Etienne Laborie

Victor Le-Minh

Thanh Duc Mai

Mehdi Ammar

Myriam Taverna

Claire Smadja
email: claire.smadja@universite-paris-saclay.fr

gAnalytical methods of antibody surface coverage and orientation on bio-functionalized magnetic beads: application to immunocapture of TNF-α

Keywords: magnetic beads, antibody, orientation, immunocapture, TNF-α, immunoassays

A rapid and simple approach to evaluate orientation and density of antibodies immobilized on magnetic beads

Introduction

Magnetic beads with their several advantages have found increasing applications in many fields including drug discovery, biomedicine, bioassays, diagnostics, genomics and proteomics [START_REF] Hou | Surface engineering of magnetic iron oxide nanoparticles by polymer grafting: Synthesis progress and biomedical applications[END_REF][START_REF] Kruszewska | How to effectively prepare a sample for bottom-up proteomic analysis of nanoparticle protein corona? A critical review[END_REF].

A typical application is peptides/proteins selective capture via magnetic separation using magnetic beads functionalized with a bio-receptor. Many recently developed diagnostic tools (e.g. lab-on-a-chip and biosensors) are based on bio-functionalized magnetic particles with antibodies [START_REF] Khizar | Magnetic nanoparticles in microfluidic and sensing: From transport to detection[END_REF]. For these purposes, several types of magnetic beads are currently available differing by their sizes, shape, surface chemistry and immobilization strategy that are crucial for antibodies grafting density and orientation. Preparation of magnetic nanoparticles with a stable surface remains of paramount importance. After the coating step bioreceptors such as antibodies will biofunctionalized the nanoparticles to target specific biomarkers for instance. A high antibody grafting density and a good orientation, which requires exposed and fully available F(ab)2 fragments on bead surface, are highly desirable to ensure high loading/capture of target analytes during the immuno-precipitation/enrichment or bioassay processes.

The monitoring of surface antibody density and orientation is therefore critical not only for capture or recognition efficiency but also to produce batches of functionalized magnetic beads of equal performance and quality.

Conventional methods to assess the antibody immobilization on beads are based either on spectrophotometric or biophysical methods. Colorimetric assays (e.g. Lowry, Bradford and BCA assays) have been shown to provide only indirect determination of protein grafting efficiency as they are mainly based on the determination of non-immobilized proteins present in the supernatant [START_REF] Liu | Protein Aand Protein G-gold nanoparticle bioconjugates as nano-immunoaffinity platform for human IgG depletion in plasma and antibody extraction from cell culture supernatant[END_REF] [START_REF] Zhang | Direct quantification of surface coverage of antibody in IgG-Gold nanoparticles conjugates[END_REF]. In addition, they do not provide any information on antibodies orientation. Besides, many biases have been reported with these methods mainly related to possible background interferences due to nanoparticles themselves [START_REF] Kozlowski | Characterizing the Surface Coverage of Protein-Gold Nanoparticle Bioconjugates[END_REF][START_REF] Walkey | Nanoparticle size and surface chemistry determine serum protein adsorption and macrophage uptake[END_REF][START_REF] Hinterwirth | Bioconjugation of trypsin onto gold nanoparticles: Effect of surface chemistry on bioactivity[END_REF]. Atomic force microscopy, surface plasmon resonance (SPR), spectroscopic ellipsometry and dual polarization interferometry [START_REF] Zhang | Direct quantification of surface coverage of antibody in IgG-Gold nanoparticles conjugates[END_REF][START_REF] Young | Study on orientation of immunoglobulin G on protein G layer[END_REF][START_REF] Song | Comparative study of random and oriented antibody immobilization as measured by dual polarization interferometry and surface plasmon resonance spectroscopy[END_REF][START_REF] Yoshimoto | Direct observation of adsorption-induced inactivation of antibody fragments surrounded by mixed-PEG layer on a gold surface[END_REF][START_REF] Fujiwara | Measurement of antibody binding to protein immobilized on gold nanoparticles by localized surface plasmon spectroscopy[END_REF] can provide indirect insight on antibodies orientation mainly by measuring antibodies dimensions/thickness on surfaces or shift angle. Recently, Lämmerhofer et al. proposed an approach for quantification of the protein surface coverage on gold nanoparticles using high performance liquid chromatography (HPLC) [START_REF] Liu | Protein Aand Protein G-gold nanoparticle bioconjugates as nano-immunoaffinity platform for human IgG depletion in plasma and antibody extraction from cell culture supernatant[END_REF][START_REF] Liu | Accurate and reliable quantification of the protein surface coverage on protein-functionalized nanoparticles[END_REF]. In this work, pepsin conjugated to gold nanoparticles is hydrolyzed (6N HCl). The released amino acids are tagged with a fluorescent dye and then analyzed by HPLC with fluorescence detection. The derivatized Glycine measurement is used to estimate the pepsin surface coverage on nanoparticles. Kozlowski et al. gave an estimate of surface coverage of dihydrofolate reductase (DHFR)-gold particle bioconjugates, using a combination of several techniques including SDS-PAGE, UV-vis spectrophotometry, dynamic light scattering and a fluorescence-based method [START_REF] Kozlowski | Characterizing the Surface Coverage of Protein-Gold Nanoparticle Bioconjugates[END_REF]. These methodologies allowed a fine protein surface coverage characterization but required several steps and a combination of several techniques. In another study relying on streptavidinmagnetic beads, Gagey-Eilstein et al. communicated a chemiluminescent test to monitor the antibody grafting rate [START_REF] Bouzas-Ramos | Controlling Ligand Surface Density on Streptavidin-Magnetic Particles by a Simple, Rapid, and Reliable Chemiluminescent Test[END_REF] and by sandwich-type immunoassay using magnetic beads grafted with antibodies have been also reported [START_REF] Näreoja | Kinetics of bioconjugate nanoparticle label binding in a sandwich-type immunoassay[END_REF][START_REF] Hua | Upconversion fluorescence immunoassay for imidaclothiz by magnetic nanoparticle separation[END_REF]. All these methods, however, did not provide information on the antibody-grafting orientation nor on the functionality and ability of antibodies to capture the biological target.

Herein we report the development of a method for quantification of antibody surface coverage and monitoring of antibody grafting orientation on magnetic beads functionalized with different functional groups or chemistries. Together with a recent publication by Shen et al [START_REF] Shen | Restricted Proteolysis and LC-MS/MS to Evaluate the Orientation of Surface-Immobilized Antibodies[END_REF], our study is one of the two pioneering works which shed light on both orientation and density of antibodies decorated on magnetic beads using chromatographic approaches. Shen et al proposed a method which used a proteolysis performed by trypsin-decorated magnetic beads to quantify the number of antibodies attached to magnetic beads. LC-MS/MS of the released tryptic peptides allowed to quantify representative peptides of either the F(ab)2 or Fc region of the antibody. However, it is important to note that the release of F(ab)2 peptides from antibodies could arise from antibodies attached via one Fab fragment leading thereby to an overestimation of the number of well oriented antibodies.

Our work relies on the use of a selective proteolysis of F(ab)2 regions of antibodies, using (IdeS) that cleaves antibodies with a unique degree of specificity in the hinge region leading to the release of Fc and F(ab)2 fragments [START_REF] Johansson | IdeS: A bacterial proteolytic enzyme with therapeutic potential[END_REF]. Our straightforward analytical approach relied on size exclusion liquid chromatography coupled to fluorescent detection (FLD) allowing F(ab)2 and Fc separation according to their molecular weight (100 and 50 kDa respectively) [START_REF] Shen | Restricted Proteolysis and LC-MS/MS to Evaluate the Orientation of Surface-Immobilized Antibodies[END_REF]. F(ab)2 and Fc will be clearly identified with this new approach. In our case the quantification of the ratio Fab / Fc is expected to be directly related to the orientation of immobilized antibodies. Indeed, after IdeS digestion, if the antibodies are grafted through their Fc fragment, F(ab)2 is released whereas when they are immobilized via Fab fragment, Fc fragment is released. The relevance and utility of the developed approach was demonstrated with a high diagnostic-value application, by quantifying the human Tumor Necrosis Factor alpha (TNF-α), a proinflammatory cytokine, serving for screening of Major Depressive Disorders (MDD) and which is involved in many inflammatory disorders such as cytokine release syndrome observed in COVID 19 [START_REF] Sasso | Continuous Monitoring of Inflammation Biomarkers During Simulated Cardiopulmonary Bypass Using a Microfluidic Immunoassay Device-A Pilot Study[END_REF][START_REF] Kumar | Recent advances in biosensors for diagnosis and detection of sepsis: A comprehensive review[END_REF][START_REF] Yndestad | Systemic inflammation in heart failure -The whys and wherefores[END_REF][START_REF] Russell | Associations between immune-suppressive and stimulating drugs and novel COVID-19-a systematic review of current evidence[END_REF][START_REF] Mehta | COVID-19: consider cytokine storm syndromes and immunosuppression[END_REF][START_REF] Slavich | From stress to inflammation and major depressive disorder: A social signal transduction theory of depression[END_REF][START_REF] Cizza | Elevated Neuroimmune Biomarkers in Sweat Patches and Plasma of Premenopausal Women with Major Depressive Disorder in Remission: The POWER Study[END_REF].

Experimental

Chemicals and reagents

IdeS FabRICATOR enzyme (5000 units) was purchased from Genovis AB (Lund, Sweden). HPLC-grade propan-2-ol and sodium di-hydrogen phosphate (NaH2PO4) were obtained from Fisher Scientific (Karlsruhe, Germany). Sodium chloride was provided from Euromedex (Souffelweyersheim, France). 32% sodium hydroxide was purchased from VWR SAS (Fontenay-sous-Bois, France). 85% phosphoric acid was obtained from Carlo Erba Reagents (Val-de-Reuil, France). EZ-Link Sulfo-NHS-LC-biotin kit and Dynabeads MyOne Carboxylated, Tosylactivated, Streptavidin T1, Pierce Protein G (1µm) magnetic beads and TNF alpha Human Uncoated ELISA Kit were purchased from Life Technologies SAS (Villebon-sur-Yvette, France). Human IgG from whole serum (10.2 mg/mL) and Amicon Ultra 0.5mL 50 kDa and 100 kDa filters for ultrafiltration were provided by Merck Millipore (Darmstadt, Germany). Phosphate buffer saline 10X (PBS), Tris-buffer saline 10X (TBS), boric acid, Tween 20, ammonium sulphate, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysulfosuccinimide sodium salt (Sulfo-NHS) were purchased from Sigma Aldrich (St. Louis, MO, USA).

Apparatus and Material

Size exclusion liquid chromatography experiments were performed using an Agilent 1260 UHPLC-1260 FLD detector (Xenon lamp) (Agilent Technologies, Santa Clara, CA, USA). An Agilent Bio SEC-3 column (3 µm particle size; 300Å pore structure; 300 mm length; 4.6 mm i.d.) was employed at temperature of 25 °C. The mobile phase (50 mM sodium phosphate, 150 mM NaCl and 10% isopropanol at pH 6.5) was isocratically pumped at 300 µL/min. Fluorescence detection (FLD) was performed at λex = 280 nm and λem = 340 nm. Data acquisition and instrument control were performed by Chemstation (Agilent).

Methods

2.3.1: Preparation of bio-functionalized magnetic beads

Model human IgG (hIgG) were grafted on beads following commercial protocols for streptavidin and protein G beads. For beads tosylactivated (Ts) and COOH the protocols have been adapted from the supplier ones). IgG concentration in the grafting solution was kept constant at 0.1 mg/mL for accurate comparison of beads grafting efficiency.

2.3.1.1: Carboxylated magnetic beads

100 µL of Dynabeads MyOne Carboxylated (10 mg/mL) were added to 4 LoBind Eppendorf vials of 1.5 mL and washed with 1 mL of 20 mM NaOH overnight on a rotating wheel (Tube rotator SB3, Stuart, UK) at 20 rpm and 4°C. Beads were then concentrated using a magnet and washed 3 times with 500 µL of PBS. Then, beads were resuspended in 120 µL of human IgG solution at 0.66 mg/mL in PBS. 200 µL of EDC (10 mg/mL) and 200 µL of Sulfo-NHS (10 mg/mL) in PBS were added to the beads suspension. Finally, volume was adjusted to 800 µL with PBS, reaching a final concentration of 0.1 mg/mL of IgG for 1 mg of beads. Vials were then placed on a rotating wheel at 20 rpm overnight at 4°C for antibody grafting and beads were immobilized on a magnet and washed 2 times with 500 µL of a solution of PBS, Tween 20 (0.05%) (PBS-Tween) and one time with 500 µL of PBS. Washing solutions were kept at 4°C until analysis.

2.3.1.2: Tosylactivated magnetic beads

10 µL of Dynabeads MyOne Tosylactivated (100 mg/mL) were added to 4 LoBind Eppendorf vials of 1.5 mL and washed with 500µL of 0.1 M sodium borate buffer pH 9.5, gently vortexed and stirred at the same time using an Eppendorf Thermomixer for 15 min C at 25°C and 650 rpm. Beads were resuspended in 60 µL of hIgG solution at 0.66 mg/mL in PBS with 60 µL of 3M ammonium borate buffer pH 9.5 and 20 µL of 0.1M sodium borate buffer. Vials containing beads were then incubated at 37°C and stirred at 650 rpm on a Thermomixer for 16h and then sonicated for 5 minutes to prevent their adsorption on the vial's walls. Beads are were immobilized on a magnet and washed 2 times with 500 µL of a solution of PBS-Tween, and one time with 500 µL of PBS. Washing solutions were kept at 4°C until their analysis.

2.3.1.3: Streptavidin magnetic beads

Biotinylation of human IgG was done using EZ-link sulfo-NHS-LC-biotin kit. A 10 mM solution of biotin was prepared by adding 224µL of ultrapure water to 1 mg of biotin. 6.66 µL of biotin solution was added to 1000 µL of hIgG solution (0.5 mg/mL) and incubated at 25°C for 30 minutes and stirred at 300 rpm on a Thermomixer at the same time. The solution was then ultrafiltrated on a 50 kDa filter cap at 4°C, 10 000 rpm for 4 minutes to get rid of biotin salts excess. 100µL of Dynabeads MyOne Streptavidin T1 (10 mg/mL) are added to 4 LoBind Eppendorf vials of 1.5 mL and washed with 1 mL of PBS-Tween solution. Beads were then immobilized on a magnet and washed 3 times with 500 µL of PBS-Tween. Beads were then resuspended in 40 µL of biotinylated hIgG solution at 0.5mg/mL and 160 µL of PBS. Vials are were then incubated at 25°C, 450 rpm for 30 minutes on a Thermomixer and beads are were immobilized on a magnet and supernatants are collected and kept at 4°C. Beads were washed 4 times with 500 µL of a solution of PBS Tween Washing solutions were kept at 4°C.

2.3.1.4: Protein G magnetic beads

100µL of Pierce Protein G Magnetic Beads (10 mg/mL) were added to 4 LoBind Eppendorf vials of 1.5 mL and washed with 500 µL of Tris-Tween solution and gently vortexed. The process was repeated one time with 1 mL of Tris-Tween. Beads were resuspended in 217 µL of human IgG solution at 0.23 mg/mL with 283 µL of Tris-Tween. Vials were then incubated at 25°C, 650 rpm for 1 hour on a Thermomixer and beads were immobilized on a magnet and supernatants were collected and kept in the fridge for analysis. Beads were then washed 2 times with 500 µL with Tris-Tween. Washing solutions were kept in the fridge for analysis.

Characterization of the hIgG grafting

Enzymatic digestion of grafted antibodies

For all digestions, 2µL aliquot of 100 U of IdeS FabRICATOR enzyme and 50 µL of phosphate buffer (100 mM, pH 7.0) were added to the sample, hIgG solution (60 µL) or hIgG grafted on magnetic beads (suspended) and incubated for 1h30 at 37°C 300 rpm on a Thermomixer. Beads suspensions were then placed on a magnet and supernatants were collected and analyzed.

Influence of beads grafting conditions on FLD signal for hIgG, F(ab)2 and Fc fragments

To avoid any bias in the measurements, the influence of beads grafting protocols (medium, incubation steps) on hIgG FLD signals was investigated. Each sample was prepared in duplicate. hIgG samples at 0.1 mg/mL were incubated following conjugation protocols corresponding to tosylactivated carboxylic and protein G beads and then analyzed by SEC-FLD.

Calibration curves hIgG

Calibration curve for whole hIgG was obtained with a 0.1 mg/mL hIgG solution in PBS.

Dilution series of hIgG, ranging from 0.05 µg to 0.7 µg, have been injected analyzed to establish the calibration curve.

hIgG fragments

Calibration samples were made from IgG stock solution submitted to the different conjugation's protocols. hIgG solutions (0.23 mg/mL) were digested with 100 U of IdeS and analyzed with by SEC-FLD. Volumes of hIgG digests from 0.5 µL to 7 µL were analyzed and a calibration curve per conjugation protocol has been built for the two fragments (F(ab)2 and Fc).

2.3.2.4: Quantitation of non-grafted hIgG in the supernatant

Antibodies remaining in the supernatant after the grafting step were first quantified. Then, washing solutions were concentrated by ultrafiltration on 100 kDa membrane. 500 µL of washing solution was added to the filter cap and centrifuge for 4 minutes at 10 000 rpm at 4°C on a Centrifuged MicroStar 17R (VWR). The process was repeated until all the solution was filtered. Retentates volumes were measured before SEC-FLD analysis.

Immuno-capture of TNF-α by biofunctionalized magnetic beads

1 mg of COOH, Ts and Protein G magnetic beads were grafted with anti-human-TNF-α monoclonal antibodies from the Invitrogen ELISA kit for human TNF-α following the procedure described previously. Five replicates of 100µg of grafted magnetic beads of each type were then incubated for 4 hours at 25°C with 5 ng of human TNF-α in 250µL of PBS (1X).

Three blank samples were also prepared using grafted magnetic beads and PBS (1X). Beads were washed with 500µL of PBS-Tween for 10 minutes at 800 rpm on a Thermomixer. Then, heated 95°C for 5 minutes in 250µL of PBS (1X) for thermal elution of TNF-α. Eluates were diluted on PBS (1X, 10X, 100X) to match immunoassay detection range. Human TNF-α contents of non-diluted and diluted eluates were quantified using Invitrogen ELISA kit for human TNF-α using duplicates. Calibration curves were built by preparing serial dilution of human TNF-α solution at 500 pg/mL with and without thermal treatment at 95°C for 5 minutes to take into account the thermal effect on TNF-α.

Results and Discussion

The first part of the study was dedicated to the development of an analytical method to evaluate the grafting efficiency on four types of beads. Two main criteria have been considered: number of antibodies grafted on the bead's surface and their orientation. For this purpose, we specifically cleaved human hIgG grafted on the beads surface under the hinge region with an enzyme, IdeS, and analyzed the digestion products by SEC-FLD analysis. If the hIgG is immobilized via its Fc region, the F(ab)2 fragment will be released in the supernatant whereas for immobilization through F(ab)2 fragment, Fc fragment will be obtained.

Influence of the grafting conditions on native and digested human IgG fluorescent detection

We first developed a SEC-Fluorescent detection (FLD) method to quantify hIgG in solution.

Analysis of human IgG in solution (0.1 mg/mL) showed two peaks (Figure 1A): the first one corresponds to a dimeric form of hIgG (~8.2 min) and the second to the monomeric hIgG (~9.2 min). As the first peak area was very small compared to that of the 2 nd one (peak area ratio: ~8), dimeric hIgG was not considered for hIgG calibration. Repeatability and intermediate precision of SEC-FLD method have been evaluated. The intra (n=3) and inter-day (n=3) RSD of the retention times (Tr) were less than 0.1%. Resolution and limit of quantification were also evaluated at 1.3 and 160 nM respectively (see Supplementary Material, method validation chapter).

Considering the antibodies grafting density, it is important to note that for each type of beads, the grafting protocol differs in terms of buffers (i.e. pH and ionic strength) thermal and/or agitation that may impact protein conformation and thereby fluorescent intensities measured.

Therefore, hIgG free in solution (0.1 mg/mL) were submitted to experimental conditions mimicking the grafting protocols applied to COOH and Ts beads. The agitation used for hIgG grafting on COOH beads (rotating wheel at 4 °C overnight) led to a slight decrease of the FLD signal (-5,2 %) compared to the reference sample of hIgG (PBS at 4 °C). The FLD signal further decrease (19%) when EDC and Sulfo-NHS were added in the reaction medium. To explain this decrease, we hypothesized that the temperature (4 °C) as well as EDC and Sulfo-NHS could increase the intra amino acids interactions favoring thereby conformation where fluorescent amino acids are hindered. For Ts beads, an increase of the FLD signal (+9,6 %) was observed with the Ts grafting protocol (stirring at 37 °C during 16h). This signal is increased by 18 % when Ts conjugation protocol is performed in borate buffer pH 9.5 (Figure 1B). The increase of the fluorescent signal could be related to a partial denaturation of the hIgG. Several studies have demonstrated that thermally induced unfolding of proteins led to the exposure of the fluorescent amino previously buried (e.g. tryptophan) increasing thereby the fluorescent signal [START_REF] Brader | Examination of thermal unfolding and aggregation profiles of a series of developable therapeutic monoclonal antibodies[END_REF]. The same type of experiments has been performed for hIgG cleaved by IdeS. Digested IgG (0.22 mg/mL) has been first analyzed with SEC-FLD, showing two peaks: 1 st peak (~9.99 min) corresponding to Fab2 fragment of 100 kDa; 2 nd peak (~10,98 min) to Fc one (50 kDa) (Figure 2). Four calibration curves of hIgG cleaved by IdeS into Fab2 (~100 kDa) and Fc (~50 kDa) fragments have been performed under the grafting conditions selected for the 4 types of beads (figure 3). A variation has been observed for Fc peaks: a 25% decrease of the curve's slope for COOH beads conditions compared to Ts beads which could lead to underestimate an unfavorable orientation (see Figure 3A andB). Therefore, we can conclude from these results that calibration curves of hIgG and its fragments have to be established for each type of beads studied (i.e. COOH, Ts, streptavidin, protein G) to evaluate accurately the antibodies grafting density and orientation.

Antibodies grafting density and orientation on magnetic microbeads

We then evaluated the grafting density and hIgG orientation on the four types of beads: carboxylated, tosylated, protein G and streptavidin. For this purpose, magnetic beads bearing hIgG have been digested by IdeS and the fragments released analyzed by SEC-FLD.

F(ab)2 and Fc quantitation.

We first verified that IdeS did not overlap with peaks corresponding to hIgG fragments in our experimental conditions by analysing IdeS (5 U) as a control. Indeed, molecular weight of Ides is approximately 37 kDa wich might interfere with hIgG fragments detection. For IdeS, two peaks were observed at 10.08 min (dimer) and 11.0 min (monomer) with retention times close to F(ab)2 and Fc ones which could lead to overestimate peak areas related to Fc and, into a lesser extent, to Fab2 (see Figure S1 in Electronic Supplementary Material). Therefore, to accurately quantify the amount of F(ab)2 and Fc released after the digestion of hIgG grafted on magnetic beads, a calibration curve for IdeS (from 1 U to 14 U) was drawn using different injection S1, Supplementary material).

Grafting density and hIgG orientation on the different types of beads

To estimate the percentage of hIgG grafted (%), washing fractions and supernatants were analyzed and Equation 1 was used to estimate this value:

% 𝑔𝑟𝑎𝑓𝑡𝑒𝑑 = 𝑚 𝑖 -(𝑚 𝑤𝑎𝑠ℎ𝑖𝑛𝑔 + 𝑚 𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡 ) 𝑚 𝑖 𝐸𝑞. 1
where mi is the initial quantity of hIgG per mg of beads before grafting, mwashing and msupernatant are the quantity of hIgG found after the grafting step in washing solutions and supernatants respectively. A calibration curve of non-digested hIgG in solution in PBS was performed to quantify remaining hIgG in the supernatant and washing solution (see Figure S2 in Supplementary Material).

From the percentage values evaluated for the different beads (Figure 4A), we can conclude that protein G beads can graft more IgG than other beads (95.1% of injected IgG are grafted), with COOH beads being the less efficient with a lower density of IgG grafted on their surface (73.8%).

The number of hIgG per bead has been also evaluated by considering the average mass of an hIgG (150 kDa), the bead's density (1.8 g/cm 3 for Ts, COOH and streptavidin beads, 2.0 g/cm 3 for protein G beads) and average diameter of the beads (1 µm), using the following equation:

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝐼𝑔𝐺 𝑚𝑜𝑙𝑒𝑐𝑢𝑙𝑒𝑠 𝑝𝑒𝑟 𝑏𝑒𝑎𝑑 = % 𝑔𝑟𝑎𝑓𝑡𝑒𝑑 ×𝑚 𝑖 𝑚 𝐼𝑔𝐺 × 𝑚 𝑏𝑒𝑎𝑑 1 𝑚𝑔 𝑜𝑓 𝑏𝑒𝑎𝑑𝑠 (Eq.2)
where %grafted is the proportion of grafted hIgG on the beads, mi the initial mass (µg) of hIgG incubated with 1 mg of beads for grafting, mIgG the mass (µg) of one hIgG molecule, mbead the mass (µg) of the corresponding bead.

According to Figure 4B, protein G beads grafted a high number of hIgG (~70 000 molecules per bead), followed by Ts beads (~56 000 IgG molecules per bead), streptavidin beads (~25 000 molecules) and finally COOH ones (~12 000). We can note that despite a higher quantity of hIgG employed for carboxylic beads (79 µg) compared to protein G (49.91 µg) the amount of hIgG grafted is better for proteins G beads. The same observation can be made for Ts beads, where better grafting compared to carboxylic beads is observed despite the smaller concentration of hIgG used (39,6 µg). These results suggest that the grafting efficiency is not only related to the amount of hIgG in the immobilization solution but also to the surface chemistry.

The hIgG orientation has been evaluated by IdeS digestion and the analysis of released fragments. Figure 5A displays the different chromatograms obtained for COOH, Ts, streptavidin and protein G beads. The peak areas and notably Fab2/Fc ratio highlighted the differences between beads in terms of number of hIgG grafted and hIgG orientations. From Figure 5A, we have observed that protein G beads lead to a larger peak of F(ab)2 compared to other beads. The orientation ratio (Fab2/(Fab2+Fc) showed that protein G beads also exhibit the best orientation (~73.8% of antibodies grafted by their Fc fragment) (Figure 5B) compared to Ts (31.3%), streptavidin (9.9%) and carboxylic beads (5.2%). We can conclude from these results that protein G led not only to more grafted antibodies but also to a higher proportion of well orientated immobilized hIgG. This could be due to the surface chemistry of carboxylic and Ts beads that led to a random immobilization of hIgG via amino (carboxylic and Ts) and sulfhydryl groups (Ts). Therefore, only a fraction of the F(ab)2 binding sites will be potentially available for biorecognition. The better orientation ratio observed for Ts compared to carboxylic beads could be related to the hydrophobic surface of tosyl beads favoring hIgG orientation through the more hydrophobic Fc region. In contrast, for protein G, immobilization through Fc fragment allow a better orientation with a higher proportion of F(ab)2 available.

IdeS digestion efficiency on the magnetic beads

To ensure that the better peak area ratio Fab2/Fc observed in Protein G was related to a better orientation and not to a higher enzymatic digestion efficiency, IdeS digestion yield has been estimated (Equation 3).

% 𝑑𝑖𝑔𝑒𝑠𝑡𝑒𝑑 = 𝑚 𝑑𝑖𝑔𝑒𝑠𝑡𝑒𝑑 𝑚 𝑖 -(𝑚 𝑤𝑎𝑠ℎ𝑖𝑛𝑔 + 𝑚 𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡 ) 𝐸𝑞. 3
Digestion efficiency %digested introduced in Eq.3 is directly correlated to biologically available hIgG, where mi is the initial quantity of hIgG per mg of beads before grafting, mwashing (non-covalent) for Streptavidin beads [START_REF] Foddai | Maximizing capture efficiency and specificity of magnetic separation for mycobacterium avium subsp. paratuberculosis cells[END_REF]. In addition, Streptavidin beads rely on a biotinylation procedure prior to antibody grafting on beads. This additional step may decrease the efficiency of the whole conjugation procedure as biotinylation will not be total. Finally, protein G beads offer the best results in terms of antibody orientation and density on the surface. Moreover, they are particularly easy to graft with ~1h30 to complete the whole procedure compared to 18h for Ts and 30h for COOH beads, which makes them the best candidate for antibody grafting and immunocapture.

Concerning ProtA/ProtG beads, these results are in agreement with Shen et al [START_REF] Shen | Restricted Proteolysis and LC-MS/MS to Evaluate the Orientation of Surface-Immobilized Antibodies[END_REF] in terms of antibody coverage and orientation ratio. In contrast, for carboxylated beads, a high difference is observed with our study. This discrepancy could be related to the digestion tool employed. Indeed, with trypsin digestion, employed by Shen et al., the peptides released from the F(ab)2 region, detected and quantified by LC-MS, could arise from antibodies attached on beads via one Fab fragment. This could lead therefore to an overestimation of the orientation ratio for COOH beads where antibodies are randomly immobilized. On Prot G/A beads, where antibodies are immobilized via Fc fragments and thereby well oriented. Our method combining IdeS digestion with SEC analysis offers size discrimination making it possible to retrieve more information to identify the fragment released (i.e. Fc (50 kDa) vs F(ab)2 (100 kDa).

We have therefore developed a direct accurate and rapid quantification method of bioavailable hIgG on magnetic beads. Our study takes into account beads grafting conditions and IdeS contribution effect on FLD signal and absorption on beads for a more accurate quantification. Finally, we compared the performance of COOH, Ts and protein G beads for capturing TNF-α, a cytokine involved in Major Depressive Disorders (MDD) and other inflammatory-related pathologies. Streptavidin beads were not considered in the study as they did not provide added value compared to carboxylated and tosylactivated beads.

Application for immunocapture of TNF-α: towards quantification of biomarkers in biological fluids

To illustrate the performance of the previously studied magnetic beads, COOH, Ts and protein G beads were grafted with monoclonal anti-human-TNF-α antibodies following the

Conclusion

In the present study, we present an original analytical method for the evaluation of human IgG grafting efficiency on 4 types of commercial magnetic beads for immunocapture applications. This method relies on an innovative combination of a highly specific digestion of hIgG by IdeS together with the high resolution of the fragments' analysis by SEC, offering an efficient determination of antibodies orientation. This work offers a comprehensive approach of the influence of grafting conditions, IdeS and bead's surface chemistry on fluorescent signal detection. All results support the high efficiency of protein G magnetic beads for a high grafting yield of human IgG (~70 000 IgG molecules per bead) and an optimal orientation of antibodies for antigen capture (75% of Fab2 fragment free). Moreover, protein G beads ease-of-use in terms of grafting conditions and time make them a powerful and convenient tool for biosensing and lab-on-a-chip applications. These findings have been illustrated with a high-value application for the immunocapture and elution of human TNF-α at 20 ng/mL, towards the monitoring of inflammatory-related pathologies. This work will be extended to other cytokines. Finally, this method can be more generally used to assess the hIgG coverage and orientation on any type of surface, relying on the high specificity and efficiency of the IdeS digestion of hIgG.

Figures captions

Fig. 1 Analysis of hIgG (0.1 mg/mL) by SEC-FLD: (A) control sample (human IgG in PBS at 4°C), (B) Influence of bead grafting conditions on FLD signal of human IgG. COOH conditions are agitation at 20 rpm on rotating wheel at 4°C overnight in PBS, with 10 mg/mL of EDC and Sulfo-NHS. Ts conditions are agitation a 650 rpm on Thermomixer at 37°C for 16h with borate and ammonium buffer pH 9.5.

Mobile phase was 50 mM sodium phosphate, 150 mM NaCl and 10% isopropanol pH 6.5.

Column: BioSEC-3, (4.6 mm × 300 mm, 300 Å, particle diameter 3 µm); Flow rate: 300 µL/min; FLD: ex=280 nm, em=340 nm Fig. 2 SEC-FLD analysis of human IgG at 0.22 mg/mL in PBS after IdeS digestion.

Experimental conditions as mentioned in

  volumes (see Fig S2, Electronic Supplementary Material) and was used to evaluate of IdeS contribution in Fab2 and Fc peak areas values. Therefore, peak areas of F(ab)2 and Fc fragments were corrected with IdeS calibration in the whole study allowing a more accurate quantification (see Table

Fig. 3

 3 Fig. 3 Calibration curves corresponding to grafting conditions, A) Fab2 fragment, B) Fc

Fig. 4 Fig. 5

 45 Fig. 4 hIgG immobilization study on beads: (A) grafting efficiency, (B) number of hIgG

Fig. 6 1 Fig. 3

 613 Fig. 6 Comparison of IdeS digestion efficiency for COOH, Ts, Streptavidin and Protein G magnetic beads

Fig. 6

 6 Fig. 6 Comparison of IdeS digestion efficiency for COOH, Ts, Streptavidin and Protein G magnetic beads

Table 1

 1 Comparison of beads efficiency for immunocapture of TNF-α (n=5 replicates per bead)

395:1033-1034

funding, project supervision, writing -original draft. We thank the "Investissement d'Avenir" program, through the "IDI 2017" project funded by the IDEX Paris-Saclay, ANR-11-IDEX-0003-02 which provided the financial support of Etienne Laborie as a fellowship, LabeX LaSIPS (ANR-10-LABX-0040-LaSIPS) and LabeX NanoSaclay, (ANR-10-LABX-0035). We thank the Doctoral school of Chemical Sciences of University Paris Saclay which provided the financial support of Victor Le-Minh as a fellowship Availability of data and material Not applicable Code availability Not applicable

Abstract

The use of magnetic beads bio-functionalized by antibodies (Ab), is constantly increasing with a wide range of biomedical applications. However, despite an urgent need for current methods to monitor Ab's grafting process and orientation, existing methods are still either cumbersome and/or limited.

In this work, we propose a new simple and rapid analytical approach to evaluate antibody orientation and density on magnetic beads. This approach relies on the cleavage by IdeS, a highly specific protease for human immunoglobulin G (hIgG), of immobilized antibodies. The F(ab)2 and Fc fragments could be then accurately quantified by Size Exclusion Chromatography (SEC)-coupled to fluorescent detection (FLD) and the ratio of these fragments was used to give insight on the IgG orientation at the bead surface. Four different commercially available magnetic beads, bearing carboxyl groups, tosyl groups, streptavidin or protein G on their surface have been used in this study. Results obtained showed that this approach ensures reliable information on hIgG orientation and beads surface coverage. Protein G magnetic beads demonstrated an optimal orientation of antibodies for antigen capture (75% of accessible Fab2 fragment) compared to tosylactivated, carboxylated and streptavidin ones. Capture efficiency of the different functionalized beads toward human TNF-α immunocapture, a biomarker of inflammation, has been also compared. Protein G beads provided a more efficient capture compared to other beads. In the future, this approach could be applied to any type of surface and beads to assess the hIgG coverage and orientation after any type of immobilization. and msupernatant are the quantity of hIgG found in washing liquids and supernatants and mdigested is the quantity of hIgG found in the digestate after IdeS digestion. Indeed, we considered that IdeS cannot fully digest every hIgG molecule grafted on the bead, which means that this method could give an underestimated quantification of hIgG available to IdeS molecules. Being a relatively large molecule (~37kDa), IdeS may not access easily to the hinge region of some hIgG molecules within the antibody layers (or multilayers), resulting in a partial proteolysis yield. Assuming that these unavailable hIgG molecules would not be available for other reactions with biomolecules (such as antigen recognition and capture), we can suppose that the digestion yield also reflects the antigen capture potential of the beads.

The results showed that IdeS digestion yield is higher for tosylactivated beads (46.3%) than protein G (34,2%) and streptavidin (37,5%). Unexpectedly, we found a very low digestion yield for COOH beads (5.6%), as depicted in Figure 6.

The lower digestion yield for protein G beads compared to the tosylactivated beads is probably related to the higher grafting density leading thereby to a steric hindrance detrimental for IdeS biological activity. Considering the better ratio Fab2/(Fab2+Fc) and the lower digestion yield of IdeS for protein G beads, these results confirm the better orientation of hIgG on this kind of beads.

Therefore, the overall results showed that protein G beads exhibit the best orientation ratio as well as the best grafting density. This could be explained by their ability to capture hIgG through Fc fragment, leading thereby to a more favorable orientation [START_REF] Choe | Fc-binding ligands of immunoglobulin G: An overview of high affinity proteins and peptides[END_REF]. In contrast, hIgG are randomly immobilized by covalent bonding for COOH and Ts beads and via Biotin interactions procedure previously described. 5 ng of human TNF-α were then incubated for 4 hours at 25 °C with 100 µg of grafted magnetic beads of each type. Elution of antigens was done using thermal elution in PBS at 95°C for 5 minutes in order to maximize the amount of antigen release and minimize protein denaturation [START_REF] Mai | Magneto-immunocapture with on-bead fluorescent labeling of amyloid-β peptides: towards a microfluidized-bed-based operation[END_REF]. Eluates were then prepared in three dilutions: non diluted, ten and one hundredfold diluted to ensure that recovered antigen concentration matched the immunoassay's linear range. Quantification was carried out using an ELISA kit for human TNF-α on microplate by absorbance spectrometry. A calibration curve was built using human TNF-α standard solution. The standard was also treated at 95°C for 5 minutes in order to reflect possible conformational changes of TNF-α that would affect immunoassay detection. As shown in Figure S3, thermal treatment influenced calibration curve slope with a 40% decrease in slope after thermal treatment. Quantification of eluates from the beads was therefore performed using the thermal treatment calibration curve. Results are shown in Table 1.

For 5.0 ng of human TNF-α incubated with 100µg of beads, 67.6% of TNF-α was recovered with protein G beads, but only 6.6% and 2.8% for COOH and Ts beads respectively. These results support the high antibody grafting efficiency of protein G beads in terms of both antibody coverage and orientation. For COOH and Ts beads, they suggest a very low capture ability of these beads for this application probably due to poor orientation and/or antibody density. However, it should be noted that thermal elution does not necessarily elute all the captured antigens. Nevertheless, it is still the mildest procedure for antigen elution without causing full protein denaturation (from acidic media) that would render accurate quantification with ELISA impossible. These results are in agreement with our SEC FLD study highlighting the better grafting and orientation of hIgG on protein G magnetic beads. They are therefore promising candidates for sensitive and specific detection of biomarkers like TNF-α and could be extended to other cytokines or proteins.

Table 1 Comparison of beads efficiency for immunocapture of TNF-α (n=5 replicates per bead) Fig. 5 IdeS digestion of Immobilized hIgG: A) SEC -FLD Analysis, B) Evaluation of the percentage of hIgG orientated with F(ab)2 free on the beads surface. Experimental conditions as Figure 1


Jocelyn Balansa

Abdeltif Lahfid

Jean-Claude Hippolyte

Nicolas Espurt

Pierre Henry

Séverine Caritg

Bertrand Fasentieux

Unraveling the eroded units of mountain belts using RSCM thermometry

Keywords: RSCM thermometry, Maximum temperature (T RSCM ), Balanced cross-section, Nappes, Burial

come

Introduction

Orogenic wedges result from progressive horizontal transport and stacking of structural units formed by nappes and foreland basins [START_REF] Elliott | Structural evolution in the northern part of the Moine thrust Zone, NW Scotland[END_REF][START_REF] Hardebol | Burial and temperature evolution in thrust belt systems: Sedimentary and thrust sheet loading in the SE Canadian Cordillera[END_REF][START_REF] Rosenberg | Relating orogen width to shortening, erosion, and exhumation during Alpine collision[END_REF]. The uppermost structural units of these stacks may have been totally removed by erosion in the orogens [START_REF] Malavieille | Impact of erosion, sedimentation, and structural heritage on the structure and kinematics of orogenic wedges: Analog models and case studies[END_REF]. A challenge is to quantify the volume of these missing units. The Alps represent a prime example of a modern collisional orogen whose structural evolution is still under debate (e.g., [START_REF] Dumont | Crosspropagation of the western Alpine orogen from early to late deformation stages: Evidence from the Internal Zones and implications for restoration[END_REF] and references therein). The Alpine orogen results from the subduction of the Eurasian continental margin (the Briançonnais domain) and the Piemont-Liguria Ocean beneath the Apulia microplate during the Cenozoic era [START_REF] Kerckhove | Tectonique de soubassement parautochtone des nappes de l'Embrunais-Ubaye sur leur bordure occidentale, du Drac au Verdon[END_REF]Debelmas et al. 1983a;[START_REF] Michard | The high-pressure metamorphic front of the south Western Alps (Ubaye-Maira transect, France, Italy)[END_REF][START_REF] Dumont | Structural and sedimentary records of the Oligocene revolution in the Western Alpine arc[END_REF][START_REF] Butler | Area balancing as a test of models for the deep structure of mountain belts, with specific reference to the Alps[END_REF][START_REF] Van Hinsbergen | Orogenic architecture of the Mediterranean region and kinematic reconstruction of its tectonic evolution since the Triassic[END_REF]). The Alpine thrust wedge is characterized by far-travelled stacked nappes that propagated toward the European foreland [START_REF] Kerckhove | Tectonique de soubassement parautochtone des nappes de l'Embrunais-Ubaye sur leur bordure occidentale, du Drac au Verdon[END_REF][START_REF] Bellahsen | Collision kinematics in the western external Alps[END_REF][START_REF] Rosenberg | Relating orogen width to shortening, erosion, and exhumation during Alpine collision[END_REF].

In the southwestern part of the Alpine wedge (Fig. 1), previous studies tried to constrain the thickness of the eroded nappes from regional balanced and restored cross-sections (Debelmas et al. 1983a;[START_REF] Lickorish | Sequential restoration of the external Alpine Digne thrust system, SE France, constrained by kinematic data and synorogenic sediments[END_REF][START_REF] Graham | Allochthonous salt in the sub-Alpine fold-thrust belt of Haute Provence, France[END_REF] or thermal data [START_REF] Corsini | Alpine and late-hercynian geochronological constraints in the Argentera Massif (Western Alps)[END_REF][START_REF] Bigot-Cormier | The denudation history of the Argentera Alpine External Crystalline Massif (Western Alps, France-Italy): an overview from the analysis of fission tracks in apatites and zircons[END_REF][START_REF] Labaume | Tectonic control on diagenesis in a foreland basin: combined petrologic and thermochronologic approaches in the Grès d'Annot basin (Late Eocene-Early Oligocene, French-Italian external Alps)[END_REF][START_REF] Cavailhes | Architecture et propriétées pétrophysiques des zones de failles dans une série gréso-pélitique profondément enfouie : rôle de la déformation et des interactions fluide-roche. Exemple des Grés d'Annot[END_REF][START_REF] Bellanger | Shortening of the European Dauphinois margin (Oisans Massif, Western Alps): New insights from RSCM maximum temperature estimates and 40 Ar/ 39 Ar in situ dating[END_REF][START_REF] Rosenberg | Distribution, style, amount of collisional shortening, and their link to Barrovian metamorphism in the European Alps[END_REF]). These structural and thermal studies show strong variations in the restored nappe stack geometry and shortening. It raises the following questions: Where was the front of the nappes before erosion, and what was the thickness of these nappes? Here we study and reconstruct the thermal and tectonic history of the Subalpine Digne Nappe by combining maximum temperature data (T RSCM ), assessed through Raman Spectroscopy of Carbonaceous Material (RSCM) thermometry with cross-section balancing. The goal of this study is to reliably constrain the nature and geometry of the eroded structural units of the southwestern Alpine wedge and to quantify the burial of the sedimentary pile of the Digne Nappe. To illustrate the structure and kinematics of the southwestern Alpine nappe stack, we built a 65-km-long crustal-scale balanced cross-section across the northern edge of the Valensole basin, the Digne Nappe and the Embrunais-Ubaye nappes (Digne-Barcelonnette transect; Figs. 2,3). We used geological and structural information from published geological maps and cross-sections [START_REF] Goguel | Duplicatures de la crête de la Blanche près Seyne (Basses-Alpes)[END_REF][START_REF] Kerckhove | La Zone du flysch dans les nappes de l'Embrunais-Ubaye (Alpes Occidentales)[END_REF][START_REF] Gigot | Evolution tectono-sédimentaire de la bordure septentrionale du bassin tertiaire de Digne depuis l'Eocène[END_REF][START_REF] Kerckhove | Tectonique de soubassement parautochtone des nappes de l'Embrunais-Ubaye sur leur bordure occidentale, du Drac au Verdon[END_REF][START_REF] Arlhac | Carte géologique de la France (1/50000)[END_REF]Debelmas et al., 1983a;Haccard et al. 1989a;[START_REF] Gidon | Relations entre le charriage de la Nappe de Digne et la structure de son autochtone dans la vallée du Bès (Alpes de Haute-Provence, France)[END_REF]Balansa et al. 2022), and new field data.

Structure and kinematics of the southwestern Alpine nappe stack

The frontal thrust, the Subalpine Digne Nappe, is composed of a Mesozoic (upper Triassic to upper Cretaceous) sedimentary pile thickening northeastward from 2.2-to 4.5-kmthick (Digne basin), unconformably covered locally by up to 1.2-km-thick Eocene-Oligocene strata (composed of Lutetian "Argens conglomerates", upper Eocene limestones and blue marls, lower Oligocene "Grès d'Annot", and lower Oligocene "Schistes à Blocs" toward the hinterland; Figs. 3, 4, 5;[START_REF] Campredon | Les formations paléogènes des Alpes maritimes franco-italiennes[END_REF][START_REF] Kerckhove | Tectonique de soubassement parautochtone des nappes de l'Embrunais-Ubaye sur leur bordure occidentale, du Drac au Verdon[END_REF][START_REF] Arlhac | Carte géologique de la France (1/50000)[END_REF][START_REF] Rousset | Raman microspectroscopy of soot and related carbonaceous materials: Spectral analysis and structural information[END_REF][START_REF] Ravenne | Sedimentation and Tectonics in the Upper Eocene-Oligocene Marine Basin in the Southern Alps[END_REF]Haccard et al. 1989a;[START_REF] Sinclair | Tectono-stratigraphic model for underfilled peripheral foreland basins: an alpine perspective[END_REF][START_REF] Ford | Tertiary foreland sedimentation in the Southern Subalpine Chains, SE France: a geodynamic appraisal[END_REF][START_REF] Joseph | Dynamic Controls on Sedimentology and Reservoir Architecture in the Alpine Foreland Basin. A Field Guide to the Eocene-Oligocene Grès d'Annot Turbidite System of the SE France[END_REF]. The inner part of the Digne Nappe, outcropping within the Barcelonnette window, reaches a maximum thickness of 2 km (Fig. 3; [START_REF] Kerckhove | La Zone du flysch dans les nappes de l'Embrunais-Ubaye (Alpes Occidentales)[END_REF]Debelmas et al. 1983a). The nappe is regionally detached southwesterly above middle-upper Triassic evaporites [START_REF] Gidon | Relations entre le charriage de la Nappe de Digne et la structure de son autochtone dans la vallée du Bès (Alpes de Haute-Provence, France)[END_REF]. Shortening value reaches 22 km on its frontal part (Balansa et al. 2022). Lying at the footwall of the Digne Nappe, the Barles paraautochthonous unit involves a much thinner "Provençal" upper Carboniferous to lower Cretaceous sequence unconformably overlain by Cenozoic sediments filling the Valensole foreland basin [START_REF] Gidon | Relations entre le charriage de la Nappe de Digne et la structure de son autochtone dans la vallée du Bès (Alpes de Haute-Provence, France)[END_REF][START_REF] Hippolyte | Messinian-Zanclean canyons in the Digne nappe (southwestern Alps): tectonic implications[END_REF][START_REF] Schwartz | Foreland exhumation controlled by crustal thickening in the Western Alps[END_REF]Balansa et al. 2022). The Barles para-autochthonous unit and the northern edge of the Valensole basin accommodate 10 km of shortening (Balansa et al. 2022). Hinterlandward, the Digne Nappe includes the imbricated thrust fan of the La Blanche Range (e.g., Dormillouse and Mandeysse units; Figs. 2, 3, 5;[START_REF] Goguel | Duplicatures de la crête de la Blanche près Seyne (Basses-Alpes)[END_REF][START_REF] Kerckhove | Tectonique de soubassement parautochtone des nappes de l'Embrunais-Ubaye sur leur bordure occidentale, du Drac au Verdon[END_REF]Debelmas et al. 1983b;[START_REF] Hamiti | Géometrie, cinématique et mécanismes des chevauchements synschisteux dans une région préalablement déformée. Exemple de la couverture sédimentaire à l'ouest du massif de l'Argentera (Alpes occidentales françaises[END_REF]. Shortening of the Digne Nappe has been mainly fed from a basement thrust located under the Barcelonnette zone (Debelmas et al. 1983a;[START_REF] Michard | The high-pressure metamorphic front of the south Western Alps (Ubaye-Maira transect, France, Italy)[END_REF][START_REF] Lardeaux | A crustal-scale cross-section of the south-western Alps combining geophysical and geological imagery[END_REF][START_REF] Dumont | Structural and sedimentary records of the Oligocene revolution in the Western Alpine arc[END_REF]. This basement thrust is interpreted as the northwestern continuation of the Argentera massif (Debelmas et al., 1983a;Fig. 1). Outer basement thrusts near Barles are active since the Early-Middle Miocene [START_REF] Gigot | Evolution tectono-sédimentaire de la bordure septentrionale du bassin tertiaire de Digne depuis l'Eocène[END_REF]Haccard et al. 1989b;[START_REF] Graham | Allochthonous salt in the sub-Alpine fold-thrust belt of Haute Provence, France[END_REF]Balansa et al. 2022). After the emplacement of the Digne Nappe in Late Miocene-Pliocene, shortening in the Barles basement thrust wedge continued and was responsible for folding and erosion of the overlying Digne Nappe, which created the Barles half-window [START_REF] Gigot | Evolution tectono-sédimentaire de la bordure septentrionale du bassin tertiaire de Digne depuis l'Eocène[END_REF][START_REF] Schwartz | Foreland exhumation controlled by crustal thickening in the Western Alps[END_REF]Balansa et al. 2022). These external basement thrusts could be connected northeastward at depth to the horizontal European lower crust as observed in the Cifalps transect [START_REF] Malusà | The deep structure of the Alps based on the CIFALPS seismic experiment: A synthesis[END_REF][START_REF] Nouibat | Lithospheric transdimensional ambient-noise tomography of W-Europe: implications for crustal-scale geometry of the W-Alps[END_REF].

The inner Embrunais-Ubaye nappes form a broad klippe over the Subalpine Digne Nappe, preserved between the Argentera and Pelvoux Paleozoic massifs (Fig. 1). Equivalent nappes (San Remo-Monte Saccarello nappes) are found to the southeast of the Argentera Massif [START_REF] Sanità | Structural relationships between Helminthoid Flysch and Briançonnais Units in the Marguareis Massif: A key for deciphering the finite strain pattern in the external southwestern Alps[END_REF]. The Embrunais-Ubaye nappes include an upper thrust sheet, the Parpaillon Nappe and a lower thrust sheet, the Autapie Nappe [START_REF] Kerckhove | La Zone du flysch dans les nappes de l'Embrunais-Ubaye (Alpes Occidentales)[END_REF]Figs. 2, 3, 6). They mainly consist of helminthoid flysch of Late Cretaceous to Paleocene age [START_REF] Kerckhove | La Zone du flysch dans les nappes de l'Embrunais-Ubaye (Alpes Occidentales)[END_REF]) locally involving basal tectonic slices of Briançonnais, Sub-Briançonnais and Digne Nappe units (upper Triassic to Eocene rocks; Fig. 6). The Embrunais-Ubaye flysch nappes originate from the Piemont-Liguria Ocean and their cumulate displacement is estimated to be >50 km [START_REF] Kerckhove | La Zone du flysch dans les nappes de l'Embrunais-Ubaye (Alpes Occidentales)[END_REF][START_REF] Merle | The curved translation path of the Parpaillon Nappe (French Alps)[END_REF][START_REF] Fry | Southwestward thrusting tectonics of the western Alps[END_REF][START_REF] Ford | Tertiary foreland sedimentation in the Southern Subalpine Chains, SE France: a geodynamic appraisal[END_REF].

The initial thickness of the two Embrunais-Ubaye nappes is estimated between 5 and 10 km [START_REF] Kerckhove | La Zone du flysch dans les nappes de l'Embrunais-Ubaye (Alpes Occidentales)[END_REF][START_REF] Merle | The curved translation path of the Parpaillon Nappe (French Alps)[END_REF][START_REF] Hamiti | Géometrie, cinématique et mécanismes des chevauchements synschisteux dans une région préalablement déformée. Exemple de la couverture sédimentaire à l'ouest du massif de l'Argentera (Alpes occidentales françaises[END_REF][START_REF] Lickorish | Sequential restoration of the external Alpine Digne thrust system, SE France, constrained by kinematic data and synorogenic sediments[END_REF][START_REF] Corsini | Alpine and late-hercynian geochronological constraints in the Argentera Massif (Western Alps)[END_REF][START_REF] Bigot-Cormier | The denudation history of the Argentera Alpine External Crystalline Massif (Western Alps, France-Italy): an overview from the analysis of fission tracks in apatites and zircons[END_REF][START_REF] Labaume | Tectonic control on diagenesis in a foreland basin: combined petrologic and thermochronologic approaches in the Grès d'Annot basin (Late Eocene-Early Oligocene, French-Italian external Alps)[END_REF][START_REF] Cavailhes | Architecture et propriétées pétrophysiques des zones de failles dans une série gréso-pélitique profondément enfouie : rôle de la déformation et des interactions fluide-roche. Exemple des Grés d'Annot[END_REF]. These nappes recorded an early northwestward thrusting in the Middle Eocene and a southwestward emplacement in the Early Oligocene associated with sedimentation of the Schistes à Blocs Formation [START_REF] Kerckhove | La Zone du flysch dans les nappes de l'Embrunais-Ubaye (Alpes Occidentales)[END_REF][START_REF] Merle | The curved translation path of the Parpaillon Nappe (French Alps)[END_REF][START_REF] Fry | Southwestward thrusting tectonics of the western Alps[END_REF][START_REF] Dumont | Structural and sedimentary records of the Oligocene revolution in the Western Alpine arc[END_REF]. The thrusting of the Parpaillon Nappe may have continued until the Early Miocene [START_REF] Kerckhove | La Zone du flysch dans les nappes de l'Embrunais-Ubaye (Alpes Occidentales)[END_REF][START_REF] Merle | The curved translation path of the Parpaillon Nappe (French Alps)[END_REF][START_REF] Fry | Southwestward thrusting tectonics of the western Alps[END_REF][START_REF] Mark | Tracking exhumation and drainage divide migration of the Western Alps: A test of the apatite U-Pb thermochronometer as a detrital provenance tool[END_REF]. Along the studied cross-section the erosional front is found along the northeastern slope of La Blanche Range immediately east of its imbricates (Figs. 2,3,5).

RSCM thermometry: methodology, sampling and results

The thermometric approach based on the carbonaceous material characterization using Raman spectroscopy tool provides the T RSCM recorded by the rocks, quantitatively in the range 200°C to 650°C (e.g. [START_REF] Beyssac | Raman spectra of carbonaceous material in metasediments: a new geothermometer[END_REF][START_REF] Lahfid | Evolution of the Raman spectrum of carbonaceous material in low-grade metasediments of the Glarus Alps (Switzerland)[END_REF]) and qualitatively in the range 100°C to 200°C (e.g. [START_REF] Lahfid | Evolution of the Raman spectrum of carbonaceous material in low-grade metasediments of the Glarus Alps (Switzerland)[END_REF][START_REF] Saspiturry | Paleogeothermal gradients across an inverted hyperextended rift system: Example of the Mauléon fossil rift (Western Pyrenees)[END_REF]. RSCM thermometry was successfully applied to track the thermal behavior in a large variety of geological settings (e.g., [START_REF] Labaume | Tectonothermal history of an exhumed thrust-sheet-top basin: An example from the south Pyrenean thrust belt[END_REF][START_REF] Saspiturry | Paleogeothermal gradients across an inverted hyperextended rift system: Example of the Mauléon fossil rift (Western Pyrenees)[END_REF][START_REF] Girault | The 3-D thermal Structure of the Helvetic Nappes of the European Alps: Implications for Collisional Processes[END_REF]Caldera et al. 2020).

RSCM thermometry analyses of this study were done at BRGM (French Geological Survey, Orléans) using a Renishaw inVia Reflex system and following the analysis procedure described in [START_REF] Lahfid | Evolution of the Raman spectrum of carbonaceous material in low-grade metasediments of the Glarus Alps (Switzerland)[END_REF]. The Raman parameters of the defect bands in partially or entirely disordered carbonaceous materials depend on the energy of the incident laser radiation due to resonance effects (e.g., [START_REF] Ferrari | Raman spectroscopy of amorphous, nanostructured, diamond-like carbon, and nanodiamond[END_REF]. Thus, the laser source excitation was 514.5 nm [START_REF] Lahfid | Evolution of the Raman spectrum of carbonaceous material in low-grade metasediments of the Glarus Alps (Switzerland)[END_REF]. It was focused on the sample through a Leica DM2500 microscope using a x100 lens. To avoid heating damage in the ordered carbonaceous material structure, the laser beam power did not exceed 0.5 mW at the surface of the thin section. At least 10 spectra were recorded for each sample.

RSCM analyses were carried out on 34 samples from Mesozoic to Cenozoic slates and limestones of the Digne Nappe (Fig. 2; Table 1). Representative Raman spectra are shown in Fig. 7a. Samples at less than 10 km in horizontal distance to the cross-section have been projected onto the cross-section along the computed fold axes, i.e., 112°-2° axis in the La Robine zone and 319°-5° axis in the Seyne-Barcelonnette zone (Figs. 2,3). Because the Digne Nappe is folded by the uplift of the Barles basement high, leading to variation along strike of height of the stratigraphic units, we projected the sample DIG20-28 with a dip of about 15° toward the southeast (Fig. 2). Samples have been collected far (>100 m in vertical distance) to major fault zones to avoid the effect of shear heating during thrust emplacement [START_REF] Maino | Dating shallow thrusts with zircon (U-Th)/He thermochronometry-The shear heating connection[END_REF].

According to the shapes of Raman spectra carbonaceous materials obtained in this study, we used the RA1 Raman parameter proposed in [START_REF] Lahfid | Evolution of the Raman spectrum of carbonaceous material in low-grade metasediments of the Glarus Alps (Switzerland)[END_REF] to calculate T RSCM . The RA1 parameter represents the relative area of D1 and D4 bands (area ratio RA1 = (D1 + D4) / (D1 + D2 + D3 + D4 + G)). T RSCM were calculated using this equation: T RSCM = 1217.6 × RA1 -450.66

The fitting procedure followed to determine the band area is described in [START_REF] Lahfid | Evolution of the Raman spectrum of carbonaceous material in low-grade metasediments of the Glarus Alps (Switzerland)[END_REF] and is relatively similar to that given for the study of carbonaceous soots by Sadezky et al. (2005). This procedure represented in Fig. 7b consists in using a Lorentzian profile for all bands (G band and D1, D2, D3, D4 defect bands).

T RSCM values range from 243±10°C to 349±13°C in the upper Triassic-Jurassic-upper Cretaceous sedimentary section and from 255±19°C to 328±8°C in the overlying upper Eocene-lower Oligocene sedimentary section (Figs. 2,3,4; Table 1). T RSCM values show a gradual increase with depth and towards the inner part of the orogen. In the outer zones, the T RSCM show a similar trend regarding the northeastward increase in temperature (from ~260°C in La Robine zone to ~330°C in Seyne area) compared to the increase in burial of the Jurassic section (Figs. 3,8a). In the La Blanche Range imbricate, upper Eocene-lower Oligocene strata show a similar northeastward increase in T RSCM value from 255°C to ~325°C (Figs. 2,3,5). In the Barcelonnette window, under the Embrunais-Ubaye nappes, we obtained T RSCM values ranging from 281±11°C to 332±10°C in middle-upper Jurassic and lower Oligocene strata (Figs. 2,4). Our T RSCM values >240°C are in good agreement with widespread N130°-45°NE-dipping schistosity (normal to the southwestward compression trend) in the Mesozoic-lower Oligocene sedimentary pile of the Digne Nappe (Figs. 2,4). This is also consistent with metamorphism grade evolution suggested by illite cristallinity index (semi quantitative analysis) and organic matter maturity data across the Mesozoic rocks of the Digne Nappe [START_REF] Aprahamian | Cartographie du métamorphisme faible à très faible dans les Alpes françaises externes par l'utilisation de la cristallinité de l'illite[END_REF][START_REF] Levert | Répartition géographique des minéraux argileux dans les sédiments mésozoïques du bassin subalpin : mise en évidence d'une diagenèse complexe[END_REF][START_REF] Hamiti | Géometrie, cinématique et mécanismes des chevauchements synschisteux dans une région préalablement déformée. Exemple de la couverture sédimentaire à l'ouest du massif de l'Argentera (Alpes occidentales françaises[END_REF]. Our data are also consistent with the T RSCM values obtained by [START_REF] Bellanger | Shortening of the European Dauphinois margin (Oisans Massif, Western Alps): New insights from RSCM maximum temperature estimates and 40 Ar/ 39 Ar in situ dating[END_REF] in Jurassic and Eocene-Oligocene sequences along the Embrunais valley and around the Pelvoux Massif (Fig. 1).

Apatite fission-track [START_REF] Labaume | Tectonic control on diagenesis in a foreland basin: combined petrologic and thermochronologic approaches in the Grès d'Annot basin (Late Eocene-Early Oligocene, French-Italian external Alps)[END_REF]) and extensional fault zone fluid inclusions [START_REF] Cavailhes | Architecture et propriétées pétrophysiques des zones de failles dans une série gréso-pélitique profondément enfouie : rôle de la déformation et des interactions fluide-roche. Exemple des Grés d'Annot[END_REF][START_REF] Cavailhes | Influence of fault rock foliation on fault zone permeability: The case of deeply buried arkosic sandstones (Grès d'Annot, southeastern France)[END_REF][START_REF] Cavailhes | Difference in petrophysical properties between foliated and dilatant fault rocks in deeply buried clastics: The case of the Grès d'Annot Formation[END_REF] temperature data (~200-270°C) in the lower Oligocene "Grès d'Annot" of the Digne Nappe (Restefond and Estrop areas; Fig. 2) are also close to our T RSCM (255-281°C) in these zones.

Burial and geotherm estimation

T RSCM reached in geological settings of low-grade metamorphism can be related to the effect of extensional/transtensional crustal deformation (i.e., crustal thinning during rifting associated with high thermal gradient) or compressional event (burial under thrust sheets and foreland basins associated with low thermal gradient) (e.g., [START_REF] Bellanger | Shortening of the European Dauphinois margin (Oisans Massif, Western Alps): New insights from RSCM maximum temperature estimates and 40 Ar/ 39 Ar in situ dating[END_REF][START_REF] Lahfid | From extension to compression: high geothermal gradient during the earliest Variscan phase of the Moroccan Meseta; a first structural and RSCM thermometric study[END_REF][START_REF] Saspiturry | Paleogeothermal gradients across an inverted hyperextended rift system: Example of the Mauléon fossil rift (Western Pyrenees)[END_REF]Caldera et al. 2020). Previous studies dealing with the thermal history of the Alps suggest that the Subalpine zone experienced heating during the Tethyan-Vocontian rifting then during the Oligocene-Miocene compression [START_REF] Barlier | L'évolution des Terres Noires subalpines méridionales d'après l'analyse minéralogique des argiles et la réflectométrie des particules carbonées[END_REF][START_REF] Levert | Répartition géographique des minéraux argileux dans les sédiments mésozoïques du bassin subalpin : mise en évidence d'une diagenèse complexe[END_REF][START_REF] Deville | Contrasting thermal evolution of thrust systems: An analytical and modeling approach in the front of the western Alps[END_REF][START_REF] Butler | Basement-cover tectonics, structural inheritance, and deformation migration in the outer parts of orogenic belts: A view from the western Alps, Linkages and Feedbacks in Orogenic Systems[END_REF]. Recent tomographic data of the Cifalps transect (located ~30-35 km north of the Digne-Barcelonnette transect; Fig. 1) indicate that the European crust has a regular thickness of ∼30 km below the Baronnies basin and the Digne Nappe (e.g., [START_REF] Nouibat | Lithospheric transdimensional ambient-noise tomography of W-Europe: implications for crustal-scale geometry of the W-Alps[END_REF]. In this portion of the European crust, no thickness variation, which could support pre-contractional continental thinning with high thermal conditions, is observed. However, the 255±19°C-328±8°C T RSCM recorded by the middle/upper Eocene-lower Oligocene cover of the Digne Nappe indicate that the entire sedimentary pile of the nappe attained its maximum temperature after the Early Oligocene (Figs. 2,4,5). This is consistent with previous studies in the western Alps suggesting that the Subalpine zone experienced burial/heating during the Oligocene-Miocene period [START_REF] Deville | Contrasting thermal evolution of thrust systems: An analytical and modeling approach in the front of the western Alps[END_REF][START_REF] Bellanger | Shortening of the European Dauphinois margin (Oisans Massif, Western Alps): New insights from RSCM maximum temperature estimates and 40 Ar/ 39 Ar in situ dating[END_REF]. The increase of T RSCM towards the internal part of the orogen (Fig. 3) is interpreted as resulting from tectonic burial under the Embrunais-Ubaye nappes and foreland sediments during the Oligocene-Miocene period. Therefore, we need now to define the thermal gradient to infer the thickness of the eroded pile above the Digne Nappe.

We performed a statistical model of T RSCM within the Digne Nappe as a twoparameter linear regression of horizontal distance (D1) along the cross-section and depth (Z1) relative to the top of the Mesozoic for 25 samples localized southwest of the La Blanche Range (Fig. 8). In this eroded portion of the Digne Nappe, the top of the Mesozoic pile is estimated by southeasterly remnants of unconformable middle/upper Eocene-Oligocene strata and cross-section construction, assuming that the structural thickness of the Digne Nappe is similar to its initial stratigraphic thickness (Figs. 2,3; Table 1). Our model approximates the temperature field within the Mesozoic-Cenozoic pile at the time of their maximum burial as a uniform geothermal gradient (temperature linearly increasing with Z1) combined with burial beneath a wedge of constant taper (temperature linearly increasing with D1). The regression parameters are calculated by weighted least square inversion, 1/SD 2 , where SD is the standard error on T RSCM estimated for each sample. Error bounds on the regression parameters are given for a 95% confidence interval. If the effect of burial beneath the wedge is ignored, a simple linear regression T RSCM (Z1) may be applied but this yields a poor coefficient of determination R 2 =0.437 and a low estimated gradient of 16±7°C/km (Fig. 8a).

Fit of data is greatly improved in the two-parameter linear model (Fig. 8b). The coefficient of determination is R 2 =0.916 and the best fit is:

T RSCM = 174±15°C + 25±4°C/km × Z1 + 2.1±0.4°C/km × D1
The geothermal gradient is thus estimated to 25±4°C/km and the lateral gradient along the top of the Mesozoic is estimated to 2.1±0.4°C/km. The average taper of the wedge is given by the ratio of the lateral gradient and geothermal gradient, which is equal to 0.085 and corresponds to a 4.9° taper angle (an average value of the known taper angles of the modern orogens; e.g., [START_REF] Boyer | Sedimentary basin taper as a factor controlling the geometry and advance of thrust belts[END_REF][START_REF] Meigs | Growth of the south Pyrenean orogenic wedge[END_REF]. In Fig. 8b, the adjusted depth is calculated as:

Z = Z1 + 0.0857 × D1
This adjustment accounts for burial beneath the wedge, and Z represents the depth beneath an arbitrary horizontal level (the depth of the top of the Mesozoic at D1=0). The estimated geothermal gradient represents the gradient within the Mesozoic-Cenozoic pile and thus at a depth of several kilometers. At equilibrium, the value determined by this method represents a lower bound to the average gradient between the surface and the samples because the radioactive heat production within the formation and the general increase of thermal conductivity with depth in sedimentary piles both contribute to decreasing gradient with depth. The consequences of the variation of conductivity with depth on the depth estimates remains small as this variation mostly occurs in the first 2 km where sediments are compacting. Moreover, it will mostly affect depth estimates near the toe of the wedge.

However, the heat production in the bulk of a sediment wedge cannot be neglected. With the same parameters as in [START_REF] Goffé | Effect of the chemical composition of the crust on the metamorphic evolution of orogenic wedges[END_REF], heat conductivity 2.25 W.m -1 .K -1 and radiogenic heat production 1 μW.m -3 , the average conductive gradient between the surface and a given depth z (in km) is 0.22×z °C/km above the conductive gradient at depth z. For instance, for a burial depth of 10 to 15 km, the average gradient from the surface is about 2 to 3°C/km above the gradient estimated at depth. This remains within uncertainties.

The calculated 25±4°C/km geothermal gradient at depth is coherent with a classical collisional-type gradient. It is consistent with orogen-scale numerical models, which show that for a slow convergence rate the geotherm within the frontal part of the orogenic wedge remains close to the initial geotherm assumed for the continent entering the orogen (for instance, with 4-8 mm/yr convergence rate as may be assumed for the Alps, see [START_REF] Bousquet | Kinematic, thermal and petrological model of the Central Alps: Lepontine metamorphism in the upper crust and eclogitisation of the lower crust[END_REF]. Assuming that the samples were affected by a similar post Oligocene burial history and taking into account radioactive heat production a geothermal gradient of 28±4°C/km should be considered as a reasonable average between the surface and depth to validate burial estimations along the cross-section. Considering a mean surface temperature of 10°C, we estimate the burial depth of the samples between ~9 km to the foreland and ~11 km to the hinterland (Table 1).

Palinspastic restoration of the Digne Nappe and structural implications

The cross-section was restored and balanced using "MOVE" software on the basis of the flexural-slip algorithm, which allows to maintain bed lengths and areas [START_REF] Dahlstrom | Balanced cross-sections[END_REF].

Because the T RSCM of the sedimentary pile was acquired after compaction, sediment decompaction was not performed [START_REF] Nunns | Structural restoration of seismic and geologic sections in extensional regimes[END_REF]. The burial estimation of each sample derived from T RSCM and a 28±4°C/km geothermal gradient have been placed on the restored cross-section (Fig. 9a) to constrain the shape of the prism prior to thrusting into the Subalpine zone (Early-Middle Miocene, ~16Ma). The restored envelope surface is consistent with the present wedge geometry (Fig. 9b), considering the lowest elevation in the foreland and a progressive elevation increase toward the hinterland (~5° taper angle). The restoration illustrates the initial position and geometry of the basement outcropping in the Argentera massif, which along our cross-section was probably also a structural high initially uplifted during the Mesozoic before Cenozoic shortening and inversion. It is inferred from the restoration that the inner Embrunais-Ubaye nappes travelled far southwestward (~40 km of displacement ahead of its present erosional front) along a gently northeastward-dipping basal thrust fault (Fig. 9a). The geodynamic reconstruction arising from the model is a crustal flexure as a combination of tectonic load of the Embrunais-Ubaye nappes and sedimentary load of a middle/upper Eocene-Oligocene to lower Miocene foreland basin (Fig. 9a). The T RSCM preserved in the hinterland by Mesozoic-lower Oligocene sedimentary section are interpreted as mainly resulting from tectonic burial under the Embrunais-Ubaye nappes.

According to the cross-section restoration and T RSCM data, these nappes have exceeded 11 km in thickness in the hinterland (Fig. 9a). The thickest tectonic unit was the Parpaillon Nappe.

The nappe stack thinned toward the foreland, from 5-to 3-km-thick. An additional ~3-kmthick ~50 km-wide, northeastward-dipping foreland basin is required to explain the observed T RSCM in the underlying Mesozoic-Paleogene sequence of the future Digne Nappe (Fig. 9a).

The restoration and the present thickness data suggest that the basin was composed of at least 1.3-km-thick middle/upper Eocene-lower Oligocene strata and the rest by upper Oligocene and lower Miocene strata. This early Alpine flexural foreland basin is the response to Alpine thickening and loading in the southwestern Alps between the Middle/Late Eocene and the Early Miocene. The Embrunais-Ubaye nappes propagated southwestward in this depression between the Argentera and Pelvoux structural highs during the Middle-Late Miocene [START_REF] Merle | The curved translation path of the Parpaillon Nappe (French Alps)[END_REF][START_REF] Dumont | Structural and sedimentary records of the Oligocene revolution in the Western Alpine arc[END_REF]. Thus, a now mainly eroded large middle/upper Eocene-Oligocene to lower Miocene foreland basin existed in the southwestern Alps along the study transect like that still present in the northwestern Alps [START_REF] Girault | Exhumation of the Western Alpine collisional wedge: New thermochronological data[END_REF]. Cross-section restoration also suggests that the Jurassic strata of the Barles unit (footwall of the future Digne Nappe) have been buried by only ~400-to 900-m-thick Oligocene-lower Miocene foreland strata. Although the T RSCM recorded by the Jurassic strata of the Barles units is not constrained, the lack of schistosity (excepted along localized fault zones), that contrasts with the pervasive schistosity in the Digne Nappe [START_REF] Hamiti | Géometrie, cinématique et mécanismes des chevauchements synschisteux dans une région préalablement déformée. Exemple de la couverture sédimentaire à l'ouest du massif de l'Argentera (Alpes occidentales françaises[END_REF], suggests only shallow burial of the para-autochthonous during the Oligocene-Miocene.

According to T RSCM data/burial depth estimations, the palinspastic restoration of the cross-section suggests a total horizontal crustal shortening of ~35 km (35%) for the Subalpine zone during the Middle Miocene to Present (Fig. 9). Approximately 26 km of this shortening were transferred into the Digne Nappe, the rest (9 km) into the Barles zone. We calculate a mean shortening rate, since ~16 Ma, of ~2.2 mm/yr for the Subalpine zone. These values are in accordance with the shortening values calculated by Balansa et al. (2022) for the frontal part of the Subalpine zone. The restoration also suggests that the La Blanche Range imbricates and folds (Figs. 5 and9b) in the footwall of the Embrunais-Ubaye nappes might have developed during the emplacement of the Embrunais-Ubaye nappes in the Late Oligocene-Early Miocene period. Although the La Blanche Range imbricate was not restored, its shortening is estimated greater than 7 km as suggested by [START_REF] Hamiti | Géometrie, cinématique et mécanismes des chevauchements synschisteux dans une région préalablement déformée. Exemple de la couverture sédimentaire à l'ouest du massif de l'Argentera (Alpes occidentales françaises[END_REF].

Based on low-temperature apatite fission-track and (U-Th)/He data, [START_REF] Schwartz | Foreland exhumation controlled by crustal thickening in the Western Alps[END_REF] confirmed that the northern edge of the Neogene Valensole basin and Barles unit located in the footwall of the Digne Nappe were heated up to ~100°C. For a mean geothermal gradient of 28°C/km and surface temperature of 10°C, these low-temperature thermochronology data are consistent with an upper Neogene tectonic burial of lower Miocene strata of the Valensole basin under the 3.3-km-thick Digne Nappe (Fig. 9b). This result suggests that erosion has removed a large thickness (>8 km) of the sedimentary/tectonic cover of the Digne Nappe in the Middle to Late Miocene.

Many geological and analog/numerical modeling studies showed that the dynamics of orogenic wedges is governed by complex interactions between deformation, erosion and sedimentation processes (e.g., [START_REF] Davis | Mechanics of fold-and-thrust belts and accretionary wedges[END_REF][START_REF] Baby | Etude géométrique et expérimentale d'un bassin transporté : exemple du synclinorium de l'Alto Beni (Andes centrales)[END_REF][START_REF] Avouac | Erosion as a driving mechanism of intracontinental mountain growth[END_REF][START_REF] Meigs | Growth of the south Pyrenean orogenic wedge[END_REF][START_REF] Bonnet | Surface processes versus kinematics of thrust belts: impact on rates of erosion, sedimentation, and exhumation -Insights from analogue models[END_REF][START_REF] Malavieille | Impact of erosion, sedimentation, and structural heritage on the structure and kinematics of orogenic wedges: Analog models and case studies[END_REF][START_REF] Fillon | Syntectonic sedimentation effects on the growth of fold-and-thrust belts[END_REF][START_REF] Steer | Erosion influences the seis-micity of active thrust faults[END_REF][START_REF] Erdős | First-order control of syntectonic sedimentation on crustal-scale structure of mountain belts[END_REF][START_REF] Butler | Syn-kinematic strata influence the structural evolution of emergent foldthrust belts[END_REF][START_REF] Liu | Role of erosion in creating thrust recesses in a critical-taper wedge: An example from Eastern Tibet[END_REF][START_REF] Malavieille | Deformation partitioning in mountain belts: insights from analogue modelling experiments and the Taiwan collisional orogen[END_REF]. For instance, the growth and uplift of an antiformal nappe stack can focus erosion in the internal zones. In turn, this localized erosion can favor internal deformation and the development of very active thrust accommodating large displacements [START_REF] Bonnet | Surface processes versus kinematics of thrust belts: impact on rates of erosion, sedimentation, and exhumation -Insights from analogue models[END_REF]. Based on our results along the Digne-Barcelonnette transect, we propose that the large erosional unloading of the inner Alpine wedge might have contributed to the rapid basement thrusting [START_REF] Bigot-Cormier | The denudation history of the Argentera Alpine External Crystalline Massif (Western Alps, France-Italy): an overview from the analysis of fission tracks in apatites and zircons[END_REF]) and the large displacement of the Subalpine Digne Nappe in Middle-Late Neogene (Balansa et al. 2022). The erosion of the Embrunais-Ubaye/Digne nappe stack may have provided large sediment influx toward the foreland (paleo-Durance river), leading to the formation of the Valensole basin in this segment of the Alpine orogen. Similarly, the development of the Nice basin to the southeast could have been favored by the erosion of the San Remo-Monte Saccarello flysch nappes (Fig. 1). However, the eroded volume of these nappes is not yet constrained.

Conclusion

In the southwestern French Alps along the Digne-Barcelonnette transect, the ~4-kmthick Mesozoic-Cenozoic sedimentary pile of the Subalpine Digne Nappe records T RSCM ranging from 243±10°C to 349±13°C, resulting from substantial burial in a typical ~28±4°C/km orogenic geotherm. We proposed a structural model in which the Digne Nappe has been overburdened under the 3-to 11-km-thick inner Embrunais-Ubaye nappe wedge and ~3-km-thick frontal middle/upper Eocene-lower Miocene foreland basin. The leading edge of the Embrunais-Ubaye nappes is estimated ~40 km ahead of its present erosional front. Crosssection restoration indicates that ~35 km (35%) of horizontal crustal shortening were accommodated in the Subalpine zone over the past ~16 million years, which implies a shortening rate of ~2.2 mm/yr. The removal of substantial units of the southwestern Alpine orogen might have significantly unloaded the wedge, which has undeniably influenced the Subalpine thrust wedge propagation since the Middle Miocene.

Figure captions

Fig. 1 Geodynamic setting of the southwestern Alps. Black frame: study area (Fig. 2).

Dashed black line: Digne-Barcelonnette balanced cross-section (Fig. 3). Dashed white line: crustal-scale Cifalps profile. Fig. 2 (a) Simplified structural map of the study area (modified from [START_REF] Kerckhove | Tectonique de soubassement parautochtone des nappes de l'Embrunais-Ubaye sur leur bordure occidentale, du Drac au Verdon[END_REF][START_REF] Arlhac | Carte géologique de la France (1/50000)[END_REF]Haccard et al. 1989a) showing RSCM samples with T RSCM (°C). The locations of the Digne-Barcelonnette balanced cross-section of Fig. 3 See location in Fig. 2. Samples have been projected according to mean fold axes, i.e., 112°-2° axis in the La Robine zone and 319°-5° axis in the Seyne-Barcelonnette zone (see Fig. 2).

Bedding traces are indicated by thin black lines. Cgts: Conglomerates. SB: Schistes à Blocs. 2. This is a composite cross-section using previous cross-sections from [START_REF] Goguel | Duplicatures de la crête de la Blanche près Seyne (Basses-Alpes)[END_REF], [START_REF] Kerckhove | Tectonique de soubassement parautochtone des nappes de l'Embrunais-Ubaye sur leur bordure occidentale, du Drac au Verdon[END_REF], Debelmas et al. (1983b) and GEOL-ALP website from M. Gidon. 2). A 319±11°C T RSCM is measured in lower Oligocene "Grès d'Annot" (sample UBA20-2; see details in Fig. 4c). Improved two-parameter linear model of the T RSCM . See details of parameters in Table 1 and explanations in text.

Fig. 9

Restored cross-sections of the southwestern Alpine orogenic wedge (see location in Fig. 2). The restoration is constrained with thickness estimations of the eroded units according to T RSCM data and 28±4°C/km geothermal gradient (see Table 1).

  and surficial crosssection of Fig. 5 are shown. Field photographs of Figs. 4 and 6 are also shown. (b) The direction of the Digne-Barcelonnette balanced cross-section is chosen using the mean great circle analysis of bedding plane poles (points). The cross-section has two distinct trends: N22° in the La Robine zone and ~N49° in the Seyne-Barcelonnette zone. Mean fold axes (squares) are used to project RSCM samples onto the cross-section. (c) Schistosity planes measured in the Digne Nappe. Bedding poles and schistosity planes have been projected in a Schmidt diagram, lower hemisphere. Cgts: Conglomerates. SB: Schistes à Blocs.

Fig. 3

 3 Fig.3Balanced cross-section with projected location of the RSCM samples with T RSCM (°C).

Fig. 4

 4 Fig. 4 Field photographs of sampled rocks for RSCM analyses in the Digne Nappe. T RSCM

Fig. 5

 5 Fig. 5 Surficial cross-section showing the present front of the Autapie Nappe above the La

Fig. 6

 6 Fig. 6 (a) Panoramic view of the superimposed Parpaillon Nappe, Autapie Nappe and Digne

Fig.

  Fig.2). A 319±11°C T RSCM is measured in lower Oligocene "Grès d'Annot" (sample UBA20-

Fig. 7

 7 Fig. 7 (a) Selection of representative Raman spectra of carbonaceous materials and the

  ). G: Graphite band. D: Defect band. To avoid polishing effect, Raman analyses have been performed on carbonaceous materials covered by transparent minerals as revealed here by carbonate peaks. (b) Peak-fitting of the Raman spectrum of disordered carbonaceous material from DIG 20-3 sample. This spectrum is composed of a G band and D1, D2, D3, D4 defect bands (see Sadezky et al. 2005 for band labelling). For peak-fitting, each of the five bands has a pure Lorentzian shape and no a priori restriction is imposed on the band position, FWHM or relative intensity.

Fig. 8

 8 Fig. 8 Thermal modeling of the Digne Nappe. (a) Simple linear regression of the T RSCM . (b)

Table 1

 1 Fig.8) and a surface temperature of 10°C

Acknowledgments

This project has been supported by the BRGM-RGF (French Geological Survey-Référentiel Géologique de la France) Alpes et bassins périphériques program. We thank Myette Guiomar from the Réserve Naturelle Géologique de Haute-Provence for field assistance. Cross-section balancing was performed using the MOVE Software Suite donated by Petroleum Experts Limited. Diagrams of Fig. 2 have been performed using Stereonet v. 11 by R.W. Allmendinger.


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