Here we present a simple 1D modeling of an electrochemical cell integrated with viable bacterial cells expressing intracellular enzyme which responds to added substrate and the generated product is secreted and oxidized on electrode's surface. The effect of bacteria distribution was analysed comparing two cases: 1. Suspended cells, assuming equal distribution and homogenous response and 2. Cells immobilized on beads attached to electrode's surface, forming an inhomogeneous product generation in narrow region near the electrode. The model shows that when the total number of sensing cells is kept constant, it is preferred to locate them in close proximity to the working electrodes. Although the model is an approximation, since the beads form a 3D structure where the transport is via the gaps between the beads, the 1D model explains well the main experimental results as was shown by fitting to COMSOL Mulyiphysics (TM) simulations. Assuming that the diffusion of the products towards the electrode is the dominant factor and bacteria's response is independent of its immobilization and density, we showed that as the total population gets close to the electrode the response time becomes faster, while the long-time electrochemical current reaches the same value, which depends on the total number of bacterial cells. (C) 2016 Elsevier B.V. All rights reserved.

Purified proteins offer a homogeneous population of biological nanoparticles, equipped in many cases with specific binding sites enabling the directed self-assembly of envisaged one-, two- or three-dimensional arrays. These arrays may serve as nanoscale biotemplates for the preparation of novel functional composite materials, which exhibit potential applications, especially in the fields of nanoelectronics and optical devices. This review provides an overview of the field of protein-mediated biotemplating, focussing on achievements made throughout the past decade. It is comprised of seven sections designed according to the size and configuration of the protein-made biotemplate. Each section describes the design and size of the biotemplate, the resulting hybrid structures, the fabrication methodology, the analytical tools employed for the structural analysis of the hybrids obtained, and, finally, their claimed/intended applications and a feasibility demonstration (whenever available). In conclusion, a short assessment of the overall status of the achievements already made vs. the future challenges of this field is provided.

In the current work, the synergetic effect of microbial cells and functional nanostructures, such as highly disordered Si nanowires (SiNW-forest), has been exploited to develop a novel whole-cell biosensor. E. Coli microbial cells, expressing and displaying recombinant ZZ proteins, were immobilized onto SiNW-forest electrode. The existence of the microbial cells was verified visually by scanning electron microscopy, and viable counting, and electrochemically using impedance spectroscopy (EIS). The electrochemical impedance spectra show a clear dependence on the number of the microbial cells by allowing the detection of different number of cells in the range between 10(3) and 10(7). In addition, the EIS investigation suggests further decrease in the detected cells concentration to be possible. (C) 2017 The Electrochemical Society. All rights reserved.

A fast and cost-effective whole-cell electrochemical biosensor aiming at early-stage screening of potential inhibitors of cytochrome P450 is proposed and its feasibility demonstrated. Sensing is performed by monitoring the decrease in the electrochemical signal generated by the oxidation of the product of the enzymatic reaction of cytochrome P450 BM3 expressed in E. coli cells.The system is self-maintained and does not require enzyme purification or external addition of NADPH or other cofactors. Measurements were performed simultaneously at eight chips at low positive potential of 100 mV vs. Ag/AgC1 under continuous stirring. Kinetic analysis of aniline determination by the whole-cell system was performed and the concentration of aniline for the inhibition studies was selected accordingly.Three known inhibitors - imidazole, metyrapone and 1-aminobenzotriazole (ABT) - were tested and their inhibition profiles characterized. Imidazole was found to be the most potent inhibitor of the three.To the best of our knowledge, the system developed enables for the first time rapid and cheap cytochrome P450 inhibitors detection by a disposable whole-cell electrochemical chip, combined with the advantages of a whole-cell system, without neither enzyme purification nor NADPH addition. Furthermore, the system developed also provides capability of performing aniline detection e.g. for environmental monitoring, by means of electrochemical biosensing. (C) 2015 Elsevier B.V. All rights reserved.

Microbial cells are attractive biorecognition elements for electrochemical biosensing applications. A desired configuration is the immobilization of the cells onto the transducer's surface. Here we propose the design and demonstrate the feasibility of a novel 'cells-on-beads' (COB) immobilization approach, providing simple, fast, low cost and reproducible method for the construction of viable whole-cell biochips. The proposed immobilization approach is based on controlled chemical modification of polyacrylamide porous beads resulting in positively charged microcarriers exhibiting strong adsorption capabilities to both cells and gold surfaces. As the cells are physically adsorbed to the outer surface of the beads with no further treatments, this method is particularly suited for systems integrating sensitive cells with the detection of electroactive products susceptible to diffusion limitations. Such functional beads can be stored at 4 degrees C for at least six months and deposited on the biochip on demand. The COB approach was demonstrated using Escherichia coli (E. coli) cells expressing an intracellular enzyme, cytochrome P450 BM3, and aniline as model substrate. The current signal was generated by the oxidation of the secreted enzymatic product p-aminophenol on electrode's surface at 100 mV vs Ag/AgCl. The electrochemical biochip yielded a high and clear signal within the range of tens of nanoamperes that was linearly correlated to the substrate concentration. The proposed method was characterized and optimized and its relative advantage over a suspended cells system was illustrated. (C) 2016 Elsevier B.V. All rights reserved.

The effect of the molecular surface charge on sensing sensitivity using the electrochemical impedance spectroscopy (EIS) technique in the presence of [Fe(CN)6]3−/[Fe(CN)6]4− redox couple was investigated using Avidin- biotinylated Horseradish Peroxidase as model proteins. Reducing the positive charge of the probe molecule (Avidin) by chemical modification increased the change in the real part of the system’s impedance, which improved the sensitivity of the sensor. The system’s detection limit was within the range of few nM, where at low analyte concentrations – under 5nM – the signal becomes very noisy. The work presented herein shows that the nature and amount of charge of the molecules involved – both analyte and the binding protein, have major effect on the EIS signal, which should be taken into consideration throughout the construction and use of EIS biosensor. The results of the impedimetric measurements were characterized and compared to results obtained from the widely- established ELISA biochemical method for detection of biorecognition events. A physical model explaining the results obtained is proposed.

In this paper we present a novel electrochemical sensor with a unique 3D architecture allowing for direct measurements on contact, or in close proximity, to biological samples. For biomedical applications, the all-polymer architecture can be mounted on special probes that can access the region under test with no need for biopsy as is done today with the conventional 2D electrodes. The chip consists of a biocompatible substrate comprised of an electrochemical cell with two gold electrodes (working and counter) and an Ag/AgCl quasi-reference electrode. The metal electrodes on the biochip front (sensing) side are fabricated by conventional electroplating and patterning methods. The chip itself is made from PDMS cast from a polymer master fabricated by 3D printing. The electrical communication between the biochip front and backside is enabled by through-hole via-contacts filled with conductive PDMS containing 60 wt% graphite powder. The electroactivity of working electrodes was verified by cyclic voltammetry of ferrocyanide/ferricyanide redox reaction. Amperometric in-vitro detection of the biomarker alkaline phosphatase from three different colon cancer cell lines directly in a cell culture plate while maintaining their biological environment was successfully demonstrated. The sensor exhibit stable voltammetric signatures and significant amperometric response to the enzyme in repeated tests. This approach paves the way to perform direct, non-invasive diagnostics on top of an exposed cell layer for both in-vivo and in-vitro applications. (C) 2015 Elsevier B.V. All rights reserved.

The changes in the Faradaic impedance of gold/biomolecules system due to specific binding of small molecule to a significantly larger binding protein molecule were investigated. The biotin (244.31 Da) - avidin (66000 Da) couple was used as a model for small ligand - binding protein biorecognition. The study was carried out under open circuit potential in the presence of [Fe(CN)(6)] (3/ 4) redox couple. An equivalent electrical circuit was proposed and used for the interpretation of the recorded impedance spectra.Adsorption of thiolated avidin increased the electron transfer resistance, R-ct, by a factor of about 7.5 while subsequent addition of biotin within the concentration range of 4.1-40.9 nM reduced the value of R-ct by amount proportional to the biotin concentration. The addition of biotin did not affect, however, the equivalent double layer capacitance or other equivalent circuit parameters.A simple model based on effective surface coverage by the avidin molecules and the effect of the added biotin on electron transfer through the coated surface is proposed. A model for the minimum detection limit based on the random distribution of the binding protein and its dimensions is proposed. (C) 2015 Elsevier Ltd. All rights reserved.

Novel hybrids, comprised of a biologically active protein molecule core, coated with a thin outer layer of porous metallic silver, were developed in our lab. By the conjugation of silver reducing polymer to the surface of soluble, molecular, biologically active protein molecules and subsequent addition of silver salt, electroless silver deposition, culminating in thin porous metallic coating, was directed to the surface of the protein molecules. The silver-protein hybrids thus obtained, presenting novel nanoparticles several nanometers in size, retained their solubility and biological activity.The silver coating combined with the retained biological activity of its protein core, paved the way to a series of biomedical applications of these hybrids including "wiring" of the active site of oxido-reductase enzyme to electrodes, imaging of the presence of targeted ligands displayed on cancer cell surface and antimicrobial enzymatically attenuated release of silver ions.In this presentation we shall overview the technology of protein-silver hybrid's fabrication and analytical applications of silver-glucose oxidase and silver-Avidin hybrids, followed by feasibility demonstration of using silver-glucose oxidase hybrid as novel antibacterial and antifungal agent.

ABSTRACT The use of protein crystals as a source of nanoscale biotemplates has attracted growing interest in recent years owing to their inherent internal order. As these crystals are vulnerable to environmental changes, potential applications require their stabilization by chemical crosslinking. We have previously shown that such intermolecular chemical crosslinking reactions occurring within protein crystals are not random events, but start at preferred crosslinking sites imposed by the alignment of protein molecules and their packing within the crystalline lattice. Here we propose a new working hypothesis and demonstrate its feasibility in enabling us to extricate homogeneous populations of single protein molecules that display chemical point mutations or of dimers that show homogeneous chemical crosslinking, and that have the potential for isolation of higher structures. Characterization of the crosslinking mechanism and its end products opens the way to the potential retrieval of such specific modified/intermolecular crosslinked products simply by effecting partial crosslinking at identified preferred sites, followed by time‐controlled arrest of the crosslinking reaction and dissolution of the crystals by medium exchange complemented by chromatographic purification. Biotechnol. Bioeng. 2014;111: 1296–1303. © 2014 Wiley Periodicals, Inc.

In this work we report on the development of a new approach for the electroanalytical sensing of uranium(VI) in aqueous solutions. Uranium commonly exists in aqueous solutions in the form of its oxo ion, uranyl (UO2 ). The detection of uranyl has been accomplished by us through its deposition upon reduction by two electrons to the insoluble UO2 using a bare disk gold macroelectrode and anodic stripping voltammetry (ASV). This gave unsatisfactory detection limit of ca. 1·10 M uranyl. Moreover, the evolution of hydrogen bubbles blocked the electrode surface as a result of water reduction at negative deposition potential (−0.7 V vs. Ag/AgCl). Hence, we used a bare gold microelectrode of 25 μm diameter on which the uranium precipitated at negative potential (−1.2 V). The results were encouraging due to enhanced mass-transport that increased substantially the current density; however, the sensitivity was still insufficient (ca. 1·10 M uranyl). To improve the detection limit, we have introduced a vibrating microwire as a working electrode in ASV. The vibration reduces significantly the diffusion layer (~2 μm) during the deposition step and therefore increases the amounts of UO2 deposition. Vibration of the microelectrode was achieved by attaching a standard speaker (1 W, 8 Ω) to the working electrode. Using a 5 min deposition time, −1.2 V deposition potential and vibrating the electrode at frequency of 250 Hz and amplitude of 6 V, a detection limit of ca. 1·10 M of uranyl was achieved.

The response modeling of whole-cell biochip represents the link between cellular biology and transducer output, allowing better system engineering. It provides the mathematical background for signal and noise modeling, performance prediction and data analysis. Here we describe an analytical model for whole-cell biosensors with electrochemical detection for single use, test and dispose applications. In this system the electrochemical signal is generated by the oxidation of the by-products of the reaction between an external substrate and the enzyme alkaline phosphatase. The enzyme expression can be either normal or enhanced due to the response of the biological cell to an external excitation. The electrochemical oxidation current is measured as a function of time. The model is based on the electrochemical reaction rate equations; an analytical solution is presented, compared to data and discussed.

Back ground: Deep enteroscopy for the evaluation and treatment of small bowel pathology has undergone significant technology advancements over the last ten years. Studies directly comparing single (SBE) and double balloon enteroscopy (DBE) with spiral enteroscopy (SE) are few but suggest that the three techniques are comparable. Method: Retrospective review of Spirus® small bowel endoscopy cases over 12 months Results: A single experienced endoscopist completed all cases. The patient characteristics are listed in Table 1. Two patients had strictures (one was dilated) and one had multiple arteriovenous malformations (coagulated with Argon Plasma). Two patients experienced complications. Case 1: A 77 year old female with remote radiation treatment for endometrial carcinoma being evaluated for anemia had several slightly narrowed areas; all judged to be spacious enough to accommodate the overtube except one. The last area appeared tight and the overtube was unlocked as soon as it engaged the location and the scope alone was advanced further. Surprisingly the cecum was reached without difficulty. Upon endoscope withdrawal, bleeding was noted and a perforation was suspected. A tense abdomen was decompressed with a needle, rapidly stabilizing a low blood pressure. Perforation was identified and the segment with several strictures was resected. Case 2: A 44 year old female with cirrhosis secondary to primary sclerosing cholangitis status post hepaticojejunostomy had recurrent anastomotic strictures. On SE assisted ERCP, a perforation was suspected on radiography. ERCP was completed and exam was continued under water without gas insufflation. The perforation was found on the biliary limb and was not favorable for endoclip closure. The spilled intestinal contents were suctioned and pneumoperitoneum was decompressed allowing the patient to be comfortable and stable until laparotomy. A guidewire was placed endoscopically which allowed easy identification of the site during surgery (Figure 1). Conclusion: Patients with known or occult small bowel strictures and surgically altered anatomy with possible adhesional fixation may be at a higher risk of complications and we suggest caution with use of SE in these scenarios. When perforations occur, early recognition and immediate management are of benefit. Despite suffering complications, both patients benefited from their procedures. In case 1, the resection of the diseased intestine led to resolution of anemia. In Case 2, the bile duct stricture was successfully treated and she was bridged to transplant. Table 1: Patient characteristics