Modeling the coverage of an AFM tip by enzymes and its application in nanobiosensors (original) (raw)
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Molecular Modeling of Enzyme Attachment on AFM Probes
Journal of Molecular Graphics and Modelling, 2013
The immobilization of enzymes on atomic force microscope tip (AFM tip) surface is a crucial step in the development of nanobiosensors to be used in detection process. In this work, an atomistic modeling of the attachment of the acetyl coenzyme A carboxylase (ACC enzyme) on a functionalized AFM tip surface is proposed. Using electrostatic considerations, suitable enzyme–surface orientations with the active sites of the ACC enzyme available for interactions with bulk molecules were found. A 50 ns molecular dynamics trajectory in aqueous solution was obtained and surface contact area, hydrogen bonding and protein stability were analyzed. The enzyme–surface model proposed here with minor adjustment can be applied to study antigen–antibody interactions as well as enzyme immobilization on silica for chromatography applications.
Introduction to Atomic Force Microscopy Simulation
Microscopy: Science, Technology, Applications and Education, 2010
The atomic force microscopy (AFM) is a powerful for single-molecule force experiment that can characterize physical and chemical properties of biological and polymeric matter at the nanometer scale. However, it does not reveal the molecular mechanisms behind the binding of ligands and conformational changes in biomolecules in atomic time scale. This information can only be addressed by molecular dynamics (DM) simulation, which simulates the AFM experiments through methodology called Steered Molecular Dynamics (SMD). The AFM simulation is usually obtained by integrating the mean force from an ensemble of configurations resulted from a molecular mechanics calculation. In this chapter, we shall concentrate on simulation of the atomic force spectroscopy (AFS), which procedure consist in perform a constant velocity molecular dynamics simulation, recording force and position at each time point, to reproduce and predict the atomic force curves. The AFM simulation showed to be very useful to provide qualitative and quantitative information about ligand binding pathways in enzymes and the mechanical properties of biological and synthetic polymers.
Atomic Force Microscopy as a Tool Applied to Nano/Biosensors
Sensors, 2012
This review article discusses and documents the basic concepts and principles of nano/biosensors. More specifically, we comment on the use of Chemical Force Microscopy (CFM) to study various aspects of architectural and chemical design details of specific molecules and polymers and its influence on the control of chemical interactions between the Atomic Force Microscopy (AFM) tip and the sample. This technique is based on the fabrication of nanomechanical cantilever sensors (NCS) and microcantilever-based biosensors (MC-B), which can provide, depending on the application, rapid, sensitive, simple and low-cost in situ detection. Besides, it can provide high repeatability and reproducibility. Here, we review the applications of CFM through some application examples which should function as methodological questions to understand and transform this tool into a reliable source of data. This section is followed by a description of the theoretical principle and usage of the functionalized NCS and MC-B technique in several fields, such as agriculture, biotechnology and immunoassay. Finally, we hope this review will help the reader to appreciate how important the tools CFM, NCS and MC-B are for characterization and understanding of systems on the atomic scale.
Biophysics, 2011
An approach to measure the activity of single oligomers of the heme containing enzyme cyto chrome P450 CYP102A1 (CYP102A1) by atomic force microscopy (AFM) has been developed. It was found that the amplitude of fluctuations of the height of single CYP102A1 molecules performing the catalytic cycle is twice as great as the amplitude of fluctuations of the height of the same enzymes in the inactive state. It was shown that the amplitude of height fluctuations of a CYP102A1 protein globule depends on temperature, the maximum of this dependence being observed at 22°C. The activity of a single CYP102A1 molecule in the unit amplitude of height fluctuations of a protein globule per unit time was 5 ± 2 Å/s. The elasticity of a single protein molecule was measured from the deformation of this molecule by the action of an AFM probe. The use of AFM probes of different geometry made it possible to determine the integral and local Young's mod ulus for the monomers of the protein putidaredoxin reductase from the cytochrome P450 CYP101 (P450cam) containing monooxigenase system, which were 37 ± 117 and 1 ± 3 MPa, respectively.
Use of molecular dynamics simulation in interpreting the atomic force microscopy data
Biophysics, 2010
A new approach to interpreting and refining the atomic force microscopy (AFM) data, based on comparing them with the output of computer simulated probe scanning, has been tested with lysozyme. Dis tinct AFM images were obtained experimentally for individual lysozyme monomers adsorbed from a clear aqueous solution onto a mica wafer. Two steps of simulations were performed to reproduce the environment and processes in the AFM experiment. First, we used the molecular dynamics software (NAMD) to model the structure of lysozyme adsorbed from a water solution onto a silicon oxide support (the latter was modeled manually according to its crystal structure). Second, we applied molecular mechanics to reproduce probe tip interactions with the object. As a result, we have obtained the lysozyme surface height as a function of hori zontal coordinates. Comparison with the real AFM data gave a fair fit in the shape of lysozyme molecules but a significant difference in size. Analysis of the possible causes of this discrepancy indicated that more detailed simulations of AFM imaging with fuller account of the experimental conditions are needed to reach a better correspondence. The first results of testing our approach provide sufficient information for improving the accuracy in further applications.
Methods in molecular biology (Clifton, N.J.), 2005
For investigation of laterally resolved information on biological activity, techniques for simultaneously obtaining complementary information correlated in time and space are required. In this context, recent developments in scanning probe microscopy are aimed at information on the sample topography and simultaneously on the physical and chemical properties at the nanometer scale. With the integration of submicro- and nanoelectrodes into atomic force microscopy (AFM) probes using microfabrication techniques, an elegant approach combining scanning electrochemical microscopy with AFM is demonstrated. This instrumentation enables simultaneous imaging of topography and obtainment of laterally resolved electrochemical information in AFM tapping mode. Hence, topographical and electrochemical information on soft surfaces (e.g., biological species) and polymers can be obtained. The functionality of tip-integrated electrodes is demonstrated by simultaneous electrochemical and topographical s...
Transactions of the Materials Research Society of Japan, 2014
Protein adsorption behavior was examined from viewpoint of molecular interaction force generating on material surfaces. To achieve this, the methodology to evaluate the nano-scale molecular interaction forces on the well-defined surfaces by the force-versus-distance curve measurements using atomic force microscopy (AFM) was established. Zwitterionic, cationic, anionic, and hydrophobic polymer brush surfaces were prepared as model surfaces to analyze the interaction forces operating on the surfaces. The amount of proteins adsorbed on the polymer brush surfaces was quantified by surface plasmon resonance measurement. The molecular interaction forces operating on the polymer brush surfaces were evaluated using the AFM probes modified with functional groups. On the zwitterionic polymer brush surface, molecular interaction forces were not observed, and amount of protein adsorption was little. On the other hand, cationic, anionic, or hydrophobic polymer brush surface exhibited strong molecular interaction forces, and large amount of proteins adsorbed on these surfaces. These results indicated that the preparation of material surfaces, which avoid the molecular interactions, is significant for suppression of protein adsorption.
Nano-Mechanical Methods in Biochemistry using Atomic Force Microscopy
Current Protein & Peptide Science, 2003
The atomic force microscope has been extensively used not only to image nanometer-sized biological samples but also to measure their mechanical properties by using the force curve mode of the instrument. When the analysis based on the Hertz model of indentation is applied to the approach part of the force curve, one obtains information on the stiffness of the sample in terms of Young's modulus. Mapping of local stiffness over a single living cell is possible by this method. The retraction part of the force curve provides information on the adhesive interaction between the sample and the AFM tip. It is possible to functionalize the AFM tip with specific ligands so that one can target the adhesive interaction to specific pairs of ligands and receptors. The presence of specific receptors on the living cell surface has been mapped by this method. The force to break the cooperative 3D structure of globular proteins or to separate a double stranded DNA into single strands has been measured. Extension of the method for harvesting functional molecules from the cytosol or the cell surface for biochemical analysis has been reported. There is a need for the development of biochemical nano-analysis based on AFM technology.
Designing an enzyme-based nanobiosensor using molecular modeling techniques
Physical Chemistry Chemical Physics, 2011
Nanobiosensors can be built via functionalization of atomic force microscopy (AFM) tips with biomolecules capable of interacting with the analyte on a substrate, and the detection being performed by measuring the force between the immobilized biomolecule and the analyte. The optimization of such sensors may require multiple experiments to determine suitable experimental conditions for the immobilization and detection. In this study we employ molecular modeling techniques to assist in the design of nanobiosensors to detect herbicides. As a proof of principle, the properties of acetyl co-enzyme A carboxylase (ACC) were obtained with molecular dynamics simulations, from which the dimeric form in an aqueous solution was found to be more suitable for immobilization owing to a smaller structural fluctuation than the monomeric form. Upon solving the nonlinear Poisson-Boltzmann equation using a finite-difference procedure, we found that the active sites of ACC exhibited a positive surface potential while the remainder of the ACC surface was negatively charged. Therefore, optimized biosensors should be prepared with electrostatic adsorption of ACC onto an AFM tip functionalized with positively charged groups, leaving the active sites exposed to the analyte. The preferential orientation for the herbicides diclofop and atrazine with the ACC active site was determined by molecular docking calculations which displayed an inhibition coefficient of 0.168 mM for diclofop, and 44.11 mM for atrazine. This binding selectivity for the herbicide family of diclofop was confirmed by semiempirical PM6 quantum chemical calculations which revealed that ACC interacts more strongly with the herbicide diclofop than with atrazine, showing binding energies of À119.04 and +8.40 kcal mol À1 , respectively. The initial measurements of the proposed nanobiosensor validated the theoretical calculations and displayed high selectivity for the family of the diclofop herbicides.
An Efficient Method for Enzyme Immobilization evidenced by Atomic Force Microscopy
Protein Engineering, Design & Selection, 2012
Immobilization of proteins in a functionally active form and proper orientation is fundamental for effective surface-based protein analysis. A new method is presented for the controlled and oriented immobilization of ordered monolayers of enzymes whose interaction site had been protected using the protein ligand. The utility of this method was demonstrated by analyzing the interactions between the enzyme ferredoxin-NADP 1 reductase (FNR) and its redox partner ferredoxin (Fd). The quality of the procedure was deeply evaluated through enzymatic assays and atomic force microscopy. Single-molecule force spectroscopy revealed that site-specifically targeted FNR samples increased the ratio of recognition events 4fold with regard to the standard randomly modified FNR samples. The results were corroborated using the cytochrome c reductase activity that gave an increase on surface between 6 and 12 times for the site-specifically targeted FNR samples. The activity in solution for the enzyme labeled from the complex was similar to that exhibited by wild-type FNR while FNR randomly tagged showed a 3-fold decrease. This indicates that random targeting protocols affect not only the efficiency of immobilized proteins to recognize their ligands but also their general functionality. The present methodology is expected to find wide applications in surface-based protein -protein interactions biosensors, single-molecule analysis, bioelectronics or drug screening.