Hengyang Xiang

Chenghao Xin

Zhelu Hu
email: hu.zhelu@espci.fr

Lionel Aigouy

Zhuoying Chen
email: zhuoying.chen@espci.fr

Xiaojiao Yuan

Long-term Stable NIR-SWIR Photodetector Driven by Photothermal Effect of Polypyrrole Nanostructures

Keywords: polypyrrole nanostructures, photothermal effect, solution-process, long-term stability, broadband photodetection

Polypyrrole (PPy) is a conductive polymer and widely applied in different applications owing to its broadband absorption in the UV-visible, near-infrared (NIR) and short-wave-infrared (SWIR) spectrum, excellent conductivity and strong photothermal effect. In this work, we explored for the first time the photothermal effect of polypyrrole nanoparticles (PPy-NPs) in a photothermal-induced detector structure, and developed a new type of air-stable hybrid PPy-NPs/Pt photodetector (PD) with NIR/SWIR sensitivity. By combining PPy-NPs with a platinum (Pt) resistive pattern, we fabricated PPy-NPs/Pt PDs that are sensitive to illumination in the wavelength range from 800 nm to 2000 nm. Under the illumination of  = 1.5 µm, the maximum photoresponsivity was measured to be ~ 1.3 A/W with a 131 µs photoresponse rise time. Owing to the material stability from both PPy-NPs and the Pt pattern, the current photodetectors show long-term stable photoresponsivity when they were stored in air without encapsulation. The results suggest that the PPy-NPs/Pt hybrid PDs are promising candidates for a new type of low-cost and broadband due to their multiple advantages such as free of toxic heavy-metals, air stability and solution-processing.

Introduction

Short-wave infrared (SWIR) detectors have attracted much attention because they can be used in various applications such as night vision, light detection and ranging, biological imaging, etc. [START_REF] Pradhan | Solid-State Thin-Film Broadband Short-Wave Infrared Light Emitters[END_REF][START_REF] Sicard | Shortwave Infrared Spectroradiometer for Atmospheric Transmittance Measurements[END_REF][START_REF] Hong | Near-Infrared Fluorophores for Biomedical Imaging[END_REF][START_REF] Hansen | Overview of SWIR Detectors, Cameras, and Applications[END_REF] Current detectors can be mainly classified into either photon type photodetectors or thermal type photodetectors depending on their underlying functional mechanisms. Most state-of-the-art SWIR photodetectors are photon detectors, which typically rely on narrow band gap semiconductors (such as Ge, [START_REF] Xu | InGaAs, and Colloidal Quantum Dot Photodetectors: Comparisons for Application in Silicon Photonics[END_REF] PbS, [START_REF] Saran | Lead Sulphide Nanocrystal Photodetector Technologies[END_REF] InGaAs, [START_REF] Geum | Monolithic 3D Integration of InGaAs Photodetectors on Si MOSFETs Using Sequential Fabrication Process[END_REF] InSb 8 ), and are expensive (epitaxial growth requirement) and/or environmental unfriendly. To circumvent these disadvantages, SWIR-absorbing solution-processed plasmonic gold (Au) nanoparticles have been recently proposed as a promising candidate to apply on thermal detectors for SWIR photodetection due to their remarkable plasmonic-induced photothermal effect. [START_REF] Xiang | Short-Wave Infrared Sensor by the Photothermal Effect of Colloidal Gold Nanorods[END_REF][START_REF] Xiang | Hybrid Plasmonic Gold-Nanorod-Platinum Short-Wave Infrared Photodetectors with Fast Response[END_REF][START_REF] Xiang | Nanoscale Thermal Characterization of High Aspect Ratio Gold Nanorods for Photothermal Applications at λ = 1.5 μ M[END_REF] For example, under the illumination of a  = 1.5 µm laser, hybrid photodetectors based on plasmonic Au nanorods (NRs) and its photothermal effect applied on a platinum microwire exhibited a maximum photoresponsivity of 4500 /W (in terms of resistance change (R) over illumination power (P)) or 340 mA/W (in terms of I/P). [START_REF] Xiang | Hybrid Plasmonic Gold-Nanorod-Platinum Short-Wave Infrared Photodetectors with Fast Response[END_REF] While these initial demonstrations are highly encouraging, for applications, it would be highly desirable to further improve the responsivity of such devices.

One strategy to increase the photoresponsivity of thermal SWIR detectors is to apply alternative materials with stronger photothermal effect. Herein, we propose the application of polypyrrole nanoparticles (PPy-NPs) [START_REF] Shi | A Novel Electrically Conductive and Biodegradable Composite Made of Polypyrrole Nanoparticles and Polylactide[END_REF][START_REF] Wang | Polypyrrole Nanoparticles and Dye Absorption Properties[END_REF][START_REF] Wu | High-Performance Polypyrrole Nanoparticles Counter Electrode for Dye-Sensitized Solar Cells[END_REF][START_REF] Zha | Uniform Polypyrrole Nanoparticles with High Photothermal Conversion Efficiency for Photothermal Ablation of Cancer Cells[END_REF] in a thermal photodetector structure for near-infrared (NIR) and SWIR photodetection. Polypyrrole (PPy) is a conductive polymer which has been intensively investigated during the last decades. [START_REF] Kupila | Influence of Electrode Pretreatment, Counter Anions and Additives on the Electropolymerization of Pyrrole in Aqueous Solutions[END_REF][START_REF] Macdiarmid | Polyaniline and Polypyrrole: Where Are We Headed?[END_REF][START_REF] Bereznev | Electrodeposited (Cu-In-Se)/Polypyrrole PV Structures[END_REF] Due to its broadband absorption in the UV-Vis and NIR/SWIR spectrum, excellent conductivity and photothermal effect, PPy-NPs have been applied for photocatalysis, [START_REF] Yuan | Photocatalytic Degradation of Organic Pollutant with Polypyrrole Nanostructures under UV and Visible Light[END_REF][START_REF] Yuan | Polypyrrole Nanostructures Modified with Mono-And Bimetallic Nanoparticles for Photocatalytic H 2 Generation[END_REF] supercapacitor electrodes, [START_REF] Wang | NiCo 2 S 4 Nanoparticles Anchoring on Polypyrrole Nanotubes for High-Performance Supercapacitor Electrodes[END_REF][START_REF] Wu | Enhanced Electrochemical Performances of Organ-like Ti 3 C 2 MXenes/Polypyrrole Composites as Supercapacitors Electrode Materials[END_REF] photothermal therapy (PTT) [START_REF] Yang | In Vitro and In Vivo Near-Infrared Photothermal Therapy of Cancer Using Polypyrrole Organic Nanoparticles[END_REF][START_REF] Song | Photosensitizer-Conjugated Albumin-Polypyrrole Nanoparticles for Imaging-Guided In Vivo Photodynamic/Photothermal Therapy[END_REF][START_REF] Shan | Polypyrrole-Based Double Rare Earth Hybrid Nanoparticles for Multimodal Imaging and Photothermal Therapy[END_REF][START_REF] Su | Black Phosphorus-Polypyrrole Nanocomposites for High-Performance Photothermal Cancer Therapy[END_REF] and biomedical imaging. [START_REF] Shan | Polypyrrole-Based Double Rare Earth Hybrid Nanoparticles for Multimodal Imaging and Photothermal Therapy[END_REF][START_REF] Au | Polypyrrole Nanoparticles: A Potential Optical Coherence Tomography Contrast Agent for Cancer Imaging[END_REF][START_REF] Zha | Biocompatible Polypyrrole Nanoparticles as a Novel Organic Photoacoustic Contrast Agent for Deep Tissue Imaging[END_REF][START_REF] Krug | Polypyrrole Microcapsules Loaded with Gold Nanoparticles: Perspectives for Biomedical Imaging[END_REF] For example in PTT application, [START_REF] Qin | Quantitative Comparison of Photothermal Heat Generation between Gold Nanospheres and Nanorods[END_REF][START_REF] Shi | Inorganic Nano-Carriers Based Smart Drug Delivery Systems for Tumor Therapy[END_REF][START_REF] Zhang | Magnetic Nanoparticles Coated with Polyphenols for Spatio-Temporally Controlled Cancer Photothermal/Immunotherapy[END_REF][START_REF] Zeng | Renal-Clearable Ultrasmall Polypyrrole Nanoparticles with Size-Regulated Property for Second Near-Infrared Light-Mediated Photothermal Therapy[END_REF] while both plasmonic Au-NRs and PPy-NPs can exhibit strong photothermal effect, the synthesis of PPy-NPs does not require costly noble metal elements. More interestingly, in photothermal cancer ablation studies, a stronger photothermal effect was observed on PPy-NPs than Au-NRs under equal mass concentration. [START_REF] Zha | Uniform Polypyrrole Nanoparticles with High Photothermal Conversion Efficiency for Photothermal Ablation of Cancer Cells[END_REF] These previous studies suggest PPy-NPs have great potential to be applied in a thermal detector structure for photodetection. While the photoconductivity of PPy-NPs has been used for photodetection, [START_REF] Wen | Chemically Tunable Photoresponse of Ultrathin Polypyrrole[END_REF][START_REF] Amiri | Mid-Infrared Photoconductivity of Polypyrrole Doped with Cadmium Sulfide Quantum Dots[END_REF] the strong photothermal effect of PPy-NPs is another alternative candidate for thermal (photo)detectors. [START_REF] Yang | Flexible and Reusable Non-Woven Fabric Photodetector Based on Polypyrrole/Crystal Violate Lactone for NIR Light Detection and Writing[END_REF] In a recent report by Yang et al., [START_REF] Yang | Flexible and Reusable Non-Woven Fabric Photodetector Based on Polypyrrole/Crystal Violate Lactone for NIR Light Detection and Writing[END_REF] the photothermal effect of PPy-NPs was applied on thermochromic crystals, which achieved photosensitivity to NIR by observing the intensity of the absorption spectrum from a spectrometer. Furthermore, the reaction time of such devices (which is inversely proportional to material/device light sensitivity) is no shorter than a few seconds.

Here, we successfully demonstrate the use of solution-processed PPy-NPs and their photothermal effect to achieve broadband ( = 800 nm -2000 nm) photodetection. The synthesized PPy-NPs are deposited onto a platinum (Pt) pattern to form a hybrid PPy-NPs/Pt device, which achieved a high responsivity of ~ 1.3 A/W (or 7.5×10 6 /W in terms of R/P) at  = 1.5 µm. In addition, we followed the device characteristics over storage time and found that PPy-NPs/Pt photodetectors are highly stable in the atmospheric environment even without encapsulation. This work shows that PPy-NPs are bright nanomaterials capable to provide broadband photodetection in NIR and SWIR spectra owing to its excellent photothermal effect, together with heavy-metal-free and stable-in-ambient characteristics. PPy-NPs were synthesized in soft templates which are produced by self-assembly of surfactants in a quaternary system (water, oil, surfactant and cosurfactant, chemical structure and polymerization process of PPy-NPs is shown in Figure 1a). [START_REF] Yuan | Photocatalytic Degradation of Organic Pollutant with Polypyrrole Nanostructures under UV and Visible Light[END_REF] These PPy-NPs' structural characteristics are confirmed by Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD), as shown in Figure 1b and Figure S1, respectively, consistent with previous literature reports. [START_REF] Vishnuvardhan | Characterization and a.c. Conductivity of Polypyrrole/Y 2 O 3 Composites[END_REF][START_REF] Fu | Core-Shell Structured Sulfur-Polypyrrole Composite Cathodes for Lithium-Sulfur Batteries[END_REF] The obtained PPy-NPs exhibit a uniform particle size of 50 nm in diameter (Figure 1c). The synthesized PPy-NPs are black powder, exhibiting a broadband optical absorption spectrum covering UV, visible and NIR/SWIR range (Figure 1d). Especially for the wavelength range of 800 nm to 2000 nm, PPy-NPs exhibit significant photon absorption suggesting their potential as a photosensitive material for NIR-SWIR photodetection. Figure 2a presents the optical image of a commercial negative temperature coefficient (NTC) thermistor, by which we harvest the photo-induced heat of PPy-NPs. To confirm the photothermal effect of PPy-NPs and compare it with Au-NRs, we drop-coated an aqueous solution of PPy-NPs and an aqueous solution of Au-NRs onto NTC thermistors to form Au-NRs/thermistor PD and PPy-NPs/thermistor PD, respectively. Several layers of Au-NRs and PPy-NPs are deposited on the surface of NTC thermistor (Figure 2b-2c). The absorption spectrum of PPy-NPs solution (Figure 1c) and Au-NRs solution (Figure S2) show that they have a strong photon harvest capability around 800 nm. Therefore, a  = 808 nm laser was illuminated on the NTC thermistor and hybrid devices to act as an external heat source. In these devices, NTC thermistors are sensitive to changes in ambient temperature, and detection of it will cause its resistance to change. For this typical NTC thermistor, the relationship between its resistance and temperature has been calibrated (Figure S3). Under the illumination of the  = 808 nm laser at different powers, different temperatures were then measured on Au or PPy-NPs/NTC-thermistor hybrid devices by comparison to the control device (blank NTC thermistor without NPs) (Figure 2d). For the blank NTC thermistor, when the laser power increases from 0.03 mW to 35.2 mW (power density increases from 3.75 mW/cm 2 to 4400 mW/cm 2 ), the temperature rise owing to the direct heating of the laser itself. In contrast, when a certain concentration of Au-NRs (3 g/L) or PPy-NPs (0.4 g/L) was coated onto the NTC thermistor, thanks to the photothermal effect of the NRs/NPs, a much higher temperature value can be obtained under the same laser power. Moreover, compared with the Au-NR solution for the Au-NRs/thermistor devices, even a a lower weight concentration PPy-NPs solution after dilution can be used to fabricate the PPy-NPs/thermistor devices and obtain higher temperature value at a fixed laser power. At the optimal concentration of PPy-NPs (0.8 g/L), the temperature of the PPy-NPs/thermistor device increases from room temperature (without laser) to 125 o C (under laser illumination at 35.2 mW), which is twice higher than that of 0.4 g/L PPy-NPs/thermistor device and 2.5 times higher than the measured value of 3 g/L Au-NRs/thermistor device (concentration optimization process was shown in Figure S4). From these results, it can be observed that PPy-NPs, even with a lower mass concentration applied, exhibit a stronger photothermal effect than plasmonic Au-NRs. This strong photothermal effect has been attributed to the electron phonon interactions in PPy-NPs, [START_REF] Guan | Hexagonal Polypyrrole Nanosheets from Interface Driven Heterogeneous Hybridization and Self-Assembly for Photothermal Cancer Treatment[END_REF] resulting in a much higher photothermal conversion efficiency of 34.3%, which is much better than that of Au-NRs (Calculation detail for photothermal conversion efficiency is described in Equation S1, S2 and Figure S5). These results demonstrate the PPy-NPs' outstanding photothermal conversion ability, which is consistent with previous research. [START_REF] Zha | Uniform Polypyrrole Nanoparticles with High Photothermal Conversion Efficiency for Photothermal Ablation of Cancer Cells[END_REF] In terms of device design, although hybrid NPs/thermistor photodetectors based on plasmonic Au-NRs have been implemented in the past, [START_REF] Xiang | Short-Wave Infrared Sensor by the Photothermal Effect of Colloidal Gold Nanorods[END_REF] such hybrid photodetectors have not been demonstrated with PPy-NPs. Figure 2e shows the different steps involved in the function of such hybrid devices. Firstly, PPy-NPs absorb photons from the  = 808 nm laser due to the polaronic transitions. [START_REF] Bredas | Polarons and Bipolarons in Polypyrrole: Evolution of the Band Structure and Optical Spectrum upon Doping[END_REF][START_REF] Patil | Optical Properties of Conducting Polymers[END_REF] Secondly, due to the strong electron phonon interactions, heat is generated and diffuses to the NTC thermistor in direct contact with PPy-NPs (Step 2). Finally, in Step 3, the temperature rise of the NTC thermistor leads to a change of its resistance, which is measurable by an external circuit with an applied voltage of 2 V.

Results and Discussion

Under an external DC bias of 2 V, due to a change of resistance under monochromatic illumination, current changes can be measured at each wavelength (current changes at some typical wavelengths were shown in Figure 3a, 3b), so that the presence of NIR and SWIR light (800 nm -2000 nm) can be detected. The light power on device at this wavelength window varied from 1.3 μW (at 2000 nm) to 2.8 μW (at 1300 nm). Figure 3c presents the photoresponsivity of the hybrid PPy-NPs/thermistor device under these monochromatic wavelengths irradiation. The black symbols in Figure 3c show the photodetection capability (photoresponsivity) of this type of PPy-NPs/thermistor hybrid device, plotted in terms of ΔR/P (Ω/W), where ΔR is the resistance change (Ω) and P is the incident light power (W) on the device at each monochromatic wavelength. With a fixed external applied DC voltage (2 V), the measured photoresponsivity can also be plotted as ΔI/P (A/W), where ΔI is the current change under illumination (red symbols in Figure 3c). In the wavelength range from 800 nm to 2000 nm, a photoresponsivity ranging from 0.5 A/W to 1.3 A/W was measured in the current PPy-NPs/thermistor hybrid device, which is comparable to the results obtained on commercial InGaAs PDs. [START_REF] Siala | Dilute Nitride Photodetector Arrays for Sensing Applications[END_REF] Besides monochromatic illumination with relatively low power density, two lasers with wavelengths of 808 nm and 1.5 μm laser operated at different powers were also applied separately as illumination source to the hybrid device (Figure 3d). A clear rise and drop of the measured current was associated with the on and off of the laser. For photodetection applications, besides the photoresponsivity, the photoresponse time (T) is also a key parameter to determine the scope of potential applications. T is usually defined as the time needed for the photoresponse to rise to 80% of its maximum value under illumination, e.g. from 10% to 90% or from 0% to 80%. In the above-mentioned PPy-NPs/thermistor device, it is likely due to the inefficient heat diffusion from the NPs to the glass package and then to the thermistor, the rise time was very modest (about 2 s). In order to improve this aspect, here, we designed and fabricated platinum (Pt) patterns [START_REF] Xiang | Hybrid Plasmonic Gold-Nanorod-Platinum Short-Wave Infrared Photodetectors with Fast Response[END_REF][START_REF] Miyakawa | MEMS-Based Microthruster with Integrated Platinum Thin Film Resistance Temperature Detector (RTD), Heater Meander and Thermal Insulation for Operation up to 1,000[END_REF] to harvest the photothermal effect of PPy-NPs. In this hybrid PPy-NPs/Pt photodetector, 3 µL of PPy-NPs solution was deposited directly onto the Pt pattern which was defined on a glass substrate (Figure 4a). After the solvent was dried, PPy-NPs were then evenly coated on the Pt pattern, as indicated by the SEM image in A strong photoresponsivity (in Ω/W and A/W) was measured, and the maximum value reached 7.5×10 6 Ω/W and 1.3 A/W (Figure 4d). Detectivity (D*), as another key parameter to evaluate the performance of PDs, was also calculated by following some reference equations from previous studies. [START_REF] Kumar | Scalable Synthesis of a Sub-10 Nm Chalcopyrite (CuFeS 2 ) Nanocrystal by the Microwave-Assisted Synthesis Technique and Its Application in a Heavy-Metal-Free Broad-Band Photodetector[END_REF][START_REF] Dou | Solution-Processed Hybrid Perovskite Photodetectors with High Detectivity[END_REF] The maximum D* for our PPy-NPs/Pt device can reach 1.1*10 11 Jones, which is comparable with some InGaAs PDs (D* = 1*10 10 -1*10 13 Jones) and nanostructured PDs (D* = ~ 1*10 8 -1*10 14 Jones). [START_REF] Tan | A Black Phosphorus Carbide Infrared Phototransistor[END_REF][START_REF] Xu | Integrated Structure and Device Engineering for High Performance and Scalable Quantum Dot Infrared Photodetectors[END_REF][START_REF] Mukhokosi | Device Architecture for Visible and Near-Infrared Photodetectors Based on Two-Dimensional SnSe 2 and MoS 2 : A Review[END_REF] Compared with commercial PDs [START_REF] Tan | A Black Phosphorus Carbide Infrared Phototransistor[END_REF][START_REF] Mukhokosi | Device Architecture for Visible and Near-Infrared Photodetectors Based on Two-Dimensional SnSe 2 and MoS 2 : A Review[END_REF] and some nanoparticle-based ones [START_REF] Xiang | Hybrid Plasmonic Gold-Nanorod-Platinum Short-Wave Infrared Photodetectors with Fast Response[END_REF][START_REF] Pescaglini | Hot-Electron Injection in Au Nanorod-ZnO Nanowire Hybrid Device for Near-Infrared Photodetection[END_REF][START_REF] Luo | A Surface Plasmon Enhanced Near-Infrared Nanophotodetector[END_REF][START_REF] Hoang | Interplay of Hot Electrons from Localized and Propagating Plasmons[END_REF][START_REF] Zhao | Au-PbS Core-Shell Nanorods for Plasmon-Enhanced near-Infrared Photodetection[END_REF] , this PPy-NPs/Pt hybrid PD is competitive in photodetection range, responsivity and response time, as summarized in Table 1. Notably, PPy-NPs/Pt hybrid PD reveals both faster photoresponse and slightly larger photoresponsivity than PPy-NPs/thermistor PD, although the size of Pt pattern is much smaller than that of NTC thermistor. It means that a smaller and simpler Pt pattern structure helps to improve both photoresponsivity and response time even if the number of PPy-NPs is not very large. As we described in Figure 2e and Figure 4a, during the detection process, the thermal diffusion capacity of the device plays an important role, involving the thermal diffusivity (α) and thermal resistance (R θ ), as shown in Equation ( 1) and (2), respectively. Thermal diffusivity, α, is determined by the thermal conductivity (k), density (ρ) and specific heat capacity (C) of the heat transfer surroundings/materials. In our devices, the heat transfer surroundings are mainly the glass package

(k = 1.1 W•m -1 •K -1 , ρ = 2.2 g•cm -3 , C = 840 J•kg -1 •K -1 )
for PPy-NPs/thermistor PD and the Pt pattern (k = 70 W•m -1 •K -1 , ρ = 21.45 g•cm -3 , C = 135 J•kg -1 •K -1 ) for PPy-NPs/Pt PD. By the equation 1, the α value of 2.4*10 -5 m 2 •s -1 and 5.9*10 -7 m 2 •s -1 was calculated, respectively, showing that the Pt has much better thermal diffusion rate, which contributed to the shorter response time in PPy-NPs/Pt PD. When it comes to thermal resistance (R θ ), e.g. in a PPy-NPs/Pt PD with the determined parameters of k and cross-sectional area (A), the size of the Pt pattern (Δx in the equation 2) will be the key parameter, smaller size will be easier for the thermal to diffuse and reach a balance. In addition to the high-performance photodetection capability, the current PPy-NPs/Pt hybrid PDs exhibited an excellent stability in air and a wide processing window. For example, PPy-NPs can be dispersed and stored in various solvents, such as water, ethanol and acetone. Figure 5a shows the absorption spectra of PPy-NPs dispersed in different solvents (water, ethanol, acetone) after heating at 60 o C for 6 h. No significant changes in the optical absorption were observed in these heated solutions by comparison to freshly synthesized PPy-NPs dispersed in water. Thanks to the excellent stability of PPy-NPs, the obtained PPy-NPs/Pt hybrid PDs exhibited long-term device stability when stored in air.

C k α    (1) A k x Rθ    (2)
A comparative experiment was carried out on PPy-NPs/Pt hybrid PD by comparing with Au-NRs/Pt PD (Figure 5b). The two hybrid devices were stored in air for 6 months without any encapsulation. On Au-NRs/Pt device, a rapid decline in photoresponsivity was observed within the first month of storage owing to the fusion and regrowth of Au-NRs inherent in the high surface energy of Au nanoparticles. These morphology changes were observed in the inserted SEM images in Figure 5b. During a 3-month device storage, the Au-NRs continued to age and fuse into large micron-sized particles, which resulted in the loss of their plasmonic absorption characteristics in the NIR and SWIR spectrum. By comparison, confirmed by SEM on samples over different air storage times, the PPy-NPs morphology are highly stable. As a result, after a 6-month device storage, the PPy-NPs/Pt device still maintained photoresponsivity, which was equivalent to more than 90% of the photoresponsivity measured on fresh device. The samples were confirmed by SEM under different air storage times, and the morphology of PPy-NPs was still almost identical to that observed in fresh samples. In order to further confirm the molecular structure of the PPy-NPs after six months of months in air exposure, their IR spectra was recorded by the FTIR. The characteristic IR peaks remained completely consistent with the initial sample (Figure S8), proving the outstanding stability of the PPy-NPs.

Conclusion

In summary, we successfully demonstrated the strong photothermal effect of PPy-NPs in the thermal photodetector structure, and developed an air-stabilized hybrid NPs/Pt photodetector (PD) with photoresponsivity in the NIR/SWIR spectrum window (800 nm -2000 nm). Hybrid PPy-NPs/thermistor and PPy-NPs/Pt devices were fabricated and characterized. On PPy-NPs/Pt PD, we demonstrated a fast response time of 131 μs and a high photoresponsivity of 1.3 A/W (@ V applied = 2V) under  = 1.5 μm laser illumination. The PPy-NPs/Pt PDs have a wide processing window due to the excellent stability of PPy-NPs in various solvents. The resulting PPy-NPs/Pt PDs are air stable even without encapsulation, and maintain > 90% photoresponsivity after a 6-month air storage period.

These results indicate that PPy-NPs/Pt PD is a competitive candidate for next generation of low-cost and toxic-heavy-metal-free NIR/SWIR photodetectors.

Experimental section

Synthesis of PPy-NPs

PPy-NPs were synthesized by chemical oxidation of the Py monomer confined in the oil phase of hexagonal mesophases (0.1 mM). Hexagonal mesophases are made of a mixture of sodium dodecyl sulfate (SDS) as surfactant, salted water phase (NaCl), cyclohexane as oil phase and pentanol as co-surfactant. The experimental details are as follow: 100 μL Py was added to 4 mL of an aqueous solution containing 0.1 M NaCl, and 1.6 g SDS were introduced to the obtained solution. A transparent viscous micellar solution (solution A) was obtained after vortexed and heat (50 o C for 1 h). 0.1 M FeCl3 (as oxidant) was dissolved in 4 mL of water and then 1.6 g SDS was introduced in the solution, solution B can be obtained by vortexed and heat at 50 o C for 1 h. Subsequently, 6 mL of cyclohexane and 400 μL n-pentanol (co-surfactant) were added dropwise to each micellar. The mixtures were then vortexed until a transparent and yellow translucent, birefringent gels (hexagonal mesophases) were obtained. Mixing A and B lead to a black gel. A black powder PPy-NPs was then obtained after cleaned and dried.

Synthesis of Au-NRs

Colloidal Au-NRs used in this work were synthesized by following previously described methods [START_REF] Xiang | Short-Wave Infrared Sensor by the Photothermal Effect of Colloidal Gold Nanorods[END_REF][START_REF] Ye | Using Binary Surfactant Mixtures To Simultaneously Improve the Dimensional Tunability and Monodispersity in the Seeded Growth of Gold Nanorods[END_REF] . Au nanoparticle seed solution: After mixing 5 ml of 0.5 mM HAuCl 4 solution with 5 ml of 0.2 M CTAB solution, 1 ml of 0.006 M NaBH 4 solution was added followed by vigorously stirring for 2 minutes. This solution was then aged for 30 minutes without stirring. Au-NR growth solution: CTAB (7 g) was evenly mixed with NaOL (1.234 g), and then dissolved it in 250 ml de-ionized (DI) water under stirring at 50 o C. After cooling the solution to 30 o C, 20 ml of 4.15 mM AgNO 3 solution is added into the system, keeping the whole solution at 30 o C for 15 minutes. This is followed by the addition of 250 ml of 1 mM HAuCl 4 solution with stirring for about 90 minutes (until the solution became colorless). At this stage, 1.6 ml of 12.1 M HCl solution was added under stirring for 15 minutes. 1.25ml of 0.064 M ascorbic acid solution was then added under vigorous stirring for 30s. Finally, 0.4 ml seed solution was added under rigorous stirring for 30s, and then the whole solution was kept at 30 o C for 12 hours without stirring to maintain the NR growth. After 12 hours of growth, the final product was separated by centrifugation (6k rpm for 30 minutes) and subsequent decantation of the mother liquid. DI water was then added to re-dissolve the Au-NRs. The centrifugation/decantation process was repeated for 3 times and the final Au-NR products were dissolved in DI water.

NTC-thermistor based devices fabrication and measurement

Hybrid devices involving NTC thermistors was fabricated by the following procedure: In brief, a drop (≈ 3 µL) of Au-NRs (with a concentration of 3 g/L) or PPy-NPs (with a concentration of 0.4 g/L and 0.8 g/L) in water was casted directly to coat the surface of the thermistor. After the solvent (water) was dried on the thermistor, the hybrid devices were measured under monochromatic illumination generated by a quartz halogen lamp and a computer-controlled Oriel Cornerstone monochromator (and appropriate order sorting filters). The intensity of the monochromatic illumination was calibrated by a National Institute of Standards and Technology-calibrated germanium photodiode. The monochromatic illumination was modulated by a shutter while the resistance of the hybrid and control device was measured by a computer-controlled Keithley 2634B source measurement unit (SMU). Concerning measurements under laser, the output of a fiber-coupled laser diode (λ = 808 nm) was focused onto the devices. The diameter of the laser spot is about 1 mm, which can almost cover the entire device. Neutral density filters were used to adjust the laser power, while the laser power was calibrated by a laser power meter. The laser illumination was modulated by a shutter, while the photothermal induced current and resistance was measured by a Keithley 2634B SMU. From the data sheet of the thermistor, each resistance value measured corresponds to a temperature value. When the illumination was on (or off), a resistance value of R light-on (or R light-off ) was measured and a current value of I light-on (or I light-off ) can also be measured (under an external DC voltage bias of 2 V). From R light-on (or R light-off ) and the datasheet provided by the manufactory of the thermistor, the temperature value (T light-on or T light-off ) corresponding to the resistance value can be obtained. From T light-on and T light-off , the temperature change (T light-on -T light-off ) of the hybrid device due to the illumination was obtained.

Pt-based device fabrication and measurement

A layer of adherence promoter (TI from microchemicals) and a layer of photosensitive resin (AZ5214, microchemicals) were spin-coated layer by layer on cleaned glass substrates. Pt pattern was then defined by direct laser writing lithography (Heidelberg Ins. laserwriter µPG101). After lithography, substrates were developed by dipping the sample in a bath of developer (AZ726 MIF, microchemicals) for 30 s with subsequent DI water rising and air-drying. The Ti (5 nm)/Pt (40 nm) layer was then evaporated onto the sample by an e-beam evaporator (Plassys MEB550S). Finally, the Pt-coated substrates were lifted-off by immersing the samples into a bath of acetone. To fabricate the hybrid PPy-NPs/Pt PDs, PPy-NPs water solution was deposited onto the Pt pattern at an optimized concentration of 0.8 g/L. Finally, solvent was allowed to dry in air.

To characterize the photoresponse of PPy-NPs/Pt PDs, a Fabry-Perot pigtailed laser (peak  = 1537 nm) was focused onto the Pt/Au-NRs device. The diameter of the laser spot is about 150 µm, which was focused by an extra lens. The light spot can cover the whole Pt pattern zone (90 µm * 40 µm). To characterize the response speed, the device was placed in a series circuit with an applied voltage of 2 V. During the measurements, the laser spot was modulated by a functional generator (Aim-TTi TG1006) with a laser ON/OFF frequency of 120 Hz. The resistance changes (R) of the Au-NR/Pt device was measured in the same circuit (Bias = 2 V). To verify the broadband photoresponsivity of the device, the testing conditions under a monochromatic light source was identical to the description above on NPs/thermistor PDs. For the resistance/current responsivity measurements, neutral density filters were used to adjust the laser power, while the laser power was calibrated by a laser power meter. The laser illumination was modulated by a shutter, while the photothermal induced current and resistance was measured by a Keithley 2634B SMU identical to the description above on NPs/thermistor PDs.

Optical and morphology characterizations

UV-Visible absorption spectra were measured in the air by an Agilent 5E UV-Visible-NIR spectrometer. TEM characterizations were performed with a JEOL 2010 microscope operating at 200 kV. SEM characterizations were performed by the FEI Magellan 400 system with a field emission gun

Figure 1 .

 1 Figure 1. (a) PPy polymerization by chemical oxidation; (b) FTIR spectra of the PPy-NPs with their characteristic IR peaks; (c) Transmission Electron Microscopy (TEM) image of PPy-NPs, the insert is a zoom-out TEM image for many PPy-NPs; (d) Absorption spectrum of PPy-NPs (insert image is the picture of the PPy-NPs black powder).

Figure 2 .

 2 Figure 2. (a) A negative temperature coefficient (NTC) thermistor with PPy-NPs coating, the size of NTC thermistor is ~ 1 mm. The PPy-NPs water solution and Au-NRs water solution were shown in the insert picture. SEM image of Au-NRs (b) and PPy-NPs (c). The temperature change (ΔT) as the function of laser power on the devices measured on each type of NPs decorated NTC-thermistors by comparison to the control device (without NPs) (d). (e) Device structure diagram and working process (Step 1, 2, 3) of the hybrid device with Au-NRs or PPy-NPs.

Figure 3 .

 3 Figure 3. (a-b) Photoresponse characteristics over time of a hybrid PPy-NPs/thermistor device in the form of photo-induced current changing upon exposure of monochromatic illumination (λ from 0.8 µm to 2.0 µm). The current was measured under an external applied DC voltage of 2 V. (c) The photoresponsivity (in Ω/W or in A/W) as a function of the whole wavelength window tested (800 nm -2000 nm). (d) Photoresponse characteristics over time as a function of laser power under 808 nm laser and 1.5 µm laser.

Figure 4a .

 4a The left insert of Figure4azooms in the morphology of PPy-NPs on the Pt pattern, the right insert shows a schematic diagram of the device operation briefly (Device structure in details is shown in FigureS6and S7). To characterize such hybrid PDs, a  = 1.5 μm laser was focused onto this device with its ON/OFF modulated by a function generator. As shown in Figure4b, a clear illumination-induced resistance change was recorded at different power levels. Figure4cshows the ON/OFF modulation of the hybrid PPy-NPs/Pt device under laser illumination as a rise/fall curve of 120 Hz light response, in which the response time of 131 μs rise and 203 μs fall is measured. By comparing with the above-mentioned PPy-NPs/thermistor devices, the response time of the PPy-NPs/Pt hybrid PDs here is shortened by 6 orders of magnitude.

Figure 4 .

 4 Figure 4. (a) SEM image of the hybrid PPy-NPs/Pt photodetector, the left insert is the morphology of PPy-NPs on the Pt pattern, the right insert shows a schematic diagram of the device operation briefly. (b) Photoinduced resistance change over time as the function of laser power (λ = 1.5 µm) with a laser ON/OFF modulation frequency. (c) Response time of the PPy-NPs/Pt device measured under the λ = 1.5 µm laser modulated at 120 Hz. Rise time (T rise ) is the normalized resistance (R) rising from 0 to 80%, fall time (T fall ) is the normalized R falling from 100% to 20%. (d) The photoresponsivity (in Ω/W or in A/W) as a function of laser power from 1.5 μm laser.

Figure 5 .

 5 Figure 5. (a) Absorption spectrum of PPy-NPs in different solvents (water, ethanol, acetone) and temperature (20 o C and 60 o C). (b) Long-term device stability test of PPy-NPs/Pt device and Au-NRs/Pt device, which are exposed to the atmosphere without packaging. The insert pictures are the SEM images of Au-NRs or PPy-NPs on the Pt pattern during the test (Scale bar is 200 nm).

Table 1 .

 1 Summary of photodetection range, responsivity and response time in commercial PDs and some nanoparticle-based ones.

	Materials	Response range (nm)	Peak responsivity	Response time (T rise /T fall )	Ref.
	Commercial Si PD	400-1100	0.5 A/W @ 780 nm	< 1 ns / < 1 ns	48
	Commercial InGaAs PD	800-1700	1 A/W @ 1300	< 1 ns / < 1 ns	46
	Au-NRs/ZnO-nanowire	650-850	-	0.25 s / 0.69 s	49

ACKNOWLEDGMENT H.X., X.Y. and Z.H. acknowledge the China scholarship council (CSC) for Ph.D. thesis scholarship. H.X. acknowledge the National Natural Science Foundation of China (62004101), the Fundamental Research Funds for the Central Universities (30920041117), the China Postdoctoral Science Foundation (2020M681600). SEM characterizations performed were supported by the region Île-de-France in the framework of DIM Nano-K.

electron source. FTIR (Fourier Transform Infrared) spectroscopy were measured by a Bruker Alpha-II FTTIR spectrometer.

ASSOCIATED CONTENT

Supporting Information.

The Supporting Information is available free of charge on the ACS Publications website.

Additional results on XRD of PPy-NPs; Absorption spectrum of Au-NRs solution; Relationship between resistance and temperature in NTC thermistors; Concentration optimization process of the PPy-NPs/NTC-thermistor; Photothermal conversion efficiency calculation details; Device structure of the hybrid PPy-NPs/Pt photodetector; FTIR spectra of the PPy-NPs with their characteristic IR peaks. (PDF)

AUTHOR INFORMATION

Corresponding Author

* Corresponding authors. E-mail addresses: E-mail: hu.zhelu@espci.fr; zhuoying.chen@espci.fr; yxjxjks@126.com

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes

The authors declare no competing financial interest.


Emma Cavarocchi

Marjorie Whitfield

Ahmed Chargui

Laurence Stouvenel

Patrick Lorès

Charles Coutton

Christophe Arnoult

Pietro Santulli

Catherine Patrat

Nicolas Thierry-Mieg

Pierre F Ray

Emmanuel Dulioust

Aminata Touré
email: aminata.toure@inserm.fr

The sodium/proton exchanger SLC9C1

Keywords: male infertility, asthenozoospermia, gene mutation, whole-exome sequencing, SLC9C1 -sNHE, ion channel

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Asthenozoospermia, defined by the absence or reduction of sperm motility, is observed in nearly 80% of male infertility cases, either alone or in association with other sperm defects [START_REF] Heidary | Genetic aspects of idiopathic asthenozoospermia as a cause of male infertility[END_REF] . This pathophysiological condition is induced by structural defects of the sperm flagellum and/or functional alterations impairing flagellar beating and sperm progression. Among the structural defects, a remarkable condition described as Multiple Morphological Abnormalities of sperm Flagella (MMAF) results from defective assembly of the flagellum and leads to the presence in the ejaculate of spermatozoa with short, absent, coiled, bent or irregular tails. This phenotype of asthenoteratozoospermia was previously identified as 'dysplasia of the fibrous sheath', 'short tails' or 'stump tails' [START_REF] Chemes | Dysplasia of the fibrous sheath: an ultrastructural defect of human spermatozoa associated with sperm immotility and primary sterility**Supported by grant 0934 from Consejo Nacional de Investigaciones Científicas y Técnicas[END_REF][START_REF] Escalier | Arrest of flagellum morphogenesis with fibrous sheath immaturity of human spermatozoa[END_REF][START_REF] Escalier | New fibrous sheath anomaly in spermatozoa of men with consanguinity[END_REF] , and in the last decade, numerous mutations causing this phenotype were identified (AK7, ARMC2, MAATS1, CEP135, CFAP43-44-65-69-70-251, DNAH1-2-6-8-17, QRICH2, SPEF2, TTC21A, TTC29) [START_REF] Mbango | Genetic causes of male infertility: snapshot on morphological abnormalities of the sperm flagellum[END_REF][START_REF] Touré | The genetic architecture of morphological abnormalities of the sperm tail[END_REF] . Besides MMAF, some asthenoteratozoospermic patients with milder morphological defects such as defective annulus and abnormal mitochondrial sheath were also described to carry mutations (TEKT2, SEPT12 or SLC26A8) [START_REF] Heidary | Genetic aspects of idiopathic asthenozoospermia as a cause of male infertility[END_REF][START_REF] Touré | The Testis Anion Transporter 1 (Slc26a8) is required for sperm terminal differentiation and male fertility in the mouse[END_REF][START_REF] Kuo | SEPT12 mutations cause male infertility with defective sperm annulus[END_REF] . Importantly, asthenozoospermia is also detected in the complete absence of morphological defects, suggesting the involvement of functional defects in activation and/or regulation of flagellar beating. To date, such cases of functional asthenozoospermia remain poorly characterized, and only a few mutations were identified in genes encoding for proteins with enzymatic properties (GALNTL5), seminal component (SEMG1) and ion channels (CATSPER1-2-ε, SLC26A3, VDAC2) [START_REF] Heidary | Genetic aspects of idiopathic asthenozoospermia as a cause of male infertility[END_REF][START_REF] Xu | Abnormal Hypermethylation of the VDAC2 Promoter is a Potential Cause of Idiopathic Asthenospermia in Men[END_REF][START_REF] Ray | Genetic abnormalities leading to qualitative defects of sperm morphology or function: Genetic abnormalities leading to qualitative sperm defects[END_REF][11][START_REF] Brown | Human sperm ion channel (dys)function: implications for fertilization[END_REF] . Overall, the knowledge obtained by studying those sperm pathological phenotypes provides evidence that, in addition to proper morphology and structure, sperm fertilization potential relies on proper activation of the signalling pathways regulating motility, metabolism and energy production.

Sperm functional maturation occurs after spermiation during the transit within the epididymis, where sperm cells acquire the ability to move forward, and ultimately within the female genital tract, where they acquire complete fertilization potential through a process known as capacitation [START_REF] Freitas | Signaling mechanisms in mammalian sperm motility[END_REF] . Numerous ion channels and transporters have been identified at the surface of sperm cells and conduct complex fluxes between the male and female genital tract milieus and the sperm cytoplasm [START_REF] Shukla | Ion Channels in Sperm Physiology and Male Fertility and Infertility[END_REF] . These ion exchanges activate several biochemical and electrophysiological changes that are essential for sperm motility and fertilization potential [START_REF] Visconti | Ion channels, phosphorylation and mammalian sperm capacitation[END_REF] .

Among the changes occurring within the female genital tract (i.e. capacitation), were described alkalinization of the cytoplasm, membrane hyperpolarization and protein phosphorylation cascades induced by the activation of soluble adenylate cyclase (sAC) and Protein Kinase A (PKA), which specifically target flagellar components required for sperm fertilization potential [START_REF] Dey | Signaling Enzymes Required for Sperm Maturation and Fertilization in Mammals[END_REF] .

In the present study, by performing Whole-Exome Sequencing (WES) on a patient presenting a very low progressive motility but no detectable ultrastructural defects of the axoneme, we identified a homozygous pathogenic mutation in SLC9C1. This gene, also called sNHE, encodes a sperm-specific member of the Na + /H + exchanger family involved in the regulation of pH and cell volume in a wide range of tissues [START_REF] Fuster | Traditional and emerging roles for the SLC9 Na+/H+ exchangers[END_REF] . We demonstrate here that SLC9C1 is critical for human sperm motility and that mutations in SLC9C1 are a novel cause of human asthenozoospermia.

Materials & methods

Subject and biological samples

The study was approved by the Comité de Protection des Personnes CPP Ile de France III (record number CPP02748) and performed in accordance with ethical guidelines (Declaration of Helsinki). All patient and control individuals included in the study gave their informed consent prior to their inclusion in the study. Semen samples were obtained by masturbation after 2-7 days of sexual abstinence. Semen parameters were evaluated according to the World Health Organization (WHO) guidelines [START_REF] Cooper | World Health Organization reference values for human semen characteristics* ‡[END_REF] and David's classification [START_REF] Auger | Another look at human sperm morphology[END_REF] .

WES analysis and Sanger sequencing

WES analysis was performed on genomic DNA extracted from a blood sample, as previously described [START_REF] Martinez | Whole-exome sequencing identifies mutations in FSIP2 as a recurrent cause of multiple morphological abnormalities of the sperm flagella[END_REF] . In brief, the enrichment of coding regions together with intron/exon boundaries was performed with Exon V5 kit (Agilent Technologies, Wokingham, UK). Sequencing was performed with Illumina HiSeq 2000 at the Genoscope (Evry, France). Exome-seq data were analysed using a bioinformatics pipeline developed in-house as previously described [START_REF] Martinez | Biallelic variants in MAATS1 encoding CFAP91, a calmodulin-associated and spoke-associated complex protein, cause severe astheno-teratozoospermia and male infertility[END_REF] . The homozygous mutation in SLC9C1 identified by WES was confirmed by Sanger sequencing using ABI 3130XL and SeqScape software (Applied Biosystems; Foster City, CA, USA).

Primer sequences (5'-3'): F: TCCCAACCACTTCTAAAATGTTGT, R:

TGCATTTATAAATAACACTGCCTGGT.

Transmission electron microscopy analysis

Sperm cells were collected from fresh ejaculate and washed with M2 medium (Sigma-Aldrich Co. Ltd; Irvine, UK) by centrifugation at 300g/RT for 10 minutes. Sperm cells were then fixed and embedded for semi-thin sections as previously described [START_REF] Lorès | Mutations in TTC29, Encoding an Evolutionarily Conserved Axonemal Protein, Result in Asthenozoospermia and Male Infertility[END_REF] . All sections were examined with a JEOL 1011 electron microscope (Jeol Ltd; Tokyo, Japan). Images were acquired with Digital Micrograph software coupled to a Gatan Erlangshen CCD camera.

RT-PCR analysis

Total RNA from control and patient spermatozoa (800-1000ng) was extracted using

NucleoSpin RNA kit (Macherey-Nagel; Düren, Germany) and used for reverse transcription with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific; Waltham, MA, USA) as previously described [START_REF] Lorès | Mutations in TTC29, Encoding an Evolutionarily Conserved Axonemal Protein, Result in Asthenozoospermia and Male Infertility[END_REF] . Amplicons were gel purified and sequenced (Eurofins Scientifics, France). Results were analysed with BioEdit software (Ibis Therapeutics; Carlsbad, CA, USA).

Primer sequences (5'-3'): SLC9C1-F: TTATCAGGCCTCTTACTGTTG, SLC9C1-R: TGATCAATCCCTAAACCTGGC; HPRT-F: CCTGCTGGATTACATTAAAGCACTG, HPRT-R: GTCAAGGGCATATCCAACAACAAAC.

Immunofluorescence assay

Slides were prepared by spreading 10 μL of fresh semen sample onto a Superfrost Plus slide (Menzel Glasbearbeitungswerk, GmbH & Co. KG; Braunschweig, Germany), followed by fixation 10 minutes in PBS 4% paraformaldehyde. Immunostaining was performed as previously described [START_REF] Lorès | Mutations in TTC29, Encoding an Evolutionarily Conserved Axonemal Protein, Result in Asthenozoospermia and Male Infertility[END_REF] , using commercial antibodies, which specificity was not validated.

Results

The patient presented in this study first consulted for primary infertility in 2016 at the age of 31, after two years of natural conception failure, and was followed at the reproductive biology centre of the Cochin hospital (Paris, France). Apart from a non-evolutive chronic hepatitis B diagnosed in 2008, no urogenital disease, neither lifestyle factors nor exposures potentially deleterious for fertility were reported. The couple benefited from intra-marital ICSI performed with fresh ejaculated sperm cells but frozen oocytes because the man failed to produce a semen sample on the day of oocytes pick-up of the first ICSI attempt. Seven out of nine of the thawed oocytes were injected and 3 zygotes were observed 18 hours after ICSI.

After a prolonged embryo culture, 3 embryos with a good quality, according to the Istanbul consensus 2011 classification [START_REF] Embryology | The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting[END_REF] , were obtained. Embryo transfer scheduled on day 5 was postponed due to a high endometrial thickness; two of the embryos were frozen at day 5 and the third one at day 6. To date, only one thawed embryo was transferred but the resulting pregnancy ended with a miscarriage after 5 weeks and 3 days.

The successive semen analyses performed over 3 years on the patient revealed a severe alteration of sperm progressive motility, which never exceeded 5% (normal value > 32%), contrasting with normal sperm counts and vitality rates (Table 1). The mean percentage of typical spermatozoa was slightly decreased but the patient did not present any sperm abnormalities indicative of a MMAF phenotype (the percentage of spermatozoa with short, absent and irregular calibre of flagella being under the lower reference values [START_REF] Auger | Another look at human sperm morphology[END_REF] ) (Table 1).

However, semen analyses indicated a significant proportion of spermatozoa with an excess residual cytoplasm and disorganised midpiece with occurrences of plasma membrane lysis (Figure 1A). In addition, flagellar bending was noticed together with some head anomalies (thin head) (Table 1). Acrosomal staining performed with fluorescent peanut agglutinin (PNA) on sperm sample from the patient indicated an intact acrosome on most of the spermatozoa; hence the rate of PNA negative sperm cells, indicative of premature acrosomal reaction, was 8.8% and 5.4% for control and patient, respectively. The procedure of sperm selection with density gradient centrifugation slightly increased the fraction of progressive spermatozoa (5-25%) but also induced an increase of the number of isolated sperm heads. We found that the percentage of isolated sperm heads remained very low in control samples defined by normal sperm parameters, including morphology according to WHO [START_REF] Auger | Another look at human sperm morphology[END_REF] (1% and 3% before and after selection, respectively), while it significantly increased in patient sample after density gradient centrifugation (2% and 33% before and after selection, respectively). Importantly, we noted that flagellar bending and breaking points on sperm from the patient were not limited to the connective piece but were also present along the flagella (Supplementary Figure S1). Overall, these observations suggested an important fragility of the sperm tail, amplified by the selection procedure.

We performed Transmission Electron Microscopy (TEM) analysis to precisely examine the sperm ultrastructure of the patient. In line with the observations by optic microscopy, we detected the presence of midpiece defects, in particular, an excess of residual cytoplasm around the mitochondrial sheath and plasma membrane fragility illustrated by plasma membrane lysis (Figure 1B). Importantly, no alteration of the axonemal structure was observed in patient's sperm as quantification indicated a percentage of normal transversal sections (9+2 conformation) close to that recorded in semen from control individuals (Patient: 84%; Control mean: 84.5%, n=4). Overall, the patient presented in this study was classified as a case of asthenozoospermia with a severe reduction of the proportion of progressive sperm, membrane and flagellar fragility but no major defects of the sperm flagellum ultra-structure and assembly.

We thus subsequently performed WES analysis of this patient in search for genetic variants potentially involved in the infertility phenotype. After processing and filtering the data, we identified homozygous variants with high predicted impact on the encoded proteins in three genes: c.2748+2T>C in SLC9C1, c.3583C>T in ABCB5 and c.9del in ZNF891. Among those, the variant c.2748+2T>C in SLC9C1 appeared as the best candidate considering the pattern and abundance of expression of the three genes and the frequency of the variants in the population according to public databases (Supplementary Table 1). Indeed, in contrast to the two other candidates, SLC9C1 was shown to be preferentially expressed in the testis (Illumina Body Map; The Human Protein Atlas database; Reprogenomics Viewer) and the encoded protein was previously detected in human sperm proteome [START_REF] Wang | In-depth proteomic analysis of the human sperm reveals complex protein compositions[END_REF] (Supplementary Table 1). The homozygous variant c.2748+2T>C identified in SLC9C1 impairs a consensus sequence of the splice donor site located downstream of exon 22 and was associated with a low allele frequency in the gnomAD database (4.6x10 -5 , chr3:112180562 A>G (GRCh38.p12), rs2007949663-111899409-A-G). The presence of this homozygous variant in the patient was also consistent with the familial history of infertility and the known consanguinity of his parents (cousins). The proband originated from West Africa (Guinea Conakry); he has two brothers, one of them known to be infertile, and two fertile sisters (Figure 2A). In addition, the proband has one cousin who also displays male infertility; as it was not possible to define if the cousin was from maternal or paternal side, this individual is not included in the genealogic tree (Figure 2A).

Unfortunately, we could not obtain to biological samples from the family members. In conclusion, considering all the above arguments, we retained the c.2748+2T>C variant identified in SLC9C1 by WES, which we also confirmed by Sanger sequencing on DNA sample from the proband (Figure 2B).

SLC9C1, also known as sNHE or SLC9A10, is located on chromosome 3 and comprises 29 exons (Figure 2C). Alternative splicing produces five transcript variants, three of which encoding proteins, and among them transcript SLC9C1-201 encodes for the longer isoform of 135 kDa (Figure 2C). The full SLC9C1 protein isoform comprises the ion exchange domain (Cat/H + ex), which is characteristic of SLC9 family members and is generally formed by ten to twelve transmembrane (TM) helices (Figure 2C). The protein also exhibits a unique C-terminal tail with four additional TM helices showing similarity to a Voltage-Sensing Domain (ITD/VSD) and a consensus sequence for a Cyclic Nucleotide-Binding Domain (CNBD), which is composed of several α-helices and β-sheets and protrudes towards the cytoplasmic side 25,[START_REF] Windler | The solute carrier SLC9C1 is a Na+/H+-exchanger gated by an S4-type voltage-sensor and cyclicnucleotide binding[END_REF] . These two latter features are typical of ions channels, which, unlike solute carriers, are not activated by ion gradients but display a more sophisticated regulatory mechanism dependent on the cell status 14 .

To investigate the molecular consequence of the mutation identified in SLC9C1, we performed RT-PCR analysis on semen sample from control and patient individuals using forward and reverse primers localised in exons 21 and 23, respectively. The PCR amplicon obtained for control individual was observed at the expected size of 217 bp while product amplified from the patient sample was considerably smaller (Figure 2D). The experimental normalization, operated by analysis of the housekeeping gene HPRT, did not reveal any difference in SLC9C1 transcripts amount between the patient and control individual.

Sequencing of SLC9C1 RT-PCR amplicons indicated the total absence of exon 22 in the transcripts from the patient (Figure 2E). This exon encodes for a region included in the Cyclic Nucleotide-Binding Domain (Figure 2C) and comprises 99 bp. Such exon skipping would therefore cause a 33 amino acids in-frame deletion (corresponding to amino acids 884-916).

These results confirm the deleterious impact of the identified variant.

We next performed immunofluorescence assays with two antibodies raised against different epitopes of the SLC9C1 protein: a so-called 'Central antibody', which recognizes amino-acids 600-750 of the protein, spanning the VSD, upstream from the deletion and a socalled 'C-Terminal antibody' targeting amino-acids 882-932, in the CNBD and mainly coinciding with the deleted sequence. We first observed that both antibodies detected the SLC9C1 protein along the sperm flagellum with a signal intensity decreasing towards the tail end. Importantly, in consistence with RT-PCR data which predicted the deletion of amino acids 884-916, immunofluorescence assays performed with the 'Central antibody' marked SLC9C1 protein in spermatozoa from both control and patient samples (Figure 3A), whereas the 'C-Terminal antibody' whose epitope largely overlaps with the deleted region only marked the control sperm (Figure 3A).

Lastly, we performed western blot immunodetection from control and patient sperm protein extracts. Using the 'Central antibody' we detected a band at the expected molecular weight of the SLC9C1 longer isoform (135 kDa) in both control and patient sperm samples (Figure 3B). Technical limits of the gel electrophoresis did not permit to evidence the small size difference between the normal and the deleted protein but this data firmly confirm the presence of the SLC9C1 truncated protein in sperm cells from the studied patient.

Discussion

In conclusion, we characterize an infertile patient displaying functional asthenozoospermia caused by a homozygous splice mutation in SLC9C1, which generates a 33 amino-acid deletion within the Cyclic Nucleotide-Binding Domain (CNBD). This protein domain is considered to be involved in the interaction and reciprocal modulation between SLC9C1 (sNHE) and the soluble Adenylate Cyclase (sAC) in mouse and sea urchin sperm [START_REF] Windler | The solute carrier SLC9C1 is a Na+/H+-exchanger gated by an S4-type voltage-sensor and cyclicnucleotide binding[END_REF][START_REF] Wang | A sperm-specific Na+/H+ exchanger (sNHE) is critical for expression and in vivo bicarbonate regulation of the soluble adenylyl cyclase (sAC)[END_REF] , the latter being a key element for the regulation of sperm motility and capacitation.

In mice, the disruption of Nhe-1 did not impact male fertility 28 while disruption of Slc9c1 (sNhe) as well as Nha1 and Nha2 (single and double conditional KO mice) all resulted in a phenotype of null sperm motility. Interestingly, these phenotypes were significantly rescued by sperm incubation with cAMP analogues, confirming that Na + /H + family members are required for the activation of sperm motility and capacitation through regulation of the cAMP-PKA signalling pathway 25,[START_REF] Chen | Sodium-hydrogen exchanger NHA1 and NHA2 control sperm motility and male fertility[END_REF] . SLC9C1 (sNHE) was previously described to only localise to the principal piece of the mouse sperm flagellum 25 . Zhang et al. [START_REF] Vyklicka | Dissecting the signaling pathways involved in the function of sperm flagellum[END_REF] proposed the same localisation in human sperm cells, although the staining was weak and quite heterogeneous.

Our immunostaining assays carried out with two different antibodies suggest a distinct flagellar profile in human sperm. Hence, while we detected a prominent localisation of sNHE in the principal piece of murine spermatozoa (Supplementary Figure S2), in human sperm, SLC9C1

was detected along the whole length of the flagellum.

Regarding, the molecular mechanisms of action, it is well established that intracellular pH regulation is fundamental at different steps of sperm maturation and capacitation [START_REF] Nishigaki | Intracellular pH in sperm physiology[END_REF] , and Na + /H + exchangers (NHE) play a remarkable role in sperm alkalinisation [START_REF] Martins | Physiology of Na+/H+ Exchangers in the Male Reproductive Tract: Relevance for Male Fertility1[END_REF] . SLC9C1 (sNHE) was shown to be required for pH homeostasis and suggested to promote CatSper activation following Slo3-mediated sperm hyperpolarization during mouse sperm capacitation 25,[START_REF] Chávez | SLO3 K + Channels Control Calcium Entry through CATSPER Channels in Sperm[END_REF] . In humans, to date no information is available regarding sNHE possible implication in sperm pH homeostasis. Some elements have pointed a role of the proton channel Hv1 [START_REF] Lishko | Acid Extrusion from Human Spermatozoa Is Mediated by Flagellar Voltage-Gated Proton Channel[END_REF][START_REF] Berger | Post-translational cleavage of Hv1 in human sperm tunes pH-and voltage-dependent gating: Hv1Sper in human sperm[END_REF] , although so far, no mutation in Hv1 were reported in human asthenozoospermia. Our study therefore sheds light on the essential role of sNHE and a likely similar function in pH homeostasis in humans.

Importantly, our findings are in line with a recent comparative genomic analysis, which identified the molecular trio sNHE-sAC-CatSper as an evolutionary conserved machinery for the regulation of sperm flagellar beating in Metazoa [START_REF] Romero | Comparative genomic analysis suggests that the sperm-specific sodium/proton exchanger and soluble adenylyl cyclase are key regulators of CatSper among the Metazoa[END_REF] .

The involvement of SLC9C1 in human sperm physiology has been poorly investigated, and so far only weak evidence was provided regarding its dysfunction in human asthenozoospermia 17,36 , 37 . The mutation in SLC9C1 we described here constitutes a solid demonstration of SLC9C1 involvement in the regulation of human sperm motility and fertilization potential. We show that this mutation has a drastic impact on sperm progression; however sperm motility was not totally abrogated as observed in Slc9c1 KO mouse model 25 .

This difference might be explained by the nature of the mutations in those two models; the KO mouse model totally lacks the protein while the patient carries a truncated protein, which could preserve partial functionality. In addition, while in patient sperm we observed flagellar angulation, excess residual cytoplasm and overall a flagellar fragility upon sperm selection, such defects were not reported in the KO mouse model. These observations in patient sperm are compatible with a dysregulation of the ion equilibrium and overall cell volume regulation, which were previously described to depend on channels and aquaporins activity [START_REF] Yeung | Human sperm volume regulation. Response to physiological changes in osmolality, channel blockers and potential sperm osmolytes[END_REF] .

Overall, this article provides a new evidence of the physiological importance of ion exchange regulation and pH homeostasis in the control of sperm motility and fertilization potential. The first and most remarkable evidence was provided for the CATSPER channel, for which mutations (CATSPER 1-2-ε subunits) were identified in several asthenoteratozoospermic individuals with altered fertilizing capacity and/or sperm with coiled flagella [START_REF] Brown | Human sperm ion channel (dys)function: implications for fertilization[END_REF][START_REF] Avidan | CATSPER2, a human autosomal nonsyndromic male infertility gene[END_REF] . More recently, pathogenic variants affecting members of the SLC26 family of anion exchangers, namely SLC26A3 and SLC26A8 (TAT1), were also shown to impair the functionality of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) channel, causing asthenozoospermia, associated with defective capacitation, midpiece and annulus disorganization in the case of SLC26A8 11, [START_REF] Dirami | Missense Mutations in SLC26A8, Encoding a Sperm-Specific Activator of CFTR, Are Associated with Human Asthenozoospermia[END_REF][START_REF] Wedenoja | A missense mutation in SLC26A3 is associated with human male subfertility and impaired activation of CFTR[END_REF] . The voltage-dependent anion channels VDAC2 and 3 were also identified with pathogenic defects leading to idiopathic asthenozoospermia 42 9 . The specific location of all those ion channels/transporters at the plasma membrane of sperm cells, constitutes an interesting cellular background for potential therapeutic strategies to improve sperm motility in asthenozoospermic men.

  Primary antibodies : SLC9C1 rabbit polyclonal antibody raised against the central region (Invitrogen PA5-104160; 1:100 dilution); SLC9C1 rabbit polyclonal antibody raised against the C-terminal region (Sigma SAB2106664; 1:100 dilution); mouse monoclonal anti-α-Tubulin Sigma T9026 (1:500 dilution). Secondary antibodies: goat polyclonal anti-rabbit IgG Alexa Fluor 568 (Invitrogen A-11029; 1:500 dilution) and goat polyclonal anti-mouse IgG Alexa Fluor 488 (Invitrogen A-28175; 1:500 dilution).Western blot analysis10 million sperm cells from control individual and patient were washed in M2 medium and next in PBS. Sperm cells were then subjected to sonication (20s on, 60s off, 15 cycles) in RIPA buffer [150 mM NaCl, 1% NP40 (Sigma-Aldrich Co.; St. Louis, MO, USA), 0.5% DOC, 0.1% SDS, 25 mM TrisHCL, cOmplete Protease Inhibitor Cocktail (Roche, Basel, Switzerland)].Samples were kept on ice for 30 minutes and centrifuged 13000g at 4°C for 15 minutes. The supernatant was collected and denatured in Laemmli sample buffer, prior to SDS-PAGE [8% acrylamide/bisacrylamide (40%, 37.5:1)] and transfer to nitrocellulose membranes. After a blocking step in PBS 0.1% Tween, 5% BSA or milk for SLC9C1 and β-Tubulin detection, respectively, incubation was performed with primary and secondary antibodies. Primary antibodies: rabbit polyclonal anti-SLC9C1 Invitrogen (PA5-104160; 1:500 dilution) and mouse monoclonal anti-β-Tubulin clone AA2 (Sigma 05-661; 1:2500 dilution). Secondary antibodies: rabbit polyclonal anti-mouse Ig coupled to Horse Radish Peroxydase (HRP) (Dako; 1:1000 dilution), swine polyclonal anti-rabbit Ig coupled to HRP (Dako; 1:1000 dilution).

13 (FiguresFigure 1 -

 131 Figures

Table 1 -

 1 Semen parameters and sperm morphological defects (flagellum, head and acrosome) of the patient carrying a mutation in SLC9C1 gene 485 Five semen evaluations were performed for the patient over 3 years. Values are expressed in percent, unless specified otherwise. ND: not Lower or upper reference limits (5th centiles and their 95% confidence intervals) according to the World Health Organization (WHO) standards 488 and the distribution range of morphologically abnormal spermatozoa observed in fertile individuals[START_REF] Cooper | World Health Organization reference values for human semen characteristics* ‡[END_REF][START_REF] Auger | Another look at human sperm morphology[END_REF] ; in bold characters: abnormal values.

	486
	487	determined.
	489
	490

a

(57)(58)(59)(60)(61)(62)(63)

Acknowledgement

We thank all the individuals and their families for their cooperation, as well as all the referring physicians. We thank all the technicians from the Service de Biologie de la Reproduction at the Hospital Cochin (Paris) for routine semen sample evaluation (Jacques Bras, Nathalie Chériau, Véronique Hernandorena, Jean-Claude Cambronne and Caroline Villalon). We thank the Cellular Imaging Facility of the Institut Cochin for electron microscopy procedures.

Funding

This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Centre National de la Recherche Scientifique (CNRS), the Université Paris Descartes, the University Grenoble-Alpes, the French National Research Agency (Grant MASFLAGELLA ANR-14-CE15-0002 and FLAGEL-OME ANR-19-CE17-0014 to PR, NTM and AT).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author,

[AT], upon reasonable request.

Author's roles

AT designed the study. ED and CP recruited the patient, performed clinical analysis and characterization. AC and PS carried out the ICSI procedure for the patient and his partner. NTM, CC and PR performed exome and bioinformatics analysis. EC did experimental work (RT-PCR, Sequencing, Immunofluorescence, Immunoblotting, MO analysis), data quantification and analysis. AT did TEM analysis. MW, LS and PL contributed in setting up the experimental conditions. EC and AT performed data illustrations. EC and AT analysed the data and wrote the manuscript. MW, AC, PL, CC, CA, PR, NTM, CP and ED did critical reading of the manuscript. Immunofluorescence detection of SLC9C1 protein on mouse spermatozoa using the antibody directed against the central region of SLC9C1, co-staining with α-Tubulin (green). SLC9C1 (red) preferentially localises at the principal piece of the flagella. Scale bar represents 10μm.