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Tailoring the oxygen reduction activity of hemoglobin through immobilization within microporous organic polymer#Graphene composite Ahmed B. Soliman, Rana R. Haikal, Arwa A. Abugable, Mohamed H. Hassan, Stavros G. Karakalos, Perry J Pellechia, Hamdy H. Hassan, Magdi Habeeb Yacoub, and Mohamed H. Alkordi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06146 • Publication Date (Web): 16 Jun 2017 Downloaded from http://pubs.acs.org on June 18, 2017

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ACS Applied Materials & Interfaces

Tailoring the oxygen reduction activity of hemoglobin through immobilization within microporous organic polymer‒Graphene composite Ahmed B. Soliman,a,b,† Rana R. Haikal,a Arwa A. Abugable,a,c Mohamed H. Hassan,a Stavros G. Karakalos,d Perry J. Pellechia,e Hamdy H. Hassan, *b Magdi H. Yacoub, *f and Mohamed H. Alkordi *a a

Center for Materials Science, Zewail City of Science and Technology, Sheikh Zayed Dist., 12588, Giza, Egypt. Chemistry Department, Faculty of Science, Ain-Shams University, Abbasia, Cairo, 11566, Egypt c Center of genomics, Helmy Institute, Zewail City of Science and Technology, Sheikh Zayed Dist., 12588, Giza, Egypt. d College of Engineering and Computing, Swearingen Engineering Center, University of South Carolina, Columbia, SC 29208, United States. e Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208, USA. f Heart Science Centre, Imperial College, Harefield, Uxbridge UB9 6JH, United Kingdom. † Current address: Nanochemistry and Nanoengineering, School of Chemical Engineering, Department of Chemistry and Materials Science, Aalto University, Kemistintie 1, 00076 Aalto, Finland. b

* [email protected], [email protected], [email protected] Abstract. A facile one pot, bottom up, approach to construct composite materials of Graphene and a pyrimidine-based porous-organic polymer (PyPOP), as host for immobilizing human hemoglobin (Hb) biofunctional molecules is reported. The graphene was selected due to its excellent electrical conductivity, while the PyPOP was utilized due to its pronounced permanent microporosity and chemical functionality. This approach enabled enclathration of the hemoglobin within the microporous composite through a ship-in-a-bottle process, where the composite of the PyPOP@G was constructed from its molecular precursors, under mild reaction conditions. The composite-enclathrated Fe-protoporphyrin-IX, demonstrated electrocatalytic activity towards oxygen reduction, as a functional metallocomplex yet with distinct microenvironment provided by the globin protein. This approach delineates a pathway for platform microporous functional solids, where fine tuning of functionality is facilitated by judicious choice of the active host molecules or complexes, targeting specific application.

Keywords. Oxygen-reduction reaction, hemoglobin immobilization, porous-organic polymers, graphene, hemoglobin XPS. potentially be constructed from such solids while still capable of delivering appreciable current density, targeting specific applications including implantable medical devices. Microporous solids generally possess certain characteristics underlined by their molecular 4 blueprints. These included high surface area and accessible pore systems capable of encapsulating or enclathrating functional guest molecules, and in many 5 cases enhanced chemical and physical stability. The modular nature of molecular microporous solids enabled fine tuning of their functionalities through proper choice

Introduction The oxygen reduction reaction (ORR) is currently being actively investigated due to its application in clean energy technologies, and thus novel materials that can potentially catalyze the ORR are of scientific and 1 commercial interest. Although a myriad of different 2 catalysts were recently reported for the ORR, with main focus in fuel cell technology, novel solid catalysts, specifically microporous solids, are actively being 3 investigated. Due to the high surface area intrinsic to microporous solids, miniaturized electrodes can

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of the building blocks, the guest molecules, and reaction conditions, resulting in microporous solids with 6-8 pronounced activity as heterogeneous catalysts. As previously reported, porous solids demonstrated efficient performance as heterogeneous catalysts in myriad reactions where the catalytically active sites were either functional guest molecules incarcerated within the cages of the solid, or being an integral part of the 9-10 backbone of microporous solids. However, due to poor electrical conductivity of typical microporous solids, their utilization as heterogeneous catalyst were limited to thermally-promoted conversion processes. However, recent advances made to enhance the electrical 11-15 conductivity of microporous solids, including 16 17 compositing with graphene and carbon nanotubes, opened the doors for their utilization as efficient materials 18-23 in electrochemical processes. Porous organic polymers (POPs)constructed through 24 the bottom-up approach from their molecular precursors have attracted significant interest due to a myriad of unique properties including chemical and physical stability, with demonstrated potentials for CO2 capture, storage, 5, 25-27 and sequestration. The superior electrical conductivity of graphene (G), has also attracted significant 28attention for its utilization in demanding applications. 30 In this report we present a step forward in inclusion of relatively large molecule, hemoglobin, as a functional guest within a composite of the pyrimidine-based POP (PyPOP) and graphene, in attempt to construct functional material demonstrating a true mix of properties of its three different components. Furthermore, we demonstrate the efficiency of such novel composite toward one of the actively investigated process, the oxygen reduction reaction (ORR).

alteration to the synthesis process, i.e., by including hemoglobin (Hb) within the reaction mixture, resulted in immobilization of the Hb within the composite matrix (PyPOP-Hb@G), scheme 1. The Fourier-transform infrared spectroscopy (FT-IR) conducted on the PyPOP-Hb@G confirmed the presence of the PyPOP in the composite, Fig. 1. The FT-IR spectrum showed several peaks characteristic of the PyPOP, including the internal ν C≡C -1 stretching at 2215 cm , pyrimidine ν C=N stretching at 1564 -1 cm and absence of the terminal ν C-H stretching found at -1 ~3200 cm for the starting triethynylbenzene. Due to thin coverage of the PyPOP and inclusion of the Hb within the composite, it was challenging to observe distinct changes to the FTIR spectra between the PyPOP@G and the PyPOPHb@G, with the

Results and Discussion In a one-pot reaction, the bottom-up assembly of the PyPOP was conducted through SH cross coupling reaction in presence of dispersed, non-modified graphene. This resulted in homogenous deposition of the PyPOP on top of graphene sheets, referred to here as (PyPOP@G). Simple

Figure 1. FTIR spectra for the PyPOP@G and the PyPOPHb@G.


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Scheme 1. Synthesis of the PyPOP-Hb@G ternary system with schematic presentation of potential PyPOP fragments atop graphene showing immobilized Hb units. C (gray, orange in PyPOP), N (blue), H (white) O (red). while that at 158 ppm was assigned to the C2 carbon. The 13 characteristic peaks for Hb observed in the C-CPMAS spectrum of the PyPOP-Hb@G are remarkably similar to 31 those for Hb , and thus were taken to indicate successful immobilization and maintained structural stability of the Hb under the synthesis condition utilized here. Further characterization for the composition was accomplished through elemental analysis, indicating significant changes to C and N contents in the PyPOP, PyPOP@G, and PyPOP-Hb@G, table 1. The observed trend of increased C content and decreased N content, when comparing the PyPOP to the PyPOP@G, is expected due to incorporation of purely carbon-based G into the matrix of the PyPOP. When the elemental composition of the PyPOP-Hb@G is compared to that for the PyPOP@G, an increase in N and H content was observed, in agreement with inclusion of the N-rich protein. In order to gain more information about the near-surface composition of the composite, X-ray photoelectron spectroscopy (XPS) was conducted on a sample of the composite, before and after sputtering with Ar ions. The sample sputtering was conducted in order to gain more information about the composition of the sample at different depth profiles. The signal expected for Fe 2p was challenging to record due to the low atom content of Fe in the Hb. Previous spectroscopic investigation on Hb and its pyrolyzed products in fact demonstrated the Fe peak with poor 31 signal to noise ratio. The XPS detailed spectra for C and N 1s are shown in Fig. 3 (graphs labeled before and after Ar sputtering). The C1s signal could be deconvoluted into

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Figure 2. C-CPMAS NMR spectra for the PyPOP@G and PyPOP-Hb@G (labeled lines). -1

exception of a weak band at ~ 1650 cm (more enhanced in the PyPOP-Hb@G) which could be assigned to the amide bonds within the Hb (compared to the green trace in Fig. 1 for the FTIR spectrum of Hb). Utilizing the solid-state NMR spectroscopy provided a more solid indication for Hb inclusion within the matrix of 13 the composite, Fig.2. In Fig.2, the C cross-polarization 13 magic angle ( C-CPMAS) solid-state NMR spectra for both the PyPOP@G and PyPOP-Hb@G are overlaid, demonstrating additional distinct resonances in the spectrum for the PyPOP-Hb@G. The extra observed peaks include a signal at 175 ppm, assigned for the amide carbonyl, and two additional broad regions centered at ~ 25 ppm and ~53 ppm, assigned for the aliphatic groups 31 within the Hb. The CPMAS spectrum of the PyPOP-Hb@G showed the characteristic peaks for the PyPOP including the alkyne sp carbons ( 90 ppm), the aromatic carbons (122 ppm), the aromatic C-H atoms (133 ppm), as well as two observed peaks for the pyrimidine ring. The signal at 149 ppm was assigned to the C4,6 of the pyrimidine ring,

Table 1. Elemental analysis for the G, PyPOP, PyPOP@G, and PyPOP-Hb@G.

G PyPOP PyPOP@G PyPOP-Hb@G


C% 94.30 71.55 76.40 75.98

N% 21.14 6.64 8.03

H% 2.66 2.95 3.02


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Figure 3. XPS detailed spectra for the C 1s, N 1s, and O 1s before and after sample sputtering (labeled traces) for the PyPOPHb@G. several components, a peak at (284.6 eV) corresponding to 32 the aromatic C=C, the C=N in pyrimidine at (285.5 eV) and 33, 34 C-O/C-N observed at (286.6 eV). The C1s spectrum collected after sputtering demonstrated noticeable differences in relative peak intensities. One major difference is the appearance of a new peak at (288.7 eV), 33 assigned to amide carbonyl carbon atom, or those - 33 present in deprotonated carboxylate groups COO , which indicated that Hb was successfully immobilized within the composite wrapped by a protective layer of the PyPOP and hence its characteristic XPS C1s signal was detected only after removal of few surface layers of the PyPOP through sample sputtering. Similarly, observation of the N1s peak at 402.4 eV in the sputtered sample, which can be + assigned to the amide or the –NH3 ammonium nitrogen 34,35 atoms present in the Hb, provided additional indication for the immobilization of the Hb relatively deep 36 inside the composite. The O 1s spectra recorded before and after sputtering are shown in Fig. 3, where the signal deconvolution indicated three major components. The peak at (531.5 eV)can tentatively be assigned to oxygen in 34 the carboxylate COO form. The additional two observed peaks could be ascribed to the oxygen atoms in free carboxylic acid forms, O=C-OH (532.7 eV) and O=C-OH 34 (533.9eV). Considering all of the above observed trends in C/N/O XPS spectra and the changes upon sample sputtering it is reasonable to assume immobilization of the Hb within the microporous matrix. The uniform coverage of G by the PyPOP was evident from the scanning electron microscopy (SEM) image for

Figure 4.SEM for the PyPOP-Hb@G the PyPOP-Hb@G, Fig.4. The SEM image of the PyPOPHb@G demonstrated a rough surface with no detectable segregation of G and the polymer. To establish the microporosity of the PyPOP-Hb@G, N2 sorption isotherm was measured, and further compared to those for the PyPOP and the PyPOP@G. All three compounds were found to be microporous with comparable apparent surface area, as calculated using Brunner-Emmet-Teller (BET) model, Fig. 5. The calculated BET surface area values 2 2 2 were 664 m /g and 582.7 m /g, and 445 m /g for the PyPOP, PyPOP@G, and PyPOP-Hb@G, respectively. Applying the non-local density function theory (NLDFT)model of carbon finite pores to the early adsorption points in the three samples permitted calculations of the corresponding pore size distribution (PSD), Fig.5. The PSD histograms of the PyPOP and the


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PyPOP@G were quite similar, indicating that most of the microporosity in the composite was contributed by the PyPOP. This is in agreement with the observed BET surface area of the PyPOP@G, showing similar microporosity to the PyPOP rather than the G. In the overlaid PSD histograms, differences between the PyPOP@G and the PyPOP-Hb@Gwere most noticeable for the large pores within the PyPOP-Hb@G, those at 32 Å, 42 Å, and 50~60 Å (indicated by arrows in Fig. 5). Comparing the PSD histograms revealed reduced relative abundance of such larger pores in the PyPOP-Hb@G. This rather interesting observation can be taken to indicate immobilization of the Hb (having a cross sections of 49~64 Å, as determined 37 from its crystal structure, protein databank code 1GZX) within the corresponding pores of the PyPOP. The inclusion of Hb within those larger pores might have affected the packing of the PyPOP chains, resulting in diminished pores at the 32Å, 42Å range. In order to investigate the electrocatalytic performance of the prepared PyPOP-Hb@G towards oxygen reduction reaction (ORR), several electrochemical techniques were utilized. These included cyclic voltammetry (CV)and linear sweep voltammetry (LSV). Several parameters were recently utilized for benchmarking the performance of ORR catalysts, including 38, 44 onset and half wave potentials , as well as the recoded potential (vs. RHE) at fixed current density (e.g. 25 µA.cm 2 39 ). Alternatively, the ORR catalytic performance was also assessed using the ORR corrected current values at fixed 40 potentials (e.g. 0.85V). The CVs recorded in the O2 saturated, 0.1 M KOH solution, using the PyPOP@G, Hb, and the PyPOP-Hb@G carbon paste electrode (CPEs) are shown in Fig 6. The bare CPE is known for having very low 41 activity towards the oxygen reduction reaction and demonstrated such behavior in our experiments. This was confirmed by the low current recorded in its CV, (SI). The PyPOP@G demonstrated an enhanced onset potential and current density for the ORR as compared to bare CPE. The observed enhancement in the onset potential (‒0.186 V vs. Ag|AgCl) of the PyPOP@G electrode can be attributed 42 to introducing new active sites for the ORR within the microporous PyPOP@G while the high surface area potentially contributed to the observed enhancement in collected current, Fig. 6. Testing Hb in the same electrode configuration revealed an interesting behavior, where two reduction plateaus were observed with onset potentials of ‒0.217 V and ‒0.52 V vs. Ag|AgCl. While the first cathodic process can be ascribed to an ORR, the second one can be 43 ascribed to Fe(III/II) reduction (label Pc), overlapped with another ORR. The onset of the second reduction wave falls within the range reported for the Hb-Fe(III/II) in basic

Figure 5. (a) N2 Gas sorption isotherms for the PyPOP, PyPOP@G, and PyPOP-Hb@G, (b) PSD histograms with arrows highlighting the most significant differences. 43

solution, and can be explained in light of electrochemical Fe(II/III) transformation upon reacting with oxygen and/or the peroxide produced in the potential 44 range of the first plateau. This tentative assignment was further supported by the observed CVs for the PyPOP0Hb@G, vide infra. Upon immobilization of hemoglobin within the porous matrix of the PyPOP (PyPOP-Hb@G), a considerable enhancement in the ORR activity was observed, manifested by the higher cathodic current and the lower onset potential (‒0.16 V vs. Ag|AgCl), Fig 6. The anodic shift of the onset potential (as compared to the PyPOP@G and even Hb) can be assigned to stimulating the 42 ORR active sites , presumably by the Hb, coupled to presence of oxygen reservoirs within the pores of the PyPOP.



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based electrocatalysts, non-noble metal electrocatalysts, 47 as well as to some Perovskites. The enhancement to the ORR onset potential on the PyPOP-Hb@Gcan tentatively be ascribed to the PyPOP providing localized concentration of oxygen within the microporous matrix in the immediate vicinity of the Hb, thus overcoming some unfavorable process(es) including oxygen desolvation to reach the catalytic sites. The ORR mechanism on catalytic N4-Fe species, represented by pyrolized Fe-porphyrin, was previously reported to proceed mostly through the 445 electron pathway. The enhanced ORR activity in the composite PyPOP-Hb@G can then be explained in light of promoted inner sphere electron transfer mechanism 44 (ISET). It is reasonable to assume distinct mobility of gaseous species (with relevance here is the O2 molecule) as compared to solvates migrating within the micropores of the PyPOP. An additional attractive aspect of our composite, as compared to previously reported 31, 50-51 hemoglobin based system is that our system extends the utilization of hemoglobin as a molecular electrocatalyst for the ORR to highly alkaline medium (i.e. pH>10) through the successful immobilization of such large bio-molecule within a microporous polymer matrix. In attempt to probe the ORR pathway on the PyPOPHb@G, we conducted further analyses utilizing the rotating ring disk electrode (RRDE) technique. The LSVs recorded at a fixed rotation speed of 1600 rpm were carried out in oxygen saturated 0.1M KOH, Fig 7. As control experiments, the LSVs for the CPE and the PyPOP@G were also recorded and shown in Fig. 7. The LSVs for both CPE and PyPOP@G demonstrated similar behavior toward the ORR, with enhanced disk current for the PyPOP@G, indicating an impact of the enhanced surface area of the PyPOP@G on the collected disk current. The collected ring current due to H2O2 oxidation demonstrated an enhanced production of H2O2 on the PyPOP@G as compared to the CPE. The onset for the O2 reduction wave on the PyPOP@G was also slightly shifted toward more anodic potential as compared to the CPE, Fig. 7a, and signifying facilitated ORR on the microporous support. In comparison, the PyPOP-Hb@G demonstrated an earlier onset potential for the disk current which can be associated to the presence of the hemoglobin inside the matrix. Utilizing both the ring and disk current, the portion of the current consumed to the 4-electron pathway was 52 calculated (see the trace labeled % 4 electrons) for the Hb, PyPOP@G and the PyPOP-Hb@G, Fig 7 b-d. Interestingly, the ring-current for the three samples showed retarded onset as compared to the disk current, signaling an earlier onset of the 4-electron pathway 48-49

Figure 6. CVs for the PyPOP@G, the Hb, and the PyPOP-Hb@G (labeled graphs), with inserts showing magnified portion of the CVs indicating the different onset potentials. The dashed trace in the PyPOP-Hb@G plot is the CV collected in absence of oxygen, Pc indicating the cathodic peak for Fe(III/II). Dashed vertical lines are guides to the eye for the ORR onset potentials in the three plots (matching colors). CVs were collected on a rotating disk electrode maintained at 1600 rpm, scan rate of 10 mV/s in oxygen saturated 0.1M KOH.

Furthermore, the cathodic peak PC was more clearly observed for the PyPOP-Hb@G, EPc = ‒0.676 V, as compared to the Hb, Fig. 6. This peak was also detected using the same electrode material after degassing the solution, dashed curve in Fig 6. The absence of Fe(II/III) oxidation peak in this case can be ascribed to a fast electrochemical reaction of the Hb-Fe(II) with traces of entrapped oxygen within the composite, as was previously 43 observed for Hb immobilized on CPE. Moreover, this observation further indicated an enhanced catalytic activity of our PyPOP-Hb@G system, as compared to Hb 43 immobilized on CPE in a sol-gel film. The peak Pc could thus be assigned to the one electron, pH dependent, reduction of the Hb-Fe(III) to Hb45 Fe(II). The onset potential for oxygen reduction wave at (‒0.16 V vs. Ag|AgCl, ~ 0.82 vs. RHE) for the PyPOP-Hb@G CV compares well with that reported under similar 46 as well as to the conditions on Pt/C (0.809 Vs. RHE) onset potential of the ORR catalyzed by precious metal-


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Figure 7. LSVs using the RRDE configuration (a) overlay of the disk and ring current LSVs for the CPE, PyPOP@G, and PyPOPHb@G (b) LSVs and % 4 electron for Hb, (c) for PyPOP@G, and (d) for PyPOP-Hb@G. The dashed vertical lines in b-c indicate the lagging between the onset potential for the disk and ring current. Scan rate of 10 mV/s in oxygen saturated 0.1 M KOH, disk rotation rate was fixed to 1600 rpm. (area A in the graph) in similar way to previous reports for 41 ORR on carbon paste electrode. This observation can be explained in light of a sluggish, kinetically controlled, reduction of oxygen on the CPE and the PyPOP@G. At higher overpotentials, region (B), the charge transfer step was enhanced and the ORR became more diffusioncontrolled process, i.e., the recorded current can be assigned to either diffusion or mixed controlled process. On the other hand, the cathodic current of the ORR over the Hb or PyPOP-Hb@G electrode surface varies with the disk rotation rate in the potential region (A), Fig. 8. The disk current dependence on electrode rotation is much more pronounced for the PyPOP-Hb@G as compared to Hb in the potential window below ‒0.6 V. This could be ascribed to synergism between the PyPOP and the Hb, where the PyPOP acted mostly as a gas reservoir to help maintaining steady supply of O2 within the vicinity of the Hb. The linear fitting of the Koutecky–Levich plot was then

through the ISET mechanism, as compared to the 2electron pathway to generate H2O2. As the lagging between disk and ring current was maximal in case of the PyPOP-Hb@G (difference of 80 mV, Fig 7d), we propose an earlier onset of the 4-electron ISET mechanism for the composite, as compared to each of its studied components separately. Therefore, a synergy between the PyPOP and Hb has resulted in tuning the activity of the Hb. It is hypothesized that the PyPOP acted mostly as microporous reservoir for O2, enriching its concentration near the active entrapped Hb, and thus facilitating the ORR taking place mostly on the immobilized Hb, through the most efficient 4-electron pathway. In order to further probe the ORR mechanism, several LSVs were collected utilizing the rotating-disk electrode (RDE) technique under different rotation rates, Fig. 8. Noticeably, both the CPE and the PyPOP@G did not demonstrate disk current dependence on the rotation rate



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the ORR mainly through the more efficient 4-electron pathway.

Conclusion In conclusion, a ternary system of graphene, Hb and the microporous PyPOP was constructed through a solvothermal process. The ship-in-a-bottle approach to immobilize the Hb in a covalent microporous matrix proved successful in extending its desirable molecular catalytic activity into the heterogeneous catalysis arena, while the tight windows of the PyPOP prevented leaching of Hb to the reaction medium. Moreover, the PyPOP with its microporous network acted as a reservoir of O2, thus replenishing the O2 content near the catalytically active Hb molecules, and in turn augmenting the catalytic activity of the Hb. The G sheets imbedded within the ternary system imparted needed electron transport support. Overall, a true synergy between the desirable physicochemical properties of the three components of the system was achieved, as a result of the efficient compounding strategy presented herein. Experimental: All reagents were used as received without further purification. Solvents, catalysts, and common chemicals were purchased from Sigma-Aldrich or Fisher Scientific-UK. Brominated aromatics were purchased from Combi-Blocks. Triethynylbenzene was synthesized as described 16 elsewhere. Nitrogen gas for sorption were purchased from Airliquide (N2 AlphaGaz2 (99.999%) while O2 (99.999%) was purchased from AirSupply. Graphene was purchased from Alfa-Aesar and used without prior purification. Hemoglobin human, lyophilized powder, was purchased from sigma-aldrich and used as received. Gas sorption analysis was performed on Micromeretics ASAP2020. The apparent surface areas were determined from the nitrogen adsorption isotherms collected at 77 K by applying the Brunner-Emmett-Teller (BET) and Langmuir models. Pore size analyses were performed using a slit NLDFT pore model system by assuming a carbon finite pores surface. CHN elemental analyses were conducted on ThermoScientific Flash 2000. Infra-red absorption spectra were recorded on ThermoScientific Nicolet is-10equipped with attenuated total reflectance sample stage. SEM image was acquired on a FEI Nova NanoSEM 450.

Figure 8. RDE LSVs at different rotation rates (labeled lines) for (top) CPE and PyPOP@G, (middle) Hb, and (below) the PyPOP-Hb@G, with insert showing the linear fit to the Koutecky–Levich plot. constructed, insert in Fig. 8,where the number of electrons (n) involved in the oxygen reduction reaction was 53 calculated to be 4 using Eq. 1 . This indicated that the ORR proceeded mostly via the more efficient 4-electron pathway.

(1) Overall, our findings here pointed towards significant role of the Hb fixated within the porous matrix in facilitating


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decoupling was performed with SPINAL64 modulation and a 147 kHz field strength. Free induction decays were collected with a 22 ms acquisition time over a 300 ppm spectra width with a relaxation delay of 2.0 s.

Synthesis of PyPOP@G and PyPOP-Hb@G: In a 100 mL pressure vessel, a mixture of DMF (15 mL) and Et3N (2 mL) was added, cooled in liquid nitrogen bath, degassed through three freeze-pump-thaw cycles and maintained under nitrogen atmosphere. To this prepared solution was added graphene powder (8 mg), 4,6dibromopyrimidine (23 mg, 0.1 mmol), 1,3,5triethynylbenzene (15 mg, 0.1mmol). The tube was sonicated for 30 minutes and then CuI (5 mg, 0.026 mmol), PPh3 (5 mg, 0.019 mmol) and PdCl2(PPh3)2 (5 mg, 0.014 mmol) were added. The tube was sealed with a Teflon screw and kept under a flow of nitrogen. The reaction mixture was then placed in an oil bath maintained at 80°C for 24 h. A dark olive-black solid was formed which was filtered under vacuum through a sintered glass funnel, washed with acetonitrile (ACN) and left to exchange in ACN at 60°C in sealed vial under autogenous pressure for 6 h. The material was then filtered under vacuum and left to dry at 110°C for a few minutes before conducting further analyses. The total dry weight of the solid was 30 mg, quantitative yield based on the sum of the masses for starting material excluding the mass of Br atoms. An identical synthesis was conducted for the PyPOP-Hb@G with the addition of Hemoglobin (5 mg) to the reaction mixture, isolated dry weight of 33 mg.

Electrochemical Studies: All experiments were carried out in 0.1 M NaOH solutions in a jacketed EuroCell kit provided by Gamry instruments, USA. The working electrode was RRDE with an exchangeable 5mm diameter tip; a Graphite tip (insert) was used as a back current collector, the material under investigation was thoroughly mixed with the carbon paste and packed into the electrode graphite insert. The counter electrode and the reference electrode were a platinum wire and saturated double junction Ag|AgCl reference electrodes, respectively. Prior to each experiment, the electrode was wetted and rinsing with distilled water. A fresh solution and newly packed electrode is used for each experiment. All the measurements were maintained at room temperature (25 ± 1 °C).All electrochemical measurements were conducted using a reference 3000 and interface 1000 Gamry, USA electrochemical workstations equipped with Pine instruments rotating disk and rotating ring disk shafts, respectively.

XPS measurements: X-ray photoelectron spectroscopy measurements were performed using a Kratos AXIS Ultra DLD XPS system with a monochromatic Al Ka source operated at 15 keV and 150W and a hemispherical energy analyzer. The X-rays were incident at an angle of 45° with respect to the surface normal. Samples were placed in small powder pockets on the holder and analysis was -9 performed at a pressure below 1x10 mbar. High resolution core level spectra were measured with pass energy of 40eV. The XPS experiments were performed by using an electron beam, directed on the sample, for charge neutralization. Sample sputtering was performed under Ultra High Vacuum conditions using an ion gun + mounted on the XPS analysis chamber. The Ar ions were accelerated to beam energy of 4 KeV and the raster size was selected at 6mm x 6mm for homogeneous removal of surface layers in the XPS analysis area.

Acknowledgment We acknowledge the financial support from Zewail City of Science and Technology-Center for Materials Science, Egyptian Science and Technology Development Fund (STDF, USC17-43), the Magdi Yacoub Institute (MYI)- Heart Science Center, Harefield, UK, and Biotech Nano, UK. Supporting information Elemental analysis and NLDFT fitting are available free of charge via the internet or through communication to the corresponding author. References 1. Faunce, T.; Styring, S.; Wasielewski, M. R.; Brudvig, G. W.; Rutherford, A. W.; Messinger, J.; Lee, A. F.; Hill, C. L.; deGroot, H.; Fontecave, M.; MacFarlane, D. R.; Hankamer, B.; Nocera, D. G.; Tiede, D. M.; Dau, H.; Hillier, W.; Wang, L.; Amal, R., Artificial Photosynthesis as a Frontier Technology for Energy Sustainability. Energy Environ Sci. 2013,6, 1074-1076. 2. Shao, M.; Chang, Q.; Dodelet, J.-P.; Chenitz, R., Recent Advances in Electrocatalysts for Oxygen Reduction Reaction. Chem. Rev. 2016, 116, 3594-3657. 3. Strickland, K.; Miner, E.; Jia, Q.; Tylus, U.; Ramaswamy, N.; Liang, W.; Sougrati, M.-T.; Jaouen, F.; Mukerjee, S., Highly Active Oxygen Reduction Non-Platinum Group Metal

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Solid-state NMR: Solid-state C CPMAS spectra were collected on a Bruker Avance III-HD 500 MHz spectrometer fitted with a 1.9 mm MAS probe. The spectra were collected at ambient temperature with sample rotation rate of 20.0 kHz. Cross polarization was performed with a 1 2.0 ms contact time with linear ramping on the H channel 13 1 and 62.5 kHz field on the C channel. H dipolar



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