Development of novel conductometric biosensors based on immobilised whole cell Chlorella vulgaris microalgae
Introduction
The aquatic ecosystem health is a key element in environmental issues. Indeed water is an essential resource needed in most human activities. Protecting water quality requires early warning systems for on line and in situ pollution monitoring.
Phosphorus is needed for microphyte growth of which it is often the limiting factor. Some microphyte species can survive under strongly deficient phosphorous conditions. These micro-organisms can indeed stock large amounts of phosphorous. Phosphatase enzymes can hydrolyse phosphate containing molecules in this purpose. Depending on the species and the pH, different types of phosphatases can be found namely alkaline, acid and neutral phosphatases.
Recently, research concerning biosensors has led to the development of enzyme sensors using immobilised single enzymes (Dzadevych et al., 1994, Shul’Ga et al., 1994, Wan et al., 1999, Dennisson and Turner, 1995, Turner et al., 1987). These sensors have been tested successfully for the detection of toxic compounds in environmental controls as well as food and medicine analysis.
Enzyme-based sensors are highly selective systems for the detection of toxic compounds because of their high specificity of groups of inhibitors towards a particular enzyme. For instance, alkaline phosphatase activity (APA) is known to be mainly inhibited by heavy metal ions. From this statement, developing a multi-enzymatic biosensor will allow the detection of different groups of pollutants by the same device (Arkhypova et al., 2001). However, immobilising different enzymes on a multisensor arrays is not so easy, because all enzymes must be working under the same operational conditions simultaneously. Several problems namely the enzyme stability, their high price must be overcome.
The use of micro-organisms for multi-enzymatic biosensor design can be a good solution, each algal cell containing a large number of enzymes. Moreover using a living organism gives information concerning the ecotoxicological effects of pollutants on these organisms.
This paper describes the development of a conductometric biosensor using immobilised whole cell algae, Chlorella vulgaris, for APA analysis and heavy metal detection (for example, cadmium ions). Using whole cell algae is very interesting since these cells are the primary producers of aquatic food webs (Khoshmanesh et al., 1996): any disturbances may have consequences on the upper levels. Bioassays have already been developed for the detection of C. vulgaris alkaline phosphatase activity and the influence of heavy metal ions (Durrieu et al., 2003).
Some enzymatic reactions involve either consumption or production of charged species and, therefore, lead to a global change in the ionic composition of the tested sample that can be detected with conductometric biosensors. These sensors present a number of advantages (a) thin-film electrode are suitable for miniaturisation and large-scale production using inexpensive technology; (b) they do not require any reference electrode; (c) transducers are not light sensitive; (d) the driving voltage can be sufficiently low to decrease significantly the power consumption and (e) large spectrum of compounds of different nature can be determined on the basis of various reactions and mechanisms, but this can also be considered as a disadvantage since it lowers the selectivity of the sensor.
The first steps in the development of these biosensors are presented in this paper such as the possibility to follow APA analysis with conductometric biosensors as well as the first optimisations and toxicity assays. These results are compared with measurements by bioassays.
Section snippets
Materials
The C. vulgaris strain (CCAP 211/12) was purchased from the culture collection of algae and protozoa at Cumbria, UK. The axenic algal strain was grown in the culture medium and under conditions described by the International Organisation for Standardisation (ISO, 8692, 1989). The APA measurements require a 21-day-long starvation period in the culture medium without phosphate (Fitzgerald and Nelson, 1966). The concentration in algae cultures was ≈3×107 to 5×107 cells/ml.
Bovine serum albumin (BSA)
APA measurements
The first step of this work was to confirm the possibility to detect APA with algae conductometric biosensors. The principle of operation of such biosensor is based on the following reaction:The two most widely used substrates for assays are p-nitrophenyl phosphate and 4-methylumbelliferyl phosphate, leading to the release of p-nitrophenol and 4-methylumbelliferone, respectively. As APA can be measured by bioassays using MUP, comparisons with biosensors could
Conclusion
The conductometric biosensor presented in this paper uses a new type of membranes containing immobilised whole cells.
In aquatic ecosystems, algae are the first trophic level: any disturbances could be reported to upper levels. This is one of the main interest of this work since it gave the opportunity to follow the response of a living organism to a pollutant, namely cadmium ions. An organism possesses different self-defence mechanisms which can reduce the theoretical effects of pollutants.
Acknowledgements
Part of this work was supported by France-Ukraine Bilateral Program ‘Dnipro’.
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