Electronic transduction of photostimulated binding interactions at photoisomerizable monolayer electrodes: novel approaches for optobioelectronic systems and reversible immunosensor devices

Photoisomerizable monolayers assembled onto electrode supports act as "command interfaces" for controlling the binding interactions of biomaterials with the functionalized surfaces. The light-induced binding and dissociation of the biomaterials to and from the electrodes, respectively, are electronically transduced. Two systems, including the photostimulated binding and dissociation of cytochrome c (Cyt c) and of anti-DNP antibody to and from functionalized surfaces, are discussed. The application of the systems as optobioelectronic devices and reversible immunosensors is addressed. A mixed monolayer consisting of pyridine and nitrospiropyran (1a) photoisomerizable units assembled on a Au-electrode acts as a command interface for the light-controlled association and dissociation of Cyt c to and from the monolayer. Cyt c binds to the pyridine/1a-monolayer electrode, resulting in electrical contact between the redox protein and the electrode. Photoisomerization of the mixed monolayer to the pyridine/protonated merocyanine state (1b) results in the electrostatic repulsion of Cyt c and its dissociation from the electrode support. This blocks the electrical contact between Cyt c and the electrode. By the cyclic photoisomerization of the mixed monolayer between the 1a and 1b states, reversible "ON"-"OFF" amperometric transduction of the affinity interactions between the redox protein and the interface is accomplished. Coupling of the photostimulated electrical contact between Cyt c and the electrode surface to the Cyt c-mediated bioelectrocatalyzed reduction of O(2) by cytochrome oxidase provides a means to amplify the transduced electronic signal. A photoisomerizable thiolated dinitrospiropyran (2a) monolayer, assembled on solid supports, acts as a light-active antigen interface that enables the photocontrolled binding and dissociation of anti-dinitrophenyl antibody (DNP-Ab) to and from the interface. The dinitrospiropyran (2a) layer acts as an antigen for the DNP-Ab, whereas the protonated dinitromerocyanine (2b) lacks antigen features for the DNP-Ab. By reversible photoisomerization of the monolayer between the 2a and 2b states, cyclic binding and dissociation of DNP-Ab to and from the monolayer interface is accomplished. The association and dissociation of the DNP-Ab to and from the 2a- and 2b-monolayer states are electronically transduced, using amperometric, Faradaic impedance and microgravimetric, quartz crystal microbalance analyses. The photostimulated binding of an antibody to a photoisomerizable antigen monolayer provides a novel method to design reversible immunosensor devices.     

Protein transduction assisted by polyethylenimine-cationized carrier proteins

Previously, we have reported that cationized-proteins covalently modified with polyethylenimine (PEI) (direct PEI-cationization) efficiently enter cells and function in the cytosol [Futami et al. (2005) J. Biosci. Bioeng. 99, 95-103]. However, it may be more convenient if a protein could be delivered into cells just by mixing the protein with a PEI-cationized carrier protein having a specific affinity (indirect PEI-cationization). Thus, we prepared PEI-cationized avidin (PEI-avidin), streptavidin (PEI-streptavidin), and protein G (PEI-protein G), and examined whether they could deliver biotinylated proteins and antibodies into living cells. PEI-avidin (and/or PEI-streptavidin) carried biotinylated GFPs into various mammalian cells very efficiently. A GFP variant containing a nuclear localization signal was found to arrive even in the nucleus. The addition of a biotinylated RNase A derivative mixed with PEI-streptavidin to a culture medium of 3T3-SV-40 cells resulted in remarkable cell growth inhibition, suggesting that the biotinylated RNase A derivative entered cells and digested intracellular RNA molecules. Furthermore, the addition of a fluorescein-labeled anti-S100C (beta-actin binding protein) antibody mixed with PEI-protein G to human fibroblasts resulted in the appearance of a fluorescence image of actin-like filamentous structures in the cells. These results indicate that indirect PEI-cationization using non-covalent interaction is as effective as the direct PEI-cationization for the transduction of proteins into living cells and for expression of their functions in the cytosol. Thus, PEI-cationized proteins having a specific affinity for certain molecules such as PEI-streptavidin, PEI-avidin and PEI-protein G are concluded to be widely applicable protein transduction carrier molecules.     

Manometric transduction in enzyme biosensors

The combination of enzymatic recognition and manometric transduction is explored, using enzymes that consume or evolve a gas with low solubility in aqueous media. A design is discussed whereby change in partial pressure of a gas in the headspace is related to the turnover of analyte by the enzyme. Headspace and sample volume dimensions are considered, demonstrating the influence of flux at the air-water interface. The relative importance of diffusion and reaction for the enzyme solution is shown. When enzyme kinetics dominate, the concentration gradient is low and the overall kinetics are determined by the total amount of active enzyme, reducing either enzyme concentration or enzyme layer thickness will reduce the diffusion limitation. A Teflon-enzyme composite is presented to allow a reuseable immobilised enzyme preparation and a disc with stirring magnet identified as an efficient configuration. A glucose oxidase system was tested in the monitoring of glucose consumption during fermentation. Application to other enzyme systems is discussed.     

Potential of enzyme mimics in biomimetic sensors: a modified-cyclodextrin as a dehydrogenase enzyme mimic

This paper reports the application of a dehydrogenase enzyme mimic as a biomimetic sensor. The model compound investigated was a beta-cyclodextrin (beta-CD) derivative with a nicotinamide group attached to the secondary face of a beta-CD (Fig. 1g). It was envisaged that the nicotinamide group would act as the electron transfer agent and that the cyclodextrin would provide a suitable hydrophobic cavity for the reaction to take place in. Ethanol, propranalol, dopamine and acetone were used as substrates in backgrounds of hydrophilic and hydrophobic anions. Electrochemical and fluorescence techniques were used to study the catalytic effects in solution. It was found that the size of the analyte and the hydrophobicity of the anion affected the catalytic activity of the dehydrogenase mimic. Catalytic effects were most enhanced with ethanol and dopamine in presence of larger and more strongly solvated anions, SO4(2-) and H2PO4- which are excluded from the cavity. The molecule was also immobilised in a sol-gel matrix and investigated as a sol-gel electrochemical biomimetic sensor. Concentration dependence with increasing aliquots of ethanol was observed. These results indicated that a re-usable biomimetic sensor is indeed feasible.     

Resonance transduction of low level periodic signals by an enzyme: an oscillatory activation barrier model.

The overall rate of an enzyme catalyzed reaction is determined by the activation barrier of a rate-limiting step. If the barrier is oscillatory due to the intrinsic properties of a fluctuating enzyme, this enzymatic reaction will be influenced by a low level periodic electric field through the resonance transduction between the applied field and the oscillatory activation barrier. The ATP hydrolysis activity of a highly purified, detergent solubilized Ecto-ATPase from chicken oviduct was used to test the above concept. At 37 degrees C, this activity (1,800 mumols mg-1 min-1) was stimulated up to 47% (to 2,650 mumols mg-1 min-1) by an alternating electric field (AC), with a frequency window at 10 kHz. The maximal stimulation occurred at 5.0 V (peak-to-peak) cm-1. The potential drop across the dimension of the enzyme was approximately 10 microV (micelle diameter 20 nm). The activation barrier, or the Arrhenius activation energy, of the ATP splitting was measured to be 30 kT and the maximal barrier oscillation was calculated to be approximately 2.5 kT according to the oscillatory activation barrier (OAB) model. With the optimal AC field, full impact of the electric stimulation could be effected in much less than a second. The OAB model is many orders of magnitude more sensitive for deciphering low level periodic signals than the electroconformational coupling (ECC) model, although the latter has the ability to actively transduce energy while the former does not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free energy transduction in a chemical motor model

Motor enzymes catalyse chemical reactions, like the hydrolysis of ATP, and in the process they also perform work. Recent studies indicate that motor enzymes perform work with specific biochemical steps in their catalysed reactions, challenging the classical view that work can only be performed within a biochemical state. To address these studies an alternative class of models, often referred to as chemical motor models, has emerged in which motors perform work with biochemical transitions. In this paper, I develop a novel, self-consistent framework for chemical motor models, which accommodates multiple pathways for free energy transfer, predicts rich behaviors from the simplest multi-motor systems, and provides important new insights into muscle and motor function.     

Epistasis in a model of molecular signal transduction

Biological functions typically involve complex interacting molecular networks, with numerous feedback and regulation loops. How the properties of the system are affected when one, or several of its parts are modified is a question of fundamental interest, with numerous implications for the way we study and understand biological processes and treat diseases. This question can be rephrased in terms of relating genotypes to phenotypes: to what extent does the effect of a genetic variation at one locus depend on genetic variation at all other loci? Systematic quantitative measurements of epistasis – the deviation from additivity in the effect of alleles at different loci – on a given quantitative trait remain a major challenge. Here, we take a complementary approach of studying theoretically the effect of varying multiple parameters in a validated model of molecular signal transduction. To connect with the genotype/phenotype mapping we interpret parameters of the model as different loci with discrete choices of these parameters as alleles, which allows us to systematically examine the dependence of the signaling output – a quantitative trait – on the set of possible allelic combinations. We show quite generally that quantitative traits behave approximately additively (weak epistasis) when alleles correspond to small changes of parameters; epistasis appears as a result of large differences between alleles. When epistasis is relatively strong, it is concentrated in a sparse subset of loci and in low order (e.g. pair-wise) interactions. We find that focusing on interaction between loci that exhibit strong additive effects is an efficient way of identifying most of the epistasis. Our model study defines a theoretical framework for interpretation of experimental data and provides statistical predictions for the structure of genetic interaction expected for moderately complex biological circuits.     

Nature as a model for technical sensors

Nature has developed a stunning diversity of sensory systems. Humans and many animals mainly rely on visual information. In addition, they may use acoustic, olfactory, and tactile cues for object detection and spatial orientation. Beyond these sensory systems a large variety of highly specialized sensors have evolved. For instance, some buprestid beetles use infrared organs for the detection of forest fires. The infrared sensors of boid and crotalid snakes are used for prey detection at night. For object detection and spatial orientation many species of nocturnal fish employ active electrolocation. This review describes certain aspects of the detection and processing of infrared and electrosensory information. We show that the study of natural exotic sensory systems can lead to discoveries that are useful for the construction of technical sensors and artificial control systems. Comparative studies of animal sensory systems have the power to uncover at least a small fraction of the gigantic untapped reservoir of natural solutions for perceptive problems.     

Chronopotentiometry and Faradaic impedance spectroscopy as signal transduction methods for the biocatalytic precipitation of an insoluble product on electrode supports: routes for enzyme sensors, immunosensors and DNA sensors

The biocatalyzed precipitation of an insoluble product produced on electrode supports is used as an amplification path for biosensing. Enzyme-based electrodes, immunosensors and DNA sensors are developed using this biocatalytic precipitation route. Faradaic impedance spectroscopy and chronopotentiometry are used as transduction methods to follow the precipitation processes. While Faradaic impedance spectroscopy leads to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provides the total resistance at the interfaces of the modified electrodes. A horseradish peroxidase, HRP, monolayer-functionalized electrode is used to sense H₂O₂ by the biocatalyzed oxidation of 4-chloro-1-naphthol (1), to the insoluble product benzo-4-chlorohexadienone (2). An antigen monolayer electrode is used to sense the dinitrophenyl antibody, DNP-Ab, applying an anti-antibody–HRP conjugate as a biocatalyst for the oxidative precipitation of 1 by H₂O₂ to yield the insoluble product 2. An oligonucleotide (3) functionalized monolayer electrode is used to sense the DNA-analyte (4), that is one of the Tay–Sachs genetic disorder mutants. Association of a biotin-labeled oligonucleotide to the sensing interface, followed by the association of the avidin–HRP conjugate and the biocatalyzed precipitation of 2 leads to the amplified sensing of 4. The amount of the precipitate accumulated on the conductive support is controlled by the concentration of the respective analytes and the time intervals employed for the biocatalytic precipitation of 2. The electron-transfer resistances of the electrodes covered by the insoluble product (2) are derived from Faradaic impedance measurements, whereas the total electrode resistances are extracted from chronopotentiometric experiments. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports are found. Chronopotentiometry is suggested as a rapid transduction means (a few seconds). The precautions needed to apply chronopotentiometry in biosensors are discussed.     

Mean-field Boolean network model of a signal transduction network

In this paper we provide a mean-field Boolean network model for a signal transduction network of a generic fibroblast cell. The network consists of several main signaling pathways, including the receptor tyrosine kinase, the G-protein coupled receptor, and the Integrin signaling pathway. The network consists of 130 nodes, each representing a signaling molecule (mainly proteins). Nodes are governed by Boolean dynamics including canalizing functions as well as totalistic Boolean functions that depend only on the overall fraction of active nodes. We categorize the Boolean functions into several different classes. Using a mean-field approach we generate a mathematical formula for the probability of a node becoming active at any time step. The model is shown to be a good match for the actual network. This is done by iterating both the actual network and the model and comparing the results numerically. Using the Boolean model it is shown that the system is stable under a variety of parameter combinations. It is also shown that this model is suitable for assessing the dynamics of the network under protein mutations. Analytical results support the numerical observations that in the long-run at most half of the nodes of the network are active.     

Signal transduction by a protease cascade

The dorsoventral axis of the Drosophila embryo is determined by a spatial cue generated by ovarian somatic cells. This cue is communicated to the embryo through an extracellular serine protease cascade active only on the ventral side of the embryo. Studies of the proteases and somatically expressed proteins involved in this signalling process suggest a working model for how the protease cascade is locally activated hours after the ovarian somatic cells have degenerated.     

Ultrasound assisted intensification of enzyme activity and its properties: a mini-review

Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis–Menten kinetic parameters (k_m and V_max) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.     

Photoswitching of peroxidase activity by position-specific incorporation of a photoisomerizable non-natural amino acid into horseradish peroxidase.

Horseradish peroxidase mutants containing L-p-phenylazophenylalanine (azoAla) at various positions were synthesized by using an Escherichia coli in vitro translation system. Among the 15 mutants examined, four mutants containing a single azoAla unit at the 6th, 68th, 142nd, and 179th positions, respectively, retained the peroxidase activity. The activity of the Phe68azoAla mutant was higher when the azobenzene group was in the cis form than in the trans form. On the contrary, the activity of the Phe179azoAla mutant disappeared when the azobenzene group was photoisomerized to the cis form, but recovered in the trans form. In the latter mutant, therefore, an on/off photoswitching of the peroxidase activity was attained.     

Artificial site-specific DNA-nicking system based on common restriction enzyme assisted by PNA openers

We report on the peptide nucleic acid (PNA)-directed design of a DNA-nicking system that enables selective and quantitative cleavage of one strand of duplex DNA at a designated site, thus mimicking natural nickases and significantly extending their potential. This system exploits the ability of pyrimidine PNAs to serve as openers for specific DNA sites by invading the DNA duplex and exposing one DNA strand for oligonucleotide hybridization. The resultant secondary duplex can act as a substrate for a restriction enzyme, which ultimately creates a nick in the parent DNA. We demonstrate that several restriction enzymes of different types could be successfully used in the PNA-assisted system we developed. Importantly, the enzyme cleavage efficiency is basically not impaired on such artificially generated substrates, compared with the efficiency on regular DNA duplexes. Our design originates a vast class of semisynthetic rare-cleaving DNA nickases, which are essentially absent at present. In addition, we show that the site-specific PNA-assisted nicking of duplex DNA can be engaged in a rolling-circle DNA amplification (RCA) reaction. This new RCA format demonstrates the practical potential of the novel biomolecular tool we propose for DNA technology and DNA diagnostics.     

A simple model for a regulatory enzyme

A simple model for a regulatory enzyme is described which leads to relationships between the initial velocity of the catalysed reaction and the varied concentration of a substrate that are of the non-inflected or sigmoidal varieties without a maximum. The model assumes that the most relevant measure of protein configuration (itself determining the kinetic behaviour of the enzyme) is the apparent association constant, α_i, measured for the given fractional saturation of the ligand under investigation. It is further assumed that the original state of the protein in solution, α₀, is destabilized by an increment of energy, ΔG_p⁰, that is proportional to the fractional saturation of the enzyme by ligand so that the formation of a new configurational state, a,, can be represented by ?ΔG_p⁰ = RT ln $\text{α}{}_1\text{α}{}_0$. The rate or fractional saturation equation that can be derived from this model predicts both positive and negative cooperativity. Either equation can be transformed for linear representation, provided the maximum velocity or its equivalent maximum saturation is known, and estimates of α₀ and α_i (the apparent association constants at zero and complete saturation) can be obtained thereby. A procedure is also described by which an initial estimate of the maximum velocity or saturation can be improved. The model is tested by application to a range of data in the literature and it is shown to give fits to the data comparable in quality to those provided by the model of Monod, Wyman &; Changeux (1965).     

Chemically assisted capsulectomy in the rabbit model: a new approach

Capsular contracture remains the most common adverse sequela of aesthetic and reconstructive breast surgery when breast implants are used. Capsulectomy may be technically difficult and can result in damage to the neighboring tissues. The aim of this study was to verify the efficacy of sodium 2-mercaptoethane sulfonate (mesna) as a facilitator of periprosthetic dissection when instilled locally at the time of capsulectomy. Two 40-cc textured saline implants were placed dorsally into each of 20 rabbits. After 5 months, capsulectomy was performed after the removal of the implants. Mesna was used to highlight the junction between scar and normal tissue and to help separate the tissues during the capsulectomy in one of the two capsules in each rabbit. Saline was used for the same purpose in the other. The blood loss, duration of operation, and difficulty of dissection as experienced by the surgeon were recorded during the course of the operation. The capsules were also examined histologically for their thickness and graded according to their degree of intactness at the conclusion of the procedure. The histological grading based on the intactness of the removed capsule (p = 0.005), the operating time (p = 0.003), and the subjective evaluation of the difficulty of the procedure (p = 0.003) were significantly better in the mesna group. There was no significant difference in the blood loss between the two groups. Because of its ability as a chemical dissector, mesna may be a useful aid in capsulectomy. Clinical studies to confirm this evidence are required.     

Physiologically based mathematical model of transduction of mechanobiological signals by osteocytes

Developing mathematical models describing the bone transduction mechanisms, including mechanical and metabolic regulations, has a clear practical applications in bone tissue engineering. The current study attempts to develop a plausible physiologically based mathematical model to describe the mechanotransduction in bone by an osteocyte mediated by the calcium-parathyroid hormone regulation and incorporating the nitric oxide (NO) and prostaglandin E2 (PGE2) effects in early responses to mechanical stimulation. The inputs are mechanical stress and calcium concentration, and the output is a stimulus function corresponding to the stimulatory signal to osteoblasts. The focus will be on the development of the mechanotransduction model rather than investigating the bone remodeling process that is beyond the scope of this study. The different components of the model were based on both experimental and theoretical previously published results describing some observed physiological events in bone mechanotransduction. Current model is a dynamical system expressing the mechanotransduction response of a given osteocyte with zero explicit space dimensions, but with a dependent variable that records signal amplitude as a function of mechanical stress, some metabolic factors release, and time. We then investigated the model response in term of stimulus signal variation versus the model inputs. Despite the limitations of the model, predicted and experimental results from literature have the same trends.