Development of a fluidic sensor for the detection of herbicides using thylakoid preparations immobilised on magnetic beads to aid regenerability

Abstract

Following the industrial revolution and advances in chemical science, the pollution of the environment with trace organic pollutants has been steadily increasing, which is of concern, due to their effect on the environmental and human health. Tighter legislation that has been introduced in order to minimise the release of harmful pollutants has led to the initiation of monitoring programmes. For example, drinking water suppliers are obliged to systematically monitor drinking water supplied for human consumption for a large range of pollutants. The same applies for waste water treatment facilities. The well-established standard methods of environmental waters analysis require sampling and transportation of samples to the laboratory for detailed measurements. Therefore, the timescale from sampling to reporting is not ideal, as a considerable lag occurs. There is therefore the potential for the use of in situ methods that overcome this issue. As these do not currently exist, a need to address this is identified. Biosensors are sensing devices that rely on a biologically-derived component as an integral part of their detection mechanism. Biosensors that respond to pollutants could be used for rapid, low cost, field-based pre-screening of water samples. Herbicides are considered to be the most important class of pesticides used in the E.U. Herbicides can be highly toxic for human and animal health, and increase in the application of herbicides in agriculture during recent decades has resulted in immense pollution of both soil and water. About half of the herbicides used at present in agriculture inhibit the light reactions in photosynthesis, mostly by targeting the Photosystem II (PSII) complex. A method of detecting certain classes of herbicides is therefore proposed; the photosynthesis-inhibiting herbicides act by binding to PS II, a chlorophyll– protein complex which plays a vital role in photosynthesis, located in the thylakoid membrane of algae, cyanobacteria and higher plants. The inhibition of PS II causes a reduced photoinduced production of hydrogen peroxide, which can be measured by the HRP-mediated luminol chemiluminescence reaction. The design and development of a fluidic sensor unit for the detection of such herbicides, based upon their inhibition of the hydrogen peroxide production, will employ the use of superparamagnetic beads in order to address issues of reuse and regenerability. The illumination-dependent production of hydrogen peroxide by isolated thylakoids, and its inhibition by herbicides in a concentration-dependent manner, were achieved and measured with the HRP-mediated chemiluminescence reaction with luminol in a cuvette, batch format, allowing for the detection of herbicides down to 6.0 x 10-09.The integration of the above reactions has been achieved by designing and constructing a fluidic unit that combines the herbicide-dependent production and the detection of hydrogen peroxide in a single fluidic assay by combining all the individual steps in a compact, portable format, with both HRP and thylakoids covalently coupled on superparamagnetic beads. This addresses issues of regenerability, as the beads are introduced, used and discarded following a measurement, controlled only by magnetic and flow forces. Herbicide detection was achieved to a lower LOD of 5.5 x 10-10 M. The concept development, design and construction of the fluidic unit, as well as results of the detection of herbicides with the batch assay method has been published, in a paper by the author (Talanta, 2008, vol. 77, no. 1, pp. 42-47), Considerable progress has therefore been made towards developing a system that would be suitable for automated, field deployment applications for the detection of the most frequently used classes of herbicides; the lower LOD however is not within the stringent legislated maximum permissible limits set for herbicides measured in water, in European waters. An immediate step forward would be to achieve the required lower LOD, with the unit's development into a prototype instrument that can be field deployed being the further goal.