chip biosensor by employing various insect species

Sensors and Actuators B 78 (2001) 1±5

Extending the capabilities of an antenna/chip biosensor by employing various insect species P. Schrotha, M.J. SchoÈninga,b,*, H. LuÈtha, B. Weiûbeckerc, H.E. Hummelc, S. SchuÈtzc a

Institute of Thin Film and Ion Technology, Research Centre JuÈlich GmbH, 52425 JuÈlich, Germany b University of Applied Sciences Aachen, Ginsterweg 1, 52428 JuÈlich, Germany c Institute of Phytopathology and Applied Zoology, Justus-Liebig-University Gieûen, 35390 Gieûen, Germany

Abstract Because of their remarkable sensory abilities, insect antennae are very suitable for the construction of highly sensitive biosensors. Odour concentrations down to the low ppb range can easily be detected by means of an antenna/®eld-effect transistor (FET) junction. In this work, we present measurements performed with different kinds of beetles, namely the Colorado potato beetle (Leptinotarsa decemlineata Say) and the steelblue jewel beetle (Phaenops cyanea). Their ability to speci®cally detect organic odour molecules, like 1-octen or guaiacol, at concentrations down to the ppb range can lead to interesting applications like the detection of different kinds of ®res at an early stage. # 2001 Elsevier Science B.V. All rights reserved. Keywords: BioFET; Biosensor; Insect antenna; Potato beetle; Steelblue jewel beetle; Odour detection

1. Introduction The remarkable sensory abilities of insects have attracted the interest of biologists since many years. Among about 1,000,000 known insect species, approximately 250 have been investigated and several hundred detectable organic odour substances have been ®led. During millions of years of evolution, most insect species have become specialists in smelling speci®c substances, mostly pheromones or host plant odours [1]. Different insect species have found their respective ecological niches, which are in many cases correlated to their odour spectra. The sensitivity of an insect for a certain substance can almost always be explained by its biological behaviour. For example, the Colorado potato beetle (Leptinotarsa decemlineata Say) needs the potato plant as its host. Therefore, this beetle has the ability to detect potato plants by their emitted odour molecules over a great distance. Recently, insect antennae have been used for the ®rst time in order to assemble a new kind of biosensor, a so-called biologically sensitive ®eld-effect transistor (BioFET), with a high sensitivity and selectivity for certain odours [2,3]. Here, the antenna of a potato beetle was used as a receptor and a *

Corresponding author. Tel.: ‡49-2461-612973; fax: ‡49-2461-612940. E-mail address: [email protected] (M.J. SchoÈning).

®eld-effect transistor (FET) as a transducer. Upon detecting the respective odour molecules, the antenna develops electrical dipoles along the sensilla, the small antenna organs where the odour detection takes place. These dipoles add to a sum dipole across the whole antenna. After coupling the antenna to the gate of a FET by means of an electrolyte interface Ð yielding a bioelectronic interface between the antenna and the FET Ð the antenna signals can be detected and ampli®ed by the transistor. With this kind of sensor, host plant odour concentrations (cis-3-hexen-1-ol) could already be detected down to the low ppb range [3,4]. In this work, we investigate both the sensitivities of the potato beetle (L. decemlineata Say) and the steelblue jewel beetle (Phaenops cyanea) towards guaiacol and 1-octen, two substances that are characteristic for certain kinds of ®res. Subsequently, the possible applications emerging from these experiments will be discussed. 2. Experimental The ®eld-effect transistors were prepared using standard semiconductor technology [5]. Since the transconductance of the transistor depends on the width-to-length ratio of the gate, optimised gate layouts (meander gates or U-shaped gates) were developed for the measurements. About 30 nm thermally grown silicon dioxide as well as 70 nm silicon

0925-4005/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 0 0 5 ( 0 1 ) 0 0 7 8 3 - 3

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P. Schroth et al. / Sensors and Actuators B 78 (2001) 1±5

nitride deposited by means of a plasma-enhanced chemical vapour deposition (PECVD) process were used as gate insulator materials to ensure a electrochemically stable interface between the transistor gate and the electrolyte solution. The potential at the interface silicon nitride/liquid phase is mainly determined by the pH of the solution. In order to obtain a constant potential it is therefore suf®cient to use a buffered electrolyte solution [6]. The drain and source areas of the FET were fabricated by means of photolithographic patterning and subsequent phosphor diffusion. They were contacted by conducting tracks consisting of 50 nm Ti and 100 nm Al. Except for the gates, the transistors and the conducting tracks were passivated by 1 mm of polyimide. Finally, the wafer was cut into single chips which were glued onto printed circuit boards, contacted by ultrasonic bonding and encapsulated with a conventional epoxy resin. For the experimental set-up, there are two possibilities to couple the insect antenna to the gate of the FET (Fig. 1). The ®rst way to realise the bioelectronic interface (Whole-Beetle BioFET) is to immobilise the whole beetle and to dip the tip of the antenna into the electrolyte solution (Fig. 1A). The second way (Isolated-Antenna BioFET) is to remove the antenna from the beetle and to mount it into a home-made antenna holder. Both ends of the antenna are then dipped into the electrolyte solution, establishing the electric connection to a reference electrode on the one side and to the gate of the FET on the other side (Fig. 1B). In both cases one has to assure that a suf®ciently large part of the antenna is exposed to the air and the analyte. During the measurements, a constant stream of air was blown over the antenna in order to eliminate signals from the mechanical receptors. Different concentrations of the respective odour substances were injected into the air stream at de®nite times. Guaiacol was measured between 50 ppt and 500 ppb, 1-octen between 15 and 1500 ppb. Whereas

guaiacol represents an odour substance that is emitted by burning coniferous wood, 1-octen is set free by burning brown coal. For the ®re detection measurements, the smoke was blown over the antenna at de®nite times and the respective change in the drain current was recorded. The FET was operated in the constant-voltage mode (CVM) where the gate voltage VG and the drain-source voltage VDS were adjusted to ®xed values in order to de®ne the working point of the transistor (Fig. 2). Upon smelling the respective odours, a small potential is generated within the insect antenna that is able to modulate the channel resistivity of the FET. This leads to a corresponding change in the drain current ID. Therefore, the drain current was measured at a resistance RM, ampli®ed, ®ltered by a bandpass in order to eliminate noise and drift phenomena, ADconverted and stored by a personal computer. As the sensor signal, the drain current changes contain the information about the measured odour quantities. 3. Results and discussion In Fig. 3, a typical measurement is presented that demonstrates the ability of the potato beetle to detect cis-3-hexen1-ol at concentrations between 1 ppb and 100 ppm. Fig. 3A shows the original drain current measurement with the drain current peaks obtained by applying the various odour concentrations to the antenna. Fig. 3B shows the resulting calibration curve yielding a clear dependence between the peak height of the drain current and the respective odour concentration [4]. In Fig. 4, calibration curves are depicted for two further organic odour compounds. Both guaiacol (Fig. 4A) and 1-octen (Fig. 4B) can be detected at concentrations down to about 100 ppb by the potato beetle-based BioFET. In both cases the calibration curve has a non-linear shape.

Fig. 1. Schematic of the BioFET measuring set-up containing a bioelectronic interface between the intact chemoreceptor and the field-effect transistor. The electrical contact is established via an electrolyte solution: (A) whole-beetle set-up using the complete insect; (B) isolated-antenna set-up using a single antenna.

P. Schroth et al. / Sensors and Actuators B 78 (2001) 1±5

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Fig. 2. Block diagram of the electronic set-up of the BioFET-based measuring device.

In addition, for the jewel beetle the odour substances cis3-hexen-1-ol and guaiacol were investigated (Fig. 5). While cis-3-hexen-1-ol can be measured down to about 1 ppt (Fig. 5A), guaiacol is even detectable at trace concentrations

of about 50 ppt (Fig. 5B). For the jewel beetle, the calibration curves show a nearly linear shape. Comparing the calibration curves of the potato beetle and the jewel beetle, there are two obvious differences, ®rstly,

Fig. 3. Typical sensor response of a potato beetle's antenna towards different concentrations of cis-3-hexen-1-ol: (A) drain current changes of the isolated-antenna BioFET; (B) resulting calibration curve.

Fig. 4. (A) Calibration curve of a potato beetle's antenna for different concentrations of guaiacol between 5 and 500 ppb using an isolatedantenna set-up. (B) Calibration curve of a potato beetle's antenna for different concentrations of 1-octen between 15 and 1500 ppb using an isolated-antenna set-up. For both diagrams, every data point is the mean of six measurements.

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substances like cis-3-hexen-1-ol that are emitted by potato plants over a great distance. The jewel beetle, on the other hand, lays its eggs into burned coniferous wood after a forest ®re. Consequently, it needs to smell wood ®res very sensitively. In order to realise a speci®c application of such an antenna-based BioFET, it is therefore necessary to choose the right beetle species for the respective substance to be determined. A BioFET based on the antenna of a potato beetle is able to detect various ®re-speci®c odours. The steelblue jewel beetle on the other hand could serve to detect speci®c wood-based ®res with unrivalled sensitivity, since it can not detect 1-octen as a marker for coal ®res [8]. To demonstrate the ability of this type of BioFET to detect real smoke, some preliminary measurements were performed with the antenna of a jewel beetle and burning paper and wood, respectively (Fig. 6). For paper, there is a difference between burning paper and smouldering paper (Fig. 6A). Since smouldering paper generates more smoke than burning paper, the sensor signals are signi®cantly higher. In a second experiment, the smoke of burning wood was blown over the antenna from different distances between 10 and 50 cm. A clear dependence can be achieved between the sensor signal and the distance from the ®re to the sensor. Fig. 5. (A) Calibration curve of a jewel beetle's antenna for different concentrations of cis-3-hexen-1-ol between 1 and 100 ppt using an isolated-antenna set-up. (B) Calibration curve of a jewel beetle's antenna for different concentrations of guaiacol between 50 ppt and 5 ppb using an isolated-antenna set-up. For both diagrams, every data point is the mean of six measurements.

the shape of the calibration curves of the jewel beetle is more linear than that of the potato beetle. This shape is mainly in¯uenced by the binding kinetics between the odour molecules and the odour-binding proteins in the antenna and may therefore be different for different insect species with different odour-binding proteins [7]. Secondly, the detection limits of the jewel beetle are generally lower than those of the potato beetle, or, to put it the other way, the jewel beetle signals are higher than those of the potato beetle antenna for the same odour concentrations. There are two possible explanations for this effect. Either the antenna sensitivity for the odour substances is higher or the dipole generation along the antenna is more signi®cant. The latter possibility may be supported by the fact that the shape of the jewel beetle antenna is different from that of the potato beetle. It is much thinner and a little bit longer, so that the orientation of the single dipoles may be stricter in one direction, yielding a larger sum dipole. Regarding the types of odours detectable by different beetle species, one has to take into account that every species has its own ecological niche, correlated with a number of odours that can be detected with an extremely high sensitivity. For the potato beetle, for instance, it is crucial to ®nd its host plant, the potato plant. As a result, it is able to smell

Fig. 6. Fire detection using an isolated-antenna BioFET: (A) the drain current signals of burning paper (BP) can clearly be distinguished from the signals of smouldering paper (SP); (B) the smoke of burning wood was blown towards the antenna from different distances. The respective peaks are marked 10, 30 and 50 cm.

P. Schroth et al. / Sensors and Actuators B 78 (2001) 1±5

Compared to a semiconductor gas sensor that mainly detects H2 and CO [9], a BioFET-based sensor for the detection of ®res has two main advantages, the much higher sensitivity and the better selectivity. However, its main disadvantage has to be seen in its still limited lifetime. Since the BioFET always detects a sum signal of the whole electrical antenna activity, a self-designed measuring set-up in order to detect single substances has been developed [10]. This system utilises the principle of adaptation, if a constant odour concentration is applied to the insect antenna, the signal generated in the antenna will decrease. So it is possible to employ a measuring procedure where the BioFET is ®rst calibrated with de®ned odour concentrations injected into clean air and then, secondly, with odour concentrations injected into ambient air. If the ambient air contains the respective odour in a comparable concentration range, the signals in the second calibration will be decreased due to the adaptation of the insect antenna. Thus, it is possible to selectively detect single odour concentrations even in a complex mixture of different analytes. 4. Conclusions In summary, it could be shown that by using appropriate insect species and their antenna, BioFETs can be designed for the detection of various organic substances. Fig. 7 shows a survey of the insects and odours investigated in this work. Depending on the beetle species, the odour substances can be detected in various concentration ranges yielding different possible applications, for instance in agriculture or early ®re detection. In order to enable a still broader ®eld of applications, a library with different beetle species and their odour detection

Fig. 7. List of detectable odour substances investigated in this work with their respective concentration ranges and possible applications. The number of ``‡'' indicates the sensitivity towards the respective odour in increasing order.

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spectra is just being built up and will be expanded step by step. For instance, the Colorado potato beetle (L. decemlineata) and the steelblue jewel beetle (P. cyanea) are members of the insect groups of Chrysomelidae and Buprestidae, respectively. Both groups contain about thousand different species, each with similar olfactory capabilities but, depending on their vastly differing ecology throughout the world, very different specialisation. Thus, a detailed examination of the olfactory specialisation of these insects can yield biocomponents with good compatibility to the BioFET system and a large variety of speci®cally and sensitively detected analytes. Acknowledgements The authors would like to thank K.-H. Apel, P. Epanya, U. È . Malkoc, M. Marso, A. Riemer, A. Koch, C.-D. Kohl, U Steffen and U. Windirsch for technical support and valuable discussions. This work was supported by the ``Bundesministerium fuÈr Bildung und Forschung'' (Project: Biosensoren auf der Basis intakter Chemorezeptoren). References [1] M.J. SchoÈning, P. Schroth, S. SchuÈtz, The use of insect chemoreceptors for the assembly of biosensors based on semiconductor field-effect transistors, Electroanalysis 12 (2000) 645±652. [2] S. SchuÈtz, M.J. SchoÈning, A. Riemer, B. Weiûbecker, P. KordosÏ, H. LuÈth, H.E. Hummel, Field effect transistor-insect antenna junction, Naturwissenschaften 84 (1997) 86±88. [3] P. Schroth, M.J. SchoÈning, P. Kordos, H. LuÈth, S. SchuÈtz, B. Weiûbecker, H.E. Hummel, Insect-based BioFETs with improved signal characteristics, Biosens. Bioelectron. 14 (1999) 303±308. È . Malkoc, A. Steffen, M. [4] P. Schroth, M.J. SchoÈning, S. SchuÈtz, U Marso, H.E. Hummel, P. Kordos, H. LuÈth, Coupling of insect antennae to field-effect transistors for biochemical sensing, Electrochim. Acta 44 (1999) 3821±3826. [5] M.J. SchoÈning, S. SchuÈtz, P. Schroth, B. Weiûbecker, A. Steffen, P. Kordos, H.E. Hummel, H. LuÈth, A BioFET on the basis of intact insect antennae, Sens. Actuators B 47 (1998) 235±238. [6] D.B. Sattelle, L.M. Hall, J.G. Hildebrand, Receptors for Neurotransmitters, Hormones and Pheromones in Insects, Elsevier, Amsterdam 1980, p. 261. [7] W. Hoppe, W. Lohmann, H. Markl, H. Ziegler, Biophysik, Springer, Berlin, 1982, p. 722. [8] S. SchuÈtz, B. Weiûbecker, H.E. Hummel, K.-H. Apel, H. Schmitz, H. Bleckmann, Insect antenna as a smoke detector, Nature 398 (1999) 298±299. [9] D. Kohl, J. Kelleter, A. Schwarz, H. Petig, H. Laurs, W. Hosak, VGB Kraftwerkstechnik 11 (1997) 928±932. [10] S. SchuÈtz, B. Weiûbecker, U.T. Koch, H.E. Hummel, Detection of volatiles released by deseased potato tubers using a biosensor on the basis of intact insect antennae, Biosens. Bioelectron. 14 (1999) 221±228.