Thi Xuan

Huong Le

Mathieu Etienne
email: mathieu.etienne@univ-lorraine.fr

François Lapicque

Alain Hehn

Neus Vilà

Alain Walcarius

Dr Etienne Mathieu

Local removal of oxygen for NAD + detection in aerated solution

Keywords: NAD(P)H, [Cp*Rh(bpy)Cl] +, diazonium electrografting, electrochemical oxygen filter, biosensor

The mediated electrochemical reduction of NAD(P) + in aerated solution was investigated and exploited for NAD(P)H regeneration in view of biosensing application. Oxygen was locally removed from the electrode surface with an electrochemical oxygen filter made of platinum grids positioned close to the oxygen sensitive reactions. The working electrode was a bucky paper functionalized with [Cp*Rh(bpy)Cl] + by diazonium electrografting. The local oxygen removal was first evaluated, before to be applied to deoxygenation for NAD + and NADP + reduction. A porous silica gel layer encapsulating D-sorbitol dehydrogenase was finally deposited on the top of the modified bucky paper and the resulting bioelectrode was found sensitive to the presence of D-fructose.

Introduction

β-nicotinamide adenine dinucleotide NAD + /NADH, and its phosphate derivative (NADP + /NADPH) have been widely studied for their application in electrochemical biosensor and bioelectrocatalysis [START_REF] Hollmann | Non-enzymatic regeneration of nicotinamide and flavin cofactors for monooxygenase catalysis[END_REF][START_REF] Gorton | NAD(P)-Based Biosensors[END_REF][START_REF] Hollmann | Biocatalytic Redox Reactions for Organic Synthesis: Nonconventional Regeneration Methods[END_REF][START_REF] Dominguez-Benetton | Enzymatic Electrosynthesis: An Overview on the Progress in Enzyme-Electrodes for the Production of Electricity, Fuels and Chemicals[END_REF][START_REF] Zhang | Functional electrodes for enzymatic electrosynthesis[END_REF]. Indeed, there exists a large variety of NAD-dependent redox enzymes that can catalyze the electrochemical oxidation or electrochemical reduction of a large range of substrate [START_REF] Kornberger | Electrochemical Cofactor Regeneration[END_REF].

For enzymatic catalysis, these enzymes provide a regio-and a stereoselectivity particularly that is powerful to synthetize complex molecules, with examples reported for dehydrogenase [START_REF] Kohlmann | Electroenzymatic synthesis[END_REF], P450 cytochromes with their reductase [START_REF] Hehn | Biosynthesis of coumarins in plants : a major pathway still to be unravelled for cytochrome P450 enzymes[END_REF], etc. Electrochemistry should be of interest for the regeneration of the reduced form of the cofactor. The direct electrochemical reduction of NAD(P) + to NAD(P)H is however kinetically limited, and the low potential that must be used for allowing electron transfer reactions with the nicotinamide moieties induces side reactions and dimerization and thus inactivation of the enzymatic cofactor [START_REF] Morrison | Improved strategies for electrochemical 1,4-NAD(P)H2regeneration: A new era of bioreactors for industrial biocatalysis[END_REF][START_REF] Hildebrand | Synthesis, Characterization and Application of New Rhodium Complexes for Indirect Electrochemical Cofactor Regeneration[END_REF]. For that reason, redox catalysts have been evaluated and the complex [Cp*Rh(bpy)Cl] + has been identified some decades ago to be particularly efficient for the electrocatalytic reduction of NAD(P) + to NAD(P)H [START_REF] Ruppert | Efficient indirect electrochemical in-situ regeneration of nadh:electrochemically driven enzymatic reduction of pyruvate catalyzed by D-LDH[END_REF].

Electrodes modified with this Rhodium complex have been developed, targeting specifically the electrochemical reduction of the nicotinamide cofactor, with modified conductive polymer matrices [START_REF] Cosnier | A polypyrrole [RhIIIC5Me5(bpy)Cl]+ modified electrode for the reduction of NAD+ cofactor[END_REF][START_REF] Beley | Electrochemical regeneration of nicotinamide cofactor using a polypyrrole rhodium bis-terpyridine modified electrode[END_REF][START_REF] Van Esch | Reduction of Nicotinamides, Flavins, and Manganese Porphyrins by Formate, Catalyzed by Membrane-Bound Rhodium Complexes[END_REF], and carbon nanomaterials [START_REF] Tan | Regeneration of the NADH Cofactor by a Rhodium Complex Immobilized on Multi-Walled Carbon Nanotubes[END_REF][START_REF] Lee | Graphene-Rh-complex hydrogels for boosting redox biocatalysis[END_REF]. More recently, we reported strategies of immobilization based on an initial step of diazonium electrografting [START_REF] Zhang | Covalent Immobilization of (2,2′-Bipyridyl) (Pentamethylcyclopentadienyl)-Rhodium Complex on a Porous Carbon Electrode for Efficient Electrocatalytic NADH Regeneration[END_REF][START_REF] Zhang | Molecular and Biological Catalysts Coimmobilization on Electrode by Combining Diazonium Electrografting and Sequential Click Chemistry[END_REF][START_REF] Zhang | Electrocatalytic Biosynthesis using a Bucky Paper Functionalized by [Cp*Rh(bpy)Cl] + and a Renewable Enzymatic Layer[END_REF]. A first strategy involved the electrografting of an azide containing molecule before conducting azide-alkyne Huisgen cycloaddition (click chemistry) to introduce a bipyridine group and a final complexation with the rhodium(III) metal [START_REF] Zhang | Covalent Immobilization of (2,2′-Bipyridyl) (Pentamethylcyclopentadienyl)-Rhodium Complex on a Porous Carbon Electrode for Efficient Electrocatalytic NADH Regeneration[END_REF]. We later introduced a simpler strategy by electrografting directly the bipyridine group on the surface of a bucky paper followed by a last step of complexation with rhodium [START_REF] Zhang | Electrocatalytic Biosynthesis using a Bucky Paper Functionalized by [Cp*Rh(bpy)Cl] + and a Renewable Enzymatic Layer[END_REF].

The modified electrodes have been applied to NADH regeneration and D-sorbitol biosynthesis in an oxygen depleted solution, the removal of O2 being achieved by purging the solution with N2 and maintaining a nitrogen flow during the whole experiment [START_REF] Zhang | Covalent Immobilization of (2,2′-Bipyridyl) (Pentamethylcyclopentadienyl)-Rhodium Complex on a Porous Carbon Electrode for Efficient Electrocatalytic NADH Regeneration[END_REF][START_REF] Zhang | Electrocatalytic Biosynthesis using a Bucky Paper Functionalized by [Cp*Rh(bpy)Cl] + and a Renewable Enzymatic Layer[END_REF]. The reason is that oxygen can interfere with the reduction of NAD + , which limits the accuracy of the electrochemical measurement [START_REF] Gorton | NAD(P)-Based Biosensors[END_REF], and can have deleterious effect on the catalyst or cofactor itself [START_REF] Tosstorff | Mediated electron transfer with monooxygenases -Insight in interactions between reduced mediators and the co-substrate oxygen[END_REF], which is highly problematic for biocatalytic applications.

Another major field of interest involving nicotinamide cofactors is related to biosensors [START_REF] Gorton | NAD(P)-Based Biosensors[END_REF]. In that case, the main target was the electrochemical oxidation of NADH or NADPH.

Again, the electrochemical reaction is slow and a large variety of redox mediators have been proposed, immobilized on electrode and evaluated in electrochemical biosensors [START_REF] Gorton | Electrochemistry of NAD(P) + /NAD(P)H[END_REF]. The reason to favor oxidation reactions is due to the absence of interference from molecular oxygen at the potential used for the biodetection. The interest of NAD(P) + reduction in biosensor application can only be evaluated in fully deoxygenated media.

Generally, methods for oxygen elimination can be sub-divided into four categories, (i) namely physical (purging the solution with nitrogen/argon flow in the solution), (ii) chemical (by adding oxygen reducing agent such as ascorbic acid [START_REF] Andersen | An oxygen insensitive microsensor for nitrous oxide, Sensors Actuators[END_REF] or sodium thiosulfate [START_REF] Gu | Eliminating the interference of oxygen for sensing hydrogen peroxide with the polyaniline modified electrode[END_REF]), (iii) enzymatic (using glucose as oxygen scavenger in the presence of an enzyme to reduce oxygen molecules [START_REF] Swoboda | Enzymatic Oxygen Scavenging for Photostability without pH Drop in Single-Molecule Experiments[END_REF]), and (iv) electrochemical [START_REF] Hanekamp | An electrochemical scrubber for the elimination of eluent background effects in polarographic flow-through detection[END_REF][START_REF] Bertin | Interdigitated microelectrodes for oxygen removal in N2H4sensors[END_REF]. The latter can have some significant advantages, if small volumes have to be treated without introducing additional reactive species in the analyzed solution, i.e. without introduction of oxygen scavenger.

In this work, we have investigated the possibility of coupling local oxygen removal with NAD(P) + reduction for application in biosensing. The working electrode was a bucky paper functionalized with [Cp*Rh(bpy)Cl] + . It was obtained by diazonium electrografting of aminobipyridine followed by complexation of RhCp*Cl. It was covered by an electrochemical filter that allowed for local elimination of oxygen, according to an idea that we reported very recently [START_REF] Etienne | Electrochemical Filter To Remove Oxygen Interference Locally, Rapidly, and Temporarily for Sensing Applications[END_REF]. The effectiveness of oxygen removal was first checked in a background electrolyte solution and then in the presence of NAD(P) + to evaluate its interest for reductive cofactor regeneration in aerated solutions. The electrode was finally modified by D-sorbitol dehydrogenase and was found sensitive to D-fructose thanks to the electrochemical monitoring of NAD + that was produced when D-fructose was converted to D-sorbitol.

Experimental

Chemicals

Platinum grid (Pt, 0.12 mm nominal aperture, 0.04 mm wire diameter) and polyamide -Nylon grid (39 µm wire diameter, 50 µm nominal aperture) were purchased from Goodfellow, England. Multi-walled carbon nanotubes (MWCNT, > 95%, Φ 6-9nm, L 5µm), D-fructose (99 wt %), 3-glycidoxypropyltrimethoxysilane (GPS, 98%), β-nicotinamide adenine dinucleotide hydrate (NAD + , ≥ 96.5%), β-nicotinamide adenine dinucleotide, reduced disodium salt hydrate (NADH, ≥ 97 %), pentamethylcyclopentadienylrhodium (III) chloride dimer ((RhCp*Cl2)2), 1M hydrochloric acid solution, potassium chloride (99.99 %) were obtained from Sigma-Aldrich. Tetraethoxysilane (TEOS, 98%, Alfa Aesar), poly(ethylene imine) (PEI, 50% w/v in water, Mn = 60000, Fluka), sodium dihydrogen phosphate (99.5%, Merck), dichloromethane (Carlo Erba), glassy microfiber filters (0.26 mm thickness, 70 mm diameter, Whatman ® ) were used as received. Overproduction of N-His(6) D-sorbitol dehydrogenase (NHSD) (800 U mL -1 ) was performed with Escherichia coli BL21GOLD (DE3) containing the corresponding expression-vectors pET-24a(+) (Novagen) and purification of the enzyme was performed with Histrap columns (GE Healthcare). All solutions were prepared with high purity water (18 MΩ cm) from a Purelab Option water purification system (Elga LabWater, Veolia Water STI, France).

Electrochemical device and procedure for oxygen removal from aqueous medium

The electrochemical cell was composed of four electrodes and fabricated from Teflon.

Working electrode 1 (WE1) was a bucky paper or a glassy carbon electrode (GCE) and working electrode 2 (WE2) was made of one or a combination of two platinum grids (see Figure 1). These two working electrodes were separated by a nylon grid of less than 50 µm thickness.

Glassy carbon was firstly wet-polished by SiC grinding paper (≠4000, Struers, Denmark) for 1 min, then cleaned with ethanol and distilled water under ultrasonic condition. The experiments were conducted with an Autolab PGSTAT 100 bipotentiostat in a four-electrode configuration (two working electrodes, Ag/AgCl/3M KCl reference electrode, and stainless steel counter electrode). The reaction of oxygen with the filter and the efficiency of the electrochemical removal was investigated by cyclic voltammetry. Usually, a 10 mL aqueous solution of KCl (0.1 M) was utilized as supporting electrolyte. When needed, purging the solution with N2 gas was performed at least 15 min before running experiment and N2 flow was kept over the solution during the experiment.

Electrode modification, NAD(P)H regeneration procedure, and bioelectrode fabrication

The bucky paper electrode (BP) was prepared following the reported literature [START_REF] Hussein | Fabrication and characterization of buckypaperbased nanostructured electrodes as a novel material for biofuel cell applications[END_REF]. 10 mg MWCNT was dispersed in 50 mL ethanol by ultrasonication for 5 h. Afterwards, the suspension was decanted and vacuum filtered using the PVDF membrane filter. Then, the material was dried at 50 °C overnight. Finally, the electrode was cut at the suitable size before use. The immobilization of [Cp*Rh(bpy)Cl] + (BP-Bpy-Rh) was prepared following the protocol published in a previous article [START_REF] Zhang | Electrocatalytic Biosynthesis using a Bucky Paper Functionalized by [Cp*Rh(bpy)Cl] + and a Renewable Enzymatic Layer[END_REF]. This functionalized electrode was used for NAD + or NADP + reduction by observation of the catalytic peak on BP-Bpy-Rh during NAD + /NADP + addition as schematically shown in Figure 2. Oxygen removal was achieved by applying a constant potential (-0.4 V vs. Ag/AgCl) at the oxygen filter (WE2). The catalytic response was evaluated by running cyclic voltammetry from -0.4 V to -0.9 V vs. Ag/AgCl at 5 mV s -1 scan rate.

For the detection of D-fructose, a compact cell was designed, including different layers. First, D-sorbitol dehydrogenase (DSDH) in silica gel, called here DSDH sol was deposited on a microfiber filter layer. The DSDH sol was synthesized by stirring overnight a mixture of 0.13 g GPS, 0.18 g TEOS with 0.5 mL water and 0.625 mL 0.01 M HCl [START_REF] Mazurenko | Enzymatic bioreactor for simultaneous electrosynthesis and energy production[END_REF]. The following day, the sol was diluted three times before mixing 40 µL of this medium with 20 µL PEI (20%) solution, 20 µL water and 30 µL of DSDH stock solution. The obtained DSDH gel layer was allowed to dry at 4 ℃ overnight, then was placed directly on the top of the oxygen filter. Before experiments, 1 mM NADH was mixed in 10 mL of buffer phosphate solution (PBS, 50 mM, pH 6.5). The biosensor response was evaluated by cyclic voltammetry, by introduced gradually D-fructose in the medium (in 50 mM PBS at pH 6.5).

Results and discussion

Local oxygen removal

The electrochemical reduction of oxygen at the platinum grids used to filter oxygen was investigated by cyclic voltammetry from 0 V to -0.9 V vs. Ag/AgCl at scan rate of 5 mV s -1 using 100 mM KCl as supporting electrolyte (Figure 3). In aerated solution (Fig. 3A), two regions can be distinguished in the CV: from -0.1 V to -0.4 V, the negative current plateau is attributed to oxygen reduction under diffusion control, whereas the cathodic current "wall" starting at -0.8 V is related to hydrogen evolution. Narrowing the potential window to the range from 0 to -0.5 V allowed H2 production to be avoided, with unchanged current plateau (Fig. 3A, blue curve). The filter was composed of either one or two platinum grids. The current recorded on the two-grid layer (red curve) was higher than that with a single Pt grid (black curve). This increase can be explained by the larger electrode surface area available when more platinum is used, and also the larger volume that is treated by the porous electrodes [START_REF] Mierzwa | Electrochemistry and Spectroelectrochemistry with Electrospun Indium Tin Oxide Nanofibers[END_REF], allowing a more efficient removal of oxygen. The experiment was repeated under N2 medium for two Pt grids (Fig. 3B), showing the complete vanishing of the current profile from -0.1 V to -0.4 V. This clearly demonstrates that the plateau visible in Figure 3A (0 to -0.5 V potential region) corresponds to oxygen reduction reaction. Moreover, the large current observed in the range of -0.7 V and -0.9 V attributed to hydrogen evolution was still visible. The potential at which the filter can be poised is thus comprised between the potential at which O2 is reduced (lower than -0.2 V) and the potential of hydrogen evolution (higher than -0.7V). The potential of -0.4 V was chosen as the optimal one to apply to the platinum filter in order to evaluate its ability to remove oxygen from the surface of the sensor. First evaluation of the electrochemical scrubber for oxygen removal was carried out with a glassy carbon disk acting as sensor (WE1) and the platinum grids (one or two) as oxygen filter (WE2). In such experiment, the presence of oxygen was thus detected on WE1, whereas this species is expected to be consumed on WE2. When the filter was not connected to the electrochemical cell (OFF position), large negative currents corresponding to oxygen reduction were observed at GCE surface in aerated solution, with values as high as -6 µA (-35 µA/cm 2 ) at -0.6 V (Fig. 4A). When the filter was ON, this current vanished. Figure 4B provides a closer look on the residual current in three different conditions: oxygen was effectively removed when applying a constant potential (-0.4 V) at the filter made with one Pt layer (black curve) or two Pt layers (red curve). In accordance to the higher reduction current (in absolute value) on the two Pt-layer electrode, O2 removal was more efficient than with a single Pt grid, as revealed by the less significant residual currents, lower than 0.4 µA at 0.6 V. After degassing the solution under N2 bubbling (blue curve), the current recorded on GCE (with oxygen filter WE2 on OFF position) was very similar to that obtained in aerated solution with the 2-grid layer (with oxygen filter WE2 in the ON position), which demonstrates the efficiency of the electrochemical scrubber for oxygen removal. According to a simple modeling of the electrochemical filter, the amount of residual oxygen can be rapidly lowered to less than 1 % of its initial concentration in less than few minutes [START_REF] Etienne | Electrochemical Filter To Remove Oxygen Interference Locally, Rapidly, and Temporarily for Sensing Applications[END_REF]. This result is very promising in view to conducting experiments sensitive to the presence of oxygen, such as the electrochemical detection of NAD(P) + . voltammograms recorded in 100 mM KCl (aerated solution, oxygen concentration in solution is in the range of 200-300 µM) at 5 mV s -1 in the potential region corresponding to oxygen reduction using GCE as WE1 (working electrode 1) while applying -0.4 V vs Ag/AgCl to the oxygen filter made of one or two platinum grids (acting as WE2); for comparison purpose, a CV curve was also recorded under N2 (deaerated solution).

NAD(P)H regeneration and applications to enzymatic reactions

For NAD(P) + reduction, [Cp*Rh(bpy)Cl] + (as immobilized onto bucky paper) was chosen as an efficient non-enzymatic catalyst because of its ability to lower overvoltage for the reduction of NAD(P) + to NAD(P)H [START_REF] Ruppert | Efficient indirect electrochemical in-situ regeneration of nadh:electrochemically driven enzymatic reduction of pyruvate catalyzed by D-LDH[END_REF]. By applying a suitable cathodic potential to the BP-Bpy-Rh electrode, Rh(III) centers on the surface of electrode can be reduced to Rh(I) before fast protonation from the buffer solution, and the resulting Rh-hydride species are likely to reduce NAD(P) + [START_REF] Walcarius | Factors affecting the electrochemical regeneration of NADH by (2,2′-bipyridyl) (pentamethylcyclopentadienyl)-rhodium complexes: Impact on their immobilization onto electrode surfaces[END_REF]. The catalytic response of [Cp*Rh(bpy)Cl] + was investigated by cyclic voltammetry in 10 mL PBS solution at pH 6.5, respectively with the oxygen filter on ON and OFF positions, as illustrated in Figure 5. Oxygen concentration in solution is in the range of 200-300 µM, and depends on several parameters such as ionic strength, temperature and atmospheric pressure. Potential interference can be in principle observed for this range of concentration, and for that reason, the next experiments consider the effect of oxygen and the removal of oxygen on the detection of 250 µM NAD + .

Control experiments were first performed without NAD + (Fig. 5A) using the Rh-based sensor and the electrochemical oxygen filter made with two platinum grids. Applying reduction potential (-0.4 V) to the platinum-based filter for oxygen removal (ON position) allowed the electrochemical response of Rh(III) complex to be observed at -0.65 V (black curve). A very similar voltammogram was recorded from a nitrogen-deaerated solution (red curve). Without

[Cp*Rh(bpy)Cl] + immobilized on the bucky paper, the above cathodic signal was not observed (green curve). A clear interference from oxygen was evidenced when the oxygen filter was switched OFF, via a broad current peak observed in the range from -0.4 V to -0.7 V corresponding to the reduction of dissolved oxygen (blue curve in Fig. 5A) which is now likely to easily reach the sensor surface. The presence of oxygen masked thus the smaller signal from the rhodium catalyst immobilized on the sensing electrode.

In the presence of NAD(P) + , the BP-Bpy-Rh electrode is known to provide an electrocatalytic response as the protonated Rh(I) species generated on WE1 can transfer a hydride ion to NAD(P) + for its reduction to NAD(P)H [32]. Thanks to this process, the cofactor can be regenerated and Rh(I) can be re-oxidized for long-term use. As shown in Figure 5B, the addition of 0.25 mM of NAD + to the PBS solution with the above WE2 at -0.4 V resulted in an increase of the cathodic current together with a small negative shift of the cathodic peak from -0.65 V to -0.75 V (Fig. 5B, black curve), consistent with the literature [START_REF] Yamini | Enzymatic Electrosynthesis: An Overview on the Progress in Enzyme-Electrodes for the Production of Electricity, Fuels and Chemicals[END_REF], confirming the successful reduction of NAD + to NADH. Once again, NAD + detection was repeated with degassed solution by N2 bubbling, yielding very comparable voltammograms (Fig. 5B, red curve). NAD + reduction can be performed under air atmosphere by using the electrochemical oxygen filter made of two platinum grids. When the filter was switched OFF, a similar broad current peak observed in the range from -0.4 V to -0.7 V corresponding to reduction of dissolved oxygen was again observed and hindered the electrochemical detection of NAD + (Fig. 5B, blue curve).

In addition, the interference of oxygen is not related only to this increased current that may lead to overestimation of the current due to NAD(P) + reduction. Indeed, Tosstorff et al. mentioned in their report that the reaction of the mediator with oxygen can lead to the formation of partially reduced oxygen, potentially harmful, and that the rhodium mediator [Cp*Rh(I)(bpy)H]+ was not stable in the presence of oxygen [START_REF] Tosstorff | Mediated electron transfer with monooxygenases -Insight in interactions between reduced mediators and the co-substrate oxygen[END_REF]. Beside NAD + reduction, the oxygen filter WE2 was also used to examine the possible detection of the NADP + cofactor, as illustrated in Figure 6. To this end, amounts of NADP + at 0 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 3 mM and 4 mM, were gradually added into the buffer solution (50 mM PBS, pH = 6.5) and the two Pt grids of oxygen filter were set at -0.4 V vs Ag/AgCl.

The cathodic peak rose from 10.5 µA without cofactor up to 77 µA in the presence of 4 mM NADP + (Fig. 6A). The negative shift in potential of the reduction peak with increasing NADP + concentration is indicative of an EC mechanism. The calibration curve shows a linear relationship between the peak current and the concentration from 0.5 to 3 mM (Fig. 6B).

Beyong this value the slope decreases because of the limitation in the heterogeneous electron transfer kinetics (as reported in previous work [START_REF] Zhang | Covalent Immobilization of (2,2′-Bipyridyl) (Pentamethylcyclopentadienyl)-Rhodium Complex on a Porous Carbon Electrode for Efficient Electrocatalytic NADH Regeneration[END_REF][START_REF] Zhang | Electrocatalytic Biosynthesis using a Bucky Paper Functionalized by [Cp*Rh(bpy)Cl] + and a Renewable Enzymatic Layer[END_REF][START_REF] Walcarius | Factors affecting the electrochemical regeneration of NADH by (2,2′-bipyridyl) (pentamethylcyclopentadienyl)-rhodium complexes: Impact on their immobilization onto electrode surfaces[END_REF]), and the cathodic peak is progressively shifted to more negative potentials (from -0.65 V to nearly -0.85 V vs. Ag/AgCl) with increasing the NADP + concentration. The signal reported here is the difference between the peak and the background current for each curve.

After successfully testing the reduction of NAD + and NADP + thanks to the electrochemical filter of oxygen, the functionalized BP-Bpy-Rh electrode was exploited for electrochemical detection of D-fructose. This analysis required the presence of D-sorbitol dehydrogenase (DSDH) [START_REF] Mazurenko | Enzymatic bioreactor for simultaneous electrosynthesis and energy production[END_REF]. To avoid any interaction between the rhodium catalyst and the surface functional groups of the enzyme (which could cause their degradation [START_REF] Zhang | Electrocatalytic Biosynthesis using a Bucky Paper Functionalized by [Cp*Rh(bpy)Cl] + and a Renewable Enzymatic Layer[END_REF][START_REF] Hildebrand | Stable Electroenzymatic Processes by Catalyst Separation[END_REF]), the catalyst and the enzyme have been separated by confining them in two different layers in the bioelectrode, as follows. DSDH encapsulated in a silica gel (see Section 2.3) was spread out onto a microfiber Corresponding calibration curve. The signal reported here is the difference between the peak and the background current for each curve.

During this study, we identified two potential drawbacks of this filter. The first one is the possible production of H2O2 [START_REF] Briega-Martos | Effect of pH and Water Structure on the Oxygen Reduction Reaction on platinum electrodes[END_REF] and reactive oxygen species [START_REF] Noël | Evidence for OH radical production during electrocatalysis of oxygen reduction on pt surfaces: Consequences and application[END_REF]. Even if this cannot be excluded, we did not observe in this set of experiments evidences of interference from such species on NAD + detection. Platinum is probably one of the best materials for filter fabrication due to its catalytic properties for oxygen reduction reactions, limiting the production of harmful reactive species which could originate from an incomplete reduction of O2. The second drawback is related to pH modification in the small volume of solution that is between the filter and the sensor surface. Measuring this local pH is not trivial and we have considered this question in a recent report based on simple calculations: it came out that a pH shift could be contained by using a small buffer concentration [START_REF] Etienne | Electrochemical Filter To Remove Oxygen Interference Locally, Rapidly, and Temporarily for Sensing Applications[END_REF].

Conclusion

The electrochemical reduction and detection of NAD(P) + can be successfully performed in the absence of oxygen. Here, we have demonstrated the possibility of avoiding the removal of oxygen from the entire solution volume, by providing a way to remove it only from the surface of the electrode, in order to detect this enzymatic cofactor with a sensor made of a bucky paper electrografted with [Cp*Rh(bpy)Cl] + . This was achieved by using an electrochemical filter made of porous platinum grids, separated from the sensor surface by an insulating porous layer, a nylon grid. We have shown that oxygen was efficiently removed from the solution near the sensor surface. This allowed the detection of NAD + at a concentration as low as 0.25 mM, which was not possible in the presence of oxygen. The sensor was also likely to detect NADP + in the 0.5 to 4 mM concentration range. Finally, the detection of NAD + was combined with the enzymatic activity of D-sorbitol dehydrogenase encapsulated in a silica gel overlayer and this bioelectrode was found sensitive to increasing concentrations of D-fructose from 0.25 to 4 mM.

Despite, the possible drawback associated with the system (risk of production of H2O2 and reactive oxygen species, local pH modulation, additional technical complexity that can bring more variability in the measurement), this strategy opens a new door for the development of novel bioelectrocatalytic devices implementing both an oxygen sensitive electrochemical reaction and an enzymatic system requiring the presence of molecular oxygen to operate, e.g. P450 cytochromes.

Figure 1 .

 1 Figure 1. SEM pictures of platinum grids manufactured from (A) 1 or (B) 2 Pt layers.

Figure 2 .

 2 Figure 2. Principle of NAD + reduction on [Cp*Rh(bpy)Cl] + functionalized bucky paper

2 BFigure 3 .

 23 Figure 3. Cyclic voltammograms recorded in 100 mM KCl at a scan rate of 5 mV s -1 between

Figure 4 .

 4 Figure 4. (A) Cyclic voltammograms recorded on GCE with (filter ON) and without (filter

Figure 5 .

 5 Figure 5. Cyclic voltammograms recorded at 5 mV s -1 using a [Cp*Rh(bpy)Cl] + functionalized

Figure 6 .

 6 Figure 6. (A) Cyclic voltammograms recorded at 5 mV s -1 in 50 mM PBS buffer at pH 6.5 on

1 B

 1 filter layer: this layer was placed directly on the oxygen filter (WE2), thus at a short distance from the BP-Bpy-Rh sensor surface (WE1). The effectiveness of the bioelectrochemical reaction was checked by cyclic voltammetry on WE1 whereas the 2 Pt grids of WE2 were polarized at -0.4 V vs Ag/AgCl. The bioconversion was initiated by adding D-fructose to the PBS solution containing 1 mM of NADH. NAD + species generated by the enzymatic process left the gel layer, diffused through the porous structures of WE2 and the nylon separator to reach the surface of the sensor, where they were reduced into NADH. As shown in Figure7, the cathodic current increased gradually upon increasing the D-fructose concentration up to 4 mM, to reach a saturation value near 25 µA. An apparent Km value was estimated to be 0.5 mM, i.e. in the range of previous estimation with similar immobilization protocols[START_REF] Zhang | Electrocatalytic Biosynthesis using a Bucky Paper Functionalized by [Cp*Rh(bpy)Cl] + and a Renewable Enzymatic Layer[END_REF].Moreover, even though oxygen was present in a large part of the cell solution and near the DSDH-containing layer, the oxygen filter located between the enzyme layer and the BP-Bpy-Rh sensing element allowed the effective local oxygen removal from the solution, which was beneficial for NAD + detection.

Figure 7 .-

 7 Figure 7. (A) Cyclic voltammograms recorded at 5 mV s -1 in 50 mM PBS buffer with 1 mM

Acknowledgements

This work was supported partly by the French PIA project « Lorraine Université d'Excellence », reference ANR-15-IDEX-04-LUE. We gratefully acknowledge Gert W.

Kohring from Saarland University (Saarbrucken, Germany) for providing D-sorbitol dehydrogenase.


Childérick Severac
email: childerick.severac@itav.fr

Sergio Proa-Coronado

Cécile Formosa-Dague

Adrian Martinez- Rivas

Etienne Dague
email: edague@laas.fr

Cécile Formosa
email: formosa@insa-toulouse.fr

Automation of Bio-Atomic Force Microscope Measurements on Hundreds of C. albicans Cells AUTHORS AND AFFILIATIONS

Keywords: atomic force microscopy, C. albicans, automation, cell nanomechanics, mechanobiology, force curve analysis, cell adhesion, AFM automation, Jython, JPK experiment planner

This protocol aims at automatizing AFM measurements on hundreds of microbial cells. In the first step, microbes are immobilized into PDMS stamp microstructures while in the second step, force spectroscopy measurements are performed automatically on hundreds of cells immobilized in step one.

ABSTRACT:

The method presented in this paper aim at automatizing Bio-AFM experiments and especially the recording of force curves. Using this method, it is possible to record, in 4 hours, forces curves, on 1000 cells, automatically. To maintain a 4 hours analysis time, the number of force curves per cell is reduced down to 9 or 16. The method combines a Jython based program and a strategy for assembling cells on defined patterns. The program, implemented on a commercial Bio-AFM, is able to center the tip on the first cell of the array and then to move, automatically, from cell to cell while recording force curves on each cell. Using this methodology, it is possible to access the biophysical parameters of the cells such as their rigidity, their adhesive properties etc. Thanks to the automation and the large number of cells analyzed, one can access the

behavior of the cells population. That is a breakthrough in the Bio-AFM field where data have, so far, been recorded on only a few tens of cells.

INTRODUCTION:

The main purpose of this work is to provide a methodology to perform automatic force measurements on hundreds of living cells using an atomic force microscope (AFM). It also provides a method to immobilize microbes on a PDMS microstructured stamp that is compatible with AFM experiments conducted in liquid environment. Bio-AFM is a highly specialized technology especially conceived for applications in biology and then to study living cells. It requires a trained engineer able to analyze one cell at the time. In these conditions the number of different cells that can be analyzed is rather small, typical 5 to 10 cells in 4-5 hours. However, the quantity of force measurements recorded on a single cell are usually very high and can easily reach 1000. Thus, the current paradigm of AFM force measurements on living cells is to record hundreds of force curves (FCs) but on a limited number of cells. Statistically, this approach is questionable, and raises the issue of the representativeness of the sample. Indeed, it is difficult, for example, to evaluate the heterogeneity of a cell population by measuring only a few cells, even if hundreds of measurements are recorded on these few cells. However, it is on the basis of this paradigm that major advances have been made in biophysics, microbiology and nanomedicine 1-3 . Indeed, nanometer analysis at the scale of single cells has provided new information for example on cellular nanomechanics, on the organization of transmembrane proteins, or the action mechanism of antimicrobial or anticancer drugs [4][START_REF] Puntheeranurak | Single-molecule recognition force spectroscopy of transmembrane transporters on living cells[END_REF][START_REF] Formosa | Nanoscale analysis of the effects of antibiotics and CX1 on a Pseudomonas aeruginosa multidrug-resistant strain[END_REF][START_REF] Pillet | Atomic Force Microscopy and pharmacology: From microbiology to cancerology[END_REF] . However, during these last years, several high throughput biomechanical tests conducted on cells have emerged [START_REF] Wu | A comparison of methods to assess cell mechanical properties[END_REF] , showing the scientific community interest in changing this paradigm and to access the cell population heterogeneity. These tests all rely on microfluidic systems to deform cells and optically measure their deformation under stress to obtain an indirect measure of their overall surface elasticity [START_REF] Wu | A comparison of methods to assess cell mechanical properties[END_REF] . However, an important issue with these methods is that they are mono-parametric: only cell elasticity can be probed. Moreover, they do not allow the measurement of the mechanical parameters of adherent cells, which can be limiting for the studies of non-circulating mammalian cells or biofilms for example. Approaches involving AFM have been developed by the teams of S. Scheuring 9 and M. Favre [START_REF] Favre | Parallel AFM imaging and force spectroscopy using two-dimensional probe arrays for applications in cell biology[END_REF] . The first team immobilized cells on fibronectin patterns [START_REF] Rigato | Atomic Force Microscopy Mechanical Mapping of Micropatterned Cells Shows Adhesion Geometry-Dependent Mechanical Response on Local and Global Scales[END_REF] , forcing individual cells to take the shape of the pattern. Then this team mapped the mechanical properties of a few cells to define average data, representative of 14 to 18 cells. The development carried out by the 2 nd team 10 aimed at multiplexing the measurements by parallelizing the AFM cantilevers. To our knowledge, this work in the multiplexing direction has not led to measurements on living cells. An interesting approach proposed by Dujardin's team presents an automated AFM capable of identifying cells and imaging them at the bottom of custom-made wells. Although this method does not allow for the analysis of a large population of cells it allows the automatic testing of different conditions in each well [START_REF] Dujardin | Automated multi-sample acquisition and analysis using atomic force microscopy for biomedical applications[END_REF] .

Our objective in this work was more ambitious since we wanted to measure at least 1000 cells to access not an average cell, but, on the contrary, the heterogeneity between cells. The strategy that we developed here to access cell population heterogeneity using AFM is based on the analysis of hundreds of cells on which a limited number of force curves are recorded. Compared to the "classical" approach of recording a large number of force curves on a limited number of cells, this approach should be considered as complementary since it does not provide the same information. Indeed, while the typical method allows one to probe individual cell surface heterogeneity, using our approach, we are able to access the entire cell population heterogeneity. To achieve this objective, we have combined a method consisting in directly immobilizing microbes (here the yeast species Candida albicans) into the wells of a PDMS microstructured stamp [START_REF] Formosa | Generation of living cell arrays for atomic force microscopy studies[END_REF] , and developing an original program for moving the AFM tip, automatically, from cell to cell [START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] and measuring the mechanical properties of each cell. PROTOCOL 1. Microbial cell culture 1.1. Revivify cells from a glycerol stock. NOTE: C. albicans are stored at -80 °C in glycerol stocks, on marbles. 1.1.1. Pick a marble in the -80 °C stock and rub it on yeast peptone dextrose (YPD) agar. Grow the cells for 2 days at 30 °C, before liquid cultivation. 1.2. Prepare liquid cultures. 1.2.1. Fill a culture tube with 5 mL of sterile YPD broth and add a single colony of C. albicans cells, grown on the YPD agar plate. 1.2.2. Grow the culture in static conditions at 30 °C for 20 h before harvesting by centrifugation (4000 x g, 5 min). Discard the supernatant and eliminate as biohazard waste. 1.2.3. Wash the pellets 2x with 10 mL of acetate buffer (8 mM sodium acetate, 1 mM CaCl 2 , 1 mM MnCl 2 , pH = 5.2). Centrifuge (4000xg, 5 min) in between washings. 1.2.4. Resuspend the pellet in 2 mL of acetate buffer and use this solution for cell immobilization on the PDMS stamp. NOTE: This suspension cannot be stored and should be prepared fresh for section 3. must be used extemporaneously in section 3 (sample preparation).

PDMS stamp preparation

2.1. Silicon master mold preparation 2.1.1. Draw, using computer assisted design (CAD) software, the desired microstructures. 2.1.2. If a clean room is available follow steps 2 to 12 of the previously published protol 12. Otherwise, silicon master mold can be acquired from commercial clean room facilities. 2.2. PDMS stamp molding 2.2.1. Prepare 55 g of PDMS prepolymer solution containing a mixture of 10 to 1, mass ratio, of PDMS oligomers and curing agent cross-linking agent (Table of Materials). 2.2.2. Mix and degas this solution under vacuum (in the range of 10 -1 , 10 -2 bars) until all trapped bubbles are removed from the PDMS solution (5-10 min). 2.2.3. Pour 20 g of the degassed solution on the silicon master mold and degas again (in the range of 10 -1 , 10 -2 bars). NOTE: The stamp thickness should be around 2-3 mm. 2.2.4. When all bubbles are removed, reticulate the PDMS at 80 °C during 1 h. 2.2.5. Cut the PDMS microstructured stamp with a scalpel (0.5 x 1.5 cm 2 ) in a direction parallel to the visible microstructure arrays. 2.2.6. Peel the stamp from the silicon master mold. 2.2.7. Return the stamp to exhibit the microstructures on its upper side and deposit it on a glass slide. Make sure to have the microstructures facing up away from the glass slide. Align the microstructures that can be seen on the stamp with the side of the glass slide, which will later serve as a reference for the AFM automation procedure. NOTE: At this stage, the PDMS stamp is ready for cell immobilization. The PDMS stamps can be stored on the silicon master mold for several months. When all the PDMS is removed from the master mold, a new PDMS stamp can be casted again on the master mold (to keep the master mold safe, it is possible to replicate it in polyurethane) [START_REF] Foncy | Comparison of polyurethane and epoxy resist master mold for nanoscale soft lithography[END_REF] .

Sample preparation

3.1. Cell immobilization 3.1.1. Centrifuge (500 x g, 5 min) 600 µL of the resuspended cell solution to separate the buffer from the cells.

3.1.2. Pipet 200 µL of the supernatant from step 3.1.1 onto the PDMS stamp, and degas under vacuum (in the range of 10 -1 , 10 -2 bars) for about 40 min. NOTE: This step is important to improve the cell immobilization inside the wells. Molecules from the yeast cell wall, present in the supernatant, are probably deposited on the PDMS surface during this pre-wetting step. These molecules, most probably, enhance the adhesion of the cells and contribute to the increase in the stamp filling rate. 3.1.3. After 40 min, with a pipet, remove the buffer from the PDMS surface and deposit, with a pipet, 200 µL of the cell solution from step 1.2.4 for 15 min at room temperature. 3.1.4. The cells are then placed into the microstructures of the stamp by convective/capillary assembly. For that, manually spread 200 µL of cells suspension across the stamp using a glass slide in both direction with an angle comprised between 30 and 50°.It may be necessary to pass the glass slide several times on the stamp to achieve a high filling rate. Several passes may be necessary to reach a high filling rate. NOTE: A full description of this method is available in refernce [START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] . 3.1.5. Remove the cell suspension with a pipet. Wash the stamp 3x with 1 mL of acetate buffer, pH = 5.2 to remove the cells that were not trapped. 3.1.6. Dry the back of the stamp using nitrogen flow, in order to ensure that the stamp will adhere to the dry Petri dish. 3.1.7. Finally deposit the PDMS stamp filled with cells in a Petri dish (Table of Materials) and fill it with 2 mL of acetate buffer to maintain the cells in liquid medium. 3.2. Setting the stamp on the AFM stage 3.2.1. Center the stage at 0;0 when starting AFM operations. 3.2.2. Calibrate sensitivity and spring constant of the cantilever on glass and in water as described in Unsay et al. [START_REF] Unsay | Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers[END_REF] 3.2.3. Take the Petri dish with the stamp and place it in the AFM Petri dish holder. 3.2.4. Align the stamp edge perpendicular to the Petri dish holder Y axis. NOTE: An acceptable tilt angle is under 5° as illustrated in Figure 1. 3.2.5. Place the AFM head onto the stage and be careful that the stepper motors are sufficiently extended to avoid the tip to crash on the stamp.

Running the AFM program NOTE:

The 4.1.7. Input the well dimension in the Ws variable line 248 This is known from the design of the well patterns and can be checked on the same image as the one used to verify the pitch (Figure 2). 4.1.8. Write the path to the saving directory in line 251 to save the data at the desired place. 4.1.9. Set the totalArea variable line 254 to the desire multiple "n" of 100 µm (that is the maximum scan area of the AFM used). The total number of wells that will be probed can be calculated using this value and the pitch: maximum scan area/pitch*n 2 . NOTE: In the example of Figure 3, 9 areas of 100 x 100 µm 2 will be analyzed. 4.1.10. Set the force curves matrix, row and column (3, 3 or 4, 4), recorded per well in the numScans variable line 257. NOTE: In the example of Erreur ! Source du renvoi introuvable.3, a matrix of 3 x 3 = 9 FCs will be recorded for each well. 4.1.11. Run the program, click on the start button. NOTE: The program first automatically executes a centering algorithm to better determine the center of W1 and W2 wells (step 1). It then automatically acquires the Force Curves (FCs) matrix on each well of the first scanning area (step 2). When all the wells of that area are probed, the script automatically moves the AFM tip to the first well of the next scanning area. The tip is retracted, the microscope stage moves to the next area, the tip is again approached on the stamp and the centering algorithm is executed again to re-center automatically on the first well (1') of that area (step 3). The first area is defined by the user, the second one, is on the right etc. till n is reached. n+1 area is underneath n, n+2 on the left of n+1 etc. till 2n is reached. 2n+1 is underneath 2n, and 2n+2 is on the right on 2n etc. Globally, the tip serpentine through the total area. Step 2 and 3 are repeated automatically until the total number "n 2 " of scanning areas have been probed. Figure 5 presents the flowchart of the program. It takes ~4 h to complete the program. Come back 4 hours later and start data analysis. Data can also be analyzed later. 4.2. Data analysis 4.2.1. Execute the "Copy files" python script (Copy_files_L.py, provided in Supplementary Data) to organize the FCs files into one folder. This script was developed with Pyhton 2.7 and the SciPy module. Use Video Studio Code software to open the python script. Input path to the general folder (line 34 of the script provided in supplementary data) and where it will be stored (line 37). 4.2.2. Open the AFM manufacturer data processing software to analyze the force curves. In the top menu 'File' select open 'batch of spectroscopy curves'. 4.2.3. In the batch processing window, select the process provided in Supplementary Data (StiffnessProcess.jpk-proc-force). Select the last step of the process and click on 'keep and apply to all'. All force curves will receive the same treatment. NOTE: The process uses the calibration from the FCs files to convert the deflection curves into force curves calibrated in Newton; a data smoothing algorithm is applied (average of 3 consecutives points); the baseline is translated to rest on the zero axis; the contact point is extrapolated and the FC is offset to place the contact point at coordinate (0,0); the bending of the cantilever is subtracted to the FCs, the retract slope is fitted. At the end of the data treatment, the software generates a file that contains a table giving for each FCs: its name, Young Modulus, contact point, adhesion force, slopes, etc. 4.2.4. Repeat steps 4.2.1 to 4.2.3 for all experiments. Be careful to save the data in different folders (i.e.: "…\TREATED\" and "…\UNTREATED\") 4.2.5. Use the R script provided in Supplementary Data to plot histograms and box plots and perform ANOVA statistical treatments. 4.2.5.1. To open the R script (DataAnalisys.R), use R studio software and load the files containing the information extracted with the data processing software (.tsv). 4.2.5.2. On the environment window use the "Import Dataset" button, from the list displayed select "from text (readr)" and in the new window select the Browser button and find the .tsv file. 4.2.5.3. Once the file has loaded, select the columns (stiffness and adhesion) to be included for the analysis. To run all the code, press ctrl+alt+r. NOTE: The script works with 4 datasets, consider two experiments both having untreated and treated cells. It is possible to execute blocks of the script and see how the variables change according to the functions executed.

REPRESENTATIVE RESULTS:

We used the described protocol to analyze the effect of caspofungin on the biophysical properties of the opportunistic human pathogen C. albicans in its yeast form. Caspofungin is a last chance antifungal molecule used when other drugs are ineffective because of resistance mechanisms cells develops towards antifungals. Its mechanism of action is based on the inhibition of the subunit Fks2 of the complex fks1/Fks2 responsible for the ß glucan synthesis. As ß glucans are a major component of the fungal cell wall [START_REF] Schiavone | A combined chemical and enzymatic method to determine quantitatively the polysaccharide components in the cell wall of yeasts[END_REF][START_REF] Formosa | Nanoscale Effects of Caspofungin against Two Yeast Species, Saccharomyces cerevisiae and Candida albicans[END_REF] we expected modification of the biophysical properties of the cell wall: rigidity and adhesion. Figure 6 presents typical histograms obtained when all the protocol presented above is applied.

The red histogram represents the stiffness repartition recorded on 957 native cells and the blue one on 574 caspofungin treated cells. The first interesting observation is that both histograms demonstrate a bimodal distribution of the values. This observation is possible only because we measured hundreds of cells. On smaller samples, researchers usually observe a single distribution and miss the population heterogeneity. [START_REF] Formosa | Nanoscale Effects of Caspofungin against Two Yeast Species, Saccharomyces cerevisiae and Candida albicans[END_REF][START_REF] El-Kirat-Chatel | Nanoscale analysis of caspofungin-induced cell surface remodelling in Candida albicans[END_REF] The second observation concerns the effect of caspofungin. It globally reduces the stiffness of the cells while still 2 subpopulations exist. In a last step the proposed protocol provides an ANOVA comparison of the native and treated cells as presented in Figure 7. It demonstrates that the 2 conditions have a different stiffness and that this difference is highly significant (pvalue < 0.001). This value is reached thanks to the large number of cells analyzed and provides a greater confidence in the obtained results. The adhesion has also been extracted from the automatically recorded data and we found that the adhesion force between the bare tip and native cells was of 0.64 +/-0.6 nN. In this case also 2 subpopulations were found: the first one has a mean adhesion force of 0.7 +/-1.4 nN while the second of 4.5 +/-1.5 nN. The treatment with caspofungin had unpredictable effects on the adhesion. In one experiment no effect was observed, but in another experiment, caspofungin induced a decrease in the adhesion to the tip and a reduction of the population adhesion heterogeneity. These results are extracted from Proa et al. [START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] , where they are presented in totality.

FIGURE LEGENDS:

Figure 1: Acceptable position of the micro structured stamp on the AFM stage. The tilt angle on the left pictures (up to 5°) can be handled by the program but the tilt on the right is too important (10°). This Figure has been modified from [START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] . Figure 2: Typical AFM image of a filled PDMS stamps showing the initial coordinates as W1 and W2, the size of the scanning area (Δ2), the tilt angle (θ). This Figure has been modified from [START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] . Figure 3: User input section of the script. P1 and P2 refers to the coordinates of well 1 (W1) and well 2 (W2) of Figure 2. The other parameters are the pitch in meter, the well size in meter (Ws), the directory path for saving the data, the total square area that will be probed by the automated AFM (totalArea is the length in meter of the side of the total square area) and the number of force-curves per wells (numScans). All units are in meters. Figure 4: Optical image providing an example of valuable (green dots) initial wells. The black square represents the scanning area and the red spots, initial wells that should better be discarded. This Figure has been modified from [START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] . [START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] . Figure 8: Time-position dependency of values. Histograms in the center are the original data which is divided into the different subgroups corresponding to the subpopulations founded (cyan/green). A and B: Show the presence of the two sub-populations at every hour in the experiment. C and D: show the positions of indentation; on each position it is possible to see the presence of the subpopulations (cyan/green). Subgroup organization was done using the kmeans algorithm. This Figure has been modified from [START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] . Figure 9: The safe area. An area, inside the PDMS well, has been defined as the area where the pyramidal tip does not touche the well edge while reaching the well bottom (in the cas of an empty well). This Figure has been modified from [START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] .

DISCUSSION

The main improvement provided by this methodology is a significative increase in the number of measured cells in a determined amount of time. The counterpart is a reduction of the number of points measured per cell. It means that our method is not designed to provide a detailed analyzis of a single cell. The method only applies to cells that can fit in the wells of our PDMS stamp. The stamp is quite versatile, while it contains wells of 1.5x1.5 µm2 up to 6x6 µm2. Still it is impossible to immobilize bacillus or much bigger cells. The stamp and capillary convective deposition cannot be used to immobilize mammalian cells that are much bigger (around 100µm in length). In this context, Peric et al. [START_REF] Peric | Microfluidic bacterial traps for simultaneous fluorescence and atomic force microscopy[END_REF] developed a smart microfluidic device to immobilize bacillus like Escherichia coli and bacillus subtilis. This device makes it possible to immobilize, at defined positions and under physiological conditions, bacillus. It would be very interesting to adapt our software to the particular size of this device. Tip contamination can also be a problem in this automated system. In the case of microbial cells, it is not so pregnant but it is of high importance in the case of mammalian cells. Dujardin et al. [START_REF] Dujardin | Automated multi-sample acquisition and analysis using atomic force microscopy for biomedical applications[END_REF] addressed this issue by adding, in their automated protocol, a cleaning step. This step consists in checking the laser sum and to activate the cleaning procedure if the sum is too low. The clean step consists in immersing the tip in a well filled with water or ethanol. A question that systematically arises from this automation work has been: "does the heterogeneity comes from the evolution of the cells during the experiment?". To answer this question, we plotted the stiffness results as a function of time as presented in Figure 8 A andB. It clearly demonstrates that heterogeneous stiffness values are recorded at any time during the experiment.

In the same context the question of the tip position during the measure emerged. It actually could be possible that force curves recorded on the edge of a cell would have a different stiffness from FC recorded on the top of the cells. To avoid this inconvenient we defined what we called the safe area. It is depicted in Figure 9 A and B and represents an area inside the wells where the tip will not touch the well edges during force measurement. Using this "safe area" we were sure to record FC only on cells and at the top of them. As shown in figure 8 C and D the tip position within the safe area is not responsible for the heterogeneity of the results; as we found both phenotypes for each position of the tip, in the safe area. To make sure that the values recorded at each position are homogeneous we plotted the stiffness values as a function of the position as presented in Figure 9 C and D. It shows that heterogeneous stiffness values are recorded on each position in the well which means that the observed heterogeneity is not an artifact due to the tip position in the wells. The protocol presented in this article represents a conceptual and methodological breakthrough in the field of AFM applied in life science. The large amounts of data generated are compatible with automatic analysis which will undoubtedly allow the classification of cell populations according to new criteria. The application of this protocol to protein or sugar arrays is entirely feasible and requires only a few adaptations to consider the spacing between areas of interest. It is therefore a versatile protocol that is the result of strong interdisciplinary collaboration.

ACKNOWLEDGMENTS:

We want to acknowledge FONCYCYT of CONACYT (Mexico), the ministry of Foreign affairs of France and the Université Paris 13, though the financial support of the international collaborative ECOS-NORD project named Nano-palpation for diagnosis, No. 263337 (Mexico) and MI5P02 (France). AMR would like to thank the financial support of SIP-IPN through the project No. 20195489. SPC is supported by a PhD fellowship from CONACYT (No. 288029) and IPN through the cotutelle agreement to obtain double PhD certificate (IPN-UPS). ED and CFD are researchers at Centre National de la Recherche Scientifique (CNRS).

DISCLOSURES:

The authors have nothing to disclose.

REFERENCES:

1.

Cross, S.E., Jin, Y.-S., Rao, J., Gimzewski, J.K. Nanomechanical analysis of cells from cancer patients. Nature Nanotechnology. 2 (12) Muller, D.J., Helenius, J., Alsteens, D., Dufrene, Y.F. Force probing surfaces of living cells to molecular resolution. Nature Chemical Biology. 5 (6), 383-390 (2009).

  pdf). It requires a JPK-Bruker AFM Nanowizard II or III equipped with a motorized stage and JPK desktop software version 4.3. The program has been developed under Jython (version based on python 2.7) 4.1. Data acquisition 4.1.1. Center the AFM tip on top of the left corner of the 4.5 x 4.5 µm 2 wells (corresponding to the cell size) using the AFM optical microscope. If another well size is needed, center on top left corner of the desired wells. 4.1.2. Perform a 64 x 64 force map (Z range = 4 µm, tip velocity = 90 µm•s -1 , applied force 3 to 5 nN) over a 100 x 100 µm² area. Select Force Mapping mode from the Measurement mode dropdown box. In the force control mapping panel input the following paramteres: Rel. Setpoint = 3 to 5 nN; z length 4 µm; Z movement: constant duration; extend time: 0.01s; ext. delay:0; Retr delay: 0, Delay mode: Constant Force, Sample rate 2048 Hz; Z closed loop uncheck; Grid: check Square image, Fast 100 µm, slow: 100µm, X offset: 0 µm; Y offset: 0 µm; grid angle: 0 degree; Pixels: 64x64; pixel ratio: 1:1 NOTE: A typical resulting image is shown in Figure2. This image will help measure and verify the pitch between two wells. 4.1.3. Note the coordinates of the center of the top left well (W1) and of the bottom left well (referred as W2 on Figure2). To do so, make a square box around the well. The coordinate of the center of the box appears on the left panel of the AFM software in x,y coordinates boxes. 4.1.4. To Open the automation software (Automatip_scan.py): in the JPK desktop software click on advance in the top bar menu and select open script. In the window that opens select the path toward the script file provided in Supplementary Data (Automatip_scan.py). 4.1.5. Implement W1 and W2 coordinate values in the Inputs box section of the Jython script (Erreur ! Source du renvoi introuvable.3). Input the W1 coordinates in the P1 variable line 239 of the script and the W2 coordinates in the P2 variable line 241. NOTE: The wells selected as initial coordinates (W1 and W2) should not be too close from the scanning area edge. Otherwise the centering algorithm would not execute correctly because it needs to measure the height on the PDMS surface on each side of the well. For an exemple see Figure 4. 4.1.6. Attribute the pitch value to the pitch variable line 245 of the script.

Figure 5 :

 5 Program flowchart showing the 5 steps automatically executed by the AFM.

Figure 6 :

 6 Figure 6: Histograms of the median stiffness values. A and B: Show the median results per cell for native and caspofungin treated cell. This Figure has been modified from[START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] .. Figure7: Box plots comparing native and treated with caspofungin cells. The 3 stars represent a significativity of p<0.001. The box represents 90% of the results, the central line is the median value and the vertical bars represent the range of all the data. This Figure has been modified from[START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] . Figure8: Time-position dependency of values. Histograms in the center are the original data which is divided into the different subgroups corresponding to the subpopulations founded (cyan/green). A and B: Show the presence of the two sub-populations at every hour in the experiment. C and D: show the positions of indentation; on each position it is possible to see the presence of the subpopulations (cyan/green). Subgroup organization was done using the kmeans algorithm. This Figure has been modified from[START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] . Figure9: The safe area. An area, inside the PDMS well, has been defined as the area where the pyramidal tip does not touche the well edge while reaching the well bottom (in the cas of an empty well). This Figure has been modified from[START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] .DISCUSSIONThe main improvement provided by this methodology is a significative increase in the number of measured cells in a determined amount of time. The counterpart is a reduction of the number of points measured per cell. It means that our method is not designed to provide a detailed analyzis of a single cell. The method only applies to cells that can fit in the wells of our PDMS stamp. The stamp is quite versatile, while it contains wells of 1.5x1.5 µm2 up to 6x6 µm2. Still it is impossible to immobilize bacillus or much bigger cells. The stamp and capillary convective deposition cannot be used to immobilize mammalian cells that are much bigger (around 100µm in length). In this context, Peric et al.[START_REF] Peric | Microfluidic bacterial traps for simultaneous fluorescence and atomic force microscopy[END_REF] developed a smart microfluidic device to immobilize bacillus like Escherichia coli and bacillus subtilis. This device makes it possible to immobilize, at defined positions and under physiological conditions, bacillus. It would be very interesting to adapt our software to the particular size of this device. Tip contamination can also be a problem in this automated system. In the case of microbial cells, it is not so pregnant but it is of high importance in the case of mammalian cells. Dujardin et al.[START_REF] Dujardin | Automated multi-sample acquisition and analysis using atomic force microscopy for biomedical applications[END_REF] addressed this issue by adding, in their automated protocol, a cleaning step. This step consists in checking the laser sum and to activate the cleaning procedure if the sum is too low. The clean step consists in immersing the tip in a well filled with water or ethanol. A question that systematically arises from this automation work has been: "does the heterogeneity comes from the evolution of the cells during the experiment?". To answer this question, we plotted the stiffness results as a function of time as presented in Figure8 A and B. It clearly demonstrates that heterogeneous stiffness values are recorded at any time during the experiment.

Figure 7 :

 7 Figure 6: Histograms of the median stiffness values. A and B: Show the median results per cell for native and caspofungin treated cell. This Figure has been modified from[START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] .. Figure7: Box plots comparing native and treated with caspofungin cells. The 3 stars represent a significativity of p<0.001. The box represents 90% of the results, the central line is the median value and the vertical bars represent the range of all the data. This Figure has been modified from[START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] . Figure8: Time-position dependency of values. Histograms in the center are the original data which is divided into the different subgroups corresponding to the subpopulations founded (cyan/green). A and B: Show the presence of the two sub-populations at every hour in the experiment. C and D: show the positions of indentation; on each position it is possible to see the presence of the subpopulations (cyan/green). Subgroup organization was done using the kmeans algorithm. This Figure has been modified from[START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] . Figure9: The safe area. An area, inside the PDMS well, has been defined as the area where the pyramidal tip does not touche the well edge while reaching the well bottom (in the cas of an empty well). This Figure has been modified from[START_REF] Proa-Coronado | Beyond the paradigm of nanomechanical measurements on cells using AFM: an automated methodology to rapidly analyse thousands of cells[END_REF] .DISCUSSIONThe main improvement provided by this methodology is a significative increase in the number of measured cells in a determined amount of time. The counterpart is a reduction of the number of points measured per cell. It means that our method is not designed to provide a detailed analyzis of a single cell. The method only applies to cells that can fit in the wells of our PDMS stamp. The stamp is quite versatile, while it contains wells of 1.5x1.5 µm2 up to 6x6 µm2. Still it is impossible to immobilize bacillus or much bigger cells. The stamp and capillary convective deposition cannot be used to immobilize mammalian cells that are much bigger (around 100µm in length). In this context, Peric et al.[START_REF] Peric | Microfluidic bacterial traps for simultaneous fluorescence and atomic force microscopy[END_REF] developed a smart microfluidic device to immobilize bacillus like Escherichia coli and bacillus subtilis. This device makes it possible to immobilize, at defined positions and under physiological conditions, bacillus. It would be very interesting to adapt our software to the particular size of this device. Tip contamination can also be a problem in this automated system. In the case of microbial cells, it is not so pregnant but it is of high importance in the case of mammalian cells. Dujardin et al.[START_REF] Dujardin | Automated multi-sample acquisition and analysis using atomic force microscopy for biomedical applications[END_REF] addressed this issue by adding, in their automated protocol, a cleaning step. This step consists in checking the laser sum and to activate the cleaning procedure if the sum is too low. The clean step consists in immersing the tip in a well filled with water or ethanol. A question that systematically arises from this automation work has been: "does the heterogeneity comes from the evolution of the cells during the experiment?". To answer this question, we plotted the stiffness results as a function of time as presented in Figure8 A and B. It clearly demonstrates that heterogeneous stiffness values are recorded at any time during the experiment.

  , 780-783 (2007). 2. Dague, E. et al. Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure. Journal of Molecular and Cellular Cardiology. 74, 162-172, doi: 10.1016/j.yjmcc.2014.05.006 (2014). 3.


Wim De Neys

Lapsyde

Rational rationalization and System 2 Commentary on Cushman's "Rationalization is Rational"

In this commentary, I highlight the relevance of Cushman's target paper for the popular dual process framework of thinking. I point to the problematic characterization of rationalization in traditional dual process models and suggest that in line with recent advances, Cushman's rational rationalization account offers a way out of the rationalization paradox.

MAIN TEXT

The dual process framework (e.g., [START_REF] Evans | Dual-process theories of higher cognition advancing the debate[END_REF][START_REF] Kahneman | Thinking, Fast and Slow[END_REF] has long conceived human thinking as an interplay of fast and intuitive processing ("System 1" thinking) and slower, more demanding deliberate processing ("System 2" thinking). The characterization of rationalization in this popular framework is often problematic.

On one hand, rationalization is typically conceived as epiphenomenal. It is considered as a mere "making-up-excuses-after-the-facts" in which reasoners post hoc look for justifications for an (often inappropriate) intuitively cued choice. For example, one might think here of classic reasoning and heuristics-and-biases tasks in which people fail to correct biasing intuitions but are afterwards all too eager to find reasons to support their (erroneous) intuition (e.g., [START_REF] Evans | Rationalization in a reasoning task[END_REF][START_REF] Kahneman | Thinking, Fast and Slow[END_REF]. As such, rationalization would have no functional "rational" role to play in sound reasoning.

At the same time, dual process theorists also tend to characterize rationalization as a deliberate (System 2) process. Indeed, people often spend considerable time and effort to come up with justifications and rationalize their answers [START_REF] Pennycook | What makes us think? A three-stage dual-process model of analytic engagement[END_REF]Wason & Evans, De Neys, W. (2020). Rational rationalization and System 2. Behavioral andBrain Sciences, 43, E34. doi:10.1017/S0140525X19002048 1975). This poses a puzzle. Why would we waste scarce resources on a pointless epiphenomenon? The fact that the human cognitive miser-who typically prefers to refrain from demanding deliberationnevertheless engages in it to rationalize its behavior suggests that rationalization must serve an important function [START_REF] Evans | Reflections on reflection: the nature and function of Type 2 processes in dualprocess theories of reasoning[END_REF]. Unfortunately, this functional role of rationalization has received little attention in traditional dual process models.

However, recent dual process work has started to hint at a possible role in social communication and argumentation [START_REF] Bago | The smart System 1: Evidence for the intuitive nature of correct responding on the bat-and-ball problem[END_REF]De Neys, 2017;[START_REF] Evans | Reflections on reflection: the nature and function of Type 2 processes in dualprocess theories of reasoning[END_REF]. A key observation is that reasoners not only rationalize incorrect intuitions but also correct ones. One intriguing finding comes from two-response studies in which reasoners first have to answer as fast as possible with the first response that comes to mind and afterwards can take the time to deliberate and give a final answer [START_REF] Bago | The smart System 1: Evidence for the intuitive nature of correct responding on the bat-and-ball problem[END_REF][START_REF] Newman | Rule-based reasoning is fast and belief-based reasoning can be slow: Challenging current explanations of belief -bias and base-rate neglect[END_REF]. Results indicate that sound reasoners do not necessarily need to deliberate to correct an initial erroneous intuition (e.g., "10 cents" in the infamous bat-and-ball problem), their initial intuitive response is often already correct.

However, without subsequent deliberation, they struggle to give an explicit justification of their (correct) intuitive answer [START_REF] Bago | The smart System 1: Evidence for the intuitive nature of correct responding on the bat-and-ball problem[END_REF]. In other words, good reasoners seem to intuitively know the correct response, but don't seem to know why it is correct in the absence of further deliberation. This indicates that sound reasoners do not necessarily deliberate to correct their intuition but to rationalize it and look for an explicit justification.

As [START_REF] Mercier | The enigma of reason[END_REF] have stressed, such a justification process in which we look for explicit reasons in support of our intuitions can be critical to efficiently sway others. Clearly, if I want to convince my peers that my solution to a problem is right, I will be more successful when giving them an explicit, verifiable argument than by simply telling them that I "felt" it was right [START_REF] Bago | The smart System 1: Evidence for the intuitive nature of correct responding on the bat-and-ball problem[END_REF].

Whereas the recent dual process findings (and the work of [START_REF] Mercier | The enigma of reason[END_REF] point to a possible functional role of rationalization in social persuasion, Cushman's account points to an additional contribution to our own "internal" information processing. In my opinion, such "internal" and "external" functions do not need to be mutually exclusive. However, my goal here is not to comment on the specifics of Cushman's proposal. The key point I want to highlight is that by pinpointing a rational role of rationalization, Cushman's work offers dual process theorists a possible way out of the rationalization paradox.

resources on it and still survive as a species. Cushman builds a convincing case for the rationality of rationalization. Therefore, any dual process proponent (or critic) should take note of it. My hope is that this will instigate renewed empirical research on rationalization in the dual process field.


Aleid Ghadah

Hidayahni Ahmad

Kamila Goldyn

Svetlana Mintova

Tau Chuan Ling

Eng-Poh Ng
email: epng@usm.my

Ghadah Aleid

Nurhidayahni Ahmad

Nanosized Cs-pollucite zeolite synthesized under mild condition and its catalytic behavior

Keywords: Nanocrystals, Cesium-pollucite, Template-free synthesis, Mild synthesis condition, Perkin Condensation 1

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Introduction

Nanoscale science and engineering provide unique understanding and control of matter and mostly on a fundamental level [1][2][3]. In particular, zeolite particles have been fascinating the world of science during the last two decades due to the uniform pore size and high surface area contributed by their crystalline pores and channels of molecular dimensions [4][5][6]. As a result, zeolites have been widely used in heterogeneous catalysis, ion-exchange and adsorption [7][8][9].

In recent years, scaling down the particle size of zeolites to nanometer range has become a hot topic since it creates substantial and new modification to the properties of the materials (e.g. external surface area, diffusivity, colloidal stability, electronic, etc.) that required for advanced applications such as in atomic energy production, electronics, recording materials, drug delivery, food, ceramics, paints, paper, detergents, lubricants and so on [8][9][10][11]. While more than 230 types of zeolites have been discovered today, only 17 types of nanocrystalline zeolites (*BEA [12], EDI [13], EMT [9], FAU [14,15], GIS [16], LTA [17], LTJ [18], LTL [19], MEL [20], MER [21], MFI [22], MOR [23], SOD [24], AEI [25], AFI [26], AFO [27], CHA [28]) have been synthesized so far.

Cs-pollucite zeolite, CsAlSi2O6, which has 8-membered ring (pore diameter 2.43Å) and three-dimensional channel, is a very interesting material because of its Cs + extraframework cations that is highly aprreciated in solid basic-catalyzed organic reactions [29]. In addition, pollucite is also an excellent host for immobilization of radioactive cesium [30]. Typically, pollucite is crystallized at very high temperature and pressure (700-1200 C, >1000 bar) by using solid state reaction [31]. Hence, the synthesis of Cs-pollucite is rarely reported due to its tedious and extreme synthesis condition. Furthermore, the Cs with very high basicity tends to evaporate at such high temperature and it readily corrodes the reactors.

Recently, Cs-pollucite has been successfully crystallized under hydrothermal condition from zeolite or clay (300 C, 850 bar) [32] and soil (200 C, 300 bar) [33] at much lower temperature and pressure. However, the synthesis condition is still not ideal considering its high heating pressure and temperature. In addition, the resulting crystals are very big (> 2 m). In order to reduce the crystallite size to nanometer realm, large excessive of harmful and expensive organic template might need to be added to control the nucleation over the crystal growth [34].

Mild and safer condition for the synthesis of Cs-pollucite (particularly for nanometer-sized crystals) with lower crystallization temperature and pressure still remain a challenge, and hence it is our intention to synthesize nanosized Cs-pollucite free of organic template using lower temperature and pressure. In addition, the catalytic behavior of nanosized Cs-pollucite zeolite will also be investigated in Perkin condensation of benzaldehyde with acetic anhydride under nonmicrowave instant heating.

Experimental

Synthesis of Cs-pollucite zeolite nanocrystals

Cs-pollucite zeolite nanocrystals were synthesized from a clear precursor with a molar composition of 16.5SiO2:1Al2O3:

Characterization

The crystallinity and purity of samples were analyzed by a PANalytical X'Pert PRO XRD diffractometer (CuKα radiation, λ = 1.5406 Å, scan speed of 0.2°/min, step size of 0.02). FESEM and TEM micrographs were obtained with Leo Supra 50VP and Philips CM-12 electron microscopes operated at 20 kV and 200 kV, respectively. The chemical composition of solid was determined by using a Philips PW2404 X-ray fluorescence (XRF) spectrometer. The infrared (IR) spectrum of the solid was recorded with a Perkin Elmer's System 2000 spectrometer using KBr pellet technique (KBr:sample ratio = 50:1). The porosity analysis was performed using a Micromeritics ASAP 2010 nitrogen adsorption analyzer. The sample was first degassed at 300 C for 14 h prior to sorption at -196 C. The detection and measurement of strength of basic sites was investigated using temperature-programmed desorption of carbon dioxide (CO2-TPD). The analysis was performed on a BELCAT-B instrument where the sample (ca. 80 mg) was first degassed at 400 °C overnight. The sample was then adsorbed with CO2 gas before the gas was slowly desorbed in the temperature range of 40 to 800 °C at a heating rate of 10 °C/min.

Catalytic Perkin condensation reaction under non-microwave instant heating condition

Nanosized Cs-pollucite zeolite (0.60 g or 0.13 mmol), benzaldehyde (1.0 mmol, 99%, Merck) and acetic anhydride (1.5 mmol, Merck) were added into a 10-mL quartz reaction vessel, capped and heated at 180-190 C by using a Monowave 50 non-microwave instant heating reactor (Anton Paar). The reaction solution was isolated and analyzed with a GC-FID chromatograph (Agilent/HP 6890 GC, HP-5 capillary column) and the identity of the product was confirmed with a GC-MS spectrometer (Perkin-Elmer Clarus 500 system).

Catalyst reusability study

The nanosized Cs-pollucite zeolite, which was used after the first reaction run, was washed with diethyl ether (15 mL) for 5 times and air dried. The solid was re-activated again before it was used for the subsequent cycles of catalytic reaction using the same reaction condition. The solution after the reaction was withdrawn and separated from the zeolite catalyst before it was analyzed using a GC-FID.

Results and discussion

Preparation and characterization of Cs-pollucite zeolite nanocrystals

The pressure of in the autoclave is monitored during the crystallization process. The pressure raised progressively as the temperature increased. A maximum pressure of 22 bar was recorded and the pressure remained constant when the temperature reached 180 C. As compared to previous works (700-1200 C, >1000 bar [31]; 300 C, 850 bar [32]; 200 C, 300 bar [33], the temperature and pressure used for the synthesis of Cs-pollucite in our work are much lower thus allowing safer synthesis operation.

The evolutional formation of Cs-pollucite nanocrystals is studied using XRD technique.

Initially the precursor is transparent and no solid is formed for the first 44 h. However, the solution is becoming more viscous. Hence, it indicates that polymerization of silicate and aluminate species is taking place [35]. At 47 h, further polymerization among precursor species leads to the formation of cloudy colloidal suspension. The sample after purified and dried was analyzed with XRD diffractometer and the sample is found to be an amorphous solid (Fig. 1a). Hence, further heating is needed to undergo induction process so that zeolite nuclei can be formed [36].

When the reaction is prolonged to 48 h, the XRD amorphous hump slowly disappears after 48 h (Fig. 1b). Several diffraction peaks start to appear at 2 = 24.3 [321], 26.1 [400], 30.7 [332] and 37.2 [440] revealing that Cs-pollucite zeolite nuclei has been formed in this sample [37]. The amorphous entity fully transforms into Cs-pollucite nanocrystals after 50 h of hydrothermal treatment (Fig. 1c). As shown, the XRD diffractogram shows the characteristic pattern of ANA phase and the pattern is similar to the pattern shown on ICDD card number 00-029-0407, PDF2 files [START_REF]Database of International Centre for Diffraction Data (ICDD[END_REF]. The XRD peaks are broad indicating the small size of the Cs-pollucite nanocrystals. The size of the Cs-pollucite nanoparticles is calculated using the Scherrer equation and are found to be 72.3 nm. No crystalline competing phase is observed after 100 h of heating showing that Cs-pollucite structure is thermodynamically stable. The FESEM image of Cs-pollucite nanozeolite is captured and is shown in Fig. 2. It can be seen that the sample consists of discrete and non-agglomerated nanoparticles. The shape of the crystals is in spherical form but the crystal shape cannot be confirmed yet due to very small particle size of Cs-pollucite zeolite and low resolution of FESEM image. The particle size distribution of Cs-pollucite is determined by randomly counting the 100 crystals through FESEM observations obtained in different areas. Narrow monomodal particle size distribution centered at 79.4 nm with diameter ranging from 66.9 nm to 93.2 nm is shown indicating that the nanocrystals obtained have reasonably uniform crystal sizes (Fig. 2b). The morphology of nanocrystals are further characterized with TEM analysis. The TEM image shows that Cs-pollucite nanocrystals have a similar shape to the theoretical shape of ANAtype zeolite where its shape is trapezohedron with cubic symmetry (Fig. 3a). Similar crystal shape achieved by large pollucite crystal (40 m) was also reported by Yokomori et al. where smectite and mordenite are used as aluminum and silicon sources [32]. Unlike micronsized zeolites, the nanosized Cs-pollucite can be easily dispersed in solvents like water (Fig. 3b). The suspension is colloidally stable at nominal size and no agglomeration of solid is observed for over 1 week. Hence, this colloidal stable Cs-pollucite can be potentially used in the advanced applications that require thin film deposition such as in sensors, ceramics, paints, electronics, recording materials, etc. [13,[START_REF] Mintova | [END_REF]40]. Complimentary IR spectroscopy study on the nanosized Cs-pollucite is also conducted and its IR spectrum is shown in Fig. 4a. The IR bands at 3458 and 1630 cm -1 are due to the stretching and bending modes of O-H groups of adsorbed water [41]. The strong band at 1035 cm -1 is assigned to the vibration frequency of the T-O-Si bonds (T = Si, Al) [42]. The IR bands at 774, 720 and 631 cm -1 are characteristic of highly distorted 8-rings subunits present in the pollucite zeolite [43]. Lastly, the IR vibration band at 441 cm -1 is ascribed to the bending vibration of TO4 (T = Si, Al). Nanocrystalline Cs-pollucite exhibits Type IV N2 adsorption-desorption isotherm with H1 hysteresis loop (Fig. 4b). It indicates the presence of cylindrical or tubular type pores with a narrow distribution of mesopores of 10.7 nm due to the close-packing of nanocrystals [44]. Furthermore, an external surface area of 39 m 2 /g and a total pore volume of 0.29 cm 3 /g are also measured. No microporosity is detected due to the smaller pore size of pollucite zeolite (σ = 2.43 Å) than nitrogen probe molecules (σ = 3.64 Å) [45]. Nevertheless, this nanocrystalline material can be used as a catalyst since it contains highly basic Cs + extraframework cations on the external surface.

In order to confirm the presence of Cs element and to study the chemical composition in the Cs-pollucite nanozeolite, XRF spectroscopy analysis has been carried out. In general, the Si/Al ratio of Cs-pollucite zeolite is 2.0 while its theoretical molecular formula is Cs16[Al16Si32O96] [43,46]. Interestingly, the nanosized Cs-pollucite obtained in this work is a high-silica zeolite (Si/Al ratio = 4.11) and the experimental molecular formula is calculated to be Cs15.65[Al7.78Si32.00O83.46].

Thus, the elemental analysis reveals that nanosized Cs-pollucite zeolite might have surface basicity.

The surface basicity of nanocrystalline Cs-pollucite is studied using CO2-TPD analysis (Fig. 5). Three CO2 desorption peaks are obtained after deconvolution of the TPD profile. The interaction of CO2 with the weak basic sites located at the textural pores can be observed from the CO2 desorption band at 88.1 °C. Meanwhile, the broad peak at 442 C which can be deconvoluted into two peaks (442 C and 603 C) correspond to medium-to-strong basic sites located at the external surface of the zeolite.

Catalytic reaction study

Perkin condensation of benzaldehyde with acetic anhydride is used as a model reaction to study the potential of Cs-pollucite nanozeolite as a solid base catalyst. The reaction produces only cinnamic acid as sole product throughout the study (Scheme 1). As can be seen from Fig. 6, the conversion rate is low when no catalyst is added especially at 170 C where only 29.4% of conversion is recorded after 80 min (Fig. 6a). When the reaction temperature is increased to 190 C, the conversion increases to 65.1% using the same reaction time (Fig. 6c). On the other hand, are extracted to calculate the second order rate constants. The rate constants are then used to plot Arrhenius plots to determine the activation energy (Ea) of the reaction (Fig. 7). As calculated, the Ea of non-catalyzed Perkin condensation reaction is very high (144.89 kJ/mol). However, by adding nanosized Cs-pollucite zeolite as catalyst, the Ea is significantly reduced to 40.04 kJ/mol.

Thus, this study shows that Perkin condensation of benzaldehyde is an activated catalytic reaction. Comparative catalytic performance between nanosized Cs-pollucite zeolite and homogenous base catalysts such as sodium hydroxide and potassium hydroxide (equivalent molar amount to Cs-pollucite zeolite is added) is also performed at various reaction times (10, 20, 30 and 80 min) at 190 C. As expected, potassium hydroxide gives higher conversion (90.1%) compared to sodium hydroxide (83.1%) after 80 min of reaction due to its high basicity (Fig. 8). On the other hand, nanosized Cs-pollucite zeolite performs slightly better than potassium hydroxide under the same reaction time. Catalyst reusability is a major problem in solid catalyst [47]. Hence, catalyst reusability is tested on nanosized Cs-pollucite. The catalytic activity of the recycled nanozeolite catalyst is preserved even after the fifth consecutive reaction cycle thanks to the strong bonding between the Cs + cations and the zeolite surface via electrostatic attraction forces (Fig. 9) [33]. The XRD pattern nanocrystalline Cs-pollucite zeolite after fifth cycle of catalytic reaction was obtained (Figure 10).

The XRD peaks corresponding to ANA crystalline phase remain intact and no significant loss in crystallinity is shown. Thus, it reveals that the ANA crystalline framework structure of Cspollucite is stable even after multiple cycles of catalytic reaction. The XRD observation is further supported by TEM and FESEM analysis (Fig. 11). As can be seen, the Cs-pollucite nanocrystals retain their morphology and shape while no interparticle nor intraparticle amorphization is shown in the TEM image confirming the highly crystalline nature of recycled Cs-pollucite nanocatalyst.

Hence, the results suggest that nanosized Cs-pollucite zeolite can be used as a potential solid base catalyst besides other advanced applications.

Conclusion

One-pot hydrothermal synthesis of nanocrystalline Cs-pollucite zeolite (ca. 79.4 nm) via organotemplate-free approach is reported. The ANA framework (Si/Al ratio = 4.11) can be crystallized from a clear precursor within 50 h at mild temperature (180 °C). The Cs-pollucite dispersion is colloidally stable and is active in Perkin condensation of benzaldehyde with acetic anhydride. Thus, these nanocrystals with accessible mesoporosity have great potential in advanced applications particularly in film deposition and catalytic technologies.

Fig. 1 .

 1 Fig. 1. XRD patterns of solids after (a) 47 h, (b) 48 h and (c) 50 h of hydrothermal synthesis.

Fig. 2 .

 2 Fig. 2. (a,b) SEM images and (b) plot of particle size distribution of nanosized Cs-pollucite zeolite.

Fig. 3 .

 3 Fig. 3. (a) TEM image with theoretical shape (inset), and (b) colloidal solution of nanosized Cs-

Fig. 4 .

 4 Fig. 4. (a) IR spectrum and (b) nitrogen adsorption (close symbol), desorption (open symbol) and

Fig. 5 .

 5 Fig. 5. CO2-TPD profile of nanosized pollucite.

Scheme 1 .

 1 Scheme 1. Perkin condensation of benzaldehyde with acetyl anhydride to product cinnamic acid.

Fig. 6 .

 6 Fig. 6. Perkin condensation of benzaldehyde with acetic anhydride without catalyst at (a) 170 ºC,

Fig. 7 .

 7 Fig. 7. Arrhenius plots of Perkin condensation of benzaldehyde performed (a) without catalyst and

Fig. 8 .

 8 Fig. 8. Product conversion to vanillin acid catalyzed with (a) sodium hydroxide, (b) potassium

Fig. 9 .

 9 Fig. 9. Catalyst recycling test of nanosized Cs-pollucite zeolite in Perkin condensation reaction.

Fig. 10 .

 10 Fig. 10. XRD pattern of nanosized Cs-pollucite zeolite after 5 cycles of Perkin condensation

Fig. 11 .

 11 Fig. 11. (a) SEM and (b) TEM images of nanosized Cs-pollucite zeolite after recycling test.

Acknowledgement

The financial support from RUI (1001/PKIMIA/8011012) and SATU Joint Research Scheme (RU018O-2016) grants is gratefully acknowledged. The doctoral scholarship from University of Hail, Saudi Arabia is also acknowledged.