Bula, W. P.

Dimov, N. G.

Gardeniers, J. G.E.

Guerrero, A.

Muñoz, L.

English

We report a complete design, fabrication procedure and functional tests of the first artificial gland based on a micro-machined evaporator. Presented device was used to precise release of chemicals into environment. Very high evaporation rates and temporal modulation of the chemical signal were achieved due to the temperature elevation and the geometry of the device. The ability to precise release of sex pheromones of the Spodoptera littoralis female moth allowed studies of the interactions with the live insects. As such, the presented chemoemitter pioneer a new information emission scheme utilizing the semiochemicals as a data carrier.</p

University of Twente Research Information

ARTIFICIAL GLAND FOR PRECISE RELEASE OF SEMIOCHEMICALSFOR CHEMICAL COMMUNICATIONW.P. Bula1*, N.G. Dimov1, L. Muñoz2, A. Guerrero2, J.G.E. Gardeniers11Mesoscale Chemical Systems Group, MESA+ Institute, University of Twente, Enschede, THE NETHERLANDS2Department of Biological Organic Chemistry, CSIC, Barcelona, SPAINABSTRACTWe report a complete design, fabrication procedure and functional tests of the first artificial gland based on a micro-machined evaporator. Presented device was used to precise release of chemicals into environment. Very high evapora-tion rates and temporal modulation of the chemical signal were achieved due to the temperature elevation and the geo-metry of the device. The ability to precise release of sex pheromones of the Spodoptera littoralis female moth allowedstudies of the interactions with the live insects. As such, the presented chemoemitter pioneer a new information emis-sion scheme utilizing the semiochemicals as a data carrier.KEYWORDS: artificial gland, chemoemitter, infochemistry, micro evaporatorINTRODUCTIONThere is an increasing amount of research dedicated to understanding pheromone biosynthesis pathway and the de-tection of compounds that constitute chemical communication between insects. An additional goal is to develop an ar-tificial communication system based on functional equivalents of biological machinery that allow eusocial insects to ex-change information [1]. The key component of such a system is an artificial gland module that generates validsemiochemical signals – a chemoemitter capable of transforming liquid eluents from bio- and chemical microreactors in-to precise ratios of vapour concentration of volatile compounds (e.g. sex pheromones).EXPERIMENTALThe evaporator consists of a silicon membrane (5 mm x 5 mm x 40 µm) perforated with 37636 micromachined via-holes. Rectangular microfluidic channels deliver the mixture of predefined volatile compounds from two inlets to thereservoir (0.375 µL) located under the membrane (Figures 1-2).Figure 1: Photograph of the evaporator chip (channel sidevisible)Figure 2: Schematic representation of the evaporatorchip cross-sectionThe liquid passes through membrane and evaporates from small droplets that are formed on the outlet of every via-hole (Figure 3). Two thin-film platinum heaters and 4-wire resistive temperature sensor are integrated in evaporator andwork in a PID loop in order to stabilize the temperature with variation of 30 mK. Integrated heaters dissipate up to 8Wof energy elevating the temperature of the membrane up to 250°C.The membrane of the silicon-glass evaporator was formed on the top side of the silicon wafer by anisotropic etchingin KOH solution. On the back side via-holes through the membrane were etched together with deep channels (100 µm)by deep reactive ion etching (BOSCH process) using a mask of photoresist and SiRN. After photoresist removal, theshallow channels and under-membrane cavity were etched to 15 µm with the same process. The silicon wafer was anod-ically bonded to Pyrex glass with powderblasted via-holes and semi-buried tantalum (10 nm) and Pt (200 nm) metalliza-tion layers of heaters and temperature sensor.  The silicon-glass stack was diced into separate chips of 20 mm x 15 mm.978-0-9798064-3-8/µTAS 2010/$20©2010 CBMS 671 14th International Conference onMiniaturized Systems for Chemistry and Life Sciences3 - 7 October 2010, Groningen, The NetherlandsFigure 3: Photograph of the under-membrane reservoirwith through-membrane via-holes of the evaporator withlightly (left) and highly (right) perforated membraneFigure 4: Sequence of images taken during the evapo-ration of liquid sample (dark area) from reservoirthrough perforated membraneThe efficiency of the pheromone evaporation was tested based on the dynamic headspace technique. The evaporatorsetup was adapted so that the pheromone vapour was entrapped inside a Porapak cartridge. A PMMA cover with the gasinlet and outlet was attached via PDMS pad to the membrane side of the chip. The temperature was set to 120°C and asolution of Z,E-9,11-14:OAC (10 µg/µL in hexane) was delivered to the chip by syringe pump at a flow rates in therange from 0.5 to 20 µL/min. The gas flow rate was set to 250 mL/min. The collection time varied from 7.5 to 60 min,then the Porapak cartridge was extracted with the hexane and the amount of pheromone collected relative to the collec-tion time was quantified by gas chromatography.Similarly to the headspace experiments, the evaporation device was adapted for electroantennographic assays. Theoutlet was connected to the tube delivering the stimuli to the antenna extracted from insect. All experiments were im-plemented on the same antennae and as injection flows we used 0.01 µl/min and 0.1 µl/min. For the stimuli, a solution ofZ,E-9,11-14:OAc (0.1 µg/µl in hexane) was used vs. hexane alone (control). For each stimulus, 4 replicates were madeand between the types of stimuli the system was continuously cleaned up of pheromone by passing an air flow.RESULTS AND DISCUSSIONThe evaporation tests were performed for different solvents at various temperatures (Figures 4-6). The higher theflow rate, the higher the number of via-holes through which the liquid penetrates the membrane. The maximum evapo-ration rate was determined by increase of the flow rate to such a value that the upper part of membrane was wetted. Insuch case the evaporation takes place only from the via-hole outlets. In elevated temperature, when the flow rate issmaller than the evaporation rate, the evaporation is limited by the pumping rate of chemical compounds.Figure 5: The influence of the on-chip temperature on themaximum evaporation rate for n-hexane, ethanol andwaterFigure 6: The maximum volumetric evaporation rate ofn-hexane, ethanol and hexane-ethanol solution for varioustemperaturesThe precise composition of a vapour was achieved by modulation of the flow rates of volatile compound solutionsdelivered to the chip (Figure 7). While operating at 120°C the evaporator was capable of delivering into environmentthe major compound (Z9,E11-14:OAc) of S. littoralis sex pheromone blend with an evaporation rate 10000% higherthan from a plain reference surface at room temperature.The resulting data obtained by dynamic headspace technique showed a linear relationship between the evaporationrate and the flow rate (Figure 7). Therefore, it is possible to predict the evaporation rate of the pheromone at differentflow rates.672As long as the temperature is high enough to allow solvent and pheromone co-evaporation, and as long as the phe-romone droplets are not observed on the surface of the membrane, the volumetric evaporation rate equals the volumetricflow rate of the pheromone solution. By knowing the pheromone concentration, the exact amount of compound evapo-rated can be calculated.The electroantennographic (EAG) responses are shown in Figure 8.  The EAG response obtained by using evaporatorwas much higher comparing to the classical setup (Pasteur pipette containing a Whatman filter paper with 10 µl of sti-muli adsorbed). Furthermore, the response obtained to the pheromone was significantly higher than the mechanical re-sponse (hexane). However, no difference was found between the injection of 0.01 µl/min or 0.1 µl/min of pheromone,what suggest that the antenna had reached the saturation level.Figure 7: Evaporation rate of a solution of ZE-9,11-14:OAc in hexane versus the injection flow rate. Data ob-tained from GC-MS measurements of the pheromone va-pour adsorbed in Porapack columnFigure 6: Electroantennographic responses of S. littoralismale antennae exposed to stimuli generated by a Pasteurpipette and the micromachined  evaporator.Wind tunnel experiments showed that male S. littoralis responded to the released vapour. Due to very high concen-tration of the pheromone causing the antenna saturation, insects have experienced orientation disorder, however ele-ments of behavioural response were observed.Since the evaporation rate is limited by the liquid phase delivery, the composition of the vapours can be controlled bychanging the flow rates of the pheromones pumped into the evaporator. Therefore, the evaporator connected to theoreti-cally unlimited number of microreactors synthesizing the pheromone intermediates, allows the functional mimicking ofany biosynthetic pathway of various species that utilize airborne chemical communication.The decomposition of the pheromone was not observed during the tests. The inert and robust design of the evapora-tor, together with the cleaning procedure ability, allowed the device to be operational for weeks of experimentsCONCLUSIONWe have developed the prototype of micromachined evaporator for release of the chemical compound into environ-ment. Proposed device mimics the functionality of a biological machinery that allows a female of Spodoptera littoralisto release pheromone. We envision that this kind of artificial gland leads to development of an artificial communicationsystem based on chemical signaling. Presented device allows precise dosing of semiochemicals, which is crucial for in-formation encoding based on relative ratios between compounds. Moreover, the ease of varying the pheromone flux inwide range makes the device not only functionally compatible with biological systems, but also, together with temporalmodulation of pheromone blend composition, increases the bandwidth of potential chemical communication channels.ACKNOWLEDGEMENTSThis work is supported by the EC Framework 6 IST Programme under iCHEM project (Biosynthetic InfochemicalCommunication FP6-032275).REFERENCES[1] M. Cole, J.W. Gardner, Z. Rácz, S. Pathak, A. Guerrero, L. Muñoz, G. Carot, T.C. Pearce, J. Challiss, D. Markov-ic, B.S. Hansson, S. Olsson, L. Kübler, J.G.E. Gardeniers, N. Dimov, and W. Bula, Biomimetic insect infochemicalcommunication system, Proc. The 8th Annual IEEE Conference on Sensors, Christchurch, New Zealand, (2009).CONTACT*W.P. Bula, tel: +31 53 489 3071, w.p.bula@utwente.nl673

Artificial gland for precise release of semiochemicals for chemical communication

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Artificial gland for precise release of semiochemicals for chemical communication

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