Jean Roman

Olivier Français

Nathalie Jarroux

Gilles Patriarche

Juan Pelta
email: juan.pelta@univ-evry.fr

Laurent Bacri
email: laurent.bacri@univ-evry.fr

Bruno Le Pioufle
email: bruno.le-pioufle@ens-cachan.fr

Solid-state nanopore easy chip integration in a cheap and reusable microfluidic device for ion transport and polymer conformation sensing

Solid-state nanopores have a huge potential in upcoming societal challenging applications in biotechnologies, environment, health and energy. Nowadays, these sensors are often used within bulky fluidic devices that can cause crosscontaminations and risky nanopore chips manipulations, leading to a short experimental lifetime. We describe the easy, fast and cheap innovative 3D-printer-helped protocol to manufacture a microfluidic device permitting the reversible integration of a silicon based chip containing a single nanopore. We show the relevance of the shape of the obtained channels thanks to finite elements simulations. We use this device to thoroughly investigate the ionic transport through the solid-state nanopore as a function of applied voltage, salt nature and concentration. Furthermore, its reliability is proved through the characterization of a polymer-based model of protein-urea interactions at the nanometric scale thanks to a hairy nanopore. KEYWORDS: microfluidics, solid-state nanopore, 3D-printing, ionic transport, protein-urea interactions, grafted polymer conformation, hydrodynamic simulations Resistive pulse sensing is a rapidly growing method for macromolecules sequencing, nanopar-ticle detection and analysis [START_REF] Lepoitevin | Functionalization of single solid state nanopores to mimic biological ion channels: A review[END_REF][START_REF] Shi | Nanopore Sensing[END_REF] or diagnosis [START_REF] Wang | Nanopore-based detection of circulating microRNAs in lung cancer patients[END_REF][START_REF] Lin | Direct sensing of cancer biomarkers in clinical samples with a designed nanopore[END_REF] based on the principle of the Coulter counter. [START_REF] Coulter | Means for counting particles suspended in a fluid[END_REF] An aperture made in an insulating material is placed between two voltage biased electrolytes. The ionic conductance of this aperture transiently decreases as a single nanoparticle passes through it. This electrical technique is used to characterize its size, [START_REF] Larkin | High-Bandwidth Protein Analysis Using Solid-State Nanopores[END_REF] conformation [START_REF] Plesa | Non-equilibrium folding of individual DNA molecules recaptured up to 1000 times in a solid state nanopore[END_REF] or interactions with other particles [START_REF] Varongchayakul | Sensing Native Protein Solution Structures Using a Solid-state Nanopore: Unraveling the States of VEGF[END_REF] at the single molecule scale. Two major resistive pulse sensing technique families can be considered: nanochannels and nanopores. Nanochannels feature a small aperture in regard of their length and consist of nanocapillaries, [START_REF] Steinbock | Probing DNA with micro-and nanocapillaries and optical tweezers[END_REF] molded nanochannels [START_REF] Saleh | Direct detection of antibody-antigen binding using an on-chip artificial pore[END_REF][START_REF] Halpern | Wafer-scale fabrication of nanofluidic arrays and networks using nanoimprint lithography and lithographically patterned nanowire electrodeposition gold nanowire masters[END_REF][START_REF] Angeli | Simultaneous Electro-Optical Tracking for Nanoparticle Recognition and Counting[END_REF] and track-etched channels. [START_REF] Siwy | Fabrication of a synthetic nanopore ion pump[END_REF][START_REF] Yameen | Single conical nanopores displaying pH-tunable rectifying characteristics. manipulating ionic transport with zwitterionic polymer brushes[END_REF][START_REF] Guo | Asymmetric Ion Transport through Ion-Channel-Mimetic Solid-State Nanopores[END_REF][START_REF] Balme | Ionic transport through sub-10 nm diameter hydrophobic high-aspect ratio nanopores: experiment, theory and simulation[END_REF] The micrometric length of these nanochannels makes them less sensitive to particle translocation, for the conductance drop ratio is equal to the ratio between the volume of the empty channel and the one with the particle inside. In contrast, nanopores have similar nanometric length and diameter. They consist of either synthesized [START_REF] Bacri | Ionic channel behavior of modified cyclodextrins inserted in lipid membranes[END_REF][START_REF] Geng | Stochastic transport through carbon nanotubes in lipid bilayers and live cell membranes[END_REF][START_REF] Mamad-Hemouch | Biomimetic Nanotubes Based on Cyclodextrins for Ion-Channel Applications[END_REF][START_REF] Goepfrich | Large-Conductance Transmembrane Porin Made from DNA Origami[END_REF] or proteic [START_REF] Bezrukov | Counting polymers moving through a single ion channel[END_REF][START_REF] Wang | Determining the Physical Properties of Molecules with Nanometer-Scale Pores[END_REF] channels inserted in a lipid bilayer, or of a solid-state nanopore pierced in an ultra-thin dielectric membrane. [START_REF] Varongchayakul | Sensing Native Protein Solution Structures Using a Solid-state Nanopore: Unraveling the States of VEGF[END_REF][START_REF] Angeli | Simultaneous Electro-Optical Tracking for Nanoparticle Recognition and Counting[END_REF][START_REF] Li | Ion-beam sculpting at nanometre length scales[END_REF][START_REF] Bacri | Dynamics of colloids in single solid-state nanopores[END_REF][START_REF] Cressiot | Protein Transport through a Narrow Solid-State Nanopore at High Voltage: Experiments and Theory[END_REF][START_REF] Giamblanco | Detection of protein aggregate morphology through single antifouling nanopore[END_REF][START_REF] Oukhaled | Dynamics of Completely Unfolded and Native Proteins through Solid-State Nanopores as a Function of Electric Driving Force[END_REF][START_REF] Waduge | Nanopore-Based Measurements of Protein Size, Fluctuations, and Conformational Changes[END_REF][START_REF] Larkin | Slow DNA Transport through Nanopores in Hafnium Oxide Membranes[END_REF][START_REF] Storm | Fabrication of solid-state nanopores with single-nanometre precision[END_REF] The main advantage of protein channels is the reproducibility of their structure and their sensitiv-ity. [START_REF] Baaken | High-Resolution Size-Discrimination of Single Nonionic Synthetic Polymers with a Highly Charged Biological Nanopore[END_REF][START_REF] Peraro | Pore-forming toxins: ancient, but never really out of fashion[END_REF] However, theses pores are limited by the mechanical fragility of lipid bilayers and of their internal diameter, bound to a few nanometers.

In this work, we propose biomimetic nanopores made in solid membranes using techniques of the micro-electronic industry. [START_REF] Li | Ion-beam sculpting at nanometre length scales[END_REF][START_REF] Storm | Fabrication of solid-state nanopores with single-nanometre precision[END_REF] These membranes are resistant to chemical, mechanical and heat stress. They are mainly pierced thanks to a focalized ion beam, [START_REF] Li | Ion-beam sculpting at nanometre length scales[END_REF][START_REF] Bacri | Dynamics of colloids in single solid-state nanopores[END_REF][START_REF] Oukhaled | Dynamics of Completely Unfolded and Native Proteins through Solid-State Nanopores as a Function of Electric Driving Force[END_REF][START_REF] Wanunu | microRNAs using thin nanopore sensors[END_REF][START_REF] Lee | Large apparent electric size of solid-state nanopores due to spatially extended surface conduction[END_REF] an electron beam [START_REF] Cressiot | Protein Transport through a Narrow Solid-State Nanopore at High Voltage: Experiments and Theory[END_REF][START_REF] Waduge | Nanopore-Based Measurements of Protein Size, Fluctuations, and Conformational Changes[END_REF][START_REF] Storm | Fabrication of solid-state nanopores with single-nanometre precision[END_REF] or more recently via dielectric breakdown [START_REF] Kwok | Nanopore fabrication by controlled dielectric breakdown[END_REF][START_REF] Feng | Observation of ionic Coulomb blockade in nanopores[END_REF][START_REF] Lin | Characterization of DNA duplex unzipping through a sub-2 nm solid-state nanopore[END_REF] to obtain a nanopore that matches the analyte size. Unfortunately, these membranes often bear a high surface energy. Thus, they are prone to particle or protein adsorption. [START_REF] Oukhaled | Dynamics of Completely Unfolded and Native Proteins through Solid-State Nanopores as a Function of Electric Driving Force[END_REF][START_REF] Niedzwiecki | Single-molecule observation of protein adsorption onto an inorganic surface[END_REF] Chemical, [START_REF] Wanunu | Chemically modified solid-state nanopores[END_REF] protein [START_REF] Kowalczyk | Single-molecule transport across an individual biomimetic nuclear pore complex[END_REF][START_REF] Ketterer | DNA origami scaffold for studying intrinsically disordered proteins of the nuclear pore complex[END_REF] and polymer grafting [START_REF] Giamblanco | Detection of protein aggregate morphology through single antifouling nanopore[END_REF][START_REF] Roman | Functionalized Solid-State Nanopore Integrated in a Reusable Microfluidic Device for a Better Stability and Nanoparticle Detection[END_REF][START_REF] Yin | Covalent Modification of Silicon Nitride Nanopore by Amphoteric Polylysine for Short DNA Detection[END_REF] or the use of HfO 2 membranes [START_REF] Larkin | Slow DNA Transport through Nanopores in Hafnium Oxide Membranes[END_REF] has been proved to avoid these adsorptions and to increase nanopore conductance stability over time. In this study, we choose to graft polymer chains on the membrane.

Traditional and commercially available integrating devices are typically composed of two compartments open to the air and separated by the nanopore chip (Figure S1). Toric joints are often used to prevent any current leaking. Changing the analytes in such systems can only be done by emptying completely each compartment and then filling them up with the new analyte. Thus, crosscontamination between the analytes can only be avoided by reiterating the analyte change many times. The aperture to the nanopore chip and the joints hold a dead volume hardly reachable that increase the cross-contamination risk. In this publication, we propose to insert the nanopore chip into a microfluidic device. [START_REF] Tahvildari | Integrating nanopore sensors within microfluidic channel arrays using controlled breakdown[END_REF][START_REF] Tahvildari | Manipulating Electrical and Fluidic Access in Integrated Nanopore-Microfluidic Arrays Using Microvalves[END_REF] Based on micrometric channels directly in contact with the nanopore chip, this technology allows for rapid and efficient changes of analytes without dead volumes. Furthermore theses microfluidic channels hold only about 100 µL of analytes. Whereas classical microfluidic nanopore-integrating devices are irreversibly sealed onto the nanopore chip, we show an easy way to obtain a reversible set-up, permitting to secure the valuable nanopore in between experiments.

The main difficulty of the making of PDMSbased microfluidic systems resides in the fabrication of a master mold. This is usually done using photolithography, [START_REF] Duffy | Rapid prototyping of microfluidic systems in poly(dimethylsiloxane)[END_REF] a time-consuming technique requiring a clean room. Here, we propose a new way to rapidly produce low-cost microfluidic masters thanks to a 3D-printer. [START_REF] Mcdonald | Prototyping of Microfluidic Devices in Poly ( dimethylsiloxane ) Using Solid-Object Printing the fabrication of microfluidic devices in poly ( dimethyl[END_REF][START_REF] Macdonald | Comparing Microfluidic Performance of Three-Dimensional (3D) Printing Platforms[END_REF] Thanks to finiteelements simulations we show that the shape of the channels we obtain are better suited for the analysis of biological samples than typical ones as they yield a lesser hydrodynamic resistance and a more homogeneous shear stress.

In this work, we describe a new method to make an easy to use, unsealed device. It permits to use the same device with several nanopore chips and thus different pores or pore grafting or to reuse nanopore chips on different devices. We can also rapidly change the analyte without disassembling the nanopore chip from the microfluidic system.

We use this device with a nanopore grafted with long polymer chains. [START_REF] Peleg | Morphology Control of Hairy Nanopores[END_REF] We discuss the nanopore conductance in a large range of LiCl and KCl salt concentrations in terms of ions and counter-ions transports. [START_REF] Lepoitevin | Functionalization of single solid state nanopores to mimic biological ion channels: A review[END_REF][START_REF] Balme | Ionic transport through sub-10 nm diameter hydrophobic high-aspect ratio nanopores: experiment, theory and simulation[END_REF][START_REF] Stein | Surfacecharge-governed ion transport in nanofluidic channels[END_REF][START_REF] Schoch | Transport phenomena in nanofluidics[END_REF] We discuss the conformation of the polymer chain grafted on the Si 3 N 4 surface from the evaluation of apparent radius and length of the nanopore after polymer grafting.

As a proof of concept, the device is used for the analysis of grafted long chains conformations depending on the salt used. The electrical response of the pore leads us to a simple experimental proof for an important question about the mechanism of protein unfolding induced by urea. [START_REF] Myers | Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding[END_REF][START_REF] Vanzi | Effect of the Protein Denaturants Urea and Guanidinium on Water Structure: A Structural and Thermodynamic Study[END_REF][START_REF] Bennion | The molecular basis for the chemical denaturation of proteins by urea[END_REF] The design of the mold was performed using Blender (Blender foundation), a 3D graphics software. Instructions to the 3D-printer were then generated using the slicer Cura (Ultimaker B.V., Netherlands). We use an Ultimaker 2 3D-printer (Ultimaker B.V., Netherlands) to quickly fabricate the low-cost master for the molding of the PDMS sub-units of the device. This kind of printers is called Fused Deposition Modeling printers (FDM). It allows for the 2D patterning of melted material on a surface, leading to the fabrication, layer after layer, of a 3D object. In our case, only a few layers are necessary, each layer being 100 µm thick. FDM printers can be used on any flat surface provided that the first layer of deposited material can stick to the substrate. We print the Poly(Lactic Acid) (PLA) master on a silicon wafer to benefit from its low roughness. We print at high temperature (200 o C) and at a low speed (10 mm/s) to avoid residual stress inside the printed material and optimize print adhesion. Additional modifica- tions to the instructions code were made thanks to a custom program of our own. During the print, the substrate is heated up to 80 o C to enhance print adhesion and limit the warping of printed objects due to the high temperature difference between the bottom of the object and the nozzle. Lastly, the freshly printed mold is heated up to 180 o C for a few seconds using a hot plate. This last step is crucial in order to i) smooth the shape of the printed pattern and ii) achieve a good adherence of the PLA on the silicon wafer. Printed patterns were realized down to a width of 50 µm. However we chose to work with 150 µm wide channels as it induces less hydrodynamic resistance and make for more robust masters. The three inches master contains eight of those channels, for the mounting of a total of four devices (Figure 2a).

PDMS is used because of its insulating properties as well as its fluidic sealing properties. We use Sylgard 184 (Dow Corning, U.S.A.) with a 10:1 base to curing agent ratio. After a thorough mixing, the solution is degassed at 300 mbar for 10-15min. The fabrication steps of the microfluidic device we propose is detailed in Figure 1. The device is made of two identical PDMS subunits corresponding to the cis and trans sides of the nanopore chip. The latter will be inserted in between these sub-units (Figure 1a andc). Each sub-unit is composed of two parts: one main part containing the channel and the other one a thin film for closing the channel. The first one is cast on a 3D-printed master (Figure 1a), the channels dimensions are reported on Figure 2c. This layer should be around 5 mm high to be flexible enough to easily mount the nanopore chips without breaking and still be durable. Fluidic access to the channels are made at this step using a biopsy puncher. The thin film is spin-coated down to 200 µm over a naked silicon wafer (Figure 1a). PDMS is cured at 75 o C for 1h30 (Figure 2b). Using a Basic Plasma Cleaner (Harrick Plasma, U.S.A.), the PDMS film is covalently bonded to the main part containing the channel to finalize the fabrication of each subunit (Figure 1c).

These sub-units are then partially bonded together. First, the top half of both these parts is masked with tape (red line represented in Figure 1d). Then, a second plasma activation bonds the unmasked bottom parts of the two sub-units. The upper part (dotted red line in Figure 1e) remains open for nanopore chip insertion. Fluidic access tubes are inserted in the holes we previously pierced. A talon of PDMS is cast to perfect their sealing (colored in orange in Figure 1f). For this, the device is put in a 2 in Petri dish which is then filled with PDMS. This talon also permits the device to stand for easy manipulations. Finally, using a biopsy puncher again, apertures are made through the thin layer covering the channel in place of where the nanopore chip will be inserted (Figure 2d). It will permit the analytes to reach the nanopore. To avoid any leaking during the changes of analytes, light pressure is applied to the upper part of the device thanks to two glass slates and a paper clip after nanopore mounting. This step is crucial to reversibly seal/detach the nanopore chips into/from the microfluidic device.

We use 3 mm silicon nitride chips (Nanopore solutions, Portugal) : these chips are composed of a 200 µm thick silicon substrate, covered by a 20 nm-thin Si 3 N 4 layer. The substrate is etched in its center to obtain a 15 × 15 µm 2 free-standing Si 3 N 4 membrane (Figure 3a).

Nanopores are all pierced thanks to a Titan Themis TEM mode at 8 nA. The size of the probe is adapted to the desired pore size to obtain straight walls and a more stable pore. Thanks to the high currents used and the great control over the size of TEM beams, pores from 3 to 200 nm can be obtained rapidly. [START_REF] Storm | Fabrication of solid-state nanopores with single-nanometre precision[END_REF] Immediately after piercing, the electron beam is defocused and we obtain an image of the pore. In this publication we use a 31 nm Si 3 N 4 pore (Figure 3).

PEG-7kDa (PolyPure AS, Norway) was grafted over the surface of the nanopore chip thanks to a three steps protocol already described in a previous paper [START_REF] Roman | Functionalized Solid-State Nanopore Integrated in a Reusable Microfluidic Device for a Better Stability and Nanoparticle Detection[END_REF][START_REF] Yang | Silylation of mesoporous silica MCM-41 with the mixture of Cl(CH2)3SiCl3 and CH3SiCl3: combination of adjustable grafting density and improved hydrothermal stability[END_REF] and is briefly reported in the Supporting Information.

We use Ag/AgCl electrodes directly inserted inside the channels of the device to measure the ionic current going through the pore. Current amplification was done thanks to an Axopatch 200B (Molecular devices, U.S.A.). Data was acquired with a PCIe-6251 board (National Instruments, U.S.A.) interfaced with a custom .Net based program.

Current versus voltage curves (IVs) were obtained applying 30 s long voltage steps of 25 mV between ±200 mV. The current was recorded at 10 3 samples/s and filtered thanks to the Axopatch 200B with a Bessel four poles low pass filter at 5 kHz. The average current for each voltage step is obtained from the last 15 s of the steps to avoid the expected capacitive surges induced by the changes of voltage. Finally, the slope of the IV curve yields the conductance of the pore according to the Ohm's law. Typical recordings as well as the resulting IV curves are shown in Figure 4a andb. A schematics of a typical microfluidic system for solid-state nanopore measurements is shown in Figure S1. These systems are composed of two large compartments containing the electrolytes separated by the nanopore chip. The main drawback of such a system is that the change of analytes is done without a managed fluidic flow. The operator needs to change the electrolyte many times to avoid cross-contamination. Also, these systems often contain unreachable volumes of electrolytes, namely dead volumes, which accentuate this disadvantage. The use of a grazing channel facilitates the change of electrolytes and ensures less crosscontamination. These channels are a typical solution employed in microfluidics.

Previous approaches of printing microfluidic chips [START_REF] Macdonald | Comparing Microfluidic Performance of Three-Dimensional (3D) Printing Platforms[END_REF][START_REF] Tang | Automated 3D-printed unibody immunoarray for chemiluminescence detection of cancer biomarker proteins[END_REF] or molds [START_REF] Mcdonald | Prototyping of Microfluidic Devices in Poly ( dimethylsiloxane ) Using Solid-Object Printing the fabrication of microfluidic devices in poly ( dimethyl[END_REF] with FDM printers show that the spatial resolution is limited to 200±100 µm depending on the 3D-printer used, with a roughness of 11 µm. [START_REF] Macdonald | Comparing Microfluidic Performance of Three-Dimensional (3D) Printing Platforms[END_REF] To rise up to this challenge, we chose to print the mold over a silicon wafer to take advantage of the wafer nanometric smoothness. After printing, we melt the mold using a hot plate to enhance adhesion between the printed PLA and the silicon wafer. The profile of the 3D-printed mold has been measured thanks to a Dektak profilometer (Bruker, Germany) and is reported in Figure 2b. The height and width of the channel are constant along a straight part with 213 ± 0.1 µm height and 826 ± 1 µm width measured on both profiles. Also we measured the roughness of the printed parts of the mold. We show that the heating step allows to decrease the channel roughness from R raw a = 0.21 ± 0.1 µm and R raw q = 0.25 ± 0.1 µm to R melted a = 8 ± 4 nm and R melted q = 10 ± 4 nm (calculus explained in Supporting Information, Eq. 1). This low roughness allows for more durable molds as PDMS won't clutch to it when removed.

The reversible sealing of the device has been tested thanks to a simple manometer. The device withstands to up to 2 bar without leaking. Most of the time, old devices or intensively used ones only leak from the entry point of the access tubes. Thus, measurements are still possible with small leakage.

This microfluidic device permitted to record every set of data used for a graph within 7 hours each. The study we describe in the next section has been done along 4 weeks, dismounting the same nanopore chip at the end of the day to safely store it between experiments. This shows the reliability of the device for the handling of the fragile chip.

Whereas obtaining high channels is costly and difficult with traditional photolithography methods, our method allows for the fabrication of channels up to 1 mm-high without hassle. An advantage of using high channels is their reduced hydrodynamic resistance. Thanks to this, the filling of the microfluidic system and the change of analytes in this device takes only 6 to 10 s for 1 mL injections by hand using a syringe. Also, we studied the effect of hemicylindrical channels on the fluidic flow using COMSOL Multiphysics finite elements simulations (COMSOL Group, Sweden). The flow speed and shear stress calculated for hemicylindrical, rectangular and cylindrical channels having the same cross-section area are reported in Fig- ure S2. These simulations show the hemicylindrical channel yields a hydrodynamic resistance 3% lower than the rectangular one for a straight channel. Furthermore, the shear stress is more homogeneous along the wall in the hemicylindrical channel. Such homogeneity, especially along the walls of the channel is advantageous in tissue engineering, which is quite understandable as it corresponds to the shape of vascular vessels. A cylindrical channel allows for an even more homogeneous shear stress and a lesser hydrodynamic resistance. An assembly of two hemicylindrical channels obtained thanks to a 3D-printed mold could achieve such a channel. However, in our case, hemicylindrical channels are more suited to the integration of a nanopore chip.

Grafting long polymer chains on nanopores tends to reduce their apparent size. [START_REF] Giamblanco | Detection of protein aggregate morphology through single antifouling nanopore[END_REF] Theses chains presumably occupy space inside the pore and partially block the ion flux. The conformations of those chains inside or near the pore should greatly depend on their affinity with the electrolyte. [START_REF] Milner | Polymer Brushes[END_REF] We studied a Si 3 N 4 31 nm pore grafted with a long polymer: PEG-7kDa. Those pores have been characterized intensely through the study of their conductance in KCl and LiCl buffers at various salt concentrations (Figure 4). First, we plot the current traces at fixed voltages between -200 mV and +200 mV (Figure 4a). In figure 4a, we observe long fluctuations of the current in 10 mM LiCl. This behavior is discussed in the Supporting Information.

From the average value of the current trace, we plot the rectification parameter |I(+200mV)/I(- 200mV)| according to the LiCl or KCl concentration (Figure 4b). When we increase the LiCl concentration from 1 µM to 10 mM, the rectification decreases from 1.49 to 1.17. For concentrations larger than 10 mM, this rectification remains steady (1.14 ± 0.05). In presence of KCl, the rectification remains steady (1.08 ± 0.06) whatever the concentration. Considering the Debye length is larger than 3 nm if the salt concentration is smaller than 10 mM, [START_REF] Schoch | Transport phenomena in nanofluidics[END_REF] the electroosmotic flow could drag the grafted chains along the nanopore. In presence of KCl, the K + cations are linked to the PEG chains, which are too strongly attracted to the negatively charged inner side of the nanopore to be dragged by the electroosmotic flow. In this case, we do not notice any current fluctuation.

Secondly, the average value of each current trace of the Figure 4a is used to plot the current-voltage characteristic curve (I-V curve) of the nanopore (Figure 4b). We observe a linear behavior of the ionic current versus the applied voltage : the ionic conductance through this channel follows the Ohm's law. The IV-curve slope defines the conductance G of the nanopore.

We repeat these steps changing the salt concentration (1 µM < C < 2 M) and nature (LiCl or KCl) to obtain Figure 4d. This figure shows that the pore conductance G increases with salt concentration according to two regimes. At high concentrations (C > 0.1 M), the conductance increases linearly with the salt concentration thus being governed by the bulk ionic transport. At low concentration, the conductance reaches a plateau and is dominated by the movement of counter-ions on the inner surface of the nanopore.

Pore size determination according to its ionic conductance is a frequently discussed matter. [START_REF] Lee | Large apparent electric size of solid-state nanopores due to spatially extended surface conduction[END_REF][START_REF] Ma | Entrance Effects Induced Rectified Ionic Transport in a Nanopore/Channel[END_REF][START_REF] Wen | Physical Model for Rapid and Accurate Determination of Nanopore Size via Conductance Measurement[END_REF] This conductance can be described by following simplified models. [START_REF] Schoch | Transport phenomena in nanofluidics[END_REF][START_REF] Frament | Nanopore Surface Coating Delivers Nanopore Size and Shape through Conductance-Based Sizing[END_REF] At high ionic concentration, the conductance is governed by the bulk conductivity K : G vol = (π R 2 )/L × K where R is the radius of the pore, L its length. Nevertheless, we must take the access conductance G acc = 4 R K of the nanopore into account. [START_REF] Hall | Access resistance of a small circular pore[END_REF] Considering G ionic = 1/(1/G vol + 1/G acc ), we write :

G ionic = K L π R 2 + 1 4 R (1)
In this regime, the bulk conductivity reads :

K = (µ + + µ -) e 2 c (2)
where µ + and µ -are the cation (µ K+ = 4.76 × 10 11 m 2 s -1 J -1 , µ Li+ = 2.50 × 10 11 m 2 s -1 J -1 ) and the anion (µ Cl = 4.94 × 10 11 m 2 s -1 J -1 ) mobilities respectively, e is the elementary charge and c the ion concentration (in ions per m 3 ). Now, we consider that the thickness h of the grafting is the same on the membrane and in the nanopore. [START_REF] Frament | Nanopore Surface Coating Delivers Nanopore Size and Shape through Conductance-Based Sizing[END_REF] Then, the apparent radius and length of the nanopore becomes R = R 0 -h and L = L 0 +2 h respectively after membrane coating, with R 0 and L 0 the radius and length of the pore before grafting. From this, we can deduce a relation between the nanopore dimensions after grafting :

L = L 0 + 2 (R 0 -R ).
Finally, the equations ( 1) and ( 2) become :

G ionic = (µ + + µ -) e 2 c L 0 +2 (R 0 -R ) π R 2 + 1 4 R (3)
The fit of the experimental data (Figure 4d) leads to a decrease of the apparent radius of the nanopore after grafting R LiCl = 9.4 ± 0.6 nm in LiCl buffer and R KCl = 12 ± 0.2 nm in KCl buffer.

Thus, we estimate the thickness of the decorated membrane L LiCl = 32.7 ± 1.2 nm and L KCl = 27.5 ± 0.4 nm in LiCl and KCl buffer respectively. These results show that the grafted chain thickness is h LiCl = 5.6 ± 0.6 nm or h KCl = 3 ± 0.2 nm in presence of LiCl or KCl respectively. Now, we discuss the conformation of the polymer chains grafted on the Si 3 N 4 surface assuming that there is no overlapping between grafted chains. [START_REF] Alexander | Adsorption of chain molecules with a polar head a scaling description[END_REF][START_REF] Gennes | Conformations of Polymers Attached to an Interface[END_REF][START_REF] Auroy | Characterization of the brush regime for grafted polymer layers at the solid-liquid interface[END_REF] First, we could consider that these chains have better affinity with water molecules than with the membrane. In this case, they behave according to a good solvent conformation, as it is depicted in Figure 5. In this situation, the polymer chain has a better affinity for LiCl or KCl solvent than for itself. The chain size is characterized by the Flory radius R F . [START_REF] De Gennes | Scaling Concepts in Polymer Physics[END_REF] With PEG-7kDa, we calculate R F = 7.3 nm (Figure 5a). As R F is larger than the grafted chain thickness, we can assume that the PEG-7kDa chains does not follow this regime. Then, we could consider inter- actions between polymer chains and the solvent are balanced with the ones with the membrane. In this situation, the polymer chain could be considered as an ideal one characterized by the ideal radius R 0 (Figure 5b). As R 0 = 4.4 nm, we could consider that the polymer chains behaves as ideal polymer brush only in presence of LiCl (h LiCl > R 0 ), whereas it is not the case in presence of KCl (h KCl < R 0 ). In the latter case, the PEG chains seem to be collapsed on the membrane in presence of KCl (Figure 5c). In this situation, the polymer chains are more attracted by the Si 3 N 4 surface than by the aqueous phase, and behaves as in bald solvent regime. This behavior could be due to the binding of K + cations. [START_REF] Reiner | Theory for polymer analysis using nanopore-based single-molecule mass spectrometry[END_REF][START_REF] Breton | Exploration of Neutral Versus Polyelectrolyte Behavior of Poly(ethylene glycol)s in Alkali Ion Solutions using Single-Nanopore Recording[END_REF] Then, the chains adopt a cigar-like shape, as it was previously described in. [START_REF] Reiner | Theory for polymer analysis using nanopore-based single-molecule mass spectrometry[END_REF] The PEG chains behave as positive polyelectrolytes in KCl, which increases their attractions with the negatively charged silanol groups on the membrane surface. [START_REF] Behrens | The charge of glass and silica surfaces[END_REF] These observations show that the conformation of grafted PEG chains is controlled by the electrolyte: apparent ideal chain in LiCl and apparent collapsed chain in KCl.

At low ionic concentration, the conductance is dominated by surface effects due to the presence of counter-ions on the inner surface of the nanopore. It reads :

G s = 2πR L 0 µ + e σ (4)
with σ the surface charge of the pore, µ + the ionic mobility of the positive counterions, considering that the negative charge surface is due to the the dissociation of silanol groups. [START_REF] Behrens | The charge of glass and silica surfaces[END_REF] At low salt concentrations, we can see on Figure 4 that the pore yields a lower conductance with LiCl than with KCl : G LiCl s = 5.4 ± 0.1 nS and G KCl s = 9.4 ± 0.2 nS. According to Eq. ( 4), we calculate the corresponding surface charge : σ LiCl = 46 ± 6 mC/m 2 and σ KCl = 33 ± 3 mC/m 2 . The surface charge of the grafted nanopore is not dependent on LiCl or KCl concentration. These values are in good agreement with the one measured with shorter PEG chain, [START_REF] Giamblanco | Detection of protein aggregate morphology through single antifouling nanopore[END_REF] but they are smaller than the surface charge measured with uncoated Si 3 N 4 channels σ SiN = 60 mC/m 2 . [START_REF] Stein | Surfacecharge-governed ion transport in nanofluidic channels[END_REF] This decrease could be explained by the polymer chain brush covering the inner side of the nanopore. Moreover, this screening effect could be enhanced by the formation of K + /PEG complexes in presence of KCl (decrease from 60 to 33 mC/m 2 ). [START_REF] Reiner | Theory for polymer analysis using nanopore-based single-molecule mass spectrometry[END_REF] Now, we propose to use this nanopore to characterize interactions between proteins and urea at the nanometer scale. Protein denaturation is usually performed with urea addition. Nevertheless this process is not yet well understood. Two mechanisms are usually described: [START_REF] Xia | Collapse of unfolded proteins in a mixture of denaturants[END_REF] the first one is based on the disruption of the structure of water around the protein, which increases the solubility of hydrophobic groups. [START_REF] Hammes | An Investigation of Water-Urea and Water-Urea-Polyethylene Glycol Interactions[END_REF] These bindings would lead to the entering of urea into the unfolded protein. Thus, urea would behave as a surfactant between the protein and water. [START_REF] Zangi | Urea's action on hydrophobic interactions[END_REF] The second mechanism is based on direct interactions between urea and the protein. [START_REF] Hua | Urea denaturation by stronger dispersion interactions with proteins than water implies a 2-stage unfolding[END_REF] In this work, PEG chains act as a model chain to mimic interactions of proteins with urea or water. As the electrical detection coupled with microfluidic integration allows to precise the PEG conformation, we use this method to determine the PEG chain conformation in presence of LiCl or KCl. Firstly, we consider the high salt concentration where the conductance is governed by the ionic flow. We plot IV-curves in a 0.5 M LiCl solution by increasing the urea concentration from 0 M to 9 M (Figure 6a andS5). As we do not notice current rectification, we use the slope of each IV-curve to calculate the nanopore conductance. In presence of 0.5M LiCl, we observe that the nanopore conductance decreases linearly from 52.1 ± 0.1 nS to 25 ± 2.3 nS when urea concentration goes from 0 up to 9 M (Figure 7b). As the ionic mobility is inversely proportional to the viscosity, this decrease could be due to the viscosity increase, when we increase the urea concentration.

To answer this assumption, we consider a conductance G v calculated from conductivity K of the urea solution :

G v = K K b G b (5)
with G b and K b the pore conductance and the electrolyte conductivity without urea respectively. This relation, based only on the viscosity variations, is plotted in Figure 6b with square markers. At 0.5 M LiCl, there is a good agreement between conductance measurements, plotted with circle markers, and calculated conductance G v . We observe the same behavior at 0.5 M KCl (Figure 6c). Then, we repeat these measurements at low salt concentration where the conductance is dominated by counter-ions, i.e. charge density on the inner side of the nanopore. We observe the same behavior with 10 mM KCl (Figure 6e), but the conductance does not decrease as much as expected as the viscosity increases when we add urea in presence of 10 mM LiCl (Figure 6d).

Then, we normalize the conductance G with the conductance G v calculated above (Eq. 5) to know if the conductance variations are due to viscosity or to geometrical ones :

G N = G G v (6)
Considering the regime of high ionic concentrations, we observe that the normalized conductance G N is constant G N = 0.99 ± 0.05 at 0.5 M LiCl (Figure 7a). In this regime, the conductance inside the nanopore is mainly governed by the ionic flow. We could neglect the effects due to the surface conductance of the pore. According to Eq. 3, G N is just function of the geometrical dimensions of the nanopore. Then, we define the normalized radius :

R N = R R b (7)
where R and R b represent the apparent nanopore radius with or without urea respectively, calculated from Eq. 3. In Figure 7c, this normalized radius is steady when the urea concentration increases R N = 0.99 ± 0.02. Then, we could suppose that urea has no action on the pore sizes and doesn't affect the conformation of PEG chains at high LiCl concentrations. We observe the same behavior in presence of 0.5 M KCl (Figure 7a). In this case, the normalized conductance does not vary (G N = 0.96 ± 0.07). Then, we plot the normalized radius according to Eq. 7 versus the urea concentration. It is constant R N = 0.98 ± 0.03. Then, urea seems to not affect the conformation of grafted PEG chains in presence of KCl, as it was the case with LiCl. Now, we could consider the regime of low ionic concentrations. If we plot the normalized conductance G N , we observe that it increases with urea concentration (Figure 7b). Considering that the nanopore radius is not modified by the increase of urea concentration, this increase could be due to the increase of counter-ion charge according to Eq. 4. Then, we define a normalized surface charge:

σ N = σ σ b (8)
where σ and σ b represent the nanopore charge surface with or without urea respectively, calculated from Eq. 4. σ N increases with urea concentration until 2.7 times at 9 M urea (Figure 7d). This strong increase could be explained by the high dipole moment of urea (4.56 D), which is 2.5 times higher than the water one (1.85 D). Then, we could suppose that urea could replace water inside the PEG brush and modify strongly the counter-ions distribution inside the nanopore. Now, we consider the experiments performed with 10 mM KCl. We observe that the normalized conductance of the pore doesn't increase with urea concentration : G N = 1.03±0.07 (Figure 7b). Considering that the nanopore conductance is gov- erned by the counter-ions, we plot the normalized charge surface σ N in Figure 7d. As σ N is steady σ N = 1.08 ± 0.15, the surface charge seems to not be modified by the addition of urea. Contrary to the observations performed with LiCl, urea molecules seem to not replace the water ones inside the PEG brush in presence of KCl. These observations could be explained by considering the PEG conformation. Previously, we have shown that the PEG chains are strongly attracted to the Si 3 N 4 surface in presence of KCl. Then, in that state the polymer brush behaves like in an apparent bad solvent. In this collapsed conformation, we could suppose urea can't go inside the PEG brush to replace water.

We designed and manufactured a cheap microfluidic master from an easy and fast 3D-printerbased protocol for the reversible integration of a solid-state nanopore chip. This device permits to quickly perform a large series of electrical measurements in different environments (nature and concentration of salt, urea) without disassembling the nanopore from the microfluidic sys-tem. Also, our device could be used to rapidly pierce nanopores thanks to the dielectric breakdown method [START_REF] Kwok | Nanopore fabrication by controlled dielectric breakdown[END_REF][START_REF] Feng | Observation of ionic Coulomb blockade in nanopores[END_REF][START_REF] Lin | Characterization of DNA duplex unzipping through a sub-2 nm solid-state nanopore[END_REF] to be then stored for future use. We found that, at low salt concentrations, the conductance is dominated by the movement of counter-ions on the inner surface of the nanopore and that at high concentrations, the conductance is governed by the bulk ionic transport. From electrical measurements we can deduce the conformation of the polymer chains grafted on the nanopore surface. At high ionic concentration, the polymer chains behave as ideal polymer brush or as a cigarlike shape respectively in presence of LiCl or KCl. For ideal chains, urea molecules can stand in for the water molecules leading to an increase of the apparent charge of the polymer-grafted surface. On the other hand, if the chain is collapsed, the urea molecule cannot enter into the chain and thus don't change its conformation nor its polarisability. This biomimetic system allows to enhance our understanding of interactions between urea and proteins. In our case, the PEG chains in presence of LiCl are similar to the hydrophilic domains of the protein whereas in presence of KCl they behave like the hydrophobic domains. This experimental study is in good agreement with previous simulations of protein denaturation with urea. [START_REF] Xia | Collapse of unfolded proteins in a mixture of denaturants[END_REF][START_REF] Hua | Urea denaturation by stronger dispersion interactions with proteins than water implies a 2-stage unfolding[END_REF] Nevertheless, we did not observe the penetration of urea into the hydrophobic core. [START_REF] Hua | Urea denaturation by stronger dispersion interactions with proteins than water implies a 2-stage unfolding[END_REF] The authors declare no competing financial interest.

and fluctuations with hairy nanopore; Conductance measurement in urea;

Figure 1 :

 1 Figure 1: Protocol for the realization of the nanopore chip microfluidic device (not to scale). a) (left) The printed mold is covered by PDMS. (right) A thin PDMS film is produced by spin coating b) Curing at 75 o C. c) Plasma bonding (first) d) Masking e) Plasma bonding (second) f) Tube insertion and PDMS casting

Figure 2 :

 2 Figure 2: PDMS based device for easy solid-state nanopore chip use. a) Picture of mold 3D-printed on a 3 in silicon wafer. Four devices can be assembled from the eight printed patterns. b) Profiles of a 3D-printed mold before (red) and after melting. Two profiles are presented to show the uniformity of the printed parts, their location are approximately represented by red marks on the inset. c) Side and top schematic view of the device. d) Picture of the device with a mounted nanopore chip.

Figure 3 :

 3 Figure 3: Morphology of a nanopore chip. a) Stylized side-view of a nanopore chip grafted with PEG-7kDa. b) TEM view of the pierced pore before grafting.

Figure 4 :

 4 Figure 4: Characterization of a hairy Si 3 N 4 nanopore. a) Typical sequence of current measurements used to obtain one current versus voltage (IV) plot a hairy nanopore. Each trace in this graph corresponds to a voltage ranging from -200 mV (blue) to 200 mV (red) in a 10 mM LiCl buffer. b) IV plots obtained with 1 µM to 2 M LiCl. c) Rectification |I(+200 mV )/I(-200 mV )| of the same nanopore according to the KCl and LiCl concentrations. d) Conductance of the pore at different KCl and LiCl concentrations showing the transition between surface conduction at low concentration and ionic conduction at higher concentrations. Solutions were buffered by 5 mM Hepes at pH 7.4. Experiments are performed with a 31 nm wide nanopore grafted with PEG-7kDa in a 10 mM LiCl buffer.

Figure 5 :

 5 Figure 5: Conformations of polymer chain grafted on Si 3 N 4 membrane a) behavior in good solvent regime b) in ideal solvent regime c) in bad solvent regime. N is the number of monomeric units of length a. Calculations are perform with PEG-7kDa chain (N = 159, a = 0.35 nm).

Figure 6 :

 6 Figure 6: Interactions of urea with polymer brushes in confined medium. a) IV curves obtained with urea concentrations ranging from 10 mM to 9 M at 0.5 M LiCl. Pore conductance (plotted with circle markers) versus urea concentration at b) 0.5 M LiCl, c) 0.5 M KCl d) 10 mM LiCl e) 10 mM KCl. The conductance calculated from bulk conductivity (Eq. 5) is plotted with square markers. All experiments were performed in buffered solution with 5 mM Hepes at pH 7.5.

Figure 7 :

 7 Figure 7: Normalized pore conductance as a function of urea concentration at a) 0.5 M LiCl and KCl and b) 10 mM LiCl and KCl. The dotted lines represents the average values of the conductance. None have been plotted for 10 mM LiCl. The plain black line represents the expected value of G N = 1. c) Normalized pore radius at 0.5 M LiCl and KCl at various urea concentrations. d) Normalized surface charge of the nanopore at 10 mM LiCl and KCl at various urea concentrations.

Acknowledgement This research was supported by the "DEFI CNRS Instrumentation aux limites" 2016 & 2017 and by ANR Epsilomics (ANR-17-CE09-0044-02). We thank Johanna Canovas, Rasta Ghasemi and Abdelghani Oukhaled for their assistance. This publication is dedicated to Loïc Auvray's memory.

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: Description of a traditional flow-cell; Calculation of the roughness measurement; Flow simulation in microfluidic channels; Current traces Graphical TOC Entry


Antoine Lassagne
email: antoine.lassagne@renault.com

Andras Kemeny
email: andras.kemeny@renault.com

Javier Posselt
email: javier.posselt@renault.com

Frederic Merienne
email: frederic.merienne@ensam.eu

Comparing Tangible and Fully Virtual Haptic Systems for HMI Studies in Simulated Driving Situations

Keywords: Automotive design, HMI, haptics, perception, virtual reality

The contribution of tangible and intangible feedback is compared for virtual tactile car Human-Machine Interfaces (HMI) design, to measure their performance both in static conditions and while driving. A subjectively calibrated transparent glass provides tangible passive haptics, and visual cues are used to study sensory substitutionbased intangible interactions. The results show that the performance of the subjects was significantly improved in driving conditions as they interacted faster, more accurately and with a higher satisfaction. In addition, our findings highlight that the contribution of tangible systems is significantly lower in driving conditions, raising new questions about the nature of haptic modalities in the function of the context of use. This study provides additional knowledge about the influence of dynamic environments and external tasks on haptic perception.

Introduction

Virtual Reality (VR) has many applications in automotive industry, especially in engineering design. However, haptic feedback may offer new capabilities to simulators, allowing effective interactions and improved perception to enhance the immersion [Bro99]. HMI testing is one of many use-cases that inherently requires interactions.

Passive Haptics

Passive haptic consists of opposing tangible objects, co-located with virtual objects which need haptics. Hoffman et al. [Hof98] reached a satisfactory haptic feedback by introducing physical plates into a virtual environment (VE). Carlin et al. [Car97] evaluated the possibility of curing spider phobia through a similar implementation and also provided promising results. Insko et al. [Ins01] significantly improved presence in a visual-cliff environment augmented by means of passive haptics. They all concluded that passive haptic generates appropriate haptic feedback in various situations, and they emphasize the added value of adding haptic capabilities to VE, especially passive haptic which is safe, affordable, simple to implement and requires no continuous computation.

Intangible haptics

Previous research has proposed intangible haptic systems with sensory substitution, effective in numerous uses. Realistic visual cues like inter-reflections or shadows [Hu00, Mad01, Cha10] provided significant performance improvement in interactions. Kitagawa et al. [Kit04] measured the accuracy of subjects applying force in the presence of visual and auditory non-realistic cues and showed that continuous visual cues significantly improve performance, while auditory should be discrete for intrusiveness issues. In similar studies, Petzold et al. [Pet04] concluded that despite the advantages of sensory substitution, there exists an additional cognitive burden of translating sensory information other than haptic into force.

Spatial Perception in VEs

Perception of depth and scale is not always accurate in VEs and it can deteriorate the haptic when not taken into consideration. According to different studies [Ken07, Loo96, Cut95], the environment should include a lot of surrounding objects to enhance spatial perception, and there exist important disparities between subjects. Research also demonstrated that high visual quality may improve spatial perception [Int06]. In driving simulation, motion parallax and egocentric direction are sources of information, as discussed by Kemeny et al. [Kem03]. CAVE systems bring additional sources of bias.

Tactile dashboard HMI design

Previous results compared tangible and intangible interfaces in static conditions [Las17]. It provided an evaluation of different haptic systems for tactile dashboard HMI design and highlighted several issues.

• The tangible interface significantly enhanced usability in comparison with the visual-only interface which was difficult to interact with. • A lack of robustness in human spatial perception was identified as a major obstacle to VEs interaction, especially when no tangible haptic system can provide additional space references.

Subjects were not driving during this experiment. Further studies are thus needed in a driving context to complete this evaluation. The final goal would be to implement such a system as an effective engineering tool.

Research Questions

This study aims at comparing the performances of haptic systems in different contexts in immersive VEs. We seek to study how a dynamic environment and a driving task can lead to performance differences when compared to the static situation previously studied.

Our questions of research are the following :

• To which extent a dynamic environment and a driving task modify the performance of interactive systems in immersive environments ? • Is this influence different between several haptic modalities with known performances ?

We expect major differences in comparison with a static context due to two factors.

• The cognitive burden induced by driving should force subjects to focus and may improve their performance. • We expect a continuously renewed environment to decrease the perceptual shifts observed in static situations, as demonstrated by Atkins et al. [Atk01,Atk03].

Methodology

11 subjects (males and females, Renault employees) were instructed to interact with a simple virtual tactile dashboard HMI in the CAVE while driving. The interface displayed 4 buttons with 3 scales. Subjects successively employed intangible and tangible interaction systems to proceed with 2 (intangible, tangible) * 3 (button scales) series, in a random order. During series, 20 interactions were called by the HMI (one button turns green until the subject touches it). The flow of series is described in figure 1.

As performance indicators, we measured execution duration, relative error, and two questionnaires were filled out by subjects, corresponding to tangible and intangible haptic modes.

Initial state of buttons

Color change of one button These experiments took place in Renault P3I (Industrial Immersive Integration Platform) CAVE, a 4-sided virtual reality room powered by ultra-short throw full HD Panasonic projectors. We use active stereoscopy and optical tracking with A.R.T. infrared technologies † . Interaction implementation is described below :

T 0 T 1 User action T 1 -T 0 R x R y
• Intangible feedbacks : color changes from the interface, no haptic feedback. • Tangible feedback : a passive transparent glass attached to a support is added.

In addition, a 3-finger tracked glove is used to acquire interactions.

The virtual scene was displayed by Oktal SCANeR Studio [Kem93] and placed users inside a car in driving conditions (figure 2). Logitech G25 steering wheel was dedicated to driving. Theoretical latency was under 30ms with this setup. The methodology and apparatus were identical during static studies, except for the smallest scale. 17 subjects took part in static studies, 3 of them took part in both experiments.

Subjective calibration Consistency between vision and interactions was attained by means of a subjective calibration. This calibration allowed us to bypass morphologic disparities. Subjects were instructed to successively touch 3 corners of the virtual touchscreen to map the collision engine to their perceived visual location (figure 3). When the transparent glass was in use, it was placed by subjects on what they reported was the right location. We put a particular attention on this step, as misplaced glasses had highly degraded the experiment during preparation.

Results

Student's t-Test was used for the statistical analysis of paired samples among the different conditions (tangible or intangible interactions, static or dynamic scenarios), with a significance level of 5%.

Execution duration

The data comparison of execution duration is displayed in figure 4. The static study resulted in significant performance differences between intangible and tangible systems. Driving simulation conditions provided the following results :

• Interactions with the intangible system were significantly (∼ 33%) faster while driving. • The tangible system had a lower potential for improvement as it already provided low execution duration. It still improved moderately. • Standard deviation of execution duration without tangible interface has been highly reduced.

Driving conditions led to a higher performance for both intangible and tangible studies. These conditions were more advantageous to the intangible interaction system which could almost attain the same level of performance as the tangible one.

Robustness issues -Low robustness was a major obstacle to subject interaction in static studies. Their execution duration was higher due to spatial perception issues, especially with small buttons that some subjects were unable to interact with at all. In this study, these issues were not encountered. Furthermore, we added a third, smaller button scale which did not cause difficulties to any subject. Reduced standard deviation also suggests that the system was easier to apprehend as there were less disparities between subjects.

Relative Error

The data comparison of relative errors is displayed in figure 5. Accuracy -Once more, the difficulties observed with the intangible interface in static mode seem to disappear in driving conditions. Whereas intangible haptics gave a poor performance in static mode, it nearly attains the same level of accuracy as tangible haptics in dynamic mode. Standard deviation is still significantly lower with the tangible system, which also benefited from driving conditions. Subjects precision and disparities -Standard deviation of relative errors is illustrated in figure 6. Disparities were especially reduced in intangible mode, which provided the highest values. Tangible interactions had already provided low disparities in the static context and thus had less potential for progress, nevertheless performance was also improved.

Subject judgement

All subjects reported that they encountered no difficulties to interact while driving. Their interactions were successful with the smallest button scale, and they were surprised when told that subjects had difficulties with medium scales during static studies.

A majority of the subjects reported that they preferred the intangible modality for various reasons : • Easier setup : placing the glass on the right place could be difficult and had an important influence on the experience, therefore 2 to 5 minutes were needed for each subject. • False-positives : when an interaction was failed with the tangible system, subjects felt a haptic feedback anyway. No wrong feedback was produced when the intangible system was used. • Constraints : subjects were foreseeing future usecases involving interactions with other non-flat pieces, which could not always be achieved with a transparent glass.

Discussion

Spatial perception

The main identified influence on performance is the quality of spatial perception, weakened by unreliable perceptual inputs. Inaccurate spatial perception brings inaccurate actions and a reduced confidence, which users encountered in a static context. Several parameters of this experiment led to an improved spatial perception.

Level of concentration on cues affects perception -

The driving task forced subjects to continuously concentrate on their environment to prevent a crash, whereas they could keep their eyes on the HMI during static studies. Spatial cues are distributed inside the whole VE (objects of known size, parallax, stereoscopy, etc.) and are thus more easily perceived by subjects when they focus on them. Furthermore, these cues are continuously renewed as the car progresses, and they are seen from different points of view, in different layouts. Moving the head also adds parallax cues.

Shifts in visuo-haptic consistency -Morphologic disparities are often responsible for miscalculated vision frustrums. Most of the time, the software uses mean anthropometric data to interpolate the location of the eyes from the location of the tracked 3D glasses, causing inaccurate eye location data. These disparities are valued in millimeters but can become centimeters on the screen.

The subjective calibration used in this experiment grants visuo-haptic consistency in the configuration of the calibration, when looking straight forward. However, while some subjects looked straight forward to interact, others kept their head toward the road and preferred moving only their eyes. Subjective methods are thus not satisfactory, although analytic methods are not without problems. In early experiments, we measured IPD without satisfactory visuohaptic consistency. Further studies will try to improve this aspect by means of continuous recalibration.

Choosing the best tool for HMI design in VR

The added value of tangible interfaces was not probative in the driving simulation context, whereas it significantly improved interaction performance in the static context. The subjects were executing the exact same task and the only difference was the driving conditions. Therefore, the importance of the relationship between haptic modalities and usecases/context must be emphasized. There exists no perfect haptic device for every use, but their individual advantages can be optimized for specific use-cases.

Active or passive scenario -Active and passive actions are handled differently by the brain, as studied by Mima et al. [Mim99]. Different areas and magnitudes are involved, inducing performance disparities, when subjects are acting or not.

• Haptic systems with poor performance should only be used in active scenarios to obtain satisfactory results. • Haptic systems with assumed higher performance can be used in either active or passive scenarios.

Industrial specificities -The subjects' preferences may have been influenced by their affiliation to the automotive industry. Industrial projects induce additional constraints like non-intrusiveness, high robustness, and a focus on workability rather than realism.

Since an interactive device is supposed to be used in a specific context when implemented in an industry, this context should be known with as much precision as possible before choosing a haptic device. Indeed, even a small parameter, like static or driving conditions, can have a significant influence.

Conclusions and implications

The comparison of tangible and fully virtual haptic systems for HMI studies brings new data on the contribution of haptic modalities in driving conditions. The respective performances of two haptic implementations, a sensory substitution-based intangible system and a tangible passive haptic system, were compared in driving simulations and static contexts by means of efficiency, accuracy and user satisfaction. Results show that subjects significantly improved their performance in driving conditions. Furthermore, the added value of tangible haptics was significantly lower in driving conditions. Our results bring evidence that the evaluation of haptic modalities has to be carried out in driving conditions, as their contribution may be very different in static conditions.

Figure 1 :

 1 Figure 1: Flow of a single serie. Relative errors (Rx, Ry) represent the relative offset between the location of the interaction and the actual center of the button. T 1 -T 0 is the time spent by the user to perform an interaction.

Figure 2 :

 2 Figure 2: Subject immersed in the VE.

Figure 3 :

 3 Figure 3: Subjective calibration allowing accurate visuo-haptic consistency.

Figure 4 :

 4 Figure 4: Mean execution duration difference between static and driving context for tangible and intangible systems.

Figure 5 :

 5 Figure 5: Mean relative error difference between static and driving context for tangible and intangible systems.

Figure 6 :

 6 Figure 6: Mean standard deviations of relative errors.

† http://www.ar-tracking.com/home/