Impact of Surface Modification with Gold Nanoparticles on the Bioelectrocatalytic Parameters of Immobilized Bilirubin Oxidase.

2014·0.23 MB·English

by  Pankratov, D. V.

SHOrt rePOrtS
Impact of Surface Modification with Gold
Nanoparticles on the Bioelectrocatalytic
Parameters of Immobilized Bilirubin
Oxidase
D. V. Pankratov1,2, Y. S. Zeifman2, А. V. Dudareva3, G. K. Pankratova1, M. E. Khlupova1,
Y. M. Parunova2, D. N. Zajtsev3, N. F. Bashirova3, V. O. Popov1,2, and S. V. Shleev1,2,3*
1 A.N. Bach Institute of Biochemistry of Russian Academy of Sciences, Leninsky Ave. 33, building 2,
119071 Moscow, Russia
2 National Research Center “Kurchatov Institute”, Akademika Kurchatova Sq. 1, 123182 Moscow,
Russia
3 I.G. Petrovsky Bryansk State University, Bezhitskaya St. 14, 241036 Bryansk, Russia
*E-mail: [email protected]
Received 14.12.2013
Copyright © 2014 Park-media, Ltd. This is an open access article distributed under the Creative Commons Attribution License,which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
abStract We unveil experimental evidence that put into question the widely held notion concerning the impact
of nanoparticles on the bioelectrocatalytic parameters of enzymatic electrodes. Comparative studies of the bio-
electrocatalytic properties of fungal bilirubin oxidase from Myrothecium verrucaria adsorbed on gold electrodes,
modified with gold nanoparticles of different diameters, clearly indicate that neither the direct electron transfer
rate (standard heterogeneous electron transfer rate constants were calculated to be 31±9 s-1) nor the biocatalytic
activity of the adsorbed enzyme (bioelectrocatalytic constants were calculated to be 34±11 s-1) depends on the
size of the nanoparticles, which had diameters close to or larger than those of the enzyme molecules.
KeyWordS gold nanoparticle; bilirubin oxidase; direct electron transfer; bioelectrocatalysis.
abbreViationS 3D – three-dimensional; A – real/electrochemical surface area; CV – cyclic voltammogram;
real
ET – electron transfer; DET – direct electron transfer; k – standard heterogeneous electron transfer rate con-
0
stant; kapp – apparent bioelectrocatalytic constant; MCO – blue multicopper oxidase; MvBOx – Myrothecium
cat
verrucaria bilirubin oxidase; AuNP – gold nanoparticles with diameter of n nm; AuNP /Au – gold electrode
n n
modified with AuNP before and after cycling in sulfuric acid (m-AuNP/Au u-AuNP/Au, respectively); ThLc –
n
Trametes hirsuta laccase; PBS – phosphate buffered saline; NHE – normal hydrogen electrode; SEM – scanning
electron microscopy; A – absorbance maximum; А – absorbance at the wavelength of 450 nm; Γ – enzyme
spr 450
surface concentration; j – maximum current density.
max
introduction (ThLc) fungus immobilized on the gold surface). For in-
numerous studies have reported on effective direct stance, the use of AunP and nanoporous gold helped to
electron transfer (Det) of various enzymes (including increase Det [4], whereas an extremely low bioelectro-
blue multicopper oxidase – McO) immobilized on the catalytic activity of the enzyme was observed for Det
surface of nanostructured electrodes with metal and [5] in nano/microstructured silicon chips modified with
carbon nanoparticles, carbon nanotubes, graphene, gold with the enzyme immobilized on their surface.
etc. [1–3]. An increase in the bioelectrocatalytic cur- Since the use of bioelectrodes without a nano-modified
rent when using nanostructured surfaces was regard- surface leads to a very low heterogeneous transfer rate
ed as the defining evidence behind the acceleration of and sometimes to a complete absence of Det, the rou-
the Det reaction in these studies; however, neither a tine explanation for enzyme “nanobinding” is the ori-
quantitative comparative analysis of Det based on a entation of the enzyme on the nanostructured surface,
voltammograms analysis, nor a calculation of the stand- which contributes to Det.
ard constants of the heterogeneous electron transfer Despite the fact that a possible dependence of k on
0
reaction (k ) has been performed. Moreover, there are the size of the metal or carbon nanoparticles has yet
0
serious discrepancies in the data even for a single en- to be studied and that the two opposite dependences
zyme (in particular, laccase from the Trametes hirsuta of the bioelectrocatalytic oxygen reduction current
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SHOrt rePOrtS
on the diameter of AunP on electrodes modified with Sonication was performed using a ultrasonic clean-
McO were recently demonstrated (e.g., [6]), the belief er XB2 bath (VWr International Ltd., uK). SeM was
remains that the size of AunP used for electrode na- performed on a FeI nova nanoLab 600 high-resolu-
nomodification is a very important factor that deter- tion scanning electron microscope (the netherlands).
mines et in the reactions between a redox enzyme and Spectrophotometric studies were carried out using a
the electrode surface. this study offers experimental PharmaSpec uV-1700 uV-visible spectrophotometer
results that demonstrate the unlikeliness of this hy- (china).
pothesis. nanoparticles with a diameter of 5 to 60 nm were
synthesized to study the impact of the AunP size on
eXPerimental the biocatalytic properties of MvBOx.
Materials and Methods Synthesis of gold nanoparticles
na HPO ∙2H O, naH PO ∙H O, nacl, HAucl ∙3H O, with a preset particle size
2 4 2 2 4 2 4 2
H O , H SO , naBH , and sodium citrate were pur- AunP with the expected diameter of 5 nm (AunP, Fig.
2 2 2 4 4 5
chased from Sigma-Aldrich GmbH (Germany) and 1A) were synthesized as described in [7]. 50 ml of a 250
used without further purification. Oxygen was ac- μM HAucl solution was stirred for 1 min at room tem-
4
quired from AGA Gas AB (Sweden). Buffers and perature, then 1111 μl of a 38.8 mM sodium citrate solu-
other solutions were prepared using deionized water tion was added to the initial solution, and the mixture
(18 MΩ∙cm) produced using a PureLAB uHQ II sys- was stirred for another 1 min. next, 555 μl of a freshly
tem (eLGA Labwater, uK). All experiments were per- prepared 0.075% (wt.) naBH solution in a 38.8 mM so-
4
formed at room temperature in PBS (pH 7.4) consisting dium citrate solution was poured into the reaction mix-
of a 50 mM HPO 2-/H PO - solution containing 150 mM ture and the solution was stirred for another 5 min.
4 2 4
nacl. AunP with a diameter of 20–60 nm (Fig. 1A) were
MvBOx was a gift from Amano enzyme Inc. (Ja- synthesized using sodium citrate as a reductant. 50 ml
pan). of a 250 μM HAucl solution was brought to boil under
4
electrochemical measurements were performed us- constant stirring; 750, 500, or 260 μl of a 1% (wt.) so-
ing a μAutolab type III/FrA2 potentiostat/galvano- dium citrate solution was subsequently added to obtain
stat (Metrohm Autolab BV, the netherlands) using a AunP with diameters of 20, 40, and 60 nm (AunP ,
20
three-electrode circuit with a saturated calomel refer- AunP and AunP ), respectively. After adding sodi-
40 60
ence electrode (242 mV vs. normal hydrogen electrode, um citrate, the mixture was incubated for 10 min under
nHe) and a platinum wire as an auxiliary electrode. constant stirring without heating.
А B 20 nm 40 nm 60 nm Fig. 1. A. Photos of the
colloidal solutions of syn-
thesized AuNPs; B. SEM
5 nm 20 nm 40 nm 60 nm
images of AuNP/Au
samples; C. SEM image
of AuNP /Au before
200 nm 200 nm 200 nm 40
(top) and after (bottom)
D 0.20 2 cycles in H SO : D.
C 2 4
Absorbance spectra of
AuNP suspensions with
s 0.16
nit different diameters
u
.
el 0.12
r
500 nm e, 5 nm
c 20 nm
n
a 0.08 40 nm
b 60 nm
r
o
s
b 0.04
A
0.00
400 450 500 550 600 650 700 750
500 nm
Wavelength, nm
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Table 1 | Comparative analysis of the diameters of synthe- Table 2 | Real surface area vs. nanoparticles size
sized AuNP as determined using different methods
AunP diameter, nm real surface area, cm-2
Diameter of AunP, nm
5 0.21±0.01
Determined spectropho- Determined using
expected
tometrically the SeM data
20 1.40±0.01
20 16 19±2
40 1.25±0.05
40 42-51 38±5
60 1.23±0.03
60 77 59±5
the diameter of the resulting nanoparticles was were mechanically cleaned through polishing with
evaluated spectrophotometrically in accordance with Microcloth paper (Buehler, uK) in an aluminum ox-
the procedure described in [7] using the wavelength of ide suspension with a particle size of 0.1 μm (Struers,
maximum absorbance (A , Fig. 1D). the diameter of a Denmark) to obtain a mirror surface. the electrodes
spr
AunP less than 35 nm in size was calculated using the were further washed with deionized water and elec-
A /A ratio. trochemically purified through cycling in 0.5 M H SO
spr 450 2 4
nanoparticles 20-60 nm in size were further identi- using a range of potentials from –0.1 to +1.9 V vs. nHe
fied using SeM. the samples for microscopy were pre- for 20 cycles at a scan rate of 0.1 V∙s-1, then they were
pared by applying a small amount of AunP obtained washed with water and dried in an air stream.
from diluted colloidal solutions over the flat surface of Following this, 5 μl of the solution (for concentrat-
gold electrodes (Fig. 1B). It should be emphasized that ed suspensions) or 6 μl of the solution (for an AunP
the surface structure of the electrodes used in further suspension with a particle diameter of 5 nm (AunP ))
5
studies was fundamentally different from that shown was applied to the cleaned gold electrode surface. the
in Fig. 1 due to a significantly higher amount of applied modified electrode was then dried at room tempera-
AunP and surface changes resulting from treatment ture. the AunP modification procedure was repeated
with H SO (see below); thus, it can only be used to twice for concentrated suspensions of nanoparticles
2 4
evaluate the diameters of the synthesized AunP. and 5 times for AunP . the obtained electrodes were
5
the results of a size evaluation of the nanoparticles cycled in 0.5 M H SO with the potential ranging from
2 4
produced through independent methods are shown in 0.0 to +1.9 V vs. nHe. two cycles were performed in
table 1. order to avoid desorption and/or agglomeration of na-
the data shown in Figs. 1B,D allow one to conclude noparticles on the surface (Fig. 1С) at a scan rate of
that a direct determination of the AunP size using the 0.1 V∙s-1. the electrodes were then washed with water
SeM method provides the most accurate and consistent and dried. the electrochemically active (real) surface
data suitable for a statistical evaluation of the particle area of the electrodes (A ) was calculated accord-
real
size distribution. However, this method has sensitivity ing to [8], assuming the level of the charge required
limitations; in particular, in this case it was impossible to for the reduction of gold oxide during electrochemi-
estimate the size of nanoparticles smaller than 10 nm. cal cycling under specified conditions to be equal to
All the prepared AunP solutions, except for AunP 390 ± 10 μc∙cm-2 [9].
with a diameter of 5 nm, were concentrated by centrifu- the results of the calculation of A presented in ta-
real
gation at 10,000 g for 30 min. 95% of the supernatant was ble 2 show no direct relationship between A and the
real
removed, and the AunP precipitate was re-suspended size of the AunP used for surface modification. this
using sonication. nanoparticles 5 nm in diameter could fact indirectly confirms earlier results [10] on the for-
not be concentrated using the proposed method; thus, mation of a disordered three-dimensional structure via
the diluted solution was used for further experiments. repeated cycling of the AunP/Au electrode in 0.5 M
H SO . In connection to this, two types of AunP/Au
2 4
Purification of gold electrodes and electrodes were used in further experiments: either
their modification with AuNP treated with H SO (m-AunP/Au) or without cycling
2 4
Polycrystalline gold disc electrodes (Bioanalytical Sys- (u-AunP/Au). A for u-AunP/Au electrodes was as-
real
tems, uSA) with a geometric surface area of 0.031 cm2 sumed to be equal to A for m-AunP/Au samples.
real
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Fig. 2. А) Cyclic voltammo-
0 А 0 B grams (cathodic waves) of
biomodified m-AuNP /Au
Current density, µА ----2121-55500 Current density, µА -------12221116248024 Current density, µА ----23410000 524600 0N NNNmmmm vef01dtei5o olee(r0lbnenct0 asnltoui rmtt(efo y rrmω)deo ,ade1 ott5/as) g200t. ;rri 0. oIeaBn3 nmc()s5g ,oer s VraCrt e(d t –)ceeae.a ssn cdCt ,)uah 2 yar0a ropfrctunde mldndincicci: ft --
-30 -5 0 5 10 15 20√ ω25 30 35 40 waves) of MvBOx modi-
0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 0.3 0.4 0.5 0.6 0.7 0.8 fied m-AuNP/Au (B) and
Potential vs. NHE, V Potential vs. NHE, V
u-AuNP/Au (C) electrodes
based on AuNPs of different
0 C 50 D diameters; D) Dependences
А of the calculated bioelectro-
nsity, µ-10 -1e, s40 csiaztea loyft iAc upNaPrasm. eters on the
de-20 alu30 Conditions for all CVs:
ent nt v oxygen saturated PBS, scan
Curr-30 520 N Nmm nsta20 u-AuNP/Au kkca aptp rate – 20 mV s-1, second
-40 4600 NNmm Co10 m-AuNP/Au kk0ca aptp cycle
0
-50 0
0.3 0.4 0.5 0.6 0.7 0.8 10 20 30 40 50 60
Potential vs. NHE, V AuNP size, nm
Biomodification of the surface of AunP/Au elec- values were obtained using the electrodes biomodified
trodes was carried out through direct adsorption of the with m-AunP/Au (31.4 ± 5.9 μA∙cm-2 ) and u-AunP/
enzyme for 20 min from a MvBOx solution with a pro- Au (43.4 ± 5.6 μA∙cm-2) electrodes.
tein concentration of 0.25 mg∙ml-1. the surface concen- taking these data into account, the k (31 ± 9 s-1) and
0
tration of the enzyme was assumed to be 3.0 pmol∙cm-2. kapp (34 ± 11 s-1) values were calculated. the results are
cat
the k and kapp values were calculated using the shown in Fig. 2D. the calculated k and kapp values are
0 cat 0 cat
MathcAD 14 software package and the equation: similar regardless of the AunP diameter and the elec-
trode type used. the similarity of the constants for the
j
j= max , electrodes based on m-AunP/Au and u-AunP/Au in-
⎡ F ⎤ k ⎡αF ⎤
1+exp⎢ (E−E0')⎥+ 5exp⎢ (E−EE0')⎥ dicates that the assumption of identity of the Areal (de-
⎣RT T1 ⎦ k ⎣RT T1 ⎦ spite different structures) for both types of samples does
0
not add a critical error to the calculations. the overesti-
mated constants for u-AunP/Au attest to the slightly
k [O ]
k = cat 2 . higher surface area of those electrodes compared to m-
5 K +[O ] AunP/Au, owing to the absence of AunP agglomerates
M 2
(Fig. 1C), which also reduces the A . the pronounced
real
the kinetic scheme of enzyme functioning used to bends of constant vs. AunP size profiles observed when
establish the equation was presented in [11]. using m-AunP/Au may be due to the surface modifi-
cation because of the formation of different three-di-
reSultS and diScuSSion mensional (3D) structures during the treatment of elec-
the bioelectrodes were placed in oxygenated PBS, fol- trodes in H SO . the behavior of the enzyme on these
2 4
lowed by cV recording at an electrode rotation speed heterogeneous surfaces cannot be fully described by the
of 1,500 min-1 to eliminate possible diffusion limitations single theory used in this study to calculate biocatalytic
(Fig. 2A). A pronounced bioelectrocatalytic response parameters without introducing additional corrections.
with an initial oxygen electrical reduction potential of Moreover, the formation of 3D agglomerates yields er-
about 0.75 V was registered for all the electrodes used. rors when a single value of the surface concentration of
As illustrated in Fig. 2B,C, substantially similar j the enzyme (3.0 pmol∙cm-2) is used for all the m-AunP/
max
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Au-based bioelectrodes. this fact can also explain the eters of MvBOx immobilized on the AunP/Au surface
shape of the curves. However, based on the experimen- and the nanoparticle diameter. However, it should be
tal data and simulation results, we can affirm that the noted that the results obtained in this study cannot be
bioelectrocatalytic properties of MvBOx immobilized on extrapolated to other nanobiomodified surfaces (e.g.,
the AunP/Au surface show no dependence on the nano- other nanoparticles and redox enzymes). In particular,
particle diameter. the increased electrocatalytic cur- it is of particular interest to study the impact of nano-
rent of the bioelectrodes modified with nanoparticles of particles with a diameter lower than the size of the en-
different sizes found in a number of previous studies is zyme that can promote electron transfer between the
most likely associated with an increase in the geometri- enzyme and the electrode surface. Such experiments
cal surface area rather than the acceleration of the Det will provide a more complete picture of the impact of
reactions or an increase in the bioelectrocatalytic con- nanoparticles on the bioelectrocatalytic parameters of
stants of the immobilized enzymes. oxidoreductases.
concluSionS This study was supported by the Russian Foundation
Our results have experimentally demonstrated no for Basic Research (grant № 12-04-33102-mol-a-ved).
relationship between the bioelectrocatalytic param-
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