DOI: 10.1002/((please add manuscript number))
Article type: Communication
Long-Range Proton Conduction Across Free-Standing Serum Albumin Mats
Nadav Amdursky,* Xuhua Wang, Paul Meredith, Donal D. C. Bradley, Molly M. Stevens*
Dr. N. Amdursky, Prof. M. M. Stevens
Departments of Materials, Bioengineering and Institute of Biomedical Engineering, Imperial
College London, London, SW7 2AZ, United Kingdom
E-mail: n.amdursky@imperial.ac.uk, m.stevens@imperial.ac.uk
Dr. X. Wang, Prof. D. D. C. Bradley
Department of Physics and Centre for Plastic Electronics, Imperial College London, London,
SW7 2AZ, United Kingdom
Prof. P. Meredith
Centre for Organic Photonics and Electronics, School of Mathematics and Physics, University
of Queensland, Brisbane, Queensland, Australia 4072
Keywords: Proton transfer, Protein films, Impedance Spectroscopy, Current-Voltage,
Hopping mechanism
Proton translocation is one of the fundamental processes in nature. The most well-known
examples are proton translocation across the mitochondrial inner membrane or the chloroplast
by the adenosine triphosphate synthase complex[1-3] and the proton pump activity of rhodopsin
proteins (mainly bacteriorhodopsin protein) in halobacteria.[3-5] Proton transfer (PT) is also
essential for numerous biochemical processes such as redox reactions, catalysis and
phosphorylation. In general, protons are being transported by water molecules, although the
exact mechanism of PT by bulk water is still debatable.[6-7] However, protons can be
transported laterally along membranes[3, 8-9] or by specific amino acids such as the proton
pump activity of bacteriorhodopsin.[3-5]
Beyond biological systems, solid-state proton conductors are commonly used in devices
such as batteries and fuel-cells.[10-11] In these devices, the most-used materials are inorganic
oxides,[12] metal-organic frameworks,[13] solid acid membranes,[14] ionic crystals[15] and
1
polymeric membranes, where the most common example is Nafion®.[16] Several organic
semi-conductors have been also shown to have the ability to conduct protons, which enables
them to support parallel proton and electron conductivity.[17-20] In recent years several bioorganic materials have been also proposed for protonic devices, such as polysaccharide
derivatives and melanin pigment.[21-25] While a wide diversity of materials have been shown
to sustain proton current, there are only few types of proton-conductors: oxide ions, oxoacids
(and their anions), and in some cases heterocycle molecules.[10] The common denominator for
all proton conductors, whether solid-state materials or water, is the role of a hydrogen bond
network that can support long-range proton conductivity.[6-7, 26]
In this study, we explore the use of protein-based materials as long-range proton
conductors. Due to the abundance of water molecules inside the protein structure and the
presence of charged amino acids (mainly oxoacids), proteins are good candidates for the
formation of proton conducting materials. Several studies have followed proton conductivity
across collagen,[27] keratin[28] and lysozyme layers.[29-30] Recently, Gorodetsky and coworkers[31] showed that upon drop-casting reflectin (a structural protein found in cephalopods)
and drying it between two electrodes, they could measure a proton conductivity across the
film of 0.1mS/cm at room temperature, and up to 2.6mS/cm at 65°C.
We used bovine serum albumin (BSA), one of the cheapest commercially-available
proteins, to form an electrospun mat composed of fibrillar structures that can absorb large
quantities of water. Our mat is a free-standing material that can be held and manipulated by
hand. First, we explored local (short-range) proton translocation via excited-state proton
transfer (ESPT) of a photoacid that was absorbed on the mat. Second, we explored long-range
(millimetre length-scales) proton conductance between two electrodes bridged by the BSA
mat with electrochemical impedance spectroscopy (EIS) and current voltage (I-V)
measurements. We also examined their temperature dependence and the kinetic isotope effect
(KIE). We propose that the observed proton translocation is due to an ‘over-the-barrier’
2
proton-hopping mechanism that involves the oxo-amino-acids of the protein as the main
proton hopping sites.
It has previously been shown that BSA can be electrospun to form mats.[32] Following
electrospinning, our mats were composed of fibrils with a diameter of several hundred
nanometres and large spacing between individual fibrils (Fig. 1a). After placing them in
aqueous solution, the fibrils absorbed water in a sponge-like manner, and transformed to a
thick (>1μm) fibril surface with almost no spacing between individual fibrils (Fig. 1b). By
weighing the mats before and after hydration, we could determine the swelling ratio (water
content) of the mats to be 143 ± 18% (w/w). The large amount of water within the material
distinguishes itself from other bio-organic proton conductors in the literature that contain 520% of water.[22-25] Following hydration of the mats, most of the water could be removed by
heating or placing the mats in vacuum. By thermogravimetric analysis (TGA) we estimated
the amount of the remaining water content of the dehydrated mat to be ~7% (TGA results are
provided as Fig. S1 in the Supplementary Information). Removal of these water molecules
transformed the mat from a flexible film that was easy to hold and manipulate to a highly
brittle film. This observation made it clear that water played a major role in the structure of
this material. The mats could also be placed in a variety of organic solvents and acids without
being dissolved for months (Fig. S2).
a
b
Figure 1. Morphology of the SA mat. Scanning electron microscopy images of the SA mats before (a)
and after (b) immersion in water. The scale bar represents 10 μm.
3
The large amount of water in the BSA mat along with the high ratio of charged amino
acids in the protein structure of the protein (more than one-third) have encouraged us to
explore whether the BSA mat can sustain proton conduction. To probe local PT processes in
the BSA mat, we used the photoacid of 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS, see inset
of Fig. 2a for the protonated, ROH, form). Photoacids are molecules with lower pKa values in
their electronically excited state; for HPTS, an ROH photoacid, the pKa decreases from 7.4 in
the ground state to 1.3 in its first excited singlet state. Following optical excitation, HPTS in
water undergoes an ESPT reaction, [22-23] as the proton from its hydroxyl group is transferred
to the aqueous solvent leaving behind the excited RO-* anion:
ROH*
kPT
ka
[RO¯*···H+]
diffusion
RO¯* + H+
(1)
After excitation, the proton is transferred to form an ion pair with the de-protonated molecule.
The proton can then diffuse to bulk water or recombine back with the excited anion to form
the ROH* form in what is known as geminate recombination.[33] Since the ROH* and RO¯*
forms have different emission wavelengths (for HPTS, 440 and 535nm, respectively), it is
relatively easy to follow their time-resolved and steady-state fluorescence.[34-37] In pure water,
the proton diffuses rapidly from the photoanion, which results in a predominant RO¯* species,
as can be seen in the steady-state emission spectra (Fig. 2a). On the other hand, when HPTS is
in its dry state (powder), the predominant species is the ROH* (Fig. S3), as there is no bulk
water in which to diffuse. However, when HPTS was adsorbed on the BSA mat and the mat
was dehydrated (leaving ~7% water content as measured by TGA, Fig. S1, and the
measurements were conducted under similar room temperature conditions as in the previous
experiment in Fig S3), the predominant form was still RO¯*. This unexpected finding implies
that even in the relatively dry state, HPTS can transfer a proton to nearby molecules. Since
there is no excess of water molecules in the dehydrated sample, the proton is likely transferred
to nearby carboxylates or amines or to trapped water molecules. Following the gradual
4
addition of small amounts of water to the surface (up to 300% w/w of water), we observed
only a slight change in the ROH* band intensity, which was still very different to HPTS in
bulk water. Our findings can be explained by a combination of the following explanations: (1)
the PT rate of HPTS is slower on the mat than in bulk water; (2) the protons diffuse along the
mat in lower dimensionality in comparison to bulk water; and (3) the geminate recombination
is more efficient on the mat in comparison to water.
Normalized Intensity
a 1.0
Dehydrated mat
+50% H2O
RO-*
1
1
b
c
+100% H2O
0.1
+200% H2O
0.8
+300% H2O
0.1
HPTS in water
0.6
0.01
ROH*
0.4
0.01
1E-3
0.2
0.0
1E-3
400
450
500
550
600
Wavelength [nm]
650
1E-4
0
5
10
15
20
Time [ns]
25
30
35
0
5
10
15
20
25
30
Time [ns]
Figure 2. Photo-induced PT. (a) Steady state and (b and c) time-resolved fluorescence of HPTS at the
detection wavelength of (b) RO¯* and (c) ROH* of the dehydrated mat and with the different weight
percentage (%w/w) of added water in comparison to HPTS in water. The dashed line in panel c
represents the instrument response function.
To distinguish between these possibilities, we probed the time-resolved fluorescence of
HPTS at the emission wavelengths of both RO¯* (Fig. 2b and Fig. S4a and S4b) and ROH*
(Fig. 2c and Fig. S4c and S4d). The PT rate constant (kPT) can be roughly estimated by: 𝑘𝑃𝑇 =
𝐹
𝐼RO¯
𝐼𝐹
−1
⁄ 𝐹 × 𝜏RO¯
, where RO¯⁄ 𝐹 is the ratio between the steady-state intensity of the RO¯*
𝐼ROH
𝐼ROH
and the ROH* bands (Fig. 2a) and 𝜏RO¯ is the fluorescence lifetime of the RO¯* form (Fig. 2b).
Hence we can estimate kPT to be within the range of 5.5×108s-1 in the dehydrated mat to
7.6×108s-1 in the fully hydrated mat, which is significantly different in comparison to
3.4×109s-1 for HPTS in water (Table S1). The time-resolved emission of the ROH* form (Fig.
2c) can imply the dimensionality of the proton diffusion. In bulk water, protons diffuse in 3-D
and the ROH* form decays rapidly. However, the decay of the ROH* form is much slower on
the BSA mat, even in fully hydrated samples, which might imply that protons diffuse along
5
the SA mat and not into bulk water. The time-resolved emission of the ROH* form can be
used to get an estimation of the dimensionality of the proton diffusion space, where the
fluorescent tail obeys a power-law of t-d/2, where d is the diffusion space dimensionality (see
further discussion in Supplemental Materials).[34, 36-37] By plotting a log-log plot (Fig. S5) of
the lifetime corrected ROH* decay, we could estimate the fractal space dimensionality of the
proton diffusion by linear fitting the first nanoseconds of the decay. We found that for the
highly hydrated sample (with 300% w/w water, meaning that this sample contained double
the amount of water than the measured swelling ratio of the mat) the dimensionality is 1.07
(and even lower, closer to 1, for the less hydrated samples), compared to 2.99 for HPTS in
bulk water (Table S1), meaning that the protons diffuse along the fibrils within the mats. The
role of the geminate recombination of HPTS on the BSA mat is challenging to assess
quantitatively. The geminate recombination is interdependent with proton diffusion (Eq. (1)),
where slower diffusion results in an enhanced geminate recombination.[33] Proton diffusion
along the mat, and especially the dehydrated one, is expected to be slower than water. Indeed,
as will be discussed below, proton hopping along the mat is significantly slower in
comparison to water.
The ability of HPTS to transfer protons to nearby amino acids has been observed before for
the case of HPTS bound in the binding site of natively folded human serum albumin.[38] It was
also recently shown that HPTS can transfer protons to glucosamine units of chitosan.[39] In
order to probe the role of water molecules in the diffusion of the protons, we have followed
the kinetic isotope effect (KIE) of the HPTS time-resolved measurements (Fig. S6). In water,
the KIE of HPTS is ~3 (Fig. S6a). Surprisingly, we have found no KIE for all the BSA mat
samples (Fig. S6b-f), regardless to the percentage of water in the mat. The lack of a KIE
implies that proton diffusion in bulk water is not the main proton transfer mechanism in the
mat.
6
In the previous section we showed that within the excited-state lifetime of HPTS, which
limits the measurable transport length of the proton to up to ~15nm, the protons in the BSA
mat can diffuse along the fibrillar structure of the mat with different kinetics in comparison to
water. Next, we investigated whether the mat could serve as an efficient proton conductor in a
bioelectronic device. By placing the mat on top of a gold finger electrode structure (Fig. S6),
we could examine the distance-dependent a.c. electrical impedance response (Fig. 3a) using
electrochemical impedance spectroscopy (EIS) and the d.c. response (Fig. 3b) using current
voltage (I-V) measurements. It is important to note that the measurements were conducted in
the swollen state of the mat (i.e., at ~150% (w/w) water). The EIS results, which are
represented as a Nyquist plot (the imaginary part of the impedance, Zim, as function of the real
part, Zreal), exhibit a semi-circle representative of a parallel RC circuit model (the small spur
in the low frequency domain is indicative of charge accumulation in the contacts of throughplane EIS measurement).[40] By fitting the semi-circle to an RC circuit (Table S2), we were
able to extract the resistance values; these range from 0.10 ± 0.03 to 0.81 ± 0.30MΩ for l =
0.25 and 2.5mm electrode finger separations, respectively. Taking into account the distance
between electrodes, the thickness of the mat (~75 μm) and the electrode length in contact with
the mat (~7-9mm), this corresponded to conductivity values of 41.1 and 48.6μS·cm-1,
respectively (Table S2). The EIS measurements were complemented with I-V measurements
(Fig. 3b) that showed a featureless non-ohmic behaviour, which is not affected by contact
resistance (Fig. S7). Both measurements had a similar distance decay profile (Fig. 3c,
comparing the measured EIS conductance with the current magnitude in the low bias linear
regime of the I-V curve), with an identical distance-decay constant (inset of Fig. 3c) where the
𝐴
conductance/current is proportional to 1/distance (Fig. S8, following Pouillet's law: 𝐺 = 𝜎 𝑙 ,
where G is the conductance, σ is the conductivity, A is the cross-sectional area (film thickness
7
times electrode length) and l is the distance between electrodes), which confirms that the same
charge carrier (protons) dictates the conductivity in both a.c. and d.c. measurements.
a
c
b
Figure 3. In-plane proton conductance across BSA mats. (a) a.c. EIS response (plotted on an isometric
scale) and (b) d.c. I-V sweeps for several inter-electrode separation distances, l. (c) The distance decay
of the EIS conductance (left y-axis, black squares) and the I-V current at 0.05V (right y-axis, red
squares). The inset shows the same points on a semi-logarithmic scale spanning two orders of
magnitude on both y-axes, along with fits to the same 𝑋 −0.87 dependence.
In order to examine the in-plane proton conductance mechanism across the BSA mat we
measured the kinetic isotope effect (KIE) and the temperature-dependence of the conduction
process. Similar to the ESPT results (Fig. S5), we found no KIE for the proton conductance
for both EIS (Fig. 4a) and I-V (Fig. S9) measurements (the KIE of the EIS was slightly
inverse (i.e. lower than 1) but within the error range). The EIS temperature dependence
studies for two of the measured distances (l = 0.75 and 1.5mm, Fig. 4b and S10, respectively)
showed (via an Arrhenius fit) that the proton conductance was thermally activated with Ea =
0.29 ± 0.02eV. The temperature dependence of the I-V measurements showed a similar trend
8
with consistent activation energies for the measured distances (Fig. S11). We note, however,
that conductivity measurements (a.c. and / or d.c.) on hygroscopic conductors where the
electrical response is strongly affected by the state of hydration are notoriously unreliable –
the temperature dependence can be perturbed or even masked by attendant changes in the
hydration state. The fact that in our experiments the water content of the mats was high (~
150% w/w, yielding reduced sensitivity to thermal changes), and further, that we conducted
the measurements in a fairly narrow range around room temperature, we consider that the
results are moderately robust. In addition, we note that we see an increase in conductance with
increasing temperature, whereas were the measurements dominated by dehydration, the
opposite would have been expected. Moreover, a simple Arrhenius fit to the temperature
dependent I-V data over this limited range and under circumstances where there will be some
change in the state of hydration does not allow us to draw any independent mechanistic
conclusions about the underlying transport physics. It does however provide an internally selfconsistent check of the a.c. data and conclusions.
a
D2O
400000
H2O
-ZIm []
300000
200000
100000
2.5 mm
0.25 mm
0
400000
ZReal []
b
-ZIm []
160000
120000
80000
35C
30C
25C
20C
15C
800000
ln(Conductance [-1])
0
-12.3
Ea=0.28 eV
-12.6
-12.9
3.28
3.36
40000
0
15C
35C
0
3.44
1000/T [K-1]
100000
200000
ZReal []
9
300000
400000
Figure 4. KIE and temperature dependence studies. (a) KIE of EIS where water (filled black squares)
was replaced with deuterium (open blue squares). (b) Temperature dependence of EIS across the X =
0.75mm junction. The inset shows the activation energy of the process by fitting to an Arrhenius
equation (𝐺 ∝ exp(−𝐸𝑎 /𝑘𝐵 𝑇)). The graphs are displayed on an isometric scale.
Proton conductivity within hydrated samples is commonly explained by the Grotthuss
mechanism, which describes proton hopping (diffusion) across water networks, apperently by
hopping across the H9O4+ or the H5O2+ cations.[7] In this mechanism the proton hops from one
water molecule to another, and it is expected to have activation energy of 2-3kcal/mol (~90130meV), and a KIE of 1.4.[7] Gorodetsky and coworkers[31] and Rolandi and coworkers[23, 25]
explained the high proton conductance of dry films of the relectin protein and polysaccharides,
respectively, by the Grotthuss mechanism, and also suggested that the protein film contains
water channels that support proton conductivity. In Gorodetsky‘s work,[31] similar KIE (~1.7)
values as expected for the Grotthuss mechanism have been found but with a slightly larger Ea
value (~0.2eV).[31]
In our measurements, we found the following evidences suggesting that the Grotthuss
mechanism for proton diffusion across bulk water was not the predominant mechanism for the
BSA mat conductivity:
1) The photo-induced ESPT of HPTS was efficient in the dried sample and its kinetics did
not change significantly as water was slowly added to the mat;
2) We found no KIE for both the ESPT kinetics and proton conductance; and
3) The measured activation energy for proton conductance (~0.29eV) was higher than
expected for water-mediated proton hopping.
To explain these unique observations, we propose that oxo-amino-acids have a significant role
as mediators for proton hopping along the BSA mat fibrils since:
1) The ESPT from HPTS to charged amino acids in the mat can be efficient and it should
not be affected by the hydration level of the mat;
10
2) The protons of the charged amino acids in the mat are not expected to be deuterated by
the addition of D2O, so the KIE should be 1 as observed; and
3) The activation energy in the Grotthuss mechanism is related to the hydrogen bond
strength in water.[7] The larger hydrogen bond strengths between carboxylates and
between carboxylates and amines[26] might then explain a higher activation energy.
Though it is hard to visualize or predict how exactly the network of oxo-amino-acids and
trapped water molecules are arranged in space to support the proton translocation, it is very
likely that the proton conductivity mechanism within the BSA mat can be related to lateral
proton diffusion as observed next to membrane-water interfaces,[8-9] and even to some extent
in BSA monolayers at a water-air interface.[41] This mechanism is also one of the proposed
mechanisms to justify the high proton conductivity for Nafion® films, where a water
supported network of sulfonic acid moieties might serve as the proton hopping sites,[42]
though it is important to note that the conductivity of Nafion is orders of magnitude higher
than measured here for BSA mats.
We further attempted to fit our experimental results to the theoretical paradigm of proton
hopping to estimate the hopping rate and the distribution of hopping sites in the mat. Protonhopping can be envisioned as a small polaron problem where proton donors and acceptors are
bridged by a hydrogen bond; each hopping event corresponds to a phonon-assisted tunneling
across a barrier.[43] In general, an under-the-barrier event can be distinguished from an overthe-barrier one (Fig. S12a for schematic representation). The activation energy (Ea) in an
under-the-barrier event is of the order of ℏ𝜔 (where ω is the optical phonon frequency and ℏ
is the reduced Planck constant), and the PT rate, kPT, can be expressed by the common
expression for a nonadiabatic tunneling event.[43] For an over-the-barrier event, the proton
resides on the bridge (with a lifetime of 𝜏𝑃𝑇 )[44] and the activation energy serves as the barrier
where 𝐸𝑎 > ℏ𝜔.[43] For the same donor and acceptor, an over-the-barrier event will have
11
larger Ea and will be the dominant mechanism at higher temperatures (Fig. S11b S12b for
schematic representation). Our measured activation energy (~0.29eV) in comparison to the
optical phonon frequency (estimated by infrared (IR) spectroscopy [Fig. S13] to be ~3300cm-1
i.e. with ℏ𝜔 = 65meV) is suggestive of over-the-barrier proton hopping. In this case, 𝜏𝑃𝑇 can
be expressed as:
−1
𝜏𝑃𝑇
=
𝜔
ℏ
𝐸
exp[− 𝑘 𝑎𝑇],
𝐵
(2)
where kB is the Boltzmann constant and T is temperature. Using the measured values for
−1
activation energy and phonon frequency, we estimate 𝜏𝑃𝑇
to be ~1×1010s-1 (𝜏𝑃𝑇 ~100ps), a
significantly higher value than the PT rate in water (~1.5ps), which is considered an under the
barrier PT.
We can further fit our measured conductivity values using the Nernst-Einstein relation
between conductivity () and charge carrier mobility (μ):
𝜎 = 𝑛𝑒𝜇,
(3)
where n is the charge density and e the electron charge. The charge carrier mobility is further
proportional to the diffusivity of the charge carrier:
𝜇=𝑘
𝑒
𝐵𝑇
𝐷,
(4)
with diffusion constant:
−1 2
𝐷 = 𝜏𝑃𝑇
𝑟𝑛𝑛 ,
(5)
where rnn is the average hopping distance. Combining Eqs. (3)-(5) yields the following
relation for the mat conductivity:
𝜎(𝑇) =
2 𝜏 −1
𝑛𝑒 2 𝑟𝑛𝑛
𝑃𝑇
𝑘𝐵 𝑇
.
(6)
The distance between proton donor and acceptor (rnn) for a single hopping event is determined
by the length of the hydrogen bond and it is confined to a narrow range around 2.5Å. Using
the above calculated PT lifetime (~100ps), we can estimate the charge carrier density to be 1
× 1018cm-3 (= 1.7 × 10-6mol/cm-3 = 1.7mM), which can be compared to the concentration of
12
hydronium ion at pH2.8. The 2.5Å hopping distance was chosen for cases where the proton
donor and acceptor are bridged by a hydrogen bond, as discussed for the Grotthuss
mechanism.[7] However, according to the Eigen-Weller model, a PT event can be also
mediated by up to 2 water molecules, which leads to an optimal PT distance of ~7Å.[45] On
using the latter value for rnn, the estimated charge carrier density in the mat is reduced to 1.5 ×
1017cm-3. It is also important to note that the measured conductivity values may be underestimated as they consider the entire cross-section of the mat in calculations, while the
charges most likely migrate along a narrower cross-section closer to the electrodes and along
specific pathways of the fibril structure.
In summary, we followed the in-plane PT within free-standing BSA mats by photoinduced ESPT, EIS and I-V measurements. We found that protons could be transferred along
the fibrillar structure of the mat which allows long-range (millimetre length-scales) proton
transport with one of the highest deduced conductivity values (~50µS/cm) for biological
materials. Temperature dependence and KIE measurements suggest that oxo-amino-acids
have a significant role in the PT mechanism. Our results support an over-the-barrier protonhopping mechanism and we were able to calculate the proton lifetime on the bridge (barrier)
to be in the order of 100ps. Together with the Nernst-Einstein relation, we used this lifetime
to fit our measured conductivity values and estimate the charge carrier density of the hopping
sites in the mat to be 2 × 1018cm-3.
Using proteins as materials for proton conduction opens new and exciting possibilities
for bioelectronic devices. BSA mats are biocompatible and can support cell proliferation,[46]
which makes this system ideal for the study of proton translocation between cells. Moreover,
BSA mats are highly robust and can be placed in many organic solvents and acids without
being dissolved. Together with their high proton conductivity, BSA mats could be easily used
in proton conducting devices such as fuel cells and batteries. BSA mats distinguish
themselves from other bio-organic proton conductors, and also from common inorganic
13
conductors, as they are extremely cheap (in the order of 1 GBP/g) and can be easily processed
to form electrospun mats in the metre length-scale.
Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements
We thank N. Agmon, D. Huppert, N. Stingelin and J. Riley for fruitful discussions and critical
reading. N.A. was supported by Marie Curie actions FP7 through the Intra-European Marie
Curie Fellowship ConPilus under grant agreement no. 623123. P.M. is an Australia Research
Council Discovery Outstanding Research Award Fellow and the Centre for Organic Photonics
and Electronics is a strategic initiative of the University of Queensland. M.S. acknowledges
support from the Wellcome Trust Senior Investigator Grant 098411/Z/12/Z and the ERC
Seventh Framework Programme Consolidator grant “Naturale CG” under grant agreement no.
616417.
Received: ((will be filled in by the editorial staff))
Revised: ((will be filled in by the editorial staff))
Published online: ((will be filled in by the editorial staff))
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16
[43]
H. Böttger, V. V. Bryksin, Hopping Conduction in Solids, VCH, Weinheim, 1985.
[44]
It is important to distinguish between the proton transfer rate and lifetime of the
hopping events as discussed in this section to the proton transfer rate of HPTS as
discussed above.
[45]
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17
Free-standing protein mats can transport protons over millimetre length-scales. The
results of photo-induced proton transfer and voltage-driven proton conductivity measurements,
together with temperature dependent and isotope effect studies suggest that oxo-amino-acids
of the protein play a major role in the translocation of protons via an ‘over-the-barrier’
hopping mechanism. The use of proton-conducting protein mats opens new possibilities for
bioelectronic interfaces.
Keywords: Proton transfer, Protein films, Impedance Spectroscopy, Current-Voltage,
Hopping mechanism
Dr. N. Amdursky, Prof. M. M. Stevens
Departments of Materials, Bioengineering and Institute of Biomedical Engineering, Imperial
College London, London, SW7 2AZ, United Kingdom
E-mail: n.amdursky@imperial.ac.uk, m.stevens@imperial.ac.uk
Dr. X. Wang, Prof. D. D. C. Bradley
Department of Physics and Centre for Plastic Electronics, Imperial College London, London,
SW7 2AZ, United Kingdom
Prof. P. Meredith
Centre for Organic Photonics and Electronics, School of Mathematics and Physics, University
of Queensland, Brisbane, Queensland, Australia 4072
Long-Range Proton Conduction Across Free-Standing Serum Albumin Mats
Nadav Amdursky,* Xuhua Wang, Paul Meredith, Donal D. C. Bradley, Molly M. Stevens*
-ZIm []
0.4
0.2
0.0
2.5 mm
0.25
0.0
0.5
ZReal []
H+
H
H+
H+
H+
1.0
+
H+
H+
H+
H+
H+
H+
ToC figure ((Please choose one size: 55 mm broad × 50 mm high or 110 mm broad × 20 mm
high. Please do not use any other dimensions))
18
Copyright WILEY-VCH Verlag GmbH & Co. KGaA, 69469 Weinheim, Germany, 2013.
Supporting Information
Long-Range Proton Conduction Across Free-Standing Serum Albumin Mats
Nadav Amdursky,* Xuhua Wang, Paul Meredith, Donal D. C. Bradley, Molly M. Stevens*
Materials and Methods
Electrospinning of BSA mat – BSA (Sigma-Aldrich) was used to form the mats according to
the protocol of Fleischer et al.1 BSA was dissolved in 90% TFE to a final concentration of
14% (w/v). β-mercaptoethanol was added to the solution to a final concentration of 5% (v/v).
A custom-built electrospinning system was used, where a bias of 11.5kV was applied on an
18-gauge blunt needle, while the collector was grounded. The distance between the collector
and the end of the needle was ~11cm, and the rate of injection was 0.9 mL/min.
Scanning electron microscopy (SEM) characterisation – small pieces from the mat were
coated with a thin layer of Au, and imaged with a SEM JEOL 5610LV system, at an operating
bias of 17-20kV.
Steady-state and time-resolved fluorescence – Dry BSA mat was placed in a 0.05mM
solution of HPTS for ~4h. The hydrated mat with HPTS was placed in vacuo over night for
complete dehydration. Deionised water was added to the mat in small aliquots to reach the
desired water percentage. The dehydrated mat was weighed before the experiment in order to
calculate the desired amount of water. A Fluorolog system (Horiba) with 1 nm bandpass slits
in both the entrance and exit arms was used for the steady-state measurements and the sample
was excited at 390nm. A Deltaflex system (Horiba) was used for time-resolved emission
spectroscopy with a 405nm laser diode (<100ps pulse duration) as excitation source and data
(at least 20,000 counts) were collected at 440 and 530nm.
19
Finger electrode preparation – A custom stainless steel shadow mask was fabricated with the
desired finger electrode pattern. Freshly-cleaned microscope slides were used as substrates
onto which a 100nm thickness gold pattern was evaporated through the shadow mask using a
MBraun thermal evaporator (5 x 10-7mbar) inside a nitrogen filled overpressure glovebox
system. Prior to gold deposition, a 10nm thickness chromium layer was first evaporated to act
as an adhesion promotion for the gold.
Impedance Spectroscopy – The impedance measurements were carried out using an SI 1260
impedance/gain-phase analyser (Schlumberger). At least 24h before the measurement the dry
mats were placed in deionised water. The wet mats were then placed onto the gold finger
electrode substrates (Fig. S6) and were dried with filter paper to remove excess water not
tightly bound to the surface. Micromanipulator probes were used to contact the gold
electrodes. A frequency range of 10MHz – 100Hz was used with applied a.c. bias = 100mV
and integration time = 0.5s; no d.c. bias was applied. The impedance spectra were fitted using
ZView software (© Scribner Associates, Inc), allowing extraction of the resistance and
capacitance values for each junction.
Current-Voltage measurements – The current-voltage sweeps were carried out using a
Keithley 2400 source-measure unit controlled by computer via a home written Labview
program. The same electrode format, BSA sample preparation and micromanipulator probe
set ups were used for these d.c. experiments except that one of the probes was biased while
the other was grounded. The scan rate was 20mV/s in the ±1V bias range with a voltage step
of 0.01V. The presented I-V curves are averages of forward (-1V→+1V) and reverse (+1V→1V) scans. The conductance/resistance values were extracted from these current-voltage
measurements by linearly fitting the low bias (±0.05V) region.
KIE measurements – The dry electrospun mat was placed in deuterium oxide (Sigma-Aldrich
>99.9%) for at least 24h before measurement. For the time-resolved measurements, the mat
was then dehydrated in vacuum overnight, and the measurements were conducted by adding
20
small amounts of D2O to reach the desired deuterium percentage. The subsequent sample
preparation for EIS and I-V measurements was exactly as for non-deuterated samples.
Temperature dependence – The finger pattern electrodes with BSA mats on top were placed
on a heater that was controlled by a Keithley temperature controller. The sample was
equilibrated for ~5min at each temperature point (starting from 15ºC and rising to 35ºC) and
the EIS and I-V measurements were performed as already discussed above.
Fourier Transform Infrared (FTIR) measurements – FTIR measurements used a Perkin
Elmer Spectrum 100 FTIR spectrometer with a single bounce Ge-ATR at 16°C. A
background scan was measured before the sample. The spectrum was collected in the range
900-4000cm-1, with a 1cm-1 increment. Both background and sample measurements were
averaged over 10 scans.
TGA measurements – A disc (~6 mm diameter) of the BSA mat was placed in water for
several hours following by de-hydration in vacuum over-night. The TGA measurement of the
de-hydrated mat was conducted with a Netzsch STA449 F5 Simultaneous DSC/TGA.
21
100
-7%
Weight [%]
80
60
40
20
0
0
200
400
600
800
Temperature [°C]
Figure S1. Thermogravimetric analysis of the dehydrated BSA mat. The dashed line is the
derivative of the weight loss profile.
Figure S2. BSA mats in various solvents. The mats were placed for 3 months in (from left to
right): Chloroform, Dimethyl sulfoxide, 1,1,1,3,3,3-Hexafluoro-2-propanol, Acetone, 2,2,2Trifluoroethanol, 1M HCl and Deionised water.
22
Normalized Intensity
ROH*
1.0
RO-*
Dry mat
HPTS in water
Dry HPTS
0.8
0.6
0.4
0.2
0.0
400
450
500
550
600
Wavelength [nm]
Figure S3. Steady state fluorescence spectrum of HPTS in dehydrated BSA mat, in bulk
water and in dry condition (powder on glass).
23
1.1
1
0.9
0.8
b
1
Normalized Intensity
Normalized Intensity
a
0.7
0.6
0.5
0.4
0
0.3
0
1
2
3
4
5
0
5
10
Time [ns]
c
d
20
Dry mat
+50% H2O
1
Normalized Intensity
1
Normalized Intensity
15
Time [ns]
0.1
+100% H2O
+200% H2O
+300% H2O
HPTS in water
0.01
0
0
1
2
3
4
5
0
Time [ns]
5
10
15
Time [ns]
Figure S4. Time-resolved spectra of HPTS on BSA mats. (a) Zoom area and (b) linear scale
of Fig. 2b in the main text. (c) Zoom area and (d) linear scale of Fig. 2c in the main text.
24
Table S1. Calculated fractal space dimensionality of the proton diffusion.
Calculated
kPT
Derived space
𝑰𝑭𝐑𝐎¯
𝝉𝐑𝐎¯
⁄𝑭
Sample
power-law
-1
(s )
dimensionality
𝑰𝐑𝐎𝐇
(ns)
factora
Dehydrated
2.6
4.9
5.6×108 0.500 (0.970)
1.00
BSA mat
2.9
5.1
5.9×108 0.510 (0.976)
1.02
+50% H2O
3.4
5.4
6.4×108 0.530 (0.980)
1.06
+100% H2O
8
3.8
5.4
7.1×10
0.530 (0.987)
1.06
+200% H2O
4.1
5.4
7.6×108 0.535 (0.990)
1.07
+300% H2O
HPTS in bulk
18.5
5.5
3.4×109 1.495 (0.994)
2.99
H2 O
a
The brackets display the adjusted R2 of the fit.
Intensity*exp(t/5.4)
1
0.1
Dry mat
+50% H2O
+100% H2O
+200% H2O
+300% H2O
0.01
0.1
HPTS in water
1
time [ns]
Figure S5. Log-log plot of the time-resolved kinetics, after multiplying by exp(t/5.4), where
5.4 is the radiative life time in ns, together with the fit that was used to extract the power law
factor (Table S1).
Fractal space dimensionality of the proton diffusion
The ROH* decay has a non-exponential long-time fluorescence tail with a power-law decay of
t-α. Due to the high fluorescence intensity of HPTS, one can follow the tail intensity over a
long time scale (into the nanosecond regime). The decay of the ROH* fluorescence is very
sensitive to the space dimensionality of the proton diffusion. In back-to-back publications,
Huppert, Agmon and Pines developed the numerical solution for HPTS (or any other
photoacid) kinetics.2,3 They showed that if the ROH* fluorescent decay (𝐼𝑓𝑅𝑂𝐻 ) is multiplied
25
by exp[t/τr], where τr (=5.4 ns) is the radiative decay lifetime, the factor (α) of the power-law
is proportional to d/2, where the d is the space diomensionality of the proton diffusion:
𝐼𝑓𝑅𝑂𝐻 (𝑡)exp[𝑡/𝜏𝑟 ] ~
𝜋𝑎2 𝑘𝑎 exp[−𝑉(𝑎)] −𝑑/2
𝑡
2𝑘𝑃𝑇 (𝜋𝐷)𝑑/2
where D is the proton diffusion constant, ka is the proton geminate recombination rate
constant, kPT is the proton transfer rate constant, and a is the radius of the reaction sphere,
where the reaction sphere potential, V(a), is proportional to RD/a, where RD is the Debye
radius. RD is expressed as:
𝑅𝐷 =
|𝑧|𝑒 2
𝜀𝑘𝐵 𝑇
where z is the RO- form charge (in electron charge units), e is the electron charge, ε is the
dielectric constant of the medium and kB is the Boltzmann constant.
For HPTS in bulk water the protons diffuse in 3-D from the excited HPTS molecule,
which means that the power-law factor is 3/2 (d=3). In Table S1 we summarize the calculated
space dimensionality of the proton diffusion in each mat sample containing different amounts
of water, in comparison to HPTS in bulk water. It is mportant to stress that this approximation
is an analytical solution based on the ESPT of photoacids (Eq.(1) in the main text). However,
in some cases, the experimental results cannot be well fitted using this analytical solution (for
instance, the decay of HPTS in the dehydrated mat), and the extracted value of dimensionality
should, therefore, be considered with great care.
26
HPTS in water
1
ROH in H2O
ROH in D2O
RO- in H2O
0.8
RO- in D2O
0.6
0.4
0.2
Normalized Intensity
1.0
Normalized Intensity
a
HPTS in water
0.1
0.01
1E-3
0.0
1E-4
0
4
8
12
16
20
0
4
8
Time [ns]
Dehydrated mat
Normalized Intensity
1.0
ROH in D2O
RO- in H2O
0.8
1
ROH in H2O
RO- in D2O
0.6
0.4
0.2
Normalized Intensity
b
12
16
20
24
28
24
28
24
28
Time [ns]
Dehydrated mat
0.1
0.01
1E-3
0.0
1E-4
0
4
8
12
16
0
20
4
8
Normalized Intensity
1
ROH in H2O
+50% H/D2O
1.0
ROH in D2O
-
RO in H2O
0.8
RO- in D2O
0.6
0.4
0.2
Normalized Intensity
c
12
16
20
Time [ns]
Time [ns]
+50% H/D2O
0.1
0.01
1E-3
0.0
0
4
8
12
16
20
1E-4
0
Time [ns]
4
8
12
16
Time [ns]
27
20
1
ROH in H2O
+100% H/D2O
1.0
ROH in D2O
Normalized Intensity
Normalized Intensity
d
RO- in H2O
0.8
RO- in D2O
0.6
0.4
0.2
+100% H/D2O
0.1
0.01
1E-3
0.0
1E-4
0
4
8
12
16
20
0
4
8
Time [ns]
Normalized Intensity
1
ROH in H2O
+200% H/D2O
1.0
ROH in D2O
Normalized Intensity
e
RO- in H2O
0.8
12
16
20
24
28
24
28
24
28
Time [ns]
RO- in D2O
0.6
0.4
0.2
+200% H/D2O
0.1
0.01
1E-3
0.0
1E-4
0
4
8
12
16
20
0
4
8
Time [ns]
Normalized Intensity
1
ROH in H2O
+300% H/D2O
1.0
ROH in D2O
RO- in H2O
0.8
RO- in D2O
0.6
0.4
0.2
Normalized Intensity
f
12
16
20
Time [ns]
+300% H/D2O
0.1
0.01
1E-3
0.0
1E-4
0
4
8
12
16
20
0
Time [ns]
4
8
12
16
20
Time [ns]
Figure S6. KIE of HPTS kinetics of (a) HPTS in water, (b) in the dehydrated mat and upon
the addition of (c) 50%, (d) 100%, (e) 200% and (f) 300% of D2O. The left graphs present the
data on a linear scale and the right graphs present the same data on a semi-log scale.
28
Figure S7. Image of the BSA mat on the gold finger array electrode. The bar represents 5 mm.
The Au fingers are not connected to the Au bar in the top of the image, and they are separated
from each other.
Resistance (Ohm)
2.0x109
1.5x109
1.0x109
5.0x108
0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
Distance (mm)
Figure S8. Contact resistance estimation for the BSA mats. The contact resistance was
estimated by extrapolation of the resistance to a hypothetical electrode separation distance l =
0. The deduced contact resistance is negligible for the conductance measurement across the
mat (since the resistance across the mat is significantly higher).
29
EIS
I-V
4E-10
8E-06
3E-10
6E-06
2E-10
4E-06
1E-10
2E-06
Current @0.05V [A] - I-V
Conductance [S] - EIS
1E-05
0E+00
0E+00
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
1/distance [mm-1]
Figure S9. The EIS conductance (left y-axis, black squares) and I-V current at 0.05V (right yaxis, red squares) as a function of 1/distance.
Table S2. Resistance and capacitance values as measured by EIS and fitted to the below electric
circuit.
Distance
[mm]
0.25
0.5
0.75
1
1.5
2
2.5
Calculated
Resistance [Ω]
0.98±0.30·105
1.60±0.39·105
2.52±0.98·105
3.39±1.35·105
4.72±1.81·105
6.49±2.73·105
8.08±3.04·105
Calculated
Capacitance [F]
4.32·10-12
3.68·10-12
2.98·10-12
2.33·10-12
1.50·10-12
1.28·10-12
1.14·10-12
30
Conductivity
[S·cm-1]
4.11·10-5
4.88·10-5
4.65·10-5
4.63·10-5
5.01·10-5
4.86·10-5
4.86·10-5
Electric
Circuit
150
0.25 mm
150
0.5 mm
150
0.75 mm
100
100
100
100
50
50
50
50
0
0
0
0
-50
-50
-50
-50
-100
-100
-100
-100
-150
D2O
H2O
-1
0
1
150
-150
-1
0
1
150
1.5 mm
-150
-1
0
1
150
2 mm
100
100
100
50
50
50
0
0
0
-50
-50
-50
-100
-100
-100
-150
-1
0
1
-150
-1
0
1
-150
-150
1 mm
-1
0
1
2.5 mm
-1
0
1
Bias [V]
ln(Conductance [-1])
Figure S10. I-V curve KIE data for all of the measured inter-electrode separation distances.
300000
-ZIm []
Current [nA]
150
200000
35C
30C
25C
20C
15C
-12.6
-12.8
Ea=0.31 eV
-13.0
-13.2
-13.4
3.28
3.36
3.44
1000/T [K-1]
100000
0
15C
35C
0
150000
300000
450000
600000
750000
ZReal []
Figure S11. Temperature dependence of the EIS for a 1.5 mm inter-electrode separation distance.
The inset shows the activation energy of the process by fitting to an Arrhenius equation.
31
0.3
Current [A]
0.2
0.1
b
15C
20C
25C
30C
35C
-18.2
ln(Conductance [S])
a
0.0
-0.1
-0.2
-18.4
Ea=0.29 eV
-18.6
-18.8
-19.0
-0.3
-1.0
-0.5
0.0
0.5
1.0
3.25
Bias [V]
3.30
3.35
3.40
3.45
3.50
-1
1000/T [K ]
Figure S12. (a) Temperature dependence of I-V data for a 0.75 mm inter-electrode separation
distance. (b) The carrier transport activation energy (~0.29 eV) was estimated by an Arrhenius fit (red
line) to the extracted conductance data (filled squares).
32
−1
𝜏𝑃𝑇
A
𝑘𝑃𝑇
D
A
B
log kPT
Over-barrier
Under-barrier
1/T
Figure S13. (a) Schematic representation for proton transfer mechanisms from proton donor
(D) to acceptor (A). The bottom straight arrow represents under the barrier proton transfer,
characterized by a large proton transfer rate (𝑘𝑃𝑇 ) in the order of 0.3-1×1012. The curved
arrows represent the trajectory of over the barrier proton transfer. In the latter case the proton
interacts with the barrier and resides thereon, resulting in a much longer proton lifetime (𝜏𝑃𝑇 ).
33
100
98
Intensity
96
94
92
90
88
86
84
4000 3500 3000 2500 2000 1500 1000 500
Wavenumber [cm-1]
Figure S14. FTIR spectrum of a dry BSA mat. The inset bracket identifies vibrational modes
associated with charged amino acids. The peaks around 1640 and 1530 cm-1 correspond to
amide I (C═O stretching) and amide II (C‒N stretching, N‒H bending) moities respectively.
Supplemental References
[1]
S. Fleischer, A. Shapira, O. Regev, N. Nseir, E. Zussman, T. Dvir, Biotechnol. Bioeng.
2014, 111, 1246-1257.
[2]
E. Pines, D. Huppert, N. Agmon, J. Chem. Phys. 1988, 88, 5620-5630.
[3]
N. Agmon, E. Pines, D. Huppert, J. Chem. Phys. 1988, 88, 5631-5638.
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