Byeongho Park

Heehong Yang

Tai Hwan Ha

Hyun Seo Park

Seung Ja Oh

Yong- Sang Ryu

Youngho Cho

Hyo-Suk Kim

Juyeong Oh

Dong Kyu Lee

Chulki Kim

Taikjin Lee

Minah Seo

Jaebin Choi

Young Min Jhon

Deok Ha Woo

Seok Lee

Seok Hwan Kim

Hyuk-Jae Lee

Seong Chan

Dr Hyun Seok Song
email: hssong7@kbsi.re.kr

Tai Hyun Park
email: thpark@snu.ac.kr

Dr Jae Hun Kim
email: jaekim@kist.re.kr

Prof Hyun Tai

Park

Artificial Rod and Cone Photoreceptors with Human-like Spectral Sensitivities

Keywords: color sensitivity, graphene, human-like artificial material, photoreceptor, spectral characteristic

Photo-sensitive material, which needs biologically engineered elements and delicate techniques with high efficiency, stability, and biocompatibility, has not yet been welldeveloped to replace damaged visual-systems to detect light and transfer signal into a neuron in our body. In current study, artificial nanovesicle based photo-sensitive materials were found to possess the characteristics of human-like photoreceptors for the first time. They exhibited considerably effective spectral characteristics according to each pigment. Four photoreceptors originated from human-eye with color-distinguishability were produced in HEK-293 cell and partially purified in the form of nanovesicle. Under various wavelengths of visible lights, electrochemical measurements with graphene working electrode playing an important role in lipid bilayer deposition and oxygen reduction process were performed to analyze the physiological behavior and kinetics of photoreceptors. Four nanovesicles with different photoreceptors rhodopsin (Rho), short-, medium-, and long-wave sensitive opsin 1 (1SW, 1MW, 1LW) showed remarkable color dependent characteristics, consistent with those of natural human retina. With four different light-emitting-diodes for functional verification, photoreceptors embedded in nanovesicles remarkably represented particular color sensitivity.

This work demonstrates the potential to be used as photoreceptor-based artificial retina with human-like spectral feature for successful recovery from blindness, and to be applied to lightactivated platforms in biological optoelectronic industries. Spectral characteristics of photoreceptors, the rod, and cone cells in retina that are sensitive to light have been considered to be necessary to determine the absorption of photo-pigments.

They are important for investigating optical and electrical interactions between neighboring receptors. These characteristics are also essential to understand spectral input to neural signal for color perception and the development of artificial retina. When retina in eyes interacts with photons, rhodopsin and photopsin in photoreceptors contribute to photoreceptor's output signal as photopigment through seven transmembrane G protein-coupled receptor (GPCR) and vitamin A as derivatives of retinal [1] . Each photopigment has specific color response according to its structure. Rhodopsin is specifically contrast sensitive for blue-green color while three different photopsins can highly absorb red, green, and blue colors, respectively.

Using calculation under density functional theory, photonic response [2] and wavelength dependent sensitivity [3] have been studied with various retinal isomers and their analogs.

Absorbance-based optical experiments have been performed to explore the spectral features of the photoreceptor. These studies have provided well-known reference results [4] .

Bacteriorhodopsin based photo-activity has been investigated using scanning probe technique, spin-dependent electrochemical cyclic voltammetry, and chronoamperometric measurements [5] . Such measurements have been performed using biological specimens and tissues of living organisms. These previous methods have been focused on denaturation for cell membrane, competition of ion behavior in limited outer cellular space, electrical coupling among different receptors, and cell return current caused by high resistance of basement membrane [6] .

The objective of this study was to demonstrate empirical results for the color responses of artificially grown human photoreceptors with an opto-electrochemical experiment using four types of nanovesicle-based photoreceptors including rhodopsin and photopsins.

Photoreceptors were partially purified using cell-derived nanovesicle method for signal generation [7] . Biochemical conversion of photoreceptors was then electrochemically analyzed.

Voltammetry techniques with three electrodes were utilized for the electrochemical analysis of photochemical retinal transformation in photoreceptors intercalated in a vesicle-based lipid bilayer deposited on a graphene probe as a working electrode. Graphene-and carbon-based electrodes have been employed as biosensors due to their high conductivity and binding affinity for biomaterials. Since nanovesicles stimulated by photonic response on a graphene electrode could generate reduction peak-potential shift (RPS) in linear sweep voltammetry (LSV), color-distinguishability of artificially grown photoreceptors on vesicles was explored in this study by simple droplet voltammetry measurement. Spectral characteristics of nanovesicle based photoreceptors were measured using electrochemical reaction (Figure 1). With monochromatic incident lights ranging from 420 nm to 600 nm in an interval of 10 nm, LSV results were obtained from screen printed sensor using graphene working electrode. To monitor ion channel behavior in the lipid bilayer deposited onto graphene sheets, repeated light exposure and LSV measurement were applied to the screen printed sensor with samples.

HEK-293 cells were transfected with mammalian expression vector containing human photoreceptor genes [rhodopsin (Rho), short wave sensitive opsin 1 (1SW), medium wave sensitive opsin 1 (1MW), and long wave sensitive opsin 1 (1LW) related to rod cell, blue cone, green cone, and red cone, respectively] (Figure S1a). Human photoreceptors were successfully expressed in HEK-293 cells (Figure S1b). They were mainly localized in cell membranes (Figure S1c). For visual signal processing activity, photoreceptors were bound with 11-cis-retinal using retinal-containing DMEM media. To determine the activity of photoreceptor in HEK-293 cells after 11-cis-retinal binding, calcium ion-selective Fluo-4 AM dye was used in calcium assay (Figure S2). Fluorescence emission was only detected for 11cis-retinal-bound photoreceptors. Binding between human photoreceptor and 11-cis-retinal plays a key role in photonic response as described in previous works [8] . To partially purify human photoreceptors, cells were transfected with human photoreceptor genes to produce photoreceptor nanovesicles containing receptors on the surface. To prepare photoreceptornanovesicles, optimized method described in previous studies [7a, 9] was used. Results of western blot analysis of photoreceptor-nanovesicles achieved in HEK-293 cells are shown in Screen printed electrode coated with reduced graphene oxide (RGO) as a working electrode was used to probe the opto-electrochemical response of photoreceptor-samples. As shown in Figure 3a, the working electrode had a sharp sheet structure and clean surface of typical graphene. A mushy and soft layer was observed from vesicle-deposited surface on RGObased working electrode (Figure 3b). These vesicles were added onto the RGO surface, building a single lipid bilayer. With wide view of SEM images and XPS spectral results shown in Figure S3, the deposition of the lipid bilayer onto RGO was verified specifically.

For phosphate buffered saline (PBS, buffer solution)-dropped graphene electrode, small salt particles were aggregated on the graphene surface (Figure S3b). Vesicles were settled down on the RGO surface or formed a single lipid bilayer (Figure S3c). In XPS results (Figure S3d), bare working electrode had a few oxygen bindings as RGO while nanovesicle-deposited graphene electrode provided broad and complex XPS peaks with various binding features due to bonding among lipids, proteins, and graphene. Figure 3c describes LSV results and vesicle-deposition process. As the vesicles settle down by sweeping the voltage, they form a lipid bilayer covering the graphene surface, which results in decreasing the absolute amount of current in voltammetry. During LSV measurement, charges were induced by electric potential on RGO working electrode. With the assistance of charges and oxygen-based functional groups on graphene surface, vesicles could be attracted rapidly [9b, 10] . After vesicledeposition on working electrode, vesicles were ruptured and transformed into a single lipid bilayer and the surface was covered with its thin film [11] . RPS from positive to negative in linear voltammetry with reduction peak was focused in this study since our samples showed irreversible process without oxidation peak during positive potential sweep (Figure S4).

From LSV results shown in Figure 4a, RPS approached toward smaller absolute peakpotential value in our platform using consecutive measurements, including 10 times of light exposures. RPS of most samples started from -0.5 V and finished at near -0.45 V. However, current values of reduction peak were stable at near 15.5 μA during the entire measurement.

Rho and 1LW photoreceptors represented intriguing RPS under white incident light according to exposure time, of which curves were saturated gently (Figure 4b). However, linear RPS of photoreceptor-nanovesicles was observed without any incident light (dark state, Figure 4c).

Linear RPS was also observed from PBS buffer solution, and nanovesicles without photoreceptors in the presence or absence of incident light (Figure S5). Taken together, these results demonstrated that RPS of each element (either PBS or photoreceptors) was successfully derived by using deconvolution (Figure S6). Overall results from Figure 4b,c, Figure S5, and Figure S6 clearly showed that these artificially produced and partially purified photoreceptors successfully interacted with incident light and devoted to saturation in RPS. A trend of linear shift in reduction peak was also observed with ferricyanide ([Fe(CN)6] 3-), a standard material. The shift was found to be originated from electrode surface modification (Figure S7).

The reduction peak in LSV was due to oxygen reduction with four electron processes expressed in the following equation:

2 2 2 4 4 O H O e OH      (1)
Graphene-based electrode was investigated for oxygen reduction reaction as a metal-free electro catalyst that provided several reduction potentials with various pathways for the production of peroxide [12] . Surface condition of graphene-based electrode was changed after every voltammetry measurement, leading to RPS. PBS buffer contributed considerably to the surface alteration of graphene-based electrode after O2 reduction since oxygen functional groups on the edge of graphene sheet participated in all LSV results, including steady RPS.

Curves of photoreceptor-nanovesicles with incident light were surprisingly saturated as shown in Figure 4b. Activated channels in single lipid bilayer might have played an important role in the variation of RPS since the surface property of RGO electrode to interact with oxygen in solution could be controlled by incident light (Figure 4d and4e). Many studies have reported that there are various ion channels in mammalian membrane for ion exchanges in and out of cells. They act as switches with receptor proteins such as taste receptors [13] , olfactory receptors [14] , and dopamine receptors [9c] . Stimulation of photoreceptors by incident light can arouse the flux of specific ions through the ion channel into nanovesicles as described in Figure 4d. The number of activated channels on the RGO electrode was gradually increased with channel activation by photoreceptors under incident light, resulting in a saturating trend of RPS with gradient variation. Additionally, high bonding affinity between reduced all-trans-retinals and graphene surface might have contributed to RPS, similar to results of previous studies showing that glassy carbon electrode could attribute to RPS due to chemical reaction of all-trans-retinal with hydrogen in water during voltammetry measurement (Figure 4e) [15] . Reduced all-trans retinals are easily attached onto graphene sheets with thin layers, leading to the surface modification of the electrode. Two dominant reaction activities mentioned above (channel activation and morphological change on the surface) might have generated RPS variation with the surface property change of graphene electrode. Such method can be used to explore photoreceptors' spectral characteristics specifically with facile experimental setup and to obtain amplified signal from biomaterials.

Responses of four different photoreceptors to various incident lights with various wavelengths are shown in Figure 5. Representative curves provided differences in saturation point, slope change, and degree of RPS according to the wavelength of incident light (Figure 5a-d). This can be used to understand the spectral characteristics of samples indirectly under three-dimensional diagrams. To consider our results thoroughly, these curves were subjected to saturation behavior analysis (Figure S8) [16] . The relative sensitivity was calculated against the time when it reached at the half of RPS saturation point with inverse proportion for the reaction rate. Each curve in Figure S9 was fitted and computed to obtain a value of relative sensitivity from the whole data represented in Figure 5a-d. Rho sample showed a large RPS peak near 485 nm. Likewise, 1SW, 1MW, and 1LW provided the greatest RPS peaks at 440, 530, 556 nm, respectively. Similar results have been reported in previous studies on photoreceptors [17] . While most curves were increased gradually as samples were exposed to incident light, the saturation ratio was different according to varying wavelength. Artificially grown human photoreceptors had different sensitivities to corresponding wavelengths, comparable to previous results obtained from live animal models [6b-e] . As shown in Figure 5e, four types of artificially grown photoreceptors exhibited outstanding relative sensitivities, consistent with the results of absorbance spectrum obtained from human eyes [17] . By using Gaussian fitting method [START_REF] Carroll | Proc. Natl. Acad. Sci[END_REF] , fitting graph for specific sensitivity under different wavelengths of incident light was achieved as shown in Figure 5e.

To further investigate artificial photoreceptors, color distinguishability of our samples was evaluated using the following four types of light emitting diodes (LEDs) at the colors of blue (465 nm), green (520 nm), yellow (575 nm), and red (640 nm). As shown in Figure 5f-i, each cone receptor had different sensitivity according to each LED's emission wavelength. With blue and red LEDs, specific samples such as 1SW and 1LW reacted intensely. Green and yellow LEDs stimulated two types of samples (1MW and 1LW) according to their spectral characteristics. Our artificial human photoreceptors produced from the in vitro system had highly different responses to various wavelengths, leading to human-like light sensing. These functional verification suggest that the artificial human photoreceptors can be practically used in challenging fields such as artificial retina camera and biological imaging system.

In conclusion, versatile photoreceptor-nanovesicles were developed in this study to explore spectral characteristics of artificially grown human photoreceptors with relatively simple method to enable mass-scale production. Color dependent physiological responses of these artificial biomaterials holding photoreceptors were demonstrated here for the first time. For electrochemical investigation, graphene working electrode was used to settle down nanovesicles rapidly, leading to a stable lipid bilayer on the surface. Oxygen reduction of the working electrode was changed by ion channel activation on membrane layer with photoreceptors and attachment of reduced all-trans-retinal onto graphene surface. RPS results

were influenced by the wavelength of incident light. The saturation curve of RPS was subjected to mathematical analysis to understand the complex reaction of our bio-sample.

Four types of photoreceptor-nanovesicles (Rho, 1SW, 1MW, and 1LW) showed fine sensitivity according to the wavelength of incident light. Even in the experiment with LEDs, our artificially grown photoreceptors provided color distinguishability similar to human photoreceptor cells. The approach presented herein can be applied to study physiological, biophysical, and biochemical processes in photoreceptors. It has considerable potential to be used to develop artificial retina technology.

Experimental Section

Cloning and expression of photoreceptors in HEK-293 cell. Genes of photoreceptors in pCMV6-entry vector were purchased from OriGene (USA). These photoreceptor genes were cloned into pcDNA3 mammalian expression vectors (Invitrogen, USA). Their sequences were confirmed by DNA sequencing (GenoTech, Korea). pcDNA3/Rho-EGFP and pcDNA3/1LW-DsRed were also constructed and confirmed by DNA sequencing. Human embryonic kidney (HEK)-293 cells were cultured with Dulbecco's Modified Eagles Medium (DMEM) (HyClone, USA) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco, USA), and 10 % Fetal Bovine Serum (FBS) (Gibco, USA) at 37 °C and 5 % CO2. Transfection was performed using Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer's protocol. Transfected cells were harvested with phosphate-buffed saline (PBS, pH 7.4) and lysed by sonication using Sonics Vibracell (USA) with 2 s on/off for 2 min.

Western blot analysis was performed using anti-FLAG rabbit antibody (Cell Signaling Technology, USA) as primary antibody, HRP-conjugated anti-rabbit antibody (Millipore, USA) as secondary antibody, and Luminata Forte western HRP substrate (Millipore, USA).

Formation of nanovesicles expressing photoreceptors on surface. Cells were harvested and resuspended in DMEM containing 10 μM of 11-cis-retinal and 10 μg/mL of cytochalasin B (Sigma, USA) followed by agitation at 300 rpm for 30 min. Nanovesicles were separated from cells by centrifugation at 1000×g for 10 min. The supernatant was centrifuged at 15000×g for 30 min to collect nanovesicles. After the supernatant was removed, nanovesicles were produced and stored at -80 °C. They were thawed before use. All procedures were performed in the dark. The shape of nanovesicles was observed by field emission scanning electron microscopy (SEM). These nanovesicles were applied onto poly-D-lysine-coated slide glass, washed with PBS, and fixed with 4% paraformaldehyde at room temperature for 20 min.

Fixed nanovesicles were incubated with 1 % OsO4 solution for 10 min, dehydrated with increasing concentrations of ethanol, and lyophilized with a freeze-dryer. Lyophilized nanovesicles were coated with Pt (10 nm) by sputter and visualized by SEM. Additionally, nanovesicles on graphene electrode after smooth air-dry and Pt (10 nm) coating were used for SEM images to monitor the intrinsic surface of the sample. The surface of graphene working electrode was analyzed using X-ray photoemission spectroscopy (XPS) (VG Scientific Instruments, ESCALAB 220i-XL).

Opto-electrochemical voltammetry measurements with three electrodes. Linear sweep voltammetry (LSV) was investigated by screen printed electrodes (SPE) (110GPH, Dropsens).

Graphene was used as working electrode. Carbon was used as counter electrode while silver was used as reference electrode. A small drop of solution samples was employed onto the SPE surface. Using voltammetry analytical equipment (SP1, ZIVE LAB), induced potential was swept from 1 to -1 V. The current was detected under incident light with various wavelengths ranging from 420 to 640 nm obtained from a supercontinuum white light source (SuperK CompacK, NKT Photonics). The specific wavelengths with different bandwidths were generated using Flexible Wavelength Selector (FWS-B-M, Spectrolight Inc.). As a stabilization process, LSV measurement for the graphene sheet was repeated 5 times to minimize residue on electrodes and build-up of lipid bilayer from nanovesicles on graphene working electrodes. For spectral responsivity measurement of photoreceptors, LSV measurement was performed after incident light was applied to the sample for a duration of 10 seconds. The process mentioned above was repeated 10 times to observe the overall electrochemical reaction of each photoreceptor. Relative sensitivity spectrum of all photoreceptor samples calculated using saturation behavior analysis. As functional verification, sensitivity comparison among samples using LED light sources with different emission: (f) blue, (g) green, (h) yellow, and (i) red.

Table of Contents

Artificially synthesized biological nanomaterials with four types of photoreceptors in nanovesicle show outstanding human-like spectral characteristics for visible light on a graphene working electrode for electrochemical measurement.

Keywords: color sensitivity, graphene, human-like artificial material, photoreceptor, spectral characteristic Byeongho Park 1,2, † , Heehong Yang 3, † , Tai Hwan Ha 4 , Hyun Seo Park 1 , Seung Ja Oh 1 , Yong-Sang Ryu 1 , Youngho Cho 1,5 , Hyo-Suk Kim 1,6 , Juyeong Oh For mammalian expression of receptors, these photoreceptor genes were cloned into pcDNA3 mammalian expression vector and verified with agarose gel electrophoresis (Fig. S1a). The band of target DNA was confirmed by restriction enzyme digestion (EcoRI/XhoI) and the size of target gene was observed based on its expected size. Results showed that these pcDNA3 expression vectors containing genes of photoreceptors were successfully prepared.

These photoreceptors were also successfully expressed in HEK-293 cells based on Western blot analysis (Fig. S1b). Bands of around 39 kDa were consistent with the expected sizes of these photoreceptors. Expressions of these photoreceptors on membrane surface were also monitored through CLSM images. Fig. S1c shows that fluorescences of the Rho-GFP and 1LW-DsRed are detected on the cell membrane. However, they were not observed control cells. These images demonstrated that cells transfected with Rho and 1LW expressed these receptors on the membrane surface. These results indicated that human photoreceptors were successfully expressed in HEK-293 cell and these receptors were localized on the cell-surface of membrane. For mathematical analysis, usual symbols (x, y, dx, dy, dy/dx, d 2 y/dx 2 ) were used. In Fig. S8, each continuous incremental increase, dx, in x was less dominant at increasing dy. After saturation with very large value of x, the, maximum y was generated in the graph. As x was increased, fractional changes dx/x and dy/y were decreased. The positive slope (dy/dx) was steadily decreased. The second derivative (d 2 y/dx 2 ) was also steadily decreased. It remained negative: (d 2 y/dx 2 ) = -|d 2 y/dx 2 |.

From the general differential equation depending on specific integer of A and B, various natural phenomena could be described in the following:

Figure 2a .

 2a Figure 2a. Bands of four different photoreceptors were clearly observed with expected sizes.

FiguresFigure 1 .

 1 Figures

  Figure 1. Schematic and opto-electrochemical experimental setup of three-electrode system

Figure 2 .

 2 Figure 2. Preparation and characterization of human photoreceptors with 11-cis retinal. (a)

Figure 3 .

 3 Figure 3. SEM images of graphene electrodes (a) before and (b) after vesicle deposit. (c)

Figure 4 .

 4 Figure 4. (a) Overall linear sweep potential result on graphene based and screen printed

Figure 5 .

 5 Figure 5. RPS results of (a) 1SW, (b) Rho, (c) 1MW, and (d) 1LW as a function of measuring

  Figure S1. Cloning and expression of human photoreceptors. (a) Agarose gel electrophoresis

Figure S2 .

 S2 Figure S2. Calcium flux assays of HEK-293 cells expressing photoreceptors using Fluo-4

Figure S3 .Figure S4 .

 S3S4 Figure S3. SEM images. (a) Bare working electrode surface, (b) PBS dropped and dried

Figure S5 .Figure S6 .Figure S7 .

 S5S6S7 Figure S5. RPS of (a) PBS and (b) vesicle without photoreceptor in the presence or absence

Figure S8 .

 S8 Figure S8. Voltammetry results according to exposing time by saturation behavior analysis

Figure S9 .

 S9 Figure S9. Curves of RPS in (a) Rho and (b) 1LW under different wavelength of incident

Human-like Spectral Sensitivities of Artificial Rod and Cone Photoreceptors Supplementary Figures Figure S1.

  1,2 , Dong Kyu Lee 1 , Chulki Kim 1 , Taikjin Lee 1 , Minah Seo 1 , Jaebin Choi 1 , Young Min Jhon 1 , Deok Ha Woo 1 , Seok Lee 1 , Seok Hwan Kim 7 , Hyuk-Jae Lee 5 , Seong Chan Jun 2 , Hyun Seok Song 8,9,* , Tai Hyun Park 3,* and Jae HunKim 1,*

Acknowledgements

This research was supported by the National Research Foundation funded by the Korean government (MSIT) (NRF-2018R1A2B3004498). This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement No 682286). This study was also supported by KIST Institutional Program (2E27270), Samsung Research Funding Center of Samsung Electronics under Project Number SRFC-IT1701-02, and Korea Basic Science Institute (D37410).

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Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Supporting Information

Human-like Spectral Sensitivities of Artificial Rod and Cone Photoreceptors

Byeongho Park 1,2, † , Heehong Yang 3, † , Tai Hwan Ha 4 , Hyun Seo Park 1 , Seung Ja Oh 1 , Yong-Sang Ryu 1 , Youngho Cho 1,5 , Hyo-Suk Kim 1,6 , Juyeong Oh 1,2 , Dong Kyu Lee 1 , Chulki Kim 1 , Taikjin Lee 1 , Minah Seo 1 , Jaebin Choi 1 , Young Min Jhon 1 , Deok Ha Woo 1 , Seok Lee 1 , Seok Hwan Kim 7 , Hyuk-Jae Lee 5 , Seong Chan Jun 2 , Hyun Seok Song 8,9,* , Tai Hyun Park 3,* and Jae Hun Kim 1,*

Supplementary Methods

Confocal laser scanning microscopy (CLSM).

Plasmids pcDNA3/Rho-EGFP and pcDNA3/1LW-DsRed were transfected into HEK-293 cells, respectively. Cells were fixed with 4% paraformaldehyde for 10 min at 37 °C and incubated with PBS (0.25% Trition X-100) for 10 min. Cells were also treated with DAPI (Thermo Fisher Scientific, USA) and washed with PBS. Fluorescences were detected using confocal microscopy (Olympus, USA).

Calcium flux assay of photoreceptors in HEK-293 cell. Transfected cells were incubated

with 5 μM of fluo-4 AM (Molecular Probes, USA), 2.5 mM probenecid, and 10 μM of 11-cisretinal (Santa Cruz Biotechnology, USA) in Ringer's solution (140 mM NaCl, 1 mM MgCl2, 1.8 mM CaCl2, 5 mM KCl, 5 mM glucose, 10 mM HEPES (pH 7.4)) at 37 °C for 1 hr. After three times of washing with Ringer's solution and further incubation for 30 min to remove nonspecific binding and allow complete de-esterification of AM esters, cells were imaged with a fluorescence microscope. All experiments were performed in the dark.


Patrice Berthod
email: patrice.berthod@centraliens-lille.org

Zohra Himeur
email: zohra.himeur5@etu.univ-lorraine.fr

Influence of Co content on the high-temperature oxidation performance of (Ni,Co)-25Cr-0.4C-6Ta cast alloys

Keywords: Nickel-based alloys, Cobalt-based alloys, Chromium carbides, Tantalum carbides, High temperature oxidation, Thermodynamic calculations

Tantalum carbides (TaC) are classical strengthening particles, like chromium carbides, in equiaxed cast Cr-rich cobalt-based alloys. When they are of a eutectic nature and in quantity high enough, they may be of great interest for applications at elevated temperature, due to their favorable script-like morphology and their high stability at high temperature. Unfortunately they cannot be so easily obtained in Cr-rich nickel-based alloys of similar composition. This can be resolved by adding cobalt in substitution to nickel but such chemical modification may change the high temperature oxidation behavior in the wrong direction. In this work several alloys, all containing 25%Cr, 0.4%C and 6% Ta (wt.%), were elaborated and studied at a high temperature: 1127°C. It appears that the 27wt.% Co content is high enough to allow obtaining primary carbides which are essentially TaC. The alloys rich enough in Co containing only tantalum carbides are clearly disadvantaged in term of oxidation resistance at high temperature by the high Co content and by the absence of interdendritic chromium carbides known to act as useful Cr reservoirs. Among the six studied alloys these are the three ones for which Co/Ni >1 that demonstrated starts of catastrophic oxidation before 24 hours of exposure to air.

INTRODUCTION

Primary carbides belong to the earliest metallurgical means employed for strengthening the cast metallic alloys used at high temperature under mechanical stresses [START_REF] Sims | The Superalloys[END_REF][START_REF] Bradley | Superalloys: A technical guide[END_REF]. Beside chromium carbides, tantalum carbides can play a special role, for example in the MarM-509 alloy [START_REF] Morrow | [END_REF][4][5]. Even if many MC carbides may be obtained in conventionally cast Ni-based [6] or Co-based [7] alloys, TaC carbides keep an interest high enough to be still employed in numerous superalloys [START_REF] Donachie | Superalloys: A Technical Guide[END_REF]. Primary carbides are principally used in equi-axed polycrystalline alloys and these ones often contain also high contents in chromium to resist oxidative chemical aggression at high temperature [START_REF] Young | High Temperature Oxidation and Corrosion of Metals[END_REF]. Chromium is a potential competitor for tantalum in the formation of carbides. Tantalum is not necessarily a carbide-former stronger than chromium and, in some cases, chromium carbides may precipitate preferentially to TaC. For example, during the solidification of chromium-rich cobalt-based [START_REF] Berthod | [END_REF] and iron-based [11] alloys, TaC forms at the expense of Cr 7 C 3 or Cr 23 C 6 . It is the contrary for Cr-rich nickel-based alloys [12]. Cobalt and nickel are often associated to one another in commercial alloys. For example Co can be present in nickel alloys for solid solution strengthening purpose and Ni can be added to cobalt alloys to extend the temperature range of thermodynamic stability of the high temperature austenitic face centered cubic crystalline structure of cobalt. Other possible types of association of nickel and cobalt aim to the better high temperature oxidation behavior brought by nickel to a cobalt matrix by expediting chromium diffusion towards the oxidation front, or to the stabilization of the TaC carbides induced by cobalt addition to a nickel matrix [13].

The aim of the present work is to explore how the behavior in oxidation at high temperature of carbidescontaining nickel-chromium cast alloys evolves when more and more cobalt is added to stabilize tantalum carbides more and more efficiently.

EXPERIMENTAL

To investigate how the behavior in oxidation at high temperature of cast equi-axed nickel-based alloys, rich in chromium, strengthened by carbides and containing a high tantalum content, may evolve when nickel is increasingly substituted by cobalt, six alloys of the Ni-Co-25Cr-0.4C-6Ta were elaborated. Their targeted chemical compositions are displayed in Table 1. These alloys were synthesized by classical foundry, by melting together parts of pure elements (Ni, Co, Cr and Ta from Alfa Aesar, and graphite, purity higher than 99.9% in weight). This was achieved in the water cooled copper crucible of a CELES high frequency induction furnace (50kW, about 100Hz) in which the molten alloys also solidified. All the alloys were kept in the obtained as-cast state, without any postsolidification heat treatment, in order to stay in the usual conditions of use of many of the commercial alloys (e.g. Mar-M322 or 509 of which the present alloys can be considered the simplified versions [START_REF] Sims | The Superalloys[END_REF][START_REF] Bradley | Superalloys: A technical guide[END_REF][START_REF] Donachie | Superalloys: A Technical Guide[END_REF].

The six obtained ingots, all weighing about 40 grams, were each cut in four parts. One of them was kept for preparing the sample devoted to the as-cast microstructures control and a second one was specially prepared for the oxidation tests at high temperature. The chosen temperature and duration were 1127°C (1400K) and 24 hours (a day). The samples were placed in a muffle furnace (Nabertherm) and exposed to laboratory air at the targeted temperature.

The obtained oxidized samples were first covered by an extra-thin gold coating. They were then immersed in a 50°C-heated Watt's bath. They staid located between two nickel coupons which played the role of anodes. The samples were polarized at a high potential allowing a constant electrical current corresponding to the 1.6A × dm -2 current density known to allow a good conditions of coating. After 2 hours of electrolytic coating, the samples were surrounded by metallic nickel shell thick enough to thereafter protect the external scale during the cutting necessary to the preparation of cross-sections. Cutting of each Ni-coated oxidized sample in two halves was carried out using a metallographic Buehler Delta Abrasimet 5000 metallographic saw. The obtained halves were embedded in a cold resin mixture (ESCIL). These cross-sectional samples prepared from the oxidized alloys and the samples for as-cast microstructure observation were polished using SiC papers from 240-grit to 1200-grit. They were then washed and ultrasonically cleaned to remove all possible attached SiC particles. Final polishing, carried out using textile supporting very fine (1µm) hard particles, led to mirror-like states.

Metallographic examinations were done using a scanning electron microscope (SEM, JEOL, JSM-6010LA model) equipped with an Energy Dispersion Spectrometer (EDS). The most used mode for imaging was Back Scattered Electrons one (BSE). The applied voltage was 20kV. The EDS full frame analyses were carried out at ×250 on three locations (random positioning) to control the general chemical compositions of the alloys. Spot analyses were performed to identify the different phases present in the alloys, and the different oxides seen in the external scales and the internal oxides appeared in the subsurface. Some thermodynamic calculations were also carried out. For that one used the Thermo-Calc software (version N ) [START_REF]Foundation for Computational Thermodynamics[END_REF] and a database. This database was the SSOL database [START_REF]SGTE Solutions Database[END_REF] which was enriched with additional binary and ternary systems involving Ta, element which was missing in the original database. This enriched version which was then used contained the descriptions of the Ta-Ni, Ta-Co, Ta-Cr and Ta-C binaries, and of the Co-Ta-C, Ni-Ta-C, Cr-Ta-C ternaries.

RESULTS

Chemical compositions and microstructures of the alloys in their as-cast conditions

As this can be verified by the EDS analysis results presented in Table 2, the obtained chemical compositions well meet the targeted ones. However one must note that the Cr contents are a little higher than what it was wished. This is also true for the Ta contents which are greater than expected. This phenomenon is usual when full frame EDS is used for microstructures in which a part of tantalum is involved in coarse TaC carbides. Nevertheless one can consider that the Ta contents in alloys are themselves close to the wished ones. EDS does not operate for carbon since this one is a light element and, in addition, present in very low quantity (wished contents of only 0.4%wt.%). This is by considering the densities of the obtained carbide network which allows estimate that the 0.4wt.%C is well respected in all alloys. Periodically performed spark spectrometry analysis of alloys of this family, elaborated with the same protocol, constantly confirms that the targeted carbon contents are always obtained without any loss.

The rather rapid solidification and solid state cooling induced crystallization and subsequent solid state transformations which did not really respect the phase diagram (Figure 1). Indeed, it was expected that the NiCo0 and NiCo1 alloys will contain no TaC but only chromium carbides. For the NiCo2 and NiCo3 alloys it was expected that they contain both chromium carbides and TaC. To finish the NiCo4 and NiCo5 alloys were expected to contain only TaC. In fact, it appears that the as-cast microstructures of the two Ni-richest alloys (NiCo0 and NiCo1: Figure 2) contain significant parts of tantalum carbides (white particles when observed with a SEM in BSE mode), beside the chromium carbides (dark particles) which are notwithstanding more present than the TaC. Tantalum carbides seem to be more present than chromium carbides in the NiCo2 alloy (Figure 3) although they ought to remain the minor part of the carbide population, the chromium carbides being expected to be the major part. The NiCo3 alloy contains almost only tantalum carbides. In this alloy the chromium carbides are present only as rare small particles (Figure 3). The NiCo4 and NiCo5 alloys (Figure 4) present microstructures which do not contain chromium carbides, and their carbide network is then only constituted of tantalum carbides. Concerning the new phases which ought to appear during cooling below about 1000°C according to the phase diagram (e.g. the Ni 3 Ta ntermetallic compound), they were not observed. These small disagreements with what was predicted by thermodynamic calculations can be easily explained by the rapid cooling during both solidification (e.g. segregation of Ta rejected in the liquid at the solid/liquid interface) and solid state cooling (no time for nucleation and growth of the new phases).

Oxidation products and subsurface modifications: qualitative observations

During the twenty-four hours spent at 1127°C, all alloys suffered from oxidation by the laboratory air. In all cases a continuous oxide scale developed all around the samples. Internal oxidation also happened and an outer zone of alloy subsurface lost the carbides which were initially present there. Impoverishments in chromium and in tantalum occurred in subsurface These surface and subsurface modifications are illustrated by SEM/BSE micrographs taken on the prepared cross-sections: in Figure 5 for the two Ni-richest alloys (NiCo0 and NiCo1), in Figure 6 for the two intermediate alloys (NiCo2 and NiCo3) and in Figure 7 for the two Co-richest alloys (NiCo4 and NiCo5). In all these figures the upper micrograph gives a general view of the oxidized surface and of the deteriorated subsurface. These zones are more detailed by the higher magnification micrograph displayed bellow.

Two types of oxides formed for the three first alloys (NiCo0, NiCo1 and NiCo2) which are richer in nickel than in cobalt. These oxides are first chromia (Cr 2 O 3 ), which represents the greatest part and which is exclusively present in the external scale, and second a complex oxide which involves chromium and tantalum simultaneously (CrTaO 4 ). The greatest part of this second oxide is present in the subsurface, principally in the outer half of the carbide-free zone, close to the alloy/external scale interface. In addition, this complex oxide is also present as a wavy oxide film immersed in the chromia external scale.

The same oxides, chromia and the complex oxide of chromium and are also present in the three last alloys, NiCo3, NiCo4 and NiCo5, which are richer in cobalt than in nickel. As for the former alloys, chromia is present, again as an external scale, but another oxide, of a MO type is also present. This one involves of Ni and Co together when Ni s still present in rather high quantity (case of NiCo3). In the case of the two Co-richest alloys, it involves only Co. This MO oxide is present too as an external oxide separating chromia and air. These (Co,Ni)O and CoO oxides, visible thanks to their tint paler than the chromia's one, are known to be not so protective as Cr 2 O 3 . A third oxide can also be found beside chromia and the MO oxides: the spinel CoCr 2 O 4 . It is mainly present at the chromia/CoO interface.

Oxidation products and subsurface modifications: quantitative results

Even if it was obvious that oxide spallation occurred during the cooling after the end of isothermal oxidation, it was possible to assess the thickness of the external oxide scale. This one was measured in ten distinct locations, representative of the general state of the oxidized surface of each sample. The average and standard deviations are given in the second column of Table 3 (the first results column). One can see that the average oxide thickness globally tends to increase when the cobalt content increases. In fact, one can consider that the external scales developed over the three nickel-richest alloys are of the same thickness. This one is rather low: 5 to 6 µm. The three other alloys indisputably constitute another group, which is characterized: first by a much thicker external oxide scales (average thickness equal to 3 or 5 times the ones obtained for the three first alloys), and second, regard to the much by a worse thickness homogeneity as revealed by the high values of standard deviation.

The depth over which alloys had lost their carbides is obviously also strongly dependent on the cobalt content (Table 3, third column, e.g. second results column). Rather high for the nickel-based NiCo0 alloy, the average value (issued from ten values too) decreases of one third, from 30 to 20µm globally. From the NiCo2 alloy to the NiCo3 one, the depth of carbides disappearance is suddenly doubled (typical 40µm). By enrichment in Co, the depth of carbide-free zone decreases again, to reach almost the carbide-free depth of the NiCo0 alloy. One must also note that the values of standard deviation are higher.

The local changes in the alloy in chemical composition due to oxidation can be illustrated by the concentrations profiles acquired from the external oxide scale / alloy interface. They are presented in Figure 8 for the major elements, and in Figure 9 for the minor elements, only three of the six alloys. Each of them represents one of the three kinds of alloys: the Ni-richest ones, the medium ones, and the Co-richest ones. The profiles of Cr depletion (Figure 8) are often constituted of an outer part characterized by a stationary Cr content followed by a rather high gradient. This outer part corresponds more or less to the carbide-free zone in the case of the two Ni-richest alloys. Deeper there is a second gradient, which starts from the frontier between the carbide-free zone and the inner part of alloy in which carbides are still present. This second gradient is low in the case of the Ni-richest alloys. For the Corichest alloys the second Cr gradient is more marked and it becomes low or inexistent only much deeper, where the alloy not affected by oxidation starts. When observing these Cr-concentration profiles one can see that the chromium content in extreme surface of the sample, i.e. very close to the scale/alloy interface, tends decreasing when the global cobalt content in alloy increases. Up to ten EDS spot analyses were carried out in the alloy in extreme surface, all along the interface with the external scale. The average and standard deviation values were calculated for the obtained Cr contents. These ones are presented in Table 4. The average chromium content in extreme surface obviously decreases when the cobalt content in the alloy increases. This confirms the tendency shown by the concentration profiles. One can also say that there are to typical levels, 16-17wt.%Cr for the three Ni-richest alloys and 12-13wt.%Cr for the three Co-richest ones.

DISCUSSION

The studied alloys belong to the same family as the alloys recently studied by Z. Himeur [13]. As for these later ones, the as-cast microstructures of the present alloys do not respect exactly the microstructures predicted by Thermo-Calc. This is not surprising. Indeed solidification and solid state cooling were fast, consequence of the use of a water-cooled copper crucible. This notably led to the rejection of the heavy tantalum atoms in the liquid close to the solidification front, with consequently the appearance of interdendritic zone rich in segregated Ta. The subsequent time spent at high temperature between solidification and the end of cooling was not long enough to chemically homogenize the matrix. The chemical heterogeneity at the microstructure size scale inherited from this segregation phenomenon can be a first possible reason of the appearance of tantalum carbides in the cobalt-free NiCo0 alloy despite Thermo-Calc did not predict their presence. However, one can consider the bottom halves of the ×250 SEM/BSE micrographs presented in Figures 567, for having a general view of the cross-sections of the oxidized samples. In these zones far from surface, and therefore where the microstructure is too deep for having been affected by oxidation, it appears that a significant part of the TaC appeared during solidification staid stable during the rather long exposure at 1127°C. Furthermore, the 1127°C-stabilized microstructure of the NiCo1 alloy (bottom part of the corresponding ×250 micrograph (Fig. 5, top left) only contains TaC as carbide phase. This was not expected since thermodynamic calculations predicted the presence of chromium carbides only, with eventually very little quantity of TaC. The NiCo2 and NiCo3 alloys, which ought to contain significant quantities of chromium carbides and of tantalum carbides, contained only few chromium carbides and essentially tantalum carbides. After exposure at 1127°C for 24 hours their carbide population was composed of TaC exclusively (Figure 6, bottom micrographs, bottom halves). Concerning the two last alloys, NiCo4 and NiCo5, Thermo-Calc calculations led to the presence of only TaC. This was effectively verified experimentally, in the as-cast state as well as in the part of subsurface not affected by oxidation.. To finish one can note that the surface fractions of TaC in the as-cast microstructures seem qualitatively similar to one another between the four cobalt-richest alloys (free or almost free of chromium carbides) and between all alloys excepted NiCo0 concerning the microstructures stabilized at 1127°C (carbides exclusively TaC).

To summarize, these observations, which are often in disagreement with the thermodynamic calculations, demonstrate that the used database obviously needs to be improved to deliver more correct results in presence of Ta. Consequently one must use only the experimental results, which lead to consider that:

-the difference in high temperature oxidation behavior between the NiCo0 and NiCo1 alloy can be due to the difference in nature of the carbide population as well as to the presence of 14wt.%Co -the differences in oxidation behavior between the other alloys should be exclusively attributed to the Co content in the matrix.

The oxidation runs were carried out at a rather elevated temperature, of the same level as for the solutioning treatment usually applied during one or two hours to the commercial precipitation-hardened superalloys, prior to the precipitation treatment. Therefore the present alloys maybe considered as already chemical homogenized for the major part of the oxidation process. After cross-sectional metallographic preparation one observed and characterized the oxidation products which formed and the microstructure consequences of oxidation in subsurface. It was found that the oxide scales formed on the NiCo0 alloy and the NiCo1 alloy were of the same constitution and of the same average thickness. The carbide-free zone was significantly deeper in the NiCo0 alloy than in the NiCo1 alloy. The latter observation may be interpreted as follows: during the whole oxidation, the chromium carbides existing permanently in the NiCo0 alloy dissolved over an increasing depth from the scale/alloy interface; this phenomenon is due to the local decrease in Cr in the neighbor matrix (Figure 8) subsequently to the Cr diffusion toward the oxidation front. For the same reason, but this time concerning tantalum (Figure 9), the neighbor tantalum carbides dissolved and the Ta atoms well diffused along the interdendritic spaces free of chromium carbides. This effectively may lead to a rather deep carbide-free zone and to the presence of many CrTaO 4 oxides in the outer part of this carbide-free zone. What occurred in the NiCo1 alloy was probably different: the tantalum carbides, more present than in the NiCo0 alloy, had a little grown by profiting from the carbon atoms released by the disappearing chromium carbides, and they were present in subsurface for a longer time. Indeed, the diffusion of the Ta atoms is not as easy as the chromium atoms and this resulted in less deep carbide-free zone forthe same duration at 1127°C.

The NiCo2 alloy globally behaved as the NiCo1 one, with as results sensibly the same average scale thickness and the same carbide-free depth. However one can note a decrease of about 1wt.% of the average Cr content in extreme surface. This possibly means that the volume diffusion of Cr in the matrix may be not so easy as for the two former alloys. This is a possible effect of an important presence of cobalt (27wt.%Co) substituting nickel in the matrix.

By enriching more the alloy with cobalt, drastic changes occurred. With about 41wt.%Co against 27wt.%Ni, the next alloy, NiCo3, is no longer nickel-based but cobalt-based. This change in base is obviously accompanied by a sudden change in behavior. Probable consequence of less easy volume Cr diffusion, this alloy lost its total chromia-forming behavior before reaching the end of the 24 hours stage. Consequently other oxides formed in addition to the Cr 2 O 3 and CrTaO 4 which were the only ones to form in the same conditions for the three Ni-richer alloys. Indeed (Co,Ni)O was observed. This loss of the chromia-forming behavior is due to the decrease in Cr content in extreme surface of about 3wt.%Cr more. Less slowed down by multi-constituted oxide scale less protective than chromia, oxidation was faster. This had as result a migration of Cr and Ta toward the oxidation front, and consequently a more extended carbide-free zone. The NiCo4 and NiCo5 alloys behaved worse than the NiCo3 alloy. In their case, the difficult Cr diffusion induced a more dangerous exposure of the outer parts of alloys to oxidation. This led to a generalized oxidation of Co together with chromium. Greater parts of CoO and of spinel CoCr 2 O 4 oxides were obtained, and an inward penetration of oxidation started, as revealed by the irregular alloy/oxides frontier.

CONCLUSION

So, the increase in cobalt content induced a stabilization of the TaC carbides. Unfortunately, at the same time this hindered more and more Cr migration toward the oxidation front. Both grain boundary / interdendritic diffusion were impacted by the absence of the interdendritic chromium carbides and by the less easy diffusion in volume through the matrix. The two moderately Co-added alloys, NiCo1 and NiCo2, may present interesting mechanical properties at high temperature since they contain a strengthening network of interdendritic tantalum carbides, despite their still rather intrinsically weak Ni-rich matrix (compared to a cobalt-based one), without conceding the good high temperature oxidation behavior of a real chromium-rich Ni-based alloy. The high temperature oxidation resistance of the three other alloys, which contain more cobalt than nickel, must be improved to allow exploiting the reinforcement by TaC. This can be done globally by adding several wt.%Cr points, either in the whole alloy (enriching in Cr its chemical composition) or only in the subsurface (by depositing chromium to diffuse over a limited depth, as allowed by some Chemical Vapor Deposition techniques). Although that the available database was obviously not efficient enough to correctly describe the metallurgical states of these quinary alloys, thermodynamic calculations in the cobalt-rich half part of the isopleth section (Figure 10) would show qualitatively that some microstructural consequences, either globally (Cr-enriched alloy) or locally (Cr-enriched subsurface) ought to be taken into account for their global high temperature properties.

2 : 5 :FIGURE 1 .FIGURE 2 .FIGURE 3 .FIGURE 4 . 3 FIGURE 5 .

 25123435 FIGURE 1. {25Cr-6Ta-0.4C}-isopleth section of the Ni-Co-Cr-C-Ta diagram computed by Thermo-Calc and position of the six studied alloys

FIGURE 6 .

 6 FIGURE 6. Cross-sectional examination of the oxide scales and subsurfaces of the two medium alloys (SEM/BSE; top: ×250; bottom: ×1000).

FIGURE 10 .

 10 FIGURE 10. Effect of the addition of 5 wt.%Cr on the {xCr-0.4C-6Ta, wt.%}-isopleth section of the Ni-Co-Cr-Ta-C diagram (top: 25wt.%Cr, bottom: 30wt.%)

TABLE 1 .

 1 Targeted compositions for the six alloys of the study (in wt.%).

	Alloys	Ni	Co	Cr	Ta	C
	"NiCo0"	Bal.	0	25	6	0.4
	"NiCo1"	Bal.	13.7	25	6	0.4
	"NiCo2"	Bal.	27.4	25	6	0.4
	"NiCo3"	27.4	Bal.	25	6	0.4
	"NiCo4"	13.7	Bal.	25	6	0.4
	"NiCo5"	0	Bal.	25	6	0.4

TABLE 2 .

 2 Obtained chemical compositions (in wt.%); average and standard deviation of three full-frame SEM/EDS measurements carried out at the ×250 magnification)

	Alloys	Ni	Co	Cr	Ta	C
	"NiCo0"	Bal.	/	25.5 ±0.1	6.5 ±0.2	"0.4"*
	"NiCo1"	Bal.	14.3 ±0.2	25.4 ±0.4	5.6 ±0.9	"0.4"*
	"NiCo2"	Bal.	27.2 ±0.2	25.4 ±0.1	6.5 ±0.2	"0.4"*
	"NiCo3"	25.4 ±0.3	Bal.	25.7 ±0.4	6.9 ±0.2	"0.4"*
	"NiCo4"	12.7 ±0.5	Bal.	26.2 ±0.2	6.6 ±0.8	"0.4"*
	"NiCo5"	/	Bal.	25.3 ±0.2	7.2 ±0.5	"0.4"*

*: carbon contents not controlled by EDS but assumed to be well respected according to the practical knowledge for the elaboration protocol for many alloys before, and confirmed by the obtained density of carbides

TABLE 3 .

 3 Average and standard deviations of the thickness of the external oxide scale (first results column) and of the carbide-free depth (second results column) for the six alloys after 24h-oxidation at 1127°C

	Alloys	Oxide scale thickness (µm)	Carbide-free zone depth (µm)
	"NiCo0"	5.9 ±1.6	28.4 ±2.8
	"NiCo1"	6.0 ±2.3	18.1 ±4.8
	"NiCo2"	4.9 ±0.7	18.7 ±1.5
	"NiCo3"	16.4 ±3.9*	41.9 ±7.2
	"NiCo4"	24.4 ±4.8*	36.0 ±7.7
	"NiCo5"	28.6 ±22.6**	29.0 ±6

*,**: high or very high values of standard deviation due to very heterogeneous oxidation behavior all along the surface

TABLE 4 .

 4 Average and standard deviation for the chromium content in alloy close to the external scale/alloy interface (three measurements)

	Alloys	Chromium content in extreme surface (wt.%)
	"NiCo0"	16.8 ±0.6
	"NiCo1"	16.9 ±0.1
	"NiCo2"	16.1 ±0.4
	"NiCo3"	13.0 ±3.3
	"NiCo4"	13.1 ±0.3
	"NiCo5"	12.4 ±0.3

Ta 1 :

 1 LIQ + FCC matrix + Cr 7 C 3