Theo Mota

Ana C R Vitta

Alicia N Lorenzo-Figueiras

Carla P Barezani

Carlos L Zani

Claudio R Lazzari

Lile ´ia Diotaiuti

Lynne Jeffares

Bjo ¨rn

Bohman 1¤

Marcelo G Lorenzo
email: marcelo@cpqrr.fiocruz.br

A Multi-species Bait for Chagas Disease Vectors

Background: Triatomine bugs are the insect vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. These insects are known to aggregate inside shelters during daylight hours and it has been demonstrated that within shelters, the aggregation is induced by volatiles emitted from bug feces. These signals promote inter-species aggregation among most species studied, but the chemical composition is unknown.

Methodology/Principal Findings: In the present work, feces from larvae of the three species were obtained and volatile compounds were identified by solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). We identified five compounds, all present in feces of all of the three species: Triatoma infestans, Panstrongylus megistus and Triatoma brasiliensis. These substances were tested for attractivity and ability to recruit insects into shelters. Behaviorally active doses of the five substances were obtained for all three triatomine species. The bugs were significantly attracted to shelters baited with blends of 160 ng or 1.6 mg of each substance.

Conclusions/Significance: Common compounds were found in the feces of vectors of Chagas disease that actively recruited insects into shelters, which suggests that this blend of compounds could be used for the development of baits for early detection of reinfestation with triatomine bugs.

Introduction

The flagellate parasite Trypanosoma cruzi (Chagas), the etiological agent of Chagas disease, is transmitted to humans by vectors of the subfamily Triatominae. In part as a result of the distribution of vectors, Chagas disease occurs exclusively in Latin America where it is estimated that 90 million people are at risk of transmission, while 12 million are already infected [START_REF] Coura | Epidemiology, control and surveillance of Chagas disease: 100 years after its discovery[END_REF]. The primary vector in the Southern Cone of South America is Triatoma infestans [START_REF] Zeledo ´n | Chagas disease: An ecological appraisal with special emphasis on its insect vectors[END_REF][START_REF] Cortez | New insights on the spread of Triatoma infestans from Bolivia-Implications for Chagas disease emergence in the Southern Cone[END_REF], however other species such as Panstrongylus megistus and Triatoma brasiliensis, play an important role in transmission in some regions of Brazil [START_REF] Patterson | On the genus Panstrongylus Berg 1879: Evolution, ecology and epidemiological significance[END_REF][START_REF] Costa | The epidemiologic importance of Triatoma brasiliensis as a Chagas disease vector in Brazil: a revision of domiciliary captures during 1993-1999[END_REF]. Control of the sylvatic vector species at domiciliary ecotopes is particularly difficult as they readily invade households from wild ecotopes where they cannot be controlled with available methods [START_REF] Patterson | On the genus Panstrongylus Berg 1879: Evolution, ecology and epidemiological significance[END_REF][START_REF] Costa | The epidemiologic importance of Triatoma brasiliensis as a Chagas disease vector in Brazil: a revision of domiciliary captures during 1993-1999[END_REF].

Triatomine bugs are obligate haematophagous insects, which feed mostly on the blood of birds and mammals. During daylight hours, these insects are usually found aggregated inside protected shelters which they leave after dusk in search of food [START_REF] Lorenzo | Activity pattern in relation to refuge exploitation and feeding in Triatoma infestans (Hemiptera: Reduviidae)[END_REF]. Triatomine aggregation behavior inside shelters is well documented and is mediated by chemical signals and thigmotaxis [START_REF] Lorenzo | The spatial pattern of defaecation in Triatoma infestans and the role of faeces as a chemical mark of the refuge[END_REF]. Previously two chemical signals have been implicated in the aggregation of T. infestans inside the shelters: one released from feces [START_REF] Lorenzo | The spatial pattern of defaecation in Triatoma infestans and the role of faeces as a chemical mark of the refuge[END_REF][START_REF] Schofield | Assembly pheromone of Triatoma infestans and Rhodnius prolixus nymphs (Hemiptera-Reduviidae)[END_REF][START_REF] Figueiras | Aggregation in the hematophagous bug Triatoma infestans: Chemical signals and temporal pattern[END_REF] and another present in their cuticle [START_REF] Figueiras | Aggregation in the haematophagous bug Triatoma infestans: A novel assembling factor[END_REF], however no definitive chemical identifications were carried out.

Pheromones are substances used by organisms to transfer information between two or more members of the same species. These can be single chemical compounds or blends of several components. To date, the pheromone blend emitted by triatomine feces has not been fully described for any species. Aggregation mediated by a fecal pheromone was demonstrated in several triatomine species, including P. megistus and T. brasiliensis [START_REF] Cruz-Lopez | Aggregation pheromone in five species of Triatominae (Hemiptera: Reduviidae)[END_REF][START_REF] Figueiras | Aggregation behaviour and interspecific responses in Rhodnius prolixus Stal[END_REF][START_REF] Figueiras | Aggregation behaviour and interspecific responses in three species of triatominae[END_REF][START_REF] Pires | Aggregation behaviour in Panstrongylus megistus and Triatoma infestans: inter and intraspecific responses[END_REF][START_REF] Vitta | Aggregation mediated by faeces and footprints in Triatoma pseudomaculata (Heteroptera : Reduviidae), a Chagas disease vector[END_REF][START_REF] Vitta | The use of aggregation signals by Triatoma brasiliensis (Heteroptera : Reduviidae)[END_REF]. T. infestans deposit their feces in and around shelters and the volatiles emitted from fecal depositions act as chemical landmarks helping the bugs to locate their refuges [START_REF] Lorenzo | The spatial pattern of defaecation in Triatoma infestans and the role of faeces as a chemical mark of the refuge[END_REF]. It has been demonstrated that only dry feces of T. infestans promote aggregation, while fresh feces induces rejection [START_REF] Figueiras | Aggregation in the hematophagous bug Triatoma infestans: Chemical signals and temporal pattern[END_REF]. Feces become attractive three hours after being deposited and attract bugs for up to 10 days [START_REF] Figueiras | Temporal change of the aggregation response in Triatoma infestans[END_REF]. Triatoma infestans, Rhodnius prolixus and Triatoma mazzoti showed changes in their responses to the fecal aggregation signal depending on their nutritional status [START_REF] Schofield | Assembly pheromone of Triatoma infestans and Rhodnius prolixus nymphs (Hemiptera-Reduviidae)[END_REF][START_REF] Figueiras | Temporal change of the aggregation response in Triatoma infestans[END_REF][START_REF] Ondarza | Evidence for the presence of sex and aggregation pheromones from Triatoma mazzottii (Hemiptera: Reduviidae)[END_REF]. In fact, recently fed T. infestans do not aggregate in response to this signal until 8-10 hours after feeding [START_REF] Figueiras | Temporal change of the aggregation response in Triatoma infestans[END_REF].

Four compounds were previously identified in polar solvent extracts of feces of T. infestans and T. mazzottii [START_REF] Lopez | Chemical investigation of aggregation behavior of Triatoma bugs (Hemiptera: Reduviidae)[END_REF]; however no behavioral response was reported. Subsequently it was demonstrated that adult females and fifth instar larvae of T. infestans were attracted to blends of the fecal compounds 4-methylquinazoline and 2,4-dimethylquinazoline [START_REF] Alzogaray | Behavioural Response of Triatoma infestans (Klug) (Hemiptera: Reduviidae) to Quinazolines[END_REF]. In addition it has been reported that ammonia from humidified feces attracts larvae in bioassays [START_REF] Taneja | Ammonia attracts the haematophagous bug Triatoma infestans: behavioural and neurophysiological data on nymphs[END_REF]. To the best of our knowledge no previous studies have demonstrated aggregation behavior in response to synthetic compounds.

Several authors have reported the occurrence of cross-aggregation responses to feces in diverse triatomine species [START_REF] Cruz-Lopez | Aggregation pheromone in five species of Triatominae (Hemiptera: Reduviidae)[END_REF][START_REF] Figueiras | Aggregation behaviour and interspecific responses in Rhodnius prolixus Stal[END_REF][START_REF] Figueiras | Aggregation behaviour and interspecific responses in three species of triatominae[END_REF][START_REF] Pires | Aggregation behaviour in Panstrongylus megistus and Triatoma infestans: inter and intraspecific responses[END_REF][START_REF] Vitta | Aggregation mediated by faeces and footprints in Triatoma pseudomaculata (Heteroptera : Reduviidae), a Chagas disease vector[END_REF][START_REF] Vitta | The use of aggregation signals by Triatoma brasiliensis (Heteroptera : Reduviidae)[END_REF]. The characterization of a common aggregation signal may allow for the development of chemical baits for monitoring multiple vector species.

In the present report, we first aimed to identify common and readily obtainable compounds that promote cross species aggregation in triatomines. For this, we identified volatile compounds present in the feces of T. infestans, P. megistus and T. brasiliensis. The behavior-modifying capacity of the volatiles common to all species was subsequently tested with larvae of each of the three species. Finally, we evaluated the potential of artificial shelters baited with a blend of these fecal volatiles for promoting the aggregation of larvae of each species. We show that a synthetic blend of substances is capable of recruiting bugs into shelters, mimicking the effects of the natural aggregation signals.

Materials and Methods

Insects

Panstrongylus megistus and T. infestans colonies originated from insects captured at domestic and peridomestic refuges in Minas Gerais state, while that of T. brasiliensis came from insects from similar ecotopes in Ceara ´state, Brazil. These colonies were kept for at least ten years in a rearing chamber (4.561.6 mt) with controlled temperature and a 12:12 hour light:dark illumination cycle provided by artificial lights (4 fluorescent lamps, cold white light, 6400K, 40W).

As previous reports have shown that all developmental stages of triatomines make use of fecal aggregation pheromones [START_REF] Figueiras | Aggregation in the hematophagous bug Triatoma infestans: Chemical signals and temporal pattern[END_REF][START_REF] Cruz-Lopez | Aggregation pheromone in five species of Triatominae (Hemiptera: Reduviidae)[END_REF], we chose to use immature bugs both for our chemical and behavioral experiments because they are readily available. Triatomine larvae also have the additional benefit of not emitting alarm or sexual pheromones which could interfere in case experiments were performed with adult insects [START_REF] Manrique | Chemical communication in chagas disease vectors. Source, identity, and potential function of volatiles released by the metasternal and brindley's glands of Triatoma infestans adults[END_REF].

Identification of volatile substances from samples of feces

Feces from third and fourth instar larvae of T. infestans, P. megistus and T. brasiliensis were collected separately in 2 ml glass vials (12632 mm standard autosampler vials, Sigma-Aldrich) by gently pressing the abdomens of bugs with forceps. A solid phase microextraction (SPME) fiber (PDMS/DVB Stableflex, 65 mm, Supelco, Sigma-Aldrich) was exposed to the headspace of the samples for 30 min at room temperature prior to analysis by gas chromatography with mass-spectrometric detection (GC-MS Shimadzu 17A coupled to a Shimadzu 5050A). The desorption time in the injection port of the GC was 1 min. Helium was used as carrier gas (30 cm/s). GC injector and transfer line temperatures were 240uC and 250uC, respectively. The ionization energy was 70 eV. The oven program was 80uC for 1 min and 5uC/min to 240uC using a SupelcoWax-10 column (30 m60.25 mm i.d.60.25 mm film; Supelco, USA). Tentative identification of volatiles was based on the comparison of retention indices and mass spectra with data from the literature and a spectral library (NIST-02). All tentative identifications were confirmed by coinjections with authentic standards.

Samples of volatiles of T. infestans and P. megistus were first obtained three hours after the collection of feces (i.e., from fresh feces) and afterwards, every 24 hours during the subsequent five days (dry feces). The samples of T. brasiliensis were obtained during days 0 (fresh feces), 1, 3 and 5 (dry feces). In all cases, samples of feces were collected from bugs that had been fed one week earlier.

The vials with samples of feces were left open in a closed room during the study, with each species sampled separately. The relative abundance of each compound was determined using the area under the peak of the chromatogram. Empty vials without feces, placed in the same room, were used as blanks.

Behavioral responses to common compounds identified in feces

The common substances identified in samples of feces of T. infestans, P. megistus and T. brasiliensis were selected for behavioral tests. Standards of acetamide (Fluka), 2,3-butanediol (Supelco), acetic acid (Fluka), 3-methylbutyric acid and hexanoic acid (Sigma-Aldrich) were at least 98% pure. Individual compounds were tested with each vector species in decadic steps in doseresponse assays ranging from 1 pg to 100 mg. Solutions of 2,3butanediol, acetic acid, 3-methylbutyric acid and hexanoic acid were prepared in dichloromethane (Nanograde, Mallinckrodt) and acetamide in distilled water.

All experiments were made at 2562uC and 65610% R.H. using a circular glass arena (14 cm ) where the bottom was lined with filter paper. Two pieces of flat filter paper (161.5 cm) were placed on opposite halves of the arena, one impregnated with the test solution (test) and the other impregnated with pure solvent (control). These two papers were positioned 1 cm from the edge of the arena and separated by approximately 10 cm [START_REF] Figueiras | Aggregation in the hematophagous bug Triatoma infestans: Chemical signals and temporal pattern[END_REF][START_REF] Figueiras | Aggregation behaviour and interspecific responses in three species of triatominae[END_REF][START_REF] Pires | Aggregation behaviour in Panstrongylus megistus and Triatoma infestans: inter and intraspecific responses[END_REF].

Two control series of tests (32 assays per series) were performed for each species. i) two pieces of clean filter paper on opposite sides of the arena, in order to test for environmental asymmetries. ii) one clean filter paper against a filter paper impregnated with dichloromethane or distilled water, in order to test for solvent

Author Summary

Chagas disease is a parasitic infection affecting approximately 12 million people, and is considered to be one of the most severe burdens for public health in Latin America. Control of the disease is based on attempted elimination of domestic populations of triatomine bugs, the insects transmitting the disease to humans, by means of insecticide spraying. Currently, vigilance programs monitoring triatomine reinfestation processes in houses are performed by manual search for bugs. Effective and sustainable new methods allowing continuous monitoring of domestic triatomine populations are required. Based on the fact that the insects hide in dark refuges that are marked by volatile signals emitted in their feces, we screened the feces of three species for volatile compounds common to these prominent vectors. The potential for these odors to promote triatomine aggregation was evaluated and we present evidence that a synthetic blend of these substances is capable of recruiting bugs into shelters, mimicking the natural pheromone. This blend may be used to develop a bait to monitor triatomine reinfestation processes in a similar manner as is used commonly for the monitoring of agricultural pests. effects on behavior. For each species, we performed 32 tests with each dose of the five compounds tested. In these experiments, individual insects, third instar larvae starved for 1164 days post ecdysis, were used per test and discarded afterwards. All insects were tested during the light phase of their daily cycle, maximizing their tendency to respond to chemical signals related to shelter search. The results were analyzed by means of a binomial test.

An individual insect was placed in the center of the arena using a small inverted plastic container that avoided it to climb due to its smooth surface. After 10 minutes, the insect was released by means of a string that allowed raising the container from a distance without disturbing it. One hour later, the position of the insect was recorded. Triatomines are typically found motionless when displaying aggregation inside shelters [START_REF] Lorenzo | The spatial pattern of defaecation in Triatoma infestans and the role of faeces as a chemical mark of the refuge[END_REF] and given that we aimed to evaluate the potential role of these substances for promoting aggregation, only motionless insects were considered in our analyses.

Testing blends of compounds in artificial shelters

A square glass arena (1 m 2 ) lined with filter paper was used for these tests (Figure 1A). Two artificial shelters made from a piece of corrugated cardboard (20610 cm), folded to generate a 10 cm 2 shelter with two lateral slits of approximately 0.5 cm in height [START_REF] Lorenzo | The spatial pattern of defaecation in Triatoma infestans and the role of faeces as a chemical mark of the refuge[END_REF], were placed on opposite sides of the arena (Figure 1). In one of the shelters, a piece of filter paper (466 cm) impregnated with a blend of the five compounds was introduced, while the other shelter contained a piece of filter paper treated with solvent as control. This shelter design (Figure 1B) is proven to successfully recruit triatomine bugs [START_REF] Lorenzo | Activity pattern in relation to refuge exploitation and feeding in Triatoma infestans (Hemiptera: Reduviidae)[END_REF][START_REF] Lorenzo | The spatial pattern of defaecation in Triatoma infestans and the role of faeces as a chemical mark of the refuge[END_REF][START_REF] Lorenzo | Temperature and relative humidity affect the selection of shelters by Triatoma infestans, vector of Chagas disease[END_REF][START_REF] Mota | Lack of segregation between two species of Chagas disease vectors[END_REF], which tend to enter the shelters due to their strong thigmotaxis and intense negative phototaxis [START_REF] Reisenman | Circadian control of photonegative sensitivity in the haematophagous bug Triatoma infestans[END_REF].

Fifth instar larvae starved for 1164 days post ecdysis were used in this experiment. A group of 30 insects was released in the center of the arena two hours before the beginning of the scotophase. The illumination of the experimental room was set to a 12:12 LL/DD regime. Two hours after the end of the scotophase, the shelters were carefully removed from the arena and the number of bugs inside each of them was recorded.

Three doses of each of the five compounds (16 ng, 160 ng, and 1.6 mg) were applied in the test shelter in three separate series of assays. These tests were performed separately for T. infestans, P. megistus and T. brasiliensis. Eight replicates were performed for each dose tested with each of the three species. Since our goal was to compare the aggregation inside baited vs unbaited shelters, insects found outside refuges were excluded from the statistical analysis because they were not aggregating. It is important to highlight that in triatomines the decision to aggregate or to remain dispersed is influenced by factors such as thigmotaxis and phototaxis, i.e. factors other than the presence of a bait inside a shelter. Therefore, an additional phenomenon would have been evaluated if all insects present in the arena were included in the analysis. The effect of the different doses of compounds on the distribution of insects in baited and unbaited refuges was analyzed by means of a Generalized Linear Model (GLM) using a logistic regression adapted to the binomial nature of the response variable. This analysis was followed by a Wilcoxon signed rank test with continuity correction in order to compare shelter choice data obtained with each concentration and species against a random choice between shelters. Both tests were performed in R software 3.0.2 (R Core Team, 2013).

Results

Identification of volatile substances from samples of feces

Five compounds were common to the feces of all species studied: acetamide, 2,3-butanediol, acetic acid, 3-methylbutyric acid and hexanoic acid. The relative abundance and proportion of these five compounds varied during the five days of sampling for the three species: T. infestans (Figure 2A), P. megistus (Figure 2B) and T. brasiliensis (Figure 2C).

In T. infestans the abundance of acetic acid and 2,3-butanediol increased markedly 24 h after sample collection before decreasing between the first and second day. 3-Methylbutyric acid was initially present in fresh feces and its abundance decreased until only traces were detectable after 72 h. A relatively constant low abundance of hexanoic acid was detected across the five days of sampling. Only traces of acetamide were detected (Figure 2A).

In samples of feces of P. megistus acetic acid was more abundant at the first and second days of sampling, a result which was consistent with our findings in T. infestans samples. This was the most abundant compound during all five days. 3-Methylbutyric acid was the second most abundant compound. For P. megistus, hexanoic acid, 2,3-butanediol and acetamide were detected as traces (Figure 2B).

In T. brasiliensis all compounds were consistently detected in very low amounts over all five days, except 2,3-butanediol, for which the abundance decreased steadily from the first to the fifth day (Figure 2C).

Behavioral responses to the commonly occurring compounds

Each compound was tested individually for each species. All substances were capable of attracting the three species studied with the exception of 2,3-butanediol which did not induce any effect on P. megistus bugs (Table 1). Control tests evaluating two clean filter papers or a solvent control vs a clean filter paper presented a random distribution (Binomial test, N.S.).

Blend baited artificial shelters

The proportion of insects of each species found inside blend baited and control shelters is presented in Figure 3. The proportion of insects remaining outside refuges at the end of the experiments varied according to the species. The GLM with binomial error revealed a significant effect of the bait dose on the choice for a refuge by the three species (T. infestans: z = 3.11, P = 0.00187, residual deviance = 12.940 on 22 degrees of freedom; T. brasiliensis: z = 2.403, P = 0.0162, residual deviance = 25.084 on 22 degrees of freedom and P. megistus: z = 3.653, P = 0.000259, residual deviance = 13.669 on 22 degrees of freedom). Refuges associated with a mixture of 16 ng of each compound promoted the aggregation of only as many triatomines as clean shelters (Wilcoxon signed rank test, NS, Figure 3). Conversely, shelters associated with mixtures of 160 ng or 1.6 mg of each compound promoted a significantly stronger aggregation on T. infestans (160 ng V = 36; P = 0.0078; 1.6 mg, V = 36, P = 0.014), T. brasiliensis (160 ng, V = 36, P = 0.0078; 1.6 mg, V = 28, P = 0.022) and P. megistus (160 ng, V = 28, P = 0.022; V = 36; P = 0.014) larvae, than clean shelters.

Discussion

As initially hypothesized, the present study identified volatile compounds common to feces of three species of triatomine vectors. We combined these five substances in a blend that was capable of attracting bugs of the three species into shelters.

In contrast to previous studies we focused on the identification of compounds that were readily available and common to all species. This approach was favored as it is expected to reduce the production cost of chemical baits. We found that the presence of five common compounds was consistent, but their abundance was highly variable throughout the sampling period in all cases.

Previously it has been demonstrated that feces, despite this changing proportion of volatiles over time, are attractive for up to 10 days [START_REF] Figueiras | Temporal change of the aggregation response in Triatoma infestans[END_REF]. The low, dynamic abundance and high volatility of the fecal compounds warranted SPME as the sampling method.

Out of the five substances selected for behavioral testing, three have not previously been identified in triatomine feces: 3methylbutyric acid, hexanoic acid and 2,3-butanediol. Acetic acid and acetamide have been identified in the fecal samples of T. infestans and T. mazzotii [START_REF] Lopez | Chemical investigation of aggregation behavior of Triatoma bugs (Hemiptera: Reduviidae)[END_REF], however the biological activity of these compounds was not assessed. The results from our behavioral experiments suggest that they are all constituents of the aggregation signal from triatomine feces and reinforce the hypothesis that this aggregation signal is involved in the marking of shelters by triatomines [START_REF] Lorenzo | The spatial pattern of defaecation in Triatoma infestans and the role of faeces as a chemical mark of the refuge[END_REF][START_REF] Schofield | Assembly pheromone of Triatoma infestans and Rhodnius prolixus nymphs (Hemiptera-Reduviidae)[END_REF][START_REF] Figueiras | Aggregation in the hematophagous bug Triatoma infestans: Chemical signals and temporal pattern[END_REF]. Whether substances other than those reported here play a role in triatomine aggregation remains unclear; nevertheless this is the first report of a chemical blend identified in triatomine feces acting as an aggregation signal for Chagas disease vectors. The results obtained in shelter experiments with all the species studied in this work were very similar, which is consistent with the proposed low specificity of aggregation signals from feces of triatomines [START_REF] Cruz-Lopez | Aggregation pheromone in five species of Triatominae (Hemiptera: Reduviidae)[END_REF][START_REF] Figueiras | Aggregation behaviour and interspecific responses in Rhodnius prolixus Stal[END_REF][START_REF] Figueiras | Aggregation behaviour and interspecific responses in three species of triatominae[END_REF][START_REF] Pires | Aggregation behaviour in Panstrongylus megistus and Triatoma infestans: inter and intraspecific responses[END_REF][START_REF] Vitta | The use of aggregation signals by Triatoma brasiliensis (Heteroptera : Reduviidae)[END_REF]. Therefore, we suggest that our five substance blend could be applied as a general triatomine bait suitable for areas with several sympatric species. Table 1. Doses of acetamide, 2,3-butanediol, acetic acid, 3-methylbutyric acid and hexanoic acid significantly attractive for T. infestans, P. megistus and T. brasiliensis. Currently, the detection of domiciliary infestations in control programs is performed by manual search for triatomine bugs and/ or colonization signals, such as feces, eggs and exuviae [START_REF] Dias | O controle da doenc ¸a de Chagas no Brasil[END_REF]. In cases of low infestation, the use of chemical dislodging agents, e.g., 0.2% tetramethrin, has been introduced to induce insects to abandon their shelters and become exposed [START_REF] Sucam | Manual de normas te ´cnicas da campanha de controle da doenc ¸a de Chagas[END_REF]. In Argentina, regularly monitored cardboard boxes that offer shelter to bugs in the walls of houses or in their peridomestic structures, have been used [START_REF] Go ´mez-Nu ´n ˜ez | Desarrollo de un nuevo me ´todo para evaluar la infestacio ´n intradomiciliaria por Rhodnius prolixus[END_REF][START_REF] Wisnivesky-Colli | A new tool for continuous detection of the presence of triatomine bugs, vectors of Chagas disease, in rural households[END_REF]. This type of un-baited refuge does not contain glue or insecticide in order to capture or kill visiting insects; instead the bugs find them by chance and generally choose them as a shelter due to their physical properties. The association of these devices with baits, such as the volatile mixture developed in this work, may significantly increase their detection sensitivity. Furthermore, addition of glue [START_REF] Rojas De Arias | Post-control surveillance of Triatoma infestans and Triatoma sordida with chemicallybaited sticky traps[END_REF], insecticide or pathogenic microorganisms [START_REF] Pedrini | Control of pyrethroid-resistant Chagas disease vectors with entomopathogenic fungi[END_REF] may allow transforming these devices into sensitive control tools representing a detection/capture device highly specific for triatomines. One particularly relevant application is the detection of dispersing individuals of sylvatic species that frequently reinvade houses from wild environments after insecticide spraying [START_REF] Dias | Reinfestac ¸a ˜o do Municı ´pio de Bambuı ´por triatomı ´neos transmissores da doenc ¸a de Chagas[END_REF][START_REF] Forattini | Aspectos ecolo ´gicos da tripanossomı ´ase americana: VIII-Domiciliac ¸a ˜o de Panstrongylus megistus e sua presenc ¸a extradomiciliar[END_REF][START_REF] Silveira | Chagas disease: the epidemiological and control aspects[END_REF][START_REF] Rolo ´n | First report of colonies of sylvatic Triatoma infestans (Hemiptera: Reduviidae) in the Paraguayan Chaco, using a trained dog[END_REF][START_REF] Ceballos | First finding of melanic sylvatic Triatoma infestans (Hemiptera: Reduviidae) colonies in the Argentine Chaco[END_REF][START_REF] Dorn | Triatoma dimidiata (Latreille, 1811): A review of its diversity across its geographic range and the relationship among populations[END_REF][START_REF] Carbajal De La Fuente | Flight dispersal of the Chagas disease vectors Triatoma brasiliensis and Triatoma pseudomaculata in northeastern Brazil[END_REF]. It is important to highlight that under the low density of current infestations in most geographic locations, detection of triatomines is extremely difficult. This limitation may affect the utilization of our mixture that needs to be evaluated under field conditions. We suggest that a long-lasting formulation which would allow a cumulative sampling of bug presence may increase the chances of effective use.

In a broader context, chemical baits based on pheromones or host odors have been proposed as cost effective and environmentally benign alternative tools for detection and control of several pest insects [START_REF] Kline | Semiochemical, traps/targets and mass trapping technology for mosquito management[END_REF][START_REF] Vale | Odour-baited targets to control tsetse flies, Glossina spp.(Diptera: Glossinidae) in Zimbabwe[END_REF][START_REF] El-Sayed | Potential of ''Lure and Kill'' in Long-Term Pest Management and Eradication of Invasive Species[END_REF]. For triatomines, an odor mixture luring the bugs into detection devices may similarly constitute a practical, economical and environmentally friendly method to monitor infestations by Chagas disease vectors. Further experiments should allow the development of a slow-release formulation for the blend, as well as demonstrate its effectiveness under field conditions. Ultimately, our blend could be developed into more advanced control tools for Chagas disease vectors, which is especially relevant where colonies have developed resistance to current insecticides [START_REF] Rojas De Arias | Post-control surveillance of Triatoma infestans and Triatoma sordida with chemicallybaited sticky traps[END_REF][START_REF] Pedrini | Control of pyrethroid-resistant Chagas disease vectors with entomopathogenic fungi[END_REF].

Figure 1 .

 1 Figure 1. Schematic drawing showing: A-the experimental arena used in the shelter assays and, B-a picture of an artificial refuge. doi:10.1371/journal.pntd.0002677.g001

Figure 2 .

 2 Figure2. Abundance of odors detected in feces of triatomine bugs. Abundance of acetic acid, 2,3-butanediol, 3-methylbutyric acid, acetamide and hexanoic acid identified in the feces of larvae during five days of sampling. A-T. infestans, B-P. megistus and C-T. brasiliensis. Only traces of acetamide were detected in feces of T. infestans. doi:10.1371/journal.pntd.0002677.g002

Figure 3 .

 3 Figure 3. Recruitment of bugs to shelters baited with synthetic blends. Mean number of larvae of A-T. infestans, B-P. megistus and C-T. brasiliensis found inside experimental and control shelters after overnight assays in which mixtures of 16 ng, 160 ng or 1.6 mg of each compound were tested in association with one (test shelter) of two shelters offered. Error bars represent the standard errors of the means. Asterisks indicate significant differences in Wilcoxon signed rank test. doi:10.1371/journal.pntd.0002677.g003

PLOS Neglected Tropical Diseases | www.plosntds.org

February 2014 | Volume 8 | Issue 2 | e2677

Acknowledgments

Lina Lundborg and Prof. Anna-Karin Borg-Karlson, Royal Institute of Technology, Stockholm, are thanked for assistance with chemical analysis.

The authors would like to acknowledge the financial support of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), FAPEMIG, INCT Entomologia Molecular, Univ. of Buenos Aires, FIOCRUZ, CAPES-SETCIP and Sven and Dagmar Sahle ´ns stiftelse. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author Contributions


Damien Thuau

Mamatimin Abbas

Sylvain Chambon

Pascal Tardy

Guillaume Wantz

Philippe Poulin

Lionel Hirsch

Isabelle Dufour

Cédric Ayela

Sensitivity enhancement of a flexible MEMS strain sensor by a field effect transistor in an all organic approach

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

I. INTRODUCTION

Continuous demand for flexible electronics technology has been the driving initiator for the development of organic devices such as polymeric MEMS and organic field effect transistors (OFET) [1]. Today, organic MEMS and OFET exhibit performances comparable to costly and brittle traditional amorphous silicon-based electronic devices. In fact, the use of low Young's modulus polymer for cantilever devices instead of conventional silicon-based materials has been reported to improve surface stress sensitivity [2]. Polymer microcantilever based MEMS have been proven to be efficient devices for physical, chemical and biological sensing applications [3][4][5]. One of the main challenges in the conception of successful organic MEMS concerns the integrated electromechanical transduction which converts physical quantity (i.e. strain) into an electrical measurable quantity (i.e. current). In contrast to external optical detection, integrated electromechanical transduction schemes are often preferred nowadays opening the way for the development of portable electronic devices. Specifically, piezoresistive based nanocomposite readout technique has been shown to be a sensitive and appropriate transduction method for organic MEMS sensors with sensitivities ranging from unit to hundreds [6][7][START_REF] Seena | Proc. 23rd IEEE Int. Conf. MEMS Tech. Dig[END_REF][START_REF] Thuau | [END_REF]. Previous studies on piezoresistive responses of particle modified polymers have given valuable information on the parameters (type and shape of the fillers and conduction mechanisms) that influence the material' sensitivity [10][11][12]. In fact, sensitivity enhancement of the sensor as well as lowering both its limit of detection and resolution have gathered a large amount of research interest.

Particularly, in the present case of strain sensing, when a force is applied to the cantilever it induces a deflection of the cantilever associated to a longitudinal strain of the material. The level of strain is often on the order of 0.001 to 0.1% making the onset of its detection extremely difficult to achieve. From these observations, our focus has been directed towards the enhancement of sensitivity and limit of detection of piezoresistive organic MEMS devices. The original principle developed in this work consists in using an OFET-based device in order to amplify the output sensitivity (gauge factor) of the piezoresistive organic MEMS sensor. To this purpose, the organic MEMS sensor has been connected to the gate of an OFET with the aim of converting the resistance variations of the nanocomposite strain sensor into a current signal, thus amplifying the sensitivity of the strain sensor utilizing the intrinsic gain of the OFET in an all organic approach.

MATERIALS AND METHOD

We used Graphistrength Epoxy Master batch pellets from Arkema which consist in 25 wt% of multi-walled-carbon nanotubes (MWNTs) made via catalytic chemical vapor deposition (CCVD) dispersed in an epoxy type matrix. The nanocomposite has been prepared by mixing the SU-8 epoxy photoresist with the pellets using a high shear mixer, Silverson L4RT at 5000 rpm for 60 min in an ice bath. The U-shaped piezoresistive organic cantilevers were fabricated in a low-cost, two-step process. The micro-machining process used in the framework of this work does not require the high-cost semiconductor manufacturing equipment used for silicon micro-fabrication and thus, reduces drastically the fabrication cost of the MEMS devices. This process requires only a spin coater to deposit the material and a vinyl cutting machine to pattern the structure. The CNT/SU-8 piezoresistive solution was spin coated on a sheet of 100 mm thick Polyethylene terephthalate (PET) and soft baked at 95°C for 5 min. Then, like SU/8, the CNT-SU/8 thin film was cross-linked by exposure to UV (365 nm) radiation but at a higher dose (250 mJ.cm -2 ) combined with a post-exposure bake step at 65°C for 1 min and 120°C for 10 min followed by final hard baked of 150°C for 15 min. Afterwards, the resonators were patterned simply using a cutting plotter machine (Graphtec Craft ROBO Pro). The final dimensions of the U-shaped structures composed of a PET layer covered with the CNT/SU-8 nanocomposite were 2800 m long, 400 m wide and 118 m thick (inset FIG. 1.a). The piezoresistive sensitivity of the MEMS has been electromechanically characterized. The cantilever was bent by applying a force at the cantilever's tip using a microprobe (MicroBot, Imina Technologies SA) while measuring the resistance changes of the piezoresistor. FIG. 1 shows the evolution of the resistance as a function of applied strain, . The strain experienced by the cantilever due to an applied force at the cantilever tip is defined as:

(1)

With h and L being the thickness and length of the cantilever respectively, and  the deflection at the tip of the cantilever.

The sensitivity of the MEMS sensor defined by its gauge factor (the slope of the curve) has been calculated to be 8.3 at low strain level ( < 0.6%) and is in agreement with reported sensitivity values of hybrid piezoresistive MEMS sensors [START_REF] Thuau | [END_REF].

Previous studies have attempted to describe the complicated interplay of the different mechanisms behind the piezoresistive behavior of the CNT network [10][11][12]. It has been noticed that piezoresisitive nanocomposites containing a CNT wt% concentration close to the percolation threshold exhibit higher sensitivity suggesting that the piezoresistive effect is essentially governed by changes of the nanotube connectivity. According to a previous study, a 2wt% CNT/SU-8 nanocomposite was chosen for the following measurements [START_REF] Thuau | [END_REF]. In the present case, we focused our attention on the enhancement of the sensitivity of our sensor at low strain values. At such strain level, ( < 0.6%), the changes of resistivity of the hybrid nanocomposite are linear and typically modeled using a linear scaling rule. As expected, the resistance of the CNT/SU-8 thin layer increased linearly as a function of the applied strain. In this sense, we designed an experimental set-up where a piezoresistive organic MEMS has been mounted in a half Wheatstone bridge configuration and connected to the gate of an OFET as shown in FIG. 1.b. In response to applied strain the resistance of the MEMS piezoresistive transducer changed and consequently altered the gate voltage (V GS ) of the OFET. As a result, the strain applied to the cantilever is correlated to the variations of the drain current (I DS ) of the OFET.

The p-channel type OFET device was prepared on glass substrate. 100nm thick Al has been evaporated through shadow mask (13mm×1mm) as gate contact. 30 nm Al 2 O 3 layer has been formed via anodization to act as dielectric layer. 15 nm of Poly(1vinyl-1,2,4-triazole) (PVT) was spin coated on the oxide surface as passivation layer providing high capacitance and low interface trap density [13,14]. Then, 30 nm of dinaphtho[2,3-b:2',3'-f]thieno[3,2-b]thiophene (DNTT) was thermally vacuum evaporated (7.10 -7 mbar) as active layer at a deposition rate of 0.1 Ǻ.s -1 . DNTT was chosen as it has high mobility and air stability [15]. Finally, 100 nm of Au source/drain contacts have been deposited on the active layer through shadow mask forming a channel with length of 50 μm and width of 2.

RESULTS AND DISCUSSION

The electrical measurements have been performed at room temperature under ambient atmospheric conditions using a keithley 4200 semiconductor parameter analyzer. It is worth noting that an optimization of the switching characteristics and the mobility of the OFET have been performed in order to get a sufficient gain, and therefore amplify the sensitivity of the MEMS. Initially, Fullerene (C 60 ) was been chosen as active layer. Nevertheless the C 60 based devices showed an I ON/OFF ratio slightly higher than 10 2 . Consequently, C 60 was substituted by DNTT as active layer leading to a high current ratio over a small variation of gate voltage. These transfer characteristics make it rather sensitive and lead to an amplification of the strain sensitivity of nearly a factor 4. Transfer (I DS versus V GS ) and output (I DS versus V DS ) characteristics of the present OFET have been measured as shown in FIG. 2. The sealed device exhibited excellent stability against ambient and repetitive testing conditions. Transfer characteristics were identical after 3 days in air (supplementary information). Almost no hysteresis was observed, a prerequisite for sensing applications. The OFET showed low gate leakage current and good switching characteristics (I GS = 1.10 -11 A at V GS = -10 V and I ON/OFF = 10 6 ). The gate leakage was in the range of tens of picoamperes validating the choice of Al 2 O 3 /PVT as dielectric layer. The output characteristics of the OFET exhibited a linear increase in the drain current at low drain bias (inset of FIG. 2.a). At high drain biases, proper saturation of drain current was observed proving the good operating of the device. In linear regime, the carrier mobility at the dielectric/semiconductor interface (), can be expressed as:

(2)

Where C diel is the gate dielectric capacitance per unit area, W is the channel width, and L is the channel length of the transistor. Here, the width and length of the transistor channel are 50 m and 2.5 mm respectively. The drain voltage was set to -2V and the dielectric capacitance was measured at 127 nF/cm². A high mobility of 0.45 cm².V -1 .s -1 has been obtained at Similarly to the characterization of the intrinsic piezoresistive MEMS device, 0.3% of tensile strain was applied to the cantilever connected to the OFET in a half Wheatstone bridge configuration (FIG. 1.b) where the resistance R 1 was chosen to be identical to the piezoresistor value (PZR) at rest, namely 6138 k. This aimed to obtain thereafter the largest variations of I DS of the OFET and hence the highest sensitivity. Subsequently, variations of I DS for different V GS were recorded in order to find the best operating point. Each relative change of drain current between the cantilever at rest and under 0.3% of applied strain have been recorded for the nine operating points and their sensitivities plotted as a function of gate voltage as shown by FIG. 2.a. The highest sensitivity has been found for gate voltage in the range -3 to -4.5V where the slope of the transfer characteristics curve is steeper. As a result, a low gate voltage of -4V was chosen for all the following measurements. Under this optimized condition, an increase by a factor 3.7 (gauge factor jumped from 8.3 to 33.8) has been observed for the sensitivity of the integrated transduction of mechanical strain, compared to the direct detection of changes of resistance in the nanocomposite, as shown in FIG. 2.b. Furthermore, the linearity of the response of the piezoresistive strain sensor has not been altered by adding the OFET. Also, a low limit of detection (LOD) of 24 ppm has been estimated in terms of strain transduction efficiency. This LOD of the strain measurement made with the MEMS coupled with the OFET is twice better than the LOD of the resistance changes of the MEMS itself. The LOD of the fabricated MEMS combined with an OFET has been estimated by calculating the standard deviation of the drain current for 1000 points at rest position. The LOD could then be determined by taking three times the standard deviation and dividing it by the sensor' sensitivity.

V GS = -10V.
While in situ strain sensing capability of carbon nanotubes based composite strain sensors has been widely reported, here we present real-time measurements that are required for a large number of applications. We develop a real time sensor which can track instantaneously the force applied either by steps (FIG. 3 represent tensile strain of 1800 and 900 ppm while arrows pointing up or down show when tensile deformation steps have been applied to or taken away from the cantilever, respectively. The OFET has been found to display significant changes of I DS that correlated well with strain level applied. When a force is applied on the cantilever, the top surface of the cantilever (where the piezoresistive transducer is located) experiences tensile strain resulting in a rapid increase of I DS . Conversely, when the force is taken away from the cantilever, rapid strain relaxation occurs and thus I DS decreases to reach its initial value.

The electrical current responses for the cyclic tensile strain deformation have consistent shape, where I DS closely follows the steps of tensile strain applied as shown in FIG. 3.b. This result demonstrates the reversibility and repeatability of the OFET+MEMS strain sensor. It is worth noting that excellent response time (estimated below 500ms) and the absence of hysteresis after several cycles of tensile strain applied to the organic MEMS proves the powerful combination of these organic electronics devices as sensitive strain sensors.

CONCLUSION

The concept of using an OFET to enhance the sensing performance of an organic piezoresistive MEMS sensor has been demonstrated. The OFET has been used to convert the resistance change signal into an amplified current signal consequently improving the sensitivity and the limit of detection of the sensor. High performance OFETs were realized with I ON/OFF around 10 6 and mobility of 0.45 cm²/Vs at V GS = -10 V. Cycling experiments were performed and proved the high sensitivity, reversibility and reproducibility of strain sensing. At low deformation, the drain current (-I DS ) changes linearly with applied strain to the sensors which allows quantitative detection. The sensitivity of the coupled sensor and OFET was improved by a factor 3.7 compared to the direct measurement of the change in resistance (gauge factor jumped from 8.3 to 33.8) and a limit of detection as low as 24 ppm was determined for the strain. In addition, the ability to sense strain in real time with no hysteresis is very promising for biological and chemical sensing applications. The coupling of polymeric MEMS and OFET paves the way for the development of further integrated fully organic sensors.

SUPLEMENTARY INFORMATION

See supplemental material namely the stability of the transfer characteristics of the OFET as well as a video of the device measuring strain in real-time can be viewed.

4 FIG. 1

 41 FIG. 1.a) Relative change of resistance of the piezoresistive MEMS cantilever as a function of applied strain; inset: schematic of the MEMS strain sensor deformed by tensile strain using a micro-probe, b) Experimental set-up, and c) schematic of the structure of the DNTT-based p-channel OFET device with Al 2 O 3 /PVT as dielectric layer

FIG. 2

 2 FIG. 2. a)Transfer characteristic of the OFET (black curve) and relative change of I DS as a function of V GS for a strain of  = 0.3% (red curve); inset: output characteristic of the OFET between initial position and under 0.3% of applied strain at different V GS ranging from -1.5V to -10V by steps of -0.75V, b) Amplification of the sensitivity of the piezoresistive MEMS using an OFET

7 FIG. 3 .

 73 FIG. 3. Real time responses of I DS as a function of time under applied strain by a) steps of 900 and 1800 ppm for blue and red arrows respectively and b) at constant speed (trapezoidal shape)

ACKNOWLEDGEMENTS

This study was carried out in the framework of the Project PODCAST, and supported financially by the Grant: ANR-12-JS03-0003. We would like to acknowledge also the LabEx AMADEus, ANR-10-LABX-0042-AMADEUS along with the program of Excellence Initiative IdEx Bordeaux, Grant N° ANR-10-IDEX-0003-02.


Georges Dubourg
email: georges.dubourg@ims-bordeaux.fr

I Dufour

C Pellet

C Ayela

Optimization of the performances of SU-8 organic microcantilever resonators by tuning the viscoelastic properties of the polymer

Keywords: organic microresonator, SU-8, viscoelastic properties, Q-enhancement

This paper describes a promising method to improve the performances of organic resonant microcantilevers. This method is based on the tuning of the viscoelastic properties of the material by controlling internally the temperature of the structure. Actuation of the SU-8 free-standing microstructures has been integrated by means of electromagnetic forces while a static resistive thermal effect, obtained by applying a DC current in a conducting path, has been used to control the temperature of the structure. Investigation of the electromagnetic and thermal influence on the static and dynamic behavior has shown that the thermal effect dominates the electromagnetic one in static mode and inversely in dynamic mode. As a result, by applying a constant heating of the structure, the viscoelastic properties of polymers have been tuned, and the quality factor of SU-8 microcantilevers has been thereby enhanced by a factor 2 in air and 2.25 in liquid.

Introduction

Free-standing microcantilevers are widely used as micro-and-nanoelectromechanical systems (MEMS and NEMS). More specifically, silicon cantilevers have shown great potential for sensing applications such as, molecular recognition [START_REF] Burg | Vacuum-Packaged Suspended Microchannel Resonant Mass Sensor for Biomolecular Detection[END_REF][START_REF] Burg | Weighing of biomolecules, single cells and single nanoparticles in fluid[END_REF] or virus detection [START_REF] Hwang | Nanomechanical microcantilever operated in vibration modes with use of RNA aptamer as receptor molecules for label-free detection of HCV helicase[END_REF]. However, a recent emerging alternative concerns the introduction of polymers as structural layer in MEMS resonators [START_REF] Schmid | Electrostatically actuated nonconductive polymer microresonators in gaseous and aqueous environment[END_REF], actuators [5] and spring structures [START_REF] Bachmann | Fabrication and characterization of folded SU-8 suspensions for MEMS applications[END_REF] due to the ease of their processing, their low cost, their low temperature requirement during fabrication, and the possible tuning of mechanical properties such as Young modulus, a quality criterion hard to be achieved when using silicon. When organic MEMS are used in static mode, one of the advantages is their softness resulting in large deflections for small external applied forces. This specific property has been used for the fabrication of sensors used for chemical or biological applications [START_REF] Fritz | Translating Biomolecular Recognition into Nanomechanics[END_REF][START_REF] Calleja | Polymeric Mechanical Sensors with Integrated Readout in a Microfluidic System[END_REF][START_REF] Calleja | Polymeric Cantilever Arrays for Biosensing Applications[END_REF] and micromanipulators for cell handling [START_REF] Jager | Microfabricating Conjugated Polymer Actuators[END_REF][START_REF] Chronis | Electrothermally Activated SU-8 Microgripper for Single Cell Manipulation in Solution[END_REF]. In dynamic mode, it is well-known that the quality factor Q is an important parameter for the resonance characteristics. The quality factor determines the sharpness of the resonant peak and thus the frequency stability of the resonator. Therefore, a good quality factor is crucial for most resonator applications. In this context, the low quality factor limits the actual performance of organic MEMS resonators since losses in organic materials induce a non-negligible material damping. This raises the question of how the quality factor of polymer resonant microcantilevers can be improved. Recently, various methods have been proposed. For instance, in order to counteract the negative effect of the relative low quality factor, an electronic positive feedback has been used for polymer microcantilevers corresponding to an artificial increase of Q [START_REF] Schmid | Electrostatically actuated nonconductive polymer microresonators in gaseous and aqueous environment[END_REF]. Other promising methods consist in using microcantilevers in the in-plane modes including longitudinal and lateral ones [START_REF] Castille | Longitudinal vibration mode of piezoelectric thick-film cantilever-based sensors in liquid media[END_REF][START_REF] Cox | Characteristics of laterally vibrating resonant microcantilevers in viscous liquid media[END_REF] or introducing a microchannel into the cantilever to reduce the fluid damping and thus, to improve the quality factor for applications in liquid media [START_REF] Burg | Suspended microchannel resonators for biomolecular detection[END_REF]. Recently, the fabrication of asymmetric anchors or electrodes has been proposed to enhance the sensitivity of microcantilevers operating in the flexural modes [START_REF] Sharma | Unconventional Low-Cost Fabrication and Patterning Techniques for Point of Care Diagnostics[END_REF][START_REF] Johnson | Expression of picogram sensitive bending modes in piezoelectric cantilever sensors with nonuniform electric fields generated by asymmetric electrodes[END_REF]. However, the fabrication of such devices generally requires more complex fabrication steps, reducing the simplicity and low cost criteria associated to organic MEMS. The control of the mechanical properties of silicon cantilevers by self-oscillations can be also realized [START_REF] Hohberger | Self-oscillation of micromechanical resonators 4th IEEE Inter[END_REF] but this method is not adapted to organic structures due to their different mechanical behavior.

In this context, the understanding of intrinsic dissipation effects occurring in polymer materials and its influence on the damping behavior of polymer microresonators is of great interest since this behavior affects directly the Q-factor. Investigations on origins of damping in SU-8 microcantilevers have demonstrated that material damping due to polymer viscoelastic properties is the main loss mechanism [START_REF] Schmid | Damping mechanisms of single-clamped and prestressed double clamped resonant polymer microbeams[END_REF]. The influence of the temperature on the viscoelastic properties of SU-8 has been also reported [START_REF] Schmid | Damping mechanisms of single-clamped and prestressed double clamped resonant polymer microbeams[END_REF]. In the present work, a simple method to precisely control the viscoelastic properties of SU-8 by inducing an internal heating is proposed in order to improve the quality factor of SU-8 resonators. This is achieved by patterning a U-shaped conducting path on top of the cantilever surface, allowing a constant static resistive heating thanks to a DC current flowing through the conducting path. Also, an integrated dynamic actuation of the cantilevers is provided by electromagnetic forces due to its simple implementation, the only criterion being an alternative current flowing into the conducting path and an external magnetic field. With this approach, the performances of resonant SU-8 microcantilevers have been evaluated and the dependence of the viscoelastic properties of SU-8 on Q-factor is reported. For a better control of the viscoelastic properties, the influence of the electrothermal, electrothermomechanical and electromagnetic effects on the static and the dynamic behavior of the structures has been widely investigated. As a result, the viscoelastic properties of the polymer have been successfully controlled thermally and the quality factor of SU-8 resonators has been significantly improved in air and in liquid media, where values of Q similar to those of silicon-based microcantilevers have been obtained.

Materials and methods

Fabrication process

The specific polymer used in this study is SU-8, an epoxy-based negative photoresist (SU-8® 3000) from MicroChem, that ensures different functions: structural, support and adhesive layer. SU-8 was chosen for its excellent processability, and its properties which have been intensively studied [START_REF] Lorenz | High-aspect-ratio, ultrathick, negative-tone near-UV photoresist and its applications for MEMS[END_REF]. The fabrication process of the microcantilever is based on a method inspired from wafer bonding. It consists in structuring simultaneously two wafers where the cantilever structures are first patterned upside down on a silicon carrier wafer and then clamped on a receiver wafer as reported in our previous work [START_REF] Dubourg | Collective fabrication of all-organic microcantilever chips based on a hierarchical combination of shadow-masking and wafer-bonding processing methods[END_REF]. First, a silicon carrier substrate has been coated with an Omnicoat® sacrificial layer (from MicroChem) to allow the release of the microstructures after the transferring step. Then, SU-8 microstructures and supporting layers have been successively spin-coated and patterned by photolithography. In parallel, the SU-8 transferring layer is structured on another silicon substrate. Next, both substrates are bonded together by a thermal wafer-bonding method. Afterwards, the freestanding organic structures are released by chemical etching of the Omnicoat® sacrificial layer. To ensure the integrated actuation of the organic structures, a gold path has been deposited between the sacrificial layer and the SU-8 structural one. Standard methods for gold patterning such as lift-off or etching are not appropriate in our case since the Omnicoat® sacrificial layer does not resist to the solvents used for the protective resists patterning generally used in such processes. Hence, an alternative method based on shadow-masking has been chosen since it does not require any chemical etching step. This method combines deposition and patterning in one step via thermal evaporation of gold through polymeric microstencils made of SU-8 following the method described in [START_REF] Dubourg | Collective fabrication of all-organic microcantilever chips based on a hierarchical combination of shadow-masking and wafer-bonding processing methods[END_REF]. The resulting gold conducting path, characterized by a thickness of 100nm, and integrated on the organic structures (cf. Fig. 1) allows the integrated actuation and the thermal control of the SU-8 cantilevers as described in the next section. To finish the process, the free-standing structures are mounted on a PCB. The conducting lines of the PCB are wire-bonded to pads present on the structure. With this process, matrices of organic cantilever resonators have been successfully realized, as shown on Fig. 1.

Experiments

Polymer microstructures can be actuated by several methods including electrostatic [START_REF] Schmid | Electrostatically actuated nonconductive polymer microresonators in gaseous and aqueous environment[END_REF], electromagnetic [START_REF] Im | Electromagnetic actuation and microchannel engineering of a polymer micropen array integrated with microchannels and sample reservoirs for biological assay patterning[END_REF] or electrothermal actuation [START_REF] Zhang | An Electro thermal SU-8 Cantilever, Micro Actuator based on Bimorph Effect[END_REF]. In this paper, we propose an electromagnetic dynamical actuation coupled with an integrated resistive heating by electrothermal effects. The dynamic actuation of the polymer-based microcantilevers is generated by an electromagnetic Laplace force. For this, the structures are mounted above an external magnet, and the Laplace force is then exerted by an alternative electrical current (AC) flowing through the gold path deposited on the structure. Fig. 2 shows a schematic of the cantilever actuated by the Laplace force (Fig. 2). This configuration offers the possibility to obtain the torsional and flexural resonant modes. The dynamic characteristics of the organic microcantilevers have been then recorded with a laser-Doppler vibrometer MSA-500 from Polytec measuring nanometer magnitude displacements. A continue electrical current (DC) can be also applied on the conducting path which can generate a static Laplace force and a heating of the resistor by Joule effect. In our case, the temperature rise associated with the resistive heating has been used for a precise control of the temperature in the cantilever. For instance, an applied DC power of 3.5mW gives an internal resistive heating of the structure of 90°C (this temperature results from Comsol simulation and thermal resistor measurements). This temperature rise induces also a thermomechanical deflection of the structure due to the bimorph effect from the Au conducting path coated on top of structured SU-8. This effect can be added to the microcantilever deflection resulting from the Laplace Force. The microcantilever deflection due to both static effects has been measured thanks to an interferometry optical profiler NT9080 from Veeco. By using both approaches simultaneously, the organic microstructures can be used at different resonant frequencies while applying an initial resistive heating of the organic material.

Results and discussion

Dynamic characterization of the microstructures

To obtain a reliable organic microresonator, it is important to evaluate the actual performances of the microstructures. An accurate dynamic characterization of the organic microcantilevers including mechanical resonant frequencies and quality factors is thus required.

Resonant frequencies

Cantilever resonant frequencies have been obtained by applying an alternative current (I max =40µA) to the gold conducting path, that is low enough to prevent from a dynamic electrothermal effect. The mechanical properties of the material defined by the Young's modulus of the structured layer have been then deduced from the out-of-plane flexural resonant modes using Eq. ( 1) [START_REF] Blevins | Formulas for natural frequency and mode shape[END_REF].

(1) E= (4π

2 f 2 n L 4 12ρ)/(λ 4 n h 2 )
In this Eq., E represents the cantilever effective Young's modulus, f n is the resonance frequency of the n th mode, L and h are respectively the length and thickness of the microcantilever, ρ represents the mass density of the material, and λ n the eigenvalue of the n th resonance mode. Fig. 3 shows the Young's modulus evolution measured for different flexural resonant modes. First, a wide panel of out-of-plane resonant modes, including torsional and flexural ones can be observed due to the high flexibility of the organic structures. In some cases (data not shown), the first fifteen flexural modes and torsional modes have been observed with the vibrometer. For the flexural modes, the low values of Young's modulus obtained are in agreement with the literature [START_REF] Ferrell | Microfabrication and nanomechanical characterization of polymer microelectromechanical system for biological applications[END_REF], validating the method used for its calculation. A slight increase of E can be also observed with the frequency. In fact, it is known that SU-8 provides a viscoelastic behavior [START_REF] Wouters | Comparison of methods for the mechanical characterization of polymers for MEMS applications[END_REF], meaning that the mechanical properties of the material are frequency dependent and can be expressed by a complex modulus:

(2) E* = E'+iE''

Where E', also called storage modulus, is the real part of E*, in phase with the strain. E'', the loss modulus, is the imaginary part of E*, out-of-phase with the strain. E' represents the stiffness of the material, and E'' the loss due to material damping. It is thus noteworthy to consider the material viscoelastic properties which strongly influence the quality factor of the organic microresonators, as shown in the next paragraphs.

Quality factors

The ability of resonant structures to be used as highly mass sensitive devices depends mainly on their quality factor which defines losses in the structure. Thereby, the quality factor of SU-8 microresonators has been determined by using the -3dB bandwidth of the resonant frequency spectra. From Fig. 4, it can be seen that the organic cantilevers have relatively low quality factors in air, decreasing with the frequency. For instance, the quality factor of a structure characterized by a length of 380µm decreases from 30 at low frequencies to around 10 at high frequencies (above 1MHz). Quality factor depends on several loss sources including losses from the clamped edge, the surface losses, the air damping and internal material damping. But, in the case of organic microresonators, the actuation of the structures induces vibrations of a viscoelastic material. Losses due to viscoelastic material damping can be expressed by Eq. ( 3) [START_REF] Schmid | Damping mechanisms of single-clamped and prestressed double clamped resonant polymer microbeams[END_REF]:

(3) 1/Q viscoelast. = E''/E' = tan δ
Where tan δ, the loss tangent, represents the phase angle between stress and strain. Furthermore, previous works [START_REF] Schmid | Damping mechanisms of single-clamped and prestressed double clamped resonant polymer microbeams[END_REF] have proved that the dominant damping mechanism is the viscoelastic material damping for SU-8 microresonators. Thereby, the quality factor of the organic microstructures can be expressed by Eq. ( 4):

(4) Q = E'/E'' =1/tan δ
As shown by Eq. 4, the quality factor of SU-8 microresonators is inversely proportional to the loss tangent. The tuning of viscoelastic properties of the material could offer the possibility to enhance the quality factors of the organic microcantilevers and thus, their performances as microresonators.

3.2

Optimization of the performances of SU-8 microresonators

Method

Previous investigations on the viscoelastic behavior of SU-8 have shown the influence of the temperature on the viscoelastic properties and that the loss tangent decreases with the temperature to a minimum peak [START_REF] Schmid | Damping mechanisms of single-clamped and prestressed double clamped resonant polymer microbeams[END_REF]. Above this temperature (around 90°C), the loss tangent increases representing the glass transition. As the quality factor is inversely proportional to the loss tangent (see Eq. 4), a maximum quality factor could be observed at the temperature corresponding to the minimum peak of the loss tangent. The thermal tuning of the viscoelastic properties of the SU-8 material by controlling internally the temperature of the structure would be an efficient method to improve the quality factors of the organic SU-8 microresonators. For this to be achieved, the conducting path integrated on the top surface of the microcantilevers previously described, allows an internal resistive heating of the organic microstructure. Hence, the organic microcantilevers can be used at the resonance frequency while a constant heating can be applied by adding an offset to the sinusoidal signal. Therefore, by using an electrothermal static effect, the viscoelastic properties of the SU-8 material can be accurately tuned to enhance the performances of the organic microresonators. However, this method requires a precise knowledge on the influence of the electromagnetic and the electrothermal effect on the static and the dynamic behavior of the microcantilevers.

Static behavior of SU-8 microcantilevers

To precisely control the temperature of the SU-8 microcantilevers, the electrothermal effect is chosen for its simplicity and efficiency. But, due to different thermal expansion coefficients between SU-8 (52ppm/K) and gold (14ppm/K), a static electrothermomechanical actuation of the structures should occur. Thus, the deflection of the cantilevers due to resistive heating has been investigated by applying a constant voltage across the resistor, corresponding to the U-shaped gold conducting path. The maximum DC voltage that can be applied over the resistor of 263Ω before causing damage of the microstructure and avoiding hysteresis effects is around 2V corresponding to a structure heating above the glass transition temperature of the polymer. Also, to clearly discriminate the influence of the electromagnetic and the electrothermomechanical static actuation, measurements with and without magnet have been performed. In this context, to evaluate the influence of the electromagnetic actuation on the global deflection of the microcantilevers, measurements have been performed in order to have Laplace force up and then down by applying respectively positive and negative voltages. The resulting deflections caused by the applied power in these different conditions are presented in Fig. 5, in the case of a microcantilever characterized by a length of 380µm.

The measured deflections of the structure, presented on Fig. 5, are similar when only the electrothermomechanical actuation is provided and when a Laplace force is generated, either up or down. The discrepancy between the different measurements is mainly due to uncertainty caused by the high transparency of SU-8, making optical measurements hard to be achieved. Thus, from this figure, it is clear that the static electrothermomechanical actuation dominates the electromagnetic static actuation. The theoretical electrothermomechanical results performed with Finite Element Modeling (Comsol) confirm the trend of the deflection obtained experimentally. Moreover, below 1.5mW, the deflection of the structure is nearly zero, meaning that the thermal effect can be neglected below this power value. This result is in agreement with previous observations [START_REF] Zhang | An Electro thermal SU-8 Cantilever, Micro Actuator based on Bimorph Effect[END_REF].

Consequence on dynamic behavior

In dynamic mode, the integrated actuation can result from the electrothermomechanical or/and the electromagnetic effect. In the case of electromagnetic actuation, an AC current having an angular frequency ω results in Laplace deflection oscillating at ω. On the contrary, in the case of electrothermomechanical actuation, an AC current (without DC offset) characterized by an angular frequency of ω generates temperature oscillations in the cantilever at 2ω leading to thermomechanical deflection also at 2ω [START_REF] Lee | Lorentz force actuation of a heated atomic force microscope cantilever[END_REF]. Thus, as the Laplace actuation has the same frequency as the current oscillation, this leads to a thermomechanical response equal to twice the actuation frequency. To evaluate the influence of both actuation schemes in dynamic mode, measurements have been performed with and without magnet. From Fig. 6, it can be seen that the magnitude of the resonant peak observed with the magnet is ten times higher than the one measured without magnet. As the thermomechanical frequency is equal to twice the actual resonant frequency f [START_REF] Lee | Lorentz force actuation of a heated atomic force microscope cantilever[END_REF], this means that the thermomechanical response should be at 2f. Thus, the peak observed without magnet cannot result from the thermomechanical actuation but from an electromagnetic actuation caused by Earth's magnetic field. Moreover, no response has been observed at 2f (with and without magnet), showing that the amplitude of the AC current applied (I max =30µA) is too low to generate enough power allowing a measurable thermomechanical deflection. Under this condition, considering the applied power, the electrothermal effects can be neglected in dynamic and the electromagnetic actuation becomes particularly appropriate for the dynamic mode. Moreover, as the oscillating deflection of highly flexible organic structures can be achieved at very low current density without heating the structure, the temperature of the structure can be precisely tuned by the static electrothermal effect only.

Otherwise, an increase of the temperature due to an internal heating affects the mechanical properties of the SU-8 material and thus the resonant frequencies of the structures (see Eq. ( 1)). Therefore, this effect has to be considered in the dynamic behavior. From Fig. 7, a decrease of the resonant frequency as a function of the applied DC power and thus the temperature can be observed. One could think that initiating a static deflection into the cantilevers would increase their stiffness and thus their resonant frequencies. But in present case, the specific decrease of the resonant frequency is due to the softening of the SU-8 material caused by a decrease of E' with the temperature [START_REF] Wouters | Comparison of methods for the mechanical characterization of polymers for MEMS applications[END_REF]. Moreover, the shape of the curves observed in Fig. 7 is close to the thermal behavior of the elastic part of the complex Young's modulus [START_REF] Wouters | Comparison of methods for the mechanical characterization of polymers for MEMS applications[END_REF], confirming the thermal dependence of the SU-8 cantilevers with the one of E' of SU-8. Indeed, an important drop of the frequency can be observed between 3 and 5mW which should correspond to the beginning of the glass transition of SU-8.

Q-factor optimization in air

It has been seen previously that the viscoelastic properties of SU-8 depend on the temperature. By analyzing previous works from the literature [START_REF] Schmid | Damping mechanisms of single-clamped and prestressed double clamped resonant polymer microbeams[END_REF], the quality factor could show a maximum peak at a temperature below the one of the material glass transition. To study the influence of the structure heating on the quality factor, an offset voltage has been applied into the conducting path. One has to remind that although the static electrothermomechanical actuation of the cantilevers has been studied in this work, the main idea was to induce precisely an internal heating into the SU-8 material. Fig. 8 shows the quality factor measurements for different DC powers applied to a microcantilever characterized by a length of 380µm. For each step, measurement stability is reached ensuring a constant temperature in the structure. The evolution of the quality factor is as predicted and in agreement with the thermal evolution of the loss tangent reported in [START_REF] Schmid | Damping mechanisms of single-clamped and prestressed double clamped resonant polymer microbeams[END_REF]. Indeed, the quality factor increases until a maximum peak found at 90°C and decreases above this temperature which corresponds to the beginning of the glass transition of the material. In this case, the glass temperature is not exceeded. Irreversible modifications of the materials properties due to an internal heating are thus avoided ensuring repeatability of the measurements. An increase up to 100% of the quality factor is observed for the second resonant flexural mode. But, it has been observed that the resonant frequency decreases with the applied DC power (see Fig. 7); therefore, it can be assumed that the Q rise is not due to the increase of the stiffness of the organic structure (as often reported to improve the quality factor of resonators [START_REF] Zhu | Enhanced detection resonance frequency shift of a piezoelectric microcantilever sensor by a DC bias electric field in humidity detection[END_REF]). This rise is caused by a larger thermal decrease of the loss modulus, E'', in comparison to the decrease of the storage modulus, E', resulting in an increase of the E'/E'' ratio and therefore Q, until 3.7mW. Also, from Fig. 8 it can be seen that the quality factor of the second mode increases more than the first mode since an increase of 25% and 100% has been observed for the first and the second mode respectively.

Q-factor optimization in liquid

In liquid, losses due to the fluid damping dominate over internal material losses for silicon based microstructures. In air, Q-factors up to 1500 have been measured for the first out-of-plane bending mode [START_REF] Beardslee | Geometrical Optimization of Resonant Cantilevers Vibrating in In-Plane Flexural Modes, Sensors[END_REF]. In water, Q-factors of silicon-based microcantilevers rarely exceed 2-20 due to the substantial viscous damping caused by the fluid. However in the case of an organic microresonator, the effects of viscoelastic material damping have to be considered too. The tuning of the viscoelastic properties of the organic structures could have the potential to enhance the quality factor of the cantilevers in liquid. It is thus interesting to investigate the effects of the initial heating of the structure on its quality factor in liquid media. The liquid used in this study is dodecane, a non-conducting fluid characterized by a viscosity of 1.55cP and a density of 746kg/m 3 at 25°C. Fig. 9 shows the evolution of the quality factor of an SU-8 organic microstructure characterized by a length of 380µm for increasing DC power values.

A maximum peak of 120% can be observed for the second flexural resonant mode, for an applied power of 3.5mW corresponding to a temperature of 90°C, as already observed in air. Although in liquid the losses due to the environment cannot be neglected, the influence of the material damping remains important. In our case, the quality factor increases from 4.8 to 10.8, the latter one being similar to the values obtained for silicon cantilevers. But a greater rise can be observed in liquid (120%) than in air (100%). In liquid, losses due to fluid damping have to be considered. These losses decrease with the density and the viscosity of the fluid [START_REF] Vancura | Analysis of resonating microcantilevers operating in a viscous liquid environment[END_REF][START_REF] Vancura | Characterization of magnetically actuated resonant cantilevers in viscous fluids[END_REF]. Moreover, the viscosity and the density of the dodecane decrease with the temperature [START_REF] Caudwell | The Viscosity and Density of n-Dodecane and n-Octadecane at Pressures up to 200MPa and Temperatures up to 473K[END_REF]. Therefore, the structure heating reduces the fluid damping and thus increases also the quality factor. The combined decrease of both material and fluid damping leads to a more substantial rise of the quality-factor in liquid than in air where only the material damping decrease is considered. However, by simply integrating a conducting path onto SU-8 organic microcantilevers, used as internal heater, a significant improvement of their quality factor is successfully achieved. These results potentiate organic MEMS resonators as good candidates for future biochemical mass sensitive applications in liquid media.

Conclusion

A promising method for the improvement of the performances of organic microcantilever resonators has been demonstrated. This method consists in precisely controlling the viscoelastic properties responsible of the low quality factor of the polymer devices by applying a constant heating of the structures. For this, an electrothermal effect is generated simply by incorporating a conducting path on top of the SU-8 structural layer. But the conducting path has been used also for an electromagnetic dynamic actuation by mounting the cantilever chips above a magnet. The dynamic characterization of these organic structures has been achieved to evaluate the performances of organic resonators including mechanical resonant frequencies and quality factors. Low Young's modulus values of highly flexible organic structures have been obtained thanks to the flexural resonant frequencies. Investigations on the electrothermal and electromagnetic effect on the static and dynamic behavior of the cantilevers have been realized. This study has shown that the static resistive heating is an efficient method to control the temperature of the structure while the electromagnetic actuation is an appropriate method to create integrated structure oscillations. With this approach, the performances of the organic microcantilever resonators have been successfully improved. The quality factor of the resonant microcantilevers has been enhanced by 100% in air and 120% in liquid. These structures have thus the potential to be used for biosensing applications in liquid media. Future works will aim to find polymer materials having tunable viscoelastic properties allowing this method to be used at lower temperatures.

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