Immunosensors proposed for detection of
The detection of various types of microbial agents with harmful effects on human population is required in different situations including civil rescue and security units, homeland security, military operations in field, protection of public buildings and transportation systems including airports, metro and railway stations. Such situations need quickly responding, but sufficiently specific detection systems which could be satisfied by portable, rapid and simple instrumentation based on the bioanalytical detection principles (Lim et al., 2005; Gooding, 2006). For bioagents as microbes, viruses and toxins, various types of immunochemical devices seem to be preferred for the early response, good sensitivity and continuous monitoring capabilities. The detection occurs on the phenotype level, thus no extraction of the genetic material from the agent is required, which is the case for methods based on the polymerase chain reaction PCR (Paddle, 1996; Iqbal et al., 2000).
Our efforts in the biodection area started few years ago; the research on the electrochemical immunosensors for bioagents developed from the previous projects focused on the enzyme-based detection of chemical agents (Krejčí et al., 2008). The principle was amperometric biosensor with immobilized cholinesterase, its inhibition was indication of the presence of target compounds (organophosphate nerve agents) in the surrounding air. From the technological point of view, the biosensor employed thick film based sensors produced by screen-printing; the bioanalytical module was easily exchangable. The whole device BioNA was small enough (~ 0.5 kg) for hand-held use and it allowed for several hours of continuous operation. The acquired experience was further directed to the development of electrochemical immunosensors for bioagents detection.
The detection of bioagents was originally purely military-oriented task due to the long-lasting historical development of biological warfare agents (BWA). The common classification of bioagents comes from the Centers for Disease Control and Prevention (CDC, www.cdc.gov). BWA are classified in categories A, B and C. The category A contains the most danger agents suitable for easy dissemination and rapid transmition among persons resulting in high mortality; the following microbes are on this list:
2. Tularemia and Francisella tularensis
2.1. Description of the bacterium and the disease
Tularemia belongs to diseases of wild animals as hares, rabbits and rodents; it can be spread by ticks, flies and mosquitoes. The infection can also be obtained from contaminated food, water supply and soil. Occasionally, humans become infected, too. The most frequent disease manifestations are ulceroglandular, glanular, oculoglanular, oropharyngeal, pneumonic, typhoidal and septic, the onset of tularemia is quite fast and symptoms as high fever 38-40 C, body aches and dry cough can be observed. For disease treatment, antibiotics as streptomycin and gentamicin are widely recommended and tetracycline and chloramphenicol are alternatives (Enderlin et al., 1994).
2.2. Assay methods
Significant efforts exist towards rapid detection of
3. Immunosensing of
For bioanalytical detection of
Properties of the existing immunosensors for detection of
The direct measurement seems very attractive as signal is generated in real time - immediately after addition of the sample, and no additional reagents are required. However, as shown in Table 1, most direct devices provide detection capabilities only for microbial contents above 105 CFU/ml; a better sensitivity of assays was demonstrated for some of the indirect devices, where the use of sandwich assay formats with enzyme- or fluorophore-labelled secondary antibodies provides higher specificity and improved detection limits around 104 and even 103 CFU/ml. On the other hand, these formats employ additional immunoreagents (Fig. 1B) and also more complex manipulation. The limits of detection required for sufficiently sensitive assay of microbial agents in the form of bioaerosols in air are hard to achieve; a partial improvement can be expected due to the collection systems capturing microbes from the air to the liquid phase (cyclones), though this was not yet demonstrated for
|Principle / Assay details||LOD (CFU/ml)||Length (min)||Reference|
|optical bidiffractive grating biosensor / D ID M R||3•E10 4||50||O'Brien et al., 2000|
|RAPTOR, fiber optic biosensor / ID M R||1•E10 5||10||Anderson et al., 2000|
|fluorescence immunosensor / ID M R||5•E10 5||15||Taitt et al., 2002|
|piezoelectric immunosensor / D (IgM clusters)||5•E10 6||35||Pohanka & Skl adal, 2005|
|optial protein chip, sandwich / ID M||2•E10 6||60||Huelseweh et al., 2006|
|magnetic biosensor, sandwich, freq. mixing / ID, R||1•E10 4||"/ 60||Meyer et al., 2007|
|piezoelectric immunosensor / D||1•E10 5||5||Pohanka & Skl adal, 2007|
|electrochemical immunosensor / ID, M||1000||25||Skl adal et al., 2006|
4. Immunosensor for detection of tularemia
As it was mentioned above, the detection of cells of
The immunosensors developed for this purpose in our group will be described in the following text. Again, the formats of such assys can be direct (shown in Fig. 1C) and indirect where the captured anti-
4.1. Immunization of mice
As an animal model for tularemia, a group of female mice BALB/c was used (specific pathogen free, supplied by BioTest Konárovice, Czech Rep.). Mice were immunized by
4.2. Direct piezoelectric immunosensor
The piezoelectric quartz crystals with gold electrodes (10 MHz, International Crystal Manufacturing) were modified with a monolayer of cystamine, to which the
The crude non-purified (only diluted 10-times) sera collected from the infected mice on days 1, 3, 5, 7, 10 and 14 after inoculation were measured. NMS from healthy mice and CMS from mice immunized by
The obtained results were evaluated using the t-test (IMS vs. NMS, both measured on the specific immunosensor with immobilized LVS antigen, n = 3). The results measured on the 1st day after inoculation can be classified as positive with the probability level of 0.95, results from the following days (3rd and higher) were always detected with the probability level of 0.99. The RSD values for the NMS and IMS (day 14) samples were 2.3% and 2.4% for intra-day measurements (n = 5).
4.3. Indirect amperometric immunosensor
The amperometric immunosensor was based on the gold screen-printed 4-channel electrode array (AC8, BVT Technologies),
Thus generated signal traces are shown in Figure 4, left part, for the blank serum (non-infected mouse) and from sera obtained from infected mice taken in the indicated days after infection. The responses of sera (decrease of current) from individual days are shown in the right part together for both
The blank signal for NMS varied near around 21 nA without exhibiting any pronounced trend; similar but slightly higher response was observed for the control serum; CMS provided a higher signal (22 to 25 nA) in comparison with NMS. The IMS samples taken one day after immunization demonstrated a signal above 23 nA which was continuously increasing in the following days and resulted in the maximal response of 41 nA 21 days after immunization.
Significantly higher signals from IMS were measured on the 5th day after immunization (32.2 ± 1.6 nA) in comparison with CMS (25.0 ± 1.9 nA). Statistically, distinguishing IMS and CMS in one day after immunization was questionable; the probability of difference was on the level 0.60 (t-test). In the following sampling on days 2 and 4, the probability grew up to the levels 0.75 and 0.89, respectively, and starting on the 5th day, the probability level was above 0.99.
The developed amperometric immunosensor provided good reliability and sensitivity of assays. A small amount of 2.5 μl sample was based on appropriately diluted 0.1 μl of original sera. Consequently, this technique can be applied in field laboratories. The instrumentation used for measurements (Figure 5) is fully portable and battery operated (> 10 hours after full charge). After insertion of the strip sensor to the cell, the measuring sequence is fully automated using a script-based programming. The flow-through format was adopted due to better precision of assays; if the measurement will be based on a drop of substrate mixture, further miniaturization can be feasible. An important parameter of the assay is the speed of measurement. Here, the limiting step was measurement of the output signal (5 min). The other assay steps such as preincubation with sample and regeneration can be parallelized and include unlimited number of biosensors. Thus, considering four measuring spots per the strip, up to ten measuring cycles corresponding to 40 assays can be realized within one hour.
4.4. Correlation of results from piezoelectric and amperometric immunosensors
To compare evaluation of sera originated from infected mice, the results obtained from both piezoelectric and amperometric immunosensors were plotted in Figure 6. For a straightforward
comparison, data from both systems were normalized; the results were divided by the maximal observed response to be within the 0 to 1 range. A linear correlation was obtained (R = 0.901), however, the slope of the linear regression was not equal to 1 as well as the intercept value was significantly different from 0. Obviously, this is due to the higher proportion of the binding fraction of serum immunoglobulins able to recognize a wider group of microbial antigens even before the infection with
5. Future trends
Despite the promising results allowing rather fast identification of infection with tularemia, the straightforward detection and identification of bioagents remains challenging.
In real situations, the monitoring of air for presence of danger bioagents should be carried out with sufficiently low limits of detection. For this reason, sampling of the monitored air should be realized with the help of a cyclone system, which captures particles from air and concetrates them in a small volume of solution. Thus obtained sample can be subsequently analyzed with either direct or indirect immunosensor (Figure 7). For detection of bioagents, the amperometric detector should be preferred, as the use of enzyme labels provides significantly enhanced sensitivity compared to direct protocols (Table 1). The system based on the developed immunosensor detector ImmunoSMART and a commercially available cyclone SASS 2300 (Research International) is shown in Figure 8. A program controlling both subsystems was developed in order to allow synchronized operation.
For model detection of microbes in bioaerosols, the completely safe strain of
The amperometric and piezoelectric immunosensors suitable for assay of
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