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Journal ArticleDOI

PENTEL Actin-G Immunoelectrode: Immunoassay at the Tip of a Pencil

01 Jan 2015-Electroanalysis (John Wiley & Sons, Ltd)-Vol. 27, Iss: 1, pp 166-176
TL;DR: The design and assay methodology of an actin-G immunoelectrode for the quantitation of muscle protein, actin is presented and it is suggested that capture antibody is randomly dispersed over the surface, exposing both Fc and actin binding sites.
Abstract: Here we present design and assay methodology of an actin-G immunoelectrode for the quantitation of muscle protein, actin. The immunoelectrode was assembled using a proprietary pencil material composed of a polymer-resin and graphite composite as an antibody support and electronic conductor. An effective immobilisation procedure for the capture antibody and surface blocking protein is cited based on a two-step physisorption chemistry. BSA coverage was demonstrated to reduce non-specific interactions to below 8 % of the base polymer-graphite binding capacity. Immunoassays performed with the actin immunoelectrode utilise an o-benzoquinone-diimine electrochemistry which is formed in-situ by interfacial peroxidase oxidation. Cyclic voltammetry data revealed the immunoelectrode surface to be porous to the diimine signalling molecule, while remaining inaccessible to macromolecules of the ELISA. The molecular structure and poly-functional nature of the immunoelectrode was further investigated using an anti-antibody peroxidase conjugate. The findings suggest that capture antibody is randomly dispersed over the surface, exposing both Fc and actin binding sites. A saturation current density – concentration curve was analysed by Langmuir adsorption theory and an estimate for the antibody binding affinity derived (Ka≈1×105 M−1). The actin immunoelectrode was applied as a peroxidase detector in a double-antibody immunoassay. The electrode shows a dose-response characteristic which is most sensitive to actin over the concentration range 1–100 ng mL−1. Immunoelectrode sensitivity for actin is of the order of 5 nA mm−2/ng mL−1 (10–100 ng mL−1) with a limit of detection estimated at 10 ng mL−1 and measurement precision RSD 7.1 % (100 ng mL−1 actin). At higher concentrations of actin, >100 ng mL−1 the immunoelectrode response is more complex. A high-dose hook characteristic becomes evident which arises from macromolecular steric interactions and other binding site limitations at the electrode surface.

Summary (3 min read)

1 Introduction

  • Actin is a ubiquitous structural protein of eukaryote cells with important roles in chemo-mechanical processes associated with motility and muscle contraction [1,2].
  • The study looks at the immunochemical characteristics of an anti-actin functionalised electrode (actin-G immunoelectrode) operating as a sensitive peroxidase detector based on o-benzoquinone-diimine redox electrochemistry.
  • Inevitably, this aspect of the immunoassay introduces a further degree of complexity to the detection of actin, since the conjugate recognises both exposed Fc orientations of the capture antibody as well as the presence of specifically bound secondary antibody to captured actin.

2.1 Materials

  • Hi-polymer Super 0.5 mm HB re-fill pencil lead, C505 12 leads/box was supplied by Pentel Pencil Co, Japan.
  • Solvent for polymer dispersion was tetrahydrofuran, 99.5 % (LabScan Analytical Sciences).
  • Immunoassay reagent solutions were prepared with phosphate buffer saline tablets (PBS, pH 7.4) purchased from Sigma Life Science.
  • Actin from bovine skeletal muscle was supplied as a lyophilised solid 1 mg .

2.2 Electrochemistry

  • Electrochemistry studies were conducted using a CHI660c Electrochemical Workstation (CH Instruments, USA).
  • For cyclic voltammetry measurements a single-compartment, three-electrode electrochemical cell (8 mL capacity) was employed, with a platinum coil counter electrode (0.5 mm 10 mm) and a fritted Ag/ AgCl j3 M KCl reference.
  • Working electrodes were composed of the polymer-graphite pencil leads sealed with a polycarbonate lacquer, leaving an exposed electrode tip of ca. 3 mm.
  • Electrochemical assays for peroxidase activity bound to polymer-graphite and immunoelectrode surfaces were performed in phosphate buffered solution (0.1 M, pH 6.0) containing KCl (0.1 M), and substrates; ophenylenediamine (1.0 mM) and hydrogen peroxide (1.0 mM).
  • Cathodic diimine and background currents were normalised for immunoelectrode area and quoted as current density, j= I/A.

2.3 Surface Imaging

  • SEM analysis was undertaken on vertical and transverse sections of Pentel pencil leads (JEOL LV-6390) to study the microscopic structure of the electrode surface.
  • Transverse sections of Pentel pencil leads were analysed by mounting the leads horizontally on a carbon adhesive tab.
  • Vertical sections were mounted in an amine-cured epoxy resin, which were ground and polished to a 1mm finish with diamond suspension.
  • Epoxy mounted specimens were thin-film coated with Au/Pd using a SC7620 minisputter coater (Quorum Technologies) before electron microscope imaging.

2.4 Pencil Electrodes

  • The utility of commercial pencil leads as analytical electrodes was recognised several years ago with some of the earliest physicochemical characterisation studies in Japan on materials sourced from Mitsubishi and Pentel Pencil Companies [5, 6].
  • It should be noted that in many cases the inherent electrochemical properties of commercial pencil leads define the extent of their usefulness.
  • Pencil graphite rods were partially coated with a poly film to define the geometric area of the electrode.
  • Graphite rods coated with polycarbonate were then heat cured at 60 8C for 2 hours.
  • Surface roughness would thereby impart considerably greater microscopic area to the electrode than calculated by geometric measurements.

2.5 Actin-G Immunoelectrode

  • This work outlines an effective physisorption technique for the capture antibody onto a polymer-graphite material used for pencil leads.
  • The antibody coating procedure is similar to that for plate preparation in ELISA, where carbonate or phosphate buffer reagents are applied for efficient protein immobilisation onto polystyrene microwells.
  • SEM images of polymer-graphite lead composites: (1) Pencil lead held in the mechanical pencil system; (2) polymer-graphite rod with polycarbonate insulation; (3–4) shafts of pencil leads identifying edge deformations and surface striae; (5) Surface of the electrode at X200 magnification detailing the approx.
  • This initial treatment also leaves a loosely formed monolayer of capture antibody and BSA molecules adhered to the polymer-graphite surface, held largely by weak polar-ionic interactions.
  • Sets of electrodes coated with BSA alone were prepared in a similar manner by exposing the polymer-graphite material to a protein solution followed by washing with PBS-T and storing under refrigeration conditions.

2.6.1 Nonspecific Adsorption

  • Adsorption characteristics of the anti-antibody-peroxidase conjugate onto BSA functionalised polymer-graphite surfaces were compared to the conjugate s interactions with the base polymer-graphite material [10, 11].
  • Similar treatments were followed for adsorption studies on the polymer-graphite material.
  • The electrode was transferred to an electrochemical cell holding a platinum auxiliary electrode and silver reference electrode and electrolyte composed of phosphate buffer (K2HPO4/KH2PO4 0.1 M, KCl 0.1 M, pH 6.0, 2 mL) and peroxidase substrates, o-phenylenediamine (1.0 mM) and hydrogen peroxide (1.0 mM).
  • After a period of one minute, cyclic voltammetry measurements were run to detect diimine formation and determine peroxidase activity at the electrode surface.

2.6.2 Specific Binding

  • The binding of anti-rabbit antibody-peroxidase conjugate to the actin immunoelectrode was investigated in a series of assays to elucidate capture antibody orientation.
  • Here, immunoelectrodes and BSA electrodes were exposed to buffer solutions of antibody-peroxidase-conjugate (200 mL, 1000 ngmL 1) for 1 hour at 25 1 8C (set of 5 per vial) after which electrodes were dip-washed with PBS-T (pH 7.4, 3 times) and the peroxidase activity measured by cyclic voltammetry.
  • The electrochemical assay method applies BSA electrode controls with immunoelectrode measurements because of the concentration dependence of conjugate non-specific binding.

2.6.3 ELISA

  • Actin immunoassays with actin-G immunoelectrode as detector involved the following procedure: (i) actin immunoelectrode (set of 5 per vial), incubated with actin-G antigen (200 mL, standards: 0–1000 ng mL 1) for 1 hour at 25 1 8C.
  • This was followed by PBS-T dip-wash (3 times).
  • (ii) actin-bound immunoelectrodes were then incubated with anti-actin antibody (secondary antibody) for 1 hour 25 1 8C, and again washed with PBS-T. (iii) actin immunoelectrode is then incubated with anti-antibody peroxidase conjugate (200 mL, 1000 ngmL 1) and again washed with PBS-T. Immunoelectrodes were then transferred to an electrochemical cell for peroxidase activity measurement.
  • A set of BSA-functionalised electrodes as controls followed the same routine and were utilized for background assessment.

3.1 Diimine Electrochemistry

  • Ortho-Phenylenediamine electrochemistry at polymergraphite electrodes is complex, displaying several electron transfer processes and chemical reactions leading to insitu generated redox species.
  • The anodic processes of o-phenylenediamine were researched previously in some detail.
  • Reverse scans display significantly less complexity, with a single and weak reduction process evident at Ep,c = +9 mV.
  • Once diimine is formed in this interfacial solution, the electrode detects the redox molecule by electroreduction.
  • This oxidation reaction is not obvious in the cyclic voltammetry of o-phenylenediamine and serves to emphasise the mechanistic differences between the peroxidase and electrode processes.

3.2 Conjugate Adsorption

  • Adsorption of the anti-antibody-peroxidase-conjugate onto polymer-graphite and BSA-functionalised surfaces was investigated to establish the degree of non-specific binding to these materials [10,11].
  • A current-concentration response of the BSA electrode to peroxidase conjugate was further elucidated by cyclic voltammetry.
  • Current densities, jp, are calculated from cathodic current and electrode area estimates.
  • A Langmuir treatment of conjugate adsorption could only be expected to yield estimates of maximum immunoelectrode output, jpmax, or a rudimentary of the affinity constant, Ka for the capture antibody-conjugate immunoreaction.
  • Once more, this could be explained by the crude adsorption model adopted for protein binding and the assumptions made equating the electrochemical parameter, jp, and surface concentration of conjugate.

3.3 Actin ELISA

  • The immunoelectrode response towards actin-G antigen in a double-antibody ELISA was studied by diimine voltammetry.
  • Figure 9 shows typical forms of the immunoelectrode signal after exposure to an actin standard (1000 ngmL 1), following incubation with the secondary antibody and anti-antibody peroxidase conjugate.
  • Cathodic shifts in Epc again appear in these voltammetric measurements which correlate with increasing surface concentration of peroxidase (data not shown).
  • The actin immunoelectrode current density reaches an optimum value for the 100 ng mL 1 actin standard, before decreasing at greater actin concentrations.
  • Such interactions involve all of the molecules in the ELISA scheme.

4 Conclusions

  • Cyclic voltammetry response of the actin immunoelectrode to actin antigen 100 ng/mL: (a) BSA electrode response; (b) actin immunoelectrode.
  • ELISA data summary in Table 2. Table 2. Actin ELISA data collated for an actin responsive immunoelectrode utilising a double-antibody protocol.
  • Plotted as current density, jp (background corrected) versus actin concentration (logarithmic scale).
  • The work illustrates how such polymer-graphite materials offer adequate physisorption of proteins and act as reliable supports for antibodies in electrochemical immunoassay.
  • This result has implications for immunoelectrode sensitivity and would infer that most capture antibodies are arranged side-on at the electrode surface.

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DOI: 10.1002/elan.201400396
PENTEL Actin-G Immunoelectrode: Immunoassay at the
Tip of a Pencil
Dhanraj Rathod,*
[a]
Susan Warren,
[c]
Brian Seddon,*
[b]
Baljit Singh,*
[b]
and Eithne Dempsey*
[a, b]
1 Introduction
Actin is a ubiquitous structural protein of eukaryote cells
with important roles in chemo-mechanical processes asso-
ciated with motility and muscle contraction [1,2]. The
molecule can transition between a 42 kDa monomeric
globular form (actin-G) and a filamentous structure
under the control of actin-binding proteins. Cardiac
muscle isoforms of globular actin are known and these
were formerly applied as biochemical markers prior to
troponin for heart diagnostic tests [3]. The occurrence of
globular actin in blood is indicative of general tissue ne-
crosis and considering the proteins abundance in muscle
offers diagnostic value for the assessment of skeletal
muscle damage.
Electronic devices possessing selective immunochemi-
cal receptors such as immunoelectrodes make ideal ana-
lytical instruments for the sensitive detection and quanti-
tation of actin where muscle performance is being moni-
tored. Immunoassay at the Tip of a Pencil was a research
exercise intended to investigate assay development
themes in connection with immunoelectrodes assembled
with proprietary pencil-lead materials as alternatives to
print or metal films. Practical aspects of the assay format
and indeed the electrode system itself are not overriding
issues in the current work, nor are method correlations
for optimising a muscle actin diagnostic test. Rather, this
preliminary research examines immuno-electrochemical
methodology and concerns the advancement of opera-
tional aspects of immunoelectrode systems.
The study looks at the immunochemical characteristics
of an anti-actin functionalised electrode (actin-G immu-
noelectrode) operating as a sensitive peroxidase detector
based on o-benzoquinone-diimine redox electrochemistry.
The work is largely concerned with aspects of actin re-
agent chemistry which is relevant to immunoelectrode-
ELISA use. Molecular stability and immunoreactivity of
Abstract: Here we present design and assay methodology
of an actin-G immunoelectrode for the quantitation of
muscle protein, actin. The immunoelectrode was assem-
bled using a proprietary pencil material composed of
a polymer-resin and graphite composite as an antibody
support and electronic conductor. An effective immobili-
sation procedure for the capture antibody and surface
blocking protein is cited based on a two-step physisorp-
tion chemistry. BSA coverage was demonstrated to
reduce non-specific interactions to below 8% of the base
polymer-graphite binding capacity. Immunoassays per-
formed with the actin immunoelectrode utilise an o-ben-
zoquinone-diimine electrochemistry which is formed in-
situ by interfacial peroxidase oxidation. Cyclic voltamme-
try data revealed the immunoelectrode surface to be
porous to the diimine signalling molecule, while remain-
ing inaccessible to macromolecules of the ELISA. The
molecular structure and poly-functional nature of the im-
munoelectrode was further investigated using an anti-an-
tibody peroxidase conjugate. The findings suggest that
capture antibody is randomly dispersed over the surface,
exposing both Fc and actin binding sites. A saturation
current density concentration curve was analysed by
Langmuir adsorption theory and an estimate for the anti-
body binding affinity derived (K
a
110
5
M
1
). The actin
immunoelectrode was applied as a peroxidase detector in
a double-antibody immunoassay. The electrode shows
a dose-response characteristic which is most sensitive to
actin over the concentration range 1–100 ngmL
1
. Immu-
noelectrode sensitivity for actin is of the order of
5nAmm
2
/ngmL
1
(10–100 ngmL
1
) with a limit of de-
tection estimated at 10 ngmL
1
and measurement preci-
sion RSD 7.1% (100 ngmL
1
actin). At higher concentra-
tions of actin, > 100 ngmL
1
the immunoelectrode re-
sponse is more complex. A high-dose hook characteristic
becomes evident which arises from macromolecular steric
interactions and other binding site limitations at the elec-
trode surface.
Keywords: Pentel · Pencil · Graphite · Immunoelectrode · Actin · ELISA · Nonspecific Binding
Special Issue ESEAC
[a] D. Rathod, E. Dempsey
Centre for Research in Electroanalytical Technologies
(CREATE), Institute of Technology Tallaght (ITT Dublin)
Tallaght, Dublin 24, Ireland.
*e-mail: Eithne.Dempsey@ittdublin.ie
[b] B. Seddon, B. Singh, E. Dempsey
MiCRA-Biodiagnostics Technology Gateway Research
Centre, CASH-Synergy Centre, Institute of Technology
Tallaght (ITT Dublin)
Tallaght, Dublin 24, Ireland.
*e-mail: Brian.Seddon@ittdublin.ie
Baljit.Singh@ittdublin.ie
[c] S. Warren
Department of Materials, University of Oxford, Begbroke
Science Park, Woodstock Road, Oxford, OX51PF, UK.
www.electroanalysis.wiley-vch.de 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2015, 27, 166 176 166
Full Paper

capture antibody (IgG) surfaces are paramount to the re-
liable operation of the electrode. These parameters are
examined with reference to physi-sorption-immobilisation
of the capture antibody for data collected during conju-
gate adsorption and actin assays.
Non-specific binding characteristics of the antibody-
peroxidase conjugate towards the polymer-graphite and
protein-modified surfaces, such as BSA monolayers, are
communicated. Interest here lies in understanding and
improving the efficiency of surface blocking in order to
control random binding events while maximising access
for the redox signalling molecule to the electrode.
The advantages and limitations of a secondary antibody
detection strategy is also under scrutiny. Actin analysis
makes use of a peroxidase-antibody conjugate which
binds to a secondary antibody (polyclonal) as shown in
the assay scheme of Figure 1. Inevitably, this aspect of the
immunoassay introduces a further degree of complexity
to the detection of actin, since the conjugate recognises
both exposed Fc orientations of the capture antibody as
well as the presence of specifically bound secondary anti-
body to captured actin. Electrochemical experiments are
conducted to assess the level of responsiveness of the
actin-G immunoelectrode to this peroxidase conjugate.
Furthermore, the implications of conjugate background
interference in quantitative actin analysis are discussed.
2 Experimental
2.1 Materials
Hi-polymer Super 0.5 mm HB re-fill pencil lead, C505 12
leads/box was supplied by Pentel Pencil Co, Japan. Film
insulating polymer, poly(propylenecarbonate) M
n
=
50 kDa is an Aldrich product. Solvent for polymer disper-
sion was tetrahydrofuran, 99.5% (LabScan Analytical
Sciences). o-phenylenediamine, (OPD) 99% m/m
(Fluka). Hydrogen peroxide, 50% v/v (Sigma-Aldrich).
All solutions were made up using deionised water (18
MW) prepared with a PureLab water system (ELGA)
and stored refrigerated until used. Immunoassay reagent
solutions were prepared with phosphate buffer saline tab-
lets (PBS, pH 7.4) purchased from Sigma Life Science.
Horseradish peroxidase (HRP) assay solutions (0.1 M,
pH 6.0) were prepared from potassium phosphate mono-
basic (K
2
HPO
4
) 98% (Sigma) and potassium phosphate
dibasic (KH
2
PO
4
), ACS reagent 99 % (Sigma), pH ad-
justed with H
3
PO
4
, 85% (Aldrich) and included potassi-
um chloride (0.1 M) 99.0 % (Sigma-Aldrich). Assay
wash buffer used was PBS-Tween 20 (PBS-T) pH 7.4,
0.05% m/v, (Fluka). Bovine serum albumin (BSA) is em-
ployed for surface blocking and immuno-reagent stabili-
sation, 96% (Sigma). Actin from bovine skeletal
muscle was supplied as a lyophilised solid 1 mg (Sigma).
Actin-G standards (0 ng mL
1
–1000 ngmL
1
) were pre-
pared in PBS-BSA (0.1% w/v pH 7.4) solutions from
a stock of 1000 ngmL
1
. Antibodies were purchased from
Santa Cruz Biotechnology Inc. USA. Rabbit polyclonal
anti-actin primary antibody (200 mg in 0.5 mL in PBS
pH 7.4) was used for actin capture and as secondary anti-
body. Goat anti-rabbit-IgG peroxidase conjugate (200 mg
in 0.5 mL in PBS pH 7.4) was used as the ELISA signal-
ling label.
2.2 Electrochemistry
Electrochemistry studies were conducted using
a CHI660c Electrochemical Workstation (CH Instru-
ments, USA). For cyclic voltammetry measurements
a single-compartment, three-electrode electrochemical
cell (8 mL capacity) was employed, with a platinum coil
counter electrode (0.5 mm10 mm) and a fritted Ag/
AgClj3 M KCl reference. Working electrodes were com-
posed of the polymer-graphite pencil leads sealed with
a polycarbonate lacquer, leaving an exposed electrode tip
of ca. 3 mm. Cyclic voltammetry was performed at a scan
rate of 100 mVs
1
over a fixed potential window of + 0.1
to 0.1 V vs Ag/AgCl. Prior to protein functionalisation,
polymer-graphite electrodes were subjected to a mild
anodic treatment in PBS (0.1 M, pH 6.0) electrolyte. This
was achieved by scanning electrode potentials between 0
and + 1.0 V vs. Ag/AgCl for 10 cycles, a process which en-
hanced the charge transfer characteristics of the carbon
material towards redox molecules as well as confirming
the low inherent electrochemistry of the composite graph-
Special Issue ESEAC
Fig. 1. Illustration of the actin immunoelectrode format exploit-
ed during ELISA. Anti-actin antibody (capture antibody) and
BSA are irreversibly immobilised onto the polymer-graphite
electrode surface. This assembly leaves inter-protein gaps which
redox molecules can traverse. The scheme shows capture anti-
body bound actin, a polyclonal secondary antibody molecule and
the signalling peroxidase conjugate prior to substrate introduc-
tion.
www.electroanalysis.wiley-vch.de 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2015, 27, 166 176 167
Full Paper

ite material. Electrochemical assays for peroxidase activi-
ty bound to polymer-graphite and immunoelectrode sur-
faces were performed in phosphate buffered solution
(0.1 M, pH 6.0) containing KCl (0.1 M), and substrates; o-
phenylenediamine (1.0 mM) and hydrogen peroxide
(1.0 mM). Peroxidase activity was determined from the
peak current of the o-benzoquinone diimine reduction
wave at the immunoelectrode using cyclic voltammetry.
4
Cathodic diimine and background currents were normal-
ised for immunoelectrode area (geometric) and quoted as
current density, j = I/A. In this work j is reported in
nAmm
2
units (equivalent to mAm
2
).
2.3 Surface Imaging
SEM analysis was undertaken on vertical and transverse
sections of Pentel pencil leads (JEOL LV-6390) to study
the microscopic structure of the electrode surface. Trans-
verse sections of Pentel pencil leads were analysed by
mounting the leads horizontally on a carbon adhesive tab.
Vertical sections were mounted in an amine-cured epoxy
resin, which were ground and polished to a 1mm finish
with diamond suspension. Epoxy mounted specimens
were thin-film coated with Au/Pd using a SC7620 mini-
sputter coater (Quorum Technologies) before electron
microscope imaging.
2.4 Pencil Electrodes
The utility of commercial pencil leads as analytical elec-
trodes was recognised several years ago with some of the
earliest physicochemical characterisation studies in Japan
on materials sourced from Mitsubishi and Pentel Pencil
Companies [5,6]. These materials are manufactured from
proprietary formulations of micron-graphite particulates
and low-temperature thermoplastic resins which are ex-
trusion processed into narrow rods. The surface composi-
tion of pencil leads may therefore be considered as
a random distribution of graphite particles interspersed
with electronically insulating polymer. Extruded graphite
composites have since found applications as electrodes in
different electroanalysis techniques, notably for studies
on metal ions [7] and pharmaceutical molecules and bio-
sensors [8,9]. Although their electronic, and in certain in-
stances electrochemical properties of pencil materials are
quite favourable, their poor tensile strength imposes prac-
tical restrictions.
Research in our laboratory has explored a series of
Pentel polymer-graphite materials for electrodes in bio-
sensor devices as alternative electronic supports to print
and thin-film metals. It should be noted that in many
cases the inherent electrochemical properties of commer-
cial pencil leads define the extent of their usefulness. In
this regard the Pentel graphite material reported in the
present study is exceptional.
Electrodes were fabricated from a thermopolymer-
graphite material used in the manufacture of commercial
fine pencil lead (Hi-Polymer HB, Pentel Pencil Company,
Japan). The leads are supplied as re-fill rods for the
SHARPLET-2 0.5 pencil system, Figure 2. Graphite rods
for this pencil measure ca 0.55 mm diameter by 60.5 mm
long and come supplied as 12 leads per box. They varied
between 0.55 mm and 0.57 mm in diameter, 0.56
0.01 mm (n = 10) as estimated by digital calipers. Electron
micrograph data of resin-potted samples provide rod di-
ameter estimates of 579 mm(n= 3). The electrochemical
characteristics of the Hi-Polymer material were formerly
investigated.
5
This polymer-graphite composite possesses
impressive chemical stability in aqueous media and excel-
lent electrical conductivity (R
rod
< 5 W). As an electrode
the material has considerable utility, no inherent electro-
chemistry, and is stable for a broad range of applied elec-
trolysis voltages (> 2 V) and current densities (ca.
1Am
2
). The material is known for its non-wetting hy-
drophobic property, which can be overcome by anodic or
oxidative chemical treatments. Owing to the organic poly-
mer content of the Hi-Polymer HB leads, the electrode
fails at high temperatures (etching at extreme current
densities) and in certain organic solvents. These charac-
teristics are not applicable in the present studies.
Pencil graphite rods were partially coated with a poly(-
propylenecarbonate) film to define the geometric area of
the electrode. This was achieved by dispersing the poly-
mer (M
n
50 kDa) in tetrahydrofuran (100 mg mL
1
). The
polycarbonate was applied to the rod using a capillary in-
sertion technique. In this procedure, polymer-solvent
(20 mL) is injected into a capillary tube (1 mm dia.).
This is followed by the insertion and removal of
a pencil rod. Stainless steel forcepts are used (HWC 110-
10, Solingen, Germany). The length of rod protruding the
capillary is estimated using digital calipers (0.01 mm reso-
lution). Graphite rods coated with polycarbonate were
then heat cured at 608C for 2 hours. This procedure left
an insulation film covering the graphite rod of film thick-
ness < 10 mm with exposed lengths of 3.00.1 mm, which
defined an electrode surface area of 5.5 0.5 mm
2
.The
polycarbonate coating, film thickness and electrode defi-
nition were examined by SEM imaging to confirm the
extent of the polymer film surface. Of interest was the in-
Special Issue ESEAC
Fig. 2. PENTEL SHARPLET-2 0.5 mechanical pencil system
with Hi-Polymer HB refill pencil leads employed in the construc-
tion of a single-measurement actin immunoelectrode.
www.electroanalysis.wiley-vch.de 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2015, 27, 166 176 168
Full Paper

tegrity of the insulation layer, its uniformity and adher-
ence to the pencil lead (Figure 3). Poly(propylenecarbon-
ate) coatings on graphite surfaces appear as glassy films.
They offer durable thick-films, chemically stable in aque-
ous solution (pH 5–8) with desirable electrical properties
(R
film
> MW). The microscopic structure of the polymer-
graphite surface is revealed in the electron microscopy
images of Figure 3. The pencil surface is composed of
a regular pattern of undulations. This structure has an ap-
proximate peak-to-peak separation of 40 mm and is an ar-
tefact of the lead manufacturing process. Surface rough-
ness would thereby impart considerably greater micro-
scopic area to the electrode than calculated by geometric
measurements. In our estimations the microscopic surface
of Pentel leads is of the order of 60% larger than the
geometric figure suggests. Electrode area measurements
by coulometry agree with the geometric calculation to
within 3.5%, since diffusion dependent charge is ana-
lysed.
2.5 Actin-G Immunoelectrode
This work outlines an effective physisorption technique
for the capture antibody onto a polymer-graphite material
used for pencil leads. The antibody coating procedure is
similar to that for plate preparation in ELISA, where car-
bonate or phosphate buffer reagents are applied for effi-
cient protein immobilisation onto polystyrene microwells.
For immunoelectrodes it is essential that a coating proto-
col offers a stable and reproducible antibody monolayer
for the immunoreaction with a specific antigen. Immu-
noelectrodes must not only have a support function for
Special Issue ESEAC
Fig. 3. SEM images of polymer-graphite lead composites: (1) Pencil lead held in the mechanical pencil system; (2) polymer-graphite
rod with polycarbonate insulation; (3–4) shafts of pencil leads identifying edge deformations and surface striae; (5) Surface of the
electrode at X200 magnification detailing the approx. 40 mm microstructure; (6) epoxy-resin mounted graphite for rod diameter meas-
urements.
www.electroanalysis.wiley-vch.de 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2015, 27, 166 176 169
Full Paper

capture antibodies, but also offer an unrestricted pathway
for redox molecules linked to the assay signalling mecha-
nism. For this reason capture molecule layers and other
surface modifications must remain porous to small mole-
cules.
The first step in the immunoelectrode preparation in-
volves immersion of polymer-graphite rods in a buffer so-
lution of anti-actin capture antibody (anti-actin rabbit
polyclonal antibody, 100 mLof1mgmL
1
) in PBS at
pH 7.4. Antibody immobilisation was carried out at 25
18C for 12 hours. Electrodes were then “block-washed”
with 5% BSA-PBS-T solution. The wash removes loosely
bound antibody while filling vacant surface sites with
inert protein. This initial treatment also leaves a loosely
formed monolayer of capture antibody and BSA mole-
cules adhered to the polymer-graphite surface, held large-
ly by weak polar-ionic interactions. Such proteins are rel-
atively easily removed from hydrophobic surfaces and as
such unreliable in immunoassay applications. To enhance
protein coating, a second step to the immobilisation pro-
cedure was found to be necessary. Actin antibody electro-
des were allowed to air dry at 2518C for 1 hour before
being stored refrigerated at 48C for 24 hours prior to im-
plementation. The combination of solution-phase physi-
sorption, air drying and cold storage improves IgG and
BSA adhesion to the polymer-graphite surface, allowing
the immunoelectrode to undergo necessary vigorous
washing steps during assays without significant loss of im-
munoreactivity. It is speculated that partial dehydration
of the antibody monolayer on the pencil material leads to
conformational changes which strengthens antibody-elec-
trode interactions. Sets of electrodes coated with BSA
alone were prepared in a similar manner by exposing the
polymer-graphite material to a protein solution followed
by washing with PBS-T and storing under refrigeration
conditions. BSA electrodes were used to assess non-spe-
cific binding of reagents and as controls in actin immuno-
assays.
2.6 Assays
2.6.1 Nonspecific Adsorption
Adsorption characteristics of the anti-antibody-perox-
idase conjugate onto BSA functionalised polymer-graph-
ite surfaces were compared to the conjugates interactions
with the base polymer-graphite material [10,11]. BSA
functionalised electrodes were incubated (set of 5 per
vial) with buffer solutions of antibody-peroxidase conju-
gate (200 mL, 1000 ngmL
1
) for 1 hour at 2518C. This
was followed by dip-washing with PBS-T 0.05% m/v
(pH 7.4, 3 times). Similar treatments were followed for
adsorption studies on the polymer-graphite material. The
electrode was transferred to an electrochemical cell hold-
ing a platinum auxiliary electrode and silver reference
electrode and electrolyte composed of phosphate buffer
(K
2
HPO
4
/KH
2
PO
4
0.1 M, KCl 0.1 M, pH 6.0, 2 mL) and
peroxidase substrates, o -phenylenediamine (1.0 mM) and
hydrogen peroxide (1.0 mM). After a period of one
minute, cyclic voltammetry measurements were run to
detect diimine formation and determine peroxidase activ-
ity at the electrode surface. Owing to the electrochemical
reactivity of the o -phenylenediamine substrate, with sig-
nificant electrooxidation beyond +0.2 V, electrode poten-
tials in peroxidase assays were scanned between + 0.1 and
0.1 V vs. Ag/AgCl.
2.6.2 Specific Binding
The binding of anti-rabbit antibody-peroxidase conjugate
to the actin immunoelectrode was investigated in a series
of assays to elucidate capture antibody orientation. Here,
immunoelectrodes and BSA electrodes were exposed to
buffer solutions of antibody-peroxidase-conjugate
(200 mL, 1000 ngmL
1
) for 1 hour at 2518C (set of 5
per vial) after which electrodes were dip-washed with
PBS-T (pH 7.4, 3 times) and the peroxidase activity mea-
sured by cyclic voltammetry. The electrochemical assay
method applies BSA electrode controls with immunoelec-
trode measurements because of the concentration de-
pendence of conjugate non-specific binding.
2.6.3 ELISA
Actin immunoassays with actin-G immunoelectrode as
detector involved the following procedure: (i) actin im-
munoelectrode (set of 5 per vial), incubated with actin-G
antigen (200 mL, standards: 0–1000 ngmL
1
) for 1 hour at
2518C. This was followed by PBS-T dip-wash (3 times).
(ii) actin-bound immunoelectrodes were then incubated
with anti-actin antibody (secondary antibody) for 1 hour
2518C, and again washed with PBS-T. (iii) actin immu-
noelectrode is then incubated with anti-antibody perox-
idase conjugate (200 mL, 1000 ngmL
1
) and again washed
with PBS-T. Immunoelectrodes were then transferred to
an electrochemical cell for peroxidase activity measure-
ment. A set of BSA-functionalised electrodes as controls
followed the same routine and were utilized for back-
ground assessment.
3 Results and Discussion
3.1 Diimine Electrochemistry
ortho-Phenylenediamine electrochemistry at polymer-
graphite electrodes is complex, displaying several electron
transfer processes and chemical reactions leading to in-
situ generated redox species. Newly formed molecules
themselves exhibit subtle oxidative electrochemistry. The
anodic processes of o-phenylenediamine were researched
previously in some detail. The oxidised species frequently
cited is the reactive o-benzoquinone-diimine. This diimine
molecule is electrochemically reduced to a di-amine or
mono-imine [7, 8]. In cyclic voltammetry the electro-oxi-
dation of o-phenylenediamine in high-buffering phos-
phate electrolyte at pH 6.0 is well defined, Figure 4. Mul-
tiple anodic reactions are prominent; a primary peak is
Special Issue ESEAC
www.electroanalysis.wiley-vch.de 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Electroanalysis 2015, 27, 166 176 170
Full Paper

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References
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Frequently Asked Questions (1)
Q1. What contributions have the authors mentioned in the paper "Pentel actin-g immunoelectrode: immunoassay at the tip of a pencil" ?

Rather, this preliminary research examines immuno-electrochemical methodology and concerns the advancement of operational aspects of immunoelectrode systems. The study looks at the immunochemical characteristics of an anti-actin functionalised electrode ( actin-G immunoelectrode ) operating as a sensitive peroxidase detector based on o-benzoquinone-diimine redox electrochemistry. Here the authors present design and assay methodology of an actin-G immunoelectrode for the quantitation of muscle protein, actin. The molecular structure and poly-functional nature of the immunoelectrode was further investigated using an anti-antibody peroxidase conjugate. The findings suggest that capture antibody is randomly dispersed over the surface, exposing both Fc and actin binding sites.