Emmanuelle Poque

Hermanus J Ruigrok

Delia Arnaud-Cormos

Denis Habauzit

Yann Chappe

Catherine Martin

Florence Poulletier De Gannes

Annabelle Hurtier

Andre Garenne

Isabelle Lagroye

André Garenne

Yves Le Dréan

Philippe Lévêque

Yann Percherancier
email: yann.percherancier@ims-bordeaux.fr

Effects of radiofrequency field exposure on proteotoxic-induced and heat-induced HSF1 response in live

Keywords: HSF1, Bioluminescence Resonance Energy Transfer, Radiofrequency, trimerisation Bioluminescence Resonance Energy Transfer radiofrequency fields. SAR: Specific Absorption Rate YFP: Yellow Fluorescent protein Wi-Fi : Wireless Fidelity

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INTRODUCTION

The massive deployment, in our environment, of radiofrequency electromagnetic fields (RF) used for wireless communication systems, raised social concerns about the potential biological and health effects of such radiations. After more than 20 years of researches, the only well-described effect of RF on biological systems is caused by dielectric-relaxation heating. Guidelines and standards have been set to protect from the health risks associated with the thermal effects of RF exposures. In other words, wireless communications emit RF fields that do not induce deleterious tissue heating [START_REF] Vecchia | 100 kHz-300 GHz)[END_REF]. The question remains however open to decipher whether or not RF exposure induces "nonthermal" effects, which ones refer to any potential biological effects that are not caused by the RF-induced increase of temperature in living matter.

In this context, the possibility that environmental RF exposure induces cellular stress responses in various cell types was evaluated considering various biochemical outputs, such as DNA integrity, apoptosis, and protein expression in several human and animal cell cultures [START_REF] Mcnamee | Radiofrequency radiation and gene/protein expression: a review[END_REF][START_REF] Vecchia | 100 kHz-300 GHz)[END_REF]. The "stress proteins", also known as heat-shock proteins (HSPs), are a group of proteins that have been reported to be affected by low-level RF exposures in some studies. Because HSPs and their associated factors are induced by a variety of stressors, they were proposed as possible biomarkers of RF exposure. Articles published before 2012 were reviewed in [START_REF] Mcnamee | Radiofrequency radiation and gene/protein expression: a review[END_REF][START_REF] Vecchia | 100 kHz-300 GHz)[END_REF]IARC 2013). A limited part of these studies reported altered expression of HSPs in certain cell lines (e.g. [START_REF] Kwee | Changes in cellular proteins due to environmental non-ionizing radiation. I. Heat-shock proteins Electro-and Magnetobiology[END_REF][START_REF] Tian | Exposure to 2.45 GHz electromagnetic fields induces hsp70 at a high SAR of more than 20 W/kg but not at 5W/kg in human glioma MO54 cells[END_REF][START_REF] Leszczynski | Non-thermal activation of the hsp27/p38MAPK stress pathway by mobile phone radiation in human endothelial cells: molecular mechanism for cancer-and blood-brain barrier-related effects[END_REF][START_REF] Miyakoshi | Effects of exposure to a 1950 MHz radio frequency field on expression of Hsp70 and Hsp27 in human glioma cells[END_REF][START_REF] Lixia | Effects of 1.8 GHz radiofrequency field on DNA damage and expression of heat shock protein 70 in human lens epithelial cells[END_REF][START_REF] Sanchez | Human skin cell stress response to GSM-900 mobile phone signals. In vitro study on isolated primary cells and reconstructed epidermis[END_REF][START_REF] Lucas | Serotonin(4) (5-HT(4)) receptor agonists are putative antidepressants with a rapid onset of action[END_REF][START_REF] Ennamany | Exposure to Electromagnetic Radiation Induces Characteristic Stress Response in Human Epidermis[END_REF]). Since 2012, eight studies reported increased HSP levels following RF exposure in vitro in PC12 pheochromocytorna cells [START_REF] Valbonesi | Effects of the exposure to intermittent 1.8 GHz radio frequency electromagnetic fields on HSP70 expression and MAPK signaling pathways in PC12 cells[END_REF], SH-SY5Y neuroblastoma cells [START_REF] Calabrò | Modulation of heat shock protein response in SH-SY5Y by mobile phone microwaves[END_REF]), RAW264.7 monocytic cells [START_REF] Novoselova | Involvement of the p38 MAPK signaling cascade in stress response of RAW 264.7 macrophages[END_REF][START_REF] López-Furelos | Exposure to radiation from single or combined radio frequencies provokes macrophage dysfunction in the RAW 264.7 cell line[END_REF], and brain tissue from exposed rats [START_REF] Yang | Exposure to 2.45 GHz electromagnetic fields elicits an HSP-related stress response in rat hippocampus[END_REF][START_REF] Kesari | Effect of 3G cell phone exposure with computer controlled 2-D stepper motor on non-thermal activation of the hsp27/p38MAPK stress pathway in rat brain[END_REF][START_REF] Sepehrimanesh | Analysis of rat testicular proteome following 30-day exposure to 900 MHz electromagnetic field radiation[END_REF][START_REF] López-Furelos | Evidence of cellular stress and caspase-3 resulting from a combined two-frequency signal in the cerebrum and cerebellum of sprague-dawley rats[END_REF]). In parallel, four studies reported no RF exposure alteration of HSP level in human corneal cells [START_REF] Miyakoshi | Effects of Exposure to 5.8 GHz Electromagnetic Field on Micronucleus Formation, DNA Strand Breaks, and Heat Shock Protein Expressions in Cells Derived From Human Eye[END_REF], the brain of young rats [START_REF] Aït-Aïssa | In situ expression of heat-shock proteins and 3-nitrotyrosine in brains of young rats exposed to a WiFi signal in utero and in early life[END_REF][START_REF] Watilliaux | Effect of exposure to 1,800 MHz electromagnetic fields on heat shock proteins and glial cells in the brain of developing rats[END_REF], and MCF10A human breast epithelial cells [START_REF] Kim | Analysis of the cellular stress response in MCF10A cells exposed to combined radio frequency radiation[END_REF]). The majority of these studies measured the expression level of HSP proteins or RNA levels and not all HSP subtypes were tested. From the results, it remains unclear whether these responses were related to specific parameters such as cell line, tissue, frequency, modulation, or were false-positives, e.g. artefacts caused by ill-defined exposure system. Additional well-characterized confirmation studies are required to further evaluate these observations. However, since HSPs activation pathways are driven mainly through protein-protein interaction and phosphorylation cascades, protein-specific approaches may provide more information on the impact of RF on HSP.

The heat shock response is typically characterized by a dramatic upregulation of all heat shock mRNA and proteins level. These biochemical phenomena are induced by heat shock factors (HSF) such as Heat shock factor 1 (HSF1) which is known as the "master regulator" of heat shock protein transcription in eukaryotes [START_REF] Gomez-Pastor | Regulation of heat shock transcription factors and their roles in physiology and disease[END_REF]. During unstressed conditions, molecular chaperones such as Hsp70, Hsp90 and TRiC/CCT interact with HSF1, repressing it by maintaining it inactive in a monomeric form. When the quantity of unfolded proteins increases, i.e. following cellular stress such as a temperature increase, the molecular chaperones bind to the misfolded proteins and dissociate from HSF1. The released HSF1 monomeric proteins undergo homotrimerization, which renders it transcriptionally active. The full activation of HSF1 trimers is also regulated by several posttranslational modifications such as phosphorylation and sumoylation. Active HSF1 trimers will accumulate into the nucleus where they bind to its DNA-responsive element, named HSE for heat shock element. These DNA sequences are found in upstream regulatory regions of HSP genes and HSF genes themselves (Dayalan [START_REF] Naidu | Regulation of the mammalian heat shock factor 1[END_REF]. Therefore, assessing HSF1 activation level in cells allows monitoring of the RF effects on the HSP-driven cellular stress response in an integrated way.

For the last twenty years, techniques based on the non-radiative transfer of energy between an energy donor and a compatible fluorescent energy acceptor emerged as powerful techniques for measuring in real-time the activity of an impressive array of proteins in live cells [START_REF] Miyawaki | Molecular spies for bioimaging--fluorescent protein-based probes[END_REF]. The key point of these techniques is the absolute reliance of the energy transfer efficiency to the molecular closeness (around 10 nm) and orientation between donor and acceptor dipoles. Thanks to these properties, RET techniques allow monitoring of both constitutive and regulated inter-and intra-molecular interactions. Based on RET techniques, we have recently demonstrated that Bioluminescence Resonance Energy Transfer (BRET) is useful for measuring proteins interactions and conformational changes in real-time and in live cells under RF exposure [START_REF] Ruigrok | Activation of the TRPV1 Thermoreceptor Induced by Modulated or Unmodulated 1800 MHz Radiofrequency Field Exposure[END_REF][START_REF] Poque | Effects of radiofrequency fields on RAS and ERK kinases activity in live cells using the Bioluminescence Resonance Energy Transfer technique[END_REF]. The present study aimed at evaluating the potential effects of 1800 MHz RF signals on HSF1 activation in live cells using a BRET-based molecular probe. We constructed and characterized an efficient HSF1 intermolecular BRET probe with which we monitored basal, temperature-induced, and chemically-induced HSF1 activity in live human HEK293T cells exposed under isothermal conditions to various signals. Specific Absorption Rate (SAR) levels of 1.5 and 6 W/kg were applied with 1800 MHz RF of Continuous Wave (CW) or Global System for Mobile (GSM)-, and WiFi-modulated signals.

MATERIAL AND METHODS

Plasmids

To generate the BRET constructs, super Yellow Fluorescent Protein 2 [START_REF] Kremers | Cyan and yellow super fluorescent proteins with improved brightness, protein folding, and FRET Förster radius[END_REF] and Renilla Luciferase II [START_REF] Loening | Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output[END_REF] were used to improve the brightness of the assay. They are referred as YFP and Luc in the rest of the manuscript. The human full-length HSF1 cDNA was a gift from Dr. NF. Mivachi (Center for Molecular Chaperone Radiobiology & Cancer Virology Group, Augusta University, GA, USA). The HSP90 expression vector was obtained by subcloning the human HSP90AA1 cDNA from the pCR-BluntII-TOPO-HSP90AA1 vector (Harvard Medical School PlasmID Repository, clone HsCD00347538) as BamH1-XhoI PCR fragment in the multi-cloning site of the vector pcDNA3.1 using the primers "hHSP90_BamHI_ATG_Sense" (TGTCTGGTACCGGATCCGCCACCATGCCTGAGGAAACCCAGACCCAAGACC) and "hHSP90_Stop_XhoI_antisense" (ATCTAGTCTAGACTCGAGCGGTTAGTCTACTTCTTCCATGCGTGATGTGTCG). The Luc-hHSF1 expression vector was obtained by subcloning the cDNA of hHSF1 as an EcoRI-XbaI PCR fragment in place of the YFP in the EcoRI-XbaI site of the pcDNA3.1 Luc-YFP vector described in [START_REF] Ruigrok | Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels[END_REF]. Using a similar strategy, the YFP-HSF1 expression vector was obtained by replacing Luc cDNA by the one coding hHSF1 in the EcoRI-XbaI site of the pcDNA3.1 YFP-Luc vector described in [START_REF] Ruigrok | Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels[END_REF]. In both case, hHSF1 cDNA was amplified by PCR using the "Fus_EcoRI_hHSF1_Sense" primer (TGTGTACCGGTGAATTCTGGTGGAGGCGGATCTATGGATCTGCCCGTGGGCCCCGGCG) and the "hHSF1-STOP-XbaI" primer (ATCTAGTCTAGACTCGAGCGGTTAGGAGACAGTGGGGTCCTTGGCTTTGG). For all configurations, the sequence joining Luc or YFP sequence to hHSF1 encodes VPVNSGGGGS as a linker. The sequence of each cDNA construct was confirmed by DNA sequencing.

Reagents

MG132 and Thapsigargin were from Sigma (Lyon, France). Coelenterazine H and Purple Coelenterazine (Nanolight Technology, Pinetop, AZ, USA) were added to a final concentration of 5 µM. Anti--actin (sc-8432) and anti-HSF (sc-17756) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-Hsp70 (SPA801) and anti-BIP (AB21685) antibodies were obtained from Stressgen (San Diego, CA, USA) and Abcam (Cambridge, UK) respectively.

Cell culture and transfections

HEK293T cells were maintained in Dulbecco's modified Eagle's medium -high Glucose (DMEM) (D6429, Sigma) supplemented with 10 % fetal bovine serum, 100 units mL-1 penicillin and streptomycin. Twenty-four hours before transfection, cells were seeded at a density of 500,000 cells per well in 6-well dishes. Transient transfections were performed using polyethylenimine (PEI, linear, Mr 25,000; catalogue number 23966 Polysciences, Inc., Warrington, PA, USA) with a PEI:DNA ratio of 4:1, as explained in [START_REF] Percherancier | Bioluminescence resonance energy transfer reveals ligand-induced conformational changes in CXCR4 homo-and heterodimers[END_REF]. For all experiments, 0.1 µg of Luc-HSF1 expression vector was co-transfected with 1.4 µg of YFP-HSF1 and 0.5 µg of HSP90 expression vectors. After overnight incubation, transfected cells were then detached, resuspended in DMEM w/o red phenol (Ref 21063-029, ThermoFisher scientific, Waltham, MA, USA) and replated at a density of 10 5 cells per well in 96-well white plates with clear bottoms (Greiner Bio one, Courtaboeuf, France) pre-treated with D-polylysine (Sigma) for reading with the Tristar2 luminometer (Berthold Technologies, Bad Wildbad, Germany) or onto 12 mm diameter glass coverslips (Knittel Glass, Braunschweig, Germany) treated with D-polylysine for the reading with the SpectraPro 2300i spectrometer (Acton Optics, Acton, MA, USA) (see below). Cells were left in culture for 24 h before being processed for the BRET assay.

Emission spectra and BRET assays

The experimental emission spectra of Luc-HSF1 (with coelenterazine H), and YFP-HSF1 were first obtained experimentally using a Cary Eclipse Fluorimeter (Agilent Technologies, Santa Clara, CA, USA) to assess the functionality of the Luc and YFP groups fused to HSF1 (Fig. S1).

BRET signals were acquired either in real time under acute RF exposure, as described in [START_REF] Ruigrok | Activation of the TRPV1 Thermoreceptor Induced by Modulated or Unmodulated 1800 MHz Radiofrequency Field Exposure[END_REF], or following RF exposure for 24h at SARs close to environmental levels, as described in [START_REF] Poque | Effects of radiofrequency fields on RAS and ERK kinases activity in live cells using the Bioluminescence Resonance Energy Transfer technique[END_REF]. In all cases, the BRET signal was determined by calculating the ratio of the light intensity emitted by the YFP (energy acceptor) over the light intensity emitted by the Luc (energy donor) according to Eq.1:

(1):

BRET = I acceptor I donor

When the BRET signals were measured in real time under RF exposure, full BRET spectra were acquired using an optical fiber linked to a Spectra Pro 2300i spectrometer (Princeton Instruments, Acton, MA, USA) equipped with a liquid-nitrogen-cooled charge-coupled device camera for recording the full visible spectrum (Acton Optics). In that case, since we were able to fully differenciate the Luc spectra from the YFP spectra using real-time spectral decomposition, Iacceptor and Idonor were calculated by integrating the area under the curves of the acceptor and the donor spectra. The BRET signal was presented as the Net BRET [START_REF] Ruigrok | Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels[END_REF]. Under that configuration, glass coverslips containing the cells were placed into a white opaque measurement chamber made of Teflon and containing 1.5 mL of saline solution (NaCl 0.145 M, KCl 5 mM, KH2PO4 4 mM, CaCl2 1 mM, MgSO4 1 mM, Glucose 10 mM) [START_REF] Ruigrok | Activation of the TRPV1 Thermoreceptor Induced by Modulated or Unmodulated 1800 MHz Radiofrequency Field Exposure[END_REF]. Coelenterazine H was added to the cells and full BRET spectra were acquired every 3 s and analyzed as explained in [START_REF] Ruigrok | Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels[END_REF].

To measure BRET signals after RF chronic exposures, transfected HEK293T cells seeded in 96well plates were exposed for 24 hours to the indicated RF exposure conditions, with the last 12 hours being in presence or absence (sham) of various concentrations of MG132. Coelenterazine H was then added to the cell culture medium at a final concentration of 5 µM and BRET assays were immediately performed using a multidetector TriStar2 LB942 microplate reader (Berthold Technologies, Bad Wildbad, Germany) and emission filters centered at 540 ± 40 nm for YFP (Iacceptor) and 480 ± 20 nm for Luc (Idonor). Due to the overlapping emission spectra of Luc and YFP, a fraction of the light detected in the YFP filter originates from the Luc emission, resulting in a contaminating signal [START_REF] Hamdan | Monitoring protein-protein interactions in living cells by bioluminescence resonance energy transfer (BRET)[END_REF]. In that configuration, the Net BRET was therefore defined as the BRET ratio of cells co-expressing Luc and YFP constructs minus the BRET ratio of cells expressing only the Luc construct in the same experiment.

Western-blot analysis

Cells were scraped from the culture plate by using Laemmli extraction buffer. Proteins were then separated on a 7.5% (w/v) polyacrylamide denaturing gel and transferred onto a nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK). Western blotting was performed as previously described [START_REF] Loison | Up-regulation of the clusterin gene after proteotoxic stress: implication of HSF1-HSF2 heterocomplexes[END_REF]). Primary antibodies were revealed using horseradish peroxidase-conjugated IgG (Amersham ECL), followed by chemiluminescence detection as recommended by the manufacturer's instructions (Merck KGaA, Darmstadt, Germany).

RF field exposure system

The RF exposure system was a tri-plate open transverse electromagnetic (TEM) cell allowing RF signals propagation [START_REF] Ruigrok | Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels[END_REF]. A vector generator (SMBV100A, Rohde & Schwarz, Munich, Germany) connected to a 10 W preamplifier and a 200 W amplifier (RF14002600-10, and RFS1800-200, RFPA, Artigues-Près-Bordeaux, France) with around 40-and 8-dB gain, respectively, were used to deliver 1800 MHz RF signals to the exposure system.

To assess the effect of acute RF effect on HSF1, HEK293T cells were exposed to RF in a Teflon chamber containing 1.5 mL of medium placed on the lower ground plate of the TEM cell, as described in [START_REF] Ruigrok | Activation of the TRPV1 Thermoreceptor Induced by Modulated or Unmodulated 1800 MHz Radiofrequency Field Exposure[END_REF]). The cell culture medium temperature was regulated using a Thermostat Plus microplate Peltier heater (Eppendorf, Hamburg, Germany) placed under the TEM cell and monitored using a fiber optic temperature probe (Luxtron-812, Lumasense Technologies, Santa Clara, USA). Temperatures and BRET data were simultaneously recorded in real time as described in [START_REF] Ruigrok | Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels[END_REF].

To measure the effect of RF chronic co-exposure with MG132 on HSF1 activity, HEK293T cells were exposed for 24h in a 96-well plate containing 200 µL of medium, as described in [START_REF] Poque | Effects of radiofrequency fields on RAS and ERK kinases activity in live cells using the Bioluminescence Resonance Energy Transfer technique[END_REF]. Investigations were carried out at two SAR levels using three 1800 MHz RF signals (CW, GSM, and Wi-Fi-modulated signals).

RF Dosimetry

The characterized exposure system was a TEM cell containing a Teflon chamber for acute exposures or a 96-well plate for chronic exposures. Numerical dosimetry of the exposure system was performed using in-house Finite Difference Time Domain (FDTD)-based software for solving

Maxwell's differential equations. Details of the numerical dosimetry can be found for the Teflon chamber in [START_REF] Ruigrok | Activation of the TRPV1 Thermoreceptor Induced by Modulated or Unmodulated 1800 MHz Radiofrequency Field Exposure[END_REF]) and for the 96-well plate in [START_REF] Poque | Effects of radiofrequency fields on RAS and ERK kinases activity in live cells using the Bioluminescence Resonance Energy Transfer technique[END_REF]).

The incident power was adjusted to apply the same average power level for all signals at 1800 MHz. For the Teflon chamber, based on the SAR efficiency (5.2±1.9 W/kg/W, mean±SD) computed in [START_REF] Ruigrok | Activation of the TRPV1 Thermoreceptor Induced by Modulated or Unmodulated 1800 MHz Radiofrequency Field Exposure[END_REF], the incident powers applied in this study were set to obtain two SAR levels. For the 96-well plate, the volume of medium was different from [START_REF] Poque | Effects of radiofrequency fields on RAS and ERK kinases activity in live cells using the Bioluminescence Resonance Energy Transfer technique[END_REF] where each well was filled with 100 µL. The dosimetry was thus carried out in this study with a 96-well plate containing 200 µL of medium and the SAR efficiency was 0.69±0.07 W/kg/W. The SAR values within the cells layer were 6.2±1.4 and 0.90±0.09 W/kg/W, for the Teflon chamber and for the 96-well plate, respectively. The two exposure levels for the whole-volume mean SAR values were 1.5 and 6.0 W/kg. Experimental dosimetry was carried out using a fiber optic temperature measurement system (Luxtron 812, LumaSense Technologies, Erstein, France.) for temperature measurements inside the Teflon chamber and inside several wells, containing 200 µL of medium, of the 96-well plate. SAR assessment from temperature measurements was described in details in [START_REF] Ruigrok | Activation of the TRPV1 Thermoreceptor Induced by Modulated or Unmodulated 1800 MHz Radiofrequency Field Exposure[END_REF]. SAR values obtained from numerical simulations and experimental measurements are in good agreement. Table 1 presents the power settings at the vector generator to obtain the required SAR values for all signals. The averaged output power delivered by the signal generator was measured using a power meter and a wideband power sensor (N1912A and N1921A, Agilent, Santa Rosa, CA, USA).

Statistical analysis

GraphPad Prism v6.00 for Windows (GraphPad Software, La Jolla, CA, USA) was used for plotting dose-response curves. Statistical analyses were performed using Anastats (Rilly sur Vienne,

France). The one sample Wilcoxon signed-rank test was used to assess the statistical significance against the null hypothesis of the difference calculated between Sham and RF exposure condition for basal BRET, MG132 potency and efficacy. P-values less than 0.05 were considered as statistically significant.

RESULTS

To build a BRET probe that can efficiently measure HSF1 activity in live cells, we took advantage of the required trimerisation step on the roadmap of HSF1 activation. We constructed the cDNA coding for N-terminally Luc-and YFP-tagged HSF1 (respectively Luc-HSF1 and YFP-HSF1).

This approach was based on the hypothesis that if HSF1 proteins tagged with a bioluminescent energy donor are co-expressed in a cell with HSF1 proteins tagged with a fluorescent energy acceptor, the resulting BRET signal might increase following HSF1 activation since trimerization brings donor and acceptor groups in close proximity (Fig. 1).

HSF1 activation is triggered by heat [START_REF] Zou | Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1[END_REF][START_REF] Hentze | Molecular mechanism of thermosensory function of human heat shock transcription factor Hsf1[END_REF]. The effect of temperature elevation on the BRET spectra measured from HEK293T cells transiently transfected with Luc-HSF1 and YFP-HSF1 was first assessed. As shown in Fig. 2A, a resonance energy transfer occurs at 25 °C between YFP-HSF1 and Luc-HSF1 since a light peak with a maximum emission at 535 nm corresponding to the acceptor reemission can be detected in addition to the Luciferase light emission that peaks at 485 nm, in agreement with the individual emission spectra of both Luc-HSF1 and YFP-HSF1 (Fig. S1). This basal energy transfer observed indicates that some HSF1 constitutive oligomers already exist in resting conditions under our experimental conditions. As expected, increasing the temperature to 45 °C for 5 min led to the increase in the amount of light emitted in the 510 -550 nm region that resulted from the transfer of energy from the luciferase to the YFP with the ensuing emission of light by the latter (Fig. 2A). To further characterize the temperature sensitivity of our HSF1-based intermolecular BRET probe, the BRET signal, defined as the ratio of light detected for YFP over the light detected for Luc (see material and methods for details), was then measured in real time while heating the cell culture from 22 to 48 °C using a Peltier heater (Fig. 2B). The initial basal BRET signal remained stable between 22 and 35 °C. It then linearly increased with the temperature and reached a short plateau between 44 and 46°C, before slightly decreasing between 46 and 48°C.

This result indicates that, under our experimental conditions, HSF1 is activated by heat with a threshold around 35 °C and a maximum activation is reached for temperatures above 40 °C as previously observed [START_REF] Zou | Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1[END_REF][START_REF] Hentze | Molecular mechanism of thermosensory function of human heat shock transcription factor Hsf1[END_REF]).

To further characterize the Luc-HSF1/YFP-HSF1 intermolecular BRET test, we assessed its sensitivity to MG132-induced proteotoxic stress. MG132 is a well-known proteasome inhibitor that has been shown to induce HSF1 activation by increasing cytosolic misfolded protein content [START_REF] Mathew | Stress-specific activation and repression of heat shock factors 1 and 2[END_REF]. As expected, MG132-induced a dose dependent increase of the BRET signal with a 3-fold maximal increase of the basal BRET signal and an EC50 in the hundred nanomolar range (pEC50 = 6.61±0.06, Fig. 3A). As control, we verified by western blot that overnight treatment with 5 µM MG132 induced overexpression of HSP70. This increase could be measured in both un-transfected and transfected cells (Fig. 3B) confirming that MG132 treatment was effective in activating HSF1, as previously described [START_REF] Mathew | Stress-specific activation and repression of heat shock factors 1 and 2[END_REF]). However, under our condition (concentration and duration of treatment), MG132 did not produce any change in the expression level of Bip1 (also known as GRP78), a well known ER stress marker, which is activated by the ER stress-related factor ATF6 but not by HSF1 [START_REF] Lee | The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress[END_REF]. Both mock-transfected HEK293T cells and HEK293T cells co-expressing Luc-HSF1 / YFP-HSF1 proteins were incubated overnight with increasing concentrations of Thapsigargin. Addition of Thapsigargin, which is a well-known competitive inhibitor of the sarco/endoplasmic reticulum Calcium ATPase (SERCA), leads to emptying the intracellular calcium stores [START_REF] Burnay | Role of the capacitative calcium influx in the activation of steroidogenesis by angiotensin-II in adrenal glomerulosa cells[END_REF]) and the subsequent triggering of endoplasmic reticulum stress following prolonged exposure [START_REF] Li | Transactivation of the grp78 promoter by Ca2+ depletion. A comparative analysis with A23187 and the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin[END_REF]). Overnight treatment with 10 nM Thapsigargin efficiently induced an ER stress as shown by the increase in BiP protein expression level (Fig. 3B). However, even at high Thapsigargin concentrations, we could only measure a low BRET increase between Luc-HSF1 and YFP-HSF1 (Fig. 3A). Similar results were obtained in Huh-7 hepatocarcinoma cell line, further reinforcing these observations (Fig. S2).

Altogether, these results indicate that the BRETsignal measured with our intermolecular BRET assay is specific of HSF1 activation by heat or proteasome inhibition.

HEK293T cells expressing Luc-HSF1 / YFP-HSF1 BRET probes were then exposed to a CW 1800 MHz RF signal at an initial temperature slightly above 35 °C that corresponds to the measured HSF1 heat activation threshold. Temperature of the cell medium and BRET signal were simultaneously recorded before and during RF exposure. Immediately after the onset of RF exposure, an increase in BRET signal was observed indicating that HSF1 was activated due to RF-induced temperature elevation (Fig. 4A). A final temperature increase of 3.5 °C was observed under exposure at a whole volume SAR around 24 W/kg for 30 min (Fig. 4B). Such RF-induced temperature increase may have masked the potential nonthermal HSF1 activation by RF exposure. In order to unravel the nonthermal HSF1 activation during RF exposure, the cell culture medium temperature was clamped using a Peltier plate. Clamping the cell-culture medium at ca. 36 °C abrogated the rise of the BRET signal which stayed stable during exposure to RF at 24 W/kg (Fig. 4A andB). The resulting BRET signal was similar with the no-RF control condition indicating that HSF1 was not activated by RF exposure at steady temperature.

The effect of RF co-exposure with MG132 on HSF1 activity was further assessed. Cells in a culture medium at 37 °C were exposed during 24 h to 1800 MHz CW, GSM or WiFi signals at two different SAR values of 1.5 W/kg and 6 W/kg. In this configuration, HEK293T cells transfected with the HSF1 BRET probe were pre-exposed to RF for 12 hours before being co-exposed for another 12 h with RF and increasing doses of MG132. Analysis of the resulting dose-response curves (Fig. 5A-F)

indicates that the basal BRET ratio of the intermolecular BRET probe Luc-HSF1 / YFP-HSF1 is slightly but reproducibly decreased, in comparison to the sham control, when cells were exposed to CW and GSM signals at 1.5 W/kg (Fig. 5G). However, the basal BRET signal was not modified when cells were exposed to CW and GSM signals at 6 W/kg SAR or exposed to WiFi signal at either 1.5 or 6 W/kg. For all signals or SAR values, RF exposure did not modify MG132 potency to activate HSF1 (Fig. 5H). However, a slight but statistically significant increase of the MG132 maximal efficacy to activate HSF1 was measured (fig. 5I). This increase was 5% for exposures to the CW signal at 1.5 and 6 W/kg and 7% for GSM at 1.5 W/kg in comparison to the sham control for each condition. No RF exposure effect was detected on MG132 maximal efficacy to activate HSF for exposures to GSM at 6 W/kg or to WiFi signal whatever SAR used.

DISCUSSION

Few studies have addressed the effect of RF on HSF1 levels and/or activity. In a very comprehensive study, [START_REF] Ohtani | Exposure time-dependent thermal effects of radiofrequency electromagnetic field exposure on the whole body of rats[END_REF] assessed the changes in the expression of Hsp and Hsf families in the cerebral cortex and cerebellum of Sprague-Dawley rats. The animals were exposed for 3 or 6 h/day to 2.14 GHz wideband code-division-multiple-access (W-CDMA) RF signals at a wholebody averaged specific SAR of 4 W/kg [START_REF] Ohtani | Exposure time-dependent thermal effects of radiofrequency electromagnetic field exposure on the whole body of rats[END_REF]). These authors found that RF exposure at 0.4 W/kg, which is the limit for the occupational exposure set by the International Commission on Non-Ionizing Radiation Protection, caused behavioral disruption in the laboratory animals.

Transcriptional analyses revealed a significant upregulation of some of the Hsp and Hsf genes, particularly in the cerebellum, for rats that were exposed to 4 W/kg for 6 h/day. No changes were measured at 0.4 W/kg or if rats were exposed at 4 W/kg for only 3 h/day. This effect was associated with an increase in core temperature by 1.0-1.5 °C with respect to the baseline, indicating a possible thermal effect of RF on hsf and hsp gene expression. In the study by [START_REF] Laszlo | The heat-shock factor is not activated in mammalian cells exposed to cellular phone frequency microwaves[END_REF], the DNAbinding activity of HSF in the hamster, mouse and human cells was determined. Acute and continuous exposures to 835.6 MHz 'frequency domain multiple access' (FDMA)-or 847.7 MHz 'code domain multiple access' (CDMA)-modulated RF at two SAR values (0.6 and ca. 5 W/kg) were studied [START_REF] Laszlo | The heat-shock factor is not activated in mammalian cells exposed to cellular phone frequency microwaves[END_REF]. Under all exposure conditions, the authors reported a lack of HSF DNA-binding ability induction in cultured mammalian cells. The only studies showing a slight effect of RF exposure on HSF gene activity and heat-shock response had been performed in the nematode C. elegans [START_REF] De Pomerai | Non-thermal heat-shock response to microwaves[END_REF][START_REF] De Pomerai | Microwave radiation can alter protein conformation without bulk heating[END_REF]. This effect has since been reinterpreted as a subtle thermal artefact caused by small temperature disparities (≤0.2 °C) between exposed and sham conditions [START_REF] Dawe | A small temperature rise may contribute towards the apparent induction by microwaves of heat-shock gene expression in the nematode Caenorhabditis Elegans[END_REF]).

In the present study, we designed and characterized an intermolecular BRET assay measuring HSF1 activation through the assessment of its trimerisation in real-time and live cells. This assay responded specifically to both MG132-induced proteotoxic stress known to trigger HSF1 activation and heat activation with thresholds similar to those reported in the literature (Fig. 2 and3). Using this BRET probe, we monitored HSF1 activation under RF exposure, CW, GSM-or WiFi-modulated signals at 1800 MHz and SAR levels of 1.5 and 6 W/kg (24 h chronic exposure) or 24 W/kg for CW (acute exposure). As expected, HSF1 was activated under RF-exposure-associated temperature elevation. To exclude specific non-thermal effects of RF, exposures at a constant temperature of 36 °C were performed under RF exposure. There were no effects of acute RF exposure on the activation state of HSF1 at a steady temperature (Fig. 4). This observation was consistent with previous experiments,

showing that HSF-sensitive reporter genes were not activated under non-thermic electromagnetic exposure [START_REF] Zhadobov | Low-power millimeter wave radiations do not alter stress-sensitive gene expression of chaperone proteins[END_REF]. At constant temperature, we tested the chemical mode of HSF1 activation using MG132. We found that RF exposure to CW signals (1.5 and 6 W/kg) and GSM signal (1.5 W/kg) only marginally increased MG132 maximal efficacy (5-7 % increase in maximal efficacy compared to the sham condition) and did not modify MG132 potency to activate HSF1 (Fig. 5). For both CW and GSM exposures at 1.5 W/kg, the slight increase in MG132 maximal efficacy correlated with a concomitant slight decrease in HSF1 basal BRET and an identical HSF1 maximal BRET level in saturated MG132 concentration. This indicates that RF slightly decreased HSF1 basal activation state without affecting the maximal capability of HSF1 to be activated under proteotoxic stress conditions. No effects were observed in GSM-modulated 1800 MHz RF exposures at 6 W/kg for 24 h on basal HSF1 activity, MG132 efficacy and potency to activate HSF1. The future deployment of the fifth-generation (5G) wireless communication will boost the rise of the Internet of Things. The consequence will be a massive increase in the number of highfrequency-powered base stations and other wireless devices everywhere in our environment. Such technologies will use carriers waves in the 3.5-5.5 and 26 GHz bands. It is important to point here that the dielectric loss of water peaks around 26 GHz at body temperature [START_REF] Buchner | The Dielectric Relaxation of Water Between 0°C and 35°C[END_REF]. This implies that RF energy will not penetrate the body as it will be absorbed by the first layer of the body with very high efficacy. At 26 GHz, the main issues to be studied in bioelectromagnetics studies become dermis and eyes. As a consequence, it will be of prime importance to assess the potential stress response induced by 5G signals in particular in these tissues using our HSF1 intermolecular BRET probes.

Beyond the scope of bioelectromagnetics, HSF1 has been validated as a powerful target and biomarker in cancer since a broad spectrum of cancer cells exhibit high levels of nuclear-active HSF1 [START_REF] Carpenter | HSF1 As a Cancer Biomarker and Therapeutic Target[END_REF]. Since HSF1 protects cells from stresses induced by chemicals, radiation, and temperature, this high level of nuclear-active HSF1 is detrimental for cancer therapies such as radiation, chemotherapy and hyperthermia [START_REF] Tabuchi | Targeting Heat Shock Transcription Factor 1 for Novel Hyperthermia Therapy[END_REF][START_REF] Dong | Inhibiting Heat Shock Factor 1 in Cancer: A Unique Therapeutic Opportunity[END_REF]. Inhibiting HSF1 in cancer cells is therefore promising to improve cancer treatment, but this intrinsically depends on the ability to discover drugs that specifically target HSF1. All existing cell-based approaches for HSF1 activity identify molecules that inhibit general transcription, translation, or upstream signaling processes and are predicted to have more 'off-target' effects. By targeting the HSF1 trimerization interface in live cell and in real time, the herein presented BRET assay can contribute to improving HSF1 drug screening. Concomitantly, HSF1 intermolecular BRET probe my also be very useful to assess the stress response induced in healthy tissue in a number of medical application techniques devoted or not to cancer treatment.

FIGURES LEGENDS

Figure 1 :

 1 Figure1: Mode of action of the intermolecular HSF1 BRET assay. In the resting state, Luc-HSF1 and YFP-HSF1 co-expressed together in cells are kept under inactive monomeric forms in the cytosol mainly due to their interaction with chaperone proteins such as HSP90. As a consequence, in the resting state, HSF1-fused Luc cannot efficiently transfer its energy to HSF1-YFP. Upon proteotoxic or heat stress, chaperone proteins interact with unfolded proteins, thereby releasing HSF1 monomers that become free to interact altogether, maximising the energy transfer between Luc and YFP groups into HSF1 trimers.

Figure 2 :

 2 Figure 2: Characterisation of the Luc-HSF1/YFP-HSF1 intermolecular BRET assay response to heat stress. A) Light emission BRET spectra of the HSF1 intermolecular BRET probe when expressed in HEK293T cells placed either at 25 °C (black circle) or 45 °C (gray circle). The light spectra of the Luc was calculated by spectral decomposition and is indicated as a dashed line. B) Evolution of the BRET signal as a function of temperature in the cell culture medium. HEK293T cells transiently coexpressing Luc-HSF1 and YFP-HSF1 proteins were heated from 22 to 47 °C using a Peltier apparatus while both BRET signal and temperature were monitored in real-time. One representative experiment out of three is presented.

Figure 3 :

 3 Figure 3: Characterisation of Luc-HSF1/YFP-HSF1 intermolecular BRET assay response to MG132-induced proteotoxic stress. A) Dose-response curves of MG132-and thapsigargin-induced changes in Luc-HSF1/YFP-HSF1 BRET signal. HEK293T cells transiently co-expressing Luc-HSF1 and YFP-HSF1 proteins cells were activated for 12 hours at 37 °C with increasing concentration of MG132 or Thapsigargin before BRET measurement. The results represent the average ± S.E.M. of 4 (MG132) and 7 (Thapsigargin) independent experiments done in duplicate. B) Western blot analysis of BIP, HSP70 and HSF1 protein levels in cellular extract of untransfected HEK293T cells (left panel) or HEK93T cells transfected with Luc-HSF1/YFP-HSF1 (right panel), that were challenged for 12 hours with either 5 µM MG132 or 10 nM Thapsigargin. CT: No treatment. MG: MG132. TG: Thapsigargin.

Figure 4 :

 4 Figure 4: Effects of CW 1800 MHz exposure on HSF1 activation. HEK293T cells expressing Luc-HSF1 and YFP-HSF1 were exposed to CW 1800 MHz signal at 24 W/kg, starting at 600 s, while BRET (Panel A) and temperature (Panel B) were monitored in real-time. Black circles: Temperature was allowed to rise under RF exposure; Grey squares: temperature was clamped at 35.8 °C using a Peltier apparatus; Open diamonds: control without RF exposure at 35.8 °C. One representative experiment out of three is presented.

Figure 5 :Supplementary Figure 1 :Supplementary Figure 2 :

 512 Figure 5: Effect of RF exposure on basal and MG132-induced HSF1 activity. A-D) HEK293T cells transfected with Luc-HSF1 / YFP-HSF1 BRET probe were sham-exposed or exposed to unmodulated CW 1800 MHz (A and B), GSM-modulated 1800 MHz (C and D) or Wi-Fi-modulated 1800 MHz (E and F) signals at either 1.5 W/kg (A, C and E) or 6 W/kg (B, D and F) for 24 hours. Cells were activated using increasing concentrations of MG132 under sham or RF exposure for the last 12 h before BRET measurement. The results in panels A-F represent the average ± S.E.M. for the effect of 8 experiments performed in duplicate. G, H and I) Box and whisker plots representing the distribution of the variations of basal BRET (G), MG132-potency (H) and MG132-maximal efficacy (I) between the RF exposed-(Expo) and sham-conditions derived for each experimental conditions plotted in A-F. Statistical significance of the derivation from the null hypothesis (no difference between sham and RF exposure) was assessed using the one-Sample Wilcoxon Signed Rank Test. n.s.: not significant; *: p<0.05; **: p<0.01.

Table 1 :

 1 Vector Generator Power Levels Settings for the three signals, two SAR levels and two exposure configurations.

			Vector Generator Power Levels Settings (dBm)
	SAR		Teflon chamber*			96-well plate**
		CW	GSM	WiFi	CW	GSM	WiFi
	1.5 W/kg	-23.1	-15.1	-23.5	-6.89	1.11	-7.34
	6.0 W/kg	-17.1	-9.1	-17.5	-0.89	7.13	-1.34
	24.0 W/kg	-11.1				Not tested
	*47.7 dB amplifier gain

**40.6 dB amplifier gain

  The same observation applied for 1800 MHz Wi-Fi signals. The minor decrease in HSF1 basal activity measured after RF exposure for 24 h at 1.5 W/kg using CW and GSM signals was inconsistent among the various signals/SAR values tested. Also, MG132 maximal efficacy to stimulate HSF1 activity was increased following 24 hr exposure to CW or GSM signals at 1.5 W/kg and CW signal at 6 W/kg. In conclusion, our study showed (i) no evidence that RF exposures under isothermal conditions affect HSF1-trimerisation and activation in response to proteotoxic or thermal stress, and (ii) evidence that HSF1 basal activation state might be slightly modified by RF exposure. Altogether, our results indicate that RF exposure at an environmental level or in isothermal condition does not impact HSF1 activation capability.

ACKNOWLEDGMENTS

We thank Bernard Veyret for his wise advice and proofreading of the manuscript.

AVAILABILITY OF DATA AND MATERIAL

Data available on request from YP (mail: yann.percherancier@ims-bordeaux.fr).

CODE AVAILABILITY

Not applicable

Declarations

FUNDING SOURCE

The research leading to these results received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 603794 (the GERONIMO project).

DECLARATION OF INTEREST

The authors declare no competing interests.


A Enel

A Bourrelier

J Vial

D Thiébaut

B Bourlon

A microfluidic device for digital manipulation of gaseous samples

Keywords: Digital microfluidics, gas samples preparation and analysis, silicon microfabrication, miniaturization, preconcentrator

Digital microfluidics is known for fine manipulation of sub-millimeter samples, with applications from biological sample preparation to diagnostic testing. Unfortunately, until now, it is only limited to liquid phases. In this paper, we present a new system based on a digital microfluidic platform (DMFP), which is able to digitally manipulate gaseous samples, such as alkanes from n-hexane to n-nonane. The DMFP relies mostly on interconnected micropreconcentrators (µPC) to trap and release the samples depending on their controlled temperature. We show that the DMFP is capable to perform all basic operations of digital microfluidics: trapping/releasing and moving samples, adding samples and separating samples, i.e. making a substraction. As a first example of more complex programmable use of our DMFP, we measured the breakthrough volume of alkanes on Tenax TA adsorbent. The results were consistent with tabulated values obtained with standard laboratory instruments. Such DMFP promises great possibilities for more complex programmable gas microfluidics digital devices and the development of new digital gas sample preparation and analysis methods.

Introduction

Over the past decade, microfluidics, defined as the manipulation of fluids at small scale, mainly submillimeter scale, have steadfastly progressed [1][START_REF] Ibrahim | Proc. IEEE[END_REF][START_REF] Fouillet | [END_REF][4][5][6][7][8][9][10][11][12] . It is also compounded by the understanding of the fluid mechanics at this scale, the chemical interactions and the microfabrication techniques mandatory to craft these devices. The development of microfluidics has yielded several techniques of small-scale fluid control, for example efficient and reproducible droplet generation by making two immiscible liquids flow through specific shapes. This technique is very useful: droplets are almost a closed medium, as diffusion to the surrounding fluid is very low, and allows for encapsulation of useful substances, such as enzymes for enzymatic assays 13 . Another technique emerged, among lots of others: electrowetting, more specifically electrowetting on a dielectric (EWOD). In this mode, two electrodes are used to create an electric field on a dielectric, which changes the wettability of the dielectric and so the contact angle of the droplet. This change allows for movement or immobilization of the droplet. It is then possible to make a grid with these electrodes, and move droplets at will on this grid. These two techniques paved the way for digital microfluidics: microfluidics programmable by analogy to a digital computer. This means that the droplets are commanded by a set of simple instructions, from known state to known state while a clock is used to synchronize the system. This leads to several operations such as moving, trapping, mixing or merging, storing, and extraction of samples. Most of these devices use liquid phases samples, mostly droplets in an immiscible surrounding fluid. Several devices also use bubbles surrounded by liquid 14,15 . To our knowledge, no digital microfluidic device has been made to manipulate gas samples within a carrier gas.

In the meantime, developments in miniaturization of air analysis devices have yielded micropreconcentrators [16][17][18]. These devices consist of a chamber containing an adsorbent, a heating element and a temperature probe. It is then possible to trap a gas on the adsorbent, or release it at will by changing the µPC temperature. When the compound of interest is trapped on the µPC, it does not diffuse within the surrounding gas. These characteristics make µPC a suitable building block for digital microfluidics. Digital microfluidics for gases could help to design programmable, automatic and versatile gas sample preparation and analysis systems. These systems could be free from diffusion related issues that are met in conventional instruments 19 . They could also allow sample manipulation without losses. As applications, such devices could be combined with conventional analytical techniques or open new approaches for portable miniaturized instruments.

In this work, a digital microfluidic platform (DMFP) using pumps, µPC and a detector was assembled. We showed that the digital manipulation of gas samples was possible with the DMFP, and demonstrated all elementary operations: trapping/releasing and moving a compound, merging two compounds and separating two compounds. Several alkanes, ranging from n-hexane (C6) to n-nonane (C9) were used to demonstrate these operations. These elementary operations were carried-out by controlling precisely, step by step, the state of the pumps and the temperature of the µPCs. These elementary operations were also the first step towards a more complex device, which could be used to perform high-level functions, such as separation of mixed samples.

As a first proof of concept of a more complex programmable application, the DMFP was used to measure the breakthrough volume of these alkanes on a classical adsorbent (Tenax TA). Breakthrough volume measurements with standard laboratory instruments, such as a gas chromatograph, are labor intensive, as they need several injections, one for each temperature studied. As an illustration of potential applications, it was possible to measure automatically with the DMFP the breakthrough volume on a wide range of temperatures using only one injection, by manipulating without loss the same initial gas sample.

Experimental

Components fabrication

The micropreconcentrator chip fabrication has been already described elsewhere 20,[START_REF] Bourlon | SENSORS[END_REF] . The size of the µPC chip was 21 mm x 7.6mm. On the front side of the silicon chip, 400 μm deep inlet/outlet and a central cavity were etched in silicon, and sealed with a Pyrex glass. The 6.6 µL cavity was filled with Tenax-TA adsorbent powder, a polymer of 2,6-diphenylphenol. Tenax TA is widely used for trapping volatile organic compounds from air samples. Inlet/outlet of the chip were glued to nickel capillaries. On the back side of the chip Ti/Pt thin film heaters and thermoresistive probes were deposited. Several other similar designs have been reported in the literature [START_REF] Akbar | [END_REF][23][24] .

The micro thermal conductivity detectors (µTCD) were also batch processed on 200 mm silicon wafers. The size of the µTCD chip was 9.6 mm x 5.4 mm. Two 100μm deep 200µm wide microchannels were etched in the silicon chip by anisotropic deep reactive ion etching (DRIE) following by isotropic plasma etching. Each channel contained two suspended membranes. Each membrane was made of 200 nm thin silicon nitride membrane on which a platinum thermoresistive conductor had been deposited by sputtering. The other half of microchannels was etched by wet etching on a Pyrex glass that was finally sealed on top of the silicon wafer. The µTCD was then glued and wire-bonded to a printed circuit board holder. 80 µm inner diameter and 150 µm outer diameter fused silica capillaries were then glued in the inlets/outlets of the silicon chip microchannels. The membranes were then connected electrically in a Wheatstone bridge structure. Several similar other designs have been reported in the literature [START_REF] Feng | 2017 19th International Conference on Solid-State Sensors, Actuators and Microsystems (TRANSDUCERS)[END_REF][START_REF] Narayanan | [END_REF] .

When the µTCD is functioning, the membranes are heated and thermal exchanges with the surrounding gas reach a stationary state. If the surrounding gas changes its thermal conductivity, for example by changing its chemical composition, the thermal exchanges are disrupted. This causes the membranes to change their temperatures, and their resistance. This changes the output of the Wheatstone bridge, which is monitored. The µTCD output is then related to the concentration of the sample inside the carrier gas. When samples go through the µTCD, the resulting signal has the shape of a peak. The peak's height is related to the concentration of the sample, and its area to the amount of sample.

The µTCD was checked for linearity: as expected, the area of the peaks scaled linearly with the amount of sample within the studied range. The results are available in the supplementary data.

Experimental setup

The schematics of the DMFP are presented in Figure 1. 106 107 As shown on Figure 1, the DMFP contained three µPC and one µTCD, with two pumps linked to them. The pumps were not miniaturized, but the main building bricks of the system were. The pumps may be miniaturized in the future, as such pumps have already been reported 27 . No valves were used here, however miniaturized valves have been reported 28 . Pumps were Xavitech models (V200-O2C12V) and connected to the DMFP with Tygon® tubes (1/6'' outer diameter, 1/10'' inner diameter). The carrier gas used to move samples inside the DMFP was ambient air. 1/16'' unions were used to connect the components together, with 1/16'' ferrules and nuts. The µTCD was connected between the µPC2 and µPC3 using one of its two gas channels (as analytical channel); the second µTCD gas channel was connected between µPC3 and pump 2 (as reference channel) to lower the baseline drift. The second reference connection is not shown in the Figure 1 for simplification. A fused silica capillary, 1.50m x 250µm inner diameter, was used to connect the µPC2 to the µTCD. This capillary was found to reduce artefacts on the µTCD signal. The artefacts were caused by transitory states during the heating of a µPC or the pumps starting. They appeared close to the sample peak, disturbing the baseline. The pumps and µPC were powered by a 12 V supply device, and the µTCD by a 9 V battery. An electronic setup connected to a computer through USB allowed to control the pumps, the µPC heaters as well as to acquire signal from the µTCD and the µPC temperature probes. Labview 2012 was used to program and control the sequence of states of the DMFP, as well as acquire and register the data.

Samples could be injected on either µPC1 or µPC2 by pumping with pump 1 or pump 2 respectively. Samples were prepared by injecting a few microliters of liquid sample inside a Tedlar® bag of 1 L (Supelco, 24633) filled with 5.0 nitrogen (Air Liquide). The samples used were n-hexane (C6) (Carlo Erba, 99% HPLC grade), n-heptane (C7) (99 % anhydrous, Sigma Aldrich), n-octane (C8) (≥99% anhydrous, Sigma Aldrich) and n-nonane (C9) (≥ 99 % anhydrous, Sigma Aldrich). Prior to injection, the bag was cleaned by filling it 3 times with 5.0 nitrogen and emptying with a vacuum pump. After injection of the sample in the bag, the bag was let at room temperature for 15 minutes to equilibrate. For the injection of samples, the Tedlar® bag was connected to the inlet of the T-junction. The connection was made with a 1/16'' stainless steel capillary. Several samples were prepared: C6 5 ppm, C7 10 ppm, C7 5 ppm, C9 5 ppm, C7-C9 5 ppm, C6 1 ppm, C7 1 ppm, C8 1 ppm, C9 1 ppm. Mass flow rates inside the DMFP were around 0.30 mL/min.

Results and discussion

The manipulation of gaseous samples relies mostly on the adsorption/desorption of compounds using adsorbent packed in the µPCs. The compounds are trapped on the adsorbent and released when the µPC is heated. Using pumps, it is possible to control the gas flow and thus the samples displacement.

Trapping and preconcentration

The ability of our µPC to trap compounds was checked using n-hexane (5 ppm). C6 was first loaded on µPC2 before starting the experiment. Table 1 presents the DMFP program used for this experiment. On step 1, the sample moved from µPC2 to µPC3. On step 2, the Tedlar bag containing C6 5 ppm was plugged to the injector: supplementary C6 was loaded on µPC1. The bag was then plugged out and the injector closed. On step 3, the supplementary C6 moved from µPC1 to µPC3. On step 4, the sample moved to µPC2.

Results are shown on Figure 2. As shown in Figure 2, µPCs are very effective at concentrating compounds, trapping them and releasing them on demand. During each step 3, the height and area of the C6 peak was the same, indicating the amount of C6 loaded during step 2 was constant. During each step 1 and step 4, the height and area of the n-hexane peak rose, meaning the amount of C6 trapped on µPC3 was increasing. This is consistent with the fact that C6 was trapped on the µPC3 and could be released on demand, and that more C6 was added to the DMFP at each step 2.

Controlled movements of samples

The sample, C7 (10 ppm), was loaded on µPC2, and then moved back and forth 20 times between µPC2 and µPC3. Table 2 shows the DMFP program used for this experiment. It consisted of two steps: step 1 consisted in heating µPC2 with pump 2 on: the sample was carried to µPC3. Step 2 consisted in heating µPC3 with pump 1 on: the sample was carried to µPC2. Figure 3 shows that the sample could be moved 20 times without significant losses over time due to diffusion, or non-efficient trapping. Indeed, the peaks obtained during the 20 steps 1 looked similar, with an area distribution of 4583 ± 683 mV*s. The peaks obtained during the 20 steps 2 also looked similar: their area distribution was 7063 ± 1226 mV*s. This means the µPC was effective at trapping compounds, and managed to release them efficiently when heated. However, one can notice a slight variation in the height of the peaks obtained during step 1 and the height of the peaks obtained during step 2 was observed, certified by a statistical test. This variation could be attributed to the fused silica capillary between PC2 and the µTCD: its volume was 75 µL, compared to the 1.5 µL capillary volume between PC3 and the µTCD. The sample diffused more when traveling through the 75 µL, causing a decrease in local concentration and thus a decrease in the µTCD peak signal. The amplitude of this variation was low and did not hinder the conclusions. This could be investigated and solved in a future setup if needed.

Mixing two different compounds

It was also possible to use this device to mix two compounds. To demonstrate this, µPC1 and µPC2 were loaded with C9 (5 ppm) with C7 (5 ppm), respectively. The compounds were added on µPC3 and then moved back and forth between µPC2 and µPC3. The Table 3 shows the DMFP program used for this experiment. The results are shown on Figure 4. went through the µTCD a signal peak was observed. The peaks observed at 600 s, 800 s and 900 s were a combination of the peaks observed at 80 s (C7), and the peak observed at 480 s (C9). To move compounds, the µPCs were heated to 150°C.

Figure 4 shows that the two compounds, C7 and C9, produced, visually, different peaks when they went through the µTCD. C7 went through the µTCD on step 1; C9 went through on step 2. On step 3 both of them were added on µPC3, the resulting peak being the sum of the two individual peaks. This meant that the addition was successful, and the two compounds could move together during steps 4 and 5: they did not separate on their own.

Separating different compounds

Using the DMFP it was also possible to separate two different compounds. To show this, C7-C9 5 ppm was loaded on µPC1, and then moved on µPC3 during a preliminary step, which is not shown.

A three-step procedure was then performed: on step 1, µPC2 and µPC3 were heated to 180°C: the sample moved from µPC3 to µPC1. On step 2, µPC2 was set to 115°C and the mix C7-C9 moved from µPC1 to µPC3. As the mix went through µPC2, the separation was performed: 115°C was hot enough for C7 to move through, but too cold for C9, which was not volatile enough to be displaced. C9 was trapped on µPC2. On step 3, µPC2 was then heated to 180°C to release C9. The DMFP program used for this experiment is shown in Table 4. The results are shown on Figure 5.

Figure 5 : Separation of C7 and C9. C7 and C9 were first loaded on µPC1. A preliminary step was done to move the sample from µPC1 to µPC3. This step is not shown. Three steps were done: on step 1, the C7-C9 mix was pumped to µPC1, µPC2 and µPC3 were heated to 180°C. On step 2, µPC2 was set to 115°C and the mix went to µPC2. 115°C was hot enough for C7 to go through µPC2 and not be trapped: it went through the µTCD to µPC3. C9 is not volatile enough and was trapped on µPC2. On step 3, µPC2 was heated to 180°C: C9 was released and went to µPC3 through the µTCD. Steps 1, 2 and 3 were then repeated twice.

Figure 5 shows that the separation was effective: by keeping µPC2 at 115°C C7 was able to go through but C9 was trapped. C9 was only released by heating µPC2 at 180°C. Three successive successful separations showed the process was repeatable. As co-elutions between C7 and C9 might be possible, control experiments were done by analysing C7 5 ppm and C9 5 ppm with the same program shown in Table 4. Results are shown on Figure 6. Figure 6 shows that the separation was quite effective, as C9 was totally trapped on µPC2 during step 2. However, the separation was not complete: a small amount of C7 went through the µTCD during step 3, meaning it was trapped on µPC2 during step 2. By measuring the peak area, around 11% of the total C7 amount was trapped on µPC2 during step 2 and 89% of the amount went through. The DMFP could not perform a perfect single-step separation, but it is still capable of separating different compounds, which is one of the basics operations of digital microfluidics. The performances could be enhanced by repeating the separation step.

Measuring breakthrough volumes

The DMFP was used to measure breakthrough volumes (BV) on n-alkanes ranging from n-hexane to nnonane (C9). With standard laboratory instruments, BV studies requires multiple samplings, injections and analyses, as the sample is lost after each analysis. The DMFP used the same sample that was digitally manipulated to gather in about 10 minutes the data needed for one BV measurement at a specific temperature. As the BV was assessed automatically for one compound with the DMFP on a wide range of temperatures in one experiment, the experiment duration was about 3 hours. The BV is the volume of carrier gas needed to elute 50% of the analyte through the adsorbent, at a specific temperature. It is expressed in liters/ grams of adsorbent. The BV is an intrinsic value, which can be tabulated. It characterizes the strength of the interaction between one compound and one adsorbent. At constant flow rate of carrier gas, the BV only changes with temperature for a couple analyte/adsorbent: as temperature increases, the BV decreases due to the adsorption equilibrium shifting towards the desorption of the analyte. The BV tables are useful to assess which adsorbent to use for a specific target, and the range of temperatures in which the target is trapped or released.

During these experiments, the sample was loaded on µPC1. The experiments then consisted in two steps: at step 1, µPC1 was heated to 150°C and µPC2 to a selected temperature, ranging from 150°C to 70°C. Pump 2 was switched on for 300 s. At step 2, µPC3 and µPC2 were heated to 150°C. Pump 1 was switched on for 60 s. Every time step 1 was repeated, µPC2 temperature was lowered by 5°C. Table 5 shows the DMFP program used for this experiment. if it appeared, it meant C6 came back to µPC1 from µPC3. It was only possible if C6 managed to reach µPC3 during step 1 and went through µPC2.

As temperature of µPC2 was decreased during the experiment, breakthrough became less pronounced. This breakthrough efficiency loss is shown in step 2: the peak observed during step 2 became less and less intense. For low temperatures, breakthrough did not occur and there was no peak observed during step 2, such as, in the C6 case shown on Figure 7, the steps done below 75°C. During this experiment, the setup was not opened, meaning only a single sample was loaded and then travelled during the duration of the whole experiment.

For several alkanes the experiment did not yield values for every temperature in the range, as the BV was too high compared to the volume pumped through the µPC. BV values were normalized by subtracting the dead volume, measured at the start of every experiment by doing a breakthrough at 180°C with the sample. At this temperature, analytes were not retained on the adsorbent. Data were then fitted according to Kroupa et al. [START_REF] Kroupa | 30 Hydrocarbon Breakthrough Volumes for Adsorbent Resins[END_REF] . They proposed a two or three parameters model for the dependence of the BV with the temperature. One of the parameters is directly related to the adsorption enthalpy of the compound on the adsorbent, which is an intrinsic characteristic of the compound/adsorbent interaction.

Figure 8 shows the measured breakthrough volume for C6, C7, C8 and C9. Figure 8 shows that the fit, according to the model proposed by Kroupa et al. [START_REF] Kroupa | 30 Hydrocarbon Breakthrough Volumes for Adsorbent Resins[END_REF] , of the BV as a function of the temperature was in good agreement with the measured data for all of the analysed samples. It was also possible to measure the adsorption enthalpy of C6 on Tenax at 25°C : the measured value of

Figure 1 :

 1 Figure 1 : DMFP schematics and pictures of the elementary bricks. The arrows indicate flow direction.

Table 1 :

 1 Program of the DMFP used for the preconcentration experiment. On every step 2 a Tedlar bag containing C6 in nitrogen was connected to the injector to load C6 on µPC1.

Figure 2 :

 2 Figure 2 : Concentration of 5 ppm n-hexane in nitrogen on µPC2.The 4 steps are indicated by the numbers on the figure: Step 1 was the movement of the sample from µPC2 to µPC3. Step 2 was the loading of the sample on µPC1. Step 3 was the movement of the sample from µPC1 to µPC3. Step 4 was the movement of the sample from µPC3 to µPC2. Step 1,2,3,4 were repeated 4 times in total. Each time the compound went through the µTCD a signal (peak) is produced. The height of the C6 peak increased on every cycle, meaning C6 was effectively being concentrated.

Figure 3

 3 Figure 3 shows the TCD signal collected during a sequence of 20 movements of the sample.

Figure 3 :

 3 Figure 3 : Controlled movement of n-heptane 10 ppm between µPC2 and µPC3. Two steps were done: on step 1, µPC2 was heated to 150°C for 30 s and n-heptane went to µPC3. µPC2 cooled down for 40 s. On step 2, µPC3 was heated to 150°C and n-heptane went to µPC2. µPC3 cooled down for 40s. See the inset for a more detailed view of the steps. Steps 1 and 2 were then repeated 20 times. A signal peak was observed every time n-heptane went through the µTCD.

Figure 4 :

 4 Figure 4 : Addition of n-heptane 5 ppm with n-nonane 5 ppm. The peaks are labelled on the figure. Each time a compound

Figure 6 :

 6 Figure 6: Separation of C7 5 ppm and C9 5 ppm. The program is the same as shown on Table4. For the black trace, both C7 5 ppm and C9 5 ppm were loaded on µPC1. For the blue trace, only C7 5 ppm was loaded. For the red trace, only C9 5 ppm was loaded.

Figure 7

 7 Figure7shows a typical breakthrough experiment.

Figure 7 :

 7 Figure 7: Breakthrough curve of 1 ppm n-hexane in nitrogen. The baseline drift and its offset were corrected. Every intense peak observed at step 2 showed the movement of C6 to µPC1 from µPC3, meaning breakthrough occurred. See the inset: the peak observed at 545 s was the breakthrough of C6 through µPC2 at 150°C during step 1. The peak observed at 817 s was the return of C6 from µPC3 to µPC1 during step 2. Every time step 1 was repeated, µPC2 temperature was lowered by 5°C.

Figure 7

 7 Figure7shows that breakthrough happened over a wide range of temperatures, as evidenced by a peak observed during step 1. The peak observed during step 2 was a second proof of a breakthrough:

Figure 8 :

 8 Figure 8: Breakthrough volume measured for a) C6, b) C7, c) C8 and d) C9. Experiments were made in duplicate.

Table 2 :

 2 Program of the DMFP used for the controlled movements experiment.

Table 3 :

 3 Program used for the C7-C9 addition experiment.

Table 4 :

 4 DMFP program used for the C7-C9 separation experiment.

Table 5 :

 5 DMFP program used for the C6 breakthrough experiment.

Data were also compared with tabulated values found on SIS website 30 : a slight deviation was observed from tabulated values. This deviation was consistent with Kroupa et al. findings [START_REF] Kroupa | 30 Hydrocarbon Breakthrough Volumes for Adsorbent Resins[END_REF] : they attributed the deviation to a fitting error in SIS interpolations.

Conclusion

The digital microfluidic platform presented in this study allows step by step programmable digital manipulations of gas samples without losses. All elementary operations (trapping/releasing, moving, mixing, and separating samples) have been demonstrated using n-hexane to n-nonane alkanes. Moreover, by programming a succession of elementary operations, it is possible to perform more complex operations and applications. This could be a first step towards digital chromatography, since the elementary operations performed by the DMFP presented here can be related to a single theoretical plate as in Martin and Synge theory [START_REF] Martin | [END_REF] . As a first illustration, the measurements of breakthrough volumes of gases have been performed. The results were in agreement with the tabulated values obtained with standard laboratory instruments, and showed good agreement with fundamental values, such as the adsorption enthalpy of the gases on the adsorbent.

Beyond this first DMFP made of three µPC, other DMFP with more complex network of µPC could be developed in the future. With the development of a more complex DMFP and gas manipulation algorithm, this work could lead to new miniaturized digital systems and methods for gas sample preparation and analysis. These new methods of sample handling would be suitable for portable gas analysis systems, but also conventional gas analysis systems. For example, a digital sample handler could be used to extract compounds within a certain volatility range from a gas sample prior to injection in a conventional gas chromatograph. As another example, a system similar to the DMFP presented here could be used as an online miniaturized trap for chromatographic applications in a similar fashion to thermal modulators for comprehensive bi-dimensional gas chromatography.