Develop Concept

Proteins are built up from amino acids (AA) that are composed of a central carbon atom bonded to a hydrogen atom, an amino group (N-terminal), a carboxyl group (C-terminal) and a side chain (R) with covalent bond as shown in Figure 1.2. The presence of ionized amino and the carboxyl (COO-_{) group produces a predominant} dipolar which can be then rotated. Although, typically there are 20 amino acids in nature, their distinctive structures are due to the presence of various non-polar and polar amino as well as charged acid side chains. The specific protein sequence formed by its amino acids are connected together to form polypeptide polymer chains and build up the primary structure of a protein. Accordingly, each polypeptide also has various non-polar and polar amino acid side chains and produces dipolar. Although the peptide bond is polar, it cannot rotate freely, thus, its motion is restricted [21]. However, these polypeptides are folded into a three-dimensional (3D) structure by a variety of intramolecular interactions, such as electrostatic force within the side chain sequence which characterizes the active proteins that affect the final protein conformation [1]. Commonly, in the 3D complex structure of the protein, however, the non-polar side chains (hydrophobic residues) are stifled inside the protein core, while the polar and charged groups are distributed on the protein surface and considered as the predominant group to be in contact with other predominantly protein groups, and with the aqueous medium interact in a complex process to perform various cell functions. The structural rearrangement of the protein changes its molecular shape, due to the bond rotation, without breaking the covalent bonds defines the protein conformation [34].

Figure 1.2. The primary protein structure, (a) amino acid structure, and (b) amino acids are connected together by a covalent linkage called a peptide bond to form polypeptide polymer chains and to build up the primary structure of a protein.

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Proteins involve their amino acid and are therefore, a dynamic molecular structure, and the distribution of high and low charges on a side chain with degrees of polarity in their sequence domain, along with hydrophobic residues, displays a heterogeneous structure. Thus the proteins comprise various conductivities and permittivity (determining the interaction with the electrical field) allows proteins to assemble with other proteins along-with a large conformational change involving structural rearrangement in response to intercellular and/or extracellular signalling to perform required cell functions [1, 36].Living organisms themselves produce electric currents that stimulate intercellular signalling and extracellular signals to regulate all biological processes. These signals enable the cell to sense and amplify small changes in the ion concentration [37, 38] of specific molecules, to produce and metabolize energy contained in molecules in its environment, to communicate with other cells in its neighbourhood, to move, to maintain its structure, to regulate its growth in response to signals in its surroundings, to speed up chemical reactions, to sense if it has been infected by a virus, to replicate itself when it is appropriate to do so and to detoxify and/or transport poisonous molecules out of it [3, 21]. The organism’s cells that respond to internal signals, can also respond to external signals such as various forms of radiation and electric forces that could similarly induce conformation changes to enhance or inhibit cell functions. One of the specific factors that could attenuate and destroy the pathogens in the body using the application of electrical field is due to complex biological effects in which change of the charge distribution of the proteins active molecules takes place. Therefore, when an electrical pulse with a given frequency is applied, the redistribution and alignment of the proteins charged molecule and its polar molecule in response to an applied external electrical field can be a useful mechanism that interrupts the communication between HIV-1 virus and the host cell. The frequency-dependent dielectric present in proteins involves the orientation and polarization of its randomly orientated polar molecules and of its charged molecules, making them rotate, move, align and polarize in response to the low frequency electrical force. This could induce an antiviral state for a period of time. Interference polarization within proteins could disturb the interaction between both sides of predominantly charged and/or polar host cell proteins and of the HIV-1 infective envelope and its protein particles.

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The HIV-1 and host cell interaction process is characteristically dominated by charge–charge interactions to induce conformational changes, in order to control host cell protein functions, and to stimulate different signals that are vital for virus cellular processes and replication. Since HIV-1 viral attachment and fusion requires stable and sustained intractions with the target cell, the intraction of the HIV-1 envelope proteins domain region with the coreseptor domain region, is considerably diminished, and hence, the dynamic forces of a virological synapse (cell-to-cell interaction to allow cell- to-cell transmission) [39] are possibly inadequate to maintain the attachment of the HIV-1 virus for a sufficient period of time. Such stable interactions can be only provided by the immunological synapse (signaling proteins for cell activation) [40]. Similarly, the applications of the electrical stimulation could disturb the binding process of its effective factors that mediate the passage of its large macromolecular infected particles and its RNA through NPC. Thus the HIV-1 virus cannot conjugate with the target cells, disturbing its life cycle, and suggests that the mechanisms of the inactivation signal induces virus death. This may disturb the life cycle of the HIV-1 virus and hence, its replication, with no potential risks and harm to the host cells compared to the pharmacological approach.

The type, intensity and duration of the electrical stimulation force must be selected carefully to stimulate an appropriate action and must not be so strong that it would produce undesirable responses. It is well known that high frequency waveforms have enough energy to break any cell’s chemical bonds (ionization), while in lower frequency waveforms, the energy has a non-ionizing effect that breaks down the chemical bonds [41, 42]. Considering proteins as dielectric materials [43] when applying a low frequency electrical pulse, proteins present a large low frequency permittivity, while at high frequencies dielectric loss takes place. Furthermore, applying a low intensity electrical field enhances ion transport across the cell membrane, due to the fact that nano scale pores form in the cell membrane temporarily, while at a high intensity, the electrical field causes dielectric break down; the cell is unable to recover from the pore formation process, causing in cell death. Thus, high intensity electrical fields can be used in electroporation therapies for treating a variety of cancerous pathologies [1, 44]. However, naturally, the variability of the dielectric properties of proteins is important for the biological function of a protein [43, 45].

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The movement of the charged molecule and the free ions, in response to an applied external electrical field can also be a useful mechanism to induce an action comparable to the normal cell endogenous electrical field. An endogenous field, which induces variation in membrane potential, due to the movement of the charged residues in the intrinsic plasma membrane proteins, as well as free ions, has importance in biological processes, such as wound healing and tissue regeneration. Since the surface charge can change with the pathophysiological state of the cell [36], thus inducing a small electric current can also enhance the normal cell functions by inducing a variation in membrane potential, and affect subcellular mechanisms, particularly the intracellular Ca2+ concentration or other voltage-dependent ionic activities [46]. An additional notable feature of this electrical stimulation is its cellular specificity. Therefore, the parameters of the generated electrical pulse can be designed and selected in order to stimulate only one particular type of tissue, such as blood, nerve, muscles, urine, leaving the others unaffected [47]. Moreover, in the electrical stimulation approach, the type of waveform must be carefully considered. Using a periodical waveform with certain stimulation frequencies can interact with the periodical intrinsic oscillators of the biological cell networks, which enhances their intrinsic oscillatory activity [48], a phenomenon that describes the natural frequency of the human body, which is an essential process in the living organism that enhances cell communication [49]. Since disease can disrupt the biochemical systems in biological cells, this inhibits the normal protein synthesis in lymphocytes, therefore, applying certain stimulation frequencies can enhance the biochemical parameters of the blood and normalize protein synthesis in lymphocytes [50].

Studies on frequency dependent dielectric properties of human blood, covering a frequency range from 1 Hz to 40 GHz, using broadband dielectric spectroscopy, have also shown no evidence of harmful effects at low frequencies [51]. The modes and the duration of the electrical stimulation pulse should be taken into consideration. Clinically, using the long duration Monopolar mode leads to charge accumulation at the electrode-skin site, causing muscle contractions which may damage the tissue, while using the long duration bipolar mode of operation, each pulse is followed by a pulse of reversed polarity which ensures charge balancing and hence, prevents damage that may occur at the electrode-skin interface [3, 44].

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Theoretically, and in my opinion, the periodical low frequency square waveform with wide pulses can block the interaction between the virus and the domain regions of the host cells for a sufficient period of time, therefore, the HIV-1 virus cannot mutate and develop resistance in the absence of the host cell proteins as in traditional pharmaceutical approaches, so that the HIV-1 virus will then vanish. Interestingly, using an application of periodical low frequency bipolar square waveform signal and low voltage electrical field can, therefore, induce enhancement and/or inhibition effect, prompting an antiviral state for a period of time, but is not expected to seriously disturb the host cell protein structure and its conformation state. Indeed, the protein molecules have non-polar hydrophobic amino acid residues building up the core, and the polar and charged amino acid residues are mostly located on the surface of the molecule, and there is no rotation around the peptide bond, thus the back bone of the protein does not rotate freely and only the polar or charged site chains rotate [1, 21]. These electrically induced protein transformations can be studied invivo/in-vitro as blood-cell treatment and as anti-HIV-1 electro-therapy. This may offer an antiviral therapy to target the most devastating pathogen in human history (more details in chapter 6).

Figure 1.3 (a) and (b) summarizes and shows the development and design steps of the biomedical intgrated circuit for electro- therapy device of this research project.

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(b) Schematic diagram shows that advances in 1) microelectronics technology involving new CMOS biomedical IC design, 2) bio-electro-chemistry science including cellular function, electro-active biological cells and their responses, and 3) knowledge of the disease condition, concerning the underlying mechanisms of the biological cells and disease state (HIV-1 and host cell engage predominantly by protein charge-charge interaction), are combined in this research project, in order to develop the concept and design a biomedical device capable of communicating with electro-active biological cells and how this could be utilized for biomedical treatment applications.

Figure 1.3: The development and design steps of electro-therapy concept and device of this research project, (a) block diagram, and (b) Schematic diagrams.

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1.4 Research Goals

The goal of this research project is to combine advances in engineering (involving new CMOS technology) and biology (including the electro-active body medium) as well as the underlying mechanisms of the biological cells and disease state (HIV-1 and host cell engage predominantly by electrostatic interactions) to develop the concept and introduce a biomedical device capable of communicating with body tissue and cells, for biomedical treatment benefits. A corresponding goal is the characterization of this concept in-vitro, and, if possible, the device in-vivo, to investigate the effect of low frequency and low voltage electrical pulse on human blood cellular proteins, to stimulate a potential reaction that can induce a short-term, or long- term anti-HIV-1viral state. Besides, this research project can develop knowledge for advanced technological electro-medical treatment devices, their design, structure and applications. The research project therefore, has the following aspects:

1. Concept development for anti-HIV-1 electro-therapy.

2. Biomedical IC Design. A novel low power and low frequency non-invasive biomedical device comprising a dual-band WFG with ultra-wide low-frequency tuning range and an active-electrode-pair will be developed, based on a new 130nm IBM CMOS technology.

3. Bio-electrochemistry experimental work. A theoretical analysis, experiment design and performance will carried out in in-vitro environments to examine the influences of the periodical low frequency bipolar square waveform signal and low voltage electrical field on human blood cells.