Georgios N Kalantzopoulos

Fredrik Lundvall

Knut Thorshaug

Anna Lind

Ponniah Vajeeston

Iurii Dovgaliuk

Bjørnar Arstad

David S Wragg

Helmer Fjellvåg

Factors Determining Microporous Material Stability in Water: The Curious Case of SAPO-37

Keywords: SAPO-37, hydration, water adsorption, in situ SR-XRD, parametric Rietveld refinement, DRIFTS, DFT calculations

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Introduction

Zeolites are used in catalytic, ion exchange and separation processes in several industries, and due to their acidic and shape-selective properties they play a major role in crude oil cracking. [START_REF] Weckhuysen | Recent advances in zeolite chemistry and catalysis[END_REF] The search for zeolite structures made with large pore windows for enhanced accessibility to their acid sites, led to the employment of the faujasite zeolites and variants of the material such as zeolites X, Y and the ultra-stable Y (USY) by a variety of industries. Today, more than 40% of total crude oil conversion takes place within the walls of a microporous catalyst with the faujasite structure. [START_REF] Martino | Catalysis for oil refining and petrochemistry, recent developments and future trends[END_REF] Silicoaluminophosphates (SAPOs) are a special class of zeolites where a framework of tetrahedrally oxygen-coordinated Al and P has been partially substituted by Si creating a Brønsted acid site. [START_REF] Lok | Silicoaluminophosphate molecular sieves: another new class of microporous crystalline inorganic solids[END_REF] Due to its highly crystalline structure, large pore opening and high surface area, SAPO-37 is a key material in processes such as the isomerization of ndecane, [START_REF] Mostad | Comparison of the iso-structural H-SAPO-37 and H-faujasite as catalysts for the isobutane/2-butene alkylation[END_REF] isobutene/2-butene alkylation, [START_REF] Smith | A quantitative description of the active sites in the dehydrated acid catalyst HSAPO-34 for the conversion of methanol to olefins[END_REF] o-xylene isomerization [START_REF] Ojo | Synthesis and properties of SAPO-37[END_REF] or as gas adsorbent [START_REF] Verboekend | Synthesis, characterisation, and catalytic evaluation of hierarchical faujasite zeolites: milestones, challenges, and future directions[END_REF] . SAPO-37, has the same faujasite (FAU) topology as found in zeolites X, Y and the USY ultra-stable Y (USY). It is a highly crystalline microporous material possessing a 3-D network where tetrahedrally coordinated T atoms (T = Al, P or Si) compose sodalite cages (SOD) linked by double 6-rings (d6r: meaning two rings of 6 T-atoms). The 3-D arrangement of the SOD cages form large 12-membered rings and create the faujasite supercage (FAU). We can describe the local T-atom environment as Si-substituted AlPO4 tetrahedra (Si substituting P and/or Al) with Si, Al and P being homogenously distributed around the framework. [START_REF] Sierra De Saldarriaga | Investigations into the nature of a silicoaluminophosphate with the faujasite structure[END_REF] The structure can also be visualized as a tetrahedral array of SOD cages interconnected by d6rs. This interconnected array of channels and cages forms the characteristic cubic structure of faujasite that crystallizes in space group Fd3 ̅ m with a typical cubic unit cell dimension of a = 24.345 Å for SAPO-37. The 12-ring has a diameter of 11.2 Å (distance across diametrically opposite oxygen atoms) allowing molecules with diameter of up to 7.3 Å to diffuse freely through the channels. [START_REF] Baerlocher | Database of Zeolite Structures[END_REF] However, due to molecular vibration, molecules possessing critical diameters of up to 9.5 Å can also penetrate the faujasite porous network at moderate temperatures. 10 SAPO-37 is synthesized with a dual-template method. Tetramethylammonium hydroxide (TMA) is used to form the SOD cages and tetrapropylammonium hydroxide (TPA) is used for the assembly of the large FAU supercage. [START_REF] Briend | Thermal stability of tetrapropylammonium (TPA) and tetra methylammonium (TMA) cations occluded in SAPO-37 molecular sieves[END_REF] Removal of these templates requires higher temperatures than usual, when compared to other SAPO synthetic zeolites. [START_REF] Kalantzopoulos | SAPO-37 microporous catalysts: revealing the structural transformations during template removal[END_REF] By itself, TMA combusts at a moderate temperature (473 K). In the case of SAPO-37 however, it is not until the FAU supercage is almost cleared of TPA template that TMA can begin its combustion. This takes place between 690 and 945 K, inducing unusual local geometric responses to the thermal events. [START_REF] Kalantzopoulos | SAPO-37 microporous catalysts: revealing the structural transformations during template removal[END_REF] During this procedure, the Brønsted acid sites are created turning the material from a template-stabilized structure into a crystalline, catalyst framework. [START_REF] Sierra De Saldarriaga | Investigations into the nature of a silicoaluminophosphate with the faujasite structure[END_REF][START_REF] Briend | Thermal stability of tetrapropylammonium (TPA) and tetra methylammonium (TMA) cations occluded in SAPO-37 molecular sieves[END_REF][START_REF] Kalantzopoulos | SAPO-37 microporous catalysts: revealing the structural transformations during template removal[END_REF] A critical parameter that promotes structural changes in SAPOs during real-life applications is the presence of humidity. [START_REF] Aramburo | X-Ray Imaging of SAPO-34 Molecular Sieves at the Nanoscale: Influence of Steaming on the Methanol-to-Hydrocarbons Reaction[END_REF][START_REF] Sastre | Mechanisms of silicon incorporation in aluminophosphate molecular sieves[END_REF] Water is an irreplaceable and frequently also an unavoidable component of reallife applications, [START_REF] Olsbye | Conversion of Methanol to Hydrocarbons: How Zeolite Cavity and Pore Size Controls Product Selectivity[END_REF] and its effect on the local structure of the framework needs to be carefully studied and understood. Although template-free SAPO-37 has exceptional hydrothermal stability at high temperatures (T>1000 K), it has surprisingly poor stability below 345 K. [START_REF] Briend | Thermal and hydrothermal stability of SAPO-5 and SAPO-37 molecular sieves[END_REF] In 1987, de Saldarriaga et al. observed that exposure of template-free SAPO-37 to atmospheric humidity at temperatures close to RT induces a shrinkage of the unit cell. [START_REF] Sierra De Saldarriaga | Investigations into the nature of a silicoaluminophosphate with the faujasite structure[END_REF] Later, Briend et al. reported that the material begins contracting and loses crystallinity below 345 K at a rate that increases the closer the temperature is to RT. [START_REF] Briend | Thermal and hydrothermal stability of SAPO-5 and SAPO-37 molecular sieves[END_REF] To date, the mechanism behind this unforeseen crystallinity loss has not been experimentally revealed.

One early suggestion for explaining the poor, low temperature hydrothermal stability was that unfavorable P-O-P bonds would form during the templates removal process. Yet to date, all the NMR studies have unanimously shown only one type of phosphorous local environment in the as-calcined SAPO-37 framework: a phosphorous atom coordinated to four aluminum atoms (P(4Al)) [START_REF] Sierra De Saldarriaga | Investigations into the nature of a silicoaluminophosphate with the faujasite structure[END_REF][START_REF] Briend | Thermal stability of tetrapropylammonium (TPA) and tetra methylammonium (TMA) cations occluded in SAPO-37 molecular sieves[END_REF][START_REF] Briend | Thermal and hydrothermal stability of SAPO-5 and SAPO-37 molecular sieves[END_REF][START_REF] Corma | Hydrothermal Stability and Cracking Behavior of Silicoaluminophosphate Molecular Sieve-37 with Different Silicon Contents[END_REF][START_REF] Buchholz | Successive steps of hydration and dehydration of silicoaluminophosphates H-SAPO-34 and H-SAPO-37 investigated by in situ CF MAS NMR spectroscopy[END_REF] as expected by charge balance considerations. In an attempt to systematically assess the hydrothermal stability of SAPO-37 Corma et al. observed at least six types of hydroxyl groups at 573 K that were formed during the templates removal process. 17 Among these, terminal SiO-H and PO-H were detected and remained present up to the end limit of the applied heat treatment at 873 K. Out of the four other hydroxyl species two were considered to be bridging Si-OH-Al directed towards the supercage and to the sodalite cages, while two types of hydroxyls were associated with remaining organic templates. When the authors performed partial template removal at 673 K (removing the TPA + from the FAU supercage but keeping the majority of TMA + in the SOD cage) the material preserved its crystallinity in presence of atmospheric humidity for substantially longer time. They suggested that calcination at 873 K would favor the condensation of some Si-OH and P-OH species leading to formation of Si-O-Si and P-O-P bonds. The latter is highly susceptible to hydrolysis upon exposure to water at near ambient temperature. However, they made no direct experimental observation of P-O-P species. These observations will be discussed in light of our new data.

Although the loss of long-range order in SAPO-37 has been known since the first reports of the material, no data are reported on how hydration affects the specific surface area (SSA), pore volume and mean pore diameter. Furthermore, the fine interplay between water and the SAPO-37 framework has never been elucidated. One hypothesis is that water adsorption dynamics should play a role, [START_REF] Briend | Thermal and hydrothermal stability of SAPO-5 and SAPO-37 molecular sieves[END_REF] but relevant experimental data is lacking. The reasons are partially due to the metastability of SAPO-37 in presence of water at temperatures below 345 K and partially due to lack of advances in the field of water adsorption assessment. The recent advances in minimizing the error propagation and significantly enhancing the reproducibility in sorption assessment by the Sieverts technique, [START_REF] Sieverts | Information on the occlusion and diffusion of gases through metals[END_REF] which stem from the boom in hydrogen storage research, [START_REF] Broom | The accuracy of hydrogen sorption measurements on potential storage materials[END_REF] have now opened new opportunities for detailed insight.

Other SAPO frameworks such as SAPO-34, a key industrial catalyst in the methanol-to-olefins (MTO) conversion, are also affected by humidity at low temperatures but the destabilization progresses at very slow rates. [START_REF] Arstad | Structural changes in SAPO-34 due to hydrothermal treatment. A NMR, XRD, and DRIFTS study[END_REF] SAPO-37 on the other hand, shows rapid and drastic loss in hydrothermal stability at low temperatures. [START_REF] Briend | Thermal and hydrothermal stability of SAPO-5 and SAPO-37 molecular sieves[END_REF] As a highly crystalline material, [START_REF] Kalantzopoulos | SAPO-37 microporous catalysts: revealing the structural transformations during template removal[END_REF] that does not form intergrowths like SAPO-34 [START_REF] Kalantzopoulos | Insitu flow MAS NMR and synchrotron PDF analyses of the local response of the Brønsted acidic site in SAPO-34 during hydration at elevated temperatures[END_REF][START_REF] Sławiński | Intergrowth structure modelling in silicoaluminophosphate SAPO-18/34 family[END_REF] , but shares the same active chemistry [START_REF] Buchholz | Successive steps of hydration and dehydration of silicoaluminophosphates H-SAPO-34 and H-SAPO-37 investigated by in situ CF MAS NMR spectroscopy[END_REF] , SAPO-37 makes an excellent model material to study the effect of humidity on SAPO frameworks in general.

In this study we present and discuss data on how water accumulates on the SAPO-37 surface and propose a mechanism of how the water concentration grows within the structure of the framework. High time-and space-resolution synchrotron radiation powder X-ray diffraction (SR-PXRD) reveals, for the first time, the local transformations connected with hydration in each of the SAPO-37 cage types. We evaluate the SAPO-37 water adsorption capacity above and below the critical temperature of 345 K while monitoring the changes in SSA, pore volume, pore size distribution and crystallinity before and after each measurement. The presence of surface defects after calcination and the local interaction of water with the SAPO-37 framework during hydration were probed by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). Density functional theory (DFT) calculations confirmed the preferential adsorption sites of water in the structure that were revealed by SR-PXRD. We also investigate the effect of SAPO-37 topology and of possible surface defects with respect to the material stability, and critically discuss our results in view of the existing literature.

Experimental

Material Synthesis. SAPO-37 was synthesized according to literature procedures. [START_REF] Franco | Crystallization kinetics of SAPO-37[END_REF] Details are provided in the S.I.

In situ Synchrotron Radiation Powder X-ray Diffraction (SR-PXRD) Hydration. In situ SR-PXRD measurements were performed at the Swiss-Norwegian beamlines (station BM01) [put ref . 26] at The European Synchrotron Radiation Facility (ESRF) in Grenoble, France. A fresh as-synthesized SAPO-37 sample was placed in a 0.5 mm quartz capillary and mounted in a flow cell that provides controlled atmosphere. For calcination, the sample was heated up to 1073 K at 2 K min -1 in flowing synthetic air using a hot air blower. For hydration experiments, nitrogen was passed through a water-filled bubbler kept at ambient temperature. Temperature calibration was performed by measuring a sample of high purity Ag and Al2O3. The SR-PXRD patterns (exposure time 6 s) were collected using a Dectris Pilatus 2M photon counting pixel area detector [25][START_REF] Dyadkin | A new multipurpose diffractometer PILATUS@SNBL[END_REF] and integrated with the SNBL Bubble software. [START_REF] Fjermestad | Mechanistic Comparison of the Dealumination in SSZ-13 and the Desilication in SAPO-34[END_REF] The wavelength, λ = 0.71210 Å was calibrated from individual runs with LaB6.

Powder X-ray Diffraction (PXRD).

In-house powder X-ray diffraction (PXRD) data were recorded at ambient atmosphere and temperature on a Bruker D8-A25 instrument using monochromatic CuKα1 radiation (λ = 1.5406 Å) from a fine-focus sealed tube source and a LynxEye XE position sensitive detector operated in transmission geometry.

X-ray Diffraction Data

Processing. The patterns were analyzed by parametric Rietveld refinement [START_REF] Stinton | Parametric Rietveld refinement[END_REF] using TOPAS 29 to extract the unit cell parameters, framework atom positions, isotropic thermal displacement parameters (B-iso) and water occupancy as a function of temperature. A single B-iso parameter was refined for all oxygen atoms. Diamond [START_REF] Brandenburg | 3.2k; Crystal Impact GbR[END_REF] and Vesta [START_REF] Momma | VESTA 3 for three-dimensional visualization of crystal, volumetric and morphology data[END_REF] were used for illustrating the crystal structures. Adobe Photoshop CS6 was used for the blending of the different time-frame illustrations in Figure S7.

Water and Nitrogen Adsorption Measurements.

Nitrogen and water sorption measurements were performed on a BelSorp Max instrument at 77 K for nitrogen and at 353 and 323 K for water. Prior to each sorption measurement (nitrogen or water) annealing took place for 2 h at 423 K in He atmosphere to remove any residual gas species from the porous material. All measurements and heat treatments took place sequentially to avoid any exposure of the samples to atmospheric air.

The measurement procedure to assess the hydration / water adsorption effect on template-free SAPO-37 was:

N2 PXRD measurements took place before the first N2 adsorption and after each subsequent N2 adsorption measurement. Given the notorious instability of the material upon exposure to humidity, the transfer and loading times from the calcination oven to the sorption instrument or the XRD apparatus capillary were restricted to less than 30 minutes and under low relative humidity atmosphere (< 50%). To avoid unnecessary exposure of the material to humidity between the two water adsorption measurements, two cells were run in parallel; cell 1 underwent the whole multi-step experiment whereas cell 2 was removed after the second nitrogen adsorption measurement for the assessment of the structural degradation via PXRD. The timeline evolution of this multi-step experiment is provided in the S.I (Figure S8).

The total SSA was extracted from the nitrogen adsorption isotherms via the Brunauer-Emmett-Teller (BET) method according to the literature. [START_REF] Shearer | Defect Engineering: Tuning the Porosity and Composition of the Metal-Organic Framework UiO-66 via Modulated Synthesis[END_REF] Non-local density functional theory (NLDFT) calculations of the pore size distribution (PSD) were performed using the commercial BELMaster software from MicrotraBEL Corp. The NLDFT calculation method was applied on the adsorption branch using the N2 physisorption data collected at 77 K. For the calculations a cylindrical pores model was assumed.

The evaluation of the number of H2O molecules per surface unit took place using equation 1, where NA is the Avogadro number, wt% is the gravimetric adsorption, MWgas is the molecular weight of the adsorbed fluid and SSA is the specific surface area obtained by BET. [START_REF] Antoniou | Methane storage in zeolite-like carbon materials[END_REF][START_REF] Antoniou | Naphthalene-based periodic nanoporous organosilicas: II. Hydrogen and methane adsorption and physicochemical study[END_REF][START_REF] Kalantzopoulos | Resistance to the transport of H-2 through the external surface of as-made and modified silicalite-1 (MFI)[END_REF][START_REF] Kalantzopoulos | Enhanced hydrogen and methane storage of hybrid mesoporous organosilicas[END_REF][START_REF] Kalantzopoulos | Hydrogen storage in ordered and disordered phenylene-bridged mesoporous organosilicas[END_REF] The values of the SAPO-37 occupiable i and accessible ii surface area were obtained by the literature. 38

𝑁 𝑚𝑜𝑙𝑒𝑐 𝑛𝑚 2 = 𝑁 𝐴 × 𝑤𝑡% 10 20 × 𝑀𝑊 𝑔𝑎𝑠 × 𝑆𝑆𝐴 (1)
In order to gain a deeper insight into adsorbant-adsorbent interaction, the experimentally obtained data were fitted according to the Tόth model 39 (equation 2), where wt%max is the asymptotic maximum storage capacity, K is the equilibrium constant, P is the pressure and t is a parameter introduced by Tόth in order to describe the level of homogeneity of the sample surface.

𝑤𝑡% = 𝑤𝑡% 𝑚𝑎𝑥 𝐾𝑃 (1 + (𝐾𝑃) 𝑡 ) 1 𝑡 ⁄ (2)

Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS).

We performed DRIFTS measurements on template-free SAPO-37 under similar conditions as the in-situ SR-PXRD and the water adsorption measurements. The experimental details and spectra can be found in the S.I.

i Area that can be occupied by a water molecule assuming a radius of 1.4 Å. ii The continuous area that can be accessed by a water molecule, excluding pores/cavities too small to host a water molecule.

Computational Details.

Quantum-mechanical calculations via the density functional theory (DFT) method were performed on the template-free SAPO-37 framework. The computational details and main results can be found in the S.I.

Results

In situ SR-PXRD hydration.

Recently we studied the template removal of SAPO-37 in situ revealing the sub-unit cell transformations that take place in the framework during calcination, resulting in a highly crystalline material [START_REF] Kalantzopoulos | SAPO-37 microporous catalysts: revealing the structural transformations during template removal[END_REF] . Building on the template removal study, we exposed the same powder sample to a variety of atmospheres at low (313 K) temperature. From these experiments we evaluated the low temperature stability of SAPO-37 in argon, atmospheric air (40-50% relative humidity) and in water-saturated Ar, under constant (313 K) and variable temperature conditions. A schematic timeline summarizing all the different experimental conditions is presented in the S.I. (Figure S2).

In the first part of the experiment, the template-free material was exposed to inert atmosphere (100% Ar) at 313 K for 190 min. In those conditions, no changes can be detected in the diffraction patterns (Figure S3). The structure is stable and retains its crystallinity over time. Then we switched to atmospheric air (40-50% relative humidity at 313 K) for about 100 min. In those conditions the diffraction patterns look macroscopically unchanged (Figure S4 top). However, a closer analysis reveals that after a short induction period, the SAPO-37 framework undergoes a small unit cell volume loss that progresses at the rate of 1 Å 3 /min (Figure S4 bottom). It is interesting to note that despite the absence of easily visible changes in the diffraction patterns, the degree of crystallinity (DoC) is profoundly affected. The DoC was calculated comparing the relative area of the strongest reflections (111 and 222) in each pattern with their corresponding area in the diffraction pattern of the fresh, template-free material according to equation 3.

Degree of crystallinity, 𝐷𝑜𝐶

= 𝑎𝑟𝑒𝑎 ℎ𝑘𝑙 𝑝𝑒𝑎𝑘,𝑖 𝑎𝑟𝑒𝑎 ℎ𝑘𝑙 𝑝𝑒𝑎𝑘,0 • 100% (3)
where hkl = 111 and 222 of PXRD pattern i of the hydrated material, compared to hkl = 111 and 222 of the starting PXRD pattern o obtained from the completely dry, template-free framework. In the last part of the experiment, the temperature was increased from 313 to 473 K at 5 K min -1 while maintaining the hydration feed (Figure 2). During the first 1.5 min (T = 313-323 K), there is peak intensity loss and a shift of the reflections towards higher values. After 1.5 min (T = 323 -333 K) the changes become more rapid and pronounced, as the hydrated material responds to heating. This transition over the first 3 minutes is accompanied by a dramatic loss in peak intensity. Furthermore, the loss of peak intensity is strongly correlated with an increase in the background in the 2θ = 6 -15° range, indicating the gradual formation of a poorly ordered phase. Further increase in temperature leaves only the first two reflections (111 and 022) visible.

For this section of the experiment it was possible to follow the transition via parametric Rietveld refinement for the first 4. From the crystallinity and cell volume variation vs. time plots, it is easily noticeable that crystallinity loss and change in the cell volume are two independent phenomena since different diffraction peaks have different DoC loss profiles. In essence, a reflection corresponding to a small d spacing will have a different response to inclusion, adsorption or accumulation of water molecules compared to a reflection from a larger d spacing that would be influenced by the accumulative effect of water in different sub-unit cell regions. Large cages would have slower response to water accumulation compared to the narrower parts of the unit cell, such as the SOD cages or the d6rs. In order to describe the geometric response of different domains of the SAPO-37 unit cell during the first seconds of hydration under heating (Figure 2), we have plotted selected T-O-T angles, T-T and O-O distances for the FAU supercages, the SOD cage and the d6r bridging units (Figure 3). As a typical T-O-T angle in the FAU cage we selected a T-O-T linkage in the d6r with the oxygen atom pointing towards the supercage (Figure 3 .77 Å (48% extension) and from 5.15 to 7.46 Å (45% extension), respectively (Figure 3e). Finally, in Figure 4c we see that there is a substantial expansion within the d6r and particularly in the SOD-d6r interface until reaching the breaking point (on selected T-O-T bonds). This T-O-T angle is the first part of SAPO-37 to respond to the increasing temperature during hydration, becoming wider already 20 s within the experiment. Overall, the T-O-T angle widens from 140 to 154° (Figure 3f). Simultaneously, the T-T distance between two SOD cages increases from 2.57 to 2.82 Å (9.7% extension) and has a similarly fast response as the T-O-T angle (Figure 4f). The T-O length increases from 1.87 to 2.19 Å (Figure 4f). The starting value (1.87 Å) is already 15% longer than the nominal T-O of the template-free SAPO-37 (average T-O length in our sample at RT, 1.625 Å). As the hydration continues and the temperature increases this length reaches a value that is 17% higher with respect to the beginning of the experiment (and 35% with respect to the average T-O length of the dry, template-free framework). This is the largest bond length variation found in the framework during this experiment. As such, it is a clear indication that this point in the framework is critical during the structural collapse.

From the above analysis, we can observe that each building unit has a slightly different time response to the temperature change during hydration. The smallest volume (d6r), responds to heating first, followed by a contraction of the large FAU supercage and the distortion of the SOD cage. This can be explained if we assume that water is primarily concentrated in the SOD cage and in the SOD-d6r interface. Note that the structural changes in the FAU supercage are primarily due to the geometric responses occurring to each of the six surrounding SOD cages and the d6rs. A graphical illustration comparing the dry and hydrated framework at selected time-frames up to the point of the selected T-O bond failure is shown and discussed in detail in Figure S7.

Water Adsorption on SAPO-37

In order to further understand water's role in the destabilization of the SAPO-37 framework, we performed water adsorption measurements above and below the critical temperature of 345 K. Measurements were carried out first at 353 K and subsequently at 323 K where the interaction of water with the framework is anticipated to be very strong. Specific surface area and crystallinity were assessed by nitrogen physisorption measurements and PXRD at defined stages throughout the experiment, as described in the S.I. (Figure S8).

At 2.75 kPa the material surface retains 7.1 H2O molecules per unit cell, corresponding to a surface density of 0.31 H2O molecules per nm 2 in both sample cells (Figure 4). By comparison, water adsorption on SAPO-37 at 323 K shows a multi-step surface filling mechanism (Figure 4). During the very first adsorption steps (p < 0.05 kPa) the material uptakes 5.0 H2O molecules per unit cell, that is comparable to the total capacity at 353 K and 2.75 kPa. As the pressure increases, the adsorption continues linearly until a second adsorption step is seen in the pressure region 0.2 -0.5 kPa. A small shoulder can be observed at 1 kPa before the adsorption capacity reaches a quasi-saturation plateau. Above that pressure the adsorption continues linearly and at a substantially lower rate (3.8 H2O molecules per kPa). Compared to the isotherm at 353 K, the number of H2O molecules per unit cell at 323 K is an order of magnitude higher, and the H2O molecules per surface unit increases by a factor of seven (Figure 4).

In order to obtain more quantitative information the isotherms were fitted with the Tόth model (equation 2). The model was selected due to its ability to describe the adsorbant coverage over a variety of adsorbent surfaces and porous structures [START_REF] Kalantzopoulos | Resistance to the transport of H-2 through the external surface of as-made and modified silicalite-1 (MFI)[END_REF][START_REF] Kalantzopoulos | Hydrogen storage in ordered and disordered phenylene-bridged mesoporous organosilicas[END_REF] and complex hybrid materials that offer a variety of adsorption sites within the same framework. [START_REF] Kalantzopoulos | Enhanced hydrogen and methane storage of hybrid mesoporous organosilicas[END_REF] The water adsorption isotherm at 353 K shows a multi-step adsorption mechanism with a total uptake of 33 wt% (Figure S9, at S.I.) corresponding to 64 H2O molecules per unit cell (Figure 4). According to the Tόth model fitting, the surface has a theoretical capacity of 95 H2O molecules per unit cell at these pressure and temperature conditions. Note, the multi-step adsorption leads to a comparatively poor fit of the Tόth model and the maximum theoretical capacity should be regarded as a rough estimation that will not be discussed further. Details in the results are reported in Table S1.

Diffuse Reflectance Infrared Fourier Transform Spectroscopy (DRIFTS).

To obtain complementary data to PXRD when SAPO-37 interacts with water we performed DRIFTS measurements, and the overall interpretation and analyses of these data are in agreement with our in situ SR-PXRD and the ex-situ structural analysis of the samples after water adsorption. After the in situ calcination of the as-synthesized material under synthetic air in the DRIFTS sample cell, the spectrum shows all the key features reported by Corma et al. [START_REF] Corma | Hydrothermal Stability and Cracking Behavior of Silicoaluminophosphate Molecular Sieve-37 with Different Silicon Contents[END_REF] (error with Error! Reference source not found.S13a in the S.I.). Band assignment can be found in the S.I.

The band positions observed in template-free SAPO-37 and after annealing at 423 K are the same, but their intensities are somewhat lower in the latter. This suggests that 423 K is a sufficient temperature for dehydration of the framework although there are some minor irreversible material changes likely due to interaction with water. After exposure to H2O at 323 K (error with Error! Reference source not found.S13d), the bands assigned to -OH groups in the sodalite cage and supercage are barely observed in the spectra, suggesting that the cages have collapsed. Heating to 423 K and then 723 K does not regenerate the SAPO-37 (Figure S13e). The main bands observed in the final material coincide with the bands assigned to P-OH and Si-OH in template-free SAPO-37. From spectra (error with Error! Reference source not found. S13) we infer that the structure deterioration is more pronounced at 323 K than at 353 K due to the less resolved bands.

Quantum-mechanical calculations via the density functional theory (DFT) method.

We performed DFT calculations on template-free SAPO-37 framework in order to assess the effect of topology and local geometry on the enthalpy of water adsorption and to identify the areas within the SAPO-37 framework with highest affinity to water. The computational details and more detailed results can be found in the S.I.

According to the calculations the H2O adsorption energy value for the SOD cage site and the FAU supercage site are -212 and -13 kJ/mol respectively. This difference in magnitude of the adsorption energy clearly indicates that the H2O absorption in SOD cage is much more energetically favorable than in the FAU supercage.

Figure 6 shows the calculated water density in the SAPO-37 framework and in SOD cages. The water density in the SOD cages is clearly higher compared to the water density in the FAU environment. Interestingly, the water molecules in the FAU cages have higher concentration along the channels interconnecting the FAU cages. The calculated distribution of the H2O-H2O molecules distance in the material is shown in the Figure S14. The largest fraction of water molecule pairs has a calculated average distance that is less than 1.8 Å, indicating a medium strength H-bonding. [START_REF] Steiner | The Hydrogen Bond in the Solid State[END_REF] Most of them (> 60 %) were found to be concentrated in the SOD cage.

Discussion

Structural Transformations induced on the SAPO-37 framework during low temperature hydration.

Our results indicate that as the dry and template-free SAPO-37 is being hydrated, water molecules accumulate within the narrowest internal domains of the framework, namely the SOD cages and the d6r units. The Brønsted acidic centers (one per SOD cage and 1.7 per FAU cage on average) and the P-OH and Si-OH moieties (which are known to increase the hydrophilicity of the surface [START_REF] Zhang | Factors that Determine Zeolite Stability in Hot Liquid Water[END_REF] ) can act as local anchors for water molecules to the framework enhancing the adsorbent/adsorbate interaction. The material has a pronounced water adsorption capacity which is much greater at 323 K than 353 K (observed by H2O adsorption and corroborated by DFT calculations). This suggests that at low temperatures there is a significantly increased chance for water to be in close contact with the surface in the SOD cage or the d6r. At high loadings, the properties of this surface water layer approach those of bulk water. The high concentration of water around the acid centers and defects, combined with the confining geometry of the framework result in the structural destabilization of the SAPO-37 framework. Adding thermal energy to a highly hydrated SAPO-37 accelerates the breakdown of the material.

The geometrical characteristics of the SOD-d6r-SOD interface provide an explanation as to why the structure ruptures at the T-O2-T moiety. This interface includes three adjacent 4-rings (Figure 7), where the T-O4-T angle is particularly strained in the hydrated form of the material, compared with the dry template-free structure (166 ° vs. 144° dry, O4: red spheres marked with green dashed circles in Figure 7). As the SOD cages distort during heating and hydration due to the massive uptake of water, increased pressure is applied to this area of the framework, observed as the dramatic change in the T-O4-T angle from 166 to 101° in a matter of seconds before the structure collapses. To accommodate this change in angle, the T-O2 bond of the strained 4-ring stretches from 1.87 to 2.19 Å in a concerted motion before it finally breaks (O2: red spheres marked with black dashed lines in Figure 7).

Mechanism of Water Attack on SAPO-37.

Based on our experiments, we propose a mechanism for how water accumulates and attacks the SAPO-37 framework resulting in its rapid, complete disruption. Water behaves quite differently in the relatively large FAU cage compared to the more confined SOD cage and the d6r. The surface of the FAU cage has a smaller curvature than the SOD cage as well as a lower concentration (approximately half) of acidic centers per nm 2 . As the template-free framework is exposed to water molecules, some will interact weakly with the surface of the FAU cage. Other will enter the more confined and acidic SOD cage and d6r. Once a water molecule finds itself within the SOD cage or d6r, it is highly likely to get "trapped" there because of the local environment. This is strongly supported by our calculations showing that this part of the framework has a 𝛥𝛨 𝑎𝑑𝑠 factor of 16 higher than the FAU cages.

From our experiments and calculations it is clear that water accumulates initially in the small confined spaces in the SOD cages and the d6rs. This water space-filling procedure takes place rapidly and with great consequences. In the confined environment of a SOD cage or d6r entity the tightly packed cluster of water molecules is rather stable and should be able to deprotonate the corresponding acidic sites as it has been shown that clusters of 3-5 water molecules are able to deprotonate a zeolitic Brønsted site. [START_REF] Kletnieks | Adsorbate Clustering and Proton Transfer in Zeolites: NMR Spectroscopy and Theory[END_REF] This results in a highly acidic solution localized in the SOD/d6r domain. As it has been shown elsewhere, zeolite frameworks, and in particular those with Si/Al < 5, are highly susceptible to destabilization in an acidic environment. [START_REF] Yamanaka | Acid stability evaluation of CHA-type zeolites synthesized by interzeolite conversion of FAU-type zeolite and their membrane application for dehydration of acetic acid aqueous solution[END_REF] This is further supported by the fact that the SAPO-34 framework dissolves in 1 M HCl acid within minutes. [START_REF] Arstad | Methanol-to-hydrocarbons reaction over SAPO-34. Molecules confined in the catalyst cavities at short time on stream[END_REF] The change in the local pH could easily and quickly introduce local defects that will destabilize the framework leading eventually to the loss of long range order by e.g. attacking and O2 O4 O2 O4 destabilizing the Al-O-P bonds. In contrast, the FAU cages are far too large to form water clusters with the size and stability to initiate chemical attack in the framework and consecutive destabilization in a rate faster than that, which occurs in the SOD and d6r domains.

Conclusions

We have performed a comprehensive multi-technique study on the effect of hydration on the SAPO-37 framework and investigated the role that material topology and surface properties play on the material destabilization. In particular, we have followed the hydration of the framework with high time and spaceresolution in situ SR-PXRD at constant and dynamic temperature conditions up to the point of multiple bond ruptures in the unit cell. In addition, we have measured water adsorption and collected DRIFTS spectra above and below the critical temperature of 345 K, the threshold temperature below which the framework substantially degrades. Finally, DFT calculations have provided a valuable support to the extensive experimental study.

SAPO-37 is stable in dry air but shows reduced stability in atmospheric air that is reversibly proportional to the temperature and the degree of humidity. As the hydration progresses we can observe the coexistence of a dry and hydrated framework for a very brief period. Longer exposure to water at 313 K results in loss of the reflections corresponding to d spacing that is smaller than water molecule's kinetic diameter. Monitoring the degree of crystallinity in the 111 and 222 peaks revealed a different response to the hydration for the FAU and the SOD cages: Relatively confined areas fill up with water first whereas changes in the larger cage progress more slowly as a cumulative response to the water diffusion, adsorption and local geometry.

Water adsorption experiments confirmed the strong interaction that H2O has with the framework below 345 K. Interestingly, SAPO-37 can adsorb an order of magnitude more water molecules at 323 K than at 353 K. The exposure to water creates a substantial decrease in the SSA and is related to how close the material's temperature is to the RT. Additionally, the higher the water content within the framework, the broader the pore size distribution becomes. This pronounced material deterioration at lower temperatures was confirmed by the DRIFTS measurements. Finally, DFT calculations revealed that the SOD cage internal surface has a much higher affinity to water than the FAU supercage, having a calculated H2O adsorption energy value of -212 vs. -13 kJ/mol for the FAU, an astonishing factor of 16 times higher.

In conclusion the notorious instability of SAPO-37 can be explained as the cumulative contribution of topological, physical and chemical events leading to an array of rapidly evolving cascading effects. The narrow spaces in the SOD and d6r will physically trap water molecules. This preferred accumulation of water increases the mechanical strain leading to the rupture of the d6r which were found to be the weakest structural element of the framework. As the water concentration increases, bulk-like water starts forming locally. When it gets in contact with the Brønsted acidic sites it forms the highly acidic H3O + which autocatalyzes the destruction of the SAPO framework.

Our work shows how advancements in XRD instrumentation can provide data of exceptionally high quality with excellent time resolution. Detailed analysis of the large datasets obtained from experiments of this type can furthermore be analyzed using methods such as parametric Rietveld refinement to give new insight to highly dynamic and subtle features previously difficult to observe or measure.

The conceptual insight that non-homogeneous sorption of molecular species will induce dynamic features with dramatic consequences at a molecular and atomic level is very important for further work. We expect that the methods used, and the mechanisms found for SAPO-37 will be more broadly applicable in other framework materials. The work also provides a framework for developing more advanced models for gassolid reactions.

Figure 1 :

 1 Figure 1: 2D film plots of the evolution of the template-free SAPO-37 diffraction patterns during the first hydration period at 313 K (λ = 0.71210 Å). Selected diffraction patterns highlighting critical differences between the dry and hydrated material are shown in the top panel. The co-existence of the wet and hydrated SAPO-37 phases is illustrated in the dashed inset.

Figure 2 :

 2 Figure 2: 2D film plots of the evolution of the template-free SAPO-37 diffraction patterns during the second hydration period in the temperature range 313 -473 K. Selected diffraction patterns illustrating the evolution at critical time frames are shown in the top panel. The wavelength is λ = 0.71210 Å.

  25 min, up to 353 K (vide infra). Above this temperature we could only monitor the structural degradation via parametric peak fitting of the 111 and 022 reflections. The DoC and unit cell volume loss (assuming that the SAPO-37 unit cell is still present) are shown at FigureS6. Another interesting observation made in those conditions is that the (111) degree of crystallinity is affected more strongly than (222) degree of crystallinity (𝛥 111 𝐷𝑜𝐶 = 46.1 % 𝑣𝑠 𝛥 222 𝐷𝑜𝐶 = 32.4 %) pointing towards a rupture of the FAU cages and consecutive loss of the long range order.

Figure 3 :

 3 Figure 3: for the FAU supercages (a), the SOD cages (b) and the d6r bridging units (c). Selected T-O-T angles, T-T and O-O distances for the FAU supercage (a), the SOD cage (b) and the d6r bridging units (c). The variation of the selected T-O-T angles, T-T and O-O distances are illustrated in figures (d)-(f) for the FAU supercage, the

  (a)). Rather than monitoring the bond length of a given T-O pair we followed the variation of the FAU cage opening diameter (12-ring) using the distance of two diametrically opposite T-T and O-O pairs (dashed blue lines in Figure4(a)). In the SOD cage we selected a T-O-T linkage shared between the FAU and the SOD cages (Figure3(b)). As representative T-T and O-O distances of the SOD cage, we selected T-and O-atoms belonging to the 6-ring interface between SOD and FAU cages for monitoring. In the d6r bridging units we monitored a T-O-T linkage (green) shared between the 4-ring of the SOD cage and the d6r unit, a T-T pair representing the distance between the two SOD cages (dashed blue line and along the d6r) and finally an individual T-O bond length of the same entity used in the T-O-T angle (Figure 3(c)). For the latter, an equivalent position is shown in blue on Figure 3(c) for the sake of clarity.During the first 60 s of heating in humid Ar from 313 -473 K at 10 K min -1 there is practically no structural response to the heating, probably due to a delayed thermal response in the experimental setup. After this initial delay, there are considerable and rapid changes occurring in each individual domain of the unit cell. In the FAU supercage the T-O-T angle increases substantially from 95 to 132° in less than 45 s (Figure3d). Simultaneously, the T-T and O-O distances have completely opposite individual responses. The T-T distance decreases from 10.4 to 9.75 Å, whereas the neighboring O-O distance shows a substantial increase from 7.7 to 9.7 Å which effectively is a consequence of widening of the aforementioned T-O-T angle. In the SOD cage, the monitored T-O-T angle decreases from 166 to 101° in a matter of 40 s. At the same time, both the T-T and O-O distances increase substantially from 6.60 to 9

2 Figure 4 :

 24 Figure 4: Number of H2O molecules per SAPO-37 unit cell and H2O molecules concentration per surface area unit during H2O adsorption at 353 and 323 K. The isotherms are fitted with the Tόth model. The purpose of running two parallel sample cells (cell 1 & 2) is illustrated in Figure S8 in the S.I.

Figure 6 :

 6 Figure 6: Water molecules embedded into the whole SAPO-37 framework as viewed along the 111 direction (left). Spheres highlight the water concentrated only in the SOD cages (right).

Figure 7 :

 7 Figure 7: Excerpt of a completely dry, template-free SAPO-37 structure (left) and heavily hydrated SAPO-37 structure (right) highlighting the SOD-d6r-SOD interface. Selected O2-and O4-atoms are marked with black and green dashed circles, respectively. A triplet of adjacent 4-rings (4r) is indicated with blue planes. The internal volume of the SOD cages are indicated with yellow spheres (T-atom: blue, O-atom: red).

Acknowledgements

The research leading to these results was funded by the project "Catlife: Catalyst transformation and lifetimes by in-situ techniques and modelling", P#233848" of the Research Council of Norway. Partial support was received by the Department of Chemistry at UiO and by the Interreg project MAX4ESSFUN -UiO-007 (project#190980) of the Research Council of Norway. We acknowledge the use of the Norwegian centre for X-ray diffraction and scattering (RECX). PV acknowledges the Research Council of Norway for providing the computer time (under the project number NN2875k) at the Norwegian supercomputer. The authors thank the project team at BM01 from the Swiss-Norwegian Beamlines at the ESRF, France for their skillful assistance.


Pouyan Razmshoar

S Hajir Bahrami

Mohammad Rabiee

Isaac A M Frias

Marie Hangouet

Marie Martin

François Bessueille

Abdelhamid Errachid

Nicole Jaffrezic-Renault

S Hajir Bahrami

An impedimetric immunosensor based on PAMAM decorated electrospun polystyrene fibers for detection of interleukin-10 cytokine

Keywords: Immunosensor, Fibers, Plasma, Polystyrene, Polyamidoamine (PAMAM), Interleukin-10

Introduction

Cytokine storms are defined as excessively high and exaggerated cytokine production originating from a variety of viral and non-infectious disorders, causing severe inflammatory responses and organ damage. [START_REF] Han | Profiling serum cytokines in COVID-19 patients reveals IL-6 and IL-10 are disease severity predictors[END_REF] Interleukin-10 (IL-10) is an anti-inflammatory cytokine produced by T helper 2 cells that plays an important role in immunoregulation and inflammation management. [START_REF] Udomsinprasert | Circulating levels of interleukin-6 and interleukin-10, but not tumor necrosis factor-alpha, as potential biomarkers of severity and mortality for COVID-19: systematic review with meta-analysis[END_REF][START_REF] Islam | Elevated interleukin-10 levels in COVID-19: potentiation of pro-inflammatory responses or impaired anti-inflammatory action?[END_REF] IL-10 has been considered as a viable therapeutic and diagnostic tool for a variety of diseases, including inflammatory autoimmune diseases. [START_REF] Nessark | Capacitance Polypyrrole-based Impedimetric Immunosensor for Interleukin-10 Cytokine Detection[END_REF][START_REF] Lee | A novel biosensor based on hafnium oxide: Application for early stage detection of human interleukin-10[END_REF] Recent studies with coronavirus (COVID-19) patients' non-survivors have reported elevated concentrations of interleukin-6 (IL-6) and IL-10 cytokines compared with survivors owing to hyperinflammation [START_REF] Lu | Changes of serum IL-10, IL-1β, IL-6, MCP-1, TNF-α, IP-10 and IL-4 in COVID-19 patients[END_REF][START_REF] Zimecki | The potential for Lactoferrin to reduce SARS-CoV-2 induced cytokine storm[END_REF][START_REF] Azmy | Cytokine profiles before and after immune modulation in hospitalized patients with COVID-19[END_REF].

The findings from 102 COVID-19 patients reveal that the median level of IL-10 cytokine in their serum, (5.37 pg/mL) was 37% higher than the median level in healthy serum (3.91 pg/mL) [START_REF] Han | Profiling serum cytokines in COVID-19 patients reveals IL-6 and IL-10 are disease severity predictors[END_REF]. Salivary IL-10 cytokine levels in COVID-19 and post-COVID-19 patients have been reported to be 10.01 ± 2.05 pg/mL and 9.08 ± 1.17 pg/mL, respectively. These findings suggest that elevated IL-10 concentrations could be utilized to predict the severity of COVID-19 [START_REF] De Oliveira Santos | Impact of SARS-CoV-2 on saliva: TNF-⍺, IL-6, IL-10, lactoferrin, lysozyme, IgG, IgA, and IgM[END_REF].

The enzyme-linked immunosorbent assay (ELISA) is regularly used as a standard laboratory test for cytokine detection. Despite its great sensitivity and precision, this approach is expensive, time-consuming, and requires the analysis of professional specialists. [START_REF] Bahri | Capacitance electrochemical biosensor based on silicon nitride transducer for TNF-α cytokine detection in artificial human saliva: Heart failure (HF)[END_REF][START_REF] Baraket | A fully integrated electrochemical biosensor platform fabrication process for cytokines detection[END_REF] Hence, more accessible, highly sensitive, and selective diagnostic methods for the detection of inflammatory cytokines are urgently needed to enable early therapeutic intervention and improve patient quality of life. [START_REF] Pruna | A lowcost and miniaturized potentiostat for sensing of biomolecular species such as TNF-α by electrochemical impedance spectroscopy[END_REF] Electrochemical immunosensors, as opposed to these suggested techniques, have attracted a lot of interest because of their significant features, such as ease of use, swiftness, portability, time and cost effectiveness, and excellent sensitivity. [START_REF] Karaman | Sensitive sandwich-type electrochemical SARS-CoV-2 nucleocapsid protein immunosensor[END_REF] These immunological responses triggered by particular probe-target complexes are characterized by legible current signals. The signal amplification strategy is the most widespread and effective way for increasing the sensitivity of electrochemical immunosensors. In this regard, several approaches can be followed to improve the electron transport efficiency and surface area of the substrate while keeping the immunological molecule's activity. [START_REF] Karaman | Electrochemical neuron-specific enolase (NSE) immunosensor based on CoFe2O4@ Ag nanocomposite and AuNPs@ MoS2/rGO[END_REF] Multiple nanomaterials have been employed in the creation of electrochemical immunosensors and improve their performance as a result of the quick development of nanomaterial breakthroughs due to their numerous benefits, including high stability, strong electrocatalytic activity, and ease of modification. [START_REF] Boyacıoğlu | A novel electrochemical kidney injury molecule-1 (KIM-1) immunosensor based covalent organic frameworks-gold nanoparticles composite and porous NiCo2S4@ CeO2 microspheres: the monitoring of acute kidney injury[END_REF][START_REF] Karaman | Electrochemical cardiac troponin I immunosensor based on nitrogen and boron-doped graphene quantum dots electrode platform and Ce-doped SnO2/SnS2 signal amplification[END_REF][START_REF] Yola | Carbohydrate antigen 19-9 electrochemical immunosensor based on 1D-MoS2 nanorods/LiNb3O8 and polyoxometalate-incorporated gold nanoparticles[END_REF] The ability to electrospun fibers (ESFs) mats with a high surface area, mechanical flexibility, good surface functionalization, and interconnected porosity, which helps in the stabilization of higher receptors with better contact with the surrounding medium and access to the target analyte, underpins their use in the production of 3-D interfaces for electrochemical nano-biosensors. [START_REF] Yang | Recent insights into functionalized electrospun nanofibrous films for chemo-/bio-sensors[END_REF][START_REF] Razmshoar | Novel platform based on polystyrene electrospun nanofibrous mats doped with PAMAM dendritic polymer for enhanced immunosensing[END_REF][START_REF] Santos | Advances in functional polymer nanofibers: From spinning fabrication techniques to recent biomedical applications[END_REF][START_REF] Adabi | An electrochemical immunosensor based on electrospun carbon nanofiber mat decorated with gold nanoparticles and carbon nanotubes for the detection of breast cancer[END_REF] Among the various fiberforming technologies, electrospinning is known as an efficient, low-cost, simple, and versatile tool which uses electrostatic forces to generate polymer nanofibers (NFs) of different morphologies and diameters from solutions, suspensions, or melts [START_REF] Santos | Advances in functional polymer nanofibers: From spinning fabrication techniques to recent biomedical applications[END_REF]. Normally, in the electrospinning synthesis of NFs, the solution is extruded at a constant feed rate through the capillary to the tip of the spinneret, resulting in a pendant liquid drop due to surface tension. When an electric field is applied, electrostatic repulsion of surface charges of the same sign on the polymer droplet occurs, causing it to deform into a cone shape (known as a Taylor cone). By increasing the electric field and the dominance of electric force over surface tension, a charged polymer jet emerges from the cone, undergoing stretching, elongation, solvent evaporation, and solidification in the distance between the nozzle and the collector. [START_REF] Santos | Advances in functional polymer nanofibers: From spinning fabrication techniques to recent biomedical applications[END_REF][START_REF] Xue | Electrospinning and electrospun nanofibers: Methods, materials, and applications[END_REF][START_REF] Ogueri | Nanofiber technology for regenerative engineering[END_REF] Our major objective in this project was to create a new ESF-based transducer for the detection of AgIL-10 by directly electrospinning PS ESFs on the surface of a gold electrode. PS was chosen as a polymer for its high mechanical characteristics, great shape stability in aqueous media without swelling, and low cost. [START_REF] Rezaei | Multifactorial modeling and optimization of solution and electrospinning parameters to generate superfine polystyrene nanofibers[END_REF] However, PS alone cannot offer an ideal interface because it lacks the surface-active groups and sufficient hydrophilicity needed for biological recognition elements' covalent attachment. [START_REF] Razmshoar | Novel platform based on polystyrene electrospun nanofibrous mats doped with PAMAM dendritic polymer for enhanced immunosensing[END_REF] For this reason, plasma treatment, a process that alters the surface of polymers, increase their surface energy, and their hydrophilicity while maintaining their advantageous bulk features intact [START_REF] Morelli | Utilizing Radio Frequency Plasma Treatment to Modify Polymeric Materials for Biomedical Applications[END_REF][START_REF] Sathish | Air Plasma-Enhanced Covalent Functionalization of Poly(methyl methacrylate): High-Throughput Protein Immobilization for Miniaturized Bioassays[END_REF], was utilized to produce carboxyl groups on the PS fiber surface before decorating the fibers with the second generation (G2) of PAMAM dendritic polymer. PAMAM dendrimers with huge peripheral groups have been employed as branching polymeric macromolecules in the construction of different biosensors to readily interact with a large variety of bioreceptors, boosting their sensitivity, and improving their detection limit. [START_REF] Li | Carbon nanotube-polyamidoamine dendrimer hybrid-modified electrodes for highly sensitive electrochemical detection of microRNA24[END_REF][START_REF] Miodek | E-DNA Sensor of Mycobacterium tuberculosis Based on Electrochemical Assembly of Nanomaterials (MWCNTs/PPy/PAMAM)[END_REF] Grafting highly branched PAMAM polymer onto the fiber's surface can overcome the drawbacks of PS fibers, such as their hydrophobicity and lack of functional groups. Furthermore, the synergistic properties of electrospun fibers and highly branched polymers to create immunosensors with high capture antibody immobilization and increased sensitivity would be beneficial.

In a previous report, our group employed electrospinning to create a composite nanofiber from PS and PAMAM dendritic polymers in a one-step process by blending their solutions and using it as a 3-D interface to detect TNF-alpha cytokines. [START_REF] Razmshoar | A novel electrochemical immunosensor for ultrasensitive detection of tumor necrosis factor α based on polystyrene -PAMAM dendritic polymer blend nanofibers[END_REF] As novelty, the benefit of decorating PAMAM on the PS fiber surface avails higher accessibility to PAMAM amine groups due to their larger loading on the fiber surface at considerably lower PAMAM concentrations. To create a strong covalent bond between the anti-IL-10 and the fiber surface, glutaraldehyde coupling chemistry was used to create a bond between the PAMAM dendritic polymer groups and the amine groups in the Fc region of the anti-IL-10. Schematic 1 depicts all stages of the immunosensor fabrication. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the electrochemical characteristics of the developed ESFsbased immunosensor at each stage and AgIL-10 impedimetric detection, respectively. Schematic.1.

Experimental

Chemicals and reagents

The reagents, instruments, and characterizations sections were thoroughly discussed in the supplementary information.

Immunosensor preparation 2.2.1. Preparation of PS ESFs

PS granules were dissolved in DMF and stirred overnight to make a 25 wt.% electrospinning solution. A vertical electrospinning setup (Spraybase®, Ireland) was used for producing ESFs, which included a polytetrafluoroethylene (PTFE) tube to transfer the polymer solution from the pump to the nozzle. The electrospinning parameters, including voltage, feed rates, and nozzle to collector distance, were set at 27 kV, 0.25 mL/h, and 20 cm, respectively, with these parameters having been previously optimized. [START_REF] Razmshoar | Novel platform based on polystyrene electrospun nanofibrous mats doped with PAMAM dendritic polymer for enhanced immunosensing[END_REF] To achieve the highest level of similarity between the electrodes in terms of the thickness and amount of ESFs deposited, fibers were electrospun on gold electrodes for 1 min each by placing each on a specific location of aluminum foil. Before electrospinning, the gold electrodes were cleaned in an ultrasonic bath for 15 min in an acetone solution and then in an ethanol solution and were rinsed with ultrapure water at each stage and at the end. Then they were dried with a mild stream of nitrogen (N2) and then placed in a UV-ozone ProCleanerTM for 30 min to eliminate any possible organic contamination. This process results in a gold electrode with a hydrophilic feature, which increases not only fiber adherence to the electrode surface but also electrode wettability and electron transfer. Then the electrodes containing ESFs were stored at room temperature (RT) for subsequent use.

Air plasma treatment

Gold electrodes modified with ESFs were treated with a frequency/low pressure (13.56 MHz, 50 W) plasma generator (Pico PCCE, Diener Electronic, Ebhausen, Germany) to produce carboxylic acid groups on the surface of the PS ESFs. By the time the electrodes were placed in the center of the chamber, the pressure within the chamber was reduced to 0.2 mbar by pumping to remove the air. The chamber was then filled with filtered air for 1 min before plasma was applied at a pressure of 0.2 mbar for 10 s. To find the optimal plasma time, five different plasma timings (10 s, 20 s, 30 s, 60 s, and 120 s) were tested.

PAMAM decoration of PS ESFs

The plasma-generated carboxylic acid groups were activated by carbodiimide chemistry to decorate the surface of the fibers with PAMAM. Gold electrodes containing PS-plasma (10 s) treated ESFs were dipped in a 2 mL solution of EDC (0.4 M)/NHS (0.1 M) mixture in deionized water for 1 h. The electrodes were immediately immersed in an aqueous PAMAM solution for 4 h, rinsed with deionized water, and then dried at RT. To determine the optimal dendrimer concentration, three PAMAM solution concentrations of 0.5, 1, and 2 wt.% were investigated.

Covalent immobilization of antibodies and blocking steps

The PAMAM amine groups on the PS ESFs surface were activated to form a covalent bond with the Fc region of anti-IL-10 using glutaraldehyde as a coupling agent. For this purpose, the electrodes were put in a desiccator containing glutaraldehyde saturated vapor for overnight. Then, 20 μL of anti-IL-10 (10 μg/mL) was kept in contact with the ESF-modified electrodes for 1 h at 4 °C. After that, the immunosensor was carefully rinsed with PBS (10 mM, pH 7.4) and the blocking treatment was done by incubating it in 40 μL of BSA 2% in PBS solution (10 mM, pH 7.4) for 1 h at 4 °C to reduce non-specific binding and deactivate the unbounded sites on the ESFs surface.

Detection of AgIL-10 in PBS buffer and in artificial saliva (AS)

The ESFs based immunosensor was immobilized in the electrochemical cell. The immunosensor was incubated in 40 μL of different concentrations of AgIL-10 standard solution in PBS or AS for 30 min at RT, followed by PBS washing to eliminate unbound AgIL-10 cytokine. The electrochemical cell was then filled with PBS (10 mM, pH 7.4) containing 5 mM K3[Fe(CN)6]/K4[Fe(CN)6] at each detection to record an EIS response. The electrolyte solution in the cell was withdrawn and the cell was washed with PBS solution after each EIS measurement.

Characterizations

Instrumentation and procedures used for the physicochemical characterization are presented in the supplementary information.

Electrochemical measurements

Instrumentation and procedures used for the electrochemical measurements are presented in the supplementary information.

Results and discussion

Choosing the optimal plasma time

Morphological analysis

Plasma-induced chemical changes are typically accompanied by morphological changes. It should be highlighted that extremely harsh surface treatment results in uneven surface etching, which has a significant influence on the immobilization and performance of bio-interface. The surface morphology influences the activity of immobilized biomolecules as well as the biophysical and chemical aspects of the biointerface, therefore picking the optimal time for plasma is crucial to minimize undesirable ion-assisted chemical etching and damage of PS fibers. [START_REF] North | Plasma-based surface modification of polystyrene microtiter plates for covalent immobilization of biomolecules[END_REF] Five different times (10 s, 20 s, 30 s, 60 s, and 120 s) were tested to find the best plasma time for causing the least amount of damage to the PS ESFs surface and generating the most functional groups. The SEM images of ESFs are shown in Fig. S1. PS ESFs produced from electrospinning solution with a concentration of 20 wt.% had a bead-on-strings morphology, therefore a PS solution with concentration of 25 wt.% was chosen to enhance polymer chain entanglement and produce fibers with a uniform morphology and reproducible characteristics (Fig. S1A).

According to the SEM images (Fig. S1 B-D), there are no signs of damage to the PS ESFs for 10 s, 20 s, and 30 s of plasma, but after that, the ESFs diameter begins to decrease (Fig. S1E andF). The mean diameter of pure PS ESFs was 1805 ± 336 nm, while the diameter after 60 s of plasma decreased to 1271 ± 253 nm.

The ESFs structure was destroyed and the fibers were charred after 120 s of plasma. Consequently, using plasma to reduce fiber diameter for some specific purposes may be deemed a technique. Because PS ESFs were utilized as an interface in this study, their strength was especially crucial during the washing and immobilization processes. As a result, preserving their strength was prioritized, and SEM evaluation led to the selection of 10 s for plasma.

wettability analysis

Contact angle measurements were conducted to assess the involvement of plasma treatment in the creation of functional groups on the surface of PS ESFs and their hydrophilic characteristics. According to the results (Fig. S2), the contact angle of the PS ESFs after 10 s of plasma was 0°, while the contact angle of pure PS ESFs was 145° ± 2.5°. The formation of COOH, OH, C=O, and C-O, leads to the contact angle decreasing.

[31] The production of singly and doubly bonded C-O functionality is based on the removal of hydrogen from tertiary C-H bonds or benzene rings, followed by oxygen substitution in free radical sites. [START_REF] Lock | Surface composition, chemistry, and structure of polystyrene modified by electron-beam-generated plasma[END_REF] A duration of 10 s was chosen as the optimal plasma time since it reduced the contact angle to 0° and caused the least amount of damage to the ESFs. It should be mentioned that fiber hydrophilicity is a key feature for the homogenous immobilization of antibodies on the fiber surface, as well as improved electron transfer between the electrolyte and the electrode.

Evaluation of fiber-based immunosensor

FE-SEM characterization

FE-SEM microscopic analysis of the fibers' surface before and after the plasma indicated that a plasma time of 10 s was appropriate since there were no signs of fracture or cracking on the surface of the fibers (Fig. 1A-B). The significant difference between before and after the plasma is the rise in nanoporosity size on the surface of the fibers, which increases surface area and enhances potential interaction with the environment [START_REF] Dufay | Effect of Cold Plasma Treatment on Electrospun Nanofibers Properties: A Review[END_REF]. The fibers' morphology changed to peach skin after being decorated with PAMAM (Fig. 1C). The average diameter of PS-plasma 10 s-PAMAM 2 wt.% ESFs was 1565 ± 290 nm (Fig. 1D).

fluorescence microscopy

Fluorescence microscopy of PS ESFs revealed that plasma-modified PS fibers had autofluorescence (4.26 ± 1) at 488 nm wavelength excitation as compared to pure PS (Fig. 2A-B). A high level of autofluorescence has been proven in thermoplastic polymers, including PS, particularly between the wavelengths of 300 -400 nm. A recent study has shown that thermal and solvent treatment increases the levels of autofluorescence of PS plate and changes its wavelength to longer wavelengths. [START_REF] Young | Assessment of enhanced autofluorescence and impact on cell microscopy for microfabricated thermoplastic devices[END_REF] Here, the PS ESFs have no autofluorescence at 488 nm wavelength excitation, whereas autofluorescence and spots of different sizes are observed on the PS-plasma 10 s ESFs. The autofluorescence of PS fibers has been attributed to excimers resulting from polymer chain reorganization, which have longer wavelengths than the monomer emission peak. It should be noted that due to the high interest in the use of ESFs in various fields, particularly optical immunosensors, and the proximity of the fluorophores wavelength used for antibody conjugation to the plasma treated PS wavelength, serious consideration should be given to their autofluorescence after plasma to avoid false positive results. [START_REF] Young | Assessment of enhanced autofluorescence and impact on cell microscopy for microfabricated thermoplastic devices[END_REF] Fig. 2C-E shows images of fibers after being decorated with various concentrations of PAMAM. Because PAMAM has fluorescence properties at 488 nm excitation [START_REF] Razmshoar | Novel platform based on polystyrene electrospun nanofibrous mats doped with PAMAM dendritic polymer for enhanced immunosensing[END_REF], the fluorescence increase in PS-plasma 10 s-PAMAM ESFs could be noticed when PAMAM concentration increases. The quantity of grafted PAMAM at the fiber surface is larger in the PSplasma 10s-PAMAM 2 wt.% sample than in the other samples. The average fluorescence intensity for each of the mentioned ESFs was obtained by preparing 5 distinct samples and assessing 5 different points of each sample. Fig. 2F shows the fluorescence intensities of different ESFs samples, according to which the fluorescence intensities for the 0.5, 1, and 2 wt.% dendrimer samples were 30.5 ± 4.4, 47.2 ± 6.7, and 60.8 ± 5.5, respectively. Because of its higher fluorescence properties, PS-plasma 10 s-PAMAM 2 wt.% was chosen as the best sample, and all immunosensors were prepared with this concentration. Fig. 2.

FTIR analysis

ATR-FTIR analysis were done in order to more accurately evaluate the chemical composition of PS ESFs during the surface modification and immunosensor fabrication processes. The bands at 3080-3024 cm -1 , 2920 cm -1 , and 2846 cm -1 in the PS spectra are attributable to aromatic C-H stretching vibration absorption and the presence of CH2, respectively. The peaks at 1939 cm -1 , 1869 cm -1 , 1801 cm -1 , and 1741 cm -1 are associated with overtone aromatic. The C=C stretching vibration absorption is seen at 1600 cm -1 , while CH2 bending vibrations are seen at 1492 cm -1 and 1451 cm -1 (Fig. 3Aa). [START_REF] Razmshoar | Novel platform based on polystyrene electrospun nanofibrous mats doped with PAMAM dendritic polymer for enhanced immunosensing[END_REF] A comparison of spectra of PS and PS-plasma 10 s shows two new peaks at 3454 cm -1 and 1726 cm -1 , which correspond to OH and C=O from carboxylic acids, respectively (Fig. 3Ab). Fig. S3. depicts a schematic representation of the likely PS structure following plasma treatment. [START_REF] Suganya | Plasma Surface Modified Polystyrene and Grafted with Chitosan Coating for Improving the Shelf Lifetime of Postharvest Grapes[END_REF] According to previous study, the major mechanism for plasma treatment is an oxidative assault on the benzene ring followed by ring-opening to create aldehyde/carboxylate species. Oxidation of PS to a higher oxidation state and carbonyl/carboxyl species formation has been confirmed by IR-visible sum frequency generation (SFG) spectra. [START_REF] Zhang | Effects of UV irradiation and plasma treatment on a polystyrene surface studied by IR-visible sum frequency generation spectroscopy[END_REF] The spectrum from PS-plasma 10 s-PAMAM 2 wt.% has amide bands of PAMAM νNH at 3293 cm -1 , νCO (amide I band) at 1652 cm -1 , δNH and νCN (amide II band) at 1553 cm -1 . [START_REF] Razmshoar | A novel electrochemical immunosensor for ultrasensitive detection of tumor necrosis factor α based on polystyrene -PAMAM dendritic polymer blend nanofibers[END_REF] It should be noted that the peak of carboxylic acid is decreased due to covalent bonding with PAMAM. Also, PS peaks are still present in the spectrum (Fig. 3Ac).

Previous studies have noted the N-H stretching vibration peak in the PAMAM dendrimer at 3293 cm -1 , which increased with increasing dendrimer concentration. [START_REF] Razmshoar | Novel platform based on polystyrene electrospun nanofibrous mats doped with PAMAM dendritic polymer for enhanced immunosensing[END_REF][START_REF] Lu | Unexpected strong blue photoluminescence produced from the aggregation of unconventional chromophores in novel siloxane-poly (amidoamine) dendrimers[END_REF] In the spectra of PS-plasma 10 s-PAMAM 2 wt.%, a broad peak in the range of 3100 cm -1 to 3600 cm -1 is observed, which consists of two OH peaks in the region of 3454 cm -1 formed by plasma modification and the amide bands νNH of PAMAM at 3293 cm -1 , which are overlapped. When the two spectra in Fig. 3B are compared, the tip of the peak in the PS-plasma 10 s-PAMAM 2 wt.% spectra is placed near the N-H stretching vibration peak, confirming the presence of this peak of PAMAM in the spectrum.

The spectrum of PS-plasma 10 s-PAMAM 2 wt.%-glutaraldehyde before and after anti-IL-10 (10 μg/mL) immobilization is shown in Fig. 3Ca andb, respectively. Fig. 3Ca shows the characteristic peak at 1715 cm -1 for glutaraldehyde and also the peaks at 1662 cm -1 and 1552 cm -1 of the amide bands of PAMAM in the spectrum. After anti-IL-10 antibody immobilization, the glutaraldehyde peak at 1715 cm -1 decreased significantly, indicating a covalent bond between the antibody and glutaraldehyde (Fig. 3 Cb). [START_REF] Mahmoudifard | Ammonia plasma-treated electrospun polyacrylonitryle nanofibrous membrane: The robust substrate for protein immobilization through glutaraldhyde coupling chemistry for biosensor application[END_REF][START_REF] Chuang | Enhancing of intensity of fluorescence by DEP manipulations of polyaniline-coated Al2O3 nanoparticles for immunosensing[END_REF] However, it should be noted that due to the peak overlap of PAMAM and anti-IL-10 amine groups, it is not possible to distinguish between them.

UV-vis analysis

Fig. 3D shows the UV-visible absorption spectrum of the supernatant after washing PS-plasma 10 s-PAMAM-glutaraldehyde with washing buffer, PBS solution (10 mM, pH 7.4) for 5 min. An absorption peak is observed at 235 nm, which is connected to the carbon -carbon double bond π -π * transition of aldehyde groups. [START_REF] Purss | Effect of "glutaraldehyde" functionality on network formation in poly(vinyl alcohol) membranes[END_REF][START_REF] Tang | Acetylcholinesterase-reduced graphene oxide hybrid films for organophosphorus neurotoxin sensing via quartz crystal microbalance[END_REF] According to the findings, the amount of unreacted glutaraldehyde in the supernatant is inversely linked to the concentration of PAMAM, so the PS-plasma 10 s-PAMAM 2 wt.%glutaraldehyde supernatant has the lowest amount of glutaraldehyde adsorption. This shows that increasing the PAMAM concentration while also increasing the number of amine groups on the fiber surface induces more glutaraldehyde molecules to establish stronger covalent bonds with them.

Electrochemical characterization

The ESFs modified-gold electrode in stepwise fabrication process of ESFs based immunosensor was evaluated by CV measurement in PBS (10 mM, pH 7.4) containing 5 mM [Fe(CN)6] 3-/4-because of any alterations in the bio-interface physically hinder the electrochemical behavior of the redox probe. As shown in Fig. 4A, the bare gold electrode presents well-defined anodic/cathodic peaks of [Fe(CN)6] 3-/4-and a reversible voltammogram with peak-to peak separation (ΔE P ) of ~182.5 mV, with a peak maximum intensity (I pa = 115.6 μA). After electrospinning of the PS fibers on the electrode, the oxidation and redox peaks of the redox probe almost disappeared (I pa = 14.6 μA). Electron transfer from the electrolyte to the gold electrode surface happens as a result of tunneling through the porosity of the fibrous mats. The lack of conductivity and high hydrophobicity of the PS ESFs block the diffusion of redox probe to the electrode surface. Plasma modification for 10 s significantly increased the amperometric response of the electrode containing PS ESFs (I pa = 113.5 μA). The formation of oxygen-containing groups after plasma is the main factor contributing to the increasing mobility of the redox probe towards the electrode. [START_REF] Lee | Flexible electrochemical biosensors based on O2 plasma functionalized MWCNT[END_REF] Modification of the fiber surface with PAMAM increased oxidation/reduction current (I pa = 135.1 μA) and decreased ΔE P from ~210.5 to ~186.5 mV, which indicates the successful attachment of PAMAM at the PS fiber surface [START_REF] Razmshoar | A novel electrochemical immunosensor for ultrasensitive detection of tumor necrosis factor α based on polystyrene -PAMAM dendritic polymer blend nanofibers[END_REF]. The pKa of the peripheral functional groups (-NH3 + , pKa ∼ 9.52) gave the PAMAM a protonated interface in aqueous media at neutral pH. [START_REF] Bustos | Enantioselective recognition of alanine in solution with modified gold electrodes using chiral PAMAM dendrimers G4.0[END_REF] Positively charged PAMAM accumulates the ferro/ferricyanide redox probe on its surface because of its electrostatic interaction. The electron-rich PAMAM surface amine groups and the electron-deficient interior create an electric field that accelerates electron transfer on the electrode's surface. [START_REF] Zhou | Novel Ce (III)-metal organic framework with a luminescent property to fabricate an electrochemiluminescence immunosensor[END_REF] Fig. 4B depicts the role of PAMAM in increasing current response; when dendrimer concentration rose from 0.5 to 2 wt.% due to increased dendrimer presence at the fiber surface, the current increased from (I pa = 121.7 μA) to (I pa = 135.1 μA). The results were compatible with the fluorescence images in section 3.2.2. Due to improved electron transfer and a larger presence of amine groups on the surface, the concentration of 2 wt.% was determined as the best concentration for immunosensor preparation.

The CV responses of the PS-plasma 10 s-PAMAM 2 wt.%-glutaraldehyde ESFs electrode at different scan rates (10 to 100 mVs -1 ) are shown in Fig. S4A. Increasing the scan rate, which in turn reduces the penetration layer, increases the current peak and ΔE P . Fig. S4B shows the square root of the scan rate versus anodic and cathodic peak currents �I pa (μΑ) = 14.8739 v 1/2 �(mV/s) 1/2 � + 9.2634. R 2 = 0.99� and �I pc (μΑ) = -14.7623 v 1/2 �(mV/s) 1/2 � -12.4408. R 2 = 0.99�, respectively. Fig. S4C shows the square root of the scan rate versus anodic and cathodic peak potentials, �E pa (V) = 0.0083 v 1/2 �(mV/s) 1/2 � + 0.2549. R 2 = 0.99� and, �E pc (V) = -0.0087 v 1/2 �(mV/s) 1/2 � + 0.1888. R 2 = 0.99�, respectively. Peak currents and peak potentials of developed ESFs-based electrodes have a linear relationship with the square root of the scan rate (square of regression coefficient of 0.99), demonstrating that the electron transfer process in developed immunosensor is mostly based on a diffusion-controlled process. [START_REF] Li | A nanostructured conductive hydrogelsbased biosensor platform for human metabolite detection[END_REF] The electro-active surface area provided by the ESFs developed immunosensor was determined to be 0.149 cm 2 by the Randles-Sevcik equation I p = 268600AD 1/2 n 3/2 Cv 1/2 in which I P is the redox peak current (A), A is electroactive surface area (cm 2 ), D is the diffusion coefficient ( D = 7.60 × 10 -6 , at 25 °C), n is the number of electrons transferred in the redox reaction (n = 1), C is the concentration of the reactant ( 5mM Fe(CN)6 3-/4-) (mol/cm 3 ), and v is the scan rate (V/s). [START_REF] He | Electrochemical Peptide Biosensor Based on in Situ Silver Deposition for Detection of Prostate Specific Antigen[END_REF][START_REF] Zhu | Cyclic voltammetry measurements of electroactive surface area of porous nickel: peak current and peak charge methods and diffusion layer effect[END_REF] For bare gold electrode, the electro-active surface area of 0.127 cm 2 was measured, indicating the role of fibers in 17.3% increasing the available surface-active area.

To evaluate ESFs based electrode's capability as an effective bio-interface, 40 μL of different concentrations of anti-IL-10 were immobilized on the surface of the PS-plasma 10 s-PAMAM 2 wt.%-glutaraldehyde ESFs electrode at 4 °C for 1 h and the electrode characterized with CV measurement in PBS (10 mM, pH 7.4) containing 5 mM [Fe(CN)6] 3-/4-with a scan rate of 60 mV s -1 (Fig. 4C). A drop in redox current was seen following the immobilization of the antibody on the fiber surface due to the insulating characteristics of antibodies and this was a valid indicator of successful grafting on the fiber surface. Increasing the antibody concentration further reduced the anodic peak intensity, confirming the higher immobilized density of the antibody at the extraordinarily high available surface area provided by the fibers.

The activity of the anti-IL-10 on the surface of the fibers and the performance of the immunosensor before and after antigen incubation were also examined with CV in PBS (10 mM, pH 7.4) containing 5 mM [Fe(CN)6] 3-/4-at the final stage of immunosensor preparation (Fig. 4D). The current of [Fe(CN)6] 3-/4-on PS-plasma 10 s-PAMAM 2 wt.% ESFs electrode was (I pa = 130.4 μA). After incubating 10 pg/mL of AgIL-10 on the electrode surface, the redox current (I pa = 112.8 μA) were lower than the PS-plasma 10 s-PAMAM 2 wt.%-anti-IL-10-BSA current (I pa = 124.0 μA). This decrease is due to immune complexes formed by Anti-IL-10 and Ag-IL-10, which could obstruct electron transfer to the electrode surface as an insulating layer and confirms the developed ESFs immunosensor's high affinity for Ag-IL-10 cytokine.

The results of the developed immunosensor's stability after 20 consecutive cycles at a scan rate of 60 mVs -1 are shown in Fig. S4D. The covalent bond between anti-IL-10 and glutaraldehyde, as well as the strong fiber adhesion to the electrode surface, resulted in constant redox peaks and peak potential separation, with relative standard deviations (RSDs) of 0.68 % and 0.87 % for I pa and I pc , respectively. [START_REF] Rezaei | An electrochemical immunosensor for cardiac Troponin I using electrospun carboxylated multi-walled carbon nanotube-whiskered nanofibres[END_REF]

Electrochemical detection of AgIL-10 in PBS

EIS was employed as a sensitive approach to assess antibody-antigen interactions at the immunosensor interface and to quantitatively determine AgIL-10 in PBS and AS. The presented EIS results in the Nyquist diagram for AgIL-10 detection from 1 pg/mL to 50 pg/mL in PBS (10 mM, pH 7.4) are shown in Fig. 4E, where the semicircle at higher frequencies refers to the electron transfer process and the linear section at low frequencies belongs to the diffusion process. The gray semicircle corresponds to the immobilization of 10 μg/mL of anti-IL-10 on the surface of the PS-plasma 10 s-PAMAM 2 wt.%-glutaraldehyde ESFs electrode for 1 h at 4 °C and shows low R ct , which is consistent with the CV results and demonstrates the role of PAMAM in fast electron transfer and easily accessible redox probe to the electrode surface. As expected, after immobilization of each concentration of the AgIL-10 for 30 min at RT (20 ± 1 °C), the diameter of semicircles increased owing to immunorecognition of AgIL-10 by surface-fixed antibodies that can hinder redox probe charge transfer as an electrical contact barrier. Furthermore, the results show that the proposed immunosensor has a high affinity for AgIL-10 with rising R ct , due to the increased antigen steric hindrance on the electroactive interface for redox probe and restriction of the Faradaic reaction. [START_REF] Palanisamy | Label-Free Bimetallic in Situ-Grown 3D Nickel-Foam-Supported NH2-MIL-88B(Fe2Co)-MOF-based Impedimetric Immunosensor for the Detection of Cardiac Troponin i[END_REF] The electrochemical parameters were derived after each detection by fitting the Nyquist plots with the Randle's equivalent circuit, where R s corresponds to the ohmic electrolyte resistance, Q CPE to constant phase element, R ct to charge transfer resistance, and 𝒲𝒲 to Warburg impedance (inset of Fig. 4E). The calibration diagram of immunosensor obtained from the impedance fitting data based on ΔR/R ,�R antigen -R antibody �/R antibody , versus different concentrations of AgIL-10 (1, 5, 10, 20, 35, and 50 pg/mL) is shown in Fig. 4F. The immunosensor showed two linear correlation in the ranges from 1 pg/mL to 10 pg/mL with a linear regression equation of ∆R/R = (0.03188 ± 0.0018)[IL -10] + 0.21685 (±0.01) and a correlation coefficient of R 2 = 0.99. The second range of 10 pg/mL to 50 pg/mL was similarly adjusted to a linear regression equation of ∆R/R = (0.00838 ± 0.0008)[IL -10] + 0.45164 (±0.01), with R 2 = 0.98. The smaller change in redox current at higher Ag-IL-10 concentrations, which led the calibration curve to be separated into two segments, is due to anti-IL-10 antibody saturation and a reduction of free antibodies available on the surface of the electrospun fibers to form an immunocomplex. The immunosensor has a limit of detection (LOD) of 1 pg/mL based on 3 S m � , where S represents the residual standard deviation and m denotes the slope of the regression line.

The inter-and intra-assay precisions of the results obtained using the designed immunosensor were studied.

The inter-assay reproducibility of ESFs immunosensor was performed using five independent electrode prepared in the same manner for assessing samples containing 10 pg/mL of AgIL-10. While for the intraassay, the same designed immunosensor was used five times to detect 10 pg/ml of AgIL-10. The RSD of inter-and intra-assay was around 3.18% and 0.79%, respectively. This result indicated that the developed ESFs immunosensor's precision, reproducibility, and repeatability were desirable.

Table 1 compares the analytical performance of the ESFs-based immunosensor to that of other immunosensors for the detection of IL-10 that have been reported to date. To our knowledge, this is the first study to use ESFs as an interface to detect IL-10. The increased surface area provided by the fibers permitted a significantly higher number of antibodies on the surface (100 μg/mL) and provided a comparable detection range and extremely low detection limit to previous studies.

Fig. 4.

Table 1.

Interference study

To evaluate the specificity of the immunosensor, an aliquot of 20 μL of anti-IL-10 (10 μg/mL) was fixed on the ESF modified-gold electrode at 4 °C for 1 h and 40 μL of TNF-alpha antigen with different concentrations (1, 5, 10, and 20 pg/mL) was incubated with the ESF modified-gold electrode at RT for 30 min. The results of the interference studies in Fig. 5A show that the developed immunosensor is selective for AgIL-10.

Electrochemical detection of AgIL-10 in AS

The function of the immunosensor for AgIL-10 detection in AS as a complex medium and natural saliva simulator was assessed by EIS. The EIS concepts and immunosensor fabrication procedures were the same as mentioned before. Initially, the likelihood of any unspecific absorption by AS was examined by incubating 40 μL of it on the surface of the ESFs electrode (without AgIL-10). Fig. 5B shows the 3 times incubation of AS on the ESFs immunosensor. After the first incubation of AS, an increase in R ct was observed. This impact was detected in all AS measures in this investigation and is thus considered a constant. The matrix effect, also known as unspecific adsorption, has been discovered in prior investigations. [START_REF] Bellagambi | Electrochemical biosensor platform for TNF-α cytokines detection in both artificial and human saliva: Heart failure, Sensors Actuators[END_REF] Re-incubation of AS (second and third semicircles) did not result in increased impedance or undesired absorption. It should be emphasized that the immunosensor surface was thoroughly washed with PBS between each incubation step. Fig. 5C shows the Nyquist plot for the detection of AgIL-10 in AS. After 3 times incubation of AS and maintaining a consistent R ct , the AS containing various concentrations of AgIL-10 (10, 25, 50, and 100 pg/mL) was applied to the fiber surface for 30 min at RT each time. The detection of AgIL-10 in AS as a complex medium is highlighted by increasing the Nyquist plot semicircle each time. Furthermore, the EIS signal displayed a linear relationship with IL-10 concentrations ranging from 10-100 pg/mL and a LOD of 4,63 pg/mL, with a regression equation of ∆R/R = (0.00483 ± 0.0002)[IL -10] + 0.00382 (±0.008) and a correlation coefficient of R 2 = 0,96 (Fig. 5D).

Fig. 5.

Conclusion

In this study, PAMAM polymer was used to decorate the air plasma-modified PS electrospun fibers' surface,

Figure Captions

Schematic.1. The schematic depicts the step-by-step preparation procedure for the developed immunosensor. Polypyrrole modified silicon nitride (Si3N4) Casting EIS 1-10 pg/mL 0.347 pg/mL [START_REF] Nessark | Capacitance Polypyrrole-based Impedimetric Immunosensor for Interleukin-10 Cytokine Detection[END_REF] Graphene interdigitated electrodes (IDEs) Aerosol-jet-printed EIS 0.1-2 ng/mL 46 pg/mL [START_REF] Parate | Aerosol-Jet-Printed Graphene Immunosensor for Label-Free Cytokine Monitoring in Serum[END_REF] One-dimensional photonic crystal (1DPC) PIAD BSW 250 pg/mL 110 pg/mL [START_REF] Occhicone | Enhanced Fluorescence Detection of Interleukin 10 by Means of 1D Photonic Crystals[END_REF] PAMAM decorated plasma-treated electrospun PS fibers Electrospinning EIS 1-50 pg/mL 1 pg/mL This work LOD -limit of detection; mPDGs -magnetic poly (divinylbenzene-co-glycidyl methacrylate) colloidal particles; HfO2 -hafnium oxide; ALDatomic layer deposition; EIS -electrochemical impedance spectroscopy; LSPR -localized surface plasmon resonance; MHDAmercaptohexadecanoic acid; NPs -nanoparticles; PTFE -polytetrafluoroethylene; SERS -surface-enhanced Raman spectroscopy; PIAD -plasma ion-assisted deposition technology; BSW -Bloch surface waves; PAMAM -polyamidoamine; PS -polystyrene.

Fig. 1 .

 1 Fig. 1.

Fig. 3 .

 3 Fig. 3.

resulting in the development of a 3 -

 3 D open-porous interface and an impedimetric immunosensor with very high sensitivity to the IL-10 cytokine. The formation of carboxylic acid functional groups on fiber surfaces, a rise in hydrophilicity, and the creation of autofluorescence characteristics in PS fibers were all induced by 10 s of air plasma. Increased PAMAM concentration on the fiber surface causes more glutaraldehyde molecules to form covalent bonds with them. Anti-IL-10 capture antibody was successfully immobilized on the PS fiber surface by glutaraldehyde coupling chemistry on the amine functional groups of PAMAM.The immunosensor showed ameliorated stability due to covalent bonding between PAMAM amine groups and anti-IL-10 using a glutaraldehyde coupling agent. Modification of the fiber surface with PAMAM increased oxidation/reduction current and promoted antibody immobilization by increasing surface area.The PS-plasma-PAMAM 2 wt.% ESFs-based immunosensor is a new 3-D framework with great sensitivity and selectivity within a wide linear range of AgIL-10 detection in both PBS and AS. The proposed ESFs based interface is a promising material that can be employed in the clinical detection of cytokines or as a substrate for immunosensors development.

Fig. 1 .Fig. 2 .Fig. 4 .Fig. 5 .

 1245 Fig. 1. FE-SEM images of A) pure PS, B) PS after 10 s plasma treatment, C) PS-plasma 10 s-PAMAM 2 wt.% ESFs, and D) the diameter distribution of PS-plasma 10 s-PAMAM 2 wt.% ESFs.

Fig. 1 .

 1 Fig. 1. FE-SEM images of A) pure PS, B) PS after 10 s plasma treatment, C) PS-plasma 10 s-PAMAM 2 wt.% ESFs, and D) the diameter distribution of PS-plasma 10 s-PAMAM 2 wt.% ESFs.

Fig. 2 .

 2 Fig. 2. Fluorescence images of A) pure PS, B) PS-plasma 10 s, C) PS-plasma 10 s-PAMAM 0.5 wt.%, D) PS-plasma 10 s-PAMAM 1 wt.%, E) PS-plasma 10 s-PAMAM 2 wt.%, and F) quantification of fluorescent intensity of different samples (a) pure PS, (b) PS-plasma 10 s, (c) PS-plasma 10 s-PAMAM 0.5 wt.%, (d) PS-plasma 10 s-PAMAM 1 wt.%, and (e) PS-plasma 10 s-PAMAM 2 wt.%.

Fig. 4 .

 4 Fig. 4. A) CVs of different stages of immunosensor preparation, B) CV of PS-plasma10 s-PAMAM with different concentrations electrode, C) effect of different immobilized anti-IL-10 concentrations (10, 20, 40, 50, and 100 μg/mL) on reducing the redox current, D) evaluation of anti-IL-10 activity (10 μg/mL) on the fiber surface, E) Nyquist plots of PS-plasma10 s-PAMAM 2 wt.%-glutaraldehyde-anti-IL-10 ESFs modified gold electrode of AgIL-10 detection ranging from 1 to 50 pg/mL, and F) calibration curves of the ESFs based immunosensors for the detection of AgIL-10.

Fig. 5 .

 5 Fig. 5. A) Selectivity resualts of the developed immunosensors, B) Nyquist plots of three times incubation of AS (without AgIL-10) on PS-plasma 10 s-PAMAM 2 wt.% ESFs modified gold electrode, C) Nyquist plots of the immunosensor for AgIL-10 detection in AS, and D) detection calibration curves of the immunosensor toward AgIL-10 in AS.

Table 1 .

 1 Comparison of analytical characteristics of other immunosensors reported so far for IL-10 detection with the presented ESFs based immunosensor.

Table 1 .

 1 Comparison of analytical characteristics of other immunosensors reported so far for IL-10 detection with the presented ESFs based immunosensor.

	Immunosensors fundamentals	Development Method	Transduction technique	Linear Range	LOD	Ref.
	Epoxy-functionalized mPDGs on a glass substrate	Immobilization	Fluorescence 0.25 μg/mL	-	[50]
	HfO2 grown by ALD on silicon substrate	ALD	EIS	0.1-20 pg/mL	-	[5]
	Diazonium modified gold microelectrodes	Soft lithography	EIS	1-15 pg/mL	-	[51]
	Polypyrrole microwires (PPy-μWs)	Soft lithography	EIS	1-50 pg/mL	0.122 pg/mL [52]
	Antibody conjugated gold nanorods	Microfluidic patterning	LSPR	5-20 pg/mL	10.97 pg/mL [53]
	MHDA modified gold microelectrodes	Soft lithography	EIS	1-15 pg/mL	-	[54]
	Diazonium modified gold microelectrodes	Soft lithography	EIS	1-15 pg/mL	0.3 pg/mL	[11]
	Capture-end NPs modified PTFE membrane	Conjugation	SERS	0.01-100 pg/mL 0.1 pg/mL	[55]
	Gold LSPR strip	Nanoimprinting lithography LSPR	0.2-200 nM	-	[56]

Acknowledgements P.R. thanks the Ministry of Science, Research and Technology of the Islamic Republic of Iran for providing the scholarship. The authors acknowledge the financial support of the EU H2020 WIDESPREAD Program entitled Bionanosens grant agreement # 951887. Campus France is acknowledged for the financial support through PHC Maghreb EMBISALIM.