Programmable DARPin-based receptors for the detection of thrombotic markers

Cellular therapies remain constrained by the limited availability of sensors for disease markers. Here we present an integrated target-to-receptor pipeline for constructing a customizable advanced modular bispecific extracellular receptor (AMBER) that combines our generalized extracellular molecule sensor (GEMS) system with a high-throughput platform for generating designed ankyrin repeat proteins (DARPins). For proof of concept, we chose human fibrin degradation products (FDPs) as markers with high clinical relevance and screened a DARPin library for FDP binders. We built AMBERs equipped with 19 different DARPins selected from 160 hits, and found 4 of them to be functional as heterodimers with a known single-chain variable fragments binder. Tandem receptors consisting of combinations of the validated DARPins are also functional. We demonstrate applications of these AMBER receptors in vitro and in vivo by constructing designer cell lines that detect pathological concentrations of FDPs and respond with the production of a reporter and a therapeutic anti-thrombotic protein

Eggshell membrane: A natural substrate for immobilization and detection of DNA

Chemically modified eggshell membranes.(ESM) have been explored as potentially novel platforms for immobilization of oligonucleotides and subsequent detection of target DNA. The fibrous network of the native ESM as well those functionalized with acetic acid or n-butyl acetate has been examined by field-emission scanning electron microscopy (FESEM). The formation of surface functional moieties has been confirmed by Fourier-transform infrared spectroscopy (FTIR). DNA molecules, with an end terminal - NH2 group (at 5' end) have been immobilized on the chemically modified ESM surface. The effect of surface modification on the DNA immobilization efficiency has been investigated using fluorescence microscopy and atomic force microscopy (AFM). The above studies concurrently suggest that functionalization of ESM with n-butyl acetate causes a better homogeneity of the DNA probes on the membrane surface. On-chip hybridization of the target DNA with the surface bound capture probes has been performed on the functionalized membranes. It is observed that n-butyl acetate modification of ESM pushes the limit of detection (LOD) of the DNA sensors by at least an order of magnitude compared to the other modification method. (C) 2015 Elsevier B.V. All rights reserved

Symbiotically Augmented removal of Congo red by polyaniline/cobalt sulfide/graphite composites

The presence of Congo red dye (CR) in industrial sewage causes a serious threat to the environment. Therefore, it is imperative to develop high-performance, low-cost functional materials to mitigate such issues. During past decades, polyaniline and its composites have been recognized as an emerging candidate to remove hazardous organic effluents from water. The present work demonstrates the successful elimination of CR from water in presence of newly synthesized graphite/cobalt sulfide/PANI-based ternary composites. Several morphological or physicochemical characterization tools were adopted to confirm the formation of the ternary composite and subsequent synergistic interaction between individual elements of the composites. The experimental results delineate that a maximum of similar to 95.55% CR removal (%) was achieved after 120 min. Fast removal (similar to 5-10 min) of CR dye is observed for APS/Ternary composite system. From the fitted experimental data utilizing 1st or 2nd order rate kinetic models, it was observed that the adsorption induced degradation of CR dye and the process was chemisorptions in nature. Further, an intra-molecular diffusion model was also introduced that signifies both boundary layer diffusion or intraparticle diffusion phenomenon was responsible for CR removal. Furthermore, the cytotoxicity profile of the composite treated Congo red aqueous solution was evaluated when exposed to L929 fibroblast cells after 24 h or 72 h of exposure and the result deciphers the non-toxic nature of composite treated CR water

Blood-Glucose-Powered Metabolic Fuel Cell for Self-Sufficient Bioelectronics

Currently available bioelectronic devices consume too much power to be continuously operated on rechargeable batteries, and are often powered wirelessly, with attendant issues regarding reliability, convenience, and mobility. Thus, the availability of a robust, self-sufficient, implantable electrical power generator that works under physiological conditions would be transformative for many applications, from driving bioelectronic implants and prostheses to programing cellular behavior and patients' metabolism. Here, capitalizing on a new copper-containing, conductively tuned 3D carbon nanotube composite, an implantable blood-glucose-powered metabolic fuel cell is designed that continuously monitors blood-glucose levels, converts excess glucose into electrical power during hyperglycemia, and produces sufficient energy (0.7 mW cm(-2), 0.9 V, 50 mm glucose) to drive opto- and electro-genetic regulation of vesicular insulin release from engineered beta cells. It is shown that this integration of blood-glucose monitoring with elimination of excessive blood glucose by combined electro-metabolic conversion and insulin-release-mediated cellular consumption enables the metabolic fuel cell to restore blood-glucose homeostasis in an automatic, self-sufficient, and closed-loop manner in an experimental model of type-1 diabetes.ISSN:0935-9648ISSN:1521-409

Design of programmable post-translational switch control platform for on-demand protein secretion in mammalian cells

The development of novel strategies to program cellular behaviors is a central goal in synthetic biology, and post-translational control mediated by engineered protein circuits is a particularly attractive approach to achieve rapid protein secretion on demand. We have developed a programmable protease-mediated post-translational switch (POSH) control platform composed of a chimeric protein unit that consists of a protein of interest fused via a transmembrane domain to a cleavable ER-retention signal, together with two cytosolic inducer-sensitive split protease components. The protease components combine in the presence of the specific inducer to generate active protease, which cleaves the ER-retention signal, releasing the transmembrane-domain-linked protein for trafficking to the trans-Golgi region. A furin site placed downstream of the protein ensures cleavage and subsequent secretion of the desired protein. We show that stimuli ranging from plant-derived, clinically compatible chemicals to remotely controllable inducers such as light and electrostimulation can program protein secretion in various POSH-engineered designer mammalian cells. As proof-of-concept, an all-in-one POSH control plasmid encoding insulin and abscisic acid-activatable split protease units was hydrodynamically transfected into the liver of type-1 diabetic mice. Induction with abscisic acid attenuated glycemic excursions in glucose-tolerance tests. Increased blood levels of insulin were maintained for 12 days.ISSN:1362-4962ISSN:0301-561

A versatile bioelectronic interface programmed for hormone sensing

Abstract Precision medicine requires smart, ultrasensitive, real-time profiling of bio-analytes using interconnected miniaturized devices to achieve individually optimized healthcare. Here, we report a versatile bioelectronic interface (VIBE) that senses signaling-cascade-guided receptor-ligand interactions via an electronic interface. We show that VIBE offers a low detection limit down to sub-nanomolar range characterised by an output current that decreases significantly, leading to precise profiling of these peptide hormones throughout the physiologically relevant concentration ranges. In a proof-of-concept application, we demonstrate that the VIBE platform differentiates insulin and GLP-1 levels in serum samples of wild-type mice from type-1 and type-2 diabetic mice. Evaluation of human serum samples shows that the bioelectronic device can differentiate between samples from different individuals and report differences in their metabolic states. As the target analyte can be changed simply by introducing engineered cells overexpressing the appropriate receptor, the VIBE interface has many potential applications for point-of-care diagnostics and personalized medicine via the internet of things

Surfactant and catalyst free facile synthesis of Al-doped ZnO nanorods - An approach towards fabrication of single nanorod electrical devices

Classical models illustrate the genesis of bottom-up techniques for synthesis of pristine ZnO or Al-doped ZnO nanoparticles via surfactant aided or hydrothermal chemistry, to constitute the fundamental modules of nanotechnology and nanodevices. The present study demonstrates a facile as well as surfactant and catalyst free route for synthesis of morphology controlled Al-doped ZnO nanorods. Morphological evaluation (via FESEM and TEM characterization) clearly verifies the successful synthesis of ZnO and Al-doped ZnO nanorods. Moreover, crystallographic and FTIR studies ratified presence of multiple different planes as well as phases of ZnO and Al-doped ZnO nanostructures, eventually confirming the doping process. Further, the alteration in mode of electronic transition and surface charge of ZnO nanorods post doping with Al was witnessed from its UV-visible or photoluminescence spectra and zeta potential measurements, respectively. Electrical measurements were performed on prepared Al-doped ZnO nanorods which were fabricated as single nanorod devices. Owing to substitutional and interstitial doping, the electrical conductivity of the devices was drastically enhanced after doping. Excellent electrical attribute of the nanorods when fabricated into single nanorod device was indicative of its potential to be deployed as next generation nano-biosensors or piezo-electric devices

Programmable DARPin-based receptors for the detection of thrombotic markers

Cellular therapies remain constrained by the limited availability of sensors for disease markers. Here we present an integrated target-to-receptor pipeline for constructing a customizable advanced modular bispecific extracellular receptor (AMBER) that combines our generalized extracellular molecule sensor (GEMS) system with a high-throughput platform for generating designed ankyrin repeat proteins (DARPins). For proof of concept, we chose human fibrin degradation products (FDPs) as markers with high clinical relevance and screened a DARPin library for FDP binders. We built AMBERs equipped with 19 different DARPins selected from 160 hits, and found 4 of them to be functional as heterodimers with a known single-chain variable fragments binder. Tandem receptors consisting of combinations of the validated DARPins are also functional. We demonstrate applications of these AMBER receptors in vitro and in vivo by constructing designer cell lines that detect pathological concentrations of FDPs and respond with the production of a reporter and a therapeutic anti-thrombotic protein.ISSN:1552-4450ISSN:1552-446