Catálogo de publicaciones - libros

A novel, portable Electro-Optical (EO) Biosensor (CELAN) for the detection of extremely low concentrations of microorganisms in real-time (<25 minutes) with immediate on-the-spot interpretation of the results has been developed. The EO biosensor consists of three major subsystems: (1) a fluid-handling subsystem, (2) electro-optical device and (3) computer hardware and software with a graphical user menu. The method is based on measurement of suspended cells polarizability before and after their interaction with selective labels. Monoclonal antibodies, phages, and gold particles have been used as markers. Live vaccine strains of , strain STI, and were used as test pathogen agents. Mathematical data processing, includes accumulation of the data, their filtration and comparison of two functions and Frequency Dependence of Anisotropy Polarisability (FDPA) before and after biospecific cell-antibody interactions have been optimized

An electro-optical approach has been used for studies of microbial cells and some biological agents (antibodies and phages) binding. The electro-optical analyzer (ELUS EO), which has been developed at the State Research Center for Applied Microbiology, Obolensk, Russia, was used as the basic instrument for electro-optical measurements. Since the AC electrokinetic effects depend on dielectric properties of bioparticles, their composition, morphology, phenotype, the medium, and the frequency of applied electrical field, the electro-orientational spectra were used for discrimination of bacteria before and after selective binding with antibodies. It has been shown in the model systems that the biospecific interactions of cells as target cells with anti- antibody in the presence of K-12, and Sp7 change the electro-optical signals significantly. Thus, the determination of the presence of particular bacteria within a mixed sample may be achieved by selection and matching of antibodies specific to individual bacterium types and by comparing spectra of bacterium in the presence and in the absence of specific antibodies. The same principles were used for the investigations of bacteria — phage interactions. Biospecific binding was used between XL-1 cells and the phage M13K07. The phage-cell interaction includes the following stages: phage adsorption on the cell surface, entry of viral DNA into the bacterial cell, amplification of phage within infected host and phage ejection from the cell. In this work, we used M13K07, a filamentous phage of the family . Preliminary study had shown that combination of the EO-approach with a phage as a recognition element has an excellent potential for mediator-less detection of phage-bacteria complex formation. The interaction of with phage M13K07 induces a strong and specific electro-optical signal as a result of substantial changes of the EO properties of the XL-1 suspension infected by the phage M13K07. Integration of the electro-optical approach with a phage has the following advantages: 1) bacteria from biological samples need not be purified, 2) the infection of phage to bacteria is specific, 3) exogenous substrates and mediators are not required for detection, and 4) it is suitable for any phage-bacterium system when bacteria-specific phages are available.

Electropolymerized films have received considerable attention in the development of biosensors and biochips and are rapidly advancing. This paper reviews the recent advances and the scientific progress in the electrochemical immobilization procedures of biological macromolecules on electrodes and optical fibers via electrogenerated polymer films bearing affinity sites or photoactivable groups. The biomolecule immobilization was based on the auto-formation of avidin bridges between biotinylated films and biotinylated biomolecules. Some advances in the electrochemical transduction of immunoreactions involving, the permeability, the conductivity or the photochemical and redox properties of electropolymerized films are also presented.

Biological and chemical warfare are fields where new types of analyzers (faster, direct, smaller and cheaper than conventional methods) are demanded. In order to achieve a multibiosensor technological platform that could be used as an early warning system for biological and/or chemical warfare, we are working in the development of two different approaches. A platform based on Optoelectronics biosensors (evanescent wave detection). Two optical biosensors have been already developed: a portable Surface Plasmon Resonance Sensor (actually in commercialization) and an integrated Mach-Zehnder interferometer device. For the second sensor, the use of standard Si microelectronics technology allow the possibility for integration of optical, fluidics and electrical function on one chip in order to obtain a complete lab-on-a-chip. A multibiosensor platform based on Nanomechanical biosensors. Microcantilever biosensors are a new class of high sensitivity biosensors able of performing local, high resolution and label-free molecular recognition measurements. Moreover, nanomechanical biosensors based on microcantilevers have been recently reported as a promisingly alternative to current DNA-chips allowing real-time monitoring of DNA without need of labelling. For that reason, we are working in the development of a portable multibiosensor microsystem based on an array of microcantilevers [2] able to detect analytes with femtomolar sensitivity and ability for discerning single base variations in DNA strands.

Different new methods for the amplified and specific electronic or photonic detection of DNA will be addressed. Specific methods that will be described include:

A key factor affected for the ability to protect society against biological and chemical threats is the deployment of an automated sensor monitoring system for use in government and commercial applications. Currently available biosensors have several shortcomings that must be overcome: 1) they are slow to recognize the presence of a pathogen; 2) they are not suitable to discriminate simultaneously a full set of pathogenic vs. non-pathogenic microorganisms in the environment; 3) they cannot be monitored or operated by remote control. The effective testing of biological agents requires new biosensor technology, which should be extremely sensitive, miniaturized, reliable, fast and require less maintenance than current equipment for monitoring of wide spectrum of biological warfare agents in real time.

Recent advances in areas such as microarray technology, microelectromechanical systems, microfluidics, and microseparations present new technological possibilities for producing fast, universal, extremely sensitive, and inexpensive “smart” sensing systems for field application.

The paper is divided into four segments: an overview of the known challenges associated with biological agent detection; discussion of general detection requirements such as ambient environment, selectivity, sensitivity, sampling, shelf life and remote operation; overview of new portable biodetectors for field applicaton; and lastly, the summary of the future avenues for the electrochemical biosensor systems.

An immunosensor for rapid and sensitive detection of (Ft) was developed using screen-printed sensors with multiple (4) Au working electrodes. The sensing zones were modified with a biolayer of anti-Ft polyclonal antibody attached through the covalently immobilized Protein A. The sandwich assay format was employed with anti-Ft IgG-peroxidase conjugate as tracer. For electrochemical measurements, an automated portable detector was developed; it consisted of a digitally controlled potentiostat and four embedded miniperistaltic pumps. The flow-through measurement involved sequential incubations with sample (10 min), tracer (10 min) and substrate mixture (iodide and hydrogen peroxide). The enzymatically-produced iodine was detected electrochemically at - 50 mV. The limit of detection was 100 Ft cells/ml; the response was significantly higher (3x) when using the living cells compared to the heat inactivated ones.

The elaboration of highly sensitive and express methods for quantitative and qualitative detection and monitoring of chemical warfare agents (CWA), organophosphate and carbamate pesticides, compounds with delayed neurotoxicity, and pathogenic microorganisms and viruses is discussed. The application of potentiometric and amperometric biosensors, automatic biosensors discriminating the neurotoxins of different classes, is performed. The information about biosensors detecting the compounds with delayed neurotoxicity through the evaluation of “neurotoxic esterase” activity in the blood is presented. The use of immunochip technology for the detection of pathogenic microorganisms and viruses is demonstrated. The enzymatic methods of destruction of organophosphorus neurotoxins are considered as the base of new defense technology

The apparatus we would like to report is an integrated instrument for rapid (seconds or minutes) detection of neurotoxic compounds in aqueous solution (or gaseous samples like confined air) and genotoxic compounds. The portable instrument can measure in the range of ppm to ppb in seconds or minutes compounds potentially genotoxic or neurotoxic. Neurotoxic devices are based mainly on the use of enzyme Acetylcholinesterase.

Genotoxic devices are DNA electrochemical biosensors that are realized by immobilizing oligonucleotide sequence or the calf thymus DNA on an electrode surface.

Prior to the 1990 Iraq-Kuwait conflict it was well known that Iraq had developed weapons of mass destruction but the extent of its programs were unclear. After the Iraqi defeat in the ensuing Gulf war 1991 the UN Security Council authorized the creation of UN Special Commission for Iraq (UNSCOM) with the purpose of ridding Iraq permanently of weapons of mass destruction. Several conclusions can be drawn from more than ten years of biological weapons inspections in Iraq. Firstly, UNSCOM managed to get a rather clear picture of the past weapons programs. Secondly, it was not possible for Iraq to restart a substantial program with UNSCOM being present in the country. Thirdly, a full and final and complete account of the weapons program could not be established despite the use of the best intellectual and technical capabilities available at the time.