Electrochemically Reduced Graphene Oxide on Electrochemically

Subscriber access provided by UNIV OF UTAH

Article

Electrochemically Reduced Graphene Oxide on Electrochemically Roughened Gold as Support for Horseradish Peroxidase Piotr Marcin Olejnik, Agnieszka Swietlikowska, Marianna Gniadek, and Barbara Palys J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/jp507227z • Publication Date (Web): 29 Nov 2014 Downloaded from http://pubs.acs.org on December 2, 2014

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry C is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Electrochemically reduced graphene oxide on electrochemically roughened gold as a support for horseradish peroxidase Piotr Olejnik, Agnieszka Świetlikowska, Marianna Gniadek, Barbara Pałys* AUTHOR ADDRESS Department of Chemistry, Warsaw University, Pasteur str. 1, 02-093 Warsaw, Poland KEYWORDS electrochemically roughened gold, biofuel cells, SERS support, infrared spectra, nano-porous gold, direct electrocatalysis, HRP, H2O2 detection, enzyme, nano-rough gold

ABSTRACT

Graphene oxide (GO) is composed of isolated graphene-like sheets containing various oxygen functionalities. The electrochemical reduction of GO enables the direct deposition of the graphene layer on electrode surfaces. Furthermore, a partial reduction of GO can be performed, enabling easy modification of the surface with oxygen-containing groups. In this contribution, the electrochemical reduction of GO is carried out on a gold electrode roughened by electrochemical oxidation-reduction cycles. Such a gold electrode contains nanostructures ranging in size from tens to 300 nm. The electrochemically reduced GO layer is subsequently used for horseradish peroxidase (HRP) immobilization. Two types of electrochemically reduced

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 33

GOs, partially reduced graphene oxide (ERGOP) and fully reduced graphene oxide (ERGOF), are used in this study. Although HRP immobilized on both types of surfaces directly catalyseselectroreduction of the hydrogen peroxide, the enzyme immobilized on the ERGOP layer exhibits slightly higher current values compared with those of ERGOF. In contrast, HRP adsorbed directly on the roughened gold reveals negligible activity. To evaluate the effect of the roughened gold, HRP immobilized on an ERGOF layer on glassy carbon was studied.

Additionally, infrared and Raman spectroscopy were used to investigate the composition of GO, ERGOP, and ERGOF as well as the HRP structure at the studied surfaces. The infrared results indicate a random orientation of the enzyme molecules on the bare roughened gold and ordered HRP layers on the surfaces covered with either ERGOP or ERGOF.

Introduction GO is commonly used as a substrate for chemical or electrochemical reductions to obtain graphene materials1,2. The electrochemical reduction of GO can be used to deposit graphene or graphene nano-stacks directly onto an electrode surface3,4. Such graphene deposition may influence the adsorption efficiency of biomolecules due to the increased hydrophobicity5. Furthermore, graphene or GO may also improve the electron transfer rate for bioelectrocatalytic reactions6,7. The advantage of using graphene over GO is its superior electric conductivity, while the oxygen functionalities of GO are beneficial for the immobilization of enzymes and other catalytically active molecules. The OH groups present on partially reduced GO may also be applied as mediators for electron transfer between the enzyme and the electrode surface8.


2

Page 3 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The electrochemical reduction of GO can be stopped before the reduction is complete, enabling the electrode surface to be tuned for optimal enzyme adsorption and activity8. Several factors such as pH and the applied potential range may influence the electrochemical reaction, affecting the quality of the deposited layers3 and possibly the surface functionalities. Additionally, among other factors the type of electrode material may also influence the electroreduction. In this contribution, we have studied the electroreduction of GO on an electrochemically roughened gold surface, which is created by repetitive electrochemical oxidation and reduction of a gold electrode. Such treatment generates nano gold deposits that increase the catalytic activity of the electrochemically roughened gold. The activity of such a surface toward oxygen reduction9 and the polymerization of various organic molecules10,11 has been previously reported. However, the effect of an electrochemically roughened gold surface on enzyme activity has not been studied, in contrast to corresponding studies using gold nanoparticles. The influence of gold nanoparticles on enzyme activity attracts interest because gold nanoparticles improve the electrical connection between the enzyme active site and the electrode. For example, their beneficial influence on the signal of enzymatic sensors has been reported for glucose oxidase12 and laccase13. The advantage of electrochemically roughened gold for enzyme studies is its ability to enhance Raman and infrared absorption, thus enabling the study of monolayers14-17. Related materials such as nanoporous gold (NPG)18 have been examined as possible supports for the immobilization of biomolecules19-23 and inorganic catalysts24,25 due to their high surface areas for enzyme interactions. The surface area of electrochemically roughened gold electrodes available for adsorption is comparable with that of NPG. The presence of gold nano-structures and edges on the roughened gold might contribute to enhanced enzyme activity in a similar way


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 33

as that observed for gold nanoparticles, making studies of enzymes immobilized on such surfaces interesting. Horseradish peroxidase (HRP) is a heme-containing enzyme with a variety of applications including H2O2 detection, biofuel cell cathodes, and targeted cancer therapy26-30. To obtain electrochemical sensors, HRP has been immobilized on carbon, metallic, oxide, and composite materials31-38, with either direct or mediated electrocatalysis observed, depending on the electrode material. The type of supporting material significantly influences the potential of the H2O2 electroreduction. In the case of direct electrocatalysis, the electrode potential may influence the mechanism of the bioelectrocatalytic reaction. HRP adsorbed on graphitic materials catalyzes the reduction of H2O2 at high potentials, and thus with low overpotential. However, the electrocatalytic efficiency of HRP on graphite electrodes may be limited by a random orientation of enzyme molecules31. In this contribution, we have studied HRP immobilized on bare electrochemically roughened gold and on a surface covered by electrochemically reduced GO deposited on roughened gold. Two types of electrochemically reduced GO electrodes, partially reduced graphene oxide (ERGOP) and fully reduced graphene oxide (ERGOF), were used in this study. The surface composition of the electrodes before and after HRP immobilization was studied by measuring their vibrational spectra. The electrocatalytic activity of the enzyme was also correlated with the structure and the orientation of immobilized HRP molecules, as measured by infrared spectroscopy.

Experimental


4

Page 5 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Reagents HRP (type VI, lyophilized powder, 250-330 units per mg), N-hydroxysuccinimide (NHS), and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDC) were purchased from Sigma Aldrich and used as received. GO was synthesized from graphite powder (Sigma Aldrich; 1–2 µm, synthetic) through the modified Hummers–Offeman method8,39. All of the remaining reagents used were of analytical grade and were purchased from POCH, Poland. Electrochemical roughening of the gold surface The gold electrode was thoroughly polished and cleaned in sulfuric acid and ethanol and was then ultrasonicated in MQ water. The cleaned gold electrode was placed in a standard threeelectrode cell containing 0.1 M KCl solution. Subsequently, cyclic voltammetry was performed in the range between -0.7 and 1.2 V vs. Ag/AgCl with a scan rate of 20 mV/s and a typical number of cycles of 20. After this treatment procedure the gold surface acquired a greyish color. A droplet of the GO suspension was placed on the roughened gold and left to dry. The electrochemical reduction of GO was carried out by cyclic voltammetry in the -0.4 to -1.2 V vs. Ag/AgCl range at 5 mV/s. The partially reduced ERGOP was obtained using 3 cycles, while the deeply reduced ERGOF was obtained using 10 cycles. HRP immobilization The HRP was physically adsorbed onto the electrochemically roughened gold surface. To adsorb the enzyme, 10µl of the HRP solution (1 mg/1 ml) was dropped onto the electrode and left for 30 min. The electrode was subsequently rinsed with MQ water.


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 33

Before immobilization on the electrochemically reduced GO (ERGO) layer, the electrode was kept in a solution of 10 mM NHS and 40 mM EDC for 1 h. After this activation step, the electrode was rinsed with water and immersed in a HRP solution in a phosphate buffer of pH 7.0 for 30 min. The electrode was then rinsed with MQ water for use in subsequent studies. Electrochemical experiments The

electrochemical

experiments

were

carried

out

using

a

PGSTAT204

potentiostat/galvanostat (Metrohm-Autolab, The Netherlands) with the NOVA software. A typical three electrode configuration with a gold disk electrode (2 mm diameter) as the working electrode, Ag/AgCl (with saturated KCl) as the reference electrode, and a platinum sheet as the counter electrode was used. Electron microscopy The layers deposited on the electrode surface were studied with a MERLIN field emission scanning electron microscopy (SEM) system (Zeiss, Germany). The GO suspension was examined by a transmission electron microscope (TEM) system (Zeiss, Germany). Infrared spectra The infrared spectra were recorded using a NICOLET IN10 MX infrared microscope (Thermo Scientific). The microscope was operated in reflectance mode and with the MCT detector cooled with liquid nitrogen. The spectra were collected from a square region of length 100 µm (area 0.01 mm2). For typical measurements, the spectral resolution was 4 cm-1 and 256 scans were averaged to obtain a single spectrum. The spectrum of the non-bonded enzyme was recorded in a KBr pellet using the microscope in transmission mode. Raman spectra


6

Page 7 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The Raman experiments were carried out using a NXR FT-Raman module together with a NICOLET 8700 FT-IR spectrometer (Thermo Scientific). The Raman module was equipped with a microstage that enabled the measurement of electrode spectra in reflectance mode. The instrument was operated with the 1064 nm excitation line. For typical measurements, the spectral resolution was 4 cm-1 and 128 scans were averaged to obtain a single spectrum.


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 33

Results and discussion Electroreduction of GO on nanoporous gold and spectral characteristics of the reduced layers Typical SEM images of the electrochemically roughened gold surface taken before and after the GO reduction are shown in Figure 1. The bare electrochemically roughened gold contains angular shaped nanoparticles that vary in size from a few to 300 nm (Figure 1A). A)

200 nm

B)

200 nm Figure 1. SEM images of (A) the bare roughened gold surface and (B) the ERGOP layer deposited on the roughened gold surface. The as prepared GO suspension contains isolated GO flakes, as indicated by the TEM images shown in Figure 2. After the GO electroreduction, the gold nanostructures are covered tightly by folded, reduced GO sheets (Figure 1B). The morphologies of the partially and fully reduced GO layers were found to be identical (not shown).


8

Page 9 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


200 nm

Figure 2. TEM images of GO.

The electrochemical reduction of GO was carried out in the -0.4 V to -1.2 V vs. Ag/AgCl potential range in a pH 7.0 phosphate buffer solution, and corresponding typical voltammograms are presented in Figure 3. In the first cycle a very broad reduction peak is observed at about -0.7 V. The reduction is irreversible because no counter-peak is observed in the reverse potential sweep. The current values diminish in the following cycles because the number of surface groups, which may undergo electroreduction, decreases in every cycle. A similar evolution of the cyclic voltammetry curves was observed when the electroreduction of GO was carried out on a glassy carbon electrode, although the reduction peak observed in the first cycle appeared at -1.0 V vs. Ag/AgCl8. This shift of the reduction peak potential suggests that the gold surface catalyzes the electrochemical reduction of GO.


9


0.0 -0.5 j / mA*cm-2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 33

5 4 3

-1.0 -1.5

2 -2.0 1 -2.5 -1.3

-1.1

-0.9 -0.7 -0.5 E / V vs. Ag/AgCl

-0.3

Figure 3. Cyclic voltammograms of the GO layer deposited on the roughened gold electrode. The scan rate was 5 mV/s and the scan direction is marked by arrows. The numbers of cycles are indicated.

The surface functionalities present on the GO surfaces before and after the electroreduction can be identified by their infrared spectra. Figure 4 compares typical spectra of the GO and ERGOP layers on roughened gold. The corresponding ERGOP layer was obtained using three GO reduction cycles. The GO spectra reveal intense bands at 888, 1056, and 1212 cm-1 due to the C-O-C bending, symmetric stretching, and asymmetric stretching modes of epoxide groups, respectively40. The band observed at 1730 cm-1 appears at the frequency characteristic of the C=O stretching mode of COOH groups. The C-O stretching mode of the COOH group typically appears in the 12001300 cm-1 range. In the GO spectrum, it most likely overlaps with the epoxide band at 1212 cm-1. The epoxide band may also contain a contribution from the C-O stretching mode of phenolic


10

Page 11 of 33

groups. The OH stretching band appears at 3460 cm-1, which includes contributions from both

3390

0.05

1584

1238

the COOH and the OH functionalities.

1212

Au/ERGO

0.1

3460

1730

1584

888

1056

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Au/GO 800

1300 1800 2300 2800 3300 3800 Wavenumbers / cm-1

Figure 4. Infrared spectra of electro-reduced GO layers on roughened gold before (Au/GO) and after (Au/ERGO) the electrochemical reduction.

The small shoulder at 1584 cm-1 appears at the frequency typical for the C=C stretching mode of graphene41. Its relatively low intensity compared with the bands of surface groups of GO indicates a large content of oxygen functionalities prior to the electrochemical reduction. Following the electrochemical reduction of GO, the band at 1584 cm-1 is relatively enhanced due to the removal of these oxygen functionalities. Additionally, among the bands characteristic of oxygen functionalities, the band at 1730 cm-1 disappears (Figure 4) after the electrochemical reduction, signifying that the COOH groups are removed by the reduction. The spectrum contains a band at 1238 cm-1, which is slightly shifted toward higher energies compared with the


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 33

epoxide mode at 1212 cm-1. The near disappearance of the remaining epoxide bands indicates that the remaining epoxides are most likely removed from the surface. The band at 1238 cm-1 corresponds to the C-O stretching mode of phenolic species, which was obscured by the strong epoxide absorption prior to the GO reduction. The OH stretching band appears at 3390 cm-1 with an altered intensity and spectral shape compared with the corresponding band measured prior to the reduction. This may be rationalized by the removal of the COOH groups that contributed to this band before the reduction. The OH surface groups are removed after ten cycles of GO reduction, as indicated by the negligible intensities of the OH bands in the spectrum of ERGOF (not shown). In summary, the infrared results indicate that COOH and epoxide groups are easily removed from the GO surface during the electrochemical reduction on the roughened gold electrode. Some hydroxyl species are retained after three reduction cycles, but they are removed after prolonged GO reduction. Raman spectroscopy is a versatile probe of the properties of graphene layers, enabling the evaluation of possible defects3. Figure 5 compares spectra of the GO layers on the ORC-treated gold before and after ten electrochemical reduction cycles. The main spectral features are two bands (G and D) characteristic of sp2 carbon materials42-44. The position of band D depends on the laser excitation line, shifting from 1400 to 1250 cm-1 upon changing the excitation wavelength from the visible to the near infrared43. Band D originates from vacancies, heteroatoms, or other defects in the plane of carbon atoms. Band G is present in all types of sp2 carbon materials, and its position is less sensitive to the laser excitation wavelength. The ratio of the intensities of band D to band G is a measure of the defect content in the studied samples.


12

Page 13 of 33

In the spectra presented in Figure 5, band G is observed at a typical frequency of 1590 cm-1, while band D appears at 1292 cm-1. Similar frequencies have been observed for carbon nanotubes excited with a near infrared laser. The quite high intensity of band D indicates the presence of numerous edges and defects in the GO samples. Additionally, as illustrated in Figure 5, the intensity ratio of band D to band G changes only slightly after electrochemical reduction of the layer, indicating that many defects still remain.

40

30

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20

B 10

0 4000

A

3500

3000

2500

2000

1500

1000

Wavelength / cm-1

Figure 5. Raman spectra of (A) GO and (B) ERGOF layers on an ORC-treated gold surface.

Interestingly, the overall Raman intensity of the ERGOF spectrum in Figure 5 is noticeably higher compared with the GO spectrum, even though the experimental conditions were identical for both spectra. The intensity variation might be rationalized by taking into account the fact that the SERS enhancement depends on the interaction between the gold support and adsorbed molecules15. The high SERS intensity indicates a strong interaction between the layer and the support. Therefore, it is possible that the reduction of GO to ERGOF strengthens the interaction of the layer with the gold support.


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 33

Structure of immobilized HRP Figure 6 presents characteristic SEM images of the nano-roughened gold covered with the ERGOP after the HRP bonding procedure. When the partially reduced ERGOP is replaced by the fully reduced ERGOF, the SEM images are very similar to those presented in Figure 6. The reduced GO layer strictly adheres to gold nanostructures; however, the layer is not homogenous over the whole electrode. For example, in some regions thicker stacks are observed and large white enzyme aggregates are visible on the top of the reduced GO layer.

200nm

Figure 6. SEM micrograph of the ERGOP layer on the ORC-treated gold after immobilization of the enzyme. Infrared experiments were carried out to investigate possible structural changes on the molecular scale caused by HRP immobilization. The spectra obtained from the surface were compared with the spectrum of native HRP. The final spectrum was measured using the standard procedure with a KBr pellet. Typical spectra from these measurements are presented in Figure 7.


14

amide III amide II amide I

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Absorbance

Page 15 of 33

A

B

C 800 1200 1600 2000 2400 2800 3200 3600 Wavenumbers / cm-1

Figure 7. Infrared spectra of (A) HRP from a KBr pellet, (B) HRP adsorbed on the bare roughened gold surface, and (C) HRP immobilized on the roughened gold covered with ERGOP.

The spectra of immobilized HRP were collected from an area as small as 0.01 mm2. The relatively high intensity of the immobilized HRP spectra indicates a SEIRA enhancement16. A consequence of the surface enhancement might be the “surface selection rule,” which says that motions perpendicular to the surface are enhanced. Accordingly, the ordered layers exhibit altered relative band intensities compared with randomly oriented molecules. The surface selection rule is the same for SEIRA and infrared reflectance spectra recorded from smooth metal surfaces45. For single peptides at smooth gold plates, the tilt angle with respect to a surface can be evaluated by comparison of the intensity ratio of the amide I to amide II bands in the spectrum of the monolayer with that of a randomly oriented sample, which is usually obtained from the peptide spectrum measured in a KBr pellet46-48. The orientation is difficult to evaluate for proteins for obvious reasons, but orientation changes are observable from infrared spectra49,50.


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 33

The relative intensity of the amide I band with respect to the amide II band in the HRP spectrum measured in KBr (Figure 7B) is similar to that of the spectrum of HRP adsorbed on bare roughened gold (Figure 7A), suggesting that the enzyme molecules have a random orientation on such a surface. For HRP adsorbed on the ERGOP layer (Figure 6C), the intensity of the amide I band relative to the neighboring amide II band is smaller, suggesting that HRP molecules are ordered at the ERGOP surface. The relative intensities of the amide I and amide II bands are similar for the enzyme adsorbed on ERGOF (not shown). Because the SEM micrographs point to partial aggregation of the HRP, it is likely that only molecules directly bonded to the surface are oriented in the ordered manner. The SEIRA enhancement is restricted to very short distances16; therefore, molecules in the vicinity of the electrode surface provide the largest contribution to the spectrum. The position of the amide I band is sensitive to the secondary and tertiary structure of the protein portion of the enzyme51. The amide I band of HRP is broad due to the presence of various elements with a tertiary structure. Its main component at 1658 cm-1 corresponds to the αhelices prevailing in the HRP molecule, and the component at 1645 cm-1 is due to unordered structures51. The amide I band shifts from 1658 cm-1 for the free enzyme to 1643 cm-1 after adsorption on the roughened gold. Such a shift might indicate an increase of the unordered structure at the expense of the α-helices, although an analogous red-shift of the amide I band has also been observed for laccase adsorbed on a smooth gold surface50. In the case of laccase, the band shift has been attributed with a redox state of the enzyme active site that indirectly influences the structure of the protein portion of the enzyme part. Because such a structural change did not influence the catalytic activity of the enzyme, the red-shift of the amide I band does not provide proof of enzyme decomposition. The amide I bands of HRP adsorbed on bare


16

Page 17 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


roughened gold and the surface covered by ERGOP appear at identical positions. Because the two enzyme layers differ in catalytic activity, this particular shift indicates a change of structure that is irrelevant for the enzyme activity. The amide III band is also sensitive to the structure of protein portion of the enzyme. It appears at 1301 cm-1 for the HRP spectrum measured in KBr, which is typical for alpha-helical structures52. After adsorption on both studied surfaces, the amide III band shifts to 1310 cm-1, supporting the suggestion that the immobilization of HRP on both surfaces affects the structure of the protein portion of the enzyme in a similar manner. The non-bonded HRP spectrum exhibits medium intensity bands at 1451 and 1400 cm-1, which after the immobilization become broader and weaker. The bands in the 1300-1500 cm-1 region correspond to heme motions53, and their intensity is sensitive to the oxidation and spin state of the heme iron. The weakening and broadening of these bands suggests that the oxidation state of HRP is affected by the immobilization on the roughened gold as well as on gold covered with the ERGOP surface. The change in the heme oxidation state might influence the tertiary structure of the protein portion of the enzyme, which in turn may alter the amide I and amide III band position. Such a structural change is possibly reversible because the heme oxidation state can change reversibly. H2O2 electroreduction The electrocatalytic activity of HRP was studied for the enzyme adsorbed directly on the roughened gold as well as on the roughened gold covered by ERGOP and ERGOF. The cyclic voltammetry responses of the studied electrodes are presented in Figure 8. Over the potential range studied, HRP adsorbed directly on the roughened gold electrode (see curve 1 in Figure 8) exhibits a small current due to the H2O2 reduction, similar to that observed for the electrode


17


without the enzyme (see curve 2 in Figure 8). Such a negative potential value is very close to that expected for the bare gold electrode54, suggesting that the H2O2 is reduced at the roughened gold electrode without a significant contribution from the enzyme. The electrocatalytic activity of HRP deposited on both ERGOP and ERGOF is readily visible from curves 3 and 4 in Figure 8, although the enzyme immobilized on ERGOP exhibits slightly higher current values.

2

0

1

-0.2

j / mA*cm-2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 33

-0.4 -0.6

4

-0.8

3 -1 -0.6

-0.4

-0.2

0

0.2

0.4

0.6

E / V vs. Ag/AgCl

Figure 8. Cyclic voltammograms of HRP immobilized on (1) bare roughened gold, (2) ERGOP without HRP, (3) HRP immobilized on ERGOP, and (4) HRP immobilized on ERGOF in 0.5 mM H2O2 at pH=7.

The electrocatalytic activity of HRP immobilized on the bare roughened gold is clearly diminished compared with the enzyme adsorbed on the roughened gold covered by ERGOP or ERGOF surfaces. A possible explanation of the declined activity is the degradation of the HRP structure through the adsorption on the roughened gold; however, such a hypothesis is not


18

Page 19 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


supported by the infrared spectra because the bands of the tertiary structure of the enzyme adsorbed on gold and on the reduced GO are similar. Alternatively, the reason for the poor electrocatalytic activity of HRP adsorbed on gold might be a disadvantageous orientation of the enzyme molecule, with the active site located far from the gold surface. Various orientations of the enzyme on the bare and covered roughened gold surfaces are supported by the infrared results. The slightly higher current values observed for HRP on ERGOP compared with HRP on ERGOF suggest that the OH functionalities present on the former surface have a beneficial influence on the electroactivity of immobilized HRP. However, the observed differences in the electrocatalytic currents between HRP on ERGOP and HRP on ERGOF are relatively small, indicating that the effect of the OH groups is moderate. Covering the roughened gold surface by reduced GO has a bigger influence on the activity of immobilized HRP. The higher catalytic activity is likely due to the beneficial orientation of HRP molecules induced by the hydrophobic properties of reduced GO. The ERGOF layers were also studied on a glassy carbon support (ERGOF/GC) to verify the effect of the electrochemically roughened gold. The enzyme was immobilized on such electrodes using exactly the same procedure as that used for the ERGOF films deposited on roughened gold electrodes (ERGOF/AU). Figure 9 compares typical cyclic voltammograms of HRP on the ERGOF/GC with those of the ERGOF/AU electrode in the presence of H2O2.


19


0.2 0

2 j / mA*cm-2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 33

-0.2 -0.4 -0.6

1 -0.8 -0.65

-0.4

-0.15

0.1

0.35

0.6

E / V vs. Ag/AgCl

Figure 9. Cyclic voltammograms of (1) HRP immobilized on the ERGOF/AU electrode and (2) HRP immobilized on the ERGOF/GC electrode in 0.5 mM H2O2 at pH 7.

Both electrodes exhibit electrocatalytic currents due to the reduction of H2O2, but the currents observed for the layer on the roughened gold electrode are clearly higher compared with those of the HRP/ERGOP/GC electrode, corroborating the improvement of the bioelectrocatalytic signal by the roughened gold. The enhancement of bioelectrocatalytic currents due to the presence of gold nanoparticles has been reported for other enzymes12,13. In the case of HRP, metal nanoparticles influenced the potential of the bioelectrocatalytic reaction. For example, HRP immobilized on graphene functionalized with platinum or palladium nanoparticles exhibits electrocatalytic currents in at positive potentials55,56, while HRP on pristine graphene exhibits a current response at negative potentials57. The effect of the metal nanoparticles on the enzyme activity has been attributed to improved electric connectivity between the enzyme and the electrode. The higher current values observed for HRP on ERGOF/AU compared with ERGOF/GC might also be caused by the numerous gold nanostructures present on the roughened gold surface


20

Page 21 of 33

(Figure 1), although the roughened gold must be covered by the thin layer of ERGOF (or ERGOP). The difference between HRP immobilized on bare gold and the covered gold is the orientation of the HRP molecules, as revealed by the infrared results. Additionally, the ERGOF (or ERGOP) might also improve the enzyme load. The amperometric response of the HRP adsorbed on the ERGOP surface was verified at an electrode potential value of 0.0 V vs. Ag/AgCl. As illustrated in Figure 10, the current values depend linearly on the H2O2 concentration over a wide range, indicating good stability of the electrode even at quite high concentrations of H2O2. The slope of the concentration dependence is very high (25 mA cm-2 M-1), which indicates high sensitivity of the adsorbed enzyme.

25

20 j / mA*cm-2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


15

10

5

0 0

0.05 c / mol*dm-3

0.1

Figure 10. Dependence of the amperometric response of the HRP/ERGOP/AU electrode on the H2O2 concentration. The electrode potential was 0.0 V vs. Ag/AgCl. The error values are estimated from 10 independent measurements.


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 33

Conclusions HRP immobilized on ERGOP and ERGOP layers deposited on electrochemically roughened gold catalyzes the reduction of H2O2 directly without the addition of any mediator. In contrast, the enzyme adsorbed on bare electrochemically roughened gold shows negligible catalytic activity. The obtained infrared spectra suggest at least a partial orientation of the HRP molecules on the reduced GO layers. The enzyme orientation is likely a reason for the increased enzyme activity. If the electrochemically roughened gold is replaced by a glassy carbon electrode, the HRP exhibits electrocatalytic activity but with significantly lower current values. The beneficial role of the roughened gold most likely results from the increased surface area of the electrode and from the presence of gold nanostructures at the surface. A similar improvement in bioelectrocatalytic activity has been observed for other enzymes – metal nanoparticle composites.


22

Page 23 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Graphical Abstract


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 33

ACKNOWLEDGMENT This work was supported by the Polish National Science Centre, project: UMO-2011/03/BST4/00717. The study was conducted using equipment purchased under the CePT project, which is co-financed by the European Union from the European Regional Development Fund under the Operational Program Innovative Economy 2007–2013. The study was carried out at the Biological and Chemical Research Centre, University of Warsaw and was established within the project co-financed by the European Union from the European Regional Development Fund under the Operational Program Innovative Economy, 2007–2013.


24

Page 25 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


References

1

Dreyer, D. R.; Park, S.; Bielawski, C. W.; Ruoff, R. S. The chemistry of graphene oxide.

Chem. Soc. Rev. 2010, 39, 228–240. 2

Chua, C. K.; Pumera, H. Chemical reduction of graphene oxide: a synthetic chemistry

viewpoint. Chem. Soc. Rev. 2014, 43, 291-312. 3

Hilder, M.; Winther-Jensen, B.; Forsyth, D. Li, M.; MacFarlane, D.R. Direct electro-

deposition of graphene from aqueous suspensions. Phys. Chem. Chem. Phys. 2011, 13 9187-9193. 4

Zhou, M.; Wang, Y. L.; Zhai, Y. M.; Zhai, J. F.; Ren, W.; Wang, F.; Dong, S. J. Controlled

synthesis of large-area and patterned electrochemically reduced graphene oxide films, Chem. – A European J. 2009, 15, 6116-6120. 5

Zhang, Y.; Zhang, J.; Huang, X.; Zhou, X.; Wu, H.; Guo, S. Assembly of graphene oxide–

enzyme conjugates through hydrophobic interaction, Small 2012, 8, 154-159. 6

Kuila, T.; Bose, S.; Khanra, P.; Mishrab, A. K.; Kim, N. H.; Lee, J. H. Recent advances in

graphene-based biosensors. Biosens. Bioelectron. 2011, 26, 4637– 4648. 7

Shao, Y.; Wang, J.; Wu, H.; Liu, J.; Aksay, I. A.; Lin, Y. Graphene Based Electrochemical

Sensors and Biosensors: A Review. Electroanalysis 2010, 22, 1027 – 1036. 8

Swietlikowska, A.; Gniadek, M.; Palys, B. Electrodeposited graphene nano-stacks for

biosensor applications. Surface groups as redox mediators for laccase. Electrochim. Acta 2013 98, 75– 81.


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

9

Page 26 of 33

Liu, S.; Xu, H. M.; Ou, J. F.; Li Z. P.; Yang S. R.; Wang, J. Q. A feasible approach to the

fabrication of gold/polyaniline nanofiber composites and its application as electrocatalyst for oxygen reduction, Mater. Chem. Phys. 2012, 132, 500-504. 10

Palys, B. J.; Bukowska, J.; Jackowska, K. SERS of 1,8-Diaminonaphthalene on Gold, Silver

and Copper Electrodes Polymerisation and Complexes Formed with the Electrode Material. J. Electroanal. Chem. 1997, 428, 19–24. 11

Liu, Y. C. New Pathway for the Autopolymerization of Pyrrole on the Chlorine- and Gold-

Containing Complexes with Nanostructures, Langmuir 2002, 118, 9513–9518. 12

Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F.; Willner, J. F. “Plugging into Enzymes”:

Nanowiring of Redox Enzymes by a Gold Nanoparticle. Science, 2003, 299, 1877-1881. 13

Gutierrez-Sanchez, C.; Pita, M.; Vaz-Dominguez, C.; Shleev, S.; De Lacey, A. L. Gold

nanoparticles as electronic bridges for laccase-based biocathodes. J. Am. Chem. Soc. 2012, 134, 17212-17220. 14

Fleischmann, M.; Graves, P. R.; Robinson, J. The Raman Spectroscopy of the

Ferricyanide/Ferrocyanide System at Gold, β-palladium Hydride and Platinum Electrodes. J. Electroanal. Chem. 1985, 182, 87-98. 15

Lin, X. M.; Cui, Y.; Xu Y. H.; Ren, B., Tian, Z. Q. Surface-Enhanced Raman Spectroscopy:

Substrate-Related Issues. Anal. Bioanal. Chem. 2009, 394, 1729–1745.


26

Page 27 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


16

Osawa, M. Surface-Enhanced Infrared Absorption. in Kawata, S. (Ed.): Near-Field Optics

and Surface Plasmon Polaritons, Topics Appl. Phys., 2001, 81, 163–187, Springer-Verlag Berlin Heidelberg 2001. 17

Sezer, M.; Millo, D.; Weidinger, I. M.; Zebger, I.; Hildebrandt, P. Analyzing the Catalytic

Processes of Immobilized Redox Enzymes by Vibrational Spectroscopies. IUBMB Life, 2012, 64 455–464. 18

Shulga, O. V.; Jefferson, K.; Khan, A. R.; D’Souza, V. T.; Liu, J.; Demchenko, A. V.; Stine,

K. J. Preparation and Characterization of Porous Gold and Its Application as a Platform for Immobilization of Acetylcholine Esterase. Chem. Mater. 2007, 19, 3902-3911. 19

Qiu, H. J.; Xu, C. X.; Huang, X. R.; Ding, Y.; Qu, Y. B.; Gao, P. J. Adsorption of Laccase

on the Surface of Nanoporous Gold and the Direct Electron Transfer Between Them. J. Phys. Chem. C 2008, 112, 14781-14785. 20

Ding, C. F.; Li, H.; Hu, K. C.; Lin J. M. Electrochemical Immunoassay of Hepatitis B

Surface Antigen by the Amplification of Gold Nanoparticles Based on the Nanoporous Gold Electrode. Talanta 2010, 80, 1385-1391. 21

Salaj-Kosla, U.; Poller, S.; Beyl, Y.; Scanlon, M. D.; Beloshapkin, S.; Shleev, S.;

Schuhmann, W.; Magner, E. Direct Electron Transfer of Bilirubin Oxidase (Myrothecium Verrucaria) at an Unmodified Nanoporous Gold Biocathode. Electrochem. Commun. 2012, 16, 92-95.


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

22

Page 28 of 33

Du, X. Y.; Liu, X. Y.; Li, Y. F.; Wu, C.; Wang, X.; Xu, P. Efficient Biocatalyst by

Encapsulating Lipase into Nanoporous Gold. Nanoscale. Res. Lett. 2013, 8, art. no. 180. 23

Wang, X.; Liu, X. Y.; Yan, X. L.; Zhao, P.; Ding, Y.; Xu, P. Enzyme-Nanoporous Gold

Biocomposite: Excellent Biocatalyst with Improved Biocatalytic Performance and Stability. Plos One 2011, 6, art. No. e24207. 24

Xia, Y.; Huang, W.; Zheng, J. F.; Niu, Z. J.; Li, Z. L. Nonenzymatic Amperometric

Response of Glucose on a Nanoporous Gold Film Electrode Fabricated by a Rapid and Simple Electrochemical Method. Biosens. Bioelectron. 2011, 26, 3555-3561. 25

Lang, X. Y.; Fu, X. Y.; Hou, C.; Han, G. F.; Yang, P.; Liu, Y. B.; Jiang, Q. Nanoporous

Gold Supported Cobalt Oxide Microelectrodes as High–Performence Electrochemical Biosensors. Nat. Commun. 2013, 4, art. No 2169. 26

Veitch, N. C. Horseradish Peroxidase: a Modern View of a Classic Enzyme. Phytochemistry.

2004, 65, 249-259. 27

Pizzariello, A.; Stred’ansky, M.; Miertus, S. A Glucose/Hydrogen Peroxide Biofuel Cell that

Uses Oxidase and Peroxidase as Catalysts by Composite Bulk-Modified Bioelectrodes Based on a Solid Binding Matrix. Bioelectrochemistry 2002, 56, 99-105. 28

Noh, H. B.; Won, M. S.; Hwang, J.; Kwon, N. H.; Shin, S. C.; Shim, Y. B.; Conjugated

Polymers and Iron Complex as Electrocatalytic Materials for an Enzyme-Based Biofuel Cell. Biosens. Bioelectron. 2010, 25, 1735-1741.


28

Page 29 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


29

Gomez, C.; Shipovskov, S.; Ferapontova, E. E.; Peroxidase Biocathodes for a Biofuel Cell

Development. J. Renew. Sustain. Energy. 2010, 2, art. no. 013103. 30

Agnes, C.; Reuillard, B.; Le Goff A.; Holzinger, M.; Cosnier, S.; A Double-Walled Carbon

Nanotube-Based Glucose/H2O2 Biofuel Cell Operating under Physiological Conditions. Electrochem. Commun. 2013, 34, 105-108. 31

Ferapontova, E. E. Direct Peroxidase Bioelectrocatalysis on a Variety of Electrode

Materials. Electroanal. 2004, 16, 1101-1112. 32

Jonsson, G.; Gorton, L. An electrochemical sensor for hydrogen peroxide based on

peroxidase adsorbed on a spectrographic graphite electrode. Electroanal. 1989, 1, 465-468. 33

Ferapontova, E.; Puganova, E. Effect of pH on direct electron transfer between graphite and

horseradish peroxidase. J. Electroanal. Chem. 2002, 518, 20-26. 34

Zhang, L.; Cheng, H.; Zhang, H., Qu, L. Direct Electrochemistry and Electrocatalysis of

Horseradish Peroxidase Immobilized in Graphene Oxide-Nafion Nanocomposite Film. Electrochim. Acta 2012, 65, 122-126. 35

Zhang, Y.; Zhang, J.; Wu, H.; Guo, S.; Zhang, J. Glass Carbon Electrode Modified with

Horseradish Peroxidase Immobilized on Partially Reduced Graphene Oxide for Detecting Phenolic Compounds. J. Electroanal. Chem. 2012, 681, 49-55. 36

Ghamouss, G.; Ledru, S.; Ruille, N.; Lantier, F.; Boujtita, M. Bulk-modified modified

screen-printing carbon electrodes with both lactate oxidase (LOD) and horseradish peroxide


29


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 33

(HRP) for the determination of l-lactate in flow injection analysis mode. Anal. Chim. Acta 2006, 570, 158-164. 37

Yin, H. S.; Ai, S. Y.; Shi, W. J.; Zhu, L. S. A novel hydrogen peroxide biosensor based on

horseradish peroxidase immobilized on gold nanoparticles–silk fibroin modified glassy carbon electrode and direct electrochemistry of horseradish peroxidase. Sens. Actuators B: Chemical 2009, 137, 747–753. 38

Shan, D.; Li, Q.-B; Ding, S.-N.; Xu, J.-Q.; Cosnier, S.; Xue, H.-G. Reagentless biosensor for

hydrogen peroxide based on self-assembled films of horseradish peroxidase/laponite/chitosan and the primary investigation on the inhibitory effect by sulfide. Biosens. Bioelectron. 2010, 26, 536-541. 39

W.S. Hummers Jr., R.E. Offeman, Preparation of graphitic oxide, J. Am. Chem. Soc. 80

(1958) 1339. 40

G. Socrates, Infrared and Raman Characteristic Group frequencies, John Wiley & Sons,

Chichester, (2001). 41

Tan, L.; Li, X.; Ji, R.; Kar Seng Teng; Tai, G.; Ye, G.; Ye, J.; Wei, C.; Shu Ping Lau

Bottom-up synthesis of large-scale graphene oxide nanosheets. J. Mater. Chem. 2012, 22, 56765683. 42

Tuinstra, F.; Koenig, J. L. Raman Spectrum of Graphite. J. Phys. Chem. 1970, 53, 1126-

1130.


30

Page 31 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


43

Brown, S. D. M.; Jorio, A.; Dresselhaus, M. S., Dresselhaus, G. Observations of the D-band

feature in the Raman spectra of carbon nanotubes. Phys. Rev. B. 2001, 64, art. 073403 44

Ferrari, A. C.; Meyer, J. C.; Scardaci, V.; Casiraghi, C.; Lazzeri, M.; Mauri, F.; Piscanec, S.;

Jiang, D.; Novoselov, K. S.; Roth, S.; Geim, A. K. Raman Spectrum of Graphene and Graphene Layers. Phys. Rev. Lett. 2006, 97, 187401-187404. 45

Kycia, A.H.; Su, Z.F.; Brosseau, C.L.; Lipkowski, J. In situ PM-IRRAS studies of

biomimetic membranes supported at a gold electrode surface, in: A. Wieckowski, C. Korzeniewski, B. Braunschweig (Eds.), Vibrational Spectroscopy at Electrified Interfaces, Wiley, Chichester, 2013, chapter 11. 46

Xu, Z.; Brauner, J. W.; Flach, C. R.; Mendelshon, R. Orientation of Peptides in Aqueous

Monolayer Films. Infrared Reflection−Absorption Spectroscopy Studies of a Synthetic Amphipathic β-Sheet. Langmuir 2004, 20, 3730−3733. 47

Miura, Y.; Kimura, S.; Imanishi, Y.; Umemura, J. Formation of Oriented Helical Peptide

Layers on a Gold Surface Due to the Self-Assembling Properties of Peptides. Langmuir 1998, 14, 6935−6940. 48

Sek, S.; Tolak, A.; Misicka, A.; Palys, B.; Bilewicz, R. Asymmetry of Electron Transmission

through Monolayers of Helical Polyalanine Adsorbed on Gold Surfaces. J. Phys. Chem. B 2005, 109, 18433−18438.


31


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

49

Page 32 of 33

Olejnik, P.; Palys, B.; Kowalczyk, A.; Nowicka, A. Orientation of Laccase on Charged

Surfaces. Mediatorless Oxygen Reduction on Amino- and Carboxyl-Ended Ethylphenyl Groups. J. Phys. Chem. C 2012, 116, 25911−25918. 50

Olejnik, P.; Pawlowska, A.; Palys, B. Application of Polarization Modulated Infrared

Reflection Absorption Spectroscopy for electrocatalytic activity studies of laccase adsorbed on modified gold electrodes. Electrochim. Acta 2013, 110, 105– 111. 51

Holzbaur, I. E.; English, A. M.; Ismail, A. A. FTIR Study of the Thermal Denaturation of

Horseradish and Cytochrome c Peroxidases in D2O. Biochemistry-USA 1996, 35, 5488-5494. 52

Al-Azzam, W.; Pastrana, E. A.; Ferrer, Y.; Huang, Q.; Schweitzer-Stenner, R.;

Griebenow, K. Structure of Poly(Ethylene Glycol)-Modified Horseradish Peroxidase in Organic Solvents: Infrared Amide I Spectral Changes upon Protein Dehydration Are Largely Caused by Protein Structural Changes and Not by Water Removal Per Se. Biophys. J. 2002, 83, 3637-3651. 53

Iwaki, M.; Rich, P. R. An IR Study of Protonation Changes Associated with Heme-Heme

Electron Transfer in Bovine Cytochrome c Oxidase. J. Am. Chem. Soc. 2007, 129, 2923-2929. 54

Gerlache, M.; Senturk, Z.; Quarin, G.; Kauffmann, J.-M. Electrochemical Behavior of H202

on Gold. Electroanal. 1997, 9, 1088-1092. 55

Wang, Z.; Xia, J.; Guo, X.; Xia, Y.; Yao, S.; Zhang, F.; Li, Y.; Xia, L. Platinum/graphene

functionalized by PDDA as novel enzyme carrier for hydrogen peroxide biosensor. Anal. Methods 2013, 5, 483-488.


32

Page 33 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


56

Nandini, S.; Nalini, S.; Manjunatha, R.; Shanmugam, S.; Melo, J. S.; Suresh, G. S.

Electrochemical biosensor for the selective determination of hydrogen peroxide based on the codeposition of palladium, horseradish peroxidase on functionalized-graphene modified graphite electrode as composite. J. Electroanal. Chem. 2013, 689, 233-242. 57

Umasankar, Y.; Unnikrishnan, B.; Chen, S.-M.; Ting, T.-W. Graphene impregnated with

horseradish peroxidase multimer for the determination of hydrogen peroxide. Anal. Methods, 2012, 4, 3653-3660.


33

Electrochemically Reduced Graphene Oxide on Electrochemically

Recommend Documents