A capacitive field-effect sensor for the direct determination of organophosphorus pesticides
Introduction
Synthetic organophosphorus compounds—among the most toxic substances known—are used as pesticides or insecticides in agriculture and chemical warfare agents. Biosensors to detect these neurotoxins, has been an actively researched area. The majority of biosensors to date is based on the inhibition of acetylcholinesterase or butyrylcholinesterase integrated with electrochemical (potentiometric and amperometric) and optical transducers [1], [2], [3]. Although sensitive, these biosensors suffer from several limitations: poor selectivity, multi-step indirect determination, and irreversible inhibition by many compounds, to name a few. Recently, we have demonstrated the application of another enzyme, generically termed organophosphorus hydrolase (OPH), as the biological recognition element for various biosensors. This enzyme catalyses the hydrolysis of organophosphates (OPs) after releasing products that can be monitored. We have integrated this enzyme with a number of different transducers to develop biosensors that allow rapid, selective, sensitive and on-line determination of OPs [3]. Taking advantage of the fact that hydrolysis of OPs by the OPH releases proton(s), we reported a simple potentiometric biosensor in which OPH was integrated with a glass pH electrode [4]. In order to miniaturise the potentiometric biosensor, OPH was immobilised at the gate of a pH-sensitive field-effect transistor (ISFET) [5]. However, the application of miniaturised ISFET-type sensors leads to several disadvantages: instability of the required passivation layer when in permanent contact with the test sample (or electrolyte), high costs of fabrication due to photolithographical process steps, etc.
To overcome these problems, we suggest the application of a simple capacitive electrolyte–insulator–silicon (EIS) field-effect structure as transducer of an OPH-based biosensor. The layer set-up of this EIS sensor corresponds to the gate region of an ISFET, however, due to the missing photolithographic process steps, no additional passivation and encapsulation layer of the sensing area is necessary. The sensor can be easily mounted in a home-made Plexiglas cell, e.g. sealed by an O-ring. Thus, this transducer structure possesses a higher stability in the long-term than ISFET-based transducer structures and is much more cheaper in sensor preparation [6].
In this paper, a novel EIS-based sensor for the direct determination of organophosphorus pesticides by the enzyme OPH is presented. For the transducer structure, three alternative pH-sensitive materials Ta2O5, Al2O3 and Si3N4 have been used. Whereas the Ta2O5 and Al2O3 layers are grown by means of pulsed laser deposition (PLD), Si3N4 has been deposited by low pressure chemical vapour deposition (LPCVD) onto a basic structure of Al/p-Si/SiO2. To achieve the capacitive EIS biosensor, OPH has been immobilised by three different strategies on top of the pH-sensitive layer structure: (1) by covalent coupling via glutaraldehyde; (2) by cross-linking with nafion; and (3) by an adsorptive technique. Depending on the immobilisation procedure used and the pH-sensitive material applied, typical sensor characteristics, like pH sensitivity of the transducer material before and after the enzyme immobilisation, the biosensor performance towards paraoxon including the linear concentration range, the reversibility of the biosensor signal, the response time as well as the detection limit and the biosensor stability in the long-term have been investigated; the selectivity to various pesticides will be discussed.
Section snippets
Materials and processing
The different EIS structures of the layer sequences Al/p-Si/SiO2/Al2O3, Al/p-Si/SiO2/Ta2O5 or Al/p-Si/SiO2/Si3N4 were fabricated from p-type Si (18–24 Ω cm, Wacker Chemitronic) with <1 0 0> orientation. A SiO2 layer of 30 nm has been grown by means of thermal oxidation. For the preparation of the pH-sensitive materials Al2O3 and Ta2O5 (thickness: 30–50 nm), respectively, the PLD process has been employed, whereas the LPCVD technique was used to deposit the Si3N4 layer. The Al rear contact has been
Results and discussion
Fig. 3 shows a typical C/V curve of a capacitive field-effect sensor with Si3N4 as pH-sensitive material in buffer solution, pH 7, before (full line) and after modification of the Si3N4 surface by OPH enzyme deposited by cross-linking (open squares). The maximum capacitance in the accumulation range is about 45 nF. Both curves indicate a nearly identical behaviour in terms of signal shape and capacitance values. The slight deviations are probably due to small variations in the additional
Conclusions
For future research studies, the developed biosensor should be integrated in a flow-injection analysis system to demonstrate its availability for real sample monitoring. The combination of a potentiometric with an amperometric sensor, for example, will improve the sensitivity and selectivity characteristics. Both sensors can be integrated as hybrid on a single chip. In such a dual biosensor chip, the amperometric detection principle is relied on the amperometric determination of para
Acknowledgements
The authors gratefully thank F. Johnen and J. Schubert for technical support. Part of the work has been funded by the Ministerium für Schule, Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the US Environmental Protection Agency (R823663-01-0, R82816001-0), the US Department of Agriculture (99-35102-8600) and the National Science Foundation (BES 9731513).
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Co-corresponding author.