Electrocatalytic features of a heme protein attached to polymer-functionalized magnetic nanoparticles

Article abstract of DOI:10.1021/ac402421z

Direct electron-transfer and electrocatalytic kinetics of covalently attached myoglobin (MB) films on magnetic nanoparticles (MB-MNP _covalent), in comparison to the corresponding physisorbed films and individual components, are reported for the first time. MB-MNP _covalent ("-" denotes a covalent linkage) was adsorbed onto a cationic poly(ethyleneimine) layer (PEI) coated high-purity graphite (HPG) electrode. Similarly, films of myoglobin physisorbed on magnetic nanoparticles (MB/MNP_adsorbed, "/" denotes a noncovalent nature), only MB, or only MNP were constructed on HPG/PEI electrodes for comparison. The observed electron-transfer rate constants (k_s, s^-1) were in the following order: MB-MNP_covalent (69 ± 6 s ^-1), MB/MNP_adsorbed (37 ± 2 s^-1), only MB (27 ± 2 s^-1), and only MNP (16 ± 3 s^-1). The electrocatalytic properties of these films were investigated with the aid of tert-butylhydroperoxide as a model reactant, and its reduction kinetics were examined. We observed the following order of catalytic current density: MB-MNP_covalent > MB/MNP_adsorbed > only MNP > only MB, in agreement with the electron-transfer (ET) rates of MB-MNP _covalent and MB/MNP_adsorbed films. The crucial function of MNP in favorably altering the direct ET and electrocatalytic properties of both covalently bound MB and physisorbed MB molecules are discussed. In addition, the occurrence of a highly enhanced electron-hopping mechanism in the designed covalent MB-MNP_covalent films over the corresponding physisorbed MB/MNP_adsorbed film is proposed. The enhanced electron-transfer rates and catalytic current density suggest the advantages of using metalloenzymes covalently attached to polymer-functionalized magnetic nanoparticles for the development of modern highly efficient miniature biosensors and bioreactors.

Full text of DOI:10.1021/ac402421z

Article
pubs.acs.org/ac
Electrocatalytic Features of a Heme Protein Attached to Polymer-
Functionalized Magnetic Nanoparticles
Sadagopan Krishnan* and Charuksha Walgama
Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078, United States
ABSTRACT: Direct electron-transfer and electrocatalytic
kinetics of covalently attached myoglobin (MB) films on
magnetic nanoparticles (MB-MNPcovalent), in comparison to
the corresponding physisorbed films and individual compo-
nents, are reported for the first time. MB-MNPcovalent (“-”
denotes a covalent linkage) was adsorbed onto a cationic
poly(ethyleneimine) layer (PEI) coated high-purity graphite
(HPG) electrode. Similarly, films of myoglobin physisorbed on
magnetic nanoparticles (MB/MNPadsorbed, “/” denotes a
noncovalent nature), only MB, or only MNP were constructed
on HPG/PEI electrodes for comparison. The observed
electron-transfer rate constants (ks, s−1) were in the following
order: MB-MNPcovalent (69 6 s−1), MB/MNPadsorbed (37 2 s−1), only MB (27 2 s−1), and only MNP (16 3 s−1). The
electrocatalytic properties of these films were investigated with the aid of tert-butylhydroperoxide as a model reactant, and its
reduction kinetics were examined. We observed the following order of catalytic current density: MB-MNPcovalent > MB/
MNPadsorbed > only MNP > only MB, in agreement with the electron-transfer (ET) rates of MB-MNPcovalent and MB/MNPadsorbed
films. The crucial function of MNP in favorably altering the direct ET and electrocatalytic properties of both covalently bound
MB and physisorbed MB molecules are discussed. In addition, the occurrence of a highly enhanced electron-hopping mechanism
in the designed covalent MB-MNPcovalent films over the corresponding physisorbed MB/MNPadsorbed film is proposed. The
enhanced electron-transfer rates and catalytic current density suggest the advantages of using metalloenzymes covalently attached
to polymer-functionalized magnetic nanoparticles for the development of modern highly efficient miniature biosensors and
bioreactors.
btaining mechanistic insights on the electron-transfer and
catalytic properties of redox enzyme films on electrodes
applications.12−14 Among several heme proteins, myoglobin
(MB) has been a good electrochemical model protein due to its
easy commercial availability, peroxidase-like catalytic activity,
and established X-ray crystal structures.
Magnetic microsphere-antibody conjugates have been
utilized for fluorescence based detection of MB and heart-
type fatty acid-binding proteins relevant to acute myocardial
infarction.15 An MNP-based fluoroimmunoassay for detecting
serum MB has also been reported.16 In addition, the binding
properties of MB with MNP have demonstrated utility as
magnetic resonance imaging markers to visualize mice heart
and myocardium.17
Glassy carbon electrodes coated with the magnetic core−
shell Fe3O4@Al2O3 or Fe3O4@Au nanoparticles were used to
examine the direct electrochemistry of immobilized hemoglo-
bin (Hb), horseradish peroxidase (HRP), and MB.18,19 The
same group later reported the application of bifunctional
Fe3O4@ZrO2 nanoparticles20 and multifunctional heme
protein-Au-polydopamine-Fe3O4 magnetic nanoparticles21 to
evaluate the direct electrochemistry of heme proteins including
MB. Similarly, glassy carbon electrodes coated with poly-
O
can aid in the understanding of complex in vivo redox processes
as well as in the design of useful biosensors and bioreactors.1−3
Under these aspects, the direct electrochemistry of redox
enzyme films on electrodes has been extensively studied.
Moreover, developing efficient enzyme based, clean bioreactors
for the green syntheses of drug metabolites and precious fine
chemicals with high stereospecificity possess an enormous
potential to change the present paradigm of the pharmaceutical
field and boost the country’s economy. To develop efficient
electrochemical bioreactors with high catalytic efficiency in a
minimal reactor volume, elegant tailoring of remarkable enzyme
catalysts with nanomaterials is highly promising. In particular,
the role of magnetic particles (nano- and microparticles) in the
development of modern ultrasensitive electrochemical and
optical biosensors is immense, and more research avenues both
on the fundamental and applied aspects remain to be
explored.4−9
Heme proteins conjugated to magnetic nanoparticles (MNP)
have been useful in the development of highly sensitive point-
of-care biosensors10 and for the efficient stereoselective
synthesis of drug metabolites and chemical compounds.11
Additionally, the intrinsic peroxidase-like activity of MNP
enables the application in diagnostic tools and other biomedical
Received: August 1, 2013
Accepted: November 9, 2013
© XXXX American Chemical Society
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Scheme 1. MB and MNP Films Constructed on the Polymer (PEI)-coated HPG Electrodes for Direct Electrochemical
Investigation: (A) MB-MNPcovalent, (B) MB/MNPadsorbed, (C) only MB, and (D) only MNP
(dimethyldiallylammonium chloride) and Fe3O4@SiO2 mag-
netic nanoparticles were employed to adsorb MB in solution.
These electrodes were reported to be useful for peroxide
detection with high thermostability.22
experiments were performed with an Autolab rotor attached to
a rotation controller unit. A sealed electrochemical cell
employing a Ag/AgCl reference electrode (1 M KCl, CH
Instruments Inc.), a Pt-wire counter electrode, and a MB film-
coated HPG working electrode was used. PBS solutions were
purged with high-purity nitrogen for 30 min before the
voltammograms were acquired, and a continuous nitrogen
atmosphere was maintained during measurements.
Covalent Attachment of Myoglobin to Magnetic
Nanoparticles (MB-MNPcovalent). The surface lysine residues
of MB (PDB 2FRF)25 were covalently attached to the
carboxylic acid groups of polyacrylic-acid functionalized MNP
according to the manufacturer’s protocol (Chemicell GmbH
Inc.) (Scheme 1A). Briefly, a freshly prepared aqueous solution
(300 μL) containing EDC (0.35 M) and NHS (0.1 M) was
added to 2 mg of MNP and incubated for 15 min to activate the
carboxylic acid groups of MNP by converting them into N-
succinimidyl esters, which are facile leaving groups. The
activated nanoparticles were separated from the unreacted
EDC/NHS solution by application of a magnetic field. Then
the particles were washed in water and immediately suspended
in MB solution (1 mL of 3 mg mL−1 in pH 5.0 acetate buffer).
The covalent attachment of MB to MNP was conducted for 2 h
at room temperature with continuous gentle mixing. The MB-
attached particles were separated from the free MB in solution
by employing a magnetic field provided by a magnet.
Although prior reports addressed many useful sensing and
direct electrochemistry aspects of MNP on heme proteins, we
report here the first mechanistic insights with respect to the
covalent attachment and physisorption of MB onto MNP and
their comparative electrochemistry with films of either MB or
MNP alone on electrodes. In this paper, the enhanced electron-
transfer and catalytic rates for MB films covalently attached to
poly(acrylic acid)-functionalized MNP and coated on HPG/
PEI electrodes are presented. To gain insight, systematic
comparisons were made with MB films physisorbed on
poly(acrylic acid)-functionalized MNP, only MB film, or only
MNP film coated on similar electrodes. Scheme 1 illustrates the
four types of films that were constructed on HPG/PEI
electrodes and investigated in this study for the first time.
EXPERIMENTAL SECTION
■
Chemicals and Materials. Polyacrylic acid-functionalized
magnetic nanoparticles (MNP, 100-nm hydrodynamic diame-
ter) were purchased from Chemicell GmbH Inc. (Berlin,
Germany). Equine heart myoglobin (MB), tert-butylhydroper-
oxide (t-BuOOH), poly(ethyleneimine) (PEI), 1-ethyl-3-[3-
dimethylaminopropyl] carbodiimide hydrochloride (EDC), and
N-hydroxysuccinimide (NHS) were purchased from Sigma.
High-purity graphite disk electrodes (HPG, geometric area 0.2
cm2) were purchased from McMaster-Carr (Atlanta, GA). All
other chemicals were of analytical grade.
The MB-MNPcovalent conjugate was washed twice in PBS,
resuspended in a fresh PBS solution (200 μL), and then used
for electrochemical studies. Similarly MB films physisorbed on
MNP (no EDC/NHS step) (denoted as MB/MNPadsorbed
)
were prepared under similar conditions. A pH 5.0 condition
was used to make MB solutions in order to obtain a net positive
charge of MB (isoelectric points, pI = 6.8 and 7.2),26,27 and
thus facilitate its interaction with the negatively charged
poly(acrylic acid) groups (pKa 4.3)28 of MNP during the
covalent attachment or the physisorption process. Once the
MB-MNPcovalent conjugate and MB/MNPadsorbed systems were
prepared, they were resuspended in physiological pH 7.4 (PBS)
to obtain a net negative surface charge of MB-MNPcovalent and
MB/MNPadsorbed films suitable to immobilize onto positively
charged PEI (a cationic polymer) coated HPG electrodes. For
comparison, free MB solution and free MNP suspension in PBS
were prepared under similar conditions and used for electro-
chemical measurements.
To obtain steady-state reduction currents for various oxygen
concentrations, known percent mixtures of oxygen in nitrogen
were supplied to the electrochemical cell using two mass flow
controllers (Aalborg Instruments and Controls Inc., NY). The
percent oxygen was converted into concentration using Henry’s
law.23,24 Unless otherwise specified, all electrochemical
measurements were performed with an anaerobic phosphate
buffer saline (PBS) electrolyte, pH 7.4 at 25 °C, under a N2
atmosphere. The current density data presented in this work
are with respect to the geometric area of electrodes (0.2 cm2).
Instrumentation. A CH instrument 6017 electrochemical
analyzer was used to perform cyclic voltammetry (CV) studies.
To conduct peroxide reduction kinetics catalyzed by MB films,
rotating-disk electrochemistry (RDE) was used. The RDE
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Preparation of MB Films on HPG Electrodes (Scheme
1). Prior to use, HPG electrodes were freshly polished on a
P320 grit SiC paper, ultrasonicated for 30 s in ethanol, followed
by 30 s in water, and then the electrodes were dried under
nitrogen. A solution of PEI (2 mg mL−1 in water) was placed
on the polished electrode and allowed to electrostatically
adsorb for 15 min. To the PEI-adsorbed HPG electrodes
(HPG/PEI), a 15-μL suspension of MB-MNPcovalent conjugate
in PBS was added and allowed to immobilize for 30 min. The
electrodes were rinsed with water between each adsorption step
to remove weakly and unbound molecules. Similarly, the PEI-
coated HPG electrodes were used to immobilize films of MB/
MNPadsorbed or only MB or only MNP.
RESULTS
■
Quantitation of MB Bound to MNP. The amounts of
bound MB in MB-MNPcovalent and MB/MNPadsorbed films were
estimated by use of a UV−vis spectrophotometer (Thermo
Scientific, model Evolution 60S). From the difference in Soret
band-absorbance of free MB in solution at 410 nm before and
after coating to MNP, it was determined that 23 3 nmol of
MB were covalently attached to MNP in MB-MNPcovalent films
and 13 2 nmol of MB were physisorbed with MNP in MB/
MNPadsorbed films. This quantitation suggests that covalent
attachment enables the high-density loading of protein
molecules onto MNP as opposed to physisorption. This can
be attributed to the large number of available activated
−COOH groups on MNP and less sites of contact required
between MB and MNP in the covalent mode of linking (only
the surface Lys residues of MB are involved in the amide-bond
formation with activated −COOH groups of MNP).
On the other hand, the physisorption of MB with MNP may
involve contacts between multiple surface amino acid groups of
MB with the surface of MNP to form an electrostatic network.
This could induce a sterically restricted arrangement of MB
molecules on the MNP surface and hence the detected less
amount of bound MB molecules. Using the data provided by
the manufacturer (Chemicell Inc.) on the number of MNP
present per g weight (∼1.8 × 1015) and by assuming a uniform
distribution of MB molecules on MNP, we calculated that
∼3860 MB molecules could be bound per MNP in MB-
MNPcovalent conjugate and ∼2350 MB molecules could be
bound per MNP in the MB/MNPadsorbed system.
Figure 1. Background-subtracted thin-film cyclic voltammograms of
(a) MB-MNPcovalent, (b) MB/MNPadsorbed, (c) only MB, and (d) only
MNP films immobilized on PEI-coated HPG electrodes in anaerobic
PBS at 25 °C; scan rate, 0.3 V s−1.
in the range of 100−120 mV (slightly greater than the ideal
one-electron value of 90.6/n mV).30 These electrochemical
properties correspond to the nonideal quasi-reversible thin-film
surface voltammetry of MB-MNPcovalent, MB/MNPadsorbed, only
MB, or only MNP films on HPG/PEI electrodes.31−34
The average electroactive amounts ( 5−8% SD) of MB in
different films were determined to be in the following order:
MB-MNPcovalent (129 pmol cm−2) > MB/MNPadsorbed (78 pmol
cm−2) > MB film alone (36 pmol cm−2). The electroactive
amount of MNP film alone was 57 pmol cm−2. The higher
electroactive amount of MB-MNPcovalent film over the MB/
MNPadsorbed film additionally suggested a greater proportion of
connected heme centers of MB-MNPcovalent film with the
electrode surface. About 3.6-times enhancement in the
electroactive amount of MB was observed as a result of
covalent attachment to MNP over the free MB film on HPG/
PEI electrodes. This property suggested that MNP in MB-
MNPcovalent film carried a large excess of covalently attached MB
to get immobilized on HPG/PEI electrodes to give high
electroactive protein amount. The physisorbed films of MB in
the MB/MNPadsorbed system showed a smaller enhancement in
electroactive amount (by 2.2-times) over the free MB film.
Moreover, to obtain clearly measurable peak-potentials for
direct ET rate calculation from the CVs of free MB films on
HPG/PEI electrodes (Figure 1), it was deemed necessary to
use a 7 times greater amount of MB in PBS solution (161 nmol
in 200 μL of PBS) in comparison to the amount of MB (23
nmol) immobilized on MB-MNPcovalent films. This property
suggests the general advantage of using relatively inexpensive
MNP to conjugate a minimal amount of expensive metal-
loenzyme catalysts to obtain superior direct electrochemical
activity. It was found that for comparative catalytic O2 and
peroxide reduction kinetics of free MB in solution (detail
below), 23 nmol of free MB (in 200 μL of PBS) provided
measurable limiting currents, similar to the amount of bound
MB in MB-MNPcovalent films.
Electrochemical Properties. Figure 1 represents the
background subtracted cyclic voltammograms (CVs) of MB-
MNPcovalent, MB/MNPadsorbed, only MB, or only MNP films
coated on HPG/PEI electrodes. Nearly the same midpoint
potential (E°′) of −0.34 V ( 0.01) versus Ag/AgCl in PBS was
observed for all MB films (MB-MNPcovalent, MB/MNPadsorbed
,
and only MB) (N = 3 repeats). On the other hand, a more
positive E°′ of −0.27 V ( 0.01) versus Ag/AgCl was observed
for the MNP film alone (N = 3 repeats). The Fe3O4 MNP used
in this study consisted of two iron atoms in the 3+ state and
one iron atom in the 2+ state29 and hence were identified to
exhibit reversible voltammetry, however, at a more positive E°′
than MB films. The observed characteristic E°′ suggests that
the voltammetric features correspond to MB in MB-MNPcovalent
and MB/MNPadsorbed films (Figure 1).
For all the MB and MNP films examined, the peak current
versus scan rate was linear in the scan rate range of 0.02 to 2 V
s−1 and the oxidation to reduction peak-current ratio was near
1. Reduction peak potentials shifted more negative with
increasing scan rate, and peak widths at half-maximum were
Rate of Direct Electron Transfer. The rate at which an
electron transfer (ET) occurs between a protein film and an
electrode (during oxidation and reduction) can be determined
from the peak separation (ΔEp) between the measured
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reduction (Ep,red) and oxidation (Ep,ox) values with increasing
scan rate (i.e., potential ramp applied in V s−1).35 This rate
calculation for surface-bound molecules was derived by Laviron
based on the Butler−Volmer surface voltammetry, which
assumes that a redox system can be characterized by a single
ET rate constant that is the rate at the formal potential.36
A simplified relationship between the ET rate constant (ks,
s−1) and peak separation for a surface-bound redox molecule on
an electrode can be given as36
ks = (nFm × scan rate)/RT
(1)
where n is the number of electrons involved in the ET, F is
Faraday’s constant (96 485 C mol−1), R is the gas constant
(8.314 J mol−1 K−1), T is temperature in Kelvin, and m is a
variable that is inversely related to the experimental peak
separation (i.e., m ∝ 1/nΔEp) in a nonlinear fashion, as derived
by Laviron.36
Thus, for a reversible redox process (fast reduction and
oxidation), the peak separation between Ep,red and Ep,ox is small,
m becomes large, and thus ks is high. On the other hand, for a
quasi-reversible or slow redox process of a protein film on an
electrode, the peak separation increases with increasing scan
rate and ks is moderate to low.31−35 Figure 2 shows the
Figure 3. Rotating-disk catalytic oxygen-reduction voltammograms of
(A) MB-MNPcovalent, (B) MB/MNPadsorbed, (C) only MB, and (D)
only MNP films with increasing oxygen concentration, (a) 900, (b)
600, (c) 300, and (d) 0 μM in PBS at 25 °C, 1000 rpm rotation rate,
scan rate 0.1 V s−1. Voltammograms e and f in part A correspond to
voltammograms in 300 μM oxygen for (e) only HPG and (f) HPG/
PEI, which did not exhibit any catalytic currents in the absence of MB
and/or MNP in the film.
accordance with the extent of electroactively connected MB
molecules in each film as detailed above.
Catalytic Peroxide-Reduction Kinetics. Catalytic per-
oxide-reduction rates of designed MB and MNP films on HPG/
PEI electrodes were tested with t-BuOOH as a model reactant.
Heme protein films, such as MB on electrodes, can catalytically
reduce t-BuOOH to form tert-butanol.31,37 This catalytic
reduction can be monitored as an increase in limiting current
in the RDE due to the formation and subsequent electro-
•
chemical reduction of reactive MB-FeIVO into MB-FeIII in
the presence of a peroxide.31,38 Investigating the kinetics of t-
BuOOH reduction to tert-butanol by heme protein films is
straightforward and not complicated by the formation of several
radical intermediates as in hydrogen peroxide reduction and
discussed elsewhere.32,37 Figure 4 displays representative
rotating disk voltammograms for MB and MNP films with
increasing t-BuOOH concentration.
Figure 2. Direct electron-transfer rate constants (ks) of MB-
MNPcovalent, MB/MNPadsorbed, only MB, or only MNP films on PEI-
coated HPG electrodes in anaerobic PBS, 25 °C [avg SD for N = 3
repeats].
determined electron-transfer (ET) rate constants for MB-
MNPcovalent, MB/MNPadsorbed, only MB, or only MNP films on
HPG/PEI electrodes. The ks of each film was calculated from
the peak separation in CVs with increasing scan rate after
subtraction of a small nonkinetic constant peak separation
obtained at slow scan rates, similar to that reported.31−34
Catalytic Oxygen Reduction. Oxygen reduction is a
characteristic feature of heme proteins. The reduced FeII-heme
protein can bind to oxygen to form an FeII−O2 complex, which
is electrochemically reduced on the electrode to regenerate
FeIII. This generates a catalytic current in proportion to the
supplied oxygen concentration.34 Figure 3 displays the oxygen
concentration dependent, steady-state reduction currents
catalyzed by the designed MB films. The following order of
oxygen reduction current density was obtained: MB-MNPcovalent
> MB/MNPadsorbed > only MB > only MNP films. The MB-
MNPcovalent films exhibited the largest limiting oxygen-reduction
currents in comparison to other MB films. This finding is in
Figure 4. Representative steady-state catalytic reduction voltammo-
grams for (A) MB-MNPcovalent, (B) MB/MNPadsorbed, (C) only MB,
and (D) only MNP films immobilized on PEI-coated HPG electrodes
with increasing t-BuOOH concentration in oxygen free PBS, 25 °C,
scan rate 0.1 V s−1, 1000 rpm rotation rate: (a) 2.8, (b) 1.0, (c) 0.4,
(d) 0.1, and (e) 0 mM.
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We observed that the increase in limiting reduction current
with increasing concentration of t-BuOOH followed Michae-
lis−Menten kinetics (Figure 5). Electrochemical Michaelis−
MNPadsorbed film (Scheme 1 and Figure 2). The observed
enhanced ET rate indicates the crucial role of iron-oxide centers
(Fe3O4) in MNP to act as efficient ET mediators for MB
molecules, possibly via an electron-hopping mechanism.
The initial electron from the electrode can either reach the
MB-heme center or an MNP-iron center in MB-MNP films.
However, the observed direct electrochemical properties of
MB-MNPcovalent and MB/MNPadsorbed films correspond to MB
and not to MNP (Figure 1). Hence, the initial electron transfer
from the HPG/PEI electrode should be to the MB-heme
center, which can subsequently undergo a hopping mechanism
with closely arranged MB-heme centers and via MNP to distant
MB-heme centers in the film (as proposed in Scheme 2). On
the other hand, even if the initial electron from the electrode
reaches an MNP-iron center in MB-MNPcovalent film, it is
possible that a subsequent hopping mechanism and significantly
faster ET rate of MB over MNP (Figure 2) seems to have
kinetically dominated to provide the observed MB electro-
chemistry in MB-MNP films.
Lisdat et al. demonstrated the occurrence of electron-
hopping between protein redox centers in a multilayer film
consist of up to 15 protein layers assembled alternatively with
an anionic polyelectrolyte.41 Hu et al. demonstrated an efficient
electron-hopping mechanism mediated by gold nanoparticles to
surface MB-heme centers over distances of >100 nm in films on
basal plane graphite electrodes.42
Recently, a carbon ionic liquid electrode coated with carbon
coated Fe3O4 nanospindles, MB, chitosan, and another
coupling ionic liquid constructed in a stepwise manner to
form a stable film was used to study the direct electrochemistry
of cast MB.43 The authors suggested the role of Fe3O4
nanospindles to act as an electron transfer mediator between
the MB heme center and the modified electrode surface.
However, the redox property of Fe3O4 nanospindles alone in
the absence of MB was not detailed.43
Figure 5. Michaelis−Menten electrochemical kinetics for peroxide
reduction by (a) MB-MNPcovalent, (b) MB/MNPadsorbed, (c) only MNP,
and (d) only MB films for the data shown in Figure 4 (avg SD for N
= 3 repeats).
Menten kinetics relating the catalytic current (Icat) to reaction
rate parameters is given by37,39,40
nFAΓ(kcat/KM)Cs
(1/KM)Cs + 1
Icat
=
(2)
where n is the number of electrons transferred in a catalytic
reaction (n = 2 for peroxide reduction by heme proteins), F is
Faraday’s constant, A is electrode area (0.2 cm2), Γ is the
electroactive enzyme amount coated on an electrode (in nmol
cm−2), Cs is the reactant concentration in solution (in mM), kcat
is the catalytic rate constant (s−1), and KM is the Michaelis−
Menten constant. The Γ is obtained from the peak area (Q,
corresponding to the charge in coulombs) for reduction in the
cyclic voltammogram of a redox protein and using the equation,
Q = nFAΓ.31
DISCUSSION
The observed highly enhanced ET rate of MB-MNPcovalent
film over MB/MNPadsorbed film (Figure 2) in our work further
suggests the existence of additional favorable factors to offer a
better ET property in covalent MB-MNP films. The covalent
immobilization of MB to MNP provides amide bond centers in
MB-MNPcovalent films that are absent in the case of physisorbed
■
Enhanced ET Rates and Electron-Hopping. Myoglobin
covalently attached to magnetic nanoparticles in MB-
MNPcovalent films and coated on HPG/PEI electrodes exhibited
an enhanced ET rate of 2.6-fold over only MB film, 4.3-fold
over the MNP film alone, and nearly 2-fold over the MB/
Scheme 2. Long-Range Electron-Hopping Mechanism Occurring in MB-MNPcovalent Films That Is Effectively Mediated by MNP
a
and Further Facilitated by Intramolecular H-Bond Network between Amide Bonds (Connecting MB and MNP Covalently)
a
The additional role of water molecules in enhancing the long-range electron hopping, via intermolecular H-bonding between water molecules and
with the amide bond centers, is also illustrated.
E
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MB/MNPadsorbed film. The property of amide bond centers in
mediating an electron-hopping mechanism between an
electrode and helical ferrocene oligopeptide, aided by an
intramolecular H-bond network, has been recently proposed.44
A similar hopping phenomenon is projected to occur in the
designed MB-MNPcovalent films to provide the observed largest
ET rate (Figure 2). Additionally, the contribution of a H-
bonded network formed by internal amide bonds in self-
assembled monolayers on gold in attaining long-range electron
transfer processes was suggested.45
Very recently, the effectiveness of short-range H-bonds to
efficiently transport ET processes between single molecules was
demonstrated.46 Additionally, it has been shown that water
channels that follow the Brownian dynamics can mediate both
short- and long-range electron self-exchange processes between
redox protein molecules.47
enzymes to achieve catalysis with exceedingly strong affinity for
substrates and highly enhanced current density (Figure 5). The
obtained apparent KM of MB-MNPcovalent films is similar to that
reported for MB films on carbon-coated Fe3O4-nanospindles
modified carbon ionic-liquid electrodes toward reducing
trichloroacetic acid.43
The observed order of relative catalytic reduction efficiency
(104 kcat/KM in M−1 s−1) for the designed films was in the
following order (Figure 5 and eq 2): MB-MNPcovalent (14.2
0.7) ≈ MB/MNPadsorbed (14.8 1.0) > only MB (5.5 0.4) >
only MNP (5.2 0.5). The obtained value of kcat/KM of only
MB films is in good agreement with that reported for MB films
assembled with polyions on pyrolytic graphite electrodes32 and
thus validates the accuracy of measurements performed in this
study. More interestingly, the catalytic efficiency of MNP-MB
films is improved by at least 2.5-times in comparison to only
the MB film or the MNP film alone. Despite the fact that
comparable catalytic efficiency was observed between the
covalent and physisorbed MB films on MNPs, the MB-
MNPcovalent film displayed significantly higher peroxide
reduction current density over MB/MNPadsorbed film, a property
that is preferred in the design of bioreactors for chemical
synthesis and other enzymatic electrochemical devices.
In addition, we found that MB-MNPcovalent suspension in
PBS was more stable than the MB/MNPadsorbed suspension in
PBS. After 1 week of storage in PBS at 4 °C, the constructed
film of MB-MNPcovalent on HPG/PEI electrodes retained ∼85%
of the initial oxygen reduction limiting-current, while the
retention in limiting-current for MB/MNPadsorbed film was only
∼60%. Taken together, we have demonstrated that the rate of
electron transfer and catalytic efficiency of either MB or MNP
alone can be significantly amplified upon the conjugation of
both together (Figures 2−5). In addition, the covalent strategy
offered better electrochemical, catalytic, and stability properties
than the corresponding physisorbed films. This suggests the
usefulness of designing metalloenzyme-MNPcovalent films to
achieve efficient direct ET and in the development of efficient
bioreactors for fine chemical synthesis.
On the basis of the obtained results in this work and in
reference to the findings from prior studies,43−47 we propose an
electron-hopping mechanism occurring in MB-MNPcovalent films
that involve long-range electron-hopping mediated by MNP to
MB molecules and additionally facilitated by a H-bond network
formed between amide bonds (i.e., −COOH of MNP with
−NH2 groups of MB forming an amide bond as a result of
covalent attachment, as detailed above) both directly and via
water molecules present in the electrolyte buffer (Scheme 2).
Interestingly, the ET rate of MB/MNPadsorbed film appears
nearly to be the additive rates of MNP and MB films alone.
This suggests the existence of similar hopping phenomenon
occurring in these electrostatically assembled noncovalent films,
and further indicates the absence of additional hopping forces
such as from the amide bonds, as proposed to be present in
MB-MNPcovalent films (Scheme 2).
The rate of electron-transfer reported between the electrode
and MB in certain magnetic nanoparticles-based studies were
small and in the range of 3.0−3.6 s−1.18−21 This could arise
from the buried inner Fe3O4 structure of MNP with an
insulating outer oxide layer as used in several prior reports for
MB immobilization. In addition, dry-coated films of MB on
MNP-modified electrodes may have poor control over the
arrangement and stability of protein molecules in films.
Catalytic Peroxide-Reduction Efficiency. The following
order of catalytic peroxide reduction current density was
exhibited by the designed MB films (Figure 5): MB-MNPcovalent
> MB/MNPadsorbed > only MNP > only MB. The enhancements
in the reduction current density were in good agreement with
the ET rates of MB-MNPcovalent and MB/MNPadsorbed films. On
the other hand, although the direct ET rate of only MB film was
slightly greater than the MNP film alone, the observed order of
catalytic current density was opposite. This is due to the greater
proportion of electroactive concentration of MNP present in
HPG/PEI film than in MB molecules (Figure 1) under the
experimental conditions followed. This observation further
indicates the presence of an effective electron hopping
mechanism occurring between iron centers of large MNP
(100 nm hydrodynamic diameter, Chemicell Inc.) in MNP
films.
CONCLUDING REMARKS
■
The results presented above provide the first mechanistic
insights into the fundamental electrochemical and catalytic
properties of a heme redox protein when combined with
magnetic nanoparticles. The results obtained suggest that one
can favorably tune the rates of ET and electrocatalysis of a
metalloenzyme film on an electrode by simple chemical
conjugation with functionalized magnetic nanoparticles, requir-
ing only minimal enzyme amount. The large surface area of
MNP with appropriate functional groups can enable the high
density attachment of protein molecules in stable form along
with a superior electronic connectivity to the electrode suitable
for constructing novel bioreactors for chemical synthesis. We
suggest the existence of an efficient long-range electron-
hopping mechanism as the major driving force for the observed
amplified ET and catalytic rates of MNP-protein films on
electrodes. The findings of this study thus possess considerable
potential in the development of novel efficient biosensors,
nanochip-devices, bioreactors, and fundamental mechanistic
studies of other enzyme films (including redox enzymes with
buried redox centers) by connecting them efficiently to
electrode surfaces via MNP.
The apparent Michaelis−Menten constant (eq 2), KM, was
found to be 1.36 ( 0.2) mM for MB-MNPcovalent, 1.15 ( 0.05)
mM for MB/MNPadsorbed, 1.6 ( 0.2) mM for only MB, and 2.9
( 0.1) mM for only MNP film (Figure 5, N = 3 repeats). The
smaller KM values of MB-MNPcovalent and MB/MNPadsorbed films
over either MB or MNP film alone suggest the advantages of
incorporating MNP into the natural elegantly designed redox
F
dx.doi.org/10.1021/ac402421z | Anal. Chem. XXXX, XXX, XXX−XXX

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AUTHOR INFORMATION
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Corresponding Author
*E-mail: gopan.krishnan@okstate.edu.
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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The financial support by the Oklahoma State University is
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