Capacitive sensor for the allatostatin direct immunoassay
Introduction
This study investigates the possibility of direct assay of the allatostatin hormone involved in insect metamorphosis. The allatostatins are a class of peptides that inhibit the biosynthesis of the juvenile hormone in some insect species [1]. Subsequently, the juvenile hormone concentration decreases and the insect starts ageing, i.e. it is not young anymore. The allatostatins are released in the blood, where they can be measured and can give an indication about the developmental stage of the individual. In this context, the design of an allatostatin capacitive immunosensor was considered in order to replace the more expensive and time consuming radio labelled immunoassay (RIA) or enzyme linked immunosorbent assay (ELISA).
Theoretical models, derived in order to describe the antigen binding to the surface immobilised antibody [2], proved that direct measurements over the micromolar to nanomolar concentration range are feasible. The fundamental limitations of the sensor response arise from the equilibrium and kinetic characteristics of the binding reaction and from mass transport constraints.
Out of the possible direct immunoassay principles, i.e. piezoelectrical 3, 4, 5, 6, optical 7, 8, 9, 10and electrochemical 11, 12, 13, 14, 15, 16, 17, 18, 19, the electrochemical methods remain the most attractive given the simplicity of both:
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the solid state devices [20]
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the required measuring circuits
Moreover, limits of detection down to 1 ng ml−1 13, 16make these methods comparable with the ELISA tests.
Section snippets
Theoretical considerations
The direct capacitive immunosensor was introduced by Newman: when an antibody is immobilised on the surface of an electrode pair, it introduces a capacitive component in the impedimetric response of the sensor [14]. This originates from the low dielectric permittivity of the immobilised macromolecule. Further, when the pair of modified electrodes is exposed to the specific antigen, an immunocomplex will be formed because of the recognition process that takes place. Consequently, the dielectric
Solid state device
The device is realised in planar technology. Sputtered Ti/Pt (100 nm/200 nm) electrodes are patterned with a lift off process on a 1 μm thick SiO2 layer [20]. The Pt electrodes of 2×7 mm and 2 mm spaced were covered first with a Ti film and then with a TiO2 sheet, sputtered from a Ti target in oxygen plasma. The structures were cut, glued on PCB strips and the bonding pads were insulated with Grace Amicon H54 paste. The Ti/TiO2 layer insures the complete absence of pinholes.
Immunospecies
The antibody was
Characterization of the solid state device
The structure characterization was performed with a Hewlett Packard high precision RLC meter model hp 4824A, by measuring the series capacitance over a frequency expanding from 20 Hz up to 1 MHz. The signal level was of 20 mVRMS.
The very flat pattern presented in Fig. 3 illustrates a homogeneous, uniform, defectless TiO2 layer. The high capacity value allows accurate measurements and guaranties the possibility of subsequent miniaturization of the device.
After different steps of the
Discussion
The behaviour highlighted in Fig. 5(b) originates from the used measurement set-up: measurements were taken in glass tubes containing at least 1 ml solution, stirred magnetically. In such cases, the experiment can be arranged in the class of convective systems.
Subsequently, the `Nernst diffusion layer' treatment can be considered and the surface flux j|x=0 can be written as:where D is the diffusion coefficient of the immunospecies to be detected, c is the
Conclusions
In this study the feasibility of a capacitive immunosensor based on TiO2 insulated electrodes was proven. The sensitivity of such a structure was shown to be one of the best from those cited in the literature 14, 15, 16, 18. This improvement originates from the good dielectric properties of the titanium dioxide which is for the first time used in a capacitive immunosensor. The higher sensitivity allows important device miniaturization, which is a crucial requirement for an immunostructure.
The
Acknowledgements
The authors are grateful to Stephen S. Tobe for generously supplying the Dip-AST-2 antibody and to Peter Van Gerwen for the help with the ESCA investigations. Dirk Veeraert and Anca Varlan also thank the Flemish I.W.T. and respectively the Katholieke Universiteit Leuven for providing a Ph.D. scholarship.
Anca Roxana Varlan (Vartires) graduated at the Electrical Engineering Department of the Polytechnical Institute of Bucharest, Romania in 1985 and obtained a Master Degree from K.U.Leuven, Belgium in 1992. In 1996 she earned the Ph.D. Degree in Applied Sciences (Electrical Engineering) at K.U. Leuven. Her main interest focuses on the development of microelectrochemical sensors and impedance measurements for biological applications.
Jan Suls got his Masters Degree in Chemistry from the K.U. Leuven
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Anca Roxana Varlan (Vartires) graduated at the Electrical Engineering Department of the Polytechnical Institute of Bucharest, Romania in 1985 and obtained a Master Degree from K.U.Leuven, Belgium in 1992. In 1996 she earned the Ph.D. Degree in Applied Sciences (Electrical Engineering) at K.U. Leuven. Her main interest focuses on the development of microelectrochemical sensors and impedance measurements for biological applications.
Jan Suls got his Masters Degree in Chemistry from the K.U. Leuven in 1979. Since 1985 he is a delegated scientist from the Interuniversity Micro-Electronics Center (IMEC) at the ESAT laboratory of K.U. Leuven. His research interest are in technologies for planar electrochemical biosensors.
Dirk Veelaert obtained his diploma of Engineer in the Chemical and Agricultural Industries in 1992. He earned his Ph.D. Degree in Applied Biological Sciences at the K.U. Leuven in 1996. He worked on neuropeptides and other brain factors controlling development in insects. At the moment he is working at the Biozentrum of the University of Basel on genes controlling the development of the eyes.
Willy Sansen had been a full professor since 1981 at the ESAT laboratory of K.U. Leuven. During the period 1984–1990 he was head of the Electrical Engineering Department. He has been involved in design automation and in analogue integrated circuit design for telecom, consumer electronics, medical applications and sensors. He has supervised 30 Ph.D. theses and has been the author and co-author of four books and 300 papers in international journals and conference proceedings.
Arnold De Loof had been a full professor since 1977 at K.U. Leuven. Since 1981 he has been head of the Animal Physiology Department. Author or co-author of about 200 papers pubished in refereed international journals, he is involved in research regarding insect physiology, comparative endocrinology, developmental physiology.