Alex Simonian

Impedance biosensing using phages for bacteria detection: generation of dual signals as the clue for in-chip assay confirmation.

In the present work, we compare the use of antibodies (Ab) and phages as bioreceptors for bacteria biosensing by Electrochemical Impedance Spectroscopy (EIS). With this aim, both biocomponents have been immobilised in parallel onto interdigitated gold microelectrodes. The produced surfaces have been characterised by EIS and Fourier Transform Infra-Red (FTIR) Spectroscopy and have been applied to bacteria detection. Compared to immunocapture, detection using phages generates successive dual signals of opposite trend over time, which consist of an initial increase in impedance caused by bacteria capture followed by impedance decrease attributed to phage-induced lysis. Such dual signals can be easily distinguished from those caused by non-specific adsorption and/or crossbinding, which helps to circumvent one of the main drawbacks of reagentless biosensors based in a single target-binding event. The described strategy has generated specific detection of Escherichia coli in the range of 10(4)-10(7) CFU mL(-1) and minimal interference by non-target Lactobacillus. We propose that the utilisation of phages as capture biocomponent for bacteria capture and EIS detection allows in-chip signal confirmation.

Biosensors as 21st Century Technology for Detecting Genetically Modified Organisms in Food and Feed

T history of genetically modified organisms (GMOs) can be traced to the year 1971, when Ananda M. Chakrabarthy discovered a multiplasmid hydrocarbon degrading bacteria Pseudomonas putida that was capable of digesting an oil spill 2 orders of magnitude faster than four similar strains. Since then, little more than 2 decades, this landmark research paved the way for a “biotech revolution” that allowed genetic transformation of virtually all terrains of life on earth. Mainly in the agricultural sector, in the years between 1997 and 1999 as much as 70−80 million acres were quickly converted to raise genetically modified (GM) food and crops. Predominantly, >40% of the corn, >50% of the cotton, and >45% of soybean acres of land and at least 2/3rds of all the U.S. processed foods contained GMOs. What caused this dramatic revolution lies in the fact that GMOs are unique, and they were mankind-created by forceful modification of their genome through gene technology. Genetic transformation/modification occurs by alteration of an organism gene cassette (Figure 1) consisting of an expression promoter (P), a structural gene (“encoding region”), and an expression terminator (T), by inserting foreign DNA, which enables the expression of an additional protein conferring new characteristics, for example, herbicide tolerance, resistance to virus, antibiotic, and insect resistance. There are two particular sequences inserted into most transgenic plants, promoter of the 35S subunit of rRNA of the cauliflower mosaic virus (CaMV35S) and the terminator of nopaline synthase gene (TNOS) from Agrobacterium tumefaciens. These are used widely in commercial production of transgenic vegetables under the brand names such as soy Roundup Ready, the maize MaisGard, and the tomato Flavr Savr. Currently, the global status of commercialized GM crops reached 170 million hectares in a total of 29 countries, as revealed by ISAAA 2011(Figure 2). Among them the U.S. remains the top with 69 million hectares raising maize, soybean, cotton, canola, sugar beet, alpha-alpha, papaya, and squash, followed by Brazil and Argentina. Despite the great progress of technology, these modified foods have not gained worldwide acceptance in the general public because of raised consumer concerns, environmental issues, transparent regulatory oversight, and skepticism in government bureaucracies. During the early development of this field, when pesticides and other tolerant crops were introduced, it was thought to be safe and harmless for consumers. However, only over a decade, this technology has shown its true harmful implications which now have led to an ongoing debate on increasing research efforts evaluating the risks associated with the introduction of GMO into agriculture (e.g., potential gene flow to other organisms, agricultural diversity destruction, allerginicity, resistance to antibiotics, and gastrointestinal problems). Additionally, economical and moral issues with realization of contamination of non-GMOs with GMOs came into play. Therefore, several countries, including EU countries, Japan, Australia, New Zealand, Thailand, and China have implemented mandatory labeling for bioengineered foods. In the EU, strict restrictions were imposed on the import and introduction of legislation requiring mandatory food labeling in cases where more than 0.9% of the food ingredients (considered individually) are of GMO origin. However, the U.S. legislation instead opted for voluntary labeling and requested companies for U.S. Food and FDA approval before their launch into market. Consequently, 90% of the consumers have no idea what has been quietly introduced into their daily based food consumption and what impact they might cause in the near future.

Engineering of the membrane of fibroblast cells with virus-specific antibodies: A novel biosensor tool for virus detection

A novel concept for the assay of viral antigens is described. The methodological approach is based on a membrane-engineering process involving the electroinsertion of virus-specific antibodies in the membranes of fibroblast cells. As a representative example, Vero fibroblasts were engineered with antibodies against Cucumber mosaic virus (CMV) and used for the construction of an ultra-sensitive miniature cell biosensor system. The attachment of a homologous virus triggered specific changes to the cell membrane potential that were measured by appropriate microelectrodes, according to the principle of the bioelectric recognition assay (BERA). No change in the membrane potential was observed upon cell contact with the heterologous cucumber green mottle mosaic virus (CGMMV). Fluorescence microscopy observations showed that attachment of CMV particles to membrane-engineered cells was associated with membrane hyperpolarization and increased [Ca(2+)](cyt). In an additional field-based application, we were able to detect CMV-infected tobacco plants at an essentially 100% level of accuracy.

Biosensors for Detection of Genetically Modified Organisms in Food and Feed

Genetically modified organisms (GMOs) have gained momentum in improving the agricultural yield through gene transfer systems. Introduction of foreign genes into the host genome for new characteristics demonstrates great progress, however represents a potential risk for the consumers and environment sustainability. Several issues on regulatory approval, safety, and public perception raised concerns that would impact the extent to which GMOs can thrive. Therefore, there is a demand for a simple, sensitive, cost-effective, fast, and reliable detection method capable of operating on the spot. Apart from vast number of available conventional methods, such as enzyme-linked immunosorbent assays or polymerase chain reaction, biosensors are cutting-edge analytical tools that have a great promise and potential in detecting GMOs in wide range of food products, from maize flour to a cookie. In this chapter we discuss the potential application of DNA biosensors for GMO identification/detection based on optical, piezoelectric, and electrochemical transducers reported until now.

Architectures of nano-biointerfaces: relevance to future biosensing, environment and energy applications

Abstract We describe here a layer-by-layer (LbL) technology to generate multi-functional protein-based biointerfaces for construction of novel bioelectronic devices for biomedical, environment and energy applications. LbL is considered as an effective, simple and well suited technique for fabrication of unique functional biointerfaces. Upon combination of nanomaterials such as carbon nanotubes, polyelectrolytes, proteins and various other biomolecules, system fabrication based on LbL creates new insights and concepts for development of novel sensor and energy technologies. We report here on flexibility and versatility of LbL technique to design the hybrid nanobio-catalytic architectures based on interactions of anionic and cationic biomolecular layers for multiple technological applications.