Development of QCM biosensor to detect a marine derived pathogenic bacteria Edwardsiella tarda using a novel immobilisation method
Introduction
Piezoelectric devices have been proposed as probes able to continuously monitor affinity reactions (antigen-antibody, DNA hybridisation reaction, etc.), without the use of any label (Minunni et al., 1995, Guilbault et al., 1992, Skladal et al., 1994). This technology offers a real time output, simplicity of use and cost effectiveness. The first use of antigens as coating in quartz crystal microbalances (QCM) was proposed by Shons et al. (1972). Since then, many reports have been published using piezoelectric sensors for a wide range of applications in the food industry, environmental monitoring, clinical diagnostics and biotechnology. Consequently, QCM has been developed as an extremely sensitive mass sensor, capable of measuring subnanogram levels (Clark et al., 1987) by improving the detection limits using crystals of higher frequencies (>10 MHz) (Bunde et al., 1998, Lin et al., 1993) and improving the immobilisation procedure on the quartz surface (Clark et al., 1987).
Generally, conventional immobilisation methods based on adsorption or covalent binding via the amino group of the protein are not site-directed and produce a random orientation of the antibody, which results in a very low binding efficiency (Vikholm et al., 1999). The strategy chosen to create the sensing layer should enable to control both the amount and the orientation of the antibody on the transducer while preserving its bioactivity (Briand et al., 2007). Thus many published papers showed an immobilisation technique based on Staphylococcus aureus protein A coating (Babacan et al., 2000, Galli Marxer et al., 2003, Grubor et al., 2004, Kaur et al., 2004, Michalzika et al., 2005).
However, even though there is the benefit of protein A coating for a proper orientation of the antibody (Lu et al., 1996), it is not convenient to detect a particular bacteria over several samples by regeneration of a sensor chip since antibody against the bacteria should be reimmobilised to protein A for every detection. It was also known that covalent coupling of antibody increased the stability against degradation during the regeneration process (Uttenthaler et al., 1998).
Concerning the orientation of antibody for the best antigen binding capacity and efficient regeneration procedures, in this study, a direct immobilising technique was introduced using oxidised IgG over the gold electrode of the quartz crystal. After oxidation to expose the aldehyde residues of the carbohydrate moiety in the Fc region, the IgG can be easily immobilised by cross-linking through hydrazine cross-linked to the self assembled monolayer (SAM).
This study applied this methodology to detect a marine derived bacterium Edwardsiella tarda. E. tarda, which is a member of the bacterial family Enterobacteriaceae, is an economically important pathogen and is frequently found in organically polluted water. The natural reservoir of this bacterium appears to be the intestine of animals including fishes. It has been isolated from a variety of cultured fishes, including eel (Anguilla japonica), channel catfish (Ictalurus punctatus), mullet (Mugil cephalus), seabream (Evynnis japonicus), chinook salmon (Oncorhynchus tsawytscha), tilapia (Tilapia nilotica), and others (Wakabayashi and Egusa, 1973, Meyer and Bullock, 1973, Kusuda et al., 1976, Kusuda et al., 1977, Amandi et al., 1982, Kubota et al., 1982). Currently, time consuming bacterial culture followed by biochemical profiling is the most popular method for identification of an unknown bacterium including E. tarda. However, several researchers are seeking for quick and sensitive method to identify various pathogenic bacteria. Yu et al. (2004) reported that high-performance capillary electrophoresis (HPCE) has been applied to the identification, separation, and quantification of intact bacteria in marine environment. Byers et al. (2002) also reported that Aeromonas salmonicida-specific polymerase chain reaction (PCR) test have a detection limit of approximately 4 × 105 CFU/g sample.
This study investigated a new methodology recruiting oxidised IgG to cross-link to hydrazide conformed on NHS activated SAM via exposed aldehyde group in Fc region of IgG to increase affinity and stability and for the convenient regenerations. The new methodology was compared with conventional immobilisation methodologies which directly immobilise IgG by cross-linking carboxylic group or amino group at the ends and indirectly immobilise Fc region of IgG via protein G cross-linked to SAM. This study also aims to introduce QCM biosensor system to detect a fish pathogen for the first time.
Section snippets
Preparation of formalin killed cells of E. tarda and its antibody
In this study, formalin killed cells (FKC) was used to prepare antiserum and to test sensor system instead of using live bacteria for a convenience of use and safety for researchers. E. tarda was isolated from diseased fish and cultured on tryptic soy agar (TSA) plate. A single colony was inoculated into 400 ml of TSB medium and cultured overnight. To prepare FKC, formalin was added at a final concentration of 1% and the mixture was incubated for 24 h at 4 °C. FKC was washed with phosphate
Effects of immobilisation methods on IgG immobilisation ability and sensitivity
Frequency shifts following IgG immobilisation represented that all tested immobilisation methods were efficient enough to immobilise considerable amount of IgG (Fig. 1). Frequency shift was highest following N-terminal of IgG immobilisation while C-terminal immobilisation showed 30% less frequency shift after IgG immobilisation revealing that immobilisation of N-terminal of IgG is more effective than immobilisation of C-terminal of IgG. Frequency shifts were less than above two but similar to
Effects of immobilisation methods on IgG immobilisation ability and sensitivity
Many published papers showed that immobilisation techniques based on direct adsorption or on protein A coating, resulted in appropriate sensor signals, but only cross-linker procedures using thiols or the interaction between avidin and biotinylated molecules, provided a long sensor lifetime. Moreover, covalent coupling increased the stability against degradation during the regeneration process (Uttenthaler et al., 1998).
This study introduced a direct immobilising methodology accompanying right
Conclusions
In conclusion, it was demonstrated that the prepared sensor chip was able to detect E. tarda cells in dose dependent manner at detection limit of less than 50 μg and the new methodology was appeared to be more sensitive than other tested methods. The prepared sensor chip was reusable over 10 times but sensitivity was reduced after the first use even though still higher sensitivity than other chips was sustained. Some experiments can be carried out to produce more stable sensor chips or to
Acknowledgement
This study was supported by grant no. F10601206A220000100 from the Ministry of Maritime Affairs and Fisheries (MOMAF) in Korea.
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