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1 Supportng Informaton for Scalable, hgh performance, enzymatc cathodes based on nanomprnt lthography Dmtry Pankratov,2, Rchard Sundberg 3, Javer Sotres, Dmtry B. Suyatn 3,4, Ivan Maxmov 3, Sergey Shleev,2,5*, and Lars Montelus 3,4 Address: Bomedcal Scences, Health and Socety, Malmö Unversty, Malmö, Sweden, 2 A.N. Bach Insttute of Bochemstry, 907 Moscow, Russa, 3 Dvson of Sold State Physcs and The Nanometer Structure Consortum (nmc@lu), Lund Unversty, 2200 Lund, Sweden, 4 Neuronano Research Center, Medcal Faculty, Lund Unversty, Lund, Sweden and 5 Kurchatov NBICS Centre, Natonal Research Centre "Kurchatov Insttute", 2382 Moscow, Russa Emal: Sergey Shleev - sergey.shleev@mah.se * Correspondng author S
2 Supplementary data Fgure S: Schematc llustraton of the fabrcaton process of the nanomprnted plastc electrodes. A) N stamp wth ca. 00 nm features s used to transfer the pattern nto the thermoplastc fol, B) nanomprnt patternng s performed by drect mechancal contact of the stamp and the fol at T equal to 60 C and pressure of 50 bar, C) after separaton of the stamp and the polymer, the stamp nano-pattern s replcated nto the polymer, D) 00 nm thck Au layer s thermally evaporated on the mprnted polymer. S2
3 Fgure S2: Typcal CVs of Au (black curve) and NIL/Au (red curve) electrodes. Condtons: 0.5 M H 2 SO 4 ; 00 mv s scan rate; second cycle. S3
4 Addtonal AFM studes Fgure S3: Atomc force mcroscopy mages and correspondng heght profles of nanostructured Au electrodes. The mages show 2D and 3D representatons of a representatve area of the samples. Scan area: 500 nm 500 nm. Colour heght scale: 0 nm nm. The heght profles (postons hghlghted n red n the topographcal mages) show the depth of the nano-cavtes along the two dfferent drectons defned by the prmtve vectors of the hexagonal lattce defned by the nanocavtes. S4
5 Addtonal boelectrochemcal studes Fgure S4: CVs of BOx-modfed Au electrodes n ar-saturated (black curve) and O 2 -saturated (blue curve) buffers. Condtons: PBS; scan rate: 20 mv s ; second cycle. Fgure S5: CVs of BOx-modfed NIL/Au electrodes n ar-saturated (black curve) and O 2 -saturated (blue curve) buffer. Condtons: PBS; scan rate: 20 mv s ; second cycle. S5
6 Fgure S6: CVs of Au electrode (black curve) and Au electrodes modfed wth enzyme solutons of dfferent concentratons, μg ml : 0.4 (blue curve), 4 (green curve), 40 (navy curve), and 400 (red curve). Condtons: O 2 -saturated PBS; 20 mv s scan rate; second cycle. Fgure S7: CVs of a non-bomodfed NIL/Au electrode (black curve) and NIL/Au electrodes modfed wth enzyme solutons of dfferent concentratons, μg ml : 0.4 (blue curve), 4 (green curve), 40 (navy curve), and 400 (red curve). Condtons: O 2 -saturated PBS; 20 mv s scan rate; second cycle. S6
7 Ellpsometry studes The tme evoluton of the adsorbed amount, thckness, and refractve ndex of BOx layer formed on a planar Au surface were montored by means of null-ellpsometry (Fgure S8). For ths, the BOx layer was ntally formed by mmersng the Au electrodes n a BOx buffer soluton at a concentraton of 40 μg ml for 60 mn (adsorpton was also montored by means of ellpsometry, correspondng to the frst 60 mnutes of the plots n Fgure S8). Then, the ambent soluton was replaced wth proten-free buffer and the layer montored for 5 more than hours. Ellpsometry data clearly showed that the adsorbed amount of BOx on the Au surface was approxmately constant wth a value of ca. 2.6 mg m 2 2 geom (4.4 pmol cm geom or 2.6 pmol cm 2 real ) durng the whole process. a Fgure S8: a) Adsorbed amount, b) Thckness and c) Refractve ndex correspondng to the formaton (ntal 60 mn), rnsng (followng 5 mn) and stablzaton (followng 5 hours) of a BOx layer on a planar Au surface montored by null-ellpsometry. b c S7
8 Theoretcal bass of boelectrochemcal nvestgatons and modellng For proper mathematcal elaboraton of boelectrochemcal data, where a boelectrocatalytc process has a mxed knetcs regme, possble dffuson lmtatons should be excluded. In general, the rate of a boelectrocatalytc process can be descrbed by a mxed knetcs equaton (Equaton S []). ET cat ss (S) where, s the observed current, ET s the lmtng current of the heterogeneous electron transfer (ET; Equaton S2; vde nfra), cat s the lmtng current of the bocatalytc process (Equaton S3), and s the lmtng dffuson current (Equaton S4 s s at close to nfnty). ET nfa real n' F( E E k Г 0 exp RT 0 ') (S2) n ' s the number of electrons n the slow electrochemcal step, F s the Faraday constant, k 0 s the standard heterogeneous ET constant, Г s the surface concentraton of the enzyme, α s the charge transfer coeffcent, E s the electrode potental, E 0 s the equlbrum potental of the electrode process, R s the gas constant, and Т s the temperature n K. If ntramolecular ET (IET) s not a lmtng step n the enzymatc process of O 2 boelectroreducton (our prevous studes of BOx showed that ths was the case at ph 7.4 [2]), the boelectrocatalytc current ( cat ) can be expressed as the electrochemcal form of the Mchaels Menten equaton: S8
9 cat app nfarealkcat С cat oxygen (S3) C K oxygen M where app k cat s the apparent rate constant for the boelectrocatalytc process and K M s the Mchaels constant. The Levch equaton (Equaton S4) defnng steady-state current lmted by the transport of substrate molecules at a rotatng electrode can be used to estmate mass-transfer lmtatons: 2 / 3 / 6.62nFD C A ss geom 0 oxygen / 2 (S4) here, s the steady-state dffuson current, n s the total number of electrons, D s the s s dffuson coeffcent of the substrate ( cm 2 s for O 2 ), C s the bulk oxygen concentraton of oxygen, O 2 ( mol cm 3 and mol cm 3 of O 2 (ar and O 2 saturated solutons, respectvely [3]), s the knematc vscosty of the soluton (0.0 cm 2 s at 25 C, a typcal value for aqueous solutons), and s the angular frequency (n rad s ). For a boelectrocatalytc process strctly lmted by O 2 dffuson, drect dependence between С oxygen and j max should be obtaned,.e., j max should decrease by a factor of ca. 5 when the O 2 concentraton was decreased from.2 mm to 0.25 mm. For electrocatalytc currents, whch are lmted by reacton knetcs, j max can be smply expressed as the electrochemcal form of the Mchaels Menten equaton (Equaton S3). When the O 2 concentraton was decreased from.2 mm down to 0.25 mm n our studes,.e., when ar-saturated buffer was used nstead of oxygen, the maxmal current S9
10 densty (j max ), whch corresponds to boelectrocatalytc O 2 reducton, decreased by a factor of ca. 3, whch follows Equaton S3, suggestng just a mnor O 2 dffuson lmtaton. In the present work, recorded CVs were analysed usng the knetc scheme recently elaborated by Clment et al. [4]. The equatons defnng the currents are as follows: DET FA k ( P ) k )] (S5) [ 2P F 0' k k0 exp E ET RT (S6) F 0' k2 k0 exp E ET RT (S7) cat O 2 O kcat nfa (S8) K M 2 where k and k 2 are potental dependent DET rate constants defned by the Butler Volmer formalsm (Equatons S2, S6, and S7), whereas P represents the fracton of adsorbed BOx molecules wth the reduced T copper centre. The maxmal boelectrocatalytc current of O 2 electroreducton, as well as current dependence on the appled potental, can be descrbed by the followng summarsed equaton: j F exp RT j max 0' k5 F 0' E E exp E E T T k0 RT (S) Usng ths equaton, modellng studes of obtaned boelectrocatalytc sgnals were performed (Fgure S9) and basc parameters of boelectrocatalytc reducton of O 2 were calculated (Table S), assumng K M and Г are equal to 0.2 mm and 2.6 pmol cm 2 real, S0
11 respectvely. For the calculatons, the K M value was taken from our prevous studes of the enzyme mmoblsed on bare polycrystallne Au surfaces [5], whereas the Г value was obtaned from the ellpsometry studes (vde supra). Fgure S9: Expermental versus modelled voltammograms of Au (trangles) and NIL/Au (crcles) bocathodes. (ponts expermental data, lnes modelled curves). Condtons: O 2 -saturated PBS; 20 mv s scan rate; second cycle; an enzyme soluton wth a concentraton equal to 40 μg ml was used for the bomodfcaton. S
12 Table S: Calculated bo-electrocatalytc parameters of Au and NIL/Au boelectrodes. Parameter Au NIL/Au Maxmal bo-electrocatalytc current densty 8 ± 3 58 ± 6 (j cat ; A cm 2 ; ar-saturated buffer) Maxmal bo-electrocatalytc current densty 48 ± 5 86 ± (j cat ; A cm 2 ; O 2 -saturated buffer) k app cat (s ) 30 ± 4 39 ± 5 k 0 (s ) 27 ± 3 27 ± 3 S2
13 References. Sucheta, A.; Cammack, R.; Wener, J.; Armstrong, F. A. Bochemstry 993, 32, do:0.02/b0007a Shleev, S.; Andoralov, V.; Falk, M.; Remann, C. T.; Ruzgas, T.; Srnec, M.; Ryde, U.; Rulíšek, L. Electroanalyss 202, 24, do:0.002/elan Truesdale, G. A.; Downng, A. L. Nature 954, 73, 236. do:0.038/73236a0 4. Clment, V.; Zhang, J.; Frs, E. P.; Østergaard, L. H.; Ulstrup, J. J. Phys. Chem. C 202, 6, do:0.02/jp Pankratov, D.; Sotres, J.; Barrantes, A.; Arnebrant, T.; Shleev, S. Langmur 204, 30, do:0.02/la402432q S3
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