas such as cancer research and biosensors [83-85]. Wagner and coworkers [60] showed that AFS is promising in studies of cellular structures, in particular by determining the work of adhesion. This technique was used for the analysis of corneocytes, which are cellular structures present in the stratum corneum, in order to assess damage caused to the protein structure of these cells after being subjected to heating at 105°C. AFS has also been employed in the characterization and stretching studies of thin films (Langmuir–Blodgett films). Studies of force has allowed researchers to extract valuable structural and mechanical information about films that could not have been experimentally obtained otherwise [81], and to promote the measurement of rupture forces and the kinetics of interactions with the different molecules. Additionally, by measuring the attractive or repulsive forces between the tip and a sample in a nanometer scale by analysis of the force curves, the surface morphology, composition, and roughness of the sample were acquired accurately, even during the film formation. Information about wetting properties and viscoelasticity of films consisting of protein or other organic molecules can also be obtained [86]. Polesel-Maris and coworkers [82] have recently proposed a methodology that describes the functionalization of AFM tips, giving them variable hydrophilic/hydrophobic and charge characteristics, and their use for the quantification of the withdrawal forces during the unfolding of the BSA protein structures. Understanding the interactions between BSA and other molecules is of great importance in view of their great biological importance for carrying fatty acids and other hydrophobic molecules. The employment of AFM in the study of specific interactions has also been reported. The selectivity of some intermolecular interactions is essential in many events that occur in biological systems, such as recognition between a receptor and a ligand, an antibody and an antigen, and complementary strands of DNA [87]. AFS can be utilized in molecular recognition investigations to report the origin of specific and non-specific interactions/bindings and

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monitoring the process of antibody-antigen/host-guest events [83, 85] in order to correlate it to a specific molecular recognition map with an AFM topography image [87]. The investigation of specific interactions goes far beyond the study and understanding of biological systems. Specific binding can also be an important tool for the analysis and quantification of various analytes of technologic and environmental interest, so that the combination of sensors/biosensors with AFS may lead to the development of devices that are highly sensitive and selective for commercial applications on several areas. Therefore, in the next section, some aspects will be discussed relative to the potential of AFM for the detection of environmental pollutants, especially agrochemicals, which are considered a major threat to ecosystems and human health.

4. Chemical Force Microscopy and Force Spectroscopy as promising tools to detect agrochemicals At the same time that the employment of systems or arrays, that include sensors and biosensors at the nanoscale, has been growing in several areas, on environmental monitoring it is still at the basic research stage [88]. However, great advances are promised in this area, because they can be developed in order to have high sensitivity and selectivity for detection of specific pollutants. In the particular case of pollutants from agricultural activities, the nanobiosensors may be highly efficient because they can allow in real time, selective and sensitive analysis either of the soil or water. Thus, some of the main agrochemicals employed worldwide can be detected by combining highly sensitive techniques coupled with extremely selective biosensors. In this case, an excellent alternative to confer selectivity to a sensor is based on the simple analysis of the mechanism of action of an agrochemical in the target plant, since each one has a unique mechanism of action which usually involves its binding to a specific biomolecule. Generally, those biomolecules are enzymes of great importance for plant development. This alternative has been explored by our research group, in which enzymes are employed in the functionalization of cantilevers, allowing the detection of agrochemicals, especially herbicides, using Atomic Force Microscopy technique. First of all, it should be highlighted that our methodology for the development of an AFM based sensor employs the concept of molecular recognition, that in accordance to Hornyak and coworkers [89], is a binding with purpose. The purpose of this specific binding event implies on a pattern of a recognition process through a set of intermolecular interactions. The ability to accomplish specific interactions between two or more molecules happens by the means of non-covalent forces (based on host-guest/lock-key chemistry) [89]. Secondly, the molecules of interest should be immobilized in a substrate using methods of chemical modification, that in the case of AFM may consist of mica, gold or Si3N4 (Chemical Force Microscopy). The modification will prevent the disordered aggregation of molecules and address stability to the substrate in order to provide them a chemically receptive surface. Finally, CFM will allow the validation, sensibility, and replicability of the signals generated in a Force Spectroscopy analysis. The result of an analysis by Force Spectroscopy, already introduced, is a force versus distance curve or force curve. Generally, this curve is composed of an approach and retraction curve (Fig. 1a). For development of a nanobiosensor, the most important data can be extracted by the retraction curve: the adhesion force (Fadh), also called rupture force [90]. The functionalization process of the tip is extremely important for the development of a reliable and specific sensor, and its relevance reflects on the measured values of Fadh. In our preliminary studies, for example, we used the enzyme inhibitor of the agrochemical metsulfuron-methyl, acetolactate synthase (ALS). This enzyme is employed in the tip functionalization to promote the specific binding and recognition of the agrochemical molecule. A method of functionalization employing 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde was used both in the modification of the cantilever and mica, in order to promote the proper orientation of the active sites of the enzyme and enable its interaction with herbicide. Figure 3 shows a schematic representation of the functionalized cantilever (biosensor) for the herbicide metsulfuron-methyl detection. As it can be seen in Fig. 4, the functionalization step was critically important to point out the real value of the adhesion force between the host and guest molecules. The adhesion forces between the tip and substrate are significantly altered if the functionalization step is carried out. When tips were used without chemical functionalization (Fig. 4a), adhesion forces values around 60 pN were found. In contrast, the tip modification with ALS (Fig. 4b) resulted in a 150% increase in the values of adhesive force, reaching around 150 pN. In other words, the employment of chemical modification directly reflects in the recognition efficiency of the sensor.

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Fig. 3 Schematic representation of the biosensor developed for the herbicide metsulfuron-methyl detection through the formation of a specific binding to ALS enzyme. Structure of the ALS enzyme obtained from Protein Data Bank (PDB), available at: http://www.rcsb.org/pdb/explore/explore.do?structureId=1YHY

Fig. 4 (a) Interaction between the ALS enzyme and metsulfuron-methyl molecule. System: AFM tip without functionalization and functionalizated substrate (metsulfuron-methyl). (b) The specific interaction between the enzyme (ALS) and metsulfuron-methyl molecule. System: Enzyme (ALS) -functionalized AFM cantilever tip and herbicide (metsulfuron-methyl)-functionalized substrate.

The analysis of a large number of force curves represented by the histograms of Fig. 5 also shows the difference between the adhesion forces obtained with the normal tip and with the functionalized tip. Using the non-functionalized tip values of adhesion force were obtained in a range between 52 and 61 pN with an average of 58 ± 3 pN, assuming a normal distribution with standard deviation of 95%. The force curves made with the functionalized tip signaled greater adhesion forces ranging from 128 to 156 pN with an average of 142 ± 11 pN, also considering a normal distribution with standard deviation of 95%.

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Fig. 5 Histograms adjusted by a Gaussian function (gray line) associated with the force curves performed with the non functionalized [25] and functionalized (blue) tips. By comparing them is observed that the adhesion forces obtained with the normal tip were lower than those obtained with the functionalized tip.

Overall, the considerable increase in adhesion force between the tip and the substrate is due solely to the formation of a specific binding between the enzyme and the herbicide. The correct organization/arrangement of the molecules afforded by the methods of surface chemical modification, allows that the active sites are available to the specific sensing and, consequently, turns the employment of this biosensor promising for herbicides detection. The same principle can be used for detection of others agrochemicals and even others environmental pollutants, which makes the AFM a promising technique in this area and supports the development of new sensors and biosensors.

Final remarks This chapter addressed aspects related to the potentiality of Atomic Force Microscopy for detection of environmental pollutants, especially agrochemicals. A description of two different operating modes of AFM, Chemical Force Microscopy and Atomic Force Spectroscopy, and a review of their main applications reported in the literature was presented, and showed that the AFM can provide much additional information to topographical images. The combination of these two techniques with chemically modified AFM tips results in powerful nanosensors and important aspects of the employment of these sensors and biosensors were also discussed. Preliminary and unpublished results obtained by our research group, using micro-cantilevers modified with the ALS enzyme, showed the importance of the functionalization process. The analysis of force curves obtained with this nanobiosensor, developed for detection of the herbicide metsulfuron-methyl, showed an elevated adhesion force value with the substrate due to the formation of a specific interaction between the enzyme and the herbicide. Thus, functionalization methodologies that aim to promote the molecular recognition can result in highly selective sensors that, when coupled with sufficiently sensitive techniques such as AFM, can result in very efficient methods of agrochemicals detection and other environmental contaminants. Acknowledgements: The support by FAPESP, CAPES and CNPq is gratefully acknowledged.

References [1] [2] [3] [4] [5] [6]

Marchis D, Ferro GL, Brizio P, Squadrone S, Abete MC. Detection of pesticides in crops: A modified QuEChERS approach. Food Control. 2012;25:270-273. Goodland R. Environmental sustainability in agriculture: diet matters. Ecological Economics. 1997;23:189-200. Gonzalez NV, Soloneski S, Larramendy ML. The chlorophenoxy herbicide dicamba and its commercial formulation banvel((R)) induce genotoxicity and cytotoxicity in Chinese hamster ovary (CHO) cells. Mutation Research-Genetic Toxicology and Environmental Mutagenesis. 2007;634:60-68. Kale VM, Miranda SR, Wilbanks MS, Meyer SA. Comparative cytotoxicity of alachlor, acetochlor, and metolachlor herbicides in isolated rat and cryopreserved human Hepatocytes. Journal of Biochemical and Molecular Toxicology. 2008;22:41-50. Martinez A, Reyes I, Reyes N. Cytotoxicity of the herbicide glyphosate in human peripheral blood mononuclear cells. Biomedica. 2007;27:594-604. Nikoloff N, Soloneski S, Larramendy ML. Genotoxic and cytotoxic evaluation of the herbicide flurochloridone on Chinese hamster ovary (CHO-K1) cells. Toxicology in Vitro. 2012;26:157-163.

© 2012 FORMATEX

1345


[7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35]

Soloneski S, Gonzalez NV, Reigosa MA, Larramendy ML. Herbicide 2,4-dichlorophenoxyacetic acid (2,4-D)-induced cytogenetic damage in human lymphocytes in vitro in presence of erythrocytes. Cell Biology International. 2007;31:1316-1322. Soloneski S, Larramendy ML. Sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary (CHO-K1) cells treated with the insecticide pirimicarb. Journal of Hazardous Materials. 2010;174:410-415. Seccia S, Albrizio S, Fidente P, Montesano D. Development and validation of a solid-phase extraction method coupled to highperformance liquid chromatography with ultraviolet-diode array detection for the determination of sulfonylurea herbicide residues in bovine milk samples. Journal of Chromatography A. 2011;1218:1253-1259. Zheng YM, Wang K, Li T, Zhang XL, Li ZY. Synthesis, Singlet Oxygen Photogeneration and DNA Photocleavage of Porphyrins with Nitrogen Heterocycle Tails. Molecules. 16:3488-3498. Kang S, Chang N, Zhao Y, Pan CP. Development of a Method for the Simultaneous Determination of Six Sulfonylurea Herbicides in Wheat, Rice, and Corn by Liquid Chromatography-Tandem Mass Spectrometry. Journal of Agricultural and Food Chemistry. 2011;59:9776-9781. Masojidek J, Soucek P, Machova J, Frolik J, Klem K, Maly J. Detection of photosynthetic herbicides: Algal growth inhibition test vs. electrochemical photosystem II biosensor. Ecotoxicology and Environmental Safety. 2011;74:117-122. Liu R, Guan G, Wang S, Zhang Z. Core-shell nanostructured molecular imprinting fluorescent chemosensor for selective detection of atrazine herbicide. Analyst. 2011;136:184-190. Boro RC, Kaushal J, Nangia Y, Wangoo N, Bhasin A, Suri CR. Gold nanoparticles catalyzed chemiluminescence immunoassay for detection of herbicide 2,4-dichlorophenoxyacetic acid. Analyst. 2011;136:2125-2130. Byzova NA, Zherdev AV, Zvereva EA, Dzantiev BB. Immunochromatographic Assay with Photometric Detection for Rapid Determination of the Herbicide Atrazine and Other Triazines in Foodstuffs. Journal of Aoac International. 2010;93:36-43. Sharma P, Gandhi S, Chopra A, Sekar N, Suri CR. Fluoroimmunoassay based on suppression of fluorescence self-quenching for ultra-sensitive detection of herbicide diuron. Analytica Chimica Acta. 2010;676:87-92. Vladkova R, Ivanova P, Krasteva V, Misra AN, Apostolova E. Assessment of Chlorophyll Fluorescence and Photosynthetic Oxygen Evolution Parameters in Development of Biosensors for Detection of Q(B) Binding Herbicides. Comptes Rendus De L Academie Bulgare Des Sciences. 2009;62:355-360. Waggoner PS, Craighead HG. Micro- and nanomechanical sensors for environmental, chemical, and biological detection. Lab on a Chip. 2007;7:1238-1255. Fritz J. Cantilever biosensors. Analyst. 2008;133:855-863. Frómeta NR. Cantilever biosensors. Biotecnología Aplicada. 2006;23:320-323. Park J, Nishida S, Lambert P, Kawakatsu H, Fujita H. High-resolution cantilever biosensor resonating at air-liquid in a microchannel. Lab on a Chip. 11:4187-4193. Warren N, Allan IJ, Carter JE, House WA, Parker A. Pesticides and other micro-organic contaminants in freshwater sedimentary environments - a review. Applied Geochemistry. 2003;18:159-194. Prado R, Rioboo C, Herrero C, Suarez-Bregua P, Cid A. Flow cytometric analysis to evaluate physiological alterations in herbicide-exposed Chlamydomonas moewusii cells. Ecotoxicology. 2012;21:409-420. Campos-Pereira FD, Oliveira CA, Pigoso AA, Silva-Zacarin ECM, Barbieri R, Spatti EF, Marin-Morales MA, Severi-Aguiar GDC. Early cytotoxic and genotoxic effects of atrazine on Wistar rat liver: A morphological, immunohistochemical, biochemical, and molecular study. Ecotoxicology and Environmental Safety. 2012;78:170-177. Fredslund L, Vinther FP, Brinch UC, Elsgaard L, Rosenberg P, Jacobsen CS. Spatial variation in 2-methyl-4chlorophenoxyacetic acid mineralization and sorption in a sandy soil at field. Journal of Environmental Quality. 2008;37:19181928. Zhang HX, Liu L. Chemicobiological effects of herbicide MCPA-Na on plasma proteins. Molecular Biology Reports. 2012;39:2745-2751. Lee JM, Park JW, Jang GC, Hwang KJ. Comparative study of pesticide multi-residue extraction in tobacco for gas chromatography-triple quadrupole mass spectrometry. Journal of Chromatography A. 2008;1187:25-33. Sinha SN, Vasudev K, Rao MVV. Quantification of organophosphate insecticides and herbicides in vegetable samples using the "Quick Easy Cheap Effective Rugged and Safe" (QuEChERS) method and a high-performance liquid chromatographyelectrospray ionisation-mass spectrometry (LC-MS/MS) technique. Food Chemistry. 2012;132:1574-1584. Barr DB, Needham LL. Analytical methods for biological monitoring of exposure to pesticides: a review. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences. 2002;778:5-29. Nguyen TD, Yu JE, Lee DM, Lee GH. A multiresidue method for the determination of 107 pesticides in cabbage and radish using QuEChERS sample preparation method and gas chromatography mass spectrometry. Food Chemistry. 2008;110:207-213. Sinha SN, Rao MVV, Vasudev K, Odetokun M. A liquid chromatography mass spectrometry-based method to measure organophosphorous insecticide, herbicide and non-organophosphorous pesticide in grape and apple samples. Food Control. 2012;25:636-646. Mughari AR, Galera MM, Vazquez PP, Valverde RS. Coupled-column liquid chromatography combined with postcolumn photochemical derivatization and fluorescence detection for the determination of herbicides in groundwater. Journal of Separation Science. 2007;30:673-678. Lei HT, Xue G, Yu CF, Haughey SA, Eremin SA, Sun YM, Wang ZH, Xu ZL, Wang H, Shen YD, Wu Q. Fluorescence polarization as a tool for the detection of a widely used herbicide, butachlor, in polluted waters. Analytical Methods. 2011;3:2334-2340. Mou RX, Chen MX, Zhi JL. Simultaneous determination of 15 phenylurea herbicides in rice and corn using HPLC with fluorescence detection combined with UV decomposition and post-column derivatization. Journal of Chromatography BAnalytical Technologies in the Biomedical and Life Sciences. 2008;875:437-443. De Rossi A, Desiderio C. Application of reversed phase short end-capillary electrochromatography to herbicides residues analysis. Chromatographia. 2005;61:271-275.

© 2012 FORMATEX

1346


[36] Xu L, Basheer C, Lee HK. Solvent-bar microextraction of herbicides combined with non-aqueous field-amplified sample injection capillary electrophoresis. Journal of Chromatography A. 2010;1217:6036-6043. [37] Wu GH, Ji HF, Hansen K, Thundat T, Datar R, Cote R, Hagan MF, Chakraborty AK, Majumdar A. Origin of nanomechanical cantilever motion generated from biomolecular interactions. Proceedings of the National Academy of Sciences of the United States of America. 2001;98:1560-1564. [38] Ilic B, Yang Y, Craighead HG. Virus detection using nanoelectromechanical devices. Applied Physics Letters. 2004;85:26042606. [39] Xu S, Mutharasan R. Rapid and Sensitive Detection of Giardia lamblia Using a Piezoelectric Cantilever Biosensor in Finished and Source Waters. Environmental Science & Technology. 2010;44:1736-1741. [40] Binnig G, Quate CF, Gerber C. Atomic Force Microscope. Physical Review Letters. 1986;56:930-933. [41] Binnig G, Rohrer H. Scanning Tunneling Microscopy. Surface Science. 1983;126:236-244. [42] Rugar D, Hansma P. Atomic Force Microscopy. Physics Today. 1990;43:23-30. [43] Garcia R, Perez R. Dynamic atomic force microscopy methods. Surface Science Reports. 2002;47:197-301. [44] Kelley TW, Granstrom EL, Frisbie CD. Conducting probe atomic force microscopy: A characterization tool for molecular electronics. Advanced Materials. 1999;11:261-264. [45] Qi YB. Investigation of organic films by atomic force microscopy: Structural, nanotribological and electrical properties. Surface Science Reports. 2011;66:379-393. [46] Frisbie CD, Rozsnyai LF, Noy A, Wrighton MS, Lieber CM. Functional-group Imaging by Chemical Force Microscopy. Science. 1994;265:2071-2074. [47] Noy A, Frisbie CD, Rozsnyai LF, Wrighton MS, Lieber CM. Chemical Force Microscopy - Exploiting Chemically-modified Tips to Quantify Adhesion, Friction, and Functional-group Distributions in Molecular Assemblies. Journal of the American Chemical Society. 1995;117:7943-7951. [48] Noy A, Vezenov DV, Lieber CM. Chemical force microscopy. Annual Review of Materials Science. 1997;27:381-421. [49] Butt HJ, Cappella B, Kappl M. Force measurements with the atomic force microscope: Technique, interpretation and applications. Surface Science Reports. 2005;59:1-152. [50] Ito T, Grabowska I, Ibrahim S. Chemical-force microscopy for materials characterization. Trac-Trends in Analytical Chemistry. 2010;29:225-233. [51] Lang HP, Hegner M, Gerber C. Cantilever array sensors. Materials Today. 2005;8:30-36. [52] Ferreira AAP, Yamanaka H. Atomic force microscopy applied to immunoassays. Quimica Nova. 2006;29:137-142. [53] Asensio L, Gonzalez I, Garcia T, Martin R. Determination of food authenticity by enzyme-linked immunosorbent assay (ELISA). Food Control. 2008;19:1-8. [54] Volcke C, Gandhiraman RP, Gubala V, Doyle C, Fonder G, Thiry PA, Cafolla AA, James B, Williams DE. Plasma functionalization of AFM tips for measurement of chemical interactions. Journal of Colloid and Interface Science. 2010;348:322-328. [55] Kim H, Noh J, Hara M, Lee H. Characterization of mixed self-assembled monolayers for immobilization of streptavidin using chemical force microscopy. Ultramicroscopy. 2008;108:1140-1143. [56] Kim H, Park JH, Cho IH, Kim SK, Paek SH, Lee H. Selective immobilization of proteins on gold dot arrays and characterization using chemical force microscopy. Journal of Colloid and Interface Science. 2009;334:161-166. [57] Li Y, Lee J, Lal J, An L, Huang Q. Effects of pH on the interactions and conformation of bovine serum albumin: Comparison between chemical force microscopy and small-angle neutron scattering. Journal of Physical Chemistry B. 2008;112:3797-3806. [58] Fiorini M, McKendry R, Cooper MA, Rayment T, Abell C. Chemical force microscopy with active enzymes. Biophysical Journal. 2001;80:2471-2476. [59] Wagner M, Mavon A, Haidara H, Vallat MF, Duplan H, Roucoules V. From contact angle titration to chemical force microscopy: a new route to assess the pH-dependent character of the stratum corneum. International Journal of Cosmetic Science. 2012;34:55-63. [60] Dufrene YF. Atomic force microscopy and chemical force microscopy of microbial cells. Nature Protocols. 2008;3:1132-1138. [61] Dague E, Alsteens D, Latge JP, Verbelen C, Raze D, Baulard AR, Dufrene YF. Chemical force microscopy of single live cells. Nano Letters. 2007;7:3026-3030. [62] Fujihira M, Furugori M, Akiba U, Tani Y. Study of microcontact printed patterns by chemical force microscopy. Ultramicroscopy. 2001;86:75-83. [63] Noy A. Strength in numbers: Probing and understanding intermolecular bonding with chemical force microscopy. Scanning. 2008;30:96-105. [64] Dordi B, Pickering JP, Schonherr H, Vancso GJ. Inverted chemical force microscopy: following interfacial reactions on the nanometer scale. European Polymer Journal. 2004;40:939-947. [65] Kado S, Murakami T, Kimura K. Effect of intramonolayer hydrogen bonding of carboxyl groups in self-assembled monolayers on a single force with phenylurea on an AFM probe tip. Analytical Sciences. 2006;22:521-527. [66] Hlinka J, Hodacova J, Raehm L, Granier M, Ramonda M, Durand JO. Attachment of trianglamines to silicon wafers, chiral recognition by chemical force microscopy. Comptes Rendus Chimie. 2010;13:481-485. [67] Otsuka H, Arima T, Koga T, Takahara A. Rational model for chiral recognition in a silica-based chiral column: chiral recognition of N-(3,5-dinitrobenzoyl)phenylglycine-terminated alkylsilane monlayer by 2,2,2-triflioro-1-(9-anthryl)ethanol derivatives by chemical force mircoscopy. Journal of Physical Organic Chemistry. 2005;18:957-961. [68] McKendry R, Theoclitou ME, Rayment T, Abell C. Chiral discrimination by chemical force microscopy. Nature. 1998;391:566-568. [69] Wong SS, Joselevich E, Woolley AT, Cheung CL, Lieber CM. Covalently functionalized nanotubes as nanometre-sized probes in chemistry and biology. Nature. 1998;394:52-55. [70] Agrawal R, Lee K, Ponnavolu B. AFM Spectroscopy. ME 382: Micro/Nano Science and Engineering. 2004;13p. [71] Buschan B, Fuchs H, Hosaka S. Applied scanning probe methods I. Heidelberg: Springer; 2004.

© 2012 FORMATEX

1347


[72] Leite FL, Mattoso LHC, Oliveira Jr ON, Herrmann Jr PSP. The Atomic Force Spectroscopy as a Tool to Investigate Surface Forces: Basic Principles and Applications. In: Méndez-Vilas A, Díaz J, eds. Modern Research and Educational Topics in Microscopy. Badajoz: Formatex; 2007:747-757. [73] Leite FL, Zeimath EC, Herrmann Jr PSP. Análise de Minerais do Solo por Espectroscopia de Força Atômica. Embrapa. 2005;70:1-7. [74] Wiesendanger R. Scanning Probe Microscopy and Spectroscopy Methods and Applications. Cambridge, University Press. 1994; [75] Burnham NA, Colton RJ, Pollock HM. Interpretation of force curves in force microscopy. Nanotechnology. 1993;4:64-80. [76] Burnham NA, Colton RJ. Measuring the Nanomechanical Properties and Surface Forces of Materials using an Atomic Force Microscope. Journal of Vacuum Science & Technology a-Vacuum Surfaces and Films. 1989;7:2906-2913. [77] Burnham NA, Dominguez DD, Mowery RL, Colton RJ. Probing the Surface Forces of Monolayer Films with an Atomic-force Microscope. Physical Review Letters. 1990;64:1931-1934. [78] Li SP, Shi RY, Wang QL, Cai JY, Zhang SQ. Nanostructure and beta 1-integrin distribution analysis of pig's spermatogonial stem cell by atomic force microscopy. Gene. 2012;495:189-193. [79] Agnihotri A, Siedlecki CA. Adhesion mode atomic force microscopy study of dual component protein films. Ultramicroscopy. 2005;102:257-268. [80] Garcia-Manyes S, Domenech O, Sanz F, Montero MT, Hernandez-Borrell J. Atomic force microscopy and force spectroscopy study of Langmuir-Blodgett films formed by heteroacid phospholipids of biological interest. Biochimica Et Biophysica ActaBiomembranes. 2007;1768:1190-1198. [81] Polesel-Maris J, Legrand J, Berthelot T, Garcia A, Viel P, Makky A, Palacin S. Force spectroscopy by dynamic atomic force microscopy on bovine serum albumin proteins changing the tip hydrophobicity, with piezoelectric tuning fork self-sensing scanning probe. Sensors and Actuators B-Chemical. 2012;161:775-783. [82] Etchegaray A, Bueno CD, Teschke O. Identification of Microcistin LR at the Molecular Level Using Atomic Force Microscopy. Quimica Nova. 2010;33:1843-1848. [83] Morris VJ, Kirby AR, Gunning AP eds. Atomic force microscopy for biologists. London: Imperial College Press; 2004. [84] Hinterdorfer P, Schilcher K, Baumgartner W, Gruber HJ, Schindler H. A Mechanistic Study of the Dissociation of Individual Antibody-Antigen Pairs by Atomic Force Microscopy. Nanobiology. 1998;4:177-188. [85] Houmadi S, Rodriguez RD, Longobardi S, Giardina P, Faure MC, Giocondo M, Lacaze E. Self-Assembly of Hydrophobin Protein Rodlets Studied with Atomic Force Spectroscopy in Dynamic Mode. Langmuir. 2012;28:2551-2557. [86] Gao H, Zhang XX, Chang WB. Study on the interaction in single antigen-antibody molecules by AFM. Frontiers in Bioscience. 2005;10:1539-1545. [87] Ghormade V, Deshpande MV, Paknikar KM. Perspectives for nano-biotechnology enabled protection and nutrition of plants. Biotechnology Advances. 2011;29:792-803. [88] Hornyak GL, Tibbals HF, Dutta J, Moore JJ. Introduction to Nanoscience and Nanotechnology. Boca Raton, FL: CRC Press; 2008. [89] Webb HK, Truong VK, Hasan J, Crawford RJ, Ivanoya EP. Physico-mechanical characterisation of cells using atomic force microscopy - Current research and methodologies. Journal of Microbiological Methods. 2011;86:131-139.

© 2012 FORMATEX

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