Zouhour Hsine

Salma Bizid

Rym Mlika

Hélène Sauriat-Dorizon

Ayoub Haj Said

Hafsa Korri-Youssoufi
email: hafsa.kori-youssoufi@universite-paris-saclay.fr

Rym Milka

Ayoub Haj Said

Nanocomposite Based on Poly (para-phenylene)/Chemical Reduced Graphene Oxide as a Platform for Simultaneous Detection of Ascorbic Acid, Dopamine and Uric Acid

Keywords: nanocomposite, reduced graphene oxide, poly(paraphenylene), Ascorbic acid, Dopamine, Uric acid, urine

Introduction

The aim today in the domain of medical research and health care is the use of nanotechnology for design of fast, efficient and quantitative diagnosis of biological substances to manage diseases and propose efficient therapy. In this context, the human body contains plenty of biological molecules in which their monitoring concentrations is crucial to disease screening. Dopamine (DA) is one of the important biological neurotransmitters in the mammalian central nervous system for message transfer [START_REF] Lin | Simultaneous determination of dopamine, ascorbic acid and uric acid at poly (Evans Blue) modified glassy carbon electrode[END_REF]. The concentration of dopamine in the human body is low and it was found between 0.01µM-1µM [START_REF] Huang | Simultaneous electrochemical determination of dopamine, uric acid and ascorbic acid using palladium nanoparticle-loaded carbon nanofibers modified electrode[END_REF]. However, when it becomes lower than this value, it may result in neurological disorders, such as Parkinson's disease and schizophrenia [START_REF] Huang | Electrospun Carbon Nanofibers Decorated with Ag-Pt Bimetallic Nanoparticles for Selective Detection of Dopamine[END_REF]. Nomal level of AA is in mM range and high level of AA can cause gastric irritation, and its metabolites can lead to renal issues, while the deficiency of AA is responsible for the illness scurvy [START_REF] Bilal | Highly Selective and Reproducible Electrochemical Sensing of Ascorbic Acid Through a Conductive Polymer Coated Electrode[END_REF][START_REF] Pakapongpan | Highly selective electrochemical sensor for ascorbic acid based on a novel hybrid graphene-copper phthalocyanine-polyaniline nanocomposites[END_REF]. In the case of UA is the primary final product of purine metabolism to normal concentration in serum is 0.2 to 0.5mM and the abnormal level of UA both in serum and urine is the symptom of series of diseases including gout, hyperuricemia, Lesh-Nyhan syndrome, hypertension, urolithiasis, kidney and cardiovascular diseases [START_REF] Ali | Paper-based selective and quantitative detection of uric acid using citrate-capped Pt nanoparticles (PtNPs) as a colorimetric sensing probe through a simple and remote-based device[END_REF]. Consequently, remarkable attention in biomedical and analytical investigations has been investigated in the design of rapid, sensitive, selective, and quantitative method for direct detection of the coexistence of these neurotransmitters [START_REF] Fabregat | Selective Detection of Dopamine Combining Multilayers of Conducting Polymers with Gold Nanoparticles[END_REF]. The low concentration of DA in the extracellular fluid for illness patient and high in healthy one requires sensitive method able to detect these compounds in very large wide of concentration. Many methods have been used for the determination of DA, such as fluorescence quenching, mass spectrometry, and capillary electrophoresis. However most of these protocols are high-cost and they are time consuming [START_REF] Kim | Direct Detection of Tetrahydrobiopterin (BH4) and Dopamine in Rat Brain Using Liquid Chromatography Coupled Electrospray Tandem Mass Spectrometry[END_REF][START_REF] Habibi | Simultaneous Determination of Acetaminophen and Dopamine Using SWCNT Modified Carbon-Ceramic Electrode by Differential Pulse Voltammetry[END_REF] which limits their value in the case of rapid diagnosis. Electrochemical sensors are among the most convenient methods for the detection of dopamine due to their reliability for the rapid determination and their quantitative determination of the analyte [START_REF] Xue | Amperometric Detection of Dopamine in Human Serum by Electrochemical Sensor Based on Gold Nanoparticles Doped Molecularly Imprinted Polymers[END_REF][START_REF] Zhu | Electrochemical Detection of Dopamine on a Ni/Al Layered Double Hydroxide Modified Carbon Ionic Liquid Electrode[END_REF]. In addition, dopamine is an electroactive substance that makes electrochemical methods more suitable for its detection.

However, various others electroactive compounds could be present in extracellular media and interfere in their detection using electrochemical methods. The two others neurotransmitters ascorbic acid (AA) and uric acid (UA) coexist with a higher concentration (between 100 and 1000 times) than that DA in biological fluids for healthy people [START_REF] Kannan | Determination of nanomolar uric and ascorbic acids using enlarged gold nanoparticles modified electrode[END_REF]. These neurotransmitters have redox signal at the potentials range close to DA and makes individual detection of dopamine difficult.

Furthermore, these two neurotransmitters AA and UA are markers for illness [START_REF] Tian | Simultaneous determination of l-ascorbic acid, dopamine and uric acid with gold nanoparticles--cyclodextrin-graphene-modified electrode by square wave voltammetry[END_REF] when there are present at high level or very low level. Therefore, development of electrochemical system able for simultaneous detection of AA, DA and UA with sensitive and selective method for their determination in vivo analysis is subject of many researches [START_REF] Zhang | One-pot facile fabrication of graphene-zinc oxide composite and its enhanced sensitivity for simultaneous electrochemical detection of ascorbic acid, dopamine and uric acid[END_REF][START_REF] Prasad | A dual-template imprinted polymer-modified carbon ceramic electrode for ultra trace simultaneous analysis of ascorbic acid and dopamine[END_REF]. This could be obtained by modification of electrode with the materials or nanomaterials improving the sensitivity and preventing the overlapping. Various materials and nanomaterials have been studied for this purpose. Conducting polymers (CPs) with π-conjugated backbones have been widely used for enhancing the detection of AA, DA or UA due to their remarkable electronic properties [START_REF] Tang | Electrochemical synthesis of polyaniline in surface-attached poly(acrylic acid) network, and its application to the electrocatalytic oxidation of ascorbic acid[END_REF][START_REF] Zheng | Simultaneous determination of ascorbic acid, dopamine and uric acid using poly(4-aminobutyric acid) modified glassy carbon electrode[END_REF].

Electroactive polymers integrating redox system demonstrate to have catalytic effect that enhance the sensitivity toward AA, DA and UA detection [START_REF] Liu | A double signal amplification platform for ultrasensitive and simultaneous detection of ascorbic acid, dopamine, uric acid and acetaminophen based on a nanocomposite of ferrocene thiolate stabilized Fe3O4@Au nanoparticles with graphene sheet[END_REF][START_REF] Miodek | Streptavidin-polypyrrole Film as Platform for BiotinylatedRedox Probe Immobilization for Electrochemical Immunosensor Application[END_REF]. Electrodes modified graphene have been also demonstrated for simultaneous detection of AA, DA and UA [START_REF] Sun | The simultaneous electrochemical detection of ascorbic acid, dopamine, and uric acid using graphene/size-selected Pt nanocomposites[END_REF][START_REF] Su | Printed Combinatorial Sensors for Simultaneous Detection of Ascorbic Acid, Uric Acid, Dopamine, and Nitrite[END_REF]. This is related to their electronic properties, surface area as well as their ability of chemical interaction with such substrates. In addition, it' has been demonstrated that materials bearing negative charges and having an anionic form improve the selectivity of the biosensors, prevent non-specific interaction and enlarge the separation peaks between AA, DA and UA through electrostatic interaction [START_REF] Chen | Tuning Surface Charge and Morphology for the Efficient Detection of Dopamine under the Interferences of Uric Acid, Ascorbic Acid, and Protein Adsorption[END_REF][START_REF] Huang | Simultaneous electrochemical detection of dopamine and ascorbic acid at a poly(p-toluene sulfonic acid) modified electrode[END_REF].

The trends now are going for development of nanocomposite that can bring various functionality for improving electrochemical sensors. For example reduced graphene oxide combined with conjugated polymers and anionic polymer polysulfonic acid has been demonstrated as efficient electrochemical sensing of neurotransmitters [START_REF] Al-Graiti | Hybrid Graphene/Conducting Polymer Strip Sensors for Sensitive and Selective Electrochemical Detection of Serotonin[END_REF]. MoS2 nanospheres and polyaniline loaded on reduced graphene oxide were used for simultaneous detection of these neurotransmitters [START_REF] Li | Electrochemical sensor based on a three dimensional nanostructured MoS2 nanosphere-PANI/reduced graphene oxide composite for simultaneous detection of ascorbic acid, dopamine, and uric acid[END_REF].Detection limit in micro-molar range is generally obtained in these sensors and difference in oxidation potential are less than 200mV.

In this work we aim to develop sensor with enhanced electrochemical activity that improve sensitivity for electrochemical detection of small molecules without overlapping. Nanocomposite based on RGO warped with poly(para-phenylene) modified with ferrocene and acidic group was synthetized by simple methods and applied for simultaneous and individual electrochemical detection of AA, DA and UA. We demonstrate that the nanocomposite brings all the functionality for efficient electrochemical sensor for various neurotransmitters in large range and without overlapping. The RGO nanosheets provide high surface area and conducting surface favoring electron transfer ability, the redox system brings electrocatalytic activity, the anionic form prevents non-specific interaction and overlapping.

Materials and Methods

Reagents and instrumentation

The polymer intituled "poly(para-phenylene)" modified with Ferrocene and acidic group in his backbone (Fc-ac-PPP) has been synthesized and characterized according to the previously reported procedure [START_REF] Blili | Synthesis of a new p-conjugated redox oligomer: Electrochemical and optical investigation[END_REF][START_REF] Bizid | Direct Electrochemical DNA Sensor based on a new redox oligomer modified with ferrocene and carboxylic acid: Application to the detection of Mycobacterium Tuberculosis mutant strain[END_REF].

Gold electrode, graphene oxide (GO), Dimethylformamide (DMF), Ethanol, Phosphate Buffer Solution (PBS tablets), Ascorbic Acid (AA), Dopamine (DA), Uric Acid (UA) , potassium ferrocyanide ([Fe(CN)6]3-/4-), ruthenium hexamine ([Ru(NH3)6 2+/3+]) and urine samples were purchased from Sigma Aldrich (France).

The interfering substances used in this study were also purchased from Sigma Aldrich. Prior the measurements, the prepared PBS was purified by 0.22 µm syringe filters. All solutions were prepared using deionized water produced by milli-Q water system. All solutions were freshly prepared each day. The instrumentation are detailed in supplementary information

Synthesis of reduced graphene oxide (RGO)

The reduction of the graphene oxide (GO) to obtain reduced Graphene Oxide (CRGO) was made by the optimized chemical approach according the method described in the literature [START_REF] Ku Hnel | High-Quality Reduced Graphene Oxide Electrodes for Sub-Kelvin Studies of Molecular Monolayer Junctions[END_REF]. The reaction was performed in DMF at 80°C during 1h. After several centrifugation and washing step, the powder of CRGO was obtained.

Nanocomposite formation CRGO/Fc-ac-PPP

The nanocomposite was prepared by mixing Fc-ac-PPP polymer with CRGO in DMF followed by ultrasonication during 30 min to allow the attachment of the polymer on the surface of graphene nanosheets. To optimize the nanocomposite properties various ratio of CRGO/Fc-ac-PPP has been studied in order to obtain the nanocomposite with high electroactivity and electronic properties.

Therefore, hybrids with different mass ratio (0.1:5, 0.5:5, 1:5, and 2:5) were prepared.

For the sensor formation, gold surface, after polishing steps, was dropped with the nanocomposite CRGO/Fc-ac-PPP suspension followed by drying step under oven at 80°C for 45 min. The membranes based on the nanocomposite CRGO/Fc-ac-PPP with different mass ratio were characterized using CV and EIS to underline the best ratio for sensor application. Scheme 1:Chemical structure of the nanocomposite

Results and discussions

Characterization of CRGO/Fc-ac-PPP nanocomposite

The structure of CRGO was analyzed by XPS and FT-IR, CV and EIS measurements to underline the efficiency of reduction method. FT-IR spectra of GO shows various oxygen functionalities after reduction, various bands related to oxyganed species in GO disappears in the FT-IR spectrum of CRGO which confirm the deoxygenation of GO after reduction (See SI, Figure S1A). The same behavior is observed for XPS analysis. Reduction of GO removes the oxygen band as observed in XPS spectra (see SI 1; Figure S1B,C) where oxygen band was removed in carbon region for CRGO.

Electrochemical characterizations through CV and ESI show also improvement in electron transfer

ability demonstrating the restoring of aromatic system (See SI.1, Figure S2).

The nanocomposite formation CRGO/Fc-ac-PPP was obtained by mixing the CRGO and the soluble polymer in DMF. The association of the two materials could be obtained through the π-π staking interaction as demonstrated previously in the case of carbon nanotube [START_REF] Bizid | Direct E-DNA sensor of Mycobacterium tuberculosis mutant strain based on new nanocomposite transducer (Fc-ac-OMPA/MWCNTs)[END_REF]. SEMs image obtained of the surface modified with RGO, polymer and the nanocomposite show clearly the warping of graphene nanosheets with the polymer (see S2. Figure S3).

The study of ratio optimization was performed following redox signal of ferrocene in the nanocomposite by cyclic voltammery measurement of the redox ferrocene attached to the polymer and the electrical behavior through ESI (See SI. 2, Figure S4). Such analysis demonstrates that the optimum signal of redox ferrocene in the nanocomposite is obtained for 1: 5 ratio of CRGO/Fc-ac-PPP where high redox signal intensity was obtained (Figure S4A). ESI shows also a decrease of Nyquist semicircle related to charge transfer resistance (Figure S4B) demonstrating the efficient electron transfer ability of redox ferrocene in the membrane of nanocomposite.

Electrochemical Characterizations

When comparing the redox properties of electrode modified with polymers and nanocomposite with CV related to redox signal of attached ferrocene on the polymer, higher redox activity is obtained in the case of nanocomposite provided by surface area of graphene nanosheets (Figure 1A).

ESI demonstrates also decrease of diameter of semi-circle of Nyquist plot for electrode modified with nanocomposite compared to polymer (Figure 1B) corresponding to decrease of charge transfer resistance. This behavior demonstrates the efficient interaction of graphene and polymer thanks to various possibility of interaction obtained with graphene nanosheets. This could be through the π-π staking with aromatic ring or the ionic interaction from carboxylic side chain attached to the redox polymer.

Figure 1:

Electrochemical performance of the nanocomposite CRGO/Fc-ac-PPP was performed using two well-known redox systems, [Fe(CN)6] 3-/4-surface-sensitive redox marker "inner-sphere" and [Ru(NH3) ] 2+/3+ , outer-sphere redox marker which could give information in electronic properties of the nanocomposite and the density of electronic states (DOS) [START_REF] Mccreery | Advanced Carbon Electrode Materials for Molecular Electrochemistry[END_REF]. The kinetics of heterogeneous electron transfer (ks) of the redox markers were calculated within the polymer and nanocomposite.

Nicholson method based on the variation of the peak potential versus scan rate was used to calculate the ks using the dimensionless parameter ψ plotted versus scan rate (ν-1/2) [START_REF] Randviir | A cross examination of electron transfer rate constants for carbon screen-printed electrodes using Electrochemical Impedance Spectroscopy and cyclic voltammetry[END_REF] (see SI2, Figure S5). (△EP) shows decrease from 163.31 mV for the polymer to 87.39 mV for the nanocomposite, which highlights that the reversibility of redox system is improved in the case of nanocomposite. This behavior demonstrates the fast diffusion of the redox marker to the nanocomposite provided by graphene nanosheets. The calculated ks were found 5.4 10 -3 cm.s -1 and 6.4 10 -3 cm.s -1 for surfaces modified with Fc-ac-PPP and CRGO/Fc-ac-PPP respectively.

Figure 2:

In the case of CVs obtained with [Ru(NH3)] 2+/3+ , same behavior was obtained where △EP decrease from 133 to 62 mV in the case of the nanocomposite (Figure 2 C,D). This indicates the enhanced of electron transfer ability due to the increase of the conductivity of nanocomposite. Furthermore, the calculated ks with the same method shows improvement in the case of nanocomposite with value calculated from 0.035 cm.s-1 to 0.075 cm.s-1. This behavior is in agreement with improvement of the density of electronic states (DOS) of CRGO/Fc-ac-PPP thanks to electronic effect of conjugated polymer.

These results confirm that the association of Fc-ac-PPP to CRGO provides nanomaterial with large surface area and high conductivity where the electron transfer ability to redox system could lead to a sensitive surface for the electrochemical detection of AA, DA, and UA.

Electrochemical activities of AA, DA and UA on modified surfaces

To evaluate the electrochemical activity of the nanocomposite CRGO/Fc-ac-PPP toward AA, DA and UA individual and simultaneous detection of these analytes were investigated at gold electrode non modified (Au), modified with the polymer (Au/Fc-ac-PPP) and modified with the nanocomposite (Au/CRGO/Fc-ac-PPP). DPVs were performed with the 3 compounds in mixture of 10 -4 M UA and AA and 10 -5 M DA (Figure 3) and individually detection (See SI.3, Figure S6). The nanocomposite CRGO/Fc-ac-PPP depicts a very well defined redox signal with intense peak current for each analyte compared with bare Au and Au/Fc-ac-PPP. Moreover, the peak potential of the individual detection of AA, DA or UA at Au/CRGO/Fc-ac-PPP shifts to lower potential range compared to the others electrodes. This behavior proves that the kinetics of electron transfer of these analyte was faster on the nanocomposite than the other electrodes [START_REF] Taleb | Ultra-sensitive voltammetric simultaneous determination of dopamine, uric acid and ascorbic acid based on a graphene-coated alumina electrode[END_REF]. In addition, the oxidation peaks are well separated Individual detection shows the same behavior (Figure 6S), the nanocomposite Au/CRGO/Fc-ac-PPP exhibits also highest peak current intensity of AA, DA and UA oxidation compared to others electrodes.

The high performance of Au/CRGO/Fc-ac-PPP compared to Au/ Fc-ac-PPP could be related to high surface provided by the RGO as well as the electronic properties of such composite with improvement of electron transfer ability. M, 2.8 10 -10 M, 1.3 10 -8 M for AA, DA and UA respectively. These results demonstrate that the nanocomposite CRGO/Fc-ac-PPP could be applied for individual as well as simultaneously detection of AA, DA, and UA. Compared with some modified electrode published in the last few years (Table 1), Au/CRGO/Fc-ac-PPP sensor presents the lowest LOD for AA, DA and UA and the wide range for their detection and high peak separation. DPV as well as chronoamperometry could be used to follow the presence of these compounds. In addition, the fabrication process is easy compared to other presented sensors. The powerful of this sensor, beside their analytical performances, it's fabricated using organic molecules without any metallic nanoparticles which is eco-compatible. reported sensors (Table 1), various properties are improved: peak potentials separation, range of detection, LOD for the three species and simultaneous determination of AA, DA, and UA without any overlapping. The reproducibility of the sensor was evaluated by nine equally prepared electrodes and their analysis by DPV in the mixture of 10 -4 M AA, UA and 10 -5 M DA (Figure 6B). The oxidation peak potentials and current of the three species were almost the same. The RSD is calculated at 3.2 %, 2% and 2 % for AA, DA, and UA, respectively. The storage stability of the Au/CRGO/Fc-ac-PPP electrode was also investigated. The sensor was kept at ambient temperature for 15 days and then used for the simultaneous detection of AA, DA and UA (Figure 6C). AA, DA, and UA oxidation retained 99.4 %, 98.3 %, and 98.4 % of its initial current response which proves the stability of the sensor due to stability of the nanocomposite.

Determination of DA in biological fluids

In order to confirm the validity of the sensor for real application, simultaneous detection of AA, DA, UA in urine sample was performed using standard addition method. The urine sample free of neurotransmitters was diluted 10 times with PBS to prevent matrix effect. Then various urine sample were mixed with various concentration of these neurotransmitters to test the simultaneous detection were various level coexists. The urine was spiked with various concentrations of AA, DA and UA and the DPV measurement was performed in the same potential region used in PBS. Various samples were tested with various level, urine 1 and urine 2 are for normal level of AA and DA and UA disorder, urine 3 and 4 for disorders in AA, DA and UA. The redox responses obtained in urine are at the same potential as PBS solution. The recovery was calculated (Table 2) and shows value from 110% to 90% depending of the concentration of neurotransmitters. RSD less than 3 % are calculated witch prove the accuracy of the measurement.

Hence these three analytes can be easily determined at Au/CRGO/Fc-ac-PPP sensor without any

Figure 2 A

 2 Figure 2 A, B shows the CVs at different scan for [Fe(CN)6] 3-/4-at surface modified with CRGO/Fc-ac-PPP and compared to Fc-ac-PPP. The variation of anodic and cathodic peaks potentials

C

  than the others electrodes. ∆Ep was obtained 455 mV for peak separation AA-DA and 354 mV for DA-UA.

Figure 3 :

 3 Figure 3: DPV responses of the mixture of 10 -4 M AA , 10 -5 M DA , 10 -4 M UA, in 0,1M PBS (pH=7, on bare Au (a) , CRGO (b), Fc-ac-PPP (c), CRGO/Fc-ac-PPP (d).

3. 4 .

 4 Analytical performance of Au/CRGO/Fc-ac-PPP sensorTo underline the detection signal within concentration of each compounds in the presence of the others compounds, where the concentration of the target biomolecule was changed, while the concentrations of the other two biomolecules were kept constant, were analyzed by DPV with Au/CRGO/Fc-ac-PPP. Figures4A, B, Cshow the DPV curve of the mixture with increasing concentration of one biomolecule. The electrochemical response shows an increase of redox signal within the concentration were the others signals remains constant. Based on the variation of the intensity of peak current related to added concentration of biomolecules, calibration curves of AA, DA and UA were plotted versus logarithm scale of the concentration with correlation coefficients of 0.99 (Figure4A1, B1, C1). Detection limits were calculated based to signal to noise ratio 3 at 4.6 10 -8

Figure 4 :

 4 Figure 4: DPV curves of CRGO/Fc-ac-PPP in 0.1 M PBS with varying concentrations of (A) AA : 10 -7 ; 10 -6 ; 10 -5 ; 10 -4 ; 10 -3 ; 10 -2 M, (B) DA : 10 -10 ; 10 -9; 10 -8; 10 -7 ; 10 -6 ; 10 -5 ; 10 -4 M , (C) UA : 10 -7 ; 10 -6 ; 10 -5 ; 10 -4 ; 10 -3 ; 10 -2 M. The corresponding plot of Ipa vs CAA (A1) , Ipa vs CDA (B1) and Ipa vs CUA(C1).

Figure5:

  Figure5: Amperometric responses of the CRGO/Fc-ac-PPP electrode after the subsequent addition of (A) [10 -7 ; 10 -6 ;10 -5 ;10 -4 ;10 -3 ;10 -2 ] of AA at applied potential of -344.50 mV, (B) [10 -10 ; 10 -9 ;10 -8 ;10 -7 ; 10 -6 ;10 -5 ] of DA at 101.21 mV and (C) [10 -7 ; 10 -6 ;10 -5 ;10 -4 ;10 -3 ;10 -2 ] of UA at 460.51 mV solution in a 010mM PBS. The corresponding calibration curves of AA(D), DA (E), and UA(F).

  Parameters for the simultaneous Determination of AA, DA, UA based on DPV * i-t measurements using different modified electrode PMES (poly(2-(N-morpholine) ethane sulfonic acid) ,(GCE)glassy carbon electrode; (GS) graphene sheet; (GS) chitosan, (PEDOT) poly(3,4-ethylenedioxythiophene); (PANI) polyaniline; (NC@GC) hierarchical core-shell metal-organic framework; (h-ATS) mesoporous silver-doped TiO2-SnO2 nanocomposite.3.5. Interferences, stability and reproducibilityThe effect of interferences on the Au/CRGO/Fc-ac-PPP sensor response was analyzed in presence of several co-existing substances in biological fluids (Glycine, serotonin, Glutamic acid, glucose, citric acid, NaCl, KCl, KNO3, Na2SO4). Figure6Adepicts the DPVs curves of Au/CRGO/Fc-ac-PPP in PBS electrolyte containing 10 -4 M AA and UA and 10 -5 M DA after the addition of 1 mM of each interferences substances. The addition of interferences molecules doesn't affect the current of redox system of AA, DA and UA which confirms the selectivity of the sensor.

Figure 6 :

 6 Figure 6: DPV of Au/CRGO/Fc-ac-PPP in 0,1 M PBS containing 10 -4 M AA and UA and 10 -5 M in presence of glycine (red), serotin (green), glutamic acid (blue), glucose (light blue), citric acid (pink), KCl (brown); KNO3 (dark blue), Na2SO4 (purple) . reproducibility (A) and stability (B) studies at Au/CRGO/Fc-ac-PPP in 0,1 M PBS containing 10 -4 M AA and UA and 10 -5 M DA

Table 2 :

 2 interference and could be applied for diagnosis of patients suffering from carcinoid syndrome, Parkinson and hypertension[40]. These results demonstrate that the proposed sensor based on CRGO/Fc-ac-PPP nanocomposite has a potential for their application in diagnosis. Simultaneous determination of AA, DA and UA in urine sample (n=3)4. ConclusionIn this work, nanocomposite formed with reduced graphene oxide and Fc-ac-PPP polymer was formed and analyzed as sensor for individual and simultaneous electrochemical detection of AA, DA and UA. Characterization of the nanocomposite by CV, EIS and FTIR demonstrated the well distribution the warping of the polymer with RGO through π-π staking interaction which undergoes improvement of surface area, conductivity and electro-catalytic activity. In addition, the nanocomposite shows faster kinetic charge transfer than Fc-ac-PPP using an inner and outer-sphere redox probe. The gold surface modified with nanocomposite allows successful detection of AA, DA and UA detection in ternary mixture with well-separated voltammetry peaks. This new nanocomposite can either detect single species separately or determine the concentration of one analytic in the presence of the two other analytes without any overlapping. The sensor can detect various concentration ranges in urine sample which makes it a powerful platform for medical diagnosis. The easy process of this sensor fabrication made by eco-compatible organic materials and providing high analytical performances towards electroactive biomolecules could be generalized to detection of other molecules of interest.Funding: This research was funded by Minister of Higher Education for Scientific Research of Tunisia for the scholarship and by MAE France for mobility project PHC Maghreb, grant number 39382RE.

Modified electrode Dynamic range (µM) LOD (µM) Ep (mV) Ref

		AA	DA	UA	AA	DA	UA	AA-DA	DA-UA
	PMES/RGO/GCE	30-100,	0.05-100	0.1-100	0.43	0.006	0.056	-	-	[33]
						2
		*1-30
	GCE/GS/CS -		1-700	1-800	-	0.14	0.17	-	-	[34]
	GCE/RGO/ZnO	50-2350	1-70	3-330	3.77	0.33	1.08	236	132	[35]

  40. Raj, M.; Gupta, P.; Goyal, R.N.; Shim, Y.B. Graphene/conducting polymer nano-composite loaded Sensors and Actuators B. 2017, 239, 993-1002.

	screen	Sample printed	Analyte carbon sensor	Added (µM) for simultaneous Found (µM) determination Recovery % of	RSD dopamine
	Urine #1 and5-hydroxytryptamine.	AA	1000	1010	101	0.4
				DA	10	11.1 µM	111	2.1
				UA	1000	1020	102	0.5
		Urine# 2		AA	100	85	85	0.9
				DA	0,1	0.1µM	100	1.5
				UA	100	100	100	0.9
		Urine# 3		AA	10	10.5	100	0.4
				DA	0,01	0,0097	97	3.4
				UA	10	10.5	105	0.2
		Urine #4		AA	0,1	0,095	95	2.4
				DA	0,0001	0,0001	107	2.7
				UA	0,1	0,11	116	0.6

Acknowledgments: Dr. Diana Dragoe and Dr. Francois Brisset from ICMMO are acknowledged for technical support for XPS analysis and SEMs images.


Amal Essaouiba

Teru Okitsu

Rie Kinoshita

Rachid Jellali

Marie Shinohara

Mathieu Danoy

Cécile Legallais

Yasuyuki Sakai

Leclerc

Development of a pancreas-liver organ-on-chip coculture model

Keywords: Islets of Langerhans, hepatocytes, coculture, microfluidic biochips, glucose homeostasis

Advances in organ-on-chip technology allowed the recapitulation of two or more organs interaction thanks to dedicated microbioreactors interconnected by microfluidic network. Here, we developed pancreas-liver coculture model in a microfluidic biochip, using rat Langerhans islets and hepatocytes. The behavior and functionality of the model were compared to islets and hepatocytes (with/without insulin) monocultures. We confirmed the insulin, glucagon and C-peptide secretions by islets monoculture and coculture. Furthermore, C-peptide and insulin secretions were higher in coculture after 5 days of culture. The islets coculture presented downregulation of Pdx1, Glut2, Gcg, App, Ins1, Neurod, Neurog3 and Gcgr genes, compared to monocultures. In hepatic compartment, the monocultures without insulin were negative to CK18 staining and displayed a weaker production of albumin when compared to monocultures with insulin. They also presented a moderate protein expression of CYP3A2, GLUT2, INSR and modulation of several hepatic genes. In coculture model, hepatocytes displayed albumin productions similar to those in monoculture with insulin. The hepatocytes cocultures were highly positive to INSR, GLUT2, CK18 and CYP3A2 immunostaining and allowed to recover mRNA levels similar to monoculture with insulin (Gcgr, Insr, Hnf4a, Igfbp1 and Alb). The result illustrated that islets can produce insulin to supplement the culture medium and recover hepatic functionality. We believe that our model illustrated the potential of organ-on-chip technology to reproduce cross-talk between liver and pancreas.

Introduction

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by deregulation of glucose homeostasis that results from insulin deficiency or systemic insulin resistance [START_REF] Rogal | Stem-cell based organ-on-achip models for diabetes research[END_REF]. DM is one of the most prevalent and costly diseases in the world affecting approximately 463 million people worldwide (1 in 11 adults), according to the latest International Diabetes Federation (IDF) estimation (IDF official web site, 2019). In 2016, World Health Organization (WHO) estimated that 1.6 million deaths were directly caused by diabetes (WHO official web sites, 2020). The complications of diabetes are also associated with multiple medical problems such as blindness, kidney failure, cardiovascular diseases, sexual dysfunction, neuropathy and peripheral vascular disease [START_REF] Rogal | Stem-cell based organ-on-achip models for diabetes research[END_REF][START_REF] Jellali | Chapter 4 -Membrane bioreactors for bio-artificial pancreas[END_REF][START_REF] Forbes | Mechanismes of diabetic complications[END_REF]. Type 2 diabetes mellitus (T2DM, called insulinindependent diabetes) is the most common form of diabetes, representing 90% of diabetic patients (415 million people worlwide, Sadu et al., 2019;IDF official web site, 2019). T2DM is caused by glucose target-tissues insensitivity to insulin and impaired insulin secretion [START_REF] Jun | In vivo-mimicking microfluidic perfusion culture of pancreatic islet spheroids[END_REF]. Currently, there is no therapy curative for T2DM.

Most treatments help patients to control the disease and include lifestyle adjustments and drug therapy such as metformin, sulphonylureas, glitazones and GLP-1 [START_REF] King | Animal models for diabetes: Understanding the pathogenesis and finding new treatments[END_REF][START_REF] Rogal | Stem-cell based organ-on-achip models for diabetes research[END_REF][START_REF] Zbinden | Noninvasive marker-independent high content analysis of a microphysiological human pancreas-on-a-chip model[END_REF].

T2DM is a complex disease involving several organs interactions, including pancreas, liver, muscle, adipose tissues, kideney and gut [START_REF] Defronzo | Type 2 diabetes mellitus[END_REF].

One of the key features of DMT2 is the insulin resistance state characterized by a drop-down of glucose uptake by the skeletal muscle, fatty tissue and liver [START_REF] Forbes | Mechanismes of diabetic complications[END_REF]. In pancreas, the state of hyperglycemia and hyperinsulinemia triggers the increase of insulin production and beta cell proliferation as a compensatory effect of insulin resistance state. T2DM occurs when the pancreas fails to adapt to increased blood glucose levels [START_REF] Bauer | Functional coupling of human pancreatic islets and liver spheroids on-a-chip: Towards a novel human ex vivo type 2 diabetes model[END_REF]. The liver (with muscle) is one of the first organs severely affected by insulin resistance following a chronic excess of dietary energy. The liver responds to chronic systemic hyperglycemia by increasing gluconeogenesis, which is responsible for the increased basal rate of glucose production [START_REF] Rogal | Stem-cell based organ-on-achip models for diabetes research[END_REF][START_REF] Defronzo | Type 2 diabetes mellitus[END_REF]. TD2M is associated with high prevalence of hepatic comorbidities, such as non-alcoholic fatty liver disease (NAFLD, [START_REF] Perry | The role of hepatic lipids in hepatic insulin resistance and type 2 diabetes[END_REF]. NAFLD is a rapidly growing disease affecting 30 % of general population and around 90% of T2DM patients [START_REF] Schulze | A 3D microfluidic perfusion system made from glass for multiparametric analysis of stimulussecretion coupling in pancreatic islets[END_REF]. Furthermore, it is still controversial whether NAFLD is a consequence or a cause of pancreas disorders.The relevant physio-pathological models involving organ to organ interactions are critical to extract relevant biomarkers and therapeutic solutions. As a result, it is crucial of importance to reproduce both the liver and the pancreas physiologies to control and identify properly the interaction of diabetes development and its systemic relationship with liver.

Advanced new in vitro technologies can create ample opportunities for a more modern approach to toxicology and to pharmacology, to replace the traditional "black box" of animal-based and of conventional 2D in vitro-based paradigms. The animal models fail to faithfully recapitulate the human condition and lose relevance when extrapolating the results to humans [START_REF] Ghaemmaghami | Biomimetic tissues on a chip for drug discovery[END_REF]. In vitro cell cultures are mainly performed in 2D cultures using conventional multi-well plates, which do not accurately reflect the physiological in vivo microenvironment [START_REF] Capulli | Approaching the in vitro clinical trial: engineering organs on chips[END_REF]. Consequently, it is essential to improve those basic plates cultures to reproduce, as closely as possible, the characteristics found in vivo. This is the reason why many groups are developing tissue-engineering and 3D culture processes in order to provide a more appropriate micro-environment for the tissue maintenance and development.

Among those in vitro complex models, organ-on-chip technology seems to be one of the suited methods for reproducing the behavior of an organ or a group of organs, as well as the controlled physiological microenvironment (Bathia and Ingber 2014). In the last years, several teams have proposed organ-on-chip devices that reproduce various behaviors of various organs and tissues including liver, kidney, gut, lung, heart and intestines [START_REF] Kimura | Organ/body-on-a-chip based on microfluidic technology for drug discovery[END_REF]Polini et al., 2104). While many of organ-on-chip technologies have been developed and advanced for the liver, only few studies were dedicated to pancreas, and even less, to our knowledge, to the liver-pancreas interaction. Among those in vitro complex models, organ-on-chip technology has been used to reproduce in vitro pancreas-on-chip model [START_REF] Schulze | A 3D microfluidic perfusion system made from glass for multiparametric analysis of stimulussecretion coupling in pancreatic islets[END_REF][START_REF] Li | 3D-templated, fully automated microfluidic input/output multiplexer for endocrine tissue culture and secretion sampling[END_REF][START_REF] Brooks | Macro-to-micro interfacing to microfluidic channels using 3D-printed templates: application to time-resolved secretion sampling of endocrine tissue[END_REF][START_REF] Lee | Microphysiological analysis platform of pancreatic islet β-cell spheroids[END_REF]2018;[START_REF] Zbinden | Noninvasive marker-independent high content analysis of a microphysiological human pancreas-on-a-chip model[END_REF].

However, most of the current microfluidic platforms have mainly been designed for islets quality assessment for subsequent in vivo implantation [START_REF] Rogal | Stem-cell based organ-on-achip models for diabetes research[END_REF]. In parallel, few recent works also demonstrated the potential of organ-on-chip technology for complex pancreatic analysis and pancreatic islet-liver cross-talk study [START_REF] Bauer | Functional coupling of human pancreatic islets and liver spheroids on-a-chip: Towards a novel human ex vivo type 2 diabetes model[END_REF][START_REF] Zbinden | Noninvasive marker-independent high content analysis of a microphysiological human pancreas-on-a-chip model[END_REF].

Following those works, we proposed in this paper a new coculture organ on chip microfluidic technology allowing hepatocytes and pancreatic islets long term culture. We presented the hepatic perturbation by removing the insulin from the culture medium. Then, we investigate the restauration of the hepatic function thanks the interaction between the liver and the endocrine pancreas trough the circulating hormones and cofactors in the co-culture configuration.

Material and methods

Device design and fabrication

The microfluidic coculture consists of two different biochips (one for the liver, one for pancreas) serially connected. Each biochip is manufactured with two polydimethylsiloxane (PDMS) layers.

In the pancreas on chip, the micro-structured bottom layer, used for islets trapping, is composed of 600 micro-wells of 400 µm in diameter (depth of 300 µm), and spaced by 50 µm (Fig. 1A, Essaouiba et al., submitted). The second PDMS layer, with a reservoir (depth of 100 µm, Fig. 1A), is placed on top of the first layer and includes an inlet and outlet for culture medium perfusion. A micro-channels network placed at the inlet and outlet of each layer allows homogenous culture medium distribution in the biochip (Fig. 1A).

Concerning the liver on chip, we used a micro-structured bottom layer composed of microchannels and microchambers network in cell culture chamber of 1.2 cm length, 1 cm width and 100 m height (Fig. 1A). The second PDMS layer, with a reservoir (depth of 100 µm, Fig. 1A) is sealed on the top of the first layer. As described above, a micro-channels network is also presents in the inlet and outlet to ensure homogenous culture medium distribution. The design and dimensions of biochip have been described in our previous work [START_REF] Baudoin | Behaviour of HepG2/C3a cell culture in a microfluidic bioreactor[END_REF]Jellali et al., 2016).

The PDMS biochips were manufactured by replica molding process. First, photolithography was performed to create the mold masters of the bottom and top layer of the biochips using SU-8 photosensitive resin. Then, PDMS prepolymer (mixture of 10:1 base polymer curing agent; Sylgard 184, Dow Corningin) was poured onto SU-8 master and cured 2 h at 75°C. The surfaces of obtained PDMS layers were activated with reactive air plasma (1 min; Harrick Scientific) and brought together immediately to form an irreversible seal.

Rat Islets and hepatocytes isolation

Pancreatic islets and hepatocytes were isolated from male Wistar rats of 8-9 and 6 weeks old, respectively (CLEA Japan, Inc, Tokyo, Japan). The rats were housed at the University of Tokyo with a 12-h light/dark cycle at 22°C with food and water ad libitum. All animal experimentation procedures were done according the guidelines of the University of Tokyo and the Japanese Ministry of education.

Islets isolation

Pancreatic islets were isolated following the protocol described by [START_REF] Yonekawa | A new mouse model for intraportal islet transplantation with limited hepatic lobe as a graft site[END_REF][START_REF] Kiba | High-quality RNA extraction from rat pancreatic islet[END_REF]. The rats were anesthetized by isoflurane inhalation solution (Pfizer). After clamping of all irrigation blood vessels, the enzymatic solution (Liberase™ TL, Roche) was injected trough the bile duct, previously identified and clamped. After the pancreatectomy, the selective chemical digestion of the organ was done at 37°C for 30 min with the Liberase TL/ ET-K solution (ET-Kyoto solution, Otsuka Pharmaceutical). The digestion was followed by washing and purification steps using a discontinuous OptiPrep® density gradient (Merck/ Sigma-Aldrich).

Then, the islets of Langerhans were identified and hand-picked with a Pasteur pipette under a stereomicroscope (Leica S9 D) and transferred to a cold solution made of UW solution (University of Wisconsin, [START_REF] Kneteman | Islet isolation from human pancreas stored in UW solution for 6 to 26 hours[END_REF]) complemented with Miraclid (Mochika pharmacy, Japan) and Heparin (Mochika pharmacy, Japan). After the islets assessment and counting, the tissue was stored at 4 °C until culture starting.

Hepatocytes isolation

Primary hepatocytes were isolated using the two-step collagenase protocol based in Seglen et al. protocol (Rizki-Safitri et al., 2020;[START_REF] Xiao | New physiologically-relevant liver tissue model based on hierarchically cocultured primary rat hepatocytes with liver endothelial cells[END_REF][START_REF] Seglen | Preparation of isolated rat liver cells[END_REF]). Briefly, after animal anesthesia by isoflurane inhalation solution (Pfizer), the liver was perfused with buffer solution in order to washout the blood. subsequently, the liver was extracted, deposited in Dulbecco's Modified Eagle Medium (DMEM, Gibco™ -Life Technologies) and the tissue was gently disrupted. The digested tissues were filtered through 100 µm filters (cell strainer 100 μm nylon; Falcon®) and the liver cell suspensions were centrifuged thrice. The resulting pellets were mixed, suspended in Percoll (#P1644, Sigma-Aldrich) and HBSS (#H6648, Sigma-Aldrich) separating solution. Percoll isogradient centrifugation was performed to isolate dead cells and a significant portion of the nonparenchymal cells in a floating top layer that was discarded. Finally, the obtained cells were suspended in seeding medium (William's E medium (Gibco™) supplemented with 10% fetal bovine serum (FBS, Gibco™) and 100 units/mL of penicillin, 100 mg/mL streptomycin (Gibco™). Cell viability was assessed by Trypan blue dye exclusion and hepatocytes culture with a viability of more than 85% were used. The purity obtained is over 98%.

Biochip culture

The experimental setup used for culture in biochip was composed of a perfusion loop, including the culture medium tank (bubble trap), the peristaltic pump, and one or two biochips. They were interconnected by 0.65 mm interior diameter silicone/Teflon tubing (Fig. S1, supplementary file). The bubble trap contained 2 mL of culture medium and the flow rate was set at 20 µL/min. Before each experiment, the circuit (bichip, tubing and bubble trap) was sterilized by autoclaving and dried in oven.

Four groups of biochips were investigated and compared: (i) hepatocytes biochip monoculture with insulin (hepatocytes monoculture ITS +); (ii) hepatocytes biochip monoculture without insulin (hepatocytes monoculture ITS -); (iii) pancreatic islets biochip monoculture (islets monoculture) and (iv) hepatocytes/islets biochips coculture without insulin (hepatocytes/islets coculture). The detailed experimental procedure is shown in Fig. 1B.

Pancreatic islets culture in biochip (pancreas on chip)

The biochips were previously filled with culture media in order to remove the air bubbles and moisturize the circuits. The preconditioning process was carried out during one hour at 37° C in the incubator. The pancreatic islets in the preservation solution were washed with cold culture media and gently diluted in the appropriate amount. In order to minimize the islets damage, wide orifice pipette tips with low binding were used during all the handling process. After loading in the biochips, the islets were counted under microscope in order to keep a record of the islets number per biochip (≈40 islets/biochip). Then, the circuit was connected to the peristaltic pump and the perfusion started. The entire setup was continuously incubated at 37 °C in a 5% CO 2 supplied incubator. The basal culture medium used for the pancreatic islets culture was a classic RPMI 1640 medium (Gibco™) supplemented with 10% FBS (Gibco™), 100 units/mL of penicillin, 100 mg/mL streptomycin (Gibco™) and GlutaMAX™ (Gibco™) at 10 Mm.

Hepatocytes biochip culture (liver on chip)

After sterilization, biochips were coated with rat tail type 1 collagen (Corning®, 300 µg/mL in phosphate-buffered saline: PBS Gibco™) and incubated at 37°C in an atmosphere supplied with 5% CO 2 . After 1h, the collagen solution was washed using the seeding medium and the freshly isolated hepatocytes (5x10 5 cells/biochip) loaded into the microfluidic device via biochip inlet ports using a micropipette tip. To keep the seeding medium inside the culture chamber, the biochip inlet ports were closed using two syringes (containing 500 µL of seeding medium), and the biochips were placed in an incubator at 37°C and 5% CO 2 . After 24 h of static conditions to promote cell adhesion, the seeding medium was replaced by the culture medium and the biochip integrated in the perfusion experimental setup to launch dynamic culture. The primary hepatocytes culture medium was composed of William's E medium (Gibco™) supplemented with 100 units/mL of penicillin / 100 mg/mL streptomycin (Gibco™), GlutaMAX™ (Gibco™) at 10 Mm, 1% non-essential amino acids (Invitrogen), 3% Bovine Serum Albumin (Sigma), 1% Insulin-Transferrin-Selenium ITS-100X (PanBiotech), 0.1 μM Dexamethasone (Wako Pure Chemical Industries), 10 ng/ml mouse Epidermal Growth Factor (Takara Bio), 0.5 mM ascorbic acid 2-phosphate (from magnesium salt n-hydrate; Wako Pure Chemical Industries) and 20 mM HEPES (Gibco™). For the hepatocytes monoculture without insulin (monoculture ITS -), we used the same medium composition excluding ITS.

Hepatocytes/islets cocultures (pancreas/liver on chip)

The liver and pancreas biochips were prepared separately. At first, the hepatocytes were inoculated inside the liver biochip (as in section 2.3.2). After 24h of adhesion, the hepatocytes were cultivated inside the liver biochips for 24h in perfusion (this result to 48h of culture in the liver biochip including 24h for adhesion in static condition and 24h of perfusion). The pancreatic islets biochips were prepared after those 48h. The islets were inoculated in the biochips as described in section 2.3.1. After 1h at rest and the islets sedimentation, the liver perfusions were stopped and one pancreas biochip and one liver biochip were serially connected to each other to create a pancreasliver coculture model (Fig. 1A and Fig. S1, supplementary file).

The culture medium for the coculture condition is a mixture 1:1 of pancreatic islets and hepatocytes media, excluding ITS from the last one.

RTqPCR assays

Total RNAs were extracted and purified from samples using a hybrid protocol that combines Trizol™ Reagent (Life Technologies) and RNeasy Mini Kit (QIAGEN 74104) following the manufacturer's instructions. Concentrations and qualities of extracted RNAs were assessed using a BioSpec-nano (Shimadzu Scientific Instruments). Reverse-transcription into cDNA was performed from 0.5 μg of total RNA using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO).

Real-time quantitative reverse transcription polymerase chain reaction (RTqPCR) was then performed with the THUNDERBIRD SYBR qPCR Mix (TOYOBO) according to manufacturer's protocol and a StepOnePlus Real-Time PCR system (Applied Biosystems). The primer sequences of genes are shown in Table. S1 and S2. β-Actin was used as the reference gene and fresh isolated hepatocytes and pancreatic islets (days 0) as the reference sample for the normalization of gene expression data.

Immunostaining

The islets were transferred to an untreated TCPS 24 wells plate. The hepatocytes immunostaining was performed in the biochip. The islets and the hepatocytes were washed with phosphate buffer saline solution (PBS, Gibco) and fixed in paraformaldehyde 4% at 4 °C for 24h. In order to perform the immunostaining in a 3D structure, the islets were permeabilized with 1% Triton X100 in PBS for 3h at 4°C and washed 3 times with PBS for 30 min, while the hepatocytes were permeablized with 0.1% Triton X100 in PBS for 15 min. Then both, islets and hepatocytes were blocked with a gelatin buffer for 24h at 4°C. Primary antibodies were incubated at 4°C in a BSA/PBS solution for 48h and overnight for pancreatic islets and hepatocytes, respectively. After several washing steps, secondary antibodies were incubated in a BSA/PBS solution at 4°C in the dark (24h for islets and overnight for hepatocytes). Actin filaments were stained with Phalloidin-iFluor 488 Reagent (abcam). Finally, the nuclei were stained with Hoechst 33342 (H342, Dojindo) at 1/800 for 30 min at room temperature in the dark.

All the incubations and washing steps were carried out using a shaker for the islets of Langerhans immunostaining process. For hepatocytes, the biochips were cut with a scalpel removing the PDMS top layer in order to increase the resolution of the image. Observations were made with an Olympus IX-81 confocal laser-scanning microscope. The primary and secondary antibodies used are presented in Table . S3 (supplementary file).

Insulin, glucagon, C-peptide and albumin measurements

The hormones and albumin release in the culture medium samples from the different culture conditions were assessed using ELISA assays, following the manufacturer's protocol. The following kits were used: insulin (Mercodia rat Insulin ELISA kit, 10-1250-01), glucagon (Glucagon DuoSet ELISA kit, DY1249 and DuoSet ELISA Ancillary Reagent Kit 2, DY008), C-peptide (Mercodia rat C-Peptide ELISA kit, 10-1172-01) and albumin (Rat Albumin ELISA quantification set from Bethyl, Cat. No. E110-125 combined with the Enzyme Substrate, TMB, Cat. No. E102). Results were obtained with an iMark microplate reader (Bio-Rad, Osaka, Japan) set to a wavelength of 450nm.

Glucose and lactate measurement

Glucose and lactate were measured using an YSI 2950 Biochemistry Analyzer.

For that purpose, 160 µL of culture medium were inserted into the analyzer.

Measurements were based on a direct reading of L-lactate (L-lactic acid) and glucose in the culture medium by the YSI enzyme sensors, as the enzymes L-lactate oxidase, and glucose oxidase are respectively immobilized in the lactate and glucose sensors.

Statistics

All experiments were repeated at least three times. The data are presented as the mean ± standard deviations (SD) of 9 biochips (3 biochips from 3 different experiments, n=3x3). The Kruskal Wallis test was performed for the statistical analysis using GraphPad software (Prism 6). Data with P-values < 0.05 were identified as statistically significant and highlighted in the figures.

Results

Cellular morphologies analysis

The hepatocytes were successfully attached in the collagen-coated biochip after few hours of cultures. They were kept at static condition in incubator. After 24h, the hepatocytes created a confluent tissue and displayed typical cuboidal hepatocyte phenotypes (Fig. S2, supplementary file).

The hepatocytes morphology at the end of the experiments (24h in static condition and 6 days of perfusion) are presented in Fig. 1. The hepatocytes monoculture with insulin (hepatocytes monoculture ITS +) displayed typical hepatic monolayer cultures (cells with typical cuboid shape). The cell monolayer was maintained over 7 days of culture, including 24h at rest and 6 days in perfusion (Fig. 2A). Without insulin (hepatocytes monoculture ITS -), the hepatocytes cultures were heterogeneous from one biochip to another. A few hepatic cultures in biochips without insulin presented similar morphologies when compared to the hepatic culture with insulin (Fig. 2B). In most of the biochips, the hepatocytes formed some aggregated structures or degraded tissue (Fig. 2C). Finally, in the hepatocytes/islets biochips cocultures, the typical hepatic phenotypes (similar to hepatocytes monoculture ITS +) were recovered as shown in Fig. 2D.

Concerning islets cultures, we did not detect a modification of the morphologies of the pancreatic islets, when we compared the islets monocultures and the islets/hepatocytes cocultures. The islets were trapped in the microwells of the pancreas biochips, and displayed the typical spheroid shapes of islets of Langerhans (Fig. 2E andF). Furthermore, we counted the number of islets at the end of experiments (5 days of perfusion). The result showed that the number of islets collected at the end of the perfusion remained similar to the number of islets seeded .

Immunostaining analysis

Immunofluorescence staining was performed at the end of the experiment (day 5 and 7 for islets and hepatocytes, respectively). For hepatocytes, we selected to stain several hepatic markers: CYP3A2 (one of the most abundant cytochrome P450 in the liver), CK18 (differentiation marker), INSR (insulin receptor) and GLUT2 (glucose transporter). We also stained two important markers in the pancreatic islets: insulin and glucagon that are markers of -cells and -cells, respectively.

The immunostainings of the hepatic cells at day 7 are presented in the Fig. 3.

The immunostainings have demonstrated that the hepatocytes monocultures with insulin and the hepatocytes/islets cocultures led to positive cell population expressing CYP3A2 and CK18. Those results illustrated the maintenance of the differentiation of the hepatocytes in both type of cultures. On contrary, the CK18 was not expressed and the CYP3A2 was moderately expressed in hepatocytes monocultures without insulin (Fig. 3A). In parallel, the hepatocytes monocultures with insulin and the hepatocytes/islets cocultures showed an intense positive cell population to INSR and to GLUT2, whereas the expressions of those markers were weaker in hepatocytes without insulin (Fig. 3B).

In the case of pancreatic islets, the level of insulin and glucagon expression inside the islets after extraction (day 0) appeared variable from one rat to another, probably due to the rat fed state at the moment of the extractions (Fig. S3, supplementary file). Then, in pancreatic islets monocultures and islets of pancreas/liver cocultures, we found an expression of both glucagon and insulin hormones at the end of the experiments (Fig. 4). Furthermore, the levels of insulin appeared over expressed in cocultures when compared to the islets monoculture levels. The glucagon was similarly expressed, in both monoculture and coculture of islets ,as far as we did not detect any difference between the immunostaining images.

RTqPCR analysis

At the end of the experiments, we analyzed the expression levels of selected hepatic genes in the different culture conditions (monoculture ITS+, monoculture ITSand coculture). The comparison of the mRNA levels between the hepatocytes cultivated in monocultures without insulin and with insulin is presented in Fig. 5A (ratio of mRNA levels in monoculture ITS-versus monoculture ITS+). We found that the mRNA level of Alb was downregulated in monocultures without insulin (fold change, FC of 0.55). On contrary, the expression levels of Hnf4a (FC 40), Insr (FC 4.5), Igfbp1 (FC 2.5) and Pck1 (FC 10) were significantly increased. A large variability in the levels of Gcgr, Glut2 and Cyp3a2 in monocultures without insulin was measured which led to no statistical difference between the two culture conditions on those genes.

The comparison was then performed for the gene expression in hepatocytes cultivated in cocultures with pancreatic islets and the hepatocytes monoculture with insulin. As showed in Fig. 5A, the coculture contributed to recover similar mRNA levels regarding the Gcgr, Insr, Hnf4a, Igfbp1 and Alb when compared to monoculture of hepatocytes done with insulin. However, the mRNA level of Cyp3a2, of 0.1 and 0.35 respectively). Conversely the mRNA level of Pck1 remained high in coculture when compared to hepatocyte with insulin monoculture and at similar levels when compared to hepatocyte monocultures without insulin.

Finally, we investigated the effect of coculture with hepatocytes on expression levels of pancreatic islet-specific genes. The comparison of the mRNA levels is shown in Fig. 5B (ratio of mRNA levels in coculture versus islets monoculture). We found that the coculture with the hepatocytes contributed to downregulate the levels of Gcgr, Neurod, Neurog3, Glut2, Ins1, Gcg, App and Pdx1 (FC between 0.1 and 0.36). In contrast, expression of Reg3a, Ins1 and Glp1r were 5 to 15.5 times higher in the coculture compared to the islets monoculture. However, a large variability was founded for Ins1 and Glp1r in both conditions of culture.

Functional assays revealed higher insulin and C-peptide secretions in the islets in cocultures with hepatocytes when compared to islets monocultures

The basal functionality of the pancreatic islets in both culture modes (islets monoculture and coculture with hepatocytes) was investigated by the measurement of the levels of insulin, C-peptide and glucagon secretions. At the end of each experiment, the islets were counted to normalize the data. The daily secretions of the three hormones at day 3 and day 5 (end of the experiment) are presented in Fig. 6.

We found that the insulin concentration in the culture medium decreased between day 3 and day 5, in both culture conditions (monoculture and coculture).

However, the cocultures with hepatocytes contributed to maintain relative high level of production when compared to monoculture (Fig. 6A). Especially at day 5, we found production of 45 ng/islet/day and 170 ng/day/islet in monoculture and coculture respectively. Similarly, we measured a higher production of C-peptide in coculture when compared to monoculture (Fig. 6B). The secretion was about 1.5 (20 pmol/islet/day) and 5 (29 pmol/islet/day) times higher in the islets coculture with hepatocytes at day 3 and 5, respectively. The levels of glucagon remained constant in the coculture during the 5 days of experiment, close to 800 pg/islets/day, but with a large dispersion at day 3 (Fig 6C ). Conversely, the level of glucagon in islets monoculture dropped from 840 to 512 pg/islet/day between day 3 and day 5 of culture.

The hepatocytes functionality was assessed by the measurement of the levels of albumin in the three culture modes (monoculture ITS+, monoculture ITS-and coculture). The hepatocytes in monoculture with insulin and in coculture with pancreatic islets produced similar quantities of albumin (constant over time, from day 3 to day 7, the end of the experiment). Conversely, without insulin in the culture medium, the hepatocytes monoculture presented a drop of the albumin production between day 3 and day 7, as shown in Fig. 7A. This led to reach a production of albumin closed to 750100 µg/10 6 cells in hepatocytes/islets biochip coculture and in liver biochip monoculture with insulin, whereas the values drop closed to 440230 µg/10 6 cells in liver biochip monoculture without insulin.

Finally, we measured the glucose and lactate levels during the culture in the different conditions (Fig 7B). The data displayed a large dispersion, but the tendency illustrated higher glucose consumption in coculture compared to liver monoculture conditions with and without insulin. Furthermore, this consumption (in hepatocytes/islets coculture) seems to be constant between day 3 and day 7.

Concerning lactate production, we could not distinguish a significant difference between the different culture conditions, with a lactate production around 0.6-0.8 mmol/L.

Discussion

We have developed a model of coculture of rat hepatocytes with rat Langerhans islets using an organ-on-chip technology. The liver biochip results from our previously developed liver on chip technology that was already successfully applied for rat and human primary hepatocytes [START_REF] Baudoin | Investigation of the levels of expression and activity of detoxication genes of primary rat hepatocytes under various flow rates and cell densities in microfluidic biochips[END_REF]Jellali et al., 2016). We coupled this liver on chip technology with a recent update of our technology to pancreas on chip model (Essaouiba et al., submitted). The interconnection of both organ-on-chip technologies led to produce a new model of interaction between the organs that are the liver and the pancreas. The coculture of two cell/tissue types required an adapted coculture medium able to maintain their characteristics. Preliminary tests were carried out to define the culture medium adapted to islets/hepatocytes coculture: i) hepatocytes monoculture in William's E medium (standard medium for rat hepatocytes culture) and in a mixture 1:1 of William's E/RPMI 1640; ii) islets monoculture in RPMI 1640 medium (standard medium for islets culture) and in a mixture 1:1 of William's E/RPMI 1640. The performed analyzes (viability, morphology, gene expression of selected markers, albumin and insulin secretion) showed that islets and hepatocytes monocultures in a mixture of William's E and RPMI 1640 maintained their characteristics (when compared to islets and hepatocytes cultivated in their standard medium).

Consequently, a mixture 1:1 of William's E/RPMI 1640 media was chosen for the islets/hepatocytes coculture experiments.

In this study, the hepatocytes monoculture without insulin led to a downregulation of the Alb mRNA and to a weaker albumin production in culture medium. The decline of albumin production and Alb mRNA level in lack of insulin was already reported in the literature of in vitro data in rat [START_REF] Kimball | Hormonal regulation of albumin gene expression in primary cultures of rat hepatocytes[END_REF][START_REF] Essaouiba | Mathieu Danoy(2), Yannick Tauran(5), Cécile Legallais(1), Yasuyuki Sakai(4), Eric Leclerc(1,2*) Pancreas-on-chip models enhanced the mRNA levels of pancreatic islets markers and the functionality of rat Langerhans islets in in vitro cultures[END_REF] and in vivo in mice [START_REF] Chen | Insulin is required to maintain albumin expression by inhibiting forkhead box O1 protein[END_REF]. In our study, the lack of insulin also led to a weaker CYP3A2 expression and to the lack of CK18 expression at the protein level (the Cyp3a2 was also downregulated at mRNA level). Both markers are hepatic differentiation markers. It is reported that rat hepatocytes presented a reduction of about 30% of the mRNA level of CYP3A in the absence of insulin (Woodkrooft et al., 1999). Although there is no literature result about the effect of insulin on CK18 immunostaining, various reports mentioned the crosstalk between CK18 and insulin resistance such as in diabetic patients [START_REF] Civera | Relationship between insulin resistance, inflammation and liver cell apoptosis in patients with severe obesity[END_REF] or patients with non-alcoholic steatohepatitis [START_REF] Kitade | Crosstalk between angiogenesis, cytokeratin-18, and insulin resistance in the progression of nonalcoholic steatohepatitis[END_REF], in which CK18 fragments are elevated in plasma. HNF4 transcription factor is involved in the mechanism of liver differentiation via the HNF1, HNF4, PXR and CYP450 axis [START_REF] Chiang | Hepatocyte nuclear factor 4α regulation of bile acid and drug metabolism[END_REF] but it is also involved in the glucose homeostasis [START_REF] Stoffel | The maturity-onset diabetes of the young (MODY1) transcription factor HNF4alpha regulates expression of genes required for glucose transport and metabolism[END_REF], in liver pancreas interactions and into diabetes [START_REF] Lau | The molecular functions of hepatocyte nuclear factors -In and beyond the liver[END_REF]. HNF4 is targeted and repressed by insulin in hepatocytes [START_REF] Hirota | Hepatocyte nuclear factor-4 is a novel downstream target of insulin via FKHR as a signal-regulated transcriptional inhibitor[END_REF][START_REF] Xie | Down-regulation of hepatic HNF4α gene expression during hyperinsulinemia via SREBPs[END_REF] with was consistent with our result as far as HNF4A was upregulated without insulin.

The hepatocytes monoculture without insulin also presented a modulation of the Igfpb1 (Insulin-like growth factor binding proteins, up regulation of mRNA level), of the Insr (lower expression of the protein in the immunostaining and upregulation of the mRNA level), of Pck1 (mRNA upregulation). Interestingly our results are consistent with the literature reporting that the insulin inhibits Igfpb1 in liver [START_REF] Powell | Insulin inhibits transcription of the human gene for insulin-like growth factorbinding protein-1[END_REF]. Pck1 catalyzes the first step in gluconeogenesis. In silencing Pck1 mice, the insulin signaling was improved in liver (Gomez et al, 2008). Conversely insulin is also reported to reduce the mRNA level of Pck1 [START_REF] Zhang | Insulin-regulated Srebp-1c and Pck1 mRNA expression in primary hepatocytes from zucker fatty but not lean rats is affected by feeding conditions[END_REF]. Once more those data appeared consistent with our biochip findings. Finally, the overall hepatic biochip culture without insulin illustrated a consistent behavior of our hepatic model when compared to literature.

The pancreatic islets in our cocultures were able to produce insulin to counter balance the suppression of ITS. However, the level of insulin detected (about 3000 g/L) where lower than the insulin level used in "conventional" hepatocyte culture model (in the present experiment, the hepatocytes monoculture with insulin were performed at 10mg/L of insulin via ITS supplementation in the medium). That could explain why, at the mRNA level of the hepatic marker in hepatocyte coculture such as Alb, Cyp3a2, remained lower and Pck1 remained higher than the level in hepatocytes monoculture with insulin. Nevertheless, at the protein level the expression of Cyp3a2, CK18 and the production of albumin were restored in coculture. The mRNA levels of insulin related genes such as Igfbp1 and Insr were also restored, including Hnf4a. In addition, the protein expression of Insr and Glut2 appeared similar in the immunostaining in both hepatocyte monoculture with insulin and in islets/hepatocytes cocultures. Those data illustrate the functional cross talk between the pancreas and the liver. It also demonstrated the partial restoration of the expected effect of insulin on hepatocytes. Nevertheless, additional experiments, by tuning the number of isles, to increase the production of insulin would be required to confirm the full recovery of Pck1 and Cyp3a2 mRNA levels.

High glucose level normally leads to high production of insulin. In the present culture conditions, the glucose level in medium was of 2,5 mM and did not decrease strikingly between two time points of culture medium change (nb similar glucose concentration was used in all culture modes). This led to a continuous stimulation for insulin production in the pancreatic islets monoculture and during the islets/hepatocytes coculture (and thus contribute to restore the hepatic functions in the coculture by insulin secretion stimulated by glucose). Regarding the effect of hepatocyte coculture on the pancreatic islets, we found that the coculture downregulated Gcgr, Glut2, Ins1, Neurod, Neurog3, App, Gcg, Pdx1 and upregulated

Glp1r at the mRNA level. Pdx1, Neurod and Neurog3 are important markers of the differentiation of the islets [START_REF] Gu | Pancreatic β cells require NeuroD to achieve and maintain functional maturity[END_REF]Mc Grath et al., 2015). Neurod is an important gene (an insulin trans activator) required to maintain functional maturity in pancreatic beta cells, including insulin production through INS1 [START_REF] Gu | Pancreatic β cells require NeuroD to achieve and maintain functional maturity[END_REF].

Consistently, we found both downregulation of Ins1 and Neurod in coculture biochips (which is consistent with Ins1 silencing in Neurod KO mice, [START_REF] Gu | Pancreatic β cells require NeuroD to achieve and maintain functional maturity[END_REF]. Neurod KO-mice expressed Ins2 gene and are thus able to produce insulin in glucose tolerance test, which also appeared consistent with our finding in which Ins2 was over expressed in pancreas biochip coculture [START_REF] Gu | Pancreatic β cells require NeuroD to achieve and maintain functional maturity[END_REF]. Pdx1 has pivotal important gene in cells. Pdx1 is a homeobox-containing transcription factor that plays a key role in pancreatic development and adult β-cell function [START_REF] Gao | Pdx1 maintains β-cell identity and function by repressing an α-cell program[END_REF]. Depletion of Pdx1 lead to hyperglycemia in mice, to a cell reprogramming in mice islets, glucagon overexpression in Min6  cells [START_REF] Gao | Pdx1 maintains β-cell identity and function by repressing an α-cell program[END_REF].

Furthermore, Pdx1 deficient  cells led to the reduction of the transcript level of Pdx1, Ins1 and Glut2 and the maintenance of glucagon one [START_REF] Gao | Pdx1 maintains β-cell identity and function by repressing an α-cell program[END_REF]. This result was partially consistent with our dataset in which we found a concomitant downregulation of Pdx1, Ins1 and Glut2 genes in coculture.

Glucagon is produced during hypoglycemia to stimulate hepatic glucose output. In our study, the glucose concentration remained high in the culture medium, leading to detect a high insulin/glucagon ratio which is consistent with the downregulation of the levels of Gcg (glucagon) and Gcgr (glucagon receptor) mRNA.

However, we also measured high level of Glp1r mRNA. It is reported that paracrine glucagon stimulates insulin secretion through both Gcgr and Glp1r. More particularly the activity of glucagon and GLP-1 receptors were reported to be essential for  cell secretory responses via paracrine intra-islet glucagon actions to maintain appropriate insulin secretion [START_REF] Svendsen | Insulin secretion depends on intra-islet glucagon signaling[END_REF], which is consistent with our findings in coculture. Although we described those behaviors in the pancreatic tissue as a result of the hepatic coculture, we did not clearly identify the underlying mechanisms and the endocrine liver signaling driving such cross talk. As a result, additional analysis would be required to complete our investigation including metabolome and proteome analysis.

Conclusion

In this study, we proposed a new liver/pancreas interaction model in biochips to investigate the cross talks between both organs. The characteristic functions of hepatocytes/islets coculture model were evaluated comparing with those of islets or hepatocytes (with and without insulin) monocultures. The hepatocyte monoculture without insulin led to a modulation of the glucose homeostasis targets and of the hepatic differentiation markers. Conversely, the coculture with pancreatic cells producing insulin contributed to recover the hepatic function illustrating the benefice of the two organs model. For pancreatic functions, the presence of the hepatocytes in coculture model contributed to modify the islet response via the increase of the insulin production and the modification of mRNA levels of the gene involved in the insulin/glucagon homeostasis. The pancreas-liver organ on chip model presented here was able to recapitulate several physiological responses, and demonstrated the potential of our approach to reproduce and investigate complex in vivo patterns using alternative in vitro methods.

Acknowledgments

The project was funded by the French National Research Agency (ANR-16-RHUS-0005). Amal Essaouiba Ph.D grant was supported by the French ministry of higher research and education. We thank Yannick Tauran and Benedikt Scheidecke for their help in the technique's education used in this project.


Nathan Jeger-Madiot
email: nathan.jeger-madiot@espci.fr

Niclas Setterblad

Lousineh Arakelian

Valérie Vanneaux

Mauricio Hoyos
email: mauricio.hoyos@espci.fr

Jérôme Larghero

Jean-Luc Aider
email: jean-luc.aider@espci.fr

Fabrication of 2D monolayers and 3D spheroids of cells in acoustic levitation

Introduction

Today, three-dimensional (3D) cell cultures tend to replace 2D conventional method because of their more relevant tissue-mimicking characteristics. Indeed, the 3D cell architecture (spheroïd, organoïd, etc) and the microenvironment is closer to In Vivo physiological behaviour [START_REF] Fang | Three-Dimensional Cell Cultures in Drug Discovery and Development[END_REF][START_REF] Van Duinen | Microfluidic 3D cell culture: from tools to tissue models[END_REF]. The main difficulties remain in creating a scaffold compatible with the targeted cells and tissues. Bioprinting is one of the great objectives for tissue engineering. For instance, stereolithography is a 3D printing technology where the freestanding object is built layer by layer with a photosensitive polymer resin through the projection of a UV image in the top plane. In recent promising work, stereolithography has been applied to create 3D hydrogel structures to guide cells like hepatocytes [START_REF] Neiman | Photopatterning of hydrogel scaffolds coupled to filter materials using stereolithography for perfused 3D culture of hepatocytes[END_REF]. Nevertheless, ideally, scaffold-free methods are needed. We propose a new method combining microfluidic channels and the acoustic radiation force (ARF) to structure and to control contactless the shape of stem-cells aggregates.

2D stratification and shaping

In a standing wave cavity, the cells can be trapped in a central cluster. After a short transient period and using the proper parameters, the cluster becomes a circular monolayer levitating in the surrounding fluid (Fig. 1a). The control of the medium flow and of the ARF allows various manipulations. For example, it is possible to structure the circular monolayer with different 2D stratifications of particles or cells into the acoustic nodal plane. The result is a cluster with type 1 particles or cells at its centre, surrounded by a ring of type 2 particles or cells (Fig. 1b). Other layers can be added, leading to a concentric multi-annular monolayer.

Multilayer aggregation: toward 3D construction

The 2D approach can be extended to 3D. Indeed, the height of the geometry of the cavity or the acoustic frequency can be modified in order to increase the number of pressure nodes and then the corresponding number of cell aggregates. It leads to a superposition of monolayers distributed along the height of the cavity (Fig. 2a).

Changing dynamically the acoustic frequency, activating or turning off the ultrasounds, using more complex cavities allows more refined manipulations of the clusters. For instance, it becomes possible to move an aggregate from one node to another or let the aggregates sediment at the bottom of the cavity to form a multilayer aggregate over the substrate.

Long term experiment with stem cells

To confirm these concepts on cells manipulation and cells culture in acoustic levitation, long term experiments with Mesenchymal stem Cells (MSCs) have been carried out. MSCs have been maintained in acoustic levitation during 24h. Without contact with a classical petri dish and in acoustic levitation, MSC behavior tends to shape spheroids. Then, we compare the viability and the behavior of the cells cluster with a classical cells culture. The MSCs have been found alive and functional and could grow and divide in a normal way.

Conclusion

With our set of microfluidic chips, we show different ways to manipulate, structure and grow stem cells over a long period of time, from a few hours to a few days. One of the advantages of such systems are the material biocompatibility, the ease of construction and the versatility. As this approach is intrinsically "scaffold-free", it is important to find ways to organize spatially the cells. Our experiments open the path to such manipulations.

Figure 2 :

 2 Figure 2: Example of multi-nodes aggregation. (a) Cavity with 7 acoustic nodes of 10 µm red polystyrene particles. (b) Cavity with 13 acoustic nodes of hollow glass microspheres

Figure 1 :

 1 Figure 1: Examples of different patterns obtained with ultrasound and flow control. (a) Circular monolayer of 10 µm polystyrene particles. (b) 2D stratification of 10µm red and green fluorescent particles. (c) Ring monolayer of endothelial cells