Michèle Bengue

Ai-Rada Pintong
email: ai-rada.pintong@ird.fr

Florian Liégeois

Antoine Nougairède
email: antoine.nougairede@univ-amu.fr

Rodolphe Hamel
email: rodolphe.hamel@ird.fr

Julien Pompon
email: julien.pompon@ird.fr

Xavier De Lamballerie
email: xavier.de-lamballerie@univ-amu.fr

Pierre Roques
email: pierre.roques@pasteur.fr

Valérie Choumet
email: valerie.choumet@pasteur.fr

Dorothée Missé
email: dorothee.misse@ird.fr

Florian Liegeois
email: florian.liegeois@ird.fr

Favipiravir inhibits Mayaro virus infection in mice

Keywords: alphavirus, arbovirus, mayaro, favipiravir, antiviral drug

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

Introduction

Mayaro virus (MAYV) is a zoonotic arbovirus that is widespread in South America and the Caribbean islands, where it has caused sporadic outbreaks [START_REF] Lorenz | Mayaro Virus Distribution in South America[END_REF][START_REF] Tesh | Mayaro Virus Disease: An Emerging Mosquito-Borne Zoonosis in Tropical South America[END_REF]. MAYV is the etiologic agent of MAYV Fever and was first isolated in 1954 from five rural workers in southeastern Trinidad (Mayaro county), during an outbreak in which many cases of arthralgia were reported [START_REF] Anderson | Mayaro Virus: A New Human Disease Agent. II. Isolation from Blood of Patients in Trinidad, B.W.I[END_REF]. The isolation of the virus from a five-year-old boy in Haiti in 2015 indicated that MAYV had migrated to the Caribbean and was actively circulating [START_REF] Lednicky | Mayaro Virus in Child with Acute Febrile Illness, Haiti[END_REF][START_REF] Hozé | Reconstructing Mayaro Virus Circulation in French Guiana Shows Frequent Spillovers[END_REF].

MAYV is usually transmitted by the bite of an infected tree-dwelling mosquito, Haemagogus janthinomys, which is considered the main vector [START_REF] Tesh | Mayaro Virus Disease: An Emerging Mosquito-Borne Zoonosis in Tropical South America[END_REF] MAYV is a single-stranded RNA-enveloped virus that belongs to the Alphavirus genus [START_REF] Leung | Replication of Alphaviruses: A Review on the Entry Process of Alphaviruses into Cells[END_REF]. The genome encodes five structural proteins: Capsid (C), Envelope (E1, E2, E3), 6K and four nonstructural proteins (nsP1,2, 3,4) [START_REF] Rupp | Alphavirus RNA Synthesis and Non-Structural Protein Functions[END_REF]. Alphaviruses can be divided into two groups: encephalitic and arthritogenic viruses [START_REF] Suhrbier | Arthritogenic Alphaviruses-An Overview[END_REF]. The first is known for its neurotropism and the second manifests a tropism for the joints. MAYV is an arthritogenic alphavirus and symptoms of acute infection include headaches, vomiting, fever, diarrhea and rash, which are sometimes associated with myalgia [START_REF] Halsey | Mayaro Virus Infection, Amazon Basin Region, Peru, 2010-2013[END_REF]. Most importantly, debilitating arthralgia has been reported in MAYV-infected patients, similar to that reported in patients infected by other arthritogenic alphaviruses, such as Chikungunya virus (CHIKV), Sindbis virus (SINV), O'nyong nyong virus, and Ross River virus (RRV) [START_REF] Friedrich-Jänicke | Genome Analysis of Mayaro Virus Imported to Germany from French Guiana[END_REF][START_REF] Galatas | Long-Lasting Immune Protection and Other Epidemiological Findings after Chikungunya Emergence in a Cambodian Rural Community[END_REF][START_REF] Kiwanuka | O'nyong-Nyong Fever in South-Central Uganda, 1996-1997: Clinical Features and Validation of a Clinical Case Definition for Surveillance Purposes[END_REF][START_REF] Harley | River Virus Disease in Tropical Queensland: Evolution of Rheumatic Manifestations in an Inception Cohort Followed for Six Months[END_REF]. Alphavirus-induced arthralgia usually affects the joints of the hands, feet, ankles, and the extremities, and frequently progresses to polyarthritis [START_REF] Slegers | Persisting Arthralgia Due to Mayaro Virus Infection in a Traveler from Brazil: Is There a Risk for Attendants to the 2014 FIFA World Cup?[END_REF][START_REF] Taylor | Recurrent Arthralgias in a Patient with Previous Mayaro Fever Infection[END_REF][START_REF] Mcgill | Viral Infections: Alpha-Viral Arthropathy[END_REF]. In addition, rare but severe pathologies, including myocarditis, hemorrhagic and neurological complications, have been reported as a result of MAYV infection [START_REF] Diagne | Mayaro Virus Pathogenesis and Transmission Mechanisms[END_REF]. Following a mosquito bite, MAYV disseminates into the bloodstream, the spleen and the liver, and then reaches the joints, muscles and bones, where it can progress to a chronic infection [START_REF] Mourão | Mayaro Fever in the City of Manaus, Brazil, 2007-2008[END_REF]. It has been demonstrated in a mouse model that there is a correlation between the severity and chronicity of MAYV infection and age, and the clinical manifestations of arthritogenic alphavirus in human confirm this observation [START_REF] Figueiredo | Mayaro Virus Replication Restriction and Induction of Muscular Inflammation in Mice Are Dependent on Age, Type-I Interferon Response, and Adaptive Immunity[END_REF][START_REF] Murillo-Zamora | Persistent Arthralgia and Related Risks Factors: A Cohort Study at 12 Months from Laboratory-Confirmed Chikungunya Infection[END_REF]. Pro-inflammatory cytokines have been described as being associated with MAYV disease severity in patients and in mouse models. Some of these include MCP-1, IL-2, IL-9, IL-13, IL-7, VEGF, IL-17, and IP-10 [START_REF] Santiago | Long-Term Arthralgia after Mayaro Virus Infection Correlates with Sustained Pro-Inflammatory Cytokine Response[END_REF][START_REF] Santos | Animal Model of Arthritis and Myositis Induced by the Mayaro Virus[END_REF]. Several mechanisms are thought to be involved in alphavirus pathogenesis, including oxidative stress [START_REF] Camini | Oxidative Stress in Mayaro Virus Infection[END_REF][START_REF] Joubert | Chikungunya-Induced Cell Death Is Limited by ER and Oxidative Stress-Induced Autophagy[END_REF][START_REF] Remenyi | Persistent Replication of a Chikungunya Virus Replicon in Human Cells Is Associated with Presence of Stable Cytoplasmic Granules Containing Nonstructural Protein 3[END_REF][START_REF] Rodriguez | SINV Induces Oxidative Stress in Baby Hamster Kidney Host Cells[END_REF]. MAYV has been shown to regulate ROS production in the liver, which had an impact on the immune response and viral replication in a mouse model [START_REF] Camini | Oxidative Stress in Mayaro Virus Infection[END_REF]. Although few clinical data are available on liver pathology due to MAYV, hepatomegaly, has been observed in patients [START_REF] Acosta-Ampudia | An Emerging Viral Threat?[END_REF]. In mouse models, MAYV has been shown to intensively replicate in the liver [START_REF] Figueiredo | Mayaro Virus Replication Restriction and Induction of Muscular Inflammation in Mice Are Dependent on Age, Type-I Interferon Response, and Adaptive Immunity[END_REF][START_REF] Silva Caetano | Mayaro Virus Induction of Oxidative Stress Is Associated With Liver Pathology in a Non-Lethal Mouse Model[END_REF]. Along with hepatomegaly, this could induce liver function impairment and damage, which, as in liver disease, could biochemically result in elevated alanine aminotransferases (ALAT) and aspartate amino transferase (ASAT) [START_REF] Moriles | Alanine Amino Transferase[END_REF].

Despite the clinical impact of MAYV infection, no therapeutic drugs or vaccines have yet been approved for this alphaviral disease. The management of the disease relies on antipyretics, nonsteroidal anti-inflammatory drugs, and analgesics [START_REF] Abdelnabi | Antiviral Treatment Efficiently Inhibits Chikungunya Virus Infection in the Joints of Mice during the Acute but Not during the Chronic Phase of the Infection[END_REF][START_REF] De Mello | Antiviral Drug Discovery and Development for Mayaro Fever-What Do We Have so Far?[END_REF]. Antiviral strategies have been designed for alphaviral infections targeting the virus itself or host factors [START_REF] Abdelnabi | Antiviral Treatment Efficiently Inhibits Chikungunya Virus Infection in the Joints of Mice during the Acute but Not during the Chronic Phase of the Infection[END_REF], some of which use inhibitors targeting the different enzymes involved in the viral life cycle, such as viral RNA dependent-RNA Polymerase, viral capping and viral protease inhibitors [START_REF] Abu Bakar | Nonstructural Proteins of Alphavirus-Potential Targets for Drug Development[END_REF][START_REF] Delogu | In Vitro Antiviral Activity of Arbidol against Chikungunya Virus and Characteristics of a Selected Resistant Mutant[END_REF].

Antiviral studies to identify molecules that inhibit MAYV infection are scarce and have more often been conducted in vitro [START_REF] Amorim | Thieno[2,3-b]Pyridine Derivatives: A New Class of Antiviral Drugs against Mayaro Virus[END_REF][START_REF] Da Costa | Effect of Brefeldin A on Mayaro Virus Replication in Aedes Albopictus and Vero Cells[END_REF][START_REF] Carvalho | Inhibition of Mayaro Virus Infection by Bovine Lactoferrin[END_REF]. In a recent study, the efficacy of Favipiravir on MAYV infection was demonstrated in cell cultures [START_REF] Langendries | Repurposing Drugs for Mayaro Virus: Identification of EIDD-1931, Favipiravir and Suramin as Mayaro Virus Inhibitors[END_REF]. However, the antiviral effect of this molecule is yet to be studied in vivo. Several studies have shown that Favipiravir inhibits arthritogenic alphaviruses, including Western equine encephalitis virus (WEEV) and CHIKV in cells and in vivo in mice [START_REF] Abdelnabi | Antiviral Treatment Efficiently Inhibits Chikungunya Virus Infection in the Joints of Mice during the Acute but Not during the Chronic Phase of the Infection[END_REF][START_REF] Delang | Mutations in the Chikungunya Virus Non-Structural Proteins Cause Resistance to Favipiravir (T-705), a Broad-Spectrum Antiviral[END_REF][START_REF] Julander | Effect of T-705 Treatment on Western Equine Encephalitis in a Mouse Model[END_REF]. In a CHIKV-infected C57BL/6 mouse model, Favipiravir (300 mg/kg/day) was effective when administered during the acute phase of infection and not during the chronic phase after CHIKV infection. CHIKV replication was strongly suppressed in joints and serum during the acute phase [START_REF] Abdelnabi | Antiviral Treatment Efficiently Inhibits Chikungunya Virus Infection in the Joints of Mice during the Acute but Not during the Chronic Phase of the Infection[END_REF]. In AG129 CHIKV-infected mice, Favipiravir protected the mice from neurological disorders and death [START_REF] Delang | Mutations in the Chikungunya Virus Non-Structural Proteins Cause Resistance to Favipiravir (T-705), a Broad-Spectrum Antiviral[END_REF].

In this study, we evaluated the effect of Favipiravir, administered at different times during infection, on MAYV-infected C57BL/6 mice. We report here that Favipiravir, depending on the kinetics of its administration, exerts a potent antiviral effect against MAYV infection that is accompanied by a decrease in the serum levels of the MAYV-induced hepatic liver enzymes, ALAT and ASAT.

Materials and Methods

Virus, Cells and Compounds

The African green monkey epithelial kidney cell line Vero-E6, macaque fibroblasts and hamster kidney BHK-21 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, France), supplemented with 5% fetal bovine serum (FBS) (Lonza, Switzerland) and 0.5% penicillin/streptomycin. The macaque tendon fibroblasts were obtained as pre-viously described [START_REF] Roques | Paradoxical Effect of Chloroquine Treatment in Enhancing Chikungunya Virus Infection[END_REF]. Briefly, primary fibroblasts were obtained from the tendons of macaques, which were negative for the presence of CHIKV or Dengue virus infection, as assessed by antibodies and PCR evaluation, after necropsy. The tendons were minced and plated in 3 cm diameter Petri dishes saturated with FCS. The tendon pieces were immerged in DMEM, 20% FCS and the explants were cultured at 37 • C, 9% CO 2 in a water-saturated atmosphere. After 7 to 10 days, the fibroblasts came out from the explants, then the cells were passaged every 3 days in DMEM, 10% FCS. A UVE/MAYV/1954/TT/TC625 strain isolated in Trinidad and Tobago was obtained from EVAg (www.european-virus-archive.com; June 2020). Ribavirin (Sigma, France) was dissolved in DMSO (Sigma, France) at 20 mg/mL aliquoted per 70 µL and stored at -20 • C. Favipiravir, kindly provided by Toyama Chemical (Japan), was dissolved in phosphate buffered saline (PBS) at 60 mg/mL and stored at -20 • C, prior to use. Both drugs were thawed at room temperature and dilutions were performed in culture medium before usage.

Ethics Statement

The mice were housed in the Institut Pasteur's Animal facilities, according to the French and European regulations on the care and protection of laboratory animals. The Animal Ethics Committee of the Pasteur Institute (CETEA-Institut Pasteur) and the French Ministry of Higher Education and Research (MESR 00762.02) approved all the animal experiments. The macaque fibroblasts were issued from biobanked tissues obtained from euthanized macaques. These studies were approved by the regional animal care and use committee in accordance with European directive 63/2010/EU: "CREEA Ile de France Sud", Fontenay aux Roses, decision #A08-012 dated 7 July 2008.

Mouse Experiment

Four-week-old, 12-14 g, female immunocompetent C57BL/6 mice purchased from Charles River Laboratories (Saint Germain Nuelles, France) were used in the experiments. The mice were kept in cages with HEPA filters in the BSL3 animal facilities of the Pasteur Institute. They were raised in a pathogen -free environment and enjoyed free access to unlimited drinking water and food.

After anesthesia with an injectable mixture of ketamine (Merial, Lyon, France) and xylazine (Bayer, Berlin, Germany), the mice were injected subcutaneously in the right hind footpad with 10 6 pfu of MAYV in a final volume of 20 µL. The mice were monitored for weight, clinical signs and viremia. Blood was collected from the caudal vein. Swelling of the inoculated foot was assessed by measuring the height and width of the perimetatarsal area using a caliper (Mitutoyo, Japan) and was calculated as follows:

Wi f × Bi f Wc f × Bc f × 100
where W: Width, B: Breadth, if : Inoculated foot and cf : Contralateral foot [START_REF] Belarbi | Bioluminescent Ross River Virus Allows Live Monitoring of Acute and Long-Term Alphaviral Infection by In Vivo Imaging[END_REF].

In Vitro Activity of Favipiravir

Vero cells (2.5 × 10 5 cells/well), BHK21 cells (1.2 × 10 5 cells/well) or primary macaque tendon fibroblasts (passage 11, 2 × 10 5 cells/well), respectively, were seeded in ninety-six welll plates and exposed to MAYV at a MOI of 0.005 (for Vero cells) and 0.002 (for BHK21 and tendon fibroblasts) for 1 h at 37 • C. The cells were then thoroughly washed twice, and 100 µL of culture medium supplemented with the drugs at a concentration between 300 down to 2.5 µg/mL and 200 down to 5 µg/mL (8 wells per dilution) for Favipiravir and Ribavirin, respectively, was subsequently added. The selected MOI consistently infected and killed all cells in 48 h. Sixteen wells without added virus served as a negative control.

The cell cultures were incubated at 37 • C, 5% CO 2 and cell viability was monitored until 72 hours post-infection (hpi), using crystal violet staining. Viral inhibition and the 50% inhibitory concentration (IC50%) were computed using Prism software version 8.1 (GraphPad Software, La Jolla, CA, USA).

Before the determination of the IC50%, the MAYV stock titer was determined in the various cell types using crystal violet staining at 72 h post-exposure. The observed titers, obtained by using the Käber method [START_REF] Kirillov | Method of quantitative determination of vaccine activity against botulism of the mink[END_REF], were 6.8 × 10 6 ; 6.2 × 10 6 ; 3.2 × 10 6 TCID 50%/mL for the Vero-E6, BHK-21 and primary tendon fibroblasts, respectively.

In Vivo Activity of Favipiravir

The mice were randomly divided into five groups, as follows: The mice were marked on their tails, then housed in cages in groups of three to four in an isolator. Favipiravir (300 mg/kg) was administered orally for five consecutive days. The mice were monitored daily up to 7 days post-treatment and/or exposure to MAYV. Half the number of mice per group were sacrificed after 7 days and the remaining mice 14 days after MAYV infection or Favipiravir treatment.

Tissue Collection

The mice were anesthetized and injected with an intra-cardiac perfusion of PBS. The brain, heart, liver, spleen, kidney, lymph nodes, right and left quadriceps muscles and right and left ankles were collected and kept frozen at -80 • C until use. Serum was separated from the whole blood samples through centrifugation in microvette tubes (Sarstedt, Nümbrecht, Germany), according to the manufacturer's instructions.

Determination of Viral Spread in Organs and Blood

The mouse organs were dissected at 7 and 14 days post-infection (dpi) to confirm the presence of MAYV RNA and infectious viral particles. The tissues was placed in tissue homogenizing bead tubes (Precellys Lysing kits, Bertin Tech., Montigny-le-Bretonneux, France) with Dulbecco's modified Eagle's medium (DMEM). After homogenization, 100 µL of the mixture was transferred to a tube containing RNA extraction lysis buffer. The remaining homogenate was centrifuged and the supernatant was stored at -80 until use. Blood was collected and 20 µL was added to the RNA extraction lysis buffer.

The total RNA was extracted from the organs at 7 and 14 dpi and blood from 0 to 14 dpi using a NucleoSpin RNA Mini kit for RNA purification (Macherey-Nagel, Düren, Germany), according to the manufacturer's protocol. The extracted RNA was eluted in 50 µL of H 2 O and quantified; 1 µg was used for reverse transcription, using the Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega, Charbonnières-les-Bains, France). Real-time PCR was carried out using a Maxima probe/ROX qPCR master mix (Promega, France), as described previously [START_REF] Bengue | Mayaro Virus Infects Human Chondrocytes and Induces the Expression of Arthritis-Related Genes Associated with Joint Degradation[END_REF][START_REF] Diop | Differential Susceptibility and Innate Immune Response of Aedes Aegypti and Aedes Albopictus to the Haitian Strain of the Mayaro Virus[END_REF]. The following set of primers and probe were designed and used for MAYV detection: MAYV_F TGCGCCTGCCAGGAGAATGCTGT; MAYV_R TCGCCTGATGCCTTGGCCAACT; FAM-ACGTACCATTGTGTGTGCCCAAT-BHQ.

Detection of Infectious Viral Particles

MAYV titers in the blood and organs were determined by plaque assay, as previously described [START_REF] Diop | Differential Susceptibility and Innate Immune Response of Aedes Aegypti and Aedes Albopictus to the Haitian Strain of the Mayaro Virus[END_REF]. Briefly, the Vero-E6 cell line was seeded in triplicate at a concentration of 10 5 cells per well in a 24 well plate. Serial dilutions of organ homogenates and blood were prepared in DMEM. The inoculum was added to the seeded cells for 1 h and 30 min at 37 • C and 5% CO 2 while being gently shaken. After incubation , the inoculum was removed and replaced by an overlay medium containing a mix of nutriments and carboxymethyl cellulose (Lonza, Switzerland). The plates were subsequently incubated for four days, after which the cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The plates were dried and the plaques were manually determined in each well. The results are presented as plaque-forming units per g (pfu/g) of tissue.

Measurement of ASAT and ALAT Serum Levels

The measurement of aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) serum levels were performed by ELISA using aspartate aminotransferase (ASAT) (Elabscience, Houston, USA) and alanine aminotransferase (ALAT) Activity Assay Kits (Elabscience), respectively, according to the manufacturer's protocol. The results are presented as U/mL. Individual mouse data were used in duplicates.

Statistical Analyses

The statistical analyses were generated with GraphPad Prism 8 software and the experimental results were expressed as mean ± standard deviation (SD). A two-way ANOVA test was used to compare groups and controls. The p-values were statistically significant when inferior to 0.05.

Results

Favipiravir Exerts Antiviral Activity against MAYV In Vitro

The Vero-E6 and BHK-21 cells were exposed to MAYV using MOI, which eventually killed all the cells, before being treated with different concentrations of Favipiravir. The inhibition of viral replication was observed in the Vero-E6 and BHK-21 cell types at an IC50% of 0.12 ± 0.01 µM and 0.2 ± 0.001 µM, respectively. Given that tendon fibroblasts are one of the main target cells for MAYV, the same experiment was conducted with the latter cells and the inhibition of viral replication was observed at IC50% of 0.06 ± 0.03 µM (Figure 2). By contrast, Ribavirin did not exert antiviral activity against MAYV replication (data not shown).

Figure 2. Favipiravir inhibits MAYV in cell culture. Favipiravir was introduced in medium culture 1 h after MAYV exposure of the cells Vero-6, BHK-21 and primate tendon primary fibroblasts at a MOI of 0.005 and 0.002, respectively. Favipiravir MAYV Inhibitory Concentration 50% was obtained on two independent assays and in octoplicates for each dilution of the drug tested.

MAYV Is Susceptible to Favipiravir In Vivo

Favipiravir was administered to the mice to test whether the in vitro results would translate into an in vivo model by protecting the animal from MAYV infection. For this purpose, a time-course study was carried out to determine the kinetics of MAYV inhibition in C57B/L6 mice during infection (Figure 3). A non-treated group of mice exposed to MAYV (CTRL + ) and a non-infected group (CTRL -) were used as controls. Favipiravir was orally administered at 300 mg/kg/day at pre-, concurrent-and post-MAYV infection for five days and the mice were euthanatized at 7 and 14 dpi. All the mice were injected with 10 6 PFU of MAYV through the right hind paws, which was not lethal (results not shown). All the mice were weighed and their paws were measured before and every other day after the Favipiravir challenge (Figure 3A,B). A significant weight loss (p < 0.05) was noticed at 2 and 3 dpi in the CTRL + group, compared to the mice that were treated concurrently with Favipiravir and the CTRL -mice (Figure 3A). In addition, a statistically significant footpad swelling (p < 0.05, two-way ANOVA) was observed in the CTRL+ and post-treated mice, as compared to the pre-and concurrent Favipiravir-treated mice at 8 dpi (Figure 3B). There was a statistically significant decrease in MAYV RNA and infectious viral particles in the blood (Figure 4) and organs (Figure 5) in pre-(Figures 4A,D and5A,E) and concurrent Favipiravir-treated mice (Figures 4B,E and5B,F), as compared to the MAYVexposed mice (CTRL + ). These differences were more significant between the CTRL + and concurrent treated groups than between the CTRL + and pre-treated groups. In fact, at 2 dpi, there was an approximately three-log difference in the amount of viral copy numbers between the CTRL + and concurrently treated groups that remained significant up to 7 dpi. In addition, with regard to the production of infectious viral particles, this difference was observed until 4 dpi. However, from day 5 onward, in both conditions no infectious viral particles were detected in the blood (Figure 4D-F). To determine whether pre-, concurrent-and post-treatment with Favipiravir had an antiviral effect on MAYV replication, viral RNA was extracted from several organs/tissues including the brain, quadriceps muscles, and right and left ankles of the infected mice at 7 dpi and 14 dpi.

The results showed a strong decrease in viral RNA in several tissues, especially in the heart, liver, kidneys, right quadriceps muscles and ankles in the concurrently treated mice compared to the CTRL + at 7 dpi (Figure 5B). However, at 7 dpi, there was a more modest reduction in the amount of viral RNA transcripts in all of the organs, except from the left ankle, between the CTRL + and the pre-treatment groups (Figure 5A). Likewise, in the blood, we observed no difference in the number of viral RNA copies between the CTRL + and post-treatment groups; however, there was a statistically significant decrease (p < 0.05, two-way ANOVA) in viral RNA in the right quadricep muscles in the posttreated group. Interestingly, infectious viral particles were only detected in the right ankles in the CTRL + and post-treated groups; on the other hand, there were no infectious viral particles detected in the pre-and concurrent Favipiravir-treated groups at 7 dpi (Figure 5D). However, we noticed a significant decrease in infectious viral particles in the post-treated group compared to the CTRL + group (p < 0.01) (Figure 5D).

We observed that Favipiravir treatment continued to be effective until day 14 post-MAYV-exposure, particularly in the heart, ganglion and ankles, in the concurrently treated group, compared to the CTRL + group (Figure 5F). For some organs (heart, ganglion, left ankle), the difference between the CTRL + and concurrently treated groups was approximately two logs, while it was roughly one log in the kidney at 14 dpi (p < 0.01, Turkey's test). Apart from the increased number of viral particles in the right quadriceps muscle, there was no significant difference between the CTRL + mice and the post-treatment group (Figure 5G). Finally, no infectious viral particles were detected in any of the groups at 14 dpi (data not shown).

Favipiravir Decreases MAYV-Induced Transaminases

To evaluate the effect of Favipiravir on MAYV-induced hepatic damage in infected C57BL/6 mice, the serum levels of (ASAT) and (ALAT) in the pre-and concurrently treated mice were quantified at 7 and 14 dpi (Figure 6A,B). We demonstrated a significant decrease in the amount of ASAT (p < 0.01) and ALAT (p < 0.01; student's test) at 7 dpi between the pre-treated and concurrently treated mice compared to the CTRL+ group (MAYV-infected and untreated mice). This difference was also observed at 14 dpi for ASAT (p < 0.01) and was less pronounced for ALAT (p < 0.05). These results demonstrate that Favipiravir's effect on viral replication included lowering the hyper-expression of the hepatic enzymes observed in the MAYV-infected mice.

Discussion

MAYV is known to cause self-limiting illness. However, an incidence of more than 50% of arthralgia/myalgia in infected individuals was reported during several outbreaks in South America [START_REF] Mourão | Mayaro Fever in the City of Manaus, Brazil, 2007-2008[END_REF][START_REF] Santiago | Long-Term Arthralgia after Mayaro Virus Infection Correlates with Sustained Pro-Inflammatory Cytokine Response[END_REF][START_REF] Suchowiecki | Persistent Joint Pain Following Arthropod Virus Infections[END_REF]. Knowledge of MAYV's pathogenesis is based principally on results from relevant animal models and other arthritogenic alphavirus studies and is therefore not fully understood. Following a mosquito bite, MAYV disseminates in the bloodstream and, via the spleen and the liver, it reaches the joints, muscles and bones, where it can cause chronic infection [START_REF] Tesh | Mayaro Virus Disease: An Emerging Mosquito-Borne Zoonosis in Tropical South America[END_REF][START_REF] Diagne | Mayaro Virus Pathogenesis and Transmission Mechanisms[END_REF][START_REF] Dupuis-Maguiraga | Chikungunya Disease: Infection-Associated Markers from the Acute to the Chronic Phase of Arbovirus-Induced Arthralgia[END_REF]. At present, no effective treatment or antiviral drugs have been approved or commercialized. The only treatment option currently available is to reduce pain with analgesics and/or non-steroidal anti-inflammatory drugs [START_REF] Diagne | Mayaro Virus Pathogenesis and Transmission Mechanisms[END_REF][START_REF] Acosta-Ampudia | An Emerging Viral Threat?[END_REF]. The co-circulation of other arboviruses in South America and the Caribbean, such as dengue virus and CHIKV, which cause symptoms similar to those of MAYV infection, renders the clinical diagnosis of MAYV fever difficult [START_REF] Hozé | Reconstructing Mayaro Virus Circulation in French Guiana Shows Frequent Spillovers[END_REF]. In the present study, we hypothesized that because of the reported antiviral activity of Favipiravir against CHIKV [START_REF] Abdelnabi | Antiviral Treatment Efficiently Inhibits Chikungunya Virus Infection in the Joints of Mice during the Acute but Not during the Chronic Phase of the Infection[END_REF][START_REF] Delang | Mutations in the Chikungunya Virus Non-Structural Proteins Cause Resistance to Favipiravir (T-705), a Broad-Spectrum Antiviral[END_REF] and several other alphaviruses [START_REF] Julander | Effect of T-705 Treatment on Western Equine Encephalitis in a Mouse Model[END_REF][START_REF] Furuta | T-705), a Novel Viral RNA Polymerase Inhibitor[END_REF], it is Favipiravir is also likely to inhibit MAYV replication in vitro and in vivo. The other objective of this study was to determine the time of administration at which Favipiravir was most effective at decreasing viral infection in vivo.

In the present study, we show that Favipiravir, an RNA polymerase inhibitor [START_REF] Driouich | Favipiravir Antiviral Efficacy against SARS-CoV-2 in a Hamster Model[END_REF], strongly decreases MAYV replication both in vitro and in vivo.

MAYV disease is known for its debilitating effects on the joints. Interestingly, in vitro, we found that IC50% seems to be more effective in tendon fibroblast than in reference cell lines. Notably, unlike Favipiravir, Ribavirin demonstrated no anti-MAYV activity in vitro, which corroborates the results in a previously published report conducted by Langendries et al., in which several molecules, including Ribavirin and Favipiravir, were tested for their ability to protect permissive cells against MAYV in vitro [START_REF] Langendries | Repurposing Drugs for Mayaro Virus: Identification of EIDD-1931, Favipiravir and Suramin as Mayaro Virus Inhibitors[END_REF].

In vivo, our results also demonstrate that the administration of Favipiravir, both prior to and during infection, reduced viral replication and, consequently, decreased footpad swelling at the virus' inoculation site. It can be inferred that the reduction of infectious viral particles, as well as viral RNA, by Favipiravir correlates with the reduction of arthritis/arthralgia because there was a considerable reduction in footpad swelling in the pre-treated and concurrently treated mice, as compared to the CTRL + mice. However, under the conditions used in this study, this effect was mainly observed when Favipiravir was administered prior to or during infection, whereas a reduction in infectious viral particles was only observed in the right ankles of the post-treated mice. These results suggest that once the infection is established, it can no longer be controlled by Favipiravir at the administered dose and the amount of virus (10 6 PFU) used for infection. As Favipiravir targets viral RNA polymerase, it can inhibit new viral cycles; the treatment performed at 2 dpi could therefore not be effective compared to its use in the other conditions. However, we observed intracellular viral RNA at 7 dpi, which decreased at 14 dpi in each of the organs and in all of the infected mouse groups. This was probably due to the animal model used in this study, as immune-competent mice are able to control viremia efficiently at 5 dpi. In addition, the viral titration of MAYV in all the organs at 7 dpi revealed the clearance of the infectious viral particles except, in the infected right ankles of the MAYV-infected and post-treated mice. The persistence of the virus in the ankles of the MAYV-infected and post-treated groups could have been due to the proximity of the site of inoculation, viral evasion and/or host defense mechanisms.

Presumably, cellular infiltrates, such as those involving macrophages, could in part explain this persistence, given that these cells are permissive to MAYV infection, thereby contributing to the continuation of viral replication. Furthermore, even after treatment with Favipiravir, we did not observe a complete clearance of MAYV RNA in the majority of the organs. This observation could be due to the difference between the viral load of MAYV, 10 6 PFU, with which we inoculated our mice and the load used in the study by [START_REF] Abdelnabi | Antiviral Treatment Efficiently Inhibits Chikungunya Virus Infection in the Joints of Mice during the Acute but Not during the Chronic Phase of the Infection[END_REF], in which they used 10 3 PFU of CHIKV [START_REF] Abdelnabi | Antiviral Treatment Efficiently Inhibits Chikungunya Virus Infection in the Joints of Mice during the Acute but Not during the Chronic Phase of the Infection[END_REF]. The amount of MAYV used in the current study could have masked the antiviral effects of Favipiravir in the pre-and post-treated groups, thereby preventing the total clearance of MAYV RNA in the organs.

Lethal mutagenesis has recently been considered as an important antiviral strategy against RNA viruses and it is now known that Favipiravir has the ability to elicit this phenomenon by increasing the number of mutations [START_REF] Borrego | Lethal Mutagenesis of Rift Valley Fever Virus Induced by Favipiravir[END_REF][START_REF] Arias | Favipiravir Elicits Antiviral Mutagenesis during Virus Replication in Vivo[END_REF]. This mutagenic activity, as previously demonstrated with CHIKV and West Nile virus, could also explain how Favipiravir inhibits MAYV replication, as shown in our study.

A wide range of studies has demonstrated the critical role of the liver in the pathogenesis of many viruses, including alphaviruses [START_REF] Assunção-Miranda | Molecular Mechanisms Involved in the Pathogenesis of Alphavirus-Induced Arthritis[END_REF][START_REF] Camini | Antiviral Activity of Silymarin against Mayaro Virus and Protective Effect in Virus-Induced Oxidative Stress[END_REF][START_REF] Gardner | Chikungunya Virus Arthritis in Adult Wild-Type Mice[END_REF][START_REF] Labadie | Chikungunya Disease in Nonhuman Primates Involves Long-Term Viral Persistence in Macrophages[END_REF][START_REF] Nunes | Microcephaly and Zika Virus: A Clinical and Epidemiological Analysis of the Current Outbreak in Brazil[END_REF]. Camini et al. demonstrated that oxidative stress, along with liver injury, was induced by MAYV infection in the HepG2 cell line, as well as in vivo in BALB/c mice [START_REF] Camini | Oxidative Stress in Mayaro Virus Infection[END_REF][START_REF] Silva Caetano | Mayaro Virus Induction of Oxidative Stress Is Associated With Liver Pathology in a Non-Lethal Mouse Model[END_REF]. Based on these findings, we hypothesized that MAYV could replicate in the liver, thereby causing organ damage, and that this condition could be prevented or improved by the administration of Favipiravir. Our study revealed a reduction in MAYV infectious viral particles and viral RNA in the livers of preand concurrent Favipiravir-treated mice at 7 and 14 dpi, demonstrating that the drug could be beneficial in the case of alphavirus-induced liver disease.

The reduction in viral replication in the liver by Favipiravir was associated with the decrease in serum ALAT and ASAT. Interestingly, the serum levels of these transaminases were found to be considerably decreased in the pre-and concurrently treated mice, as compared to the MAYV-infected mice without treatment, indicating that Favipiravir could protect the animals from potential liver injury due to MAYV liver infection.

The mouse model used in the present study seems to be ideal to study the efficacy of drugs against MAYV infection, like the model previously developed for CHIKV [START_REF] Abdelnabi | Antiviral Treatment Efficiently Inhibits Chikungunya Virus Infection in the Joints of Mice during the Acute but Not during the Chronic Phase of the Infection[END_REF][START_REF] Varghese | The Antiviral Alkaloid Berberine Reduces Chikungunya Virus-Induced Mitogen-Activated Protein Kinase Signaling[END_REF], because the virus induces clinical signs similar to those observed in MAYV-infected patients and does not cause lethality. By contrast, immunodeficient models, such as IFN receptor-knock-out mice, known to present immature immune systems, are lethal to CHIKV [START_REF] Couderc | A Mouse Model for Chikungunya: Young Age and Inefficient Type-I Interferon Signaling Are Risk Factors for Severe Disease[END_REF], as well as MAYV [START_REF] Figueiredo | Mayaro Virus Replication Restriction and Induction of Muscular Inflammation in Mice Are Dependent on Age, Type-I Interferon Response, and Adaptive Immunity[END_REF] infection, as a consequence, they are not suitable for these studies. It would be of interest to evaluate the effect of Favipiravir administrated using various doses up to 1400 mg/kg/day, as described by Driouich et al. 2021 [START_REF] Driouich | Favipiravir Antiviral Efficacy against SARS-CoV-2 in a Hamster Model[END_REF], using the present model, when administered during the chronic phase of MAYV infection, which is more representative of the debilitating state of infected patients. Finally, it would be of interest to compare the results obtained from the present mouse model with those obtained from nonhuman primate models, which are reportedly the most effective for studying alphavirus pathogenesis and drug treatments [START_REF] Roques | Paradoxical Effect of Chloroquine Treatment in Enhancing Chikungunya Virus Infection[END_REF][START_REF] Labadie | Chikungunya Disease in Nonhuman Primates Involves Long-Term Viral Persistence in Macrophages[END_REF].

In conclusion, the limited number of available therapeutic strategies to treat arboviruses is one of the major challenges facing public health. Altogether, our data suggest that Favipiravir, a RNA-Pol inhibitor, could significantly reduce MAYV replication in a majority of the organs targeted by the virus, including the joints, which are the main site of MAYV-induced chronic arthralgia.

( 1 )

 1 Pre-treatment: mice (n = 16) treated with Favipiravir one day before MAYV infection, (2) Concurrent treatment: mice (n = 16) treated with Favipiravir one hour before MAYV infection, (3) Post-treatment: mice (n = 16) treated with Favipiravir two days after MAYV infection, (4) CTRL+: MAYVinfected mice (n = 8) without treatment and (5) CTRL-: mice (n = 8) receiving Favipiravir treatment in the absence of MAYV infection (Figure 1).

Figure 1 .

 1 Figure 1. Schematic representation of the experimental procedure.

Figure 3 .

 3 Figure 3. Favipiravir protected against body weight loss and footpad swelling in MAYV-infected mice. MAYV-infected mice (10 6 pfu) were either treated with Favipiravir at different times of infection or not treated at all. (A) Body weight and (B) footpad swelling. Numbers of mice were variable within groups as some were sacrificed at 7 dpi and the remaining at 14 dpi. * represents p < 0.05, two-way ANOVA was used for statistical analysis. Error bars indicate standard deviation.

Figure 4 .

 4 Figure 4. Cont.

Figure 4 .

 4 Figure 4. Favipiravir decreased MAYV yield in peripheral blood in pre-and concurrent-treatment. MAYV-infected C57BL/6 mice were orally pre-treated (n = 16), concurrently treated (n = 16), post-treated (n = 16), or untreated (n = 8) with Favipiravir 300 mg/kg once daily for 5 consecutive days. Viral RNA and infectious viral particles were quantified in peripheral blood samples using RT-qPCR (A-C) and plaque assay (D-F) at different dpi. Each point represents values of an individual mouse. ND (not detected) indicates absence of infectious viral particles in the plaque assay. The two-way ANOVA test and Turkey's test were used; * p < 0.05; ** p < 0.01; **** p < 0.0001. Half of the mice were sacrificed at 7 dpi and the remaining animals at 14 dpi (see Figure 5).

Figure 5 .

 5 Figure 5. Cont.

Figure 5 .

 5 Figure 5. Favipiravir treatment decreased MAYV yield in multiple organs. MAYV-infected four-week-old mice were orally pre-, concurrently and post-treated with Favipiravir 300 mg/kg once daily for 5 consecutive days, or not treated at all. Tissues were harvested and subjected to RT-qPCR and plaque assay for viral load at 7 dpi panel (A-D) and 14 dpi panel (E-G). Each treated group (n = 4-6) was compared to the MAYV-infected group (n = 5). In each tissue graph, dots in the figure correspond to an animal, except in the case of animals with undetectable viral loads (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). ND (not detected) indicates absence of infectious viral particles in the plaque assay. The bar graph is mean ± SD. Data were analyzed with the Mann-Whitney U test to test each group against the other. Each quantification value (dot) in these experiments was the mean of three replicates.

Figure 6 .

 6 Figure 6. Favipiravir decreases serum ASAT (A) and ALAT (B) levels in MAYV-infected mice. Hepatic enzyme levels were quantified in MAYV-infected C57BL/6 mice treated (n = 4) or not (n = 6-8) with Favipiravir at 7 and 14 dpi. The data are expressed as the mean ± SEM. * p < 0.05; ** p < 0.01.

Funding: This work was supported by grants from the Agence Nationale de la Recherche (ANR-17-CE15-00029). This work was also publicly funded through the French National Research Agency under the "Investissements d'avenir" program, with the reference ANR-16-IDEX-0006. Michele Bengue was supported by a fellowship from the Infectiopôle Sud foundation.

Institutional Review Board Statement:

The mouse study was conducted according to the European guidelines, and approved by the Institutional Review Board (Ethics committee CETEA) of the Institut Pasteur and the French Ministry of Higher Education and Research (MESR 00762.02, dated 20 November 2015). The studies on macaques were approved by the regional animal care and use committee, in accordance with European directive 63/2010/EU: "CREEA Ile de France Sud", Fontenay aux Roses, decision #A08-012 dated 7 July 2008.

Informed Consent Statement: Not applicable.

Data Availability Statement: Not applicable.

Conflicts of Interest:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Alexandre Flete Stmicroelectronics
email: alexandre.flete@st.com

Christophe Viallon
email: cviallon@laas.fr

Philippe Cathelin
email: philippe.cathelin@st.com

Thierry Parra
email: parra@laas.fr

A ×3 Sub-Sampling Mixer for a 77 GHz Automotive Radar Receiver in 28 nm FD-SOI CMOS Technology

Keywords: Millimeter-wave, Sub-harmonic, Passive mixer, Frequency down-conversion, Low duty cycle

This paper presents an innovative x3 sub-sampling frequency downconverter for converting signals around 77 GHz from a 26 GHz LO signal. With a LO frequency divided by 3 this topology allows drastic simplifications on the receiver's LO distribution chain. The sub-sampling mixer operation principle is described and implemented with 28-nm FD-SOI CMOS technology. Simulation results show a 1dB input-referred compression power (ICP1dB) of +3 dBm and a 14.4 dB Noise Figure (NF) with a -2.5 dB conversion gain. The mixer's LO signal shaper consumption is 36 mW on a 1.2V supply while the passive mixer core doesn't require DC power.

I. INTRODUCTION

With its ability to detect distant targets under harsh visibility conditions, the 77 GHz automotive radar plays a key role in driving safety. Using high frequencies enables a good circuit integration and a fine range resolution thanks to a wide modulation bandwidth. Nevertheless, implementing a 77 GHz LO chain to drive the mixer is difficult as it generally requires a 38.5 GHz VCO, frequency doublers and high consumption 77 GHz amplifying stages.

A solution to overcome difficulty coming with this complexity could be found with sub-harmonic mixers that use a LO frequency submultiple of the RF frequency (𝑓 𝐿𝑂 ≈ 𝑓 𝑅𝐹 𝑛 , with n a natural integer). This solution has already been considered for the first generations of 77 GHz radar receivers ( [START_REF] Jang | A 79-GHz Adaptive-Gain and Low-Noise UWB Radar Receiver Front-End in 65-nm CMOS[END_REF], [START_REF] Le | A CMOS 77 GHz radar receiver front-end[END_REF]). However, as sub-harmonic passive mixers often result in prohibitive conversion losses, the high-frequency architectures mostly rely on sub-harmonic active mixers. At lower frequencies, passive sub-sampling mixers as in [START_REF] Pekau | A 2.4 GHz CMOS sub-sampling mixer with integrated filtering[END_REF] are widespread. As this principle translates into low conversion losses and good linearity, the aim of this paper is to demonstrate that it can be applied to millimeter-wave frequencies. So, a ×3 sub-sampling passive mixer is proposed in this paper for a 77 GHz radar receiver.

Section II introduces the sub-sampling mixer principle. Then, the proposed topology is detailed in section III with a focus on its innovative mixer core and the associated 26 GHz LO signal shaping method. The implementation with the 28 nm FD-SOI CMOS technology and the simulation results are given in section IV. Finally, the conclusions of this work are presented in section V.

II. SUB-SAMPLING MIXER PRINCIPLE

In an ideal sub-sampling operation, every RF signal close to any sampling frequency harmonic (𝑛. 𝑓 𝐿𝑂 ) is translated at 𝑓 𝐹𝐼 = |𝑓 𝑅𝐹 -𝑛. 𝑓 𝐿𝑂 | with a 0 dB conversion gain. The sub-sampling operation is particularly interesting because it allows a theoretically lossless frequency conversion from any LO frequency such as: 𝑛. 𝑓 𝐿𝑂 ≈ 𝑓 𝑅𝐹 . enabling a proper voltage conversion as soon as source impedance is low enough ( 𝑅 𝑠 ≪ 𝑍 𝑖𝑛 ). If 𝐷. 𝑇 𝐿𝑂 defines the 𝑀 1,2,3,4 conducting time, the passive sub-sampling mixer's voltage conversion gain (𝐺 𝑐𝑣 ) calculation gives, with a similar approach as in [START_REF] Shahani | A 12-mW wide dynamic range CMOS front-end for a portable GPS receiver[END_REF]:

𝐺 𝑐𝑣 = 1 2 𝑠𝑖𝑛𝑐(n. 𝐷)(1 -𝑒 -𝑗𝜋𝑛 ) ()
According to [START_REF] Jang | A 79-GHz Adaptive-Gain and Low-Noise UWB Radar Receiver Front-End in 65-nm CMOS[END_REF], for odd values of 𝑛 , the subharmonic frequency conversion can be achieved with low loss if D can be small enough.

In addition, this sub-sampling mixer benefits from the passive mixer's linearity performances that can be excellent if transistors switching time is low [START_REF] Lin | Device considerations and modeling for the design of an InP-based MODFET millimeter-wave resistive mixer with superior conversion efficiency[END_REF]. Consequently, with sharp LO signal's rising and falling edges, this mixer will be highly linear.

However, at millimeter-wave frequencies, the possible choices for n are limited. The equation [START_REF] Jang | A 79-GHz Adaptive-Gain and Low-Noise UWB Radar Receiver Front-End in 65-nm CMOS[END_REF] shows that the more n increases the more D must be kept low to reach a decent conversion gain. As low duty cycles are difficult to reach at high frequencies, the higher reasonable ratio n appears to be 3 for a 77 GHz sub-sampling operation. So, a 26 GHz LO frequency has been chosen for the implementation of the 77 GHz sub-sampling mixer.

The following section demonstrates that designing a LO shaping circuit for the creation of a 26 GHz pulsed signal becomes feasible by taking advantage of nm-scaled CMOS processes. This design is addressed in the next section.

III. CIRCUIT DESCRIPTION

Even if nm-scaled CMOS technologies provides high speed transistors, their gate capacitances and parasitic elements limits the high frequency gain of complex logic gates. Nevertheless, the generation of a low duty cycle LO signal only requires inverter chains to generate two time-delayed square waveforms and an AND gate for their combining. So, using only these logic gates can be compliant with the need of a proper 26 GHz pulse shaping.

In the inverter/AND gate arrangement, the AND gate is the limiting element because of its complexity. Thus, the reachable duty Cycle is not low enough. To overcome this limitation the AND function has been merged with the mixer core, as depicted in fig. This innovative approach allows to suppress the AND gates to reach lower duty cycles and sharper LO signal's edges. As a result, only inverter chains are needed to generate the required LO waveforms for the sub-sampling mixer core. Nevertheless, since two series transistors are involved in each branch of the mixer, this topology presents a higher ON-state resistor (𝑟 𝑜𝑛 ), increasing the thermal noise. The mixing transistor sizing is essential to get a good noise/linearity trade-off. To get the best linearity from the subsampling mixer, sharp LO square signal's rising and falling edges are needed. Since the capacitive load of transistor gate contributes to soften the LO square voltage edges, a lower width of transistor lead to a better linearity. On the other hand, increasing the width helps to keep a low NF by lowering the series resistance 𝑟𝑜𝑛. The resulting transistor sizing is 20 μm/30 nm.

Implementation and simulation of the sub-sampling passive mixer topology is detailed in the following section.

IV. IMPLEMENTATION AND SIMULATION RESULTS

The 77 GHz sub-sampling passive mixer is designed in a 28nm FD-SOI CMOS process from STMicroelectronics.

The LO signal shaper supply voltage is 𝑉 𝐷𝐷 = 1.2𝑉. This value satisfies a trade-off between the necessary LO swing to get a good mixer's linearity, the LO signal shaper's consumption and the transistor's reliability. In an inverter the transistors never experience high 𝑉 𝑔𝑠 and high 𝑉 𝑑𝑠 at the same time making it less sensitive to Hot Carrier Injection, even with 1.2V supply voltage. Fig. 5 shows the output voltages of the A and B inverter chains (fig. 3). The simulated voltage conversion gain is depicted in Fig6.a. Under these conditions, the sub-sampling mixer has a simulated conversion gain of -2.5 dB. The input -1 dB compression voltage V-1dB is 0.9 𝑉 𝑝-𝑝 . Power is not used here to express mixer performance as it is not relevant regarding the high 𝑍 𝑖𝑛 of the mixer. An equivalent ICP1dB of +3 dBm is derived considering that V-1dB is delivered to a 50 Ω load.

Results of a non-linear noise simulation based on harmonic balance are presented in fig. 6.b. The NF simulated with a 50 Ω input port is 14.4 dB at a 20 MHz IF frequency.

The proposed sub-harmonic passive mixer has been implemented with a variation of the LNA presented in [START_REF] Paulin | 4.6dB/5.5dB NF and 6.5dB/21.5dB gain 2 stages and 6 stages 76-81GHz LNAs for radar application in 28nm RF CMOS FD-SOI[END_REF] in a 77 GHz front end. The simulated front end's performances are summarized and compared with other radar receivers in table 1. The simulated performances show that with a high linearity, a good conversion gain and a higher 𝑓 𝑅𝐹 𝑓 𝐿𝑂 ratio the proposed mixer appears as a favourable alternative to [START_REF] Jang | A 79-GHz Adaptive-Gain and Low-Noise UWB Radar Receiver Front-End in 65-nm CMOS[END_REF] and [START_REF] Le | A CMOS 77 GHz radar receiver front-end[END_REF].

Furthermore, with the proposed solution a 26 GHz VCO can be used without multiplying stages. The 26 GHz LO signal amplification also consumes less DC power than at 77 GHz. And finally, the logical gate LO signal shaper enables a very compact design as it does not require transformers or inductors.

V. CONCLUSION

A x3 sub-sampling passive mixer topology operating at 77 GHz with a 26 GHz LO signal frequency is proposed in this paper. This solution is based on an innovative sub-sampling mixer core associated to a 26 GHz LO signal shaper.

The sub-sampling principle allows to design sub-harmonic passive mixers benefiting from good linearity while keeping a good conversion gain. This approach also gives the opportunity to significantly simplify the LO chain from the division by 3 of the LO frequency. According to these results, the proposed architecture demonstrates that the sub-sampling mixers can be convenient to address millimeter-wave frequency applications.

Figure 1 :

 1 Figure 1: sub-sampling mixer topology The sub-sampling mixer principle is described with a double-balanced topology in fig.1. Applying a low duty cycle square wave as LO signal and presenting a capacitive load at the IF outputs turn the mixer into a voltage sampler. Transistors are used as switches closed for a very short time to sample the input voltage. Sampled values are stored by the hold capacitors 𝐶 𝐻 when all the switches are open. The mixer brings a high input impedance around each odd LO harmonics ( 𝑛. 𝑓 𝐿𝑂 ) enabling a proper voltage conversion as soon as source impedance is low enough ( 𝑅 𝑠 ≪ 𝑍 𝑖𝑛 ). If 𝐷. 𝑇 𝐿𝑂 defines the 𝑀 1,2,3,4 conducting time, the passive sub-sampling mixer's voltage conversion gain (𝐺 𝑐𝑣 ) calculation gives, with a similar approach as in [4]:

2

 2

Figure 2 :

 2 Figure 2: Mixer core with the co-integration of the AND gates The operating principle of this mixer core and the one described in fig 1, stays similar. 𝐿𝑂 2 is a delayed version of 𝐿𝑂 1 . For example, in fig.2, series transistors 𝑀 1 and 𝑀 5 are both open only when 𝐿𝑂 1+ and 𝐿𝑂 2+ are simultaneously at the high state. This operation is equivalent to a single transistor driven with a pulsed LO signal with a duty cycle 𝐷 = 𝑡 𝐿𝑂 1+ ∩ 𝐿𝑂 2+ 𝑇 𝐿𝑂

Figure 3 :

 3 Figure 3: (a) single ended LO signal shaper (b) Signal shaper's layout Fig.3 presents the single ended version of the LO signal shaper. In the proposed mixer architecture, this structure is duplicated and fed with an opposite phase sinusoidal signal to implement a differential LO signal shaper. The LO signal shaper's layout of fig.3.b shows that the transistor size is progressively increased along the inverter chains. This way, current capability of inverters is progressively increased until the required value for a proper driving of the mixer. In the LO signal shaper, the inverter chains A and B have a different number of stages to create the required time delay between both square signals. The body effect of FD-SOI technologies is quite high (~ 85 mV/V), so that the body voltage can be changed to tune the 𝑉 𝑡ℎ of transistors. This property is used in this LO signal shaper to provide an additional LO duty cycle tuning capability. In each inverter, applying a tuning voltage 𝑉 𝑡𝑢𝑛𝑒 at the NMOS transistor body and -𝑉 𝑡𝑢𝑛𝑒 at the PMOS transistor body creates a 𝑉 𝑡ℎ shift resulting in a different inverter's time delay. Finally, changing the delay between the square signals before the mixer directly changes the LO duty cycle D. Tunning voltage's values of opposite sign are chosen for the inverter chains A and B to increase the duty cycle tuning range. The overall mixer architecture and the corresponding layout are given in fig.4.

Figure 4 :

 4 Figure 4: (a) Sub-sampling mixer architecture (b) Mixer's layout

Figure 5 :

 5 Figure 5: Inverter chains output voltages Solid line and dot line waveforms correspond respectively to the minimum and the maximum time delay between A and B output signals. The maximum achievable time delay variation is ∆𝑡 𝑀𝐴𝑋 = ∆𝑡 𝐴 + ∆𝑡 𝐵 = 7.9 𝑝𝑠 = 0.2 × 𝑓 𝐿𝑂 and is obtained with 𝑉 𝑡𝑢𝑛𝑒 varying in the range [-1V, +1V]. The LO signal shaping DC power consumption is 36 mW. Harmonic-balance simulation results, with 𝑓 𝑅𝐹 = 78.02 𝐺𝐻𝑧, 𝑓 𝐿𝑂 = 26 𝐺𝐻𝑧 and 𝑓 𝐼𝐹 = 20 𝑀𝐻𝑧, are reported in fig.6. This simulation is based on a layout modelling using momentum and a Post Layout Simulation extract.

Figure 6 :

 6 Figure 6: (a) RF conversion gain (b) Noise figure.

TABLE 1 :

 1 FRONT-END'S SIMULATED PERFORMANCES AND COMPARISON

		Tech	𝒇 𝑹𝑭 /𝒇 𝑳𝑶 [GHz]	𝑮 [dB]	ICP1dB [dBm]	NF [dB]	DC Power [mW]
	LNA + this work	28 nm FD-SOI CMOS	78/26	10.2	-10	11.5	36 + 9 Mixer + [6]
	[1]	65 nm CMOS	77/38.5	16	-20	10.5	57
	[2]	65 nm CMOS	77/38.5	16	-20	13	28.5


Maria-Isabel Mendoza-Diaz

Aurélie Lecestre

Ludovic Salvagnac

Botayna Bounor

David Pech

Mehdi Djafari-Rouhani

Alain Esteve

Carole Rossi
email: rossi@laas.fr

High surface area TiO 2 photocatalyst for H 2 production through silicon micromachining

Keywords: TiO 2, photocatalyst, 3D microstructure, DRIE, H 2 production, water splitting

The development of an efficient photocatalyst capable of producing enough hydrogen for applications in everyday life under direct sunlight exposure is still challenging.

In this work, a new concept for a three-dimensional microstructured photocatalyst is proposed, in which a standard deep-reactive ion etching process allows for the optimization, fabrication and subsequent deposition of TiO 2 thin films by physical vapor deposition for H 2 production by direct water splitting. After the development of enlarged surface microstructures, the composition and morphology of the 3D TiO 2 photocatalyst were characterized by XRD, XPS, UV/Vis spectroscopy and SEM. Furthermore, the influence of the area enlargement factor on the 3D photocatalyst surface morphology and its photocatalytic performance under UV-visible irradiation was thoroughly analyzed and corroborated by electrochemical experiments. The photocatalyst exhibited an increase in H 2 production by almost a factor of 12 compared to conventional planar TiO 2 films. The H 2 production was further improved by a factor of 4 through the introduction of Au nanoparticles grown on top of the TiO 2 layer. The advantages and development of robust and hierarchical photocatalysts using microelectromechanical fabrication techniques are highlighted as potential solutions for a broad range of applications from photocatalysis, electronics, and sensing elements to 3D metamaterials.

Introduction

An attractive approach for the energy industry's transition to sustainable and clean energy is the use of solar radiation to produce hydrogen [START_REF] Barreto | The hydrogen economy in the 21st century: a sustainable development scenario[END_REF]. Among the different artificial photocatalytic processes, H 2 production through water splitting (WS) is probably the most intensely studied since H 2 exhibits a high volumetric energy density and no carbon footprint and can be either directly burned or used in fuel cells to produce electricity [START_REF] Crabtree | The hydrogen economy[END_REF][START_REF] Dotan | Decoupled hydrogen and oxygen evolution by a two-step electrochemical-chemical cycle for efficient overall water splitting[END_REF]. In 1972, pioneering work by Honda and Fujishima demonstrated that the water splitting into H 2 and O 2 could be achieved by irradiating light onto a titania (TiO 2 ) photoanode connected to a platinum cathode in an electrochemical cell [START_REF] Fujishima | Electrochemical photolysis of water at a semiconductor electrode[END_REF]. A few years later, Borgarello et al. [START_REF] Borgarello | Photochemical cleavage of water by photocatalysis[END_REF] demonstrated the feasibility of a bifunctional RuO/TiO 2 photocatalyst to achieve water splitting. The WS mechanism takes place when photoexcited electrons and holes act as reducing and oxidizing agents to produce H 2 and O 2 , respectively (concept depicted in Figure 1) [START_REF] Chen | Semiconductor-based photocatalytic hydrogen generation[END_REF]. Since these seminal works, great progress has been made to understand the microscopic processes involved in photochemical systems and in catalyst engineering [START_REF] Cure | A Beehive Inspired Hydrogen Photocatalytic Device Integrating a Carbo-Benzene Triptych Material for Efficient Solar Photo-Reduction of Seawater[END_REF][START_REF] Iachella | Water Dissociation and Further Hydroxylation of Perfect and Defective Polar ZnO Model Surfaces[END_REF].

Among a large choice of proposed materials (nitride compounds [START_REF] Maeda | Photocatalyst releasing hydrogen from water[END_REF][START_REF] Ye | A review on g-C 3 N 4 for photocatalytic water splitting and CO 2 reduction[END_REF] or composed Bi 2 WO 6 , TaON, Bi 2 O 3 , Ag 2 O, Ag 3 VO 4 [START_REF] Ye | A review on g-C 3 N 4 for photocatalytic water splitting and CO 2 reduction[END_REF][START_REF] Xu | Bi 2 O 3 cocatalyst improving photocatalytic hydrogen evolution performance of TiO 2[END_REF][START_REF] Abdi | Efficient solar water splitting by enhanced charge separation in a bismuth vanadate-silicon tandem photoelectrode[END_REF]), titanium dioxide remains the most suitable catalyst for industrial applications due to its performance, easy accessibility, nontoxicity and low price [START_REF] Fajrina | A critical review in strategies to improve photocatalytic water splitting towards hydrogen production[END_REF]. However, its solar-to-hydrogen conversion efficiency remains low (~ 0.4% [START_REF] Goto | A Particulate Photocatalyst Water-Splitting Panel for Large-Scale Solar Hydrogen Generation[END_REF]), which is still far from the optimum efficiency required for practical use in the fuel cells of modern devices. Extensive research efforts are still being devoted to specifically extending the light absorption spectra to visible light (λ > 400 nm), either by doping TiO 2 with metals or plasmonic nanoparticles (Ag or Au) [START_REF] Cure | A triptych photocatalyst based on the Co-Integration of Ag nanoparticles and carbo-benzene dye into a TiO2 thin film[END_REF][START_REF] Veziroglu | Plasmonic and non-plasmonic contributions on photocatalytic activity of Au-TiO2 thin film under mixed UV-visible light[END_REF]. Another strategy to improve the hydrogen production rate is to augment the surface-to-volume ratio by creating high surface microstructures [START_REF] Eustache | Silicon-microtube scaffold decorated with anatase TiO2 as a negative electrode for a 3D litium-ion microbattery[END_REF][START_REF] Wang | Fabrication of porous Ni-Co catalytic electrode with high performance in hydrogen evolution reaction[END_REF]. This is especially of major importance when dealing with systems in which the photocatalytic area is limited. Despite is high cost, film technologies such as physical vapor deposition (PVD), offers a good solution for fabrication of films with great adhesion, compact and resistant [START_REF] Störmer | Structure and corrosion of magnetron sputtered pure Mg films on silicon substrates[END_REF].

To the best of our knowledge, a quantification of the photocatalytic activity improvement related to the electrode surface area enlargement has never been investigated. The present study proposes to use silicon micromachining technique to fabricate well-defined threedimensional (3D) geometries of TiO 2 photocatalysts in order to relate the surface area with the H 2 production rate to drive the design of future electrodes. We designed and successfully created a well-defined and repeatable array of microcavities through a photolithography process which consists of the deep-reactive ion etching (DRIE) of silicon followed by the sputter deposition of a continuous layer of TiO 2 . After developing the fabrication process of the 3D photocatalyst, the influence of the area enlargement factor (AEF) on the morphology, quality and catalytic performance of the 3D photocatalyst was thoroughly investigated. We show that the H 2 production rate can be improved ten times compared to a planar topology.

Moreover, H 2 production is improved by a factor of four through the growth of Au nanoparticles inside the continuous TiO 2 catalytic layer, leading to a UV/visible synergistic effect, as discussed in previous works [START_REF] Mendoza-Diaz | On the UV-Visible Light Synergetic Mechanisms in Au/TiO2Hybrid Model Nanostructures Achieving Photoreduction of Water[END_REF]. This work not only proposes a fabrication process that can be easily implemented in the fabrication of silicon-based microdevices which is of fundamental and technological interest for electronics, sensing elements to 3D photonics and metamaterials; but also presents a direct understanding by firmly establishing a relation between the surface area augmentation and the photocatalytic properties of electrodevices.

Materials and methods

Silicon microstructure

A 500 µm thick, single-side-polished, 4-inch silicon p-type substrate was chosen for the fabrication of the microstructures that will serve as the experimental substrates. The technological process is composed of four main steps, as depicted in Photolithography. As a first step, a bottom anti-reflective coating layer (BARC, MicroChemicals) is deposited by spin-coating and baked at 200 °C for 1 min. Next, a positive photoresist (denoted PR; AZ-40XT, MicroChemicals, 20 µm) is spin-coated onto the silicon wafer and patterned using photolithography. This PR layer is "soft baked" from 65 to 126 °C for 1.5 min and cooled to room temperature. The PR layer is then patterned by exposing it for 10 s to UV light (A6 Gen4 Suss Microtec) shone through a patterned contact mask. The patterning of the PR layer is completed by developing the exposed PR with a MF-CD-26 developer for 120 s, followed by rinsing in deionized water and drying with N 2 . The PR layer is finally "hard baked" from 65 to 105 °C for 80 s.

Deep Reactive Etching (DRIE).

Silicon etching was performed using an AMS420 reactor manufactured by Alcatel-Adixen. Etching and passivation cycles of SF 6 and C 4 F 8 /O 2 gases are set to a constant flow rate (Table 1) and variable exposure time for each gas, the latter of which will be presented in Section 4. Finally, the SiO 2 layer is removed chemically with HF solution.

TiO 2 sputter-deposition

TiO 2 was sputter-deposited by direct current magnetron sputtering of a Ti target (99.999% purity). The pressure of the chamber was 8 × 10 -8 Pa. A gas mixture of 40% O 2 and 60% Ar was set with a pressure of 1.07 Pa. The DC power of reactive plasma was 1200 W. Three different thicknesses were sputter-deposited on the silicon microstructures, being these 270, 650 and 1000 nm (measured on flat surface), named T1, T2 and T3, respectively.

Grafting of Au nanoparticles

To improve the H 2 production rate of the TiO 2 layer deposited on the microstructures, gold nanoparticles (Au NPs) are grown on the semiconductor film as described in [START_REF] Cure | A triptych photocatalyst based on the Co-Integration of Ag nanoparticles and carbo-benzene dye into a TiO2 thin film[END_REF] and [START_REF] Veziroglu | Plasmonic and non-plasmonic contributions on photocatalytic activity of Au-TiO2 thin film under mixed UV-visible light[END_REF]. (99.9%, Technic) as a sacrificial agent (5 mL) [START_REF] Veziroglu | Plasmonic and non-plasmonic contributions on photocatalytic activity of Au-TiO2 thin film under mixed UV-visible light[END_REF][START_REF] Eustache | Silicon-microtube scaffold decorated with anatase TiO2 as a negative electrode for a 3D litium-ion microbattery[END_REF]. An annealing step under air at 200 °C for 10 min is used to remove the organic ligands that surround the NPs and thus obtain chemical bonding to the TiO 2 surface. The stabilizing agent residuals were removed by rinsing with deionized water.

Au

Characterization

The characterization methods included scanning electronic microscopy (SEM, FEI Helios 600i Nanolab), grazing incidence X-ray diffraction (GI-XRD, Bruker D8 Discover system), X-ray photoelectron spectroscopy (XPS, ESCALAB 250 X-ray photoelectron spectrometer)

and UV/Vis absorption spectroscopy (Perkin-Elmer Lambda 650 Spectrometer). For photocatalytic experiments, the samples were placed into a quartz reactor (60 mL) filled with an aqueous solution (10 mL, 35% v/v ethanol) and connected to a gas chromatography apparatus (GC, Perkin-Elmer Clarus 580) to measure the H 2 production rate. The sample was irradiated by a Xenon light lamp (Cermax® PE300B-10F) with a spectral region of 300 to 1100 nm. The quantity of released hydrogen was monitored every 6 h. The hydrogen evolution rate was normalized with respect to the light flux received by the sample, corrected for the UV absorption and calculated after 24 h of irradiation. Details are given in the Supplementary File (Figure S1).

The area enlargement factor is obtained using electrochemical measurements carried out in a three-electrode configuration using a VMP-3 (Biologic) potentiostat operated with EC-Lab software. A platinum mesh and a silver chloride electrode (Ag/AgCl) were used as counter and reference electrodes, respectively. The Si/TiO 2 photocatalysts were covered with a film of Ti/Au (50 nm and 700 nm) by sputtering. The electrochemical active surface area (EASA) of the deposited gold was then calculated using the charge associated with the reduction of gold oxide in cyclic voltammetry experiments: a value of 390 µC/cm 2 in 0.5 M H 2 SO 4 [START_REF] Trasatti | International Union of Pure and Applied Chemistry Physical Chemistry Division Commission on Electrochemistry: Real Surface Area Measurements in Electrochemistry[END_REF][START_REF] Lukaszewski | Electrochemical methods of real surface area determination of noble metal electrodes -an overview[END_REF].

The electrochemical AEF is defined as the ratio of the EASA of the microstructures to the EASA of a flat reference sample composed of silicon and 1 µm thick TiO 2 covered with a thin film of Ti/Au (50 nm and 700 nm).

Theoretical considerations

This section is devoted to the theoretical design with regard to the surface area enhancement of the optimum tunable pattern, which will be subsequently fabricated by silicon micromachining before further coating with the catalytic layer (Figure 1). The 3D photocatalyst was designed with a planar area of 1 cm². It is important that the aspect ratio can be tuned precisely in order to allow for the quantification of the H 2 production rate as a function of the total surface area.

The following patterns were studied for the microstructures (Figure 3): square microcavities (hereafter denoted as microcavities), walls, pillars, rods and hollow-rods. Each pattern is characterized by a dimensionless geometric factor, g, which is used for the AEF calculation (Equation 1). The expression of g as a function of the main geometrical parameters (a, w and depth) that correspond to each pattern is given in Figure 3. Then, the AEF can be calculated using the following equation: Equation 1where w is the width of the features (width, diameter or spacing as specified in Figure 3) and depth is the depth of the etching. Obviously, technological restrictions must be considered in the variation of the geometrical parameters a, w and depth. Aside from the limitations of the photolithography resolution (10 µm) and silicon etching processes (critical aspect ratio due to DRIE process), the major restriction comes from the magnetron sputtering technique that is used to deposit the TiO 2 thin layer. To ensure continuous sputter deposition of the TiO 2 semiconductor film on the 3D micropatterned silicon substrate, an aspect ratio ≤ 2 is imposed.

Therefore, a maximum depth of 150 µm and w of 75 µm are set to ensure this maximum aspect ratio for all the patterns. The influence of the geometrical parameter a on the AEF for the different patterns is plotted in Figures 4a-b. Note that the aspect ratio restriction also imposes the restriction a ≥ 10 µm. For example, when a equals 10 µm, a microstructure composed of pillars or rods cannot achieve an AEF greater than 4, whereas microcavities achieve the highest surface enhancement with an AEF equal to 7 the depth equals 150 µm.

The wall pattern is not an efficient design to maximize the surface-to-volume ratio since the AEF is limited to 4.5. Therefore, for 3D photocatalyst fabrication, we selected the microcavity pattern with dimensional parameters of 10 and 75 µm for a and w, respectively. Moreover, since the AEF is correlated to depth (Figure 4c), several silicon etching depths will be investigated. Indeed, the AEF is predicted to change from 3 to 7 when the microstructure depth goes from 50 µm to 150 µm.

Results and discussion

Silicon microstructures fabrication

An array of microcavities is fabricated into a 550 µm thick silicon p-type substrate using the DRIE process, as presented in Section 2. Control over the profile and depth is achieved by tuning the ratio of the exposure times of SF 6 to C 4 F 8 /O 2 gases, which corresponds to the passivation and etching steps in the DRIE process, respectively. Figure 5 shows the SEM cross-sections of the etched microcavities for different SF 6 :C 4 F 8 /O 2 exposure time ratios. Along with the etch rate, the profile is also highly influenced by the SF 6 :C 4 F 8 /O 2 exposure time ratio. Beyond 0.85, negative profiles are obtained, as observed in Figures 5a-c. After decreasing to 0.75, the sidewalls become positive with a near vertical profile angle of 90.28°

(Figure 5d). Below 0.57, positive profiles are obtained as well (Figures 5e-f). It should be noted that for each process case, the same profile geometry is observed over the entire substrate, demonstrating the uniform consistency of the micromachining process. To understand the impact of the SF 6 :C 4 F 8 /O 2 exposure time ratio on the profile, it is important to explain the etching and passivation mechanisms that are inherent to the Bosch DRIE process used in this study. During the etching step, anisotropic accelerated ions remove the fluorocarbon layer that is located only at the bottom of the cavities; next, isotropic etching takes place, removing the exposed silicon in all directions (detail in Figure S3). This produces scalloping defects [START_REF] Jansen | The black silicon method: A universal method for determining the parameter setting of a fluorine-based reactive ion etcher in deep silicon trench etching with profile control[END_REF] with trenches that extend horizontally along the walls of the cavities, creating a completely corrugated surface, as observed in 5a-d). Grass formation originates from the localized high concentration of oxide reaction products that are not removed from the surface before each new etch/passivation cycle [START_REF] Jansen | The black silicon method: A universal method for determining the parameter setting of a fluorine-based reactive ion etcher in deep silicon trench etching with profile control[END_REF]. This contamination acts as a micromask that creates a peak that is passivated and, due to isotropic etching, becomes higher with each iteration.

As a concluding remark for this technological study, an SF 6 :C 4 F 8 /O 2 exposure time ratio of 0.75 will be used in the DRIE process to fabricate the microstructures, as it permits vertical walls with no grass defects to be obtained, thus ensuring good coverage and quality of the sputter-deposited TiO 2 onto the microcavities. Note that the presence of the scalloping defect inherent to the Bosh process remains, producing a corrugated surface along the walls. The presence of such irregularities could generate a noncompact conformal TiO 2 layer, as the sputtering technique is highly dependent on the morphology and roughness of the substrate.

To remove the scalloping, a 1 µm oxide layer was grown by thermal oxidation throughout the microstructure and subsequently etched with HF. The cross-sectional images in Figure 6 show smooth edges and surfaces, which demonstrates the efficiency of the postprocessing step to remove scalloping defects while maintaining high-depth microstructures with a vertical profile. Figure S5 shows additional SEM images of the microstructures which confirm that the roughness from the bottom cavity is reduced as well, and that the depth of the trenches on the walls is barely noticeable. The etch rate diminishes from 3.0 to 2.74 µm/min when working with an aspect ratio of 2.14, i.e., up to a depth of 150 µm, which represents a reliable high etch rate.

3D photocatalyst characterization

To study the impact of the enlarged surfaces of TiO 2 on the H 2 production rate, silicon microstructures were fabricated with defined AEFs of 3, 5 and 7, which correspond to microcavity depths of 50, 100 and 150 µm. Table S2 details the measured geometrical parameters for each of the microstructures used in this section. Next, a film of TiO 2 was deposited on top. Hereafter the AEF will be denoted in the name of the samples as A1, A3, A5 and A7; similarly, the TiO 2 thickness is indicated by T1, T2 and T3, as specified on the Materials and methods Section. Note that TiO 2 was also deposited on flat silicon where the AEF is 1. Sample A1-T2 with a thickness of 270 nm will be denoted as the reference sample due to its optimum photocatalytic performance, as was previously reported [START_REF] Mendoza-Diaz | On the UV-Visible Light Synergetic Mechanisms in Au/TiO2Hybrid Model Nanostructures Achieving Photoreduction of Water[END_REF]. Table 3 summarizes the set of samples having been classified by increasing AEF and presents the TiO 2 film thickness, which will be discussed later. and Ti 3+ components in the Ti 2p 2/3 peak, are located at 457.3 and 456.5 eV; and in the 2p 1/2 peak, the components are located at 466.05 and 464.8 eV, respectively [START_REF] Silva Junior | A relationship between structural and electronic order-disorder effects and optical properties in crystalline TiO2 nanomaterials[END_REF][START_REF] Cottre | Interaction of Water with Atomic Layer Deposited Titanium Dioxide on p-Si Photocathode: Modeling of Photoelectrochemical Interfaces in Ultrahigh Vacuum with Cryo-Photoelectron Spectroscopy[END_REF]. SEM observations of the 3D photocatalysts (Figure 8) show a continuous and compact layer for all samples, i.e., microstructures with aspect ratios of 0.7:1, 1.3:1 and 2:1, corresponding to AEFs of 3, 5 and 7, respectively. The cross-sectional images in Figure 8 show the columnar growth of TiO 2, which is characteristic of the sputtering process. The grain growth was the same for all the fabricated photocatalysts, and the growth direction was merely influenced by the substrate surface position with respect to the PVD Ti target. This means that on the horizontal surfaces of the microcavities, i.e., the bottoms of the cavities, and at the top surfaces of the walls, the crystallites exhibit vertical growth (Figures 8c, f andS6-7), whereas on the vertical walls, the crystallite growth follows a diagonal direction (Figures 8b, e andS8). Figures S6 andS8 show the cross-sectional details of the film at the bottoms of the cavity, the tops of the walls and at the half-height for each microstructure.

Moreover, the grain size also changes with variations in thickness. Due to its columnar growth, the upper diameter of the grain increases. By measuring the cross-sections on the top surface of the microcavities, the average grain upper diameter is 50.8, 95.0 and 153.8 nm for thicknesses T1, T2 and T3 (270, 650 and 1000 nm). Likewise, the size of the grains located at the bottom of the cavity and on the walls changes according to the layer thickness. For example, for sample A3-T3, the layers at the half-height of the wall and bottom of the microcavities are 380 and 336 nm, and the grain diameters are 61.5 and 50.2 nm, respectively. In addition, it is evident that the TiO 2 thickness deposited on the microstructures is not uniform with respect to the depth of the microcavities. As presented in Table 3, the thickness on the horizontal surfaces decreases from the top to bottom of the microcavities by a factor of 2.4, 4.0 and 9.9 for AEF values of 3, 5 and 7, (i.e., depths of 50 µm, 100 µm and 150 µm) respectively. On the vertical surfaces, the TiO 2 thickness decreases from the top to bottom of the walls by a factor of 1.9, 3.4 and 6 for AEF values of 3, 5 and 7, respectively. Nevertheless, the TiO 2 layer remains compact and dense throughout the surface. This variation in thickness comes from the fact that, during the deposition in the sputtering process, the substrate is moving in front of the target in a parallel manner, and the atoms reach the sidewalls and corners more frequently due to the periodic high oblique angles of the incident flux.

To quantify the influence of the AEF on the photocatalytic activity, it is crucial to define an average thickness ( ) of TiO 2 to distinguish each sample. In this regard, we use Equation 2, which accounts for the thicknesses measured at the top and bottom surfaces, and the average thickness deduced along the wall height; these values are then multiplied by a surface factor (Table S3) that represents the percentage of the total microstructure surface. Details are described in the Supplementary File, Section S4.

Equation 2

Where:

are the averages of the TiO 2 thickness measured on the top, bottom surfaces and along the wall height, respectively.

is a factor defined by the AEF that represents the percentage of the total surface that is located at the horizontal top of the microstructure.

is a factor defined by the AEF that represents the percentage of the total surface that is located at the horizontal bottom of the microstructure.

is a factor defined by the AEF that represents the percentage of the total surface that is located on the walls of the microstructure.

Thus, the calculated average TiO 2 thickness for the 3D photocatalysts is reported in Table 3.

Primarily, is thicker in the 3D photocatalyst with an AEF of 3, which is attributed to the low aspect ratio of 0.7:1 of the microstructure. We also observe that the has a similar value with regard to the wall thickness in all the samples, which is attributed to the utilization of F3 in Equation 2, which represents the percentage of the total surface that is located on the walls of the microstructure, and this value is 0.67, 0.84 and 0.86 for AEF values of 3, 5 and 7, respectively. This suggests that the film quality of the overall 3D photocatalysts is determined principally by the layer thickness on the walls. Another important observation is that for AEFs 3 and 5, the bottom of the microcavities has a higher thickness than the walls, which is contrary to AEF 7, and can be attributed to the limitation of the sputter deposition technique in reaching the bottom of the microcavities.

H 2 production rate. The photocatalytic H 2 production of the 3D photocatalysts was evaluated over 24 h under UV-vis irradiation. Figure S9 shows the H 2 evolution over 24 h for each photocatalyst. Note that for the reference sample (A1-T2, flat photocatalyst), the quantity of H 2 evolution is linear over 24 h, whereas for sample A1-T3 and the 3D photocatalysts, the H 2 production remains constant only after 6 h of irradiation. Furthermore, for the different AEFs from 3 to 7, the photocatalysts with T3 thickness have a remarkedly higher H 2 production rate than T1 (i.e., increased by a factor of 57, 22 and 28 for AEFs 3, 5 and 7, respectively). This

shows that the surface area enlargement not only impacts the H 2 production rate but also prominently impacts the TiO 2 layer, which, as discussed in the previous section, has a fluctuating thickness. Therefore, it is important to consider this characteristic for the interpretation of results. When considering the photocatalysts with thickness T1 that have a = 65.17 to 108.02 nm, the highest H 2 production rate was observed for sample A5-T1 with 0.018 µmol W -1 h -1 . This represents a 5-factor increase compared to A1-T1 (100 nm thickness), followed by A3-T1 rate of 0.009 µmol W -1 h -1 . Next, considering photocatalysts with thickness T2, with a = 143.33 to 237.22 nm, sample AEF5-T2 has the highest H 2 production rate with 0.196 µmol W -1 h -1 , which represents a 5-factor increase compared to the reference sample (A1-T2 of similar thickness 270 nm), followed by A3-T2 with a rate of 0.167 µmol W -1 h -1 . Among the photocatalysts with T3 thickness, the flat A1-T3 (650 nm thickness) shows a high rate of 0.313 µmol W -1 h -1 ; nevertheless, for the purpose of comparison within the 3D photocatalysts that have a = 229.79 to 446.99 nm, it is necessary to use a reference sample with a similar thickness, such as sample A1-T2; in this regard, samples A3-T3 and A5-T3 exhibit a remarkable 12-factor and 10-factor enhancement of with H 2 production rates of 0.5 and 0.393 µmol W -1 h -1 , respectively; moreover, their H 2 evolution exhibits the fastest rate during the 24 h tests. In addition, the quantum efficiency (QE) of the optimal 3D TiO 2 photocatalysts (thickness T3) was calculated considering the spectral output of the Xe lamp. Details are given in the Supplementary File, Section 6. The QE for samples A3-T3, A5-T3 and A7-T is 8.74, 7.24 and 6.29%, respectively. Due to a low power irradiation of the lamp, comparison with literature is relatively difficult. Yet, we can deduce that the enhanced photon-to-H2 production is caused by the area enlargement of the 3D photocatalysts under the same irradiation and with the same planar area. Figure 9a plots the percentage of the H 2 production rate enhancement compared to planar topology as a function of (x-axis) and the AEF (different colored lines). The surface area enlargement clearly boosts the photocatalytic activity, which leads to a remarkable enhancement of the H 2 production rate of 1214% compared to the planar topology. A higher enhancement would be expected for the 3D photocatalysts with an AEF of 5 and 7, since the availability of the sites that conduct the water-splitting reaction is higher. However, it is the photocatalyst with an AEF = 3 that exhibits a higher photocatalytic performance; this behavior can be ascribed to the semiconductor layer uniformity. For samples A5-T3 and A7-T3, the thickness layer variation along the microcavities is high (246-1105 nm and 107-1171 nm) and the enhancement is 956 and 823%, respectively. However, for sample A3-T3, the thickness variation is lower, from 454 to 1174 nm, and the enhancement is notably higher, at 1214%. This remarkable enhancement of a 3D photocatalyst with respect to a planar topology surpassed the expected results, where the efficiency was expected to be directly proportional to the surface area. This establishes that higher efficiencies can be achieved by increasing the AEF when a nearly conformal TiO 2 layer with a minimum thickness of 200 to 300 nm is deposited. Based on the behavior of the AEF on the H 2 production rate, Figure 9b shows the predicted H 2 production rate for a conformal TiO 2 layer with a thickness of 230 nm for 3D photocatalysts when varying the AEF: the H 2 production is enhanced by factors of 4, 7 and 9

for AEF values of 3, 5 and 7, respectively, when compared to a photocatalyst with an AEF of 1. This indicates that when microstructures with a higher aspect ratio ≥1. with a TiO 2 thickness of 1 µm was used as the reference sample for the AEF EAS calculation, these results not only corroborate that the augmentation of the surface is related to the change in depth of the microcavities but also take into account the roughness and fluctuations associated with the deposited 3D structure of the TiO 2 , which therefore creates more electroactive sites where gold oxide is formed and reduced. Note that the thick Au sputtered layer has a roughness that can also contribute to the real surface area augmentation.

Therefore, the experimental AEF EAS values are higher than the calculated AEF values from Equation 1, but reflect a good correlation with the theoretical values.

The stability of the photocatalyst is also an important issue in device applications. Therefore, a cyclic H 2 evolution test was carried out for 45 h under light irradiation and under the same conditions as the H 2 production rate tests. The optimal A3-T3 sample was tested for 5 consecutive cycles of 9 h each. The H 2 evolution, shown in Figure S11, remained stable, with an average H 2 production of 89.18 µmol W -1 by the end of each cycle. Additionally, a small increase was observed after the first cycle, indicating the robustness of the 3D photocatalyst.

A final experiment was carried out to increase the H 2 production rate of the 3D photocatalysts. As previously reported [START_REF] Mendoza-Diaz | On the UV-Visible Light Synergetic Mechanisms in Au/TiO2Hybrid Model Nanostructures Achieving Photoreduction of Water[END_REF], the synthesis of Au NPs on top of the TiO 2 film allows for light absorption in the visible range (455-475 nm), which has a positive impact on the water-splitting reaction. Au NPs were grown onto the optimal samples A3-T3, A5-T3 and A7-T3. As expected, their H 2 production (Figure 10a

Conclusions

The photocatalytic performance for the H 2 production of 3D photocatalysts composed of TiO 2 and TiO 2 /Au NPs was reported as a function of semiconductor layer thickness and the aspect ratio relative to specific AEF values. We have demonstrated that, by augmenting the surface area by a factor of 3, H 2 production is enhanced by a factor of 12, due to the increase in active surface area for the water-splitting reaction. Moreover, a supplementary 4-factor H 2 production rate enhancement is achieved by chemical deposition of Au NPs due to the extension of the absorption spectra of the photocatalyst. Furthermore, the results indicate that further developments, such as atomic layer deposition techniques and controlled in situ synthesis of metal nanoparticles, can result in major enhancements for higher aspect ratio 3D

Figure 1 .

 1 Figure 1. Conceptual scheme of the 3D microstructured photocatalyst with variant AEF

Figure 2 :

 2 photolithography, silicon etching by DRIE, postprocessing obtainment of a smooth silicon

Figure 2 .

 2 Figure 2. Main process steps underlining the cross-section of the 3D photocatalyst: (1)

Figure 3 .

 3 Figure 3. 3D view of the proposed patterns: microcavities, walls, square pillars, rods and

Figure 4 .

 4 Figure 4. Impact of the geometrical parameter a on the area enlargement factor (AEF)

Figure 5 .

 5 Figure 5. SEM cross-section images of silicon microstructures obtained by DRIE with

  Figure S4. For higher concentrations of F* radicals (SF 6 :C 4 F 8 /O 2 exposure time ratio ≥ 0.85), anisotropic sputtering is favored since the passivation layer is scavenged quickly and the F* ions bounce repeatedly in the already etched trenches, removing the passivating layer and etching slightly more in each iteration, thus increasing the bottom diameter and forming a negative profile.

Figure 6 .

 6 Figure 6. SEM cross-section images of silicon microstructures after postprocessing: details

TiO 2

 2 structure, average thickness and surface morphology. Characterization of the crystalline structure, optical absorbance and chemical composition were carried out only for the reference sample, as all the photocatalysts have the same TiO 2 composition, and the variation in thickness from 270 nm to 1 µm does not significantly affect the characterized properties. The GI-XRD pattern of deposited TiO 2 (Figure 7a) shows the characteristic peaks of anatase at 2 = 25.24° for the (101) plane and of rutile at 2 = 27.39° for the (110) plane, with the composition of each phase being 77.15% and 22.85%, respectively. The absorption spectra of the TiO 2 film deposited on quartz (Figure 7b) confirm the strong absorption of TiO 2 in the UV range from 200 to 349 nm. XPS results (Figures 7cd) are consistent with XRD and UV/Vis observations. The signals in the Ti 2p and O 1s spectra have a stochiometric signature which corresponds to TiO 2 . The O 1s peak is deconvoluted into two peaks, where the main component at 530.3 eV is the typical signal of the oxygen lattice of the O 2-bound to Ti +4 in TiO 2 , and the other component at 531.02 eV can be assigned to weakly adsorbed oxygen species, subsurface low-coordinated oxygen ions, surface hydroxyl groups and/or carbonate species. The Ti 2p spectrum exhibits the characteristic spin-orbit splitting with the Ti 2p 2/3 and 2p 1/2 peaks. The contribution of the Ti 4+

Figure 7 .

 7 Figure 7. Characterization of a 270 nm sputter-deposited TiO 2 film on a flat silicon substrate

Figure 8 .

 8 Figure 8. SEM cross-sectional images of the 3D photocatalyst composed of silicon

Figure 9 .

 9 Figure 9. (a) H 2 production rate enhancement as a function of the TiO 2 average thickness

) increases significantly by factors of 3 . 3 , 3 ,

 333 photocatalysts, it is important to analyze the role of the NPs in relation to the AEF. Figure

Figure 10 .

 10 Figure 10. (a) Hydrogen evolution over 24 h for 3D photocatalyst composed TiO 2 and Au

Table 1 .

 1 Detail of DRIE main process parameters.

	Gases	Flow (l min -1 )	Power (W)
	SF 6	0.25	2500
	C 4 F 8 /O 2	0.25/0.035	2500/70
	subsequently removed. The thermal oxidation treatment is composed of four steps: first, a
	preoxidation step of 30 min at 1070 °C under O 2 (10 L/min), followed by a wet oxidation step
	for 145 min under a mixture of O 2 /H 2 (6/10 L/min), a dry-oxidation step for 60 min under O 2
	(10 L/min), and the oxidation phase by annealing for 15 min in a neutral N 2 atmosphere (10
	L/min); then, a cooling down step with a ramp of -4 °C/min down to 700 °C under N 2 .

Post-processing. To remove the scalloping trenches from the cavity walls and obtain a smooth silicon surface, the silicon microstructures undergo a thermic oxidation process that consists of growing a 1 µm thick SiO 2 layer that uniformly covers all the trenches, which is

Table 2

 2 details the DRIE parameters and their corresponding profile characteristics. The profile angle , which describes the slope of the cavity wall edge, is calculated from the bottom and top widths of the wall and the cavity depth as described in Supplementary File, At a SF 6 :C 4 F 8 /O 2 exposure time ratio of 1.67 (SF 6 :C 4 F 8 /O 2 ), the etch rate was 4.35 μm/min, but dropped to 1.75 μm/min when the SF 6 :C 4 F 8 /O 2 exposure time ratio was 0.40. This

	Section 2. When  < 90°, the profile is labeled negative (closing cavity), and when  > 90°,
	the profile is labeled positive (opening cavity).

difference in etch rates is due to the concentration of free fluorine radicals (F*) in the etching step in comparison to the concentration of free oxygen radicals (O*) that passivate the surface. Consequently, when the SF 6 :C 4 F 8 /O 2 exposure time ratio is 0.40, the etching process is overcome by the passivation step, and the quantity of SiF 4 formed in each iteration is reduced, meaning that fewer silicon atoms are removed from the substrate.

Table 2 .

 2 Etch rate, profile angle  and defects for different SF 6 and C 4 F 8 /O 2 exposure times in the DRIE process of silicon.

	Exposure

time (s) SF 6 :C 4 F 8 /O 2 Ratio Etch rate (µm/min) Profile Grass defects SF 6 C 4 F 8 /O 2 Angle  (°) Description

	2.5	1.5	1.67	4.35	89.19	Negative	No
	2.2	2.0	1.10	4.17	89.63	Negative	No
	1.7	2.0	0.85	2.65	89.79	Negative	No
	1.5	2.0	0.75	3.00	90.28	Positive	No
	2.0	3.5	0.57	2.69	90.97	Positive	Low density
	2.0	5.0	0.40	1.75	91.79	Positive	High density

Table S1

 S1 gives the dimensions of the trenches that characterize the scalloping defects for the different SF 6 :C 4 F 8 /O 2 exposure time ratios: the trench dimensions vary from 35 to 66 and 47 to 118 nm for horizontal and vertical ratios, respectively, with SF 6 :C 4 F 8 /O 2 exposure time ratios ranging from 0.40 to 1.67.For SF 6 :C 4 F 8 /O 2 exposure time ratios below 0.57, the appearance of peaks is quite evident; this defect is also known as grass. For an SF 6 :C 4 F 8 /O 2 exposure time ratio of 0.57 (Figure5e) after an etch time of 26 min, the number of peaks is ~13 per cavity, whose height and width at the half-height are 22.4 µm and 0.44 µm on average, respectively. Below an SF 6 :C 4 F 8 /O 2

exposure time ratio of 0.40, after an etch time of 80 min, the density of peaks increases drastically, as the grass covers the entire bottom surface of the cavities (Figure

5f

): this results in what it is known as black silicon. Here, the silicon grass features an average height and width of 87.6 and 0.93 µm, respectively. Upon increasing the SF 6 :C 4 F 8 /O 2 exposure time ratio above 0.75, no grass was formed (Figure

Table 3 .

 3 Details of TiO 2 thickness for the 2D and 3D photocatalysts featuring a variable AEF.

	TiO 2 film thickness (nm)

*Average calculated from the top, medium and bottom heights of the wall thickness.

  4 and a conformal TiO 2 layer are deposited, H 2 production linearly follows surface augmentation.To measure the AEF, cyclic voltammetry experiments were carried out on 3D photocatalysts previously sputtered with Au. Thus, conforming electrodes have an entirely electrochemically-active surface areas; therefore, the 3D microstructures and the surface roughness are inherent to the different TiO 2 thicknesses. The augmentation of the electrochemically active surface areas (AEF EAS ) was obtained from the integration of the reduction peak of Au located at 0.85 V vs. Ag/AgCl. Cyclic voltammograms are shown in

	Figure S10. The AEF EAS values for the AEF3 samples with thicknesses of T1, T2 and T3
	were 5.78 ± 0.06, 5.54 ± 0.16 and 6.53 ± 0.06, respectively. For the AEF5 samples with
	thicknesses of T1, T2 and T3, the AEF EAS values were 8.40 ± 0.05, 5.26 ± 0.3 and 7.57 ±
	0.04, respectively. For the AEF7 samples with thicknesses of T1, T2 and T3, the AEF EAS
	values were 8.59 ± 0.18, 9.25 ± 0.03 and 9.59 ± 0.03, respectively. Since a flat photocatalyst

Acknowledgments

The authors acknowledge the financial support from the European Research Council (H2020 Excellent Science) Researcher Award (Grant No. 832889 -PyroSafe) and from the FEDER/Occitanie Region (Grant H2VERT/HYPHY). This work was also supported by the LAAS-CNRS technology platform, a member of the Renatech network. The authors also thank Dr. Hicham Jabraoui from LAAS, University of Toulouse and Dr. Kui Tan from the Department of Materials Science and Engineering, University of Texas at Dallas, Richardson, TX (USA) for their help in preparing this work: calculation of AEF for the former and performing the XPS analysis for the latter.

photocatalysts. This work provides new insights for the microelectromechanical fabrication of specific areal surface 3D photocatalysts for light-driven H 2 production.

CRediT authorship contribution statement

Maria

Declaration of Competing Interest

The authors declare no competing financial interests.

Appendix A. Supplementary File

Electronic supplementary information available: details of the fabrication processes and characterization of methods, additional microscopic characterization and photocatalytic and electrochemical experiments, quantum efficiency calculation methods.