| Description |
The electrochemical sensors and piezoelectric resonators were adopted as two alternatives of robust, sensitive and affordable transducers suitable for construction of portable detection systems. The former approach represents a highly-sensitive immunospecific detection of bioagents using tracers based on the peroxidase enzyme label (both sandwich and competitive assay formats) providing electrochemically active products measured using the screen-printed electrochemical systems. In this way, the biosensing part of the detector is easily exchanged after positive detection and disposable use is preferred. However, the heterogeneous sensing format requires washing steps and several working solutions, which are delivered by the embedded miniperistaltic pumps. The piezoelectric biosensor provides direct measurement of the target bioagents, the sensing part is used repeatedly after regeneration of the immobilized antibody. From the construction point of view, both lines of detectors share several common elements (case, rechargeable battery, microcontroller, A/D converters, pumps), though the sensing parts are different: multipotentiostat / oscillator and counter, custom flow-through cells and biosensors. Detectors are linked to the external computer with software LabTools (common data-management and user interface, individual detector-related plugin modules). For testing purposes, the model bioagent was the bacterium Francisella tularensis (Ft) included on the lists of biowarfare agents (the LVS variant was employed), several other safe bioagent simulants were recently included, too. For immobilization of biorecognition elements, the gold-coated sensing surface of either working electrode or quartz crystal resonator was modified with a self-assembled monolayer of thiocompound which was covalently linked to the specific proteins - antibodies and antigens as lipopolysaccharide fraction (LPS). The electrochemical detector was able to detect F. tularensis starting at 100 CFU/ml, one analysis (incubations with sample and tracer, washing, addition of sample, amperometric measurement) was completed within 15 min. The piezoelectric detector with immobilized specific antibodies was suitable to indicate the presence of 105 CFU/ml F. tularensis directly within 10 min, the response of 4.8 Hz was significantly higher compared to non-specific species (108 CFU/ml E. coli and B. subtilis provided signals below 2 Hz). In the case of the piezoelectric immunosensor modified with LPS, rapid detection of anti-Ft antibodies in mice inoculated with the bioagent (0.1 LD50) was possible, specific response was obtained shortly (1-3 days) after infection. In this way, the piezoelectric immunosensor serves as suitable tool for sensitive detection of tularemic infection before appearance of any clinical disease symptoms. The developed biosensor-based devices are promising tools suitable for rapid detection of bioagents as well as other biomolecules outside of laboratory. The piezoelectric detector is already commercialized, the electrochemical detector is at the phase of realization of field tests using model agents as B. subtilis, thuringiensis and Erwinia herbicola.
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