Yiu Yiu Lee

C K C Wong

Camille Oger

Thierry Durand

Jetty Jean-Marie Galano

Chung-Yung Lee

R F I]

Taylor & Francis

J.-M Galano

-Y Lee

J.-M Galano

Chung-Yung Jetty
email: jettylee@hku.hk

Lee

Prenatal exposure to the contaminant perfluorooctane sulfonate elevates lipid peroxidation during mouse fetal development but not in the pregnant dam

Keywords: PFOS, isoprostanoids, isofuranoids, oxidative stress, PUFA )-4-F 4t -NeuroP, 4

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

Introduction

Perfl uorooctane sulfonate (PFOS, C 8 F 17 SO 3 H) is a member of the perfl uorinated chemical family and is synthetically produced for industrial use. The stable carbon -fl uorine bonds of the compound make it highly resistant to all forms of degradation and exhibit environmental persistence. Due to their unique chemical properties of water and oil repulsion and an improved reduction in surface tension compared with those of other surfactants, they are mainly used as multi-resistant coatings for a wide range of products, including paper, textile, and food packaging [1]. The extensive use of PFOS since the 1950s has resulted in substantial bioaccumulation; a wide distribution in human [START_REF] Olsen | Half-life of serum elimination of perfl uorooctanesulfonate,perfl uorohexanesulfonate, and perfl uorooctanoate in retired fl uorochemical production workers[END_REF], wildlife [3], arctic populations [START_REF] Dc | Identifi cation of long-chain perfl uorinated acids in biota from the Canadian Arctic[END_REF], and detrimental eff ects to growth and development.

The main route of PFOS exposure in humans is believed to be through the consumption of contaminated water and food. Traces of PFOS have been found in human plasma, PFOS has also been shown to cross the placenta and enter fetal circulation to aff ect early development by disrupting thyroid hormone homeostasis [7], glucose metabolism [8], and lipid metabolism [START_REF] Wan | PFOS-induced hepatic steatosis, the mechanistic actions on beta-oxidation and lipid transport[END_REF]10]. Maternal exposure to PFOS results in fetal brain accumulation during animal gestation [11]. PFOS causes peroxisome proliferation, and aff ects the enzymes and proteins that are involved in lipid metabolism, leading to metabolic disruption [START_REF] Bjork | Structure-activity relationships and [human relevance for perfl uoroalkyl acid-induced transcriptional activation of peroxisome proliferation in liver cell cultures[END_REF]. However, studies in laboratory rodents have primarily assessed blood lipid levels, including triglycerides, low-density lipoprotein, and high-density lipoprotein [START_REF] Wan | PFOS-induced hepatic steatosis, the mechanistic actions on beta-oxidation and lipid transport[END_REF]10,[START_REF] Bd | Exposure to perfl uorooctane sulfonate during pregnancy in rat and mouse. I: maternal and prenatal evaluations[END_REF], while studies in cultured cells [14] and fi sh embryos [15] have studied overall lipid peroxidation in the form of malondialdehyde (MDA). There are insuffi cient reports on the different classes and species of lipids and their oxidized products after PFOS exposure.

In the present study, we examined the levels of diff erent lipid precursors (focusing on polyunsaturated fatty acids (PUFAs)) and their oxidized metabolites in adult, maternal, and fetal mouse organs in the presence of PFOS. Our fi ndings help to elucidate the mechanism of PFOS-induced oxidative stress, which leads to detrimental eff ects on health and development.

Materials and methods

Animal handling

All animals were maintained and used in accordance with the guidelines of the Department of Health, the Government of Hong Kong Special Administrative Region. Experimental procedures were performed in accordance with the guidelines of Hong Kong Baptist University Committee on the Use of Human and Animal Subjects (Permit no. 261812). Female CD-1 mice (6 -8 weeks old) were purchased from the Laboratory Animal Service Centre of the Chinese University of Hong Kong. The mice were acclimated for one week before the experiments began. The conditions and well-being of the animals were closely monitored, and no death was recorded during the study. Mice were mated overnight and vaginal plugs were identifi ed the next day and counted as day 1 of gestation (GD1). Pregnant mice were randomized into two groups, control and PFOS treatment (3 mg/kg b.w.) groups, with n Ն 4 per group. The dose of 3 mg/kg was chosen based on a dose-dependent PFOS toxicity study, which showed that rats treated with 3 mg/kg exhibited signifi cant PFOS toxicity with moderate rate of mortality [16]. They were housed in polypropylene cages with sterilized bedding, under controlled temperature (20 -25 ° C) and humidity (35 -70%) with 12-h alternating light and dark cycles, and fed with standard lab chow (Lab-Diet 5001 Rodents Diet) and water ad libitum . Oral PFOS (98% purity, Sigma Aldrich) doses was prepared by dissolving the PFOS in dimethyl sulfoxide (DMSO; fi nal concentration, Յ 0.05%) and subsequently mixing it with corn oil. The control group was given corn oil with 0.05% DMSO. Both the control and treatment groups were fed daily via oral gavage until GD17. In a separate study, mature (non-pregnant) female mice were housed in the same setting and given PFOS (3 mg/kg b.w.) for 14 days.

Tissue collection and PFOS measurement

Maternal mice (F 0 ) were euthanized via cervical dislocation, and the unborn fetuses (F 1 ) were surgically harvested on GD17. Maternal blood was collected via cardiac puncture. Blood was allowed to clot and centrifuged at 3000 ϫ g for 15 min at 4 ° C to produce a serum sample. The collected serum was immediately stored at Ϫ 20 ° C until analysis. Maternal (liver and placenta) and fetal organs (liver, brain, and kidney) were harvested and washed with phosphate-buff ered saline containing 0.005% (w/v) butylated hydroxytoluene (BHT) and 0.005% (w/v) indomethacin. The harvested organs were snap frozen and stored at Ϫ 80 ° C until further analysis.

PFOS concentrations in maternal sera were measured as described previously [START_REF] Wan | Blood plasma concentrations of endocrine disrupting chemicals in Hong Kong populations[END_REF]. In brief, the serum sample was diluted and mixed with tetra-n-butyl ammonium hydrogen sulfate buff er and methyltert -butyl ether (MTBE). The organic phase was transferred, re-extracted with MTBE, and pooled. After drying under a stream of nitrogen, it was reconstituted with 10 mM ammonium acetate:acetonitrile (6:4, v/v) and analyzed by liquid chromatography -tandem mass spectrometry (LC -MS/MS, Agilent 1200 series, Agilent Technologies, USA). PFOS and labeled PFOS standards used for calibration were both prepared in methanol. A guard column (Zorbax Eclipse PlusC8, Agilent Technologies, USA) connected to Zorbax Eclipse Plus C8 column (100 mm ϫ 2.1 mm, 3.5 μ m, Agilent Technologies, USA) was established according to the procedure described by Zhao et al. [18] and used for the analysis.

Tissue lipid extraction

Tissue homogenization and extraction were performed as described previously [19] with modifi cations. Briefl y, tissue samples were homogenized in ice-cold Folch solution (chloroform:methanol, 2:1, v/v) with 0.01% (w/v) BHT. Aqueous 0.9% NaCl was added to allow phase separation. After centrifugation at 800 ϫ g for 10 min, at 4 ° C, the lower phase was transferred to a glass vial and the solvent was evaporated under a stream of nitrogen gas. The dried Folch extract was reconstituted with methanol and hydrolyzed overnight in the dark at room temperature with 1 M aqueous potassium hydroxide (1:1, v/v). After hydrolysis, the sample was neutralized and formic acid (pH: 4.5) was added [20]. The lipid extract mixtures were centrifuged to remove any protein precipitate before solid-phase extraction. For solid-phase extraction, 60-mg MAX columns (Oasis, Waters, USA) were used to purify the hydrolyzed samples according to the method described by Lee et al. [21].

Lipid analysis

Oxidized lipid product and fatty acid measurements [22] were performed using LC -MS/MS (1290 Infi nity, Agilent Technologies and Sciex 3200 QTRAP, Applied Biosystems, USA). A Kinetex HILIC column (30 ϫ 3 mm, 2.6 μ m, Phenomenex, USA) was used and maintained at room temperature. The mobile phase consisted of 0.1% acetic acid in water (A) and 0.1% acetic acid in methanol (B). A step-wise gradient elution from 60% B for 5 min, followed by increasing to 70% for 1 min, and 100% for a fi nal 1 min was used. The fl ow rate was set to 100 μ l/min for the fi rst 5 min, and subsequently to 200 μ l/min.

Cholesterol analysis

Cholesterol analysis was performed using the same setup for lipids with diff erent buff ers and the ESI mode [22]. The LC mobile phase of 5 mM ammonium acetate in 10% methanol (A) and 5 mM ammonium acetate in 85% methanol (B) was set to a constant 200 μ l/min fl ow rate. The mobile phase ran at a constant of 0% B phase from 0 to 4 min and gradually increased to 100% from 4 to 5 min, and then 100% B for a subsequent 2 min. The sample injection volume was 20 μ l. An ESI positive mode was applied for QTRAP 3200 triple quadrupole MS (Sciex Applied Biosystems, USA) with a source temperature of 400 ° C and capillary temperature of 250 ° C. The analytes were detected by MS/MS using MRM and the transitions were monitored as suggested by Shui et al. [23]. Cholesterol quantitation was achieved by relating the peak area with its corresponding internal standard peak. Both cholesterol and deuterated cholesterol standards were purchased from Avanti Polar Lipids (USA).

Antioxidant enzyme activity assays

Superoxide dismutase (SOD) and catalase (CAT) enzyme activities were analyzed using commercial kits from Cayman Chemical (Ann Arbor, MI, USA) according to the manufacture instructions. Briefl y, samples were homogenized and centrifuged, and the supernatants were removed for immediate analysis. Total protein concentrations in each supernatant were measured by the Bradford assay (Bio-Rad, USA) and enzyme activities were normalized to per 1 mg of total protein. One unit of SOD activity was defi ned as " the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical. " One unit of catalase activity was defi ned as " the amount of enzyme that will cause the formation of 1.0 nmol of formaldehyde per minute at 25 ° C. "

Statistical analysis

Statistical analysis was performed and fi gures were prepared using GraphPad Prism version 6.0 (GraphPad Software, CA, USA). All values were expressed as the mean Ϯ standard deviation (SD). Data were analyzed by Student ' s t-test and a p value of Ͻ 0.05 was denoted as statistically signifi cant.

Results

Fetuses mandatorily received PFOS from dams via placenta

In the study, prolonged PFOS exposure via oral administration markedly increased PFOS levels in the serum of CD-1 maternal mice (Figure 1) and was signifi cantly ( p Ͻ 0.0001) higher than that in the control group. The PFOS concentration was 41.98 Ϯ 8.20 μ g/ml in the serum of maternal mice after 16 days of treatment during pregnancy, which was approximately 175-fold higher than that in the control group. This confi rms that PFOS is delivered and retained in the body of treated pregnant mice for distribution to other organs. Due to the limited fetal blood specimens present, PFOS measurements in fetal serum were not obtained, but the transfer of PFOS from the maternal blood circulation to the fetus through the placenta is thought to be the only way for the fetus to be exposed to PFOS during gestation.

PFOS altered cholesterol and PUFA levels in the exposed fetus

To determine the eff ects of PFOS on lipid metabolism, endogenous levels of PUFAs and cholesterol were measured in both the control and PFOS-treated groups (Figure 2A -D). The organs were chosen (maternal liver, placenta, and fetal liver) based on the route of PFOS exposure. In the present study, we quantifi ed cholesterol and PUFA precursors, including arachidonic (AA, 20:4 n -6), eicosapentaenoic (EPA, 20:5 n -3), and docosahexaenoic (DHA, 22:6 n -3) acids. The levels of cholesterol and PUFA precursors in PFOS-treated fetal livers were consistently diff erent from those in the control, with signifi cant increases in AA, DHA ( p Ͻ 0.05), and cholesterol ( p Ͻ 0.001), while EPA decreased significantly ( p Ͻ 0.001). However, DHA in maternal liver and AA in placenta were both elevated ( p Ͻ 0.05) after PFOS treatment. No signifi cant changes were observed in other tissues after PFOS treatment compared with those in the control group.

PFOS induced lipid peroxidation primarily in the fetus

To probe the pathway in which PFOS participates to induce oxidative stress, non-enzymatic (Figure 3A -F) and enzymatic (Figure 4A -H) oxidized lipid mediators were quantifi ed. Non-enzymatic oxidized lipid products were measured, namely 15-F 2t -isoprostane (IsoP) and 5-F 2t -IsoP derived from AA, 8-F 3t -IsoP liberated from EPA, and 4( RS )-F 4t -neuroprostance (NeuroP) released from DHA, as well as 7 β -hydroxycholesterol and 7-ketocholesterol produced from cholesterol. Collectively, non-enzymatic oxidized lipid products were signifi cantly increased in the liver of PFOS-treated fetuses, but not in maternal liver and placenta. This indicates that PFOS treatment enhanced lipid peroxidation in developing fetuses, regardless of the precursor concentration. We also investigated enzymatically oxidized lipid products derived from the lipoxygenase (LOX) and cytochrome P450 (CYP450) pathways. Concentrations of the various hydroxyeicosatetraenoic acids (HETEs, derived from AA) released via the LOX (Figure 4A -D) and CYP450 pathways (Figure 4E -H) showed no diff erences between the two groups, except for a decrease in the 15( S )-HETE level in fetal liver and an increased 5( S )-HETE level in maternal liver in the PFOS group compared with those in the control. The concentration of 27-hydroxycholesterol was also increased in the fetal liver of the PFOS group compared with that in the control. The levels of oxidized lipid products generated by the enzymatic pathway are, therefore, inconsistent compared with the non-enzymatic pathway, showing the latter to be predominant in PFOS-associated lipid peroxidation.

Maternal antioxidant enzyme activities coped with PFOS-induced oxidative stress

Despite the substantial amount of PFOS that is delivered and retained in the maternal body, our data showed that maternal mice coped with the oxidative stress better than fetuses (Figures 3 and4). We hypothesized that the welldeveloped antioxidant enzyme system in the dam is the reason for this diff erence. Thus, we assessed the activities of the antioxidant enzymes SOD and CAT (Figure 5A -B). However, due to the limitation of organ samples, fetal livers were not available for these measurements. When PFOS and lipid peroxidation in maternal/fetal mice 5 evaluating placental antioxidant enzyme activities, we found that the activities of SOD and CAT in PFOS-treated maternal liver and placenta were not signifi cantly diff erent from those of the control.

Lipid peroxidation in fetal brain is in lesser extent than that in liver and kidney

To further evaluate the eff ects of PFOS-induced lipid peroxidation (focusing on PUFAs) in other vital organs such as brain and kidney, we conducted a separate experiment where the adult (non-pregnant) female mice and unpaired fetuses were exposed to the same dose of PFOS for a similar duration (fetuses: 17 days; adult: 14 days), and their levels of lipid precursors and oxidized products were measured. The lipid precursors analyzed in this study include AA, adrenic acid (AdA, 22:4, n -6; an elongated form of AA), EPA, and DHA. Various isoprostanoids and isofuranoids (generated under higher oxygen tension) derived from diff erent precursors were determined to assess their levels of lipid peroxidation after PFOS exposure. Table I illustrates the levels of lipid precursors and their mediators from adult and fetal mouse brain tissues. The PFOS-treated brain tissues showed signifi cant increases in AA ( p Ͻ 0.05) and AdA ( p Ͻ 0.01) but not in EPA and DHA, while the levels in fetal brain were not altered compared with those in the control group. Non-enzymatic oxidized lipid products in both adult and fetal brain tissues were not statistically signifi cant between the two groups, except for the increase in neurofuran and 8-F 3t -IsoPs ( p Ͻ 0.05) in PFOS-treated adult and fetal tissues, respectively. Similarly, enzymatically oxidized lipid products derived from the LOX and CYP450 pathways were quantifi ed in adult and fetal brain tissues (Table II). These products in PFOS-exposed adult mice brain tissues were not aff ected compared with those in control, but the fetal counterparts for 9( S )-HETE, 5( S )-HETE, and 8( S )-HETE production were signifi cantly inhibited. Identical sets of lipid precursors and oxidized lipid mediators were analyzed for adult and fetal kidneys (Table III). Seemingly, PFOS exposure appeared to have an apparent eff ect on both adult and fetal kidney lipid peroxidation. While the levels of all lipid precursors (except for DHA) and their non-enzymatic metabolites in the PFOS-treated fetal kidney increased compared with those in the control, lipid precursors and oxidized products in PFOS-treated adult kidney showed a signifi cant decrease in EPA, DHA, 5-F 2t -IsoP, and 4( RS )-4-F 4t -NeuroP, and 17( RS )-F 2t -dihomo-IsoP and 17( RS )-SC-∆ 15 -11-dihomo-IsoF from AdA, but 15-F 2t -IsoP (an overall in vivo oxidative stress biomarker) was elevated.

Finally, enzymatically oxidized lipid products in PFOStreated adult mice kidney had reduced 9( S )-, 11( S )-, 8( S )-, and 12( S )-HETE levels, whereas 5( S )-and 15( S )-HETE PFOS and lipid peroxidation in maternal/fetal mice 7 levels were increased compared with those in the control (Table IV). PFOS-treated fetal kidney showed a decreased amount of 5( S )-and 11( S )-HETEs, while 15( S )-HETE levels increased compared with the control group. Aside from these, other precursors and oxidized lipid products (Table IV) were not signifi cantly altered between the control and PFOS-treated groups.

(A) (B) (C) (D) (E) (F) (G) (H)

Discussion

The purpose of this study was to probe how PFOS aff ects lipid metabolism and induces oxidative stress by evaluating the levels of oxidized PUFAs and cholesterol products from enzymatic and non-enzymatic pathways. It is well documented that the elevation of these oxidized lipid products in vivo leads to metabolic and cognitive dysfunction and a risk of developing cardiovascular disease, diabetes, cancer, and neurodegeneration [21,24 -26].

In this study, we found that lipid precursor levels in PFOS-treated fetal, but not maternal livers were consistently elevated, with the exception of EPA. As reported by others, the increase in these lipid precursor levels is rationalized by the reduction in serum lipid levels, in which triglycerides and cholesterols are transported into the liver from the circulation and retained, leading to lipid vacuolation [27,28]. This is due to the upregulation of hepatic lipoprotein lipase and fatty acid translocase for lipoprotein traffi cking, and the uptake of free fatty acids with a simultaneous reduction in apolipoprotein-B100 level that is needed for very-low-density lipoprotein packaging and secretion [START_REF] Wan | PFOS-induced hepatic steatosis, the mechanistic actions on beta-oxidation and lipid transport[END_REF]10]. However, we noted a signifi cant reduction in EPA level in fetal liver, which may indicate a possible modifi cation of the enzymes that participate in the bioconversion of EPA to docosapentaenoic acid (DPA, 22:5 n -3) and DHA, such as Δ 5 -and Δ 6 -desaturases [29], in the presence of PFOS.

Based on previous fi ndings, prenatal exposure to PFOS in humans and rodents was approximately twice as much as what is present in the fetus, whether harvested from plasma or postnatal liver [START_REF] Luebker | Neonatal mortality from in utero exposure to perfl uorooctanesulfonate (PFOS) in Sprague-Dawley rats: dose-response, and biochemical and pharamacokinetic parameters[END_REF]31]. On average, mouse gestation is approximately 18 days, and the fetal samples used in this study were extracted on GD17, which is one day before term. Assuming that treated pregnant mice carry the same PFOS distribution ratio as their fetuses, we determined the representative oxidized lipid products derived from AA, EPA, DHA, and cholesterol via enzymatic and non-enzymatic pathways. The levels of non-enzymatic oxidized lipid products in PFOS-exposed fetal livers were consistently increased, but a similar trend was absent from maternal tissues. However, enzymatically oxidized lipid products derived from AA and cholesterol had a limited overall oxidation eff ect, with the exception of 15( S )-HETE and 27-hydroxycholesterol in fetal liver and 5( S )-HETE from maternal liver. Both 15( S )-and 5( S )-HETE are reported to have modulating roles during infl ammation [32]. Of note, F 2 -IsoPs (15-F 2t -IsoP and 5-F 2t -IsoP) derived from AA, and the " gold " standard for in vivo oxidative damage [24 -26], increased tremendously in PFOSexposed fetuses, implying that PFOS-treated fetuses were under oxidative stress. Taken together, our data suggest that PFOS aff ects fetal tissues more than maternal tissues. First, PFOS exposure consistently elevated total free PUFAs and cholesterol content in fetal liver. Additionally, lipid precursors were extensively and exclusively oxidized in fetal liver, which seemed to favor a non-enzymatic pathway rather than an enzymatic pathway. These results showed that PFOS-induced oxidative stress PFOS had larger eff ects in fetuses than in dams by releasing ROS that lead to lipid peroxidation. Subsequently, we questioned whether the PUFAs were oxidized in maternal tissues and transported to the fetus, or the accumulated PFOS within fetal tissues led to lipid peroxidation. The fact that the oxidized lipid products in maternal tissues showed insignifi cant diff erences in the presence and absence of PFOS eliminates the possibility that these oxidized products were transferred to the fetuses. Thus, we can assume that the elevation of lipid peroxidation in fetal livers was due to the presence of PFOS in the tissue.

The exclusive lipid peroxidation in fetal livers then led us to postulate that the maturity of fetal antioxidant system is not in place during early stages of fetus development, or it led to antioxidant system dysfunction that resulted in a high level of oxidative stress. It is noted that, depending on the species and organs, glutathione and antioxidant enzymes, such as SOD, CAT and glutathione peroxidase, are equally important to fetal development [26]. Regardless, SOD and CAT antioxidant enzyme activities were measured in maternal liver and placenta to test our hypothesis. Surprisingly, no signifi cant diff erences were observed in the level of each antioxidant enzyme in the presence of PFOS in both types of tissues. However, because of the sample limitations in fetuses, we remain inconclusive on whether the excess lipid peroxidation experienced in fetal tissues is due to the maturity of their antioxidant systems.

Furthermore, a recent study reports on the formation of free radicals from aqueous PFOS in the presence of iron under ultraviolet (UV) irradiation [33]. Although this is an in vitro study, reactive PFOS radicals are formed with the assistance of an exogenous UV source. High amounts of free iron are present in the fetus during pregnancy, and iron is known to facilitate lipid peroxidation, particularly through the non-enzymatic free radial reaction, to generate hydrogen peroxide, superoxide radical, and hydroxyl radical [34]. Hence, the high level of iron in developing fetuses could be a main culprit for the free radical generation in the presence of PFOS presented here. In a separate study, we analyzed the brain and kidney from adult female mice exposed to the same level of PFOS, which were then compared with the fetal organs. Adult and fetal mice exposed to PFOS showed elevated levels of F 2 -IsoPs in kidney, indicating the presence of oxidative stress. The high levels of oxidative damage found in the fetal kidney may have detrimental eff ects on developmental because F 2 -IsoPs is also reported to be an endogenous vasoconstrictor [START_REF] Roberts | Camille Oger] at 00:14 06 May 2015 PFOS and lipid peroxidation in maternal/fetal mice 11 are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism[END_REF]25]. In addition to F 2 -IsoPs, levels of oxidized lipid products derived from other precursors were increased in both adult and fetal kidney. Most notably, the isofuranoid group (IsoFs, NeuroFs, and 17( RS )-SC-∆ 15 -11-dihomo-IsoF), which is generated under high oxygen tension, was signifi cantly augmented by PFOS treatment compared with the control. The importance of the endogenous isofuranoids is unknown, but it appears to be a potentially more stable form of oxidized lipid products, in particular for the brain [START_REF] Roberts | Camille Oger] at 00:14 06 May 2015 PFOS and lipid peroxidation in maternal/fetal mice 11 are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism[END_REF]35,36]. Nevertheless, unlike the kidney, the mouse brain was not consistently aff ected, possibly because limited PFOS was taken up in the brain [37], which might have been potentially hindered by the blood -brain barrier. Although brain development matures at a much later stage than many other organs, the blood -brain barrier is a natural protector and is fully developed before birth [START_REF] Saunders | Bar-[rier mechanisms in the developing brain[END_REF].

It should be noted that this is the fi rst study to measure the precise oxidized lipid products of the non-enzymatic pathway, particularly 17( RS )-SC-Δ 15 -11-dihomo-IsoF, in mouse brain tissues. Recent measurements on 17( RS )-SC-Δ 15 -11-dihomo-IsoF levels were performed on pig and rat brain samples [35,36], which have distinctly diff erent results from our data. However, the species used, the maturity of the subjects, the region of the brain assessed (as whole brain was used in this study), and the sample sizes used might lead to the inconsistencies with the current result. Taken together, PFOS-induced lipid peroxidation seemed to aff ect tissues in the order of kidney Ͼ liver Ͼ brain.

Downloaded by [E R F I], [Camille Oger] at 00:14 06 May 2015

There are previous reports on the eff ects of prenatal PFOS exposure. Notwithstanding this, samples from these studies were mainly conducted at birth or postnatal stages. Additionally, the PFOS concentrations in the exposed dams were shown to increase steadily throughout gestation and postnatal stages [START_REF] Bd | Exposure to perfl uorooctane sulfonate during pregnancy in rat and mouse. I: maternal and prenatal evaluations[END_REF]16]. However, the amount of PFOS found in the pups was only measured at postnatal days, although they were exposed during the prenatal period. Therefore, PFOS levels were not previously investigated in paired dams and pups during gestation. To the best of our knowledge, this is the fi rst report that studies trans-generational eff ects of PFOS in mouse maternalfetal lipid metabolism during gestation.

In conclusion, we have shown, for the fi rst time, that PFOS exposure in pregnant mice induces notable lipid peroxidation in fetuses during their gestation. However, PFOS-induced fetal lipid peroxidation is organ-specifi c, and occurs primarily through the non-enzymatic lipid peroxidation pathway. Nevertheless, the detrimental eff ects of prolonged oxidative stress during gestation are not determined here. Therefore, a postnatal study needs to be employed to identify whether the eff ects on fetal development will continue at a later age due to altered lipid metabolism and if cognitive behaviors are aff ected.

Figure 1 .

 1 Figure 1. PFOS concentrations in maternal serum. Values in each column are expressed as the mean Ϯ SD. The number of sample replicates of maternal serum in the control group is n ϭ 4, and in the 3 mg/kg PFOS group it is n ϭ 4. * * * p Ͻ 0.001 compared with control.

Figure 2 .

 2 Figure 2. Concentrations of AA, EPA, DHA, and cholesterol in maternal and fetal livers and placenta. Panels represent AA (A), EPA (B), DHA (C), and cholesterol (D). Values in each column are expressed as the mean Ϯ SD. C: control; T: PFOS-treated. The number of sample replicates of maternal liver is n ϭ 4, and that of placenta and fetal liver is n ϭ 8. * p Ͻ 0.05, * * * p Ͻ 0.001 compared with control.

Figure 3 .

 3 Figure 3. Non-enzymatically oxidized lipid products of PUFAs after PFOS exposure. Panels represent F 2 -IsoPs (A -B) derived from AA, F 3 -IsoP (C) derived from EPA, F 4 -NeuroP (D) derived from DHA, and 7 β -hydroxycholesterol (E) and 7-ketocholesterol (F) derived from cholesterol. Values in each column are expressed as the mean Ϯ SD. C: control; T: PFOS-treated. The number of sample replicates of maternal liver is n ϭ 4, and that of placenta and fetal liver is n ϭ 8. * p Ͻ 0.05, * * * p Ͻ 0.001 compared with control.

Figure 4 .

 4 Figure 4. Enzymatically oxidized lipid products of AA and cholesterol. Panels represent products from the LOX (A -E) and CYP450 (F -H) pathways. Values in each column are expressed as the mean Ϯ SD. C: control; T: PFOS-treated. The number of sample replicates of maternal liver is n ϭ 4, and that of placenta and fetal liver is n ϭ 8. * p Ͻ 0.05 compared with control.

Figure 5 .

 5 Figure 5. Antioxidant enzyme activities in maternal liver and placenta. Panels represent SOD (A) and CAT (B) enzyme activities. Enzyme activities are expressed as unit per mg of total protein. Values in each column are expressed as the mean Ϯ SD. The number of sample replicates of maternal liver and placenta is n ϭ 4.

Table I .

 I Level of PUFAs and their non-enzymatic oxidized lipid products in adult and fetal mice brain after PFOS treatment.

			Brain
			Adult	Fetus
		Control	PFOS-treated	Control	PFOS-treated
	Lipid precursors
	AA ϩ	4.27 Ϯ 2.13	9.3 Ϯ 1.04 *	4.60 Ϯ 0.48	5.10 Ϯ 0.60
	AdA	75.18 Ϯ 13.40 147.15 Ϯ 5.61 * * 82.55 Ϯ 8.86	85.72 Ϯ 9.70
	EPA	6.96 Ϯ 0.38	8.69 Ϯ 1.38	47.15 Ϯ 14.69	45.27 Ϯ 15.59
	DHA	459.46 Ϯ 96.62 556.00 Ϯ 69.78 551.33 Ϯ 83.53 492.43 Ϯ 75.57
	Isoprostanoids
	15-F 2t -IsoP	9.58 Ϯ 2.67	9.56 Ϯ 3.35	1.50 Ϯ 0.42	1.38 Ϯ 0.28
	5-F 2t -IsoP 8-F 3t -IsoP	31.34 Ϯ 13.65 57.18 Ϯ 62.87 9.80 Ϯ 0.30 7.90 Ϯ 1.68	6.95 Ϯ 1.26 0.61 Ϯ 0.12	5.77 Ϯ 2.76 0.80 Ϯ 0.21 *
	4( RS )-4-F 4t -NeuroP	37.64 Ϯ 17.74 34.81 Ϯ 9.79	32.06 Ϯ 7.79	31.21 Ϯ 7.68
	17( RS )-F 2t -dihomo-IsoP	14.35 Ϯ 3.04	18.14 Ϯ 12.50	2.67 Ϯ 0.71	2.59 Ϯ 0.42
	Isofuranoids
	IsoF	3.73 Ϯ 1.30	5.50 Ϯ 1.25	0.52 Ϯ 0.17	0.50 Ϯ 0.09
	NeuroF	13.56 Ϯ 2.90	29.08 Ϯ 8.30 *	37.86 Ϯ 12.15	41.72 Ϯ 11.23
	17( RS )-SC -Δ 15 -11-dihomo-IsoF	4.47 Ϯ 1.39	4.28 Ϯ 1.20	0.39 Ϯ 0.18	0.33 Ϯ 0.16

Concentration values are expressed in mean Ϯ SD, ng/g of tissue except ϩ μ g/g tissue. * p Ͻ 0.05, * * p Ͻ 0.01 compared with control. Adult: n ϭ 4; Fetus: n ϭ 8.

Table II .

 II Level of enzymatic oxidized lipid products of AA via LOX and CYP450 pathways measured in adult and fetal brain after PFOS exposure. Concentration values are expressed in mean Ϯ SD, ng/g tissue. * p Ͻ 0.05, * * p Ͻ 0.01, * * * p Ͻ 0.001 as compared with control. Adult: n ϭ 4; Fetus: n ϭ 8.

			Brain
		Adult		Fetus
		Control	PFOS-treated	Control	PFOS-treated
	CYP450-mediated
	9( S )-HETE	13.88 Ϯ 2.00	17.80 Ϯ 4.44	14.16 Ϯ 5.37	8.99 Ϯ 1.53 *
	11( S )-HETE	68.81 Ϯ 8.30	71.88 Ϯ 12.28	58.44 Ϯ 22.71	40.21 Ϯ 10.75
	20-HETE	94.49 Ϯ 5.40	109.65 Ϯ 15.74	63.72 Ϯ 23.73	47.22 Ϯ 11.81
	LOX-mediated
	5( S )-HETE	39.42 Ϯ 12.27	46.29 Ϯ 13.78	41.87 Ϯ 9.31	26.05 Ϯ 6.53 * *
	8( S )-HETE	22.81 Ϯ 1.38	28.69 Ϯ 7.49	21.62 Ϯ 6.70	15.49 Ϯ 2.55 *
	12( S )-HETE	113.05 Ϯ 12.96	94.13 Ϯ 8.85	48.55 Ϯ 12.56	44.95 Ϯ 9.28
	15( S )-HETE	9.93 Ϯ 0.78	9.56 Ϯ 2.39	10.77 Ϯ 3.77	9.27 Ϯ 2.96

Table III .

 III Level of PUFAs and their non-enzymatic oxidized lipid products in adult and fetal mice kidney after PFOS treatment.

			Kidney
			Adult		Fetus
		Control	PFOS-treated	Control	PFOS-treated
	Lipid precursors
	AA	145.95 Ϯ 36.81 162.86 Ϯ 46.79	6.83 Ϯ 4.67	24.49 Ϯ 4.09 * * *
	AdA	0.47 Ϯ 0.06	0.39 Ϯ 0.11	0.18 Ϯ 0.11	1.33 Ϯ 0.91 * *
	EPA	485.09 Ϯ 20.60 259.23 Ϯ 10.20 * * * 43.72 Ϯ 12.36 72.64 Ϯ 16.37 * *
	DHA ϩ	4.69 Ϯ 0.27	2.82 Ϯ 0.77 *	0.40 Ϯ 0.06	0.46 Ϯ 0.10
	Isoprostanoids
	15-F 2t -IsoP	89.02 Ϯ 6.00	114.62 Ϯ 6.55 * *	12.91 Ϯ 11.81 45.40 Ϯ 18.72 * * *
	5-F 2t -IsoP ϩ 8-F 3t -IsoP	0.33 Ϯ 0.08 33.04 Ϯ 5.82	0.08 Ϯ 0.01 * * 24.77 Ϯ 3.93	0.46 Ϯ 0.04 39.95 Ϯ 6.03	5.03 Ϯ 2.95 * * * 69.78 Ϯ 10.83 * * *
	4( RS )-4-F 4t -NeuroP	68.41 Ϯ 5.75	56.70 Ϯ 1.32 *	9.06 Ϯ 8.18	20.19 Ϯ 4.16 * *
	17( RS )-F 2t -dihomo-IsoP	33.75 Ϯ 2.62	22.18 Ϯ 2.54 * *	7.66 Ϯ 6.64	25.63 Ϯ 3.59 * * *
	Isofuranoids
	IsoF	24.51 Ϯ 3.71	17.58 Ϯ 3.63	6.09 Ϯ 5.62	20.02 Ϯ 5.57 * * *
	NeuroF	43.17 Ϯ 6.45	50.80 Ϯ 8.42	16.45 Ϯ 2.55	36.29 Ϯ 8.88 * * *
	17( RS )-SC-Δ 15 -11-	18.15 Ϯ 1.85	4.54 Ϯ 0.73 * * *	3.79 Ϯ 3.25	16.15 Ϯ 2.65 * * *
	dihomo-IsoF

Concentration values are expressed in mean Ϯ SD, ng/g tissue except ϩ μ g/g tissue. * p Ͻ 0.05, * * p Ͻ 0.01, * * * p Ͻ 0.001 compared with control. Adult: n ϭ 4; Fetus: n ϭ 8.

Table IV .

 IV Level of enzymatic oxidized lipid products of AA via LOX and CYP450 pathways in adult and fetal kidney after PFOS exposure. Concentration values are expressed in mean Ϯ SD, μ g/g tissue except ϩ ng/g tissue. * p Ͻ 0.05, * * p Ͻ 0.01, * * * p Ͻ 0.001 compared with control. Adult: n ϭ 4; Fetus: n ϭ 8.

			Kidney
			Adult		Fetus
		Control	PFOS-treated	Control	PFOS-treated
	CYP450-mediated
	9( S )-HETE ϩ	151.86 Ϯ 15.60	42.87 Ϯ 1.97 * * *	51.35 Ϯ 10.91	61.24 Ϯ 15.23
	11( S )-HETE	1.86 Ϯ 0.32	0.81 Ϯ 0.14 * *	2.46 Ϯ 0.35	0.19 Ϯ 0.04 * * *
	20-HETE	3.07 Ϯ 0.78	2.64 Ϯ 0.31	8.22 Ϯ 2.39	7.61 Ϯ 3.21
	LOX-mediated
	5( S )-HETE	0.85 Ϯ 0.14	3.00 Ϯ 0.33 * * *	1.07 Ϯ 1.04	0.19 Ϯ 0.04 *
	8( S )-HETE	0.30 Ϯ 0.02	0.09 Ϯ 0.01 * * *	2.00 Ϯ 0.69	1.59 Ϯ 1.50
	12( S )-HETE	2.44 Ϯ 0.58	0.36 Ϯ 0.01 * *	15.80 Ϯ 2.03	0.02 Ϯ 0.01
	15( S )-HETE ϩ	74.79 Ϯ 21.20	266.66 Ϯ 51.73 * *	3.84 Ϯ 1.77	11.99 Ϯ 7.02 * *

Downloaded by [E R F I], [Camille Oger] at 00:14 06 May 2015

Acknowledgements

This work was supported by General Research Fund (HKBU261812) and the University of Hong Kong Seed Funding Programme for Basic Research (No. 104002391 and 104003173).

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.

Supplementary material available online

Supplementary Table I to be found at online http://informahealthcare.com/doi/abs/10.3109/10715762.2015.1027199.


Ayyaz Mahmood Paracha

Philippe Basset
email: p.basset@esiee.fr

Frédéric Marty

Adrian Vaisman Chasin

Patrick Poulichet

Tarik Bourouina

A HIGH POWER DENSITY ELECTROSTATIC VIBRATION-TO-ELECTRIC ENERGY CONVERTER BASED ON AN IN-PLANE OVERLAP PLATE (IPOP) MECHANISM

Energy converter IC (sensor signal conditioning + RFID/ZigBee tag) Antenna Sensor Distributed sensor network Fig. 1 -Project Overview: the vibration-to-electricity converter is the power source for nodes in a distributed network. Each node includes a sensor, a chip-size antenna [4] and an IC.

INTRODUCTION

Various efforts have already been made to reduce the power consumption of sensors, and to make them autonomous i.e. independent of externally attached power sources [START_REF] Roundy | Micro-electrostatic vibration-to-electricity converters[END_REF]. Hence energy should be harvested from the ambient environment to power these sensors. This concept has lot of advantages: number of wires used for the electrical connections in between the main power source and different sensors can be reduced, device's lifetime increases, cost maintenance decreases as there is no need to replace the external batteries. In addition, various chemical materials, currently being used in power sources, can be eliminated. In order to provide autonomous nodes in a sensor network (cf. Fig. 1) and to power smart-dusts [START_REF] Basset | Chip-size antennas for implantable sensors and smart dusts[END_REF], ambient vibration is a power source that is largely considered [START_REF] Beeby | Energy harvesting vibration sources for Microsystems applications[END_REF], as there exists broadband vibration spectrum in applications like automotives [START_REF] Despesse | Fabrication and Characterization of High Damping Electrostatic Micro Devices for Vibration Energy Scavenging[END_REF], air planes, etc…

The three main mechanisms to harvest energy from environmental ambient vibrations are electrostatic [START_REF] Lim | Design of a novel highly capacitive MEMS-based vibration to electricity energy converter with suspended in-plane overlap technology[END_REF][START_REF] Roundy | Micro-electrostatic vibration-to-electricity converters[END_REF][START_REF] Despesse | Fabrication and Characterization of High Damping Electrostatic Micro Devices for Vibration Energy Scavenging[END_REF], piezoelectric [START_REF] Marzencki | Design and fabrication of piezoelectric micro power generators for autonomous microsystems[END_REF] and electromagnetic [START_REF] Glynne-Jones | An electromagnetic, vibration-powered generator for intelligent sensor systems[END_REF]. There are no definite rules to make a choice among these mechanisms, as it all depends on an application's requirement. But still CMOS compatible MEMS converters, with a high power generation, are a challenge. Among above three defined types, electrostatic-based energy harvesters are more compatible with CMOS process and are considered good for miniaturization.

Electrostatic converters need two sets of electrodes. First one is attached to a moving mass called as proof mass and second one is fixed to the substrate. A huge proof mass is normally used to target a low resonance frequency. External vibrations force the mobile part to move relatively to the substrate, which results in a mechanical-to-electrical energy transduction, if a constant charge or voltage is maintained on the electrodes while the capacitance decreases [START_REF] Meninger | Vibration-to-electric energy conversion[END_REF].

A. M. Paracha, P. Basset, F. Marty, A. V. Chasin, P. Poulichet, T. Bourouina An electrostatic vibration-to-electric energy converter based on an IPOP mechanism

CONVERTER DESIGN

Vibrations with amplitude below 100 µm are often converted into electric energy thanks to a resonant combdrive variable capacitor, with either In-Plane Gap-closing Combs (IPGC) [START_REF] Despesse | Fabrication and Characterization of High Damping Electrostatic Micro Devices for Vibration Energy Scavenging[END_REF] or In-Plane Overlap Combs (IPOC) [START_REF] Meninger | Vibration-to-electric energy conversion[END_REF]. Another type of variable capacitor, based upon simple parallel plate architecture, is Out-of-Plane Gap-Closing Plates (OPGP) [START_REF] Miao | Micro-machined variable capacitors for power generation[END_REF]. Instead, recently some works have been reported to use two overlapping sets of planar electrodes with in-plane motion [START_REF] Sterken | Motion based generators for industrial applications[END_REF][START_REF] Tsutsumino | Micro seismic electret generator for energy harvesting[END_REF][START_REF] Lim | Design of a novel highly capacitive MEMS-based vibration to electricity energy converter with suspended in-plane overlap technology[END_REF]. Hence this topology is termed as In-Plane Overlap Plates (IPOP). 3-D views of the presented variable capacitors design, based on this topology, are shown in Fig. 2. The devices are based on a silicon-glass technology [START_REF] Tassetti | New tunable RF MEMS Microinductor design[END_REF]. IPOP resembles the concept of traditional parallel plate capacitor, with one set of electrodes patterned on the underside of silicon proof mass and other on top of glass wafer. By virtue of mechanical vibrations present in the environment, proof mass oscillates laterally. Hence the capacitance varies due to a change in the electrode's overlap area.

The IPOP topology has the following advantages. Large value of maximum capacitance is achievable, due to small air gap between the sets of top and bottom electrodes, leading to high power generation. The complexity of the fabrication process is reduced, as the proposed designs do not have any closely spaced comb drive architecture. So tolerance of misalignment is relatively higher as compared to other topologies which have comb drive variable capacitor. The IPOP also exhibits less fluid damping: as there is no compression of air, squeeze film damping is almost negligible.

The basic difference between the two proposed designs (cf. Fig. 2) is their proof mass. In the comb configuration (CC) 19 % of the total mass of silicon is removed from the zone present in between the electrodes to reduce the parasitic capacitance. It results in reduction of overall mass of the device, hence increasing its natural frequency as compared to the proof mass configuration (PC) of the same dimensions. Both converters have been designed to resonate at 300 Hz. Their dimensions are 11 x 6.5 x 0.9 mm 3 , without including the connection pads. Vibration amplitude is limited to +/-50 µm by the help of mechanical stoppers. This corresponds to the maximum capacitance variation. A 380 µm-thick silicon wafer is being used. Use of the thick wafer helps us in two ways. Firstly, net mass of the proof mass is increased, which helps us to reduce resonance frequency of the device, hence a low frequency vibration sources can be targeted. Secondly, out-of-plane vibrations are limited due to the high aspect-ratio springs, having 30 µm width and 380 µm height.

Fig. 3 shows FEM simulations of capacitive variation of the designs. Theoretically minimum capacitance C min should be equal to zero, but in real a parasitic capacitance is always present which is evident from the curves. The capacitance is composed of three components: the substrate parasitic capacitance C sub , fringe field capacitance C ff between the two sets of electrodes and linear capacitance C lin . To reduce C min , C sub and C ff has to be minimized. C min 's value in PC is quite higher as compared to CC. Reducing 19% of total mass from PC, results in 25% reduction of C min in CC. The non-linearities in the capacitance variations with displacements are due to the electrostatic fringe fields between top and bottom electrodes, as shown by the simulation of the capacitance's variation of face-to-face aluminum electrodes. C ff can be reduced by designing different electrode's width. However it is never desirable to have C min close to zero, because voltage will approach infinity and unwanted electrode stiction could occur. The last curve in Fig. 3 represents variation of C lin . It is calculated by the following analytical formula:

( )

x W g t L N x C F AIR r NIT F r o lin - + = ε ε ε 2 ) ( , ( 1
)
where N, ε r , ε o , L F , W F , t NIT , g AIR , and x are number of fingers, relative permittivity of dielectric, permittivity of free space, finger length, finger width, dielectric thickness, air gap present between two electrodes and lateral displacement of proof mass respectively.

A. M. Paracha, P. Basset, F. Marty, A. V. Chasin, P. Poulichet, T. Bourouina An electrostatic vibration-to-electric energy converter based on an IPOP mechanism The most important issue is the through-wafer DRIE for silicon. At ESIEE, the silicon etching is performed using Alcatel tm 601E plasma etcher. To achieve the vertical anisotropic deep plasma etching, the Bosch process is used. For our designs through-wafer silicon etching is required. But it is critical as our design has different beam widths as small as 30 µm and wide range of openings from 50 µm (gap between mechanical stopper and proofmass) to mm² holes (used to access metal contacts on a lower wafer). As thick silicon wafer is used, DRIE becomes particularly delicate because of the ARDE (Aspect Ratio Dependent Etching) and notching effects. The high aspect-ratio of the structures is obtained using a specific mixed RF/LF pulsed process allowing smooth sidewalls, perfect anisotropy and no negative effect of over-etching time [START_REF] Marty | Advanced etching based on deep reactive ion etching for silicon high aspect ratio microstructures and three dimensional micro and nano structures[END_REF]. Fig. 5 shows the microscopic view of the silicon wafer's backside after DRIE. It can be seen that the mechanical springs are fully released. The dark area shows the silicon dioxide from where the bulk silicon has been removed. Top view of the final device is shown in Fig. 6.

A. M. Paracha, P. Basset, F. Marty, A. V. Chasin, P. Poulichet, T. Bourouina An electrostatic vibration-to-electric energy converter based on an IPOP mechanism

CHARACTERIZATION

The device's mechanical characterization has been done using a vibration table from Physik Instrumente tm . During the test, a sine wave is applied on the input of a dedicated current amplifier, generating a sinusoidal displacement of the table. While sweeping the frequency, the relative displacement of the structure's proof mass had been observed with an optical profiler from Weeco tm . For an unbounded silicon wafer, the resonance frequency of the PC and CC is found to be 307 Hz, which is in accordance with the simulations done in Coventorware tm . After the wafer bonding, the resonance frequency is found to be 290 Hz for the CC and 255 Hz for the PC. The decrease in resonance frequency is due to air being trapped in between the mobile and fixed parts after the bonding. The full wafer being excited with input amplitude of +/-5 µm, Fig. 7 shows, for the two designs, the maximum value of the relative displacement of proof mass versus frequency.

Experimental values of C max and C min , when the potential of the silicon substrate is floating, are reported in Fig. 3. The maximum and minimum capacitances for the PC are 214 and 140 pF respectively. For CC, corresponding C max and C min are 214 and 80 pF. If substrate is grounded, most of the substrate parasitic capacitance is cancelled, so C max and C min in both cases are reduced by 33 pF.

The harvested energy, with an ideally lossless electronic, is given by the following equation [START_REF] Meninger | Vibration-to-electric energy conversion[END_REF]:

( ) ⎟ ⎟ ⎠ ⎞ ⎜ ⎜ ⎝ ⎛ - = min max min max 2 2 1 C C C C V E in , ( 2
)
where V in is the starting voltage of the system. Power generated is calculated by multiplying the result of above equation with twice the resonance frequency, since in a single period the device capacitance reaches C min two times. For V in equals to 5 V, the harvested power would be 0.72 and 2.6 µW for the PC and CC respectively, when the potential of the silicon substrate is floating. Hence the power densities for PC and CC are 11.26 and 40.62 µW/cm 3 respectively. When substrate is grounded, the corresponding harvested power for PC and CC becomes 0.82 µW and 3.74 µW. Thus resulting in a power density of 12.95 µW/cm 3 and 59 µW/cm 3 , for PC and CC respectively.

POWER DENSITY IMPROVEMENT

In the CC 19% of total mass of silicon is removed as compared to the PC, resulting in decrease in the parasitic capacitance, hence giving a high generated power density. But this mass reduction undesirably increases the resonance frequency of the device. So spring stiffness should be reduced to make system apt for low frequency applications. As the resonance frequencies of the most useful vibration sources lie below 300 Hz. In order to achieve a system which can work at such a low frequency, we cannot afford to loose a huge mass. Hence some improvement is required to reduce the parasitic capacitance but without sacrificing lot of mass. Electric field inside the silicon acts as a big parasitic capacitance. It is strong near the top electrodes when they are positioned at a minimum capacitance point, as there is a direct overlap of the silicon substrate and aluminum electrodes on glass wafer. So in order to reduce the parasitic capacitance, silicon between the electrodes having a high permittivity and being not highly resistive should be replaced by air. It can be done by throughwafer etching as a case of the CC but the problem is the Fig. 9 -Effect of backside DRIE on maximum and minimum capacitance (FEM simulation based result) Fig. 10 -Simulated electronic power processing circuit loss of mass. To avoid this inconvenience, we propose to etch small depth of silicon present in between the top electrodes. This step is called as Backside DRIE. Fig. 8 shows a side view of modified design. Fig. 9 shows a variation in C max and C min as a function of backside DRIE depth, based on FEM simulations, using Coventor tm . It can be concluded that through-wafer etching is not required. The value of C min decreases quite rapidly with an etched depth, and after 20 µm it almost becomes constant. At this point backside DRIE results in only 2.5% mass loss. The back side DRIE has no notable effect on the value of C max . This minor mass change improves our capacitance variation, from 64 pF as in a case of the PC and 114 pF for the CC to 156 pF. Based on these FEM simulation results we can estimate an improvement in our power density. At a resonance frequency of 300 Hz with the loss less ideal electronic circuit, and a starting voltage of 5 V, the power density becomes 76.71 µW/cm 3 . So an additional fabrication step i.e. backside DRIE of the silicon wafer has been added to our fabrication process. Before doing through-wafer top DRIE to release movable proof-mass, a depth of 20 µm silicon is etched, in a zone between the top electrodes. The remaining fabrication steps are exactly the same.

POWER PROCESSING CIRCUITRY

For an implementation of the power processing electronics, we have chosen to optimize the electronic circuit described in [START_REF] Yen | A variable capacitance vibration-to-electric energy harvester[END_REF]. It is composed of two main parts: charge pump and flyback circuitry, as shown in Fig. 10. The charge pump allows an accumulation of the electric charges. It is made up of two diodes D 1 and D 2 , and the variable capacitor C var . D 1 and D 2 function like switches, whereas C var is an energy harvesting source. When a sufficient quantity of charge is accumulated in the temporary storage C store , it is then transferred periodically to the reservoir C RES , using the flyback circuitry, whose configuration is same as of a buck chopper. This circuit includes a transistor represented by a switch, a free wheeling diode D FLY and an inductance L FLY . One condition to be respected is C RES >> C STORE in order to maintain constant voltage across load.

There are three drawbacks associated with this architecture. Firstly it works well only at high resonance frequencies above about 1 kHz. Secondly for a too small value of C min , there is no positive energy conversion. Thirdly there is a need of an additional circuit to control the switch in flyback circuitry.

For the switch we use a MOS transistor. The clock signal, used to make the transistor in a conduction state, has a significant influence on the circuit's power conversion efficiency. A set of simulations was performed to study the impact of the clock signal on the output voltage. Following values of different circuit components are kept fixed during simulations: C RES = 2 µF, R Load = 20 MΩ, L FLY = 4 mH, V in = 5 V, C max = 208 pF, C min = 47 pF and Freq = 300 Hz. Fig. 11 shows a relation of the clock's pulse width with the value of the output voltage and C STORE . The optimum value of the pulse width is around 2 µsec with a value of C STORE around 2.2 nF. If the pulse width becomes too large, the differential voltage in the inductance becomes negative and with the transistor in a conduction state, the two diodes are on. As a result a short circuit is created and there is a no transfer of charge to R LOAD . Fig. 12 shows the dependence of the output voltage on the value of a clock period. An optimum value of the clock period lies between 33 ms and 36 ms, which means ten energy conversion mechanical cycles for one feedback process. An optimum value of C STORE is 2.2 nF. Hence on the basis of these simulations we can conclude that the pulse width determines the fall of output voltage after one conduction cycle of the transistor whereas the clock period determines the peak of the output voltage. Simulations also show that for the pulse width higher then 1.5 µs, the output voltage is directly proportional to the size of Still we cannot have any optimized circuit with positive energy conversion for our device, as we are targeting very low frequencies. However it is observed that in order to have a circuitry that works well at low frequency the value of C min also needs an adjustment. For the circuit optimization at low frequencies all the circuit parameters have been kept the same as described above. The value of C STORE is chosen 2.2 nF, clock pulse width is kept 2 µsec and clock period is five times bigger then mechanical vibration period, based on above optimization results. Then frequency has been reduced and C min has been increased step by step. A successful simulation has been done for the vibration frequency of 410 Hz, and for the values of C max and C min of 368 pF and 94 pF respectively. The simulation results are shown in Fig. 13. V CVAR and V CSTORE are the voltages across C VAR and C STORE respectively. Fig. 14 is a zoom of V OUT , which shows there is positive power conversion.

CONCLUSION

We have presented a resonant electrostatic vibration-toelectricity converter using an In-Plane Overlap Plate topology. Batch CMOS-compatible process is performed on a typical silicon wafer that is eventually bonded with a glass wafer. High capacitance value, due to a vertical gap of 1.5 µm between the top and bottom electrodes, allows high energy conversion. Dimensions of the device without including the pads are 11 x 6.5 x 0.9 mm 3 . Maximum and minimum capacitance values are 181 and 47 pF respectively, if the silicon substrate is grounded. The harvested power density would be 58 µW/cm 3 at 290 Hz, for a starting voltage of 5 V and a theoretically lossless electronic. The parasitic capacitance has a strong impact on the generated power. We have partially removed the silicon substrate from the proof mass by through-wafer DRIE, in the case of the CC, to reduce it. This leads to an increase in the power density but with a considerable mass A. M. Paracha, P. Basset, F. Marty, A. V. Chasin, P. Poulichet, T. Bourouina An electrostatic vibration-to-electric energy converter based on an IPOP mechanism decrease. This is totally undesirable as the most common ambient vibrations are below 300 Hz. Hence we propose an enhancement in the fabrication process i.e. backside DRIE, which means to etch small silicon depth in a zone present between the top electrodes. It allows us to reduce parasitic capacitance without loosing a too much mass. By simulations we have shown that with a lossless electronics, starting voltage of 5 V, at a mechanical resonance frequency of 300 Hz and with Backside DRIE of 20 µm the power density becomes 76.71 µW/cm 3 . It results only in 2.5 % mass loss as compared to 19 % mass loss in the CC. Finally a power processing electronic circuitry has been studied and corresponding problems for low frequency applications have been highlighted. Also an optimization of various parameters which effect the output voltage has been discussed.

Fig. 2 -

 2 Fig. 2 -Proposed designs: a) Proof mass configuration (PC), b) Comb configuration (CC)

Fig. 3 -Fig. 4 -Fig. 5 -Fig. 6 -

 3456 Fig. 3 -FEM simulation of capacitance variation for the two presented designs

Fig. 7 -

 7 Fig. 7 -Maximum relative displacement of proof mass versus frequency with sinusoidal input excitation

Fig. 8 -

 8 Fig. 8 -Modified design with backside DRIE

Fig. 11 -Fig. 12 -Fig. 13 -Fig. 14 -

 11121314 Fig. 11-Output voltage versus pulse width

©EDA Publishing/DTIP 2007 ISBN: 978-2-35500-000-3

ACKNOWLEDGEMENTS

Authors would like to thank the SMM team in ESIEE for their extensive cooperation during the fabrication process in the clean room. This work is partially funded by the French Research Agency (ANR, Agence Nationale pour la Recherche) and is supported by the French competitiveness cluster "Ville et Mobilité Durable".


Cédric Durand

Fabrice Casset

Pascal Ancey

Fabienne Judong

Alexandre Talbot

R Quenouillere

Denis Renaud

Stéphan Borel

Brigitte Florin

Lionel Buchaillot

Silicon

Rémi Quenouillère

SILICON ON NOTHING MEMS ELECTROMECHANICAL RESONATOR

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

INTRODUCTION

Recent demand in single chip, multi-standard wireless transceivers has focused research efforts towards developing integrated Radio Frequency (RF) Micro or Nano Electro Mechanical Systems (MEMS or NEMS) in order to replace existing off-chip oscillators like quartz or ceramics. The small sizes of the electromechanical resonators and their single wafer, multi frequencies possibilities has focussed tremendous interest in the development of demonstrators using standard CMOS process.

Table 1 gives an overview of the state of the art on the MEMS electromechanical resonators. We can distinguish different resonator families: vibrating beam [START_REF] Nguyen | High-Q HF Microelectromechanical Filters[END_REF], longitudinal beam [START_REF] Koskenvuori | Active post processing method to increase the electro mechanical coupling in microresonators[END_REF], bulk square extensional plate [START_REF] Kaajakai | Square-Extensional Mode Single Crystal Silicon Micromechanical RF resonator[END_REF], elliptic [START_REF] Pourkamali | High Frequency Capacitive Micromechanical Resonators with Reduced Motional Resistance using the HARPSS Technology[END_REF] or contour [START_REF] Nguyen | 1.51GHz nanocrystalline diamond micromechanical disk resonator with material mismatched isolating support[END_REF] mode disk, rings [START_REF] Nguyen | Micromechanical "Hollow-disk" ring resonators[END_REF], solid dielectric capacitive gap resonator [START_REF] Lin | Vibrating micromechanical resonator with solid dielectric capacitive transducer gaps[END_REF] and bulk mode beam [START_REF] Ayazi | High aspect ratio SOI Vibrating Micromechanical resonators and filters[END_REF]. We can focus on the simplest case to understand how a resonator works. For a vibrating beam, we can express the mechanical resonant frequency with the following simplified equation:

2 , r n Beam R L h E A f ρ = Equation 1
A n is a coefficient depending on the harmonic of vibration. E is the Young modulus, ρ the density, h and L r respectively the thickness and the length of the beam which have an "out of plan" displacement (Figure 1). Thus, the resonant frequency depends on the material properties and on the movable mechanical structure dimensions photolithographically defined.

The vibrating beam is polarized with a DC bias V p . The RF signal is applied to the resonator. At the resonant frequency, the vibrating beam presents a maximum displacement detected by an output electrode as detailed in Figure 1. The electrode to resonator initial gap d 0 has to be as small as possible because the motional resistance R x (illustrates the level of signal transmitted to the resonator) is strongly dependant on this factor as explained in the following equation [START_REF] Lin | Vibrating micromechanical resonator with solid dielectric capacitive transducer gaps[END_REF]:

) ( 2 2 2 0 4 0 2 0 ) ( 1 airgap r P r airgap x Q S d V k R ε ε ω = Equation 2
k r is the stiffness, ω 0 the pulsation, ε 0 the air dielectric constant, ε r the material dielectric constant, S the electrode surface, and Q the quality factor. For an integrated resonator we aim to obtain a motional resistance between 50 ohms and 10kohms. The resonant frequency increases when the beam stiffness increases, so smaller resonator sizes induce a resonant frequency increase. Moreover, the smaller the gap, the smaller the motional resistance. Thus reducing the size is a major challenge for resonators development. The resonator performances described in the state of the art demonstrate the electromechanical resonators potentialities for quartz replacement. However, temperature stability, packaging, motional impedance constitutes major challenges to the integration of multi frequencies MEMS or NEMS resonators in Integer Circuits (IC).

In this paper, we will first be taking a look at the applications and specifications for such components. Then we will outline our "Front End" resonator technological developments on both capacitive and Metal Oxide Semiconductor (MOS) transistor detection demonstrators. We will conclude with the IC integration perspective from a device point of view.

STATE OF THE ART ON MEMS RESONATOR AND OPPORTUNITIES

Table 1 reveals that resonators can be classed by the vibrating direction:

• Out of plane (vertical resonators)

• In plane (lateral resonators)

Initially, people were more interested in working on vertical resonators because it was technologically easier to fabricate. The majority of recent demonstrator devices are using lateral technologies mainly because of the design flexibility: squares, disks, beams… In terms of frequency, Table 1 shows resonators with very different resonance frequencies that make devices hard to compare. So we consider the factor Frequency multiplied by Quality Factor (F x Q) as a comparison factor. Compared to the quartz F.Q factor (<60MHz, Q nearly 15000, F.Q=9x10 6 ), the state of the art analysis demonstrates the electromechanical resonators potentialities for quartz replacement.

Table 1 : Electromechanical resonators state of the art

APPLICATIONS AND SPECIFICATIONS

A simple RF transceiver architecture schematic is given in Figure 2. We can identify the major applications using resonators:

• Reference oscillator to provide stable and precise frequency reference. Today, quartz is commonly used.

• Voltage Controlled Oscillator (VCO) which can insure a controlled variation of the frequency with bias variation. LC resonators are used today.

• Filters to extract the useful signal from the noise. Today, Surface Acoustic Waves (SAW) or Bulk Acoustic Waves (BAW) filters are used.

The overview of the main applications and specifications is given in Table 2. We can make a few observations about the specifications needed for the different applications describe in Error! Reference source not found.. VCO will require frequencies as high as 2GHz with a large frequency tuning. Looking at the electromechanical resonator state of the art, we can see that the highest obtained resonant frequency is around 1.5GHz. Moreover the electromechanical resonator frequency tuning is inversely proportional to the stiffness and also the frequency. Therefore it is difficult to reach the VCO specifications. The filter applications require, in general, a large band pass whereas electromechanical resonators are characterised by a high quality factor which induces a narrow band pass. A mechanical coupling could be used to enlarge the resonator band pass [START_REF] Nguyen | High-Q HF Microelectromechanical Filters[END_REF]. But in this way, it could be difficult to reach the drastic specifications needed. So, electromechanical resonators seem to be not adapted to satisfying filtering specifications. The third main application using resonator consists in providing a stable and precise reference frequency. Today, these reference oscillators are constituted by quartz oscillators which have large size and have to be built off-chip (then reported). MEMS or NEMS electromechanical resonators are good candidates to replace quartz oscillators. Its present high frequencies compared to quartz (limited around 60MHz) and use CMOS process from an integration solution perspective. We notice a good agreement between analytic and FEM results. Displacements are amplified and not representative.

RESONATOR DESIGN AND DETECTION

Analytical design & Detection

It is well know that this displacement is in the nanometre range (nearly 10-20 nm for the beam, 1-5 nm for the disk). Due to very low displacements, capacitance variation is too low for output detection using capacitive detection technique. In this way, we investigate MOS detection. 6 shows that when dimensions of resonators decrease (frequencies increase and consequently displacements decrease), the MOS detection is more efficient compared to the capacitive one. This is why MOS detection has been integrated for our technological developments.

Cédric

SON TECHNOLOGICAL DEVELOPMENTS

Decrease sizes and gap is still a challenge for useful component. In this way, a front end resonator using the Silicon on Nothing (SON) process based on Silicon Germanium (SiGe) and Silicon (Si) epitaxy (Figure 7a) and equally used for MOS realization [10], is under development. The goal is to obtain sub 100nm gap allowing the realization of NEMS with a reduced motional resistance. To make the MOS for detection, the monocrystalline Si layer is Boron doped in situ at a level doping corresponding to the MOS channel doping, around 5.10 15 at/cm 3 . A phosphorous doping is necessary to make Source, Drain, and Gate (= resonator). Figure 9 shows the depth doping level characterization with a comparison of simulation, SIMS method and a chemical doping level revelation [START_REF] Wang | 2D ultrashallow junction characterization of MOSFET with strained silicon[END_REF], with a good agreement. The doping level is not continuous with depth because of technological constraints. The minimum doping level of around 3.10 18 at/cm 3 seems to be enough for the application. The physical revelation enables the study of lateral doping diffusion (Figure 10).

Figure 10 : Lateral doping diffusion after thermal annealing

In order to limit the high motional resistance of devices, it is desirable to make small gaps between resonators and electrodes. It is why the gap has been defined by e-beam photolithography and etched (Figure 7b & c) using a highly anisotropic plasma process. Some pictures of the technological results are presented in Figure 11 and Figure 12. 12 gives a cross section view a of 90nm gap obtained after a 400nm Si etching with an enlargement of less that 10nm due to the etching process.

After the gap etching, the SiGe (sacrificial layer) has to be etched (Figure 7d) with a high selectivity SiGe/Si and SiO 2 /SiGe. The aim is to release resonators that will be suspended. The process has been developed initially mainly for CMOS using SON technology purposes with tunnel to etch around 500nm [START_REF] Borel | Isotropic Etching of Si1-xGex Buried Layers Selectively to Si for the Realization of Advanced Devices[END_REF]. Here the process has been adapted to be able to etch longer tunnels as shown in Figure 13 and Figure 14. The process selectivity is strongly dependant of the tunnel depth. For a 1.19µm tunnel depth, the initial gap measured at 90nm has been enlarged and measured at 130nm after release. The process is under progress so as to improve selectivity.

Work is in progress in collaboration with the CEA-LETI & IEMN to obtain electrical demonstrators in the close future.

CONCLUSION

MEMS or NEMS electromechanical resonator is a very important component in order to integrate frequency reference oscillator in chips. Some developments to obtain capacitive and MOS demonstrators were presented. The electromechanical resonator state of the art highlights the importance of sub 100nm gaps and packaging. The SON process seems to be well adapted to obtain promising components. Process developments have started from electrical demonstrators' realization. Future work on packaging and IC integration will be investigated in a close future.

Figure 1 :

 1 Figure 1: Top view of an « out of plan » vibrating beam (schematic)

Figure 2 :

 2 Figure 2 : RF architecture view

Figure 3 :Figure 4 :

 34 Figure 3 : FEM modal simulation of a clamped-clamped beam Dimensions: Length:10µm, Width: 0.46µm, Height: 0.4µm Coventor: 39.0MHz Analytic: 38.8MHz Delta: 0.5 %

Figure 5 :

 5 Figure 5 : Schematic of a suspended resonator using MOS detection

Figure 6 :Figure 5

 65 Figure 6 : Comparison of MOS & Capacitive detection for a flexural mode clamped-clamped beam Figure 5 gives a schematic with the principle of MOS detection. The gate is the resonant structure.Figure6shows that when dimensions of resonators decrease (frequencies increase and consequently displacements decrease), the MOS detection is more efficient compared to the capacitive one. This is why MOS detection has been integrated for our technological developments.

Figure

  Figure5gives a schematic with the principle of MOS detection. The gate is the resonant structure. Figure6shows that when dimensions of resonators decrease (frequencies increase and consequently displacements decrease), the MOS detection is more efficient compared to the capacitive one. This is why MOS detection has been integrated for our technological developments.

Figure 7 :

 7 Figure 7 : STMicroelectronics SON process

Figure 8 :Figure 8

 88 Figure 8 : SEM pictures of Si & SiGe epitaxy

Figure 9 :

 9 Figure 9 : Depth doping level characterization after phosphorous doping

Figure 11 :

 11 Figure 11 : Top SEM view of a nano beam

Figure 12 :

 12 Figure 12 : Gap etch cross SEM picture

Figure

  Figure[START_REF] Wang | 2D ultrashallow junction characterization of MOSFET with strained silicon[END_REF] gives an idea of the precision of technological gap definition after etching. Figure12gives a cross section view a of 90nm gap obtained after a 400nm Si etching with an enlargement of less that 10nm due to the etching process.After the gap etching, the SiGe (sacrificial layer) has to be etched (Figure7d) with a high selectivity SiGe/Si and SiO 2 /SiGe. The aim is to release resonators that will be suspended.

Figure 13 :Figure 14 :

 1314 Figure 13 : Tilt SEM view of SiGe released areas

  Cédric Durand, Fabrice Casset, Pascal Ancey, Fabienne Judong, Alexandre Talbot, Rémi Quenouillère, Denis Renaud, Stéphan Borel, Brigitte Florin, Lionel Buchaillot Silicon On Nothing MEMS Electromechanical Resonator

Table 2 :

 2 Resonator applications and specifications

  Durand, Fabrice Casset, Pascal Ancey, Fabienne Judong, Alexandre Talbot, Rémi Quenouillère, Denis Renaud, Stéphan Borel, Brigitte Florin, Lionel Buchaillot Silicon On Nothing MEMS Electromechanical Resonator

		V g V g
	H Source C gc H Source C gc	Gate Channel V gint Gate Channel V gint	Drain Gate Oxide Drain Gate Oxide
		L

4.2. Capacitive versus MOS Detection

MOS detection is well adapted for the output signal acquisition

[START_REF] Abelé | Comparison of RSG-MOSFET and capacitive MEMS resonator detection[END_REF]

: Courtesy of N. Abelé

[START_REF] Abelé | Comparison of RSG-MOSFET and capacitive MEMS resonator detection[END_REF]

©EDA Publishing/DTIP 2007 ISBN: 978-2-35500-000-3