Ebook: Commercial and Pre-Commercial Cell Detection Technologies for Defence against Bioterror

Bioterrorism and biological warfare employ living agents or toxins that can be disseminated or delivered by infected individuals, insects, aerosols, and by the contamination of water and food supplies. Biological warfare agents (BWA) such as bacteria, viruses, toxins and genetically engineered species are usually characterized as invisible, odor- and taste-free. Most biological agents can be thousands of times more lethal per unit than the most lethal chemical warfare agents. Unlike chemical agents, biological agents attack people stealthily with no observable reaction until after an incubation period (1–14 days). Current disease surveillance and response systems rely on post-symptomatic reporting. However, many infectious agents such as smallpox have a long latency to clinical symptoms, thereby eluding early detection and resulting in widespread, uncontrolled contagion. Consequently, the threat of deliberate dissemination of biological agents is the most complicated and problematic of the weapons of mass destruction facing mankind today.

This volume contains papers presented at the NATO Advanced Research Workshop “Commercial and Pre-commercial Cell Detection Technologies for Defense against Bioterror – Technology, Market and Society”, held in Brno, Czech Republic in September 2006. As a response to the rapidly emerging threat of bioterrorism, the objectives of the workshop were: (i) to exchange information on commercially available technologies and equipment for defense against bioterrorism; (ii) to further the development of new biosensor system prototypes into a commercially available apparatus; and to explore human factors in BWA biosensors. During the Workshop the new commercial and pre-commercial technologies that are currently emerging in the world were presented and explained. On the other hand, we discussed the interaction of modern detection systems with society and we tried to improve the relation between the scientific community and commercial entities. As a summary, the major areas of activity during the Workshop were the following:

1) A presentation of the most advanced biosensors and biodetection system which can be found in the market or are quite close to commercialization. Systems as the BIOHAWK^TM, SASS 2000, RAPTOR, Bionas^® 2500, OWLS, or a portable SPR were presented.

2) A presentation of the advances in the research of biodetection devices as DNA and protein microchips, micro and nanophotonic sensors, CMOS microsensor chips, electrochemical arrays, physical platforms, electro optical detection, mass detection, etc.

3) A description of the latest developments in the employment of bioreceptor layers for the selective detection of BWA, as protein signatures, molecular imprinted polymers, membrane engineering (MIME), cell signatures, monoclonal antibodies, synthetic antibodies, lytic phages, among others.

4) A deep discussion of the human factor: societal issues related to sensor development and employment for BWA detection.

The editors: Laura M. Lechuga, Fred P. Milanovich, Petr Skládal, Oleg Ignatov and Thomas R. Austin.

The current work addresses the technical and social issues that must be considered when assessing the probability of infectious disease occurrence of natural or man-made origins, within a nation state or across the globe. The technical aspects of the problem relate to the ability to detect potentially subtle changes in the level of threat agents in an environment, the susceptibility of the host, and environmental conditions rendering the host vulnerable to infection. In order to determine an increase in disease emergence, it is necessary to establish a baseline of normal disease incidence in a population, the concentration of pathogens in each environmental region, and the presence of non-pathogenic organisms that are genomically or proteomically similar to pathogens. Once baselines are established for these factors, multiplexed sensors and data fusion technologies can be used to relate changes in the concentration of agents or in host susceptibility to the emergence of clinical symptoms in a community. One premise of this paper is that emergent disease is neither a function of infectious agents alone nor of susceptibility of the human or livestock target alone, but rather it is a probabilistic event dependent on multiple factors including the interaction between the pathogen and the host target. The probabilistic assessment of emergent disease requires large databases, deployed sensors, data fusion, and autonomous rapid decision making capabilities. The extensive deployment of pervasive surveillance systems can cause societal concerns regarding the balance between assuring wellness in a population while simultaneously respecting the privacy of individuals. Because emergent pandemic disease is a relatively low probability event, it is probable that a significant time lapse will occur between the gathering of information from the distributed sensors and the actual realization of pandemic disease. During this interval, societal perceptions may instigate public concern over compromised privacy, because benefits from the sensor deployment may not yet be realized. Possible adverse affects include changes in insurance rates of individuals, loss of employment opportunities, and other unanticipated negative consequences resulting from widespread data acquisition. The goal, however, of maintaining the wellness of society as a whole will require thoughtful balance between the potential loss of individual privacy and maintaining the wellness of the community.

For the development of field sensor systems with enough sensitivity and selectivity we develop photonic biosensors based on evanescent wave detection. Two technologies have been implemented in parallel: a plasmonic sensor for multianalyte real-time evaluation and an integrated optical nanosensor fabricated with silicon microelectronics technology. Both devices can be use as portable analyzers and have demonstrated an excellent performance in the immunological determination of chemical pollutants and in the detection of single mutations in DNA strands.

For the field detection of various microbial species, the portable immunosensor system was developed. The detector consists of a digital 4-channel potentiostat, flow-through system with 4 miniperistaltic pumps, microcontroller unit, rechargeable battery and flow-through cell with an exchangeable immunosensor. This compact system is controlled from external computers using either serial cable or Bluetooth wireless link. Software allows both manual operation and script-based automated measuring procedures. The experience from laboratory and field trial testing will be reported.

Screen-printed electrodes coupled to biomolecules and modern electroanalytical techniques offer an interesting opportunity for the development of sensitive, rapid low cost analytical method for warfare agents. The current paper will focus attention on the recent developments in thick film electrochemical sensors, showing also some of their possible applications as alarm systems against terrorism using methods based on AChE inhibitors.

Biological warfare agents are the most problematic weapons of mass destruction and terror. Both civilian and military sources predict that over the next decade the threat from proliferation of these agents will increase significantly. Therefore, the ability to accurately predict the dispersion, concentration, and ultimate fate of biological warfare agents released into the environment in real time is essential to prepare for and respond to a biological warfare agent release. A fusion of micro- and nanotechnologies with biosciences could significantly counter biological threat agents on the battlefield. Miniaturization of biosensor technologies has great potential for improving resolution time (speed of assay), reducing reagent use, and allowing for higher sample throughput. Fast analysis and on-chip integration of supporting electronic circuitry for signal analysis and remote control would enable sensing at a remote location. This paper describes a new biosensor technology based on combination of direct bioelectrocatalysis and multi-microchannel technology. To demonstrate direct electron transfer, glucose oxidase and PQQ-dependent glucose dehydrogenase have been selected. An electrochemical sensor, which includes biological sensing element immobilized on the surface of microchannels of a working electrode, can be used in the form of a flow-through amperometric, potentiometric, or conductometric device.

This article describes electro-optical (EO) characterization of biospecific binding between Escherichia coli XL-1 and phage M13K07. 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. The operating principle of the analyzer is based on the polarizability of microorganisms, which depends strongly on their composition, morphology, and phenotype. The principle of analysis of the interaction of E. coli with phage M13K07 is based on recording changes in the optical parameters of bacterial suspensions. 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 the infected host, and phage ejection from the cell. In this work, we used M13K07, a filamentous phage of the family Inoviridae. Preliminary study had shown that combination of the EO approach with a phage as a recognition element has excellent potential for mediatorless detection of phage–bacteria complexes. The interaction of E. coli with phage M13K07 induced a strong and specific electro-optical signal as a result of substantial changes in the EO properties of the E. coli XL-1 suspension infected by phage M13K07. The signal was specific in the presence of foreign microfloras (E. coli K-12 and Azospirillum brasilense Sp7). Integration of the electro-optical approach with a phage has the following advantages: (1) bacteria from biological samples need not be purified, (2) the phage infection of 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.

Determination of bacterial cells status during its identification is the important part of their selective analysis. Heterogeneity of cells ensemble with identical superficial properties, but a different metabolic activity, is typical for a bacteria in the nature. Electro optical phenomenon was used for cells identification and analysis of their status. Method is based on the measurement of the optical density variation in cell suspension after action on it alternative electric field.

The label-free, selective and sensitive detection of cells and viruses was successfully performed down to the nanogram and picogram range with acoustic transducers such as the quartz crystal microbalance and the surface acoustic wave resonators. Selectivity of these acoustic devices was optimized by combining them with sensitive layers exhibiting pronounced molecular recognition capabilities based on size, shape and preferably hydrogen bonding. Sensitive layers, often termed as synthetic antibodies, were generated by an innovative method of surface imprinting with bio-analytes. Atomic force microscopy (AFM) proved an excellent tool to examine the bio-imprinted polymer surfaces. Bio-imprinted layers, capable of reversibly absorbing the imprint species, opened up the possibilities to detect different types of cells, for instance, yeast and bacteria. Viruses such as the tobacco mosaic virus, the pox and the human rhinovirus, were specifically detected down to a few ng/mL. Furthermore, by imprinting with bio-analytes, the cross sensitivities can almost be neglected and distinguishing different biogeneous species becomes feasible. The synthetic antibodies yield a more characteristic response pattern than the natural ones.

A better understanding of the multifunctional cellular processing of input- and output-signals in living cells is fundamental for basic research, development of drugs and for environmental monitoring e.g. the detection of biotoxic agents. For on-line monitoring of cellular reactions we develop(ed) several Cell Monitoring Systems (CMS^®). They allow the parallel and non-invasive measurement of different parameters of living cells by the use of CMOS silicon microsensors.

To characterize modes of action of substances as well as their cytotoxic effects Bionas GmbH has developed a new screening system to allow the continuous recording of how an active substance can act (Bionas^® 2500 analyzing system). In the pharmaceutical and chemical industry as well as in environmental science it is important to acquire as much information as possible about the metabolic effects of an active substance or there cytotoxicity. With the Bionas^® 2500 analyzing system metabolically relevant data including oxygen consumption, acidification rate and the adhesion (cell impedance) of cells can be measured in parallel, on-line and label-free. Using e.g. ion-sensitive field effect-transistors (ISFET) and electrode structures it is possible to observe metabolic parameters non-invasively and continuously over longer periods of time. The system has already been established for several cell models, cell lines as well as primary cells. The strength of our system can be found in the continuous data collection during the whole application period. Dynamic, reversible and / or regenerative processes can be observed in one and the same culture. Adaptation effects through repeated addition of compounds can also be observed. A long application period (hours to days) allows more realistic compound concentrations as it is possible in short-term experiments. This advantages result in a higher information content compared to end-point-methods. It also offers the advantage that regenerative effects can be observed during the same test run.

In the present work, two biosensor models intended for determination of microbial cells have been developed. One of them uses an approach related to recognition of specific peculiarities of target cells' metabolism and is able to detect 10⁶ cells/sample. At the same time, it ensures the recognition of the detected microorganism only in the case of pure culture or qualitation of target on the background of microflora with the known metabolic pattern. Another model is based on the immunoassay technique and can be used for detection of target microorganism with the lower limit of detection of 10² cells/sample. The models' operation conditions have been optimized and preliminary conclusion concerning their application has been drawn.

In recent years there has been a rapid increase in the number of diagnostic applications based on biosensors, including live, intact cells, tissues, organs or whole organisms. In similar fashion to DNA and protein microarrays, which deliver multiplex detection via the high-density spatial arrangement of molecular recognition elements, arrays of cells at high-density can form the basis of cell-based sensors with extremely high-throughput capability. The expression of receptors of interest within these arrays could yield cell-based sensors with defined specificities. In addition, transfected cell microarrays composed of high-density arrays of mammalian cells expressing de-fined genes, could be the basis for future high-throughput cell-based protein sensing platforms.

The artificial insertion of receptor-like molecules in the cell membrane is an attractive alternative to cell transformation with genes expressing membrane-bound antibodies. This generic approach is called Molecular Identification through Membrane Engineering (MIME). Interaction of MIME cells with viral particles can trigger changes to the cell membrane potential that are measured by appropriate microelectrodes, according to the principle of the Bioelectric Recognition Assay (BERA). BERA is a biosensory method based on a unique combination of a group of cells, whose immobilization in the matrix preserves their physiological functions and measures the expression of the cell interaction with viruses, through the change in electrical properties. In this way, when a positive sample is added to the probe, a characteristic, ‘signature-like’ change in electrical potential occurs upon contact between the virus and the gel matrix. BERA has been used for the detection of viruses in humans (Hepatitis B and C viruses, herpes viruses), animals (prion protein, foot and mouth disease, blue tongue virus) and plants (tobacco and cucumber viruses) in a remarkably specific, rapid (1–2 minutes), reproducible and cost-efficient fashion. The sensitivity of the virus detection with BERA is equal or even better than with advanced immunological, cytological and molecular techniques, such as the reverse transcription polymerase chain reaction (RT-PCR).

The BERA biosensor diagnostic system is currently available as a desktop, laboratory-scale prototype that can be operated by both expert and lay users. The commercialization process of the device includes engineering for a more compact, stand-alone unit. The system comprises a consumable miniature biosensor (with integrated circuitry, an immobilization matrix and virus-specifically responding cells), a data acquisition system and a PC (desktop or laptop). One of the major advantages of BERA is the extended storability of the disposable sensors, which is also documented by other research groups. So far, more than 35000 sensors have been used for screening worldwide.

Since BERA measurements are essentially electric signals, they can be instantly evaluated by means of specific software either on site (stand alone devices) or via an Internet site. Desktop devices with Internet-based evaluation are targeted to small clinic and doctors' office screening tests in the USA and European Union. On site assays are related to portable BERA field test kits, which are ideal for clinical testing in developing countries and military applications. In this way, BERA and MIME cell sensors lay the foundation for a fully operational global biothreat monitoring network.

This paper reports the results of development and test of Laser-Based Point Detector (LBPD) biodetector for on-line identification of biological warfare materials. The test results of data evaluation of its potential to identify four microorganisms (spores of Bacillus anthracis and Bacillus cereus, vegetative cells of Brucella abortus and Escherichia coli) in pure and mixed cultures at varying concentrations on-line are presented. The LBPD was tested using wireless communications against not only simulants but pathogens as well. Identification specific pathogenic organisms on-line, in a semi-automated process without reagents achieve promising results (87%).

We describe here research directed towards early (presyndromic) diagnosis of infection. By using a mouse model and a multi-component blood protein diagnostic tool we detected cowpox infection several days in advance of overt symptoms with high accuracy. We provide details of the experimental design and measurement technique and elaborate on the long-range implication of these results.

PQQ-dependent glucose, alcohol and glycerol dehydrogenases were purified and used for the design of a number of biosensors. Platinum and carbon paste electrodes were used for the biosensor design. A number or soluble and polymer-type mediators have been tested. Biosensors possessing a direct electron transport from the active center of enzymes to the surface of the carbon paste electrode were developed.

The portable device BioNA for detection of organophosphorous and carbamate substances was developed and tested. The motivation to create such a device was people protection against terrorist attacks by chemical agents (sarin, soman, tabun, VX …) and improvement of pesticides control in developing countries.

The device consists of analytical block containing the biosensor and diffusion chamber where the toxic chemicals are transferred from sampled air to the circulating solution. The electrical and hydrodynamic circuits are connected together by insertion of the analytical block into the main body of the device.

The main idea behind the device is concept of evaluation of nerve agents presence. It is not measurement of concentration but evaluation of sample toxicity. BioNA device is not still completely finished but it is on such a technical level that it can be reliably decontaminated, it is compact and sufficiently robust to test the biosensor detection possibilities both in laboratory testing chambers and in field trials with real nerve agents spread around the device.