Molecular Identification through Membrane Engineering as a Revolutionary Concept for the Construction of Cell Sensors with Customized Target Recognition Properties: the Example of Superoxide Detection

AbstractMembrane-engineering is a generic methodology for increasing the selectivity of a cell biosensor against a target molecule, by electroinserting target-specific receptor molecules on the cell surface. We have previously reported the construction of an ultra-sensitive superoxide anion (O2•−) sensor based on immobilized cells, which have been membrane-engineered with superoxide dismutase (SOD). In the present study, we provide evidence that superoxide dismutation triggered changes to the membrane potential of membrane-engineered fibroblast cells, as confirmed by electrophysiological and fluorescence assays. In addition, by conducting selective inhibition assays, we show that electroinserted SOD molecules retained their characteristic catalytic properties. We also investigated the effect of the concentration of electroinserted SOD molecules. Finally, we increased the sensitivity of the sensor by hundredfold to a detection limit of 1 pM O2•− by changing the intensity of the electrical field during electroinsertion and the concentration of immobilized cells on the performance of the biosensor

Immobilization of Electroporated Cells for Fabrication of Cellular Biosensors: Physiological Effects of the Shape of Calcium Alginate Matrices and Foetal Calf Serum

In order to investigate the physiological effect of transfected cell immobilization in calcium alginate gels, we immobilized electroporated Vero cells in gels shaped either as spherical beads or as thin membrane layers. In addition, we investigated whether serum addition had a positive effect on cell proliferation and viability in either gel configuration. The gels were stored for four weeks in a medium supplemented or not with 20% (v/v) foetal calf serum. Throughout a culture period of four weeks, cell proliferation and cell viability were assayed by optical microscopy after provision of Trypan Blue. Non-elaborate culture conditions (room temperature, non-CO2 enriched culture atmosphere) were applied throughout the experimental period in order to evaluate cell viability under less than optimal storage conditions. Immobilization of electroporated cells was associated with an initially reduced cell viability, which was gradually increased. Immobilization was associated with maintenance of cell growth for the duration of the experimental period, whereas electroporated cells essentially died after a week in suspension culture. Considerable proliferation of immobilized cells was observed in spherical alginate beads. In both gel configurations, addition of serum was associated with increased cell proliferation. The results of the present study could contribute to an improvement of the storability of biosensors based on electroporated, genetically or membrane-engineered cells

In vitro Propagation and Assessment of Genetic Relationships of Citrus Rootstocks Using ISSR Molecular Markers

The behavior of six citrus rootstocks, Volkameriana, Citrumelo ‘Swingle’, Citrange ‘Carrizo’, Poncirus trifoliata ‘Serra’, Poncirus trifoliata ‘Rubidoux’ and Poncirus trifoliata ‘Flying Dragon’, in in vitro propagation was studied and compared for shoot proliferation and rooting. In addition, the genetic relationships among the rootstocks studied and other Citrus species, using the Inter-Simple Sequence Repeats (ISSR) molecular markers, were investigated. Nodal explants of three months old shoots were used in Murashige and Skoog medium supplemented with N6-benzyladenine (BA) for shoot proliferation and with naphthaleneacetic acid (NAA) for rooting. The rootstock Volkameriana showed a statistically significant higher number of shoots (1.81), shoot length (15.14 mm) and number of leaves per explant (5.81), while all three Poncirus trifoliata rootstocks showed the lowest numbers. The number of roots and root length per explant were evaluated at the end of the rooting phase. The rootstock ‘Swingle’ showed a higher number of roots per explant (4.2) followed by ‘Flying Dragon’ (3.93) and ‘Carrizo’ (3.23) rootstocks. The rootstocks ‘Swingle’ (140.8 mm), Volkameriana (148 mm) and ‘Flying Dragon’ (131.12 mm) had significantly higher root length per explant compared to ‘Carrizo’ (31 mm) and ‘Rubidoux’ (34.5 mm). The ISSR molecular marker technique used in the present study grouped successfully the different species, varieties and rootstocks studied, revealing their genetic variability. The genetic variability observed among the rootstocks ranged between 0.29 (Poncirus trifoliata ‘Serra’ and Citrumelo ‘Swingle’) and 0.60 (Volkameriana and Citrumelo ‘Swingle’). The response of the rootstocks studied in in vitro propagation however is not related to their genetic affinity

Comparative Study of the Electrochemical Signal of Neonicotinoids and Tetronic Acid Amides on Screen Printed Electrodes With and Without the Use Of N2a Cells.

The extensive use of pesticides in agriculture has caused significant concern in public health therefore cell-based sensors have been proved as potentially useful method for studying their effects. The objective of this work was to investigate the possibility of using carbon screen printed electrodes (SPE) in combination with the use of N2a cells for the direct voltammetric determination of 5 neonicotinoids (imidacloprid, clothianidin, thiacloprid, acetamiprid and thiamethoxam) and 3 tetronic acid amide insecticides (spiromesifen, spirodiclofen and spirotetramat). The insecticide cytotoxicity in N2a cells was determined after 30 min treatment with concentrations 3, 10, 30 and 100 μM by the propidium iodide (PI) uptake assay. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed to compare signals from plain carbon screen printed electrodes and from N2a cells

Development of a Novel, Ultra-rapid Biosensor for the Qualitative Detection of Hepatitis B Virus-associated Antigens and Anti-HBV, Based on “Membrane-engineered” Fibroblast Cells with Virus-Specific Antibodies and Antigens

A novel miniature cell biosensor detection system for the detection of Hepatis B virus (HBV)-associated antigens and anti-HBV is described. The biosensor is based on “membrane-engineered” Vero fibroblast cells immobilized in an alginate matrix. The membrane-engineering process involved the electroinsertion of anti-HBV specific antibodies (anti-HBs, anti-HBe) or antigens (HBsAg) in the membranes of the Vero cells. The attachment of a homologous antigen to the electroinserted antibody (or, respectively, of the antibody to the electroinserted antigen) triggered specific changes to the cell membrane potential that were measured by appropriate microelectrodes, according to the principle of the Bioelectric Recognition Assay (BERA). The sensor was used for screening 133 clinical blood serum samples according to a double-blind protocol. Considerably higher sensor responses were observed against HBV-positive samples, compared with responses against negative samples or samples positive for heterologous hepatitis viruses such as Hepatitis C (HCV) virus. Detection of anti-HBs antibodies was made possible by using a biosensor based on immobilized Vero cells bearing the respective antigen (HBsAg). The observed response was rapid (45 sec) and quite reproducible. Fluorescence microscopy observations showed that attachment of HBV particles to cells membrane-engineered with anti-HBs was associated with a decrease of [Ca2+]cyt. The perspectives for using the novel biosensor as a qualitative, rapid screening, high throughput assay for HBV antigens and anti-HBs in clinical samples is discussed

Systems mapping for technology development in CBRN response

Purpose This project aimed to develop an EU sociotechnical systems map to represent a harmonised concept of operations (CONOPS) as a future development platform for technologies used in multi-services emergency responses to Chemical, Biological, Radiological and Nuclear (CBRN) incidents Methodology AcciMaps were developed to locate where technologies are currently used, and opportunities for new technologies. The AcciMaps were iteratively co-designed with End Users (Fire, Ambulance, Police and Military) across three EU countries (UK, Finland and Greece). Data were collected using document analysis and interviews with senior ranking (Gold or Silver Command level) representatives of the participating end users. Findings Despite differences in terminology and between service sectors, consensus was achieved for the command structures (Gold, Silver and Bronze), and Hot Zone responders (Specialist Blue Light Responders and Blue Light Responders). A Control Room was included as the communication spine. Blue Light Responder activities were limited by their scope of practice and available equipment, for example breathing apparatus. The harmonised EU AcciMap offers a high level sociotechnical systems map of CBRN response. Critical segments have been identified which offer opportunities for technology developments that can add value in terms of response capabilities (e.g. tag and trace). Originality/ Value A large scale major CBRN incident may need cross-border and cross-professional engagement where efficient interoperability is vital. This research is the first EU consensus of a sociotechnical system map for CONOPS. It supports future research for technology development e.g. detection and decontamination equipment design and use, communication, diagnosis and response technologies

Mapping Emergency Responders’ Current Procedures in the Event of a CBRNe Incident

When a Chemical, Biological, Radiological, Nuclear or explosive (CBRNe) event occurs a time and safety critical environment instantly exists. In order for emergency services to most efficiently complete their primary task of saving lives it is essential to have effective and well-rehearsed procedures in place. This environment requires many different services to interact with one another including: Fire, Police, Health and Military personnel. Therefore, it is important that each service understand not only their role but also that of the other emergency services (JESIP, 2013). In such a scenario every second can make a difference, with tasks such as triaging, treating and decontaminating casualties all heavily reliant on a swift response. However, this has to be balanced with offering maximal health and safety conditions for the emergency service personnel (NARU, 2015). Emergency personnel from other geographical locations may also be recruited to provide further support, so having a nationally recognised standard procedure for each emergency service is essential to allow smooth interaction between regional emergency crews (NATO, 2014). By taking a Human Factors/ Ergonomics approach to the problem it is essential to first understand what is required of each actor in the system. There are many different methods that can be used to capture a system such as that described above. One example is an Accident Map (AcciMap) (Rasmussen, 1997) - this allows for the different layers in the system to be identified, the lines and methods of communication to be shown as well as any interactions within a system to be acknowledged. With this in mind the aim of a work package within the European Commission (EC) funded TOXI-Triage project (Toxi-Triage, 2016) was to establish procedures in the event of a CBRNe incident for different emergency service providers across a number of EU countries

Ergonomics systems mapping for professional responder inter-operability in chemical, biological, radiological and nuclear events

A European consensus was developed as a concept of operations (CONOPS) for cross-border, multi-professional chemical, biological, radiological and nuclear (CBRN) responses. AcciMaps were co-designed with professional responders from military, fire, ambulance, and police services in UK, Finland and Greece. Data were collected using document analysis from both open and restricted sources to extract task and operator information, and through interviews with senior staff representatives (Gold or Silver Command level). The data were represented on the Accimaps as a high level Socio-Technical Systems (STS) map of CBRN response using the themes of communication, planning, action, and reflection. Despite differences between service sectors and in terminology, a macro systems level consensus was achieved for the command structures (Gold, Silver and Bronze), and Hot Zone responders (Specialist Blue Light Responders and Blue Light Responders). The detailed tasks and technologies have been analysed using Hierarchical Task Analysis (HTA) to represent both complex response scenarios (macro) and detailed technologies (micro interfaces) for detection, diagnosis and decontamination. The outputs from these two systems mapping tools (Accimaps and HTAs) are being used in two field trials/exercises

Pesticide residue screening using a novel artificial neural network combined with a bioelectric cellular biosensor

We developed a novel artificial neural network (ANN) system able to detect and classify pesticide residues. The novel ANN is coupled, in a customized way, to a cellular biosensor operation based on the bioelectric recognition assay (BERA) and able to simultaneously assay eight samples in three minutes. The novel system was developed using the data (time series) of the electrophysiological responses of three different cultured cell lines against three different pesticide groups (carbamates, pyrethroids, and organophosphates). Using the novel system, we were able to classify correctly the presence of the investigated pesticide groups with an overall success rate of 83.6%. Considering that only 70,000-80,000 samples are annually tested in Europe with current conventional technologies (an extremely minor fraction of the actual screening needs), the system reported in the present study could contribute to a screening system milestone for the future landscape in food safety control

Morphogenetic responses of embryo culture of wheat related to environment culture conditions of the explant donor plant

Availability of immature embryos as explants to establish wheat (Triticum aestivum L.) by tissue culture can be limited by climatic factors and the lack of high quality embryos frequently hampers experimentation. This study evaluates the effects of rainfall, various temperature-based variables and sunshine duration on tissue culture response (TCR) traits including callus formation (CF), regenerating calli (RC), and number of plants per embryo (PPE) for 96 wheat genotypes of worldwide origin. The objectives of this study were to evaluate the significance of a particular climatic factor on TCR traits and to determine the period of wheat growth during which these factors were the most effective. The genotypes were grown in an experimental field during three seasons differing in meteorological conditions. The relationships between TCR traits and climatic factors within three time periods of wheat growth: 2, 6 and 10 weeks prior to embryo sampling were analysed by biplot analysis. The tissue culture traits were influenced at very different degrees by climatic factors: from 16.8% (RC) to 69.8% (CF). Donor plant environment with high temperatures and low rainfalls reduced (p lt 0.05) the tissue culture performance of wheat genotypes. Callus formation was most sensitive to the temperature based factors. The environmental conditions between flowering and the medium milk stage were the most important for CF, while RC and PPE were not particularly related to any period