**Abstract**

The design of bioelectrochemical interfaces (BEI) is an interesting topic that recently demands attention. The synergy between biomolecules and chemical components is necessary to achieve high molecular selectivity and sensitivity for the development of biosensors, synthesis of different compounds, or catalytic processes. For most BEI, the charge transfer process occurs in environments with particular chemical conditions; modeling these environments is a challenging task and requires multidisciplinary efforts. These interfaces can be composed of biomolecules, such as proteins, DNA, or more complex systems like microorganisms. Oxidoreductases enzymes are good candidates, among others, due to their catalytic activities and structural characteristics. In BEI, enzymes are immobilized on conductive surfaces to improve charge transfer processes. Covalent immobilization is the most common method to prolong lifetime or modulate the detection process. However, it is necessary to implement new methodologies that allow the selection of the best candidates for a more efficient design. Homology modeling of oxidoreductases combined with Molecular Dynamics (MD) simulation methods are alternative and already routinely used tools to investigate the structure, dynamics, and thermodynamics of biological molecules. Our motivation is to show different techniques of molecular modeling (Homology Modeling, Gaussian accelerated molecular dynamics, directed adaptive molecular dynamics and electrostatic surface calculations), and using horseradish peroxidase as a model to understand the interactions between biomolecules and gold nanoclusters (as current collector). Additionally, we present our previous studies considering molecular simulations and we discuss recent advances in biomolecular simulations aimed at biosensor design.

**Keywords:** bioelectrochemical interfaces, homology modeling, covalent immobilization, gold nanoclusters, molecular dynamics

## **1. Introduction**

For some years now, the design and construction of bioelectrochemical interfaces (BEI), ranging from electrochemical biosensors (EC) for analytical applications, biofuel cells (BFC) to the development of biocomputing systems for information processing, have been topics where the scientific community has considerable participation. The implementation and integration of biomolecules

with electronic elements or conductive surfaces to produce bioelectronic devices is a current topic and gathers great importance for developing biosensors that have an essential role in clinical applications, food quality control analysis and forensic medicine [1–3]. These applications always involve high sensitivity, low-sample volume, and low-cost production. In most BEI, charge transfer processes are carried out in environments with high ionic concentration, necessary for their function. These interfaces can be composed of macromolecules such as proteins, peptides, or more complex systems such as bacteria. Since the activity and lifetime depend on the correct interaction between the conductive surfaces (current collector) and the biomaterials, to achieve good bioelectrochemical responses, the enzymes need to improve the orientation towards the surface of the current collector [4, 5].

#### **1.1 Covalent immobilization through alkanethiol linkers**

The immobilization of various biomaterials is an important issue for BEI design, the covalent immobilization of enzymes stands out among the different enzymatic coupling strategies since the bonds formed through the linkers anchored to the current collector promotes direct charge transfer responses [6, 7]. Alcakanothiols are organic molecules widely used to establish self-assembled monolayers (SAMs) on the surface of gold electrodes [8–11]. The thiol groups chemisorb on gold electrodes forming strong thiolate-gold bonds (Au-S). The resulting monolayer may be used to design molecular scaffolds to couple enzymes [8, 12]. The main drawback of covalent immobilization systems is the risk of having a low surface concentration of enzymes that are either active or correctly oriented for direct charge transfer; which may lead to a low-efficiency bioelectrochemical response [13]. Therefore, having active enzymes and a good bioelectrochemical response becomes an important task. Aromatic molecules like 4-aminothiophenol, structurally mimics enzymatic substrates and can promote stable and direct contacts, necessary for efficient bioelectrochemical reactions.

Researches have proposed nanomaterials as support matrices for the design of BEI to increase the surface concentration of active enzymes on the current collector. During the last decade, nanomaterials, coupled with enzymes, have had significant relevance in the design of biosensors [5, 14–16]. Current advances in synthesis methodologies of these materials allow having a wide variety of nanomaterials with different sizes, shapes, surface charges, and physicochemical characteristics [4, 14, 17].

Some of these nanomaterials can be modified in different ways to improve biocompatibility. Biocompatibility refers to the biological nature events that do not interfere with those of electronic signal transduction and vice versa. Metallic nanoparticles are promising materials that increase the electroactive area and improve the sensitivity and stability of the attached enzymes on the electrodes, bringing the enzymatic active site close to the electrode (the redox cofactor should not exceed a 20 Å distance) to achieve direct charge transfer reactions [14, 18–23].

In our group, we have established the conditions to improve the electrochemical response of horseradish peroxidase (HRP) coupled to gold nanoparticles modified with the aromatic alkanothiol 4-aminotiophenol, using glassy carbon (GC) as current collector [24]. We proposed that a critical factor in HRP bioelectrochemical response is to promote conformational changes and proper enzyme orientation when coupled to the electrode. The protein conformation around the prosthetic group will determine the redox potential of the Fe3+/Fe2+ couple. The activity towards H2O2 also depends on the protein structural arrangements [25, 26]. We found that a proper environment for the enzyme activity was achieved by increasing the distance between gold nanoparticles. The voltammetric studies of the prosthetic heme group showed significant differences between the enzyme immobilized on randomly

**103**

**Figure 1.**

*Design of Bioelectrochemical Interfaces Assisted by Molecular Dynamics Simulations*

recognition or transformation of the substrate, as previously observed [27].

deposited gold nanoparticles and the enzyme immobilized on a well-dispersed gold nanoparticles deposit (**Figure 1**). The dispersed electrode improved the electrochemical response of the enzyme; this fact showed that the distance between enzymes is important, probably because longer distances decreased the steric impediments between the enzymes, and it appears to be a better immobilization strategy. With these results, we proposed that an essential part of BEI design is related to the structure of the biological systems. The electrochemical sensing is dependent on the

The advent of high-resolution and robust techniques as X-ray crystallography [28, 29] and cryo-EM [30–32] has enormously contributed to the baggage of structural information available for bioelectrochemical applications. These techniques describe the atomic positions of enzymes, as well as the conformational dynamics resulted from recognition and binding processes, allowing the prediction of the possible electrochemical behavior of enzymes immobilized on electrodes.

The use of computational tools such as molecular mechanics and quantum mechanics for the study of chemical and biological reactions, involves a mathematical treatment of a large number of particles (hundreds of thousands of atoms that build up molecules like proteins, nucleic acids, lipids, and sugars) [33, 34]. In general terms, these methods are ideal for obtaining chemical and physical properties from three-dimensional molecular models [35–37]. It is necessary to correlate the structural models with their corresponding chemical, catalytic or biological properties; towards the rationalization and design of molecules for specific applications. Mainly, molecular Dynamics (MD) methods are based on the solution of Newton's second law for all the atoms in the system, and help to predict the behavior of a biomolecule during a specific time. The integration of motion equations allows to analyze the trajectories corresponding to position, velocity, and acceleration of each particle of the system in any

*Cyclic voltammogram of HRP response at different scan rates, immobilized on GC electrode modified with gold nanoparticles functionalized with the alkanethiol molecule 4-aminothiophenol. Phosphate buffer pH 6.8.*

*DOI: http://dx.doi.org/10.5772/intechopen.93884*

**1.2 Molecular modeling methodologies**

#### *Design of Bioelectrochemical Interfaces Assisted by Molecular Dynamics Simulations DOI: http://dx.doi.org/10.5772/intechopen.93884*

deposited gold nanoparticles and the enzyme immobilized on a well-dispersed gold nanoparticles deposit (**Figure 1**). The dispersed electrode improved the electrochemical response of the enzyme; this fact showed that the distance between enzymes is important, probably because longer distances decreased the steric impediments between the enzymes, and it appears to be a better immobilization strategy. With these results, we proposed that an essential part of BEI design is related to the structure of the biological systems. The electrochemical sensing is dependent on the recognition or transformation of the substrate, as previously observed [27].

The advent of high-resolution and robust techniques as X-ray crystallography [28, 29] and cryo-EM [30–32] has enormously contributed to the baggage of structural information available for bioelectrochemical applications. These techniques describe the atomic positions of enzymes, as well as the conformational dynamics resulted from recognition and binding processes, allowing the prediction of the possible electrochemical behavior of enzymes immobilized on electrodes.

### **1.2 Molecular modeling methodologies**

*Homology Molecular Modeling - Perspectives and Applications*

**1.1 Covalent immobilization through alkanethiol linkers**

bioelectrochemical reactions.

with electronic elements or conductive surfaces to produce bioelectronic devices is a current topic and gathers great importance for developing biosensors that have an essential role in clinical applications, food quality control analysis and forensic medicine [1–3]. These applications always involve high sensitivity, low-sample volume, and low-cost production. In most BEI, charge transfer processes are carried out in environments with high ionic concentration, necessary for their function. These interfaces can be composed of macromolecules such as proteins, peptides, or more complex systems such as bacteria. Since the activity and lifetime depend on the correct interaction between the conductive surfaces (current collector) and the biomaterials, to achieve good bioelectrochemical responses, the enzymes need to improve the orientation towards the surface of the current collector [4, 5].

The immobilization of various biomaterials is an important issue for BEI design, the covalent immobilization of enzymes stands out among the different enzymatic coupling strategies since the bonds formed through the linkers anchored to the current collector promotes direct charge transfer responses [6, 7]. Alcakanothiols are organic molecules widely used to establish self-assembled monolayers (SAMs) on the surface of gold electrodes [8–11]. The thiol groups chemisorb on gold electrodes forming strong thiolate-gold bonds (Au-S). The resulting monolayer may be used to design molecular scaffolds to couple enzymes [8, 12]. The main drawback of covalent immobilization systems is the risk of having a low surface concentration of enzymes that are either active or correctly oriented for direct charge transfer; which may lead to a low-efficiency bioelectrochemical response [13]. Therefore, having active enzymes and a good bioelectrochemical response becomes an important task. Aromatic molecules like 4-aminothiophenol, structurally mimics enzymatic substrates and can promote stable and direct contacts, necessary for efficient

Researches have proposed nanomaterials as support matrices for the design of BEI to increase the surface concentration of active enzymes on the current collector. During the last decade, nanomaterials, coupled with enzymes, have had significant relevance in the design of biosensors [5, 14–16]. Current advances in synthesis methodologies of these materials allow having a wide variety of nanomaterials with different sizes, shapes, surface charges, and physicochemical characteristics [4, 14, 17]. Some of these nanomaterials can be modified in different ways to improve biocompatibility. Biocompatibility refers to the biological nature events that do not interfere with those of electronic signal transduction and vice versa. Metallic nanoparticles are promising materials that increase the electroactive area and improve the sensitivity and stability of the attached enzymes on the electrodes, bringing the enzymatic active site close to the electrode (the redox cofactor should not exceed a 20 Å distance) to achieve direct charge transfer reactions [14, 18–23]. In our group, we have established the conditions to improve the electrochemical

response of horseradish peroxidase (HRP) coupled to gold nanoparticles modified with the aromatic alkanothiol 4-aminotiophenol, using glassy carbon (GC) as current collector [24]. We proposed that a critical factor in HRP bioelectrochemical response is to promote conformational changes and proper enzyme orientation when coupled to the electrode. The protein conformation around the prosthetic group will determine the redox potential of the Fe3+/Fe2+ couple. The activity towards H2O2 also depends on the protein structural arrangements [25, 26]. We found that a proper environment for the enzyme activity was achieved by increasing the distance between gold nanoparticles. The voltammetric studies of the prosthetic heme group showed significant differences between the enzyme immobilized on randomly

**102**

The use of computational tools such as molecular mechanics and quantum mechanics for the study of chemical and biological reactions, involves a mathematical treatment of a large number of particles (hundreds of thousands of atoms that build up molecules like proteins, nucleic acids, lipids, and sugars) [33, 34]. In general terms, these methods are ideal for obtaining chemical and physical properties from three-dimensional molecular models [35–37]. It is necessary to correlate the structural models with their corresponding chemical, catalytic or biological properties; towards the rationalization and design of molecules for specific applications. Mainly, molecular Dynamics (MD) methods are based on the solution of Newton's second law for all the atoms in the system, and help to predict the behavior of a biomolecule during a specific time. The integration of motion equations allows to analyze the trajectories corresponding to position, velocity, and acceleration of each particle of the system in any

#### **Figure 1.**

*Cyclic voltammogram of HRP response at different scan rates, immobilized on GC electrode modified with gold nanoparticles functionalized with the alkanethiol molecule 4-aminothiophenol. Phosphate buffer pH 6.8.*

fraction of time [38]. Unfortunately, the simplification of considering atoms as spheres, and bonds as springs or tensors (with different parameters depending on the type of bond), makes difficult to study bond-breaking and formation, or reaction mechanisms. Quantum mechanics (QM) mathematically describes the fundamental behavior of matter on an electronic scale by solving the Schrödinger equation for each atom in the system. QM also describes the behavior of atoms and molecules, in terms of their chemical reactivity, geometry, and their optical, electrical, magnetic, and mechanical properties [39, 40]. The coupled implementation of these techniques could describe the biomolecules involved in charge transfer processes, and design strategies for efficient BEI.

## **1.3 Molecular dynamics as a tool to predict the electrochemical activity in proteins**

A challenging task in BEI design is the selection of robust biomolecules capable of tolerate non-physiological conditions, without loosing efficiency and conformation [2]. The protein folding is governed by a series of molecular interactions between the amino acids and the surrounding chemical environment [34]. Therefore, predicting their behavior with molecular models contributes to the optimization of resources before the development of BEI.

In our previous work the VP6 rotavirus capsid protein was encapsulated with the ionic polymer Nafion on GC. We demonstrated that this electrode could transfer electric charge applying an external potential, when using the redox probe potassium ferricyanide (K3[Fe(CN)6]). In parallel, we modeled by MD the electrostatic and conformational states of VP6 (**Figure 2**). This model suggested a route for ionic conductivity, where the electrostatic protein surface displayed a negative charge which interacted with the ferricyanide redox probe, promoting the charge transfer reaction. In order to show if this electrochemical activity was particular to proteins in general, under the same conditions as the experiments with VP6, bovine serum albumin (BSA), whose primary biological function is

#### **Figure 2.**

*Comparison of the cyclic Voltammogram of VP6 (pink) and BSA (blue) coupled to GC, at 20 mV/s in 0.01 M K3[Fe(CN)6], 1 M KCl.*

**105**

**Figure 3.**

*(red) mesh isosurfaces with VMD v.1.9.3 [44].*

*Design of Bioelectrochemical Interfaces Assisted by Molecular Dynamics Simulations*

to transport different molecules through the bloodstream, was tested. However, we did not observe the charge transfer reaction of potassium ferricyanide when this protein was encapsulated (**Figure 2**). These results demonstrated that VP6 protein could be used as conductive scaffold for the development of different BEI

Calculations of polarized electrostatic surface potentials from homology models of viral proteins, surprisingly showed that several capsids could display field effects as the recorded on VP6. In contrast, this effect cannot be displayed on non-charged proteins as BSA (**Figure 3**). Therefore, our previous data on viral capsids can serve as a workflow to identify candidate proteins or enzymes for construction of BEI devices. Altogether, theoretical and experimental reports on BEI [41–43] are examples of how structural information could help to elucidate the behavior of biological systems during electrochemical reactions. Nonetheless, the aforementioned studies depend on structural information from biomolecules, and certain biomolecules are very complex and highly labile, which difficult elucidation of atomic positions. However, since new protein sequences are continuously available, predictive methodologies as homology modeling becomes a valuable tool

The aim of this multidisciplinary research is to perform molecular simulations on horseradish peroxidase as a model system, to generate data that can be used as selection criteria for design and further experimental validation in BEI

*Polarized electrostatic Isosurface potentials of different viral capsids. (A) Bovine serum albumin protein, (B) rotavirus VP6 trimer capsid protein, (C) influenza virus capsid protein, (D) hepatitis B virus capsid protein, (E) HIV-1 virus capsid protein, (F) HPV virus capsid protein. All polarized electrostatic Isosurfaces were calculated with PME approximation on capsid arrangements and displayed as positive (blue) or negative* 

*DOI: http://dx.doi.org/10.5772/intechopen.93884*

applications [41].

to asset structural information.

development.

#### *Design of Bioelectrochemical Interfaces Assisted by Molecular Dynamics Simulations DOI: http://dx.doi.org/10.5772/intechopen.93884*

to transport different molecules through the bloodstream, was tested. However, we did not observe the charge transfer reaction of potassium ferricyanide when this protein was encapsulated (**Figure 2**). These results demonstrated that VP6 protein could be used as conductive scaffold for the development of different BEI applications [41].

Calculations of polarized electrostatic surface potentials from homology models of viral proteins, surprisingly showed that several capsids could display field effects as the recorded on VP6. In contrast, this effect cannot be displayed on non-charged proteins as BSA (**Figure 3**). Therefore, our previous data on viral capsids can serve as a workflow to identify candidate proteins or enzymes for construction of BEI devices. Altogether, theoretical and experimental reports on BEI [41–43] are examples of how structural information could help to elucidate the behavior of biological systems during electrochemical reactions. Nonetheless, the aforementioned studies depend on structural information from biomolecules, and certain biomolecules are very complex and highly labile, which difficult elucidation of atomic positions. However, since new protein sequences are continuously available, predictive methodologies as homology modeling becomes a valuable tool to asset structural information.

The aim of this multidisciplinary research is to perform molecular simulations on horseradish peroxidase as a model system, to generate data that can be used as selection criteria for design and further experimental validation in BEI development.

#### **Figure 3.**

*Homology Molecular Modeling - Perspectives and Applications*

transfer processes, and design strategies for efficient BEI.

optimization of resources before the development of BEI.

**in proteins**

**1.3 Molecular dynamics as a tool to predict the electrochemical activity** 

A challenging task in BEI design is the selection of robust biomolecules capable of tolerate non-physiological conditions, without loosing efficiency and conformation [2]. The protein folding is governed by a series of molecular interactions between the amino acids and the surrounding chemical environment [34]. Therefore, predicting their behavior with molecular models contributes to the

the ionic polymer Nafion on GC. We demonstrated that this electrode could transfer electric charge applying an external potential, when using the redox probe potassium ferricyanide (K3[Fe(CN)6]). In parallel, we modeled by MD the electrostatic and conformational states of VP6 (**Figure 2**). This model suggested a route for ionic conductivity, where the electrostatic protein surface displayed a negative charge which interacted with the ferricyanide redox probe, promoting the charge transfer reaction. In order to show if this electrochemical activity was particular to proteins in general, under the same conditions as the experiments with VP6, bovine serum albumin (BSA), whose primary biological function is

*Comparison of the cyclic Voltammogram of VP6 (pink) and BSA (blue) coupled to GC, at 20 mV/s in* 

In our previous work the VP6 rotavirus capsid protein was encapsulated with

fraction of time [38]. Unfortunately, the simplification of considering atoms as spheres, and bonds as springs or tensors (with different parameters depending on the type of bond), makes difficult to study bond-breaking and formation, or reaction mechanisms. Quantum mechanics (QM) mathematically describes the fundamental behavior of matter on an electronic scale by solving the Schrödinger equation for each atom in the system. QM also describes the behavior of atoms and molecules, in terms of their chemical reactivity, geometry, and their optical, electrical, magnetic, and mechanical properties [39, 40]. The coupled implementation of these techniques could describe the biomolecules involved in charge

**104**

**Figure 2.**

*0.01 M K3[Fe(CN)6], 1 M KCl.*

*Polarized electrostatic Isosurface potentials of different viral capsids. (A) Bovine serum albumin protein, (B) rotavirus VP6 trimer capsid protein, (C) influenza virus capsid protein, (D) hepatitis B virus capsid protein, (E) HIV-1 virus capsid protein, (F) HPV virus capsid protein. All polarized electrostatic Isosurfaces were calculated with PME approximation on capsid arrangements and displayed as positive (blue) or negative (red) mesh isosurfaces with VMD v.1.9.3 [44].*
