Article

pubs.acs.org/ac

Black Phosphorus Nanoparticle Labels for Immunoassays via

Hydrogen Evolution Reaction Mediation
Carmen C. Mayorga-Martinez,† Naziah Mohamad Latiff,† Alex Yong Sheng Eng,† Zdeněk Sofer,‡
and Martin Pumera*,†
†
Division of Chemistry & Biological Chemistry, School of Physical Mathematical Science, Nanyang Technological University,
Singapore 637371, Singapore
‡
Department of Inorganic Chemistry, University of Chemistry and Technology Prague, Technicka 5, 166 28, Prague 6, Czech
Republic

ABSTRACT: Black phosphorus is an emerging layered

material. Its nanoparticles show an increased bandgap when
compared to bulk materials and they are typically fabricated by
ultrasonication of macroscopic black phosphorus crystals. Here
we fabricate black phosphorus nanoparticles (BP NPs) by
solution based electrochemical exfoliation with bipolar electro-
des, which induces opposite potentials on the opposite ends of
black phosphorus macroparticles thereby leading to its
decomposition into nanoparticles. BP NPs have enhanced
catalytic effect on the hydrogen evolution reaction (HER)
relative to black phosphorus macroparticles. We utilize black
phosphorus nanoparticles as electrocatalytic tags in a
competitive immunoassay for rabbit immunoglobulin G
(IgG) detection. The detection signal is produced via nanoimpacts of the BP NPs followed by HER catalysis.

L ayered and 2D materials have been used for several sensing

and biosensing applications.1,2 Recently, layered black
phosphorus has received significant amount of attention as it
can be exfoliated, similarly to graphite to its single layer 2D
material.3−7 Black phosphorus is an elemental material with
strong electrical, magnetic, and electrochemical anisotropies with
low toxicity.8−10 This material has been suggested to play a key
role in many devices spanning across broad applications ranging
from gas sensing11−14 to nanoelectronics15−17 and has more
recently expanded into catalysis.18 Its electronic and optical
properties are further known to change with a decreasing number
of layers.19,20 Black phosphorus nanoparticles (BP NPs) or
quantum dots also demonstrate altered properties when
compared to its bulk form.4,17,19 BP NPs are typically prepared
by mechanical decomposition of millimeter or micrometer sized
grains of black phosphorus with subsequent ultrasonications.4,17
There is immense potential in the use of these nanoparticles, in a
similar manner to other nanoparticles of gold or carbon, as labels
for sensors and biosensors.
Herein, for the first time, we report a single-step solution based
electrochemical exfoliation with bipolar electrodes for exfoliation
and downsizing of the layered black phosphorus microparticles
to nanoparticles with enhanced electrocatalytic activity for the
hydrogen evolution reaction. We show that we can utilize these
BP NPs for protein detection using impact electrochemistry21,22
via hydrogen evolution mediation. The BP NPs label system of a
magnetoimmunoassay based on impact electrochemistry
detection presents a new concept in biosensing technology.
■ EXPERIMENTAL SECTION
Structural and Morphological Characterization. Phoi-
bos 100 spectrometer with monochromatic Mg X-ray radiation
source (SPECS, Germany) and UV−vis spectrometer (Shimad-
zu UV-2500) were used for perform the X-ray photoelectron and
UV−vis spectroscopy, respectively. JEOL 7600F field-emission
scanning electron microscope (JEOL, Japan) in TED mode at
accelerating voltage of 30 kV was used to obtain the scanning
transmission electron micrographs. For the dynamic light
scattering experiments, PMMA cuvettes and Zetasizer Nano
ZS (Malvern Instruments, England) were used and the
measurements were done at 20 °C.
Synthesis of BP NPs. First, black phosphorus crystal was
prepared by a reported protocol.13 In this way, 500 mg of Au/Sn
alloy was obtained by melting stoichiometric amounts gold
(99.99% purchased from Safina, Czech Republic) and tin
(99.999% from STREM, Germany) in chloroform (99.9%
from PENTA, Czech Republic), the reaction was performed
under high vacuum conditions into a glass ampule. Then, 720 mg
of red phosphorus (99.999% from Sigma-Aldrich, Czech
Republic) and 15 mg of SnI4 (99.8% from PENTA, Czech
Republic) were added to the reaction and the ampule was sealed
by using an oxygen/hydrogen torch. The synthesis of SnI4 was
performed using iodine (99.8% from PENTA, Czech Republic)
Received: June 23, 2016
Accepted: September 23, 2016

© XXXX American Chemical Society A DOI: 10.1021/acs.analchem.6b02422

Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry Article

and tin in chloroform under reflux, followed by the purification of potential of 0.01 V (root-mean-square, rms) plus −0.81 V vs
SnI4 and recrystallization from chloroform. Next, the reaction RHE polarization potential in a frequency range of 1 mHz to 100
was heated at 400 °C for 3 h using a muffle furnace. Afterwards, kHz. The overpotential for HER was measured at −10 mA cm−2
the temperature of the reaction was increased to 600 °C and held current density. The electrochemical detection of BP NPs
for 24 h followed by the furnace cooling (overnight) until it captured through magneto immunoassay was performed via
reached room temperature. For removal of white phosphorus HER by spike count of the chronoamperometry scan. The three-
formed as a side product, the obtained black phosphorus platelets component screen printed electrode (obtained from eDAQ
(∼5 mm × 2 mm) were washed with CS2. Instruments) with a magnet disc attached on the reverse side of
Subsequently, the BP crystals (0.5 mg/mL) obtained were the working electrode area was positioned in a horizontal
sonicated for 4 h for pulverization to microparticles and Na2SO4 manner; a fixed 15 μL of the magnetic bead/antirabbit IgG/
was added to the solution to reach a final concentration of 0.5 M. BPNPs-rabbit IgG complex containing 0, 2, 10, 50, 100 ng/mL of
The resulted solution was transferred to the two-platinum free IgG (prepared previously) was drop-casted onto the working
electrodes electrochemical system. The distance between the electrode area. After 1 min, 15 μL of 1 M H2SO4 was drop-casted,
platinum electrodes was 2 cm and 10 V DC potential was applied and with the help of a micropipet was gently mixed and
for 30 min. The obtained solution was left to stand for 1 week, distributed onto the three-component electrode. A reductive
and the supernatants were separated from the pellet. For the potential of −0.88 V vs RHE was applied for 100 s, and
structural characterization (XPS and STEM), the solution was chronoamperometry scans were carried out immediately there-
desalinated using dialysis membrane (cutoff below 1 kDa). after to detect all possible impact of the particles onto the
Competitive Magneto-Immuno Assay Preparation. electrode.
The 3 mg/mL magnetic beads tosyl-activated (Dynabeads M-
280 from Invitrogen, Singapore) solution was washed twice in
0.1 M sodium borate buffer pH 9.2. The borate buffer was
■ RESULTS AND DISCUSSION
In this study, we describe the utilization of BP NPs as labels for
prepared using boric acid 99.5% purchased from Sigma-Aldrich, immunoassays using impact electrochemistry as a mode of
Singapore. Then, 40 μg/mL polyclonal antirabbit IgG produced
in goat (from Sigma-Aldrich, Singapore) solution was added and, Scheme 1. Synthesis of Black Phosphorus Nanoparticles (BP
the mixture was incubated overnight at 37 °C (400 rpm). The NP) by Solution-based Electrochemical Exfoliation with
resulted conjugated (MB/antirabbit IgG) was washed with Bipolar Electrodes
tween buffer. The tween buffer was prepared using 0.01 M PBS
buffer, pH 7.4 (the PBS buffer was prepared using a tablet from
Sigma-Aldrich, Singapore). After that, the blocking step was
performed by incubation for 1 h at 25 °C (400 rpm) of MB/
antirabbit IgG complex in 5% BSA-PBS buffer. The resulting
solution was washed with PBS buffer pH 7.4. In parallel, IgG
from rabbit serum (1 μg/mL) was incubated with BP NPs during
1 h at 25 °C and 400 rpm. The pH of the BP NPs solution was
previously adjusted using PBS buffer pH 7.4. Once the rabbit
IgG/BP NPs was obtained, the conjugate was blocked by adding
15 μL of a 5% BSA solution prepared in Milli-Q water and
incubating for 20 min at 650 rpm and 25 °C. For removal excess
BP NPs, the resulted solution was centrifuged for 2 h at 4 °C
(14 000 rpm). The supernatant was removed, and the pellet was
resuspended using 80 μL of PBS buffer pH 7.4. Finally, the MB/
antirabbit IgG and rabbit IgG labeled with BP NPs were
incubated (for 1 h under 400 rpm agitation at 25 °C) in the
presence of the desired concentration of free rabbit IgG. In this
way, 80 μL of magnetic bead/antirabbit IgG complex was
separated from the supernatant and mixed with 80 μL of BP NPs-
rabbit IgG (previously conjugated) containing 0, 2, 10, 50, and
100 ng/mL of rabbit IgG. Once the conjugated species was
obtained, the washing step was performed using 0.5% tween-PBS detection mediated by hydrogen evolution reaction on BP
buffer, PBS buffer, and Milli-Q water. nanoparticles. There have been many research efforts in
Electrochemical Measurement. The electrochemical developing efficient methods for downsizing and exfoliating
measurements were performed using Autolab PGSTAT204/ layered black phosphorus crystals to BP NPs and phosphorene,
FRA32 M (Eco Chemie, Utrecht, The Netherlands). An all of which were based on mechanical forces, such as
electrochemical cell composed of glassy carbon (GC) electrode ultrasonication.4−7,17−19,23 We introduce a new method for
as the working electrode, Pt counter electrode, and an Ag/AgCl fabrication of such nanoparticles using the solution based
reference electrode were used. The efficiency of BP NPs for the electrochemical exfoliation with bipolar electrodes.24 To
hydrogen evolution reaction was evaluated using linear sweep fabricate BP NPs, we first synthesized large BP crystals.13 The
voltammetry (at a scan rate of 2 mV/s) and electrochemical exfoliation and downsizing method, consists of applying a
impedance spectroscopy (EIS). For this aim, the GC working constant DC potential of 10 V between two platinum electrodes,
electrode was modified with 3 μL of BP NPs solution by drop- see Scheme 1. Potential difference is induced on the BP particles
casting. In all the cases, the measurements were done in 0.5 M at opposite sides of the particle,23 which leads to fragmentation of
H2SO4. EIS measurements were performed using an AC the BP particles into nanoparticles.
B DOI: 10.1021/acs.analchem.6b02422
Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry Article

Figure 1. Photos of black phosphorus macroparticles (BP MPs) solution before (A) and after (B) solution based electrochemical exfoliation with bipolar
electrodes process. STEM micrographs of black phosphorus nanoparticles (BP NPs) (C).

Figure 2. Particle size distribution measured by DLS (A) and UV−vis absorption spectra (B) of black phosphorus nanoparticles (BP NPs). High-
resolution XPS spectra of black phosphorus macroparticles (BP MPs) and BP NPs (C).

Figure 1 shows the BP macroparticles solutions before (A) and
after (B) the exfoliation process. We can clearly see that the
exfoliation process occurs with the disappearance of the
characteristic dark color of the BP macroparticle solution. The
BP NPs suspension was settled after exfoliation process for a
week and the clear solution was separated from the pellet. A
dialysis membrane with a cutoff below 1 kDa was used for
desalination of the BP NPs solution. Scanning transmission
electron microscopy (STEM) was performed on the dialyzed
solution as displayed in Figure 1C, which shows the presence of
platelet-like particles of various sizes. The hydrodynamic radii of
the BP NPs were around 70 nm from the data of the dynamic
light scattering (DLS) (Figure 2A).
For evaluation of the structural and chemical changes of the
resulted BP NPs, UV−visible spectroscopy (Figure 2B) and X-
ray photoelectron spectroscopy (XPS) (Figure 2C) were
performed. The optical absorption spectra of BP NPs show
characteristic absorption bands at UV and NIR regions; similar
results were reported before in the literature.17,23 The character-
istic phosphorus and surface oxidation features17,23,25 were
observed in the high-resolution XPS spectra of BP crystals and
BP NPs, see Figure 2C.
Linear scan voltammetry (LVS) measurements were carried
out for evaluating the performance of the BP macroparticles and
C DOI: 10.1021/acs.analchem.6b02422
BP NPs for hydrogen evolution reaction (Figure 3A). The
overpotential for HER at BP NPs was −0.81 V vs the reversible
hydrogen electrode (RHE), whereas the overpotential for BP
macroparticles was seen at −1.24 V vs RHE. The polarization
curve for bare GC (glassy carbon) produced an overpotential at
−0.97 V vs RHE. These results clearly demonstrate higher
electrocatalytic activity of BP NPs compared to BP macro-
particles and GC electrode.
EIS measurements show the enhanced catalytic activity of BP
NPs by the electrode kinetics under HER operating conditions.
Figure 3B shows a dramatic decrease in charge transfer resistance
(Rct) for the BP NPs (1.39 kΩ) relative to the BP macroparticles
(37.6 kΩ). Accordingly, the EIS results show that BP NPs exhibit
lower electron transfer resistance when compared to the BP
macroparticles. The exfoliation process exposed more catalytic
edges sites8 of the BP NPs (see Figure 3B). These factors
contributed to the enhanced catalytic performance of HER
observed.
Subsequently, the competitive magneto immunoassay for
protein detection using BP NPs as a label was performed.
Scheme 2 (not to scale) presents the depiction of the complete
assay. First, tosyl-activated paramagnetic beads (MB) were
conjugated with antirabbit IgG (a). In parallel, the rabbit IgG is
labeled with BP NPs (a′) and this complex was then conjugated
Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
Figure 3. Linear scan voltammograms (A) and Nyquist plots (B) of
black phosphorus macroparticles (BP MPs) and black phosphorus
nanoparticles (BP NPs). Conditions: 0.5 M H2SO4, glassy carbon (GC)
electrode was modified with 1.5 μg of materials by drop casting.
with MBs/antirabbit IgG in the presence of free rabbit IgG at the
desired concentration to perform the competitive magneto
immunoassay (b). Once the complex MBs/antirabbit IgG/rabbit
IgG-BP NPs is formed, 15 μL of this solution was drop casted
onto the screen-printed electrode followed by addition of 15 μl of
1 M H2SO4 (c). The acid leads to denaturation of the protein
complex and release of BP NPs which consequently impacts the
surface of the electrode (d). The detection of BP NPs (and thus
of IgG concentration) was performed by the electrocatalysis of
impacting BP NPs (nanoimpact method)4,20,21,25,26 via hydrogen
evolution reaction (proton reduction) (e).
Nanoimpact electrochemistry is a method that allows for
determination of nanoparticle size, type, and concentration via
direct oxidation or reduction of the nanoparticles or via catalysis
of an electrochemical reaction (i.e., proton reduction) by particle
impacting on the electrode surface.21,22,26−33 This method was
used recently for DNA analysis, demonstrating their promising
usage in the biosensing field.26 One can directly detect black
phosphorus NPs by their oxidation from P0 to P5+ using impact
electrochemistry.4 Here, we demonstrate the nanoimpact
electrochemistry as a detection technique for protein quantifi-
cation through HER by impact of BP NPs in sulfuric acid. The
acid solution serves not only as an electrolyte but also functions
as a denaturating agent which liberates the BP NPs.
Consequently, the BP NPs are detected via catalysis of H+
reduction to H2 upon impact of BP NPs onto the electrode
surface. Number of spikes is related to the number of attached
black phosphorus nanoparticles which is in turn related to
amount of analyte. The analytical protocol is based on catalytic
events (spikes) which are catalyzed by black phosphorus
nanoparticles.
In order to perform detection of liberated BP NP via
mediation of hydrogen evolution reaction, we have set a
reduction potential of −0.88 V (vs RHE), which is slightly
D DOI: 10.1021/acs.analchem.6b02422
Article
more negative than the overpotential of HER for BP NPs (see
previous section) to perform the chronoamperometry (Figure
4A).27 When the MBs/antirabbit IgG/rabbit IgG-BP NPs are in
contact with H2SO4, the BP NPs are freed and thereby strike the
electrode surface, which generates a current response (reductive
spike) that corresponds to a proton reduction (see Figure 4A, red
line). When the concentration of free rabbit IgG increases, the
number of the spikes decrease as expected in a competitive
immunoassay. This is because the amount of the BP NPs
decreases in the magneto-immunoassay as the immunoassay is
based on competitive binding between rabbit IgG-BP NP and
rabbit IgG. The number of the spikes for the magneto-
immunoassay complex at different concentrations of free rabbit
IgG are larger than the blank control (Figure 4A, black line). To
construct a calibration curve for rabbit IgG detection, we utilized
the number of spikes in the first 40 s of chronoamperograms
(Figure 4B). Only spikes with at least twice the amplitude of the
background signal were considered as impact signals from BP
NP.27 The curve relating the number of spikes as a function of
rabbit IgG concentration shows that the spike count decreases as
protein concentration increases (Figure 4B), as expected from
competitive immunoassay. Furthermore, a calibration curve
relating the inverse of number of spikes vs rabbit IgG
concentration (see Figure 4C) shows a linear range from 2 to
100 ng/mL with an R of 0.9429. Moreover, the system shows a
low limit of detection (LOD) of 0.98 ng/mL. The reproducibility
of the assay was evaluated getting a relative standard deviation
(RSD) around 18% which indicates a good reproducibility of the
system. Reproducibility experiments are performed according to
the Bioanalytical Method Validation from Guidance for Industry
of the Food and Drug Administration (FDA) federal agency of
the United States.34 The RSD reported here represents the
average of four different free rabbit IgG concentrations, with
three replicates of the assay for each concentration.
For the selectivity evaluation, immunoassays were performed
in the absence of BP NPs and nonspecific protein (human
hemoglobin) used instead of rabbit IgG. The small spike count in
the control immunoassays observed demonstrated the high
selectivity of the system (Figure 5B).
■
CONCLUSION
Here we implemented a conceptually new approach of
fabricating black phosphorus nanoparticles that consist in
solution based electrochemical exfoliation with bipolar electro-
des, yielding nanoparticles of 40−200 nm in size with a maximum
at ∼70 nm. Black phosphorus nanoparticles showed hydrogen
evolution activity at more positive potentials than black
phosphorus macroparticles. We demonstrated the utility of
these black phosphorus nanoparticles as a label for protein
detection in a competitive immunoassay through hydrogen
evolution reaction using an optimized nanoimpact method of
black phosphorus NPs anchored to magnetic beads through
immunoassay for the quantification of rabbit IgG. This
developed system shows competitive analytical performance
compared with the systems reported for gold nanoparticles. The
concept of employing black phosphorus nanoparticles on
biomolecular labels is very attractive due to low toxicity of
black phosphorus, thus it is expected to pave its way in the
development of cost efficient biosensors for different target
detection.
Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry Article

Scheme 2. Schematic of the Competitive Magneto-Immunoassay for Protein Using BP NPs as a Tag and HER Electrocatalysis
(Proton Reduction) By Impact Electrochemistry (Spikes Count) as a Detection Techniquea

a
(a and a′) Magnetic beads (MB) conjugation with anti-rabbit IgG and tag-labelled step of rabbit IgG with BP NPs, respectively, (b) incubation of
the MB/anti-rabbit IgG conjugate with rabbit IgG/BP NPs in the presence of different concentrations of free rabbit IgG. Electrochemical detection
by nano-impact method: (c) MB-based complex drop casted onto the working electrode in the presence of 0.5 M of H2SO4 and detection (e) after
impact of the liberated BP NPs (d).

Figure 4. Chronoamperograms of the competitive magneto-immunoassay at different rabbit IgG concentrations in 0.5 M H2SO4 at −0.88 V vs RHE
(A). Curve of the spike count (B) and the inverse of spikes counts (C) as a function of different rabbit IgG concentrations.

E DOI: 10.1021/acs.analchem.6b02422
Anal. Chem. XXXX, XXX, XXX−XXX
Analytical Chemistry
Figure 5. Selectivity evaluation: enlarged chronoamperograms (A) and summary of the spike count (B) obtained for the magneto-immunoassay
performed in the presence and absence of BP NPs and in the presence of human hemoglobin.
■ AUTHOR INFORMATION
Corresponding Author
*Fax: (+65) 6791 1961. E-mail: pumera.research@gmail.com.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
M.P. was supported by Tier 2 grant (Grant MOE2013-T2-1-056;
ARC 35/13) from the Ministry of Education, Singapore. Z.S. was
supported by the Czech Science Foundation (GACR Grant No.
15-09001S).
■
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