Taking advantage
of exceptional
attributes, such as being easy-to-operate, economical, sensitive,
portable, and simple-to-construct, in recent decades, considerable
attention has been devoted to the integration of recognition elements
with electronic elements to develop electrochemical sensors and biosensors.Various
electrochemical devices, such as amperometric sensors, electrochemical
impedance sensors, and electrochemical luminescence sensors as well
as photoelectrochemical sensors, provide wide applications in the
detection of chemical and biological targets in terms of electrochemical
change of electrode interfaces.
With remarkable achievements
in nanotechnology and nanoscience,
nanomaterial-based electrochemical signal amplifications have great
potential of improving both sensitivity and selectivity for electrochemical
sensors and biosensors. First of all, it is well-known that the electrode
materials play a critical role in the construction of high-performance
electrochemical sensing platforms for detecting target molecules through
various analytical principles. Furthermore, in addition to electrode
materials, functional nanomaterials can not only produce a synergic
effect among catalytic activity, conductivity, and biocompatibility
to accelerate the signal transduction but also amplify biorecognition
events with specifically designed signal tags, leading to highly sensitive
biosensing. Significantly, extensive research on the construction
of functional electrode materials, coupled with numerous electrochemical
methods, is advancing the wide application of electrochemical devices.
For example, Walcarius et al. highlighted the recent advances of nano-objects
and nanoengineered and/or nanostructured materials for the rational
design of biofunctionalized electrodes and related (bio)sensing systems.
1
The attractiveness of such nanomaterials relies
on their ability to act as effective immobilization matrices and their
intrinsic and unique features as described above. These features combined
with the functioning of biomolecules contribute to the improvement
of bioelectrode performance in terms of sensitivity and specificity.
Our group recently presented a general overview of nanomaterial-enhanced
paper-based biosensors including lateral-flow test-strip and paper
microfluidic devices.
2
With different kinds
of nanoparticles (NPs), paper-based biosensor devices have shown a
great potential in the enhancement of sensitivity and specificity
of disease diagnosis in developing countries.
This Review focuses
on recent advances in electrochemical sensors
and biosensors based on nanomaterials and nanostructures during 2013
to 2014. The aim of this effort is to provide the reader with a clear
and concise view of new advances in areas ranging from electrode engineering,
strategies for electrochemical signal amplification, and novel electroanalytical
techniques used in the miniaturization and integration of the sensors.
Moreover, the authors have attempted to highlight areas of the latest
and significant development of enhanced electrochemical nanosensors
and nanobiosensors that inspire broader interests across various disciplines.
Electrochemical sensors for small molecules, enzyme-based biosensors,
genosensors, immunosensors, and cytosensors are reviewed herein (Figure 1). Such novel
advances are important for the development
of electrochemical sensors that open up new avenues and methods for
future research. We recommend readers interested in the general principles
of electrochemical sensors and electrochemical methods to refer to
other excellent literature for a broad scope in this area.
3,4
However, due to the explosion of publications in this active field,
we do not claim that this Review includes all of the published works
in the past two years and we apologize to the authors of excellent
work, which is unintentionally left out.
Figure 1
Schematic illustration
of electrochemical sensors and biosensors
based on nanomaterials and nanostructures, in which electrochemical
sensors for small molecular, enzyme-based biosensors, genosensors,
immunosensors, and cytosensors are demonstrated.
Nonenzymatic Sensors
The pursuit of electrochemical systems
for bimolecular detection
has received significant attention over the last two decades.
5
Electroanalysis toward small molecules is also
of importance in a variety of areas. Enzymatic sensors possess high
selectivity but suffer from limitations such as instability, complicated
modification procedures, and critical microenvironmental factors.
Such limitations stimulate the development of nonenzymatic electrochemical
sensors with simple modification procedures and good stability. Enzyme-free
electrochemical sensors have been widely used for determining the
presence of hydrogen peroxide, glucose, and dopamine. The perspectives
and current challenges of enzyme-free electrochemical sensors were
discussed by Chen et al.
6
(142 references).
Miao et al.
7
recently reviewed electrocatalysis
and electroanalysis of nickel, itsoxides, hydroxides, and oxyhydroxides
toward small molecules (85 references). Following are some examples
of nonenzymatic sensors for the detection of small molecules.
Glucose
Glucose plays an important role in metabolism.
Glucose biosensors have contributed significantly to clinical monitoring.
8,9
With regard to the electrode materials, metal, metal oxide nanostructures,
and their hybrid nanocomposites are regarded to be the most promising
materials currently used. Wang et al.
10
reviewed the progress made in recent years in the field of direct
and nonenzymatic electrochemical sensing of glucose (221 references).
Tian et al.
11
also reviewed the most recent
advances in nonenzymatic glucose sensors based on various nanomaterials
(125 references). Various nanomaterials with different shapes and
compositions were synthesized to construct novel nonenzymatic electrochemical
sensors for glucose detection.
Cao et al.
12
synthesized bimetallic PtCu nanochains through a water-based
mild chemical route, compositions of which can be conveniently tuned
at the mesoscopic scale by a facile dealloying process. Electrochemical
measurements demonstrate that the sensors made by these PtCu nanomaterials
are very sensitive and specific for glucose detection due to the wiring
of dispersed crystals, porous nanostructure, clean surface, and synergetic
electronic effects of the alloyed atoms. The resulted sensor performed
very well in the detection of glucose in serum samples. Keerthy et
al.
13
produced reduced graphene oxide (rGO)
modified with platinum nanocubes and copper oxide nanoflowers. A low
cost screen printing technology was used to fabricate such electrodes
for point-of-care (POC) glucose monitoring. These sensors were highly
specific to glucose in the presence of commonly interfering species
like ascorbic acid (AA), dopamine (DA), uric acid (UA), and acetaminophen.
It was discussed that copper oxide catalyzes glucose oxidation and
Pt NPs act as a cocatalyst to enhance the electron transfer during
the oxidation of glucose. Guo et al.
14
constructed
a Ni/CdS bifunctional Ti@TiO2 core–shell nanowire
electrode through a hydrothermal and electrodeposition method. The
resultant sensor based on the fabricated nanowire electrode exhibited
great sensitivity in the electrochemical detection of glucose oxidation.
The enhanced electrochemical performance on glucose sensing was attributed
to the high dispersity of Ni NPs and the ability to discriminate the
interfering species of CdS under the irradiation of visible light.
The ability to combine the unique properties of individual nanomaterials
provides a new and exciting frontier for the formation of novel electrodes.
Xu et al.
15
prepared α-Fe2O3 cubes in the presence of a hydrophobic iron-containing
ionic liquid (IL) under hydrothermal conditions and tested the photoelectrochemical
properties by means of the transient photocurrent responses of ITO
electrodes which were modified with the as-prepared α-Fe2O3. The photoelectrochemical
approach in the application
of the glucose sensor was investigated. A glucose photoelectrochemical
detection system based on a nonenzyme catalytic oxidation reaction
has shown promising results with high sensitivity and fast response.
Hydrogen Peroxide
The rapid and accurate detection
of hydrogen peroxide (H2O2) has practical importance
in the field of bioanalysis as well as food safety and environmental
protection.
16,17
Nagaiah et al.
18
reported a H2O2 sensor based on electrochemical
deposition of Pd–Pt and Pd–Au NPs on spectrographic
graphite. The sensitivity originates from the codeposition of Pd with
either Pt or Au enhancing electrocatalytic activity for H2O2 reduction. Bai and Jiang
19
developed a H2O2 sensor based on copper sulfide
NPs-decorated rGO. They investigated the application of this sensor
in detecting H2O2 content in human serum and
urine samples as well as H2O2 released from
human cervical cancer cells. Their results have shown satisfactory
recovery rates with good reproducibility, indicating potential applications
in medical diagnosis. A powerful enzymatic mimetics employing graphene
oxide (GO), carbon nanotubes (CNTs), and Pt nanocatalysts was fabricated
by Wang’s group.
20
The GO–CNTs–Pt
nanocomposites exhibited peroxidase-like catalysis activity and electrocatalysis
activities, that tested the colorimetric and electrochemical detection
of H2O2. Moreover, sandwiched electrochemical
immunoassays were demonstrated by using the GO–CNTs–Pt
as catalytic tags. Such innovative nanostructure showed promise in
the extensive catalysis applications in environmental, medical, industrial,
and particularly aqueous biosensing fields.
Yang et al.
21
reported the development of a microwave-assisted
strategy for the synthesis of nitrogen and boron codoped graphene
with a hierarchical framework. The resultant sensor was able to selectively
detect H2O2 in the presence of glucose and AA.
Moreover, this electrode system could be easily functionalized with
biomacromolecules to generate a cost-effective, highly sensitive,
and biocompatible sensor for a variety of applications. Tian et al.
22
reported that ultrathin g-C3N4 nanosheets were fabricated by ultrasonication-assisted
liquid
exfoliation of bulk C3N4. They demonstrated
the use of these nanosheets as an effective sensing platform for the
detection of H2O2, which has been extended to
the electrochemical detection of glucose in both buffer solution and
human serum medium.
Wu et al.
23
developed
a novel nonenzymatic
electrochemical sensor based on a p-methoxy zinc
porphyrin-C60 derivative (ZnPp-C60), which was
designed and synthesized by combining p-methoxy porphyrin
and C60 moieties with a flexible methylene chain. Combined
with theoretical information, the results showed that the ZnPp-C60 would be a novel
material for construction of a nonenzymatic
electrochemical sensor for H2O2 and nitrite
analysis in a relatively wide potential range with high sensitivity
and stability.
Cation
The sensitive and selective
detection of toxic
heavy metals coupled with a cost-effective and simple assaying procedure
has paramount importance.
24
Recently, Cui
et al.
25
developed a convenient and highly
sensitive electrochemical detection platform for detecting copper.
The sensor was fabricated on a glassy carbon electrode (GCE) through
a layer-by-layer (LBL) assembling modification with multiwall carbon
nanotubes (MWCNTs), poly(amidoamine) dendrimers, and dithiobis[succinimidyl-propionate]
encapsulated Au NPs (DSP-Au NPs). The DSP modified sensor captures
cysteamine (Cys) functionalized Ag NPs through the reaction between
DSP and Cys. The presence of Cu2+ catalyzed cystocystamine
regulated the assembly of Ag NPs on the sensor surface, resulting
in the decrease of the electrochemical stripping signal of Ag NPs.
With this strategy, the detection range of Cu2+ is 1.0–1000
nM with a detection limit of 0.48 nM. In addition, Sadhukhan and Barman
26
synthesized two-dimensional C3N4 under microwave irradiation and used it to modify
GCE for
the detection of Hg2+. Due to its graphene-like structure,
this sensor detected Hg2+ down to 0.09 nM. For a real application,
the device fabricated by Rattanarat’s group using screen-printing
MWCNTs mixed carbon inks on polyester and then modified them with
binanoparticles and ferricyanide which are supposed to enhance stripping
signals and reduce Cu2+ interference.
27
The top layer of the device contains five wax-defined channels
extending outward from an open sample reservoir. In order to achieve
high selectivity and sensitivity, each channel contains an individual
sample with pretreatment and detection zones. Under the optimized
conditions, such a paper-based device can simultaneously detect Cd2+ and Pb2+ in the
range of 5–150 μg
L–1.
Anion
Madhu et al.
28
prepared
highly porous and heteroatom-enriched activated carbon (HAC) from
banana stems. HAC was used to develop electrochemical sensors for
the detection of nitrite in the application of monitoring environmental
pollution. HAC exhibits noteworthy performance in the highly sensitive
detection of nitrite. Their method was tested to determine nitrite
in various water samples with acceptable results.
Conducting
polymer-based modified electrodes have been extensively studied as
sensing materials in the last decades.
29
Yang et al.
30
reported glassy carbon
electrodes modified with polyaniline (PAni)-coated copper hexacyanoferrate
for use as a cyclic voltammetry sensor. The application of the sensor
was investigated in the detection of sulfite, which is used as a preservative
in a variety of food and pharmaceutical products to inhibit enzymatic
and nonenzymatic browning and also used in brewing industries as an
antibacterial and antioxidizing agent.
31
They showed that the synergistic effect of these structures improved
electrocatalytic activity toward detection.
Other Species
Most conducting polymer/graphene composites
have excellent electrical conductivity. Gao et al.
32
developed electrochemically coated porous structure films
of overoxidized polypyrrole/graphene (PPyox/GR) deposited onto GCE.
This structure was successfully utilized as an efficient electrode
material for the quantitative detection of adenine and guanine. With
low background current, the permselective polymer coating improved
the selectivity and sensitivity of microelectrodes for the electropositive
purine bases.
Lin et al.
33
reported
the hybridization of poly(luminol) (PLM) and poly(neutral red) (PNR)
that is then enhanced by a conductive and steric hybrid nanotemplate
using GO and MWCNTs. The PLM–PNR–MWCNT–GO mycelium-like
nanocomposite is found to be electroactive, pH-dependent, and stable
in the electrochemical system. This nanocomposite showed electrocatalytic
activity toward NADH with a high current response and low overpotential.
It also exhibited a high sensitivity of 288.9 μA mM–1 cm–2 to NADH using amperometry.
Changes
in glutathione concentration at the cellular level may
be linked to diseases such as premature arteriosclerosis, leukemia,
and diabetes. Lee et al.
34
described a
modified GCE through electropolymerization of caffeic acid onto the
electrode surface in the presence of either CNTs or nanocarbon, affording
a nanocomposite with a high concentration of o-quinone
moiety onto the electrode, that can be used for the detection of glutathione
through an electrocatalytic process. Concentrations as low as 500
nM were detected.
Zhang et al.
35
prepared
a phosphomolybdate
functionalized graphene nanocomposite for the detection of AA, in
which polyoxometalates can irreversibly adsorb onto carbon materials
to form carbon nanocomposite structures. The amperometric signals
are linearly proportional to the AA concentration in a wide concentration
range from 1× 10–6 to 8 × 10–3 M, with a detection limit of 0.5× 10–6 M.
Recently, layered transition metal dichalcogenides have been extensively
studied due to their structural similarities with graphene and their
interesting physicochemical properties, along with their diverse exotic
electronic properties.
36,37
Narayanan et al.
38
employed ultrathin MoS2 sheet-based electrodes
for electrochemical detection of dopamine (DA) as an important neurotransmitter
in the presence of AA. The results showed that MoS2 is
expected to be a good electrode material for electrochemical sensing
applications. Sun et al.
39
reported the
Au NPs-decorated MoS2 nanosheets synthesized by electrodeposition
of Au NPs on MoS2 nanosheets, which possess better properties
than pure Au NPs and MoS2. The composite film modified
electrode showed excellent electrocatalytic activity toward the oxidation
of AA, DA, and UA with three well-resolved oxidation peaks. The peak
potential separations were large enough to individually or simultaneously
detect AA, DA, and UA.
Nitric oxide (NO) plays an important
role in physiological processes.
It has been reported that some human diseases are related to their
biological function.
40
Hunter et al.
41
utilized standard photolithographic techniques
and a nitric oxide (NO) selective xerogel polymer to fabricate an
amperometric NO microfluidic sensor. The sensor detected NO levels
in small sample volumes (∼250 μL) with low background
noise. The detection sensitivity of 1.4 pA nM–1 was
demonstrated along with the limit of detection (LOD) of 840 pM. This
sensor exhibited excellent analytical performance in phosphate buffered
saline. Moreover, the analytical performance of the device was investigated
in simulated wound fluid and whole blood. The results showed that
the sensor is able to measure NO in complicated biological samples.
This proof of concept study demonstrated the feasibility of clinical
application of this method. Metters et al.
42
reported screen printed single-walled CNTs (SWCNTs) sensors which
were fabricated upon flexible polyester substrates. The screen printed
SWCNTs sensors are benchmarked using potassium ferrocyanide (II),
DA, hydrazine, and capsaicin. By using this sensor, the detection
of capsaicin has been achieved at low micromolar levels. These electrodes
hold potential in the development of disposable and highly reproducible
sensors.
It is observed that sensors that exploit the unique
properties
of nanomaterials constitute the most rapidly expanding sensor research
area. Moving forward, several areas of research will enhance the nanostructured
sensing platform. For example, research in the storage and stability
of the sensors will improve the shelf life of functional biosensors.
Electrochemical Enzyme-Based Biosensors
Electrochemical
enzyme-based biosensors, a subclass of chemical
sensors, combine the high specificity of the enzyme with the sensitivity
of electrochemical transducers. Enzyme electrodes are electrochemical
probes with a thin layer of immobilized enzyme on the surface of the
working electrode. The enzyme is the most critical component of the
enzyme electrode since it provides the selectivity for the sensor
and catalyzes the formation of the electroactive product for detection.
In the past two years, there were some review articles that focused
on the development of various materials, techniques, and applications
of electrochemical enzyme-based biosensors. Chen et al. described
recent progress in electrochemical glucose biosensors and focused
on some problems and bottlenecks in areas of enzymatic (glucose oxidase
(GOx) based) amperometric glucose sensing (240 citations).
43
The focus of the review by de Oliveira’s
group is to present the current status and some trends in enzymatic
nanoimmobilization.
44
The recent advance
of lactate biosensors
45
and enzymatic uric
acid
46
biosensors were also systematically
discussed. In addition, Schneider and Clark presented different immobilization
strategies that have been used to create Cytochrome P450s (CYPs) biosensors,
with particular emphasis on mammalian drug-metabolizing CYPs and characterization
of CYP electrodes.
47
Recent achievements
in this area have focused on the development of a novel immobilization
strategy and study of the direct electron transfer with the assistance
of functional nanomaterials. Additionally, some other papers of interest
will also be addressed.
Immobilization Strategies
The immobilization
of enzymes
is one of the key steps in developing high-performance biosensors,
since it will affect the loading as well as the bioactivity of the
enzymes. To date, different methods have been studied to achieve efficient
enzyme immobilization, such as covalently binding enzymes onto the
substrate surface or incorporating enzymes into different matrixes.
The development of the synthesis of nanomaterials provides enormous
opportunities for tailoring their properties, thus enhancing their
functions and application in immobilization of enzymes.
A great
number of nanostructured materials with different sizes, shapes, and
compositions have been synthesized and utilized as novel electrode
materials for immobilization of desired enzymes. Due to the homogeneous
spherical shape, high conductivity, and large surface area for biomolecular
conjugation, graphite NPs, consisting of several stacked graphene
sheets, were reported to construct an enzyme biosensor to detect glucose
in real samples.
48
After modification,
GOx was linked with graphite NPs through an amide bond. This cost-effective
approach will be applied to other electrochemical biosensors. Wågberg
and co-workers reported on a novel sensing platform for H2O2 and glucose based on
the immobilization of Pd helical
carbon nanofiber (Pd-HCNF) hybrid nanostructures and GOx.
49
Small size and homogeneous distribution of the
Pd NPs in combination with good conductivity and large surface area
of the HCNFs significantly reduce the overpotential and enhance the
electron transfer rate and therefore lead to a high performance glucose
sensing platform. Malhotra and co-workers synthesized a series of
nanomaterials, such as NiFe2O4,
50
Tm2O3,
51
and Cu2O,
52
which were utilized
as electrode materials for immobilizing bienzyme (cholesterol esterase
(ChEt) and cholesterol oxidase (ChOx)). These fabricated bioelectrodes
exhibit largely improved amperometric biosensing performance toward
cholesterol.
Du and co-workers developed a series of robust
organophosphorus
pesticide (OPs) biosensors based on functional nanomaterials. As a
typical example, a novel hydrolase biosensor, based on self-assembly
of methyl parathion hydrolase (MPH) on the Fe3O4@Au nanocomposite, was developed for
sensitive and selective detection
of the OPs methyl parathion.
53
There were
several advantages of this electrochemical biosensor. First, the Fe3O4nanocomposite
provides an easy way to construct
the enzyme biosensor and renew the electrode surface simply by an
external magnetic field. The hydrolase is not poisoned by OPs and
thus is reusable for continuous measurement. Moreover, Au NPs not
only provide a large surface area, high loading efficiency, and fast
electron transfer but also stabilize the enzyme through electrostatic
interactions. The MPH biosensor shows a rapid response and high selectivity
for detection of methyl parathion, with a linear range from 0.5 to
1000 ng mL–1 and a detection limit of 0.1 ng mL–1. A nanohybrid of Au NPs, polypyrrole,
and rGO (named
as Au–PPy–rGO) was also prepared on the electrode, achieved
by electrochemical deposition of rGO with pyrrole and the introduction
of Au NPs.
54
Acetylcholinesterase (AChE)
was further encapsulated in a silica matrix and immobilized on the
Au–PPy–rGO nanocomposite by codeposition with (NH4)2SiF6. The obtained nanohybrid
of Au–PPy–rGO
not only increased the surface area of the modified electrode but
also showed excellent conductivity. AChE molecules were protected
by a biocompatible 3D porous silica matrix to prevent them from leaking
out and to retain their bioactivity. Furthermore, the fabricated AChE
biosensor displayed high stability, excellent activity, and fast response
to OPs. This assembly protocol is expected to be used for the immobilization
of various enzymes and proteins, leading to robust biosensors.
Zhang and co-workers reported a facile electrochemical biosensing
interface for sensitive glucose determination based on a Pt@BSA nanocomposite
along with the covalent adsorption of GOx.
55
The electrocatalytic activity toward oxygen reduction was significantly
enhanced due to the excellent bioactivity of anchored GOx and superior
catalytic performance of interior Pt NPs. Upon the successive injection
of glucose, the GOx-based biosensor catalyzed the oxidation of glucose
to gluconolactone in the presence of oxygen, with the reduction peak
current gradually decreased, making it suitable for glucose determination.
In addition to the simple modification of enzymes on the surface of
electrode materials, embedding them within a different matrix is widely
reported. Cosnier’s group synthesized polypyrrolic bipyridine
bis(phenantrolinequinone) Ru(II) complex/CNTs composites through electropolymerization,
which were used for efficient enzyme entrapment.
56
Their ability to oxidize NADH while immobilizing enzymes
during their electrodeposition represents a straightforward technique
to design functional bioelectrodes. In presence of NAD+, the resulting enzyme electrode
exhibits high current densities
for glucose oxidation with a detection limit of 1 μM glucose.
Kale and co-workers succeeded in immobilizing GOD in a mixture containing
silica sol–gel and poly(vinyl alcohol) composite film.
57
The sensitive nature of poly(vinyl alcohol)
and improved stabilizing effect of prehydrolyzed tetraethyl orthosilicate
created in the matrix was favorable for high sensitivity. At the same
time, the presence of Au NPs in the immobilization matrix not only
offered a biocompatible microenvironment but also efficiently improved
electron transfer between the GOx/mediator and electrode surface.
LBL assembly has been selected as a reliable method to immobilize
enzymes with preserved activity due to its simplicity and versatility.
On the basis of the electrostatic interaction, poly(allylamine hydrochloride)
(PAH) modified CNTs/Au composites were assembled with negatively charged
enzyme, horseradish peroxidise (HRP) and ChOx, to fabricate a bienzyme
biosensor for the detection of cholesterol.
58
The bienzyme biosensor showed more highly efficient electrochemical
signal transduction. In addition, CNTs and Au NPs could enhance the
electrochemical signal by catalyzing the response of H2O2 and effectively facilitate
the electron transfer due
to the good conductivity, further improving the detection sensitivity
and stability. Considering the advantages in preserving enzyme activity
of poly(ethylene imine) (PEI), Ferreira and coworkers fabricated β-galactosidase
(β-Gal) immobilized in LBL films with PEI and poly(vinyl sufonate)
on an ITO electrode modified with a layer of prussian blue (PB).
59
Lactose could be detected with an amperometric
method with a sensitivity of 0.31 μA mmol–1 cm–2 and a detection limit of 1.13 mmol
L–1, which is sufficient for detecting lactose in milk
and for clinical exams. Compared to the immobilization of enzymes
onto a substrate surface, incorporation of enzymes into a 3D matrix
has the potential to increase the enzyme loading as well as to protect
the enzyme from the surrounding environment. To increase the loading
of GOx and simplify glucose biosensor fabrication, Yang and co-workers
prepared a hydrogel from Fc modified amino acid phenylalanine, which
was utilized for the incorporation of GOx.
60
The resultant hydrogel featured good biocompatibility and contained
a significant number of ferrocene (Fc) moieties, which can be considered
as an ideal matrix to immobilize enzymes for the preparation of mediator-based
biosensors. Due to the improved enzyme loading and efficient electron
transfer, the as-prepared glucose biosensor exhibited good performance
for the electrochemical detection of glucose, such as high sensitivity,
wide linear range, short response time, and good stability. Leopold
and co-workers reported the xerogel biosensors containing composite
films of (3-mercaptopropyl)trimethoxysilane xerogel embedded with
GOx, doped with Au NPs, monolayer protected clusters (MPCs), and coated
with an outer polyurethane layer.
9
The
MPC network within the sol–gel acts as a 3D extension of the
working electrode area that allows for the biosensor’s signal
to have a decreased dependence on the diffusion of H2O2. It is found that the MPC-doped
sol–gel glucose biosensors
of this study are equal to or exceed comparable glucose biosensors
reported previously. Similarly, 3D Pt NPs/PAni hydrogel heterostructures
were also used as a novel matrix to load GOx and thus to construct
highly sensitive glucose sensors (Figure 2).
61
On the one hand, the Pt NPs/PAni hydrogel heterostructure-based
glucose sensor synergizes the advantages of both the conducting hydrogel
and the nanoparticle catalyst. On the other hand, the 3D porous structure
of the PAni hydrogel favored the high density immobilization of the
enzyme and the mass transfer of the glucose. The glucose enzyme sensor
based on this heterostructure exhibited unprecedented sensitivity
and low detection limit. Willner’s group utilized enzyme-capped
relay-functionalized mesoporous carbon NPs as effective bioelectrocatalytic
3D matrices to construct a glucose electrochemical biosensor.
62
Figure 2
(a) Schematic representation of the 3D heterostructure
of the Pt
NP/PAni hydrogel, in which the PAni hydrogel acts as a matrix for
the immobilization of the GOx enzyme and homogeneous loading of Pt
NPs. (b) A 2D scheme showing the reaction mechanism of the glucose
sensor based on the Pt NP/PAni hydrogel heterostructure. Reprinted
from ref (61).Copyright
2013 American Chemical Society.
Direct Electron Transfer (DET)
Enzyme-based electrochemical
biosensors, especially the third-generation amperometric glucose biosensors,
are fascinating because they function as the ideal biosensing model
in the absence of mediators. The direct electrical communication of
GOx can also contribute to the detection of glucose at low potentials
which are slightly positive from the redox potential of GOx. Considerable
attempts to overcome the long electron-tunneling distance were made
to realize the direct electrochemistry of enzymes.
63
Liu and co-workers employed a whole-cell biocatalyst using
a yeast surface displaying GOx and a constructed GOx-yeast/CNTs electrochemical
glucose sensing platform.
64
Direct electrochemistry
was achieved, suggesting that the host yeast cell did not have any
adverse effect on the electrocatalytic property of the recombinant
GOx. Ye and co-workers reported a DET glucose biosensor based on GOx
self-assembled on electrochemically reduced carboxyl graphene.
65
It was noted that the conductivity of the graphene
was improved because most of the oxygen-containing groups were eliminated
after electrochemical reduction. Carboxylic acid groups remained and
can effectively link with GOx. Well-defined and quasi-reversible redox
peaks could be obtained. DET of GOx was also realized in bienzyme
(glucoamylase and GOx) functionalized CNTs
66
and MnO2 decorated rGO.
67
Although
well-defined voltammetric peaks of direct electrochemistry of GOx
have been achieved in previous reports, the detection of glucose based
on the direct electron transfer of GOx has been rarely realized. However,
it is worth noting that the determination of glucose based on electroreduction
of enzyme-consuming O2 at low potentials (close to the
redox potential of GOx) should conceptually belong to the first-generation
amperometric glucose biosensors, rather than the third-generation
ones. To address this issue, Gorski and co-workers systematically
investigated the signal transduction and enzyme activity in biosensors
based on the GOx and CNTs embedded in a bioadhesive film of chitosan
(CHIT).
68
This work focused on the on the
role of DET in glucose sensing at a GOx/CNTs hybrid that was embedded
in a CHIT on the electrode surface. Two main issues including the
role of reactions relevant to the electrochemical glucose sensing
the effect of CNT on the retention and enzymatic activity of GOx in
CHIT films and in aqueous suspensions were studied. The well-defined
voltammetric peaks of direct electrochemistry of GOx were observed
regardless of CHIT. However, the DET was not the mechanistic basis
for glucose sensing at a GOx/CNT-based biosensor, indicating that
GOx molecules that were within the electron tunneling distance from
CNT were not enzymatically active toward glucose. The biosensor was
sensitive to glucose in air-equilibrated solutions based on the O2-mediated enzymatic
oxidation of glucose. The signal transduction
relied on the net drop in a biosensor current that was caused by a
decrease in a 4e– O2 reduction current
and an increase in a 2e– H2O2 reduction current. Moreover, they found that CNTs nearly
doubled
the retention of GOx in a biosensor, while CNTs significantly decreased
the average enzymatic activity of GOx.
The third-generation
biosensors based on enzyme DET were also reported in these two years.
In a typical example, Cui and co-workers utilized functionalized planar
boron-doped diamond (BDD) electrode as a biosensing platform for biomolecule
immobilization with GOx as a test model.
69
In detail, BDD was treated with KOH and functionalized with 3-aminopropyltriethoxysilane
(APTES). The free amino groups of GOx and APTES were cross-linked
by glutaraldehyde (X), a bifunctional chemical to form a stable enzyme
layer (GOx-XAPTES) on BDD. DET between the flavin adenine dinucleotide
(FAD) center of GOx and the electrode was realized by using the APTES-glutaraldehyde
conjugate as a molecular wire to form electron tunneling between the
FAD center and BDD. Amperometric responses of the GOx electrode to
glucose were illustrated in both aerated and deaerated buffer solution
to address whether the signal response to glucose can be attributed
to DET. Different from the other reports, the result confirmed the
bioelectrocatalytic activity of the electrical contacted GOx. In the
presence of glucose, glucose is oxidized by GOx in concurrence with
the biochemical reduction of the GOx FAD to FADH2. Niwa
and co-workers investigated the effects of a bare ITO film electrode
surface structure on human cytochrome (CYP3A4) by using polycrystalline
ITO and amorphous ITO film.
70
They found
DET from a human CYP layer or a CYP microsome adsorbed on ITO without
any modification could be easily realized. Because of its larger surface
area and negatively charged surface, the polycrystalline ITO film
was a suitable electrode for the adsorption of CYP proteins while
maintaining efficient DET and enzymatic activity. On this basis, the
simple ITO interface was applied to drug metabolism and inhibitor
evaluation. Wang and co-workers for the first time employed small
molecular hydrogel as a surrounding matrix to stabilize Cytochrome
c (Cyt c), further facilitating electron transfer between redox enzyme
and electrode.
71
Significantly, the third-generation
biosensors based on DET of Cyt c was successfully achieved to determine
H2O2 at an optimized potential with high selectivity
over other reactive oxygen species, oxygen, metal ions, AA, and so
on, which provided a durable platform for real-time determination
of H2O2 from live cells. In addition, Zhao et
al. investigated the effect of three kinds of nanostructured silica–phytic
acid (SiO2–PA) materials with diverse morphologies
as electrode materials including spherical SiO2–PA
(s-SiO2–PA), rod-like SiO2–PA
(r-SiO2–PA), and helical SiO2–PA
(h-SiO2–PA) on the electrocatalytic activity toward
DA detection based on the laccase biosensor.
72
Combining the direct bioelectrocatalyst, it was observed that that
the laccase/h-SiO2–PA-modified electrode showed
the best electrochemical performances because helical SiO2–PA could load more laccase
and provide more spatial freedom
in its orientation and thus facilitate DET of laccase.
Other Papers
of Interest
This two year period witnessed
some novel electrochemical detection strategies and methods in developing
new enzyme-based biosensors. Vagin and co-workers reported a single-enzyme
and membrane-free self-powered biosensor, in which both cathodic and
anodic bioelectrocatalytic reactions are powered by cholesterol. Among
them, ChOx was immobilized in a sol–gel matrix on both electrodes.
Compared to either of the two individual electrodes, the self-powered
sensor formed on the high surface-area carbon cloth electrodes, resulting
in enhanced sensitivity.
73
To overcome
the disadvantage of existing adenosine-5-triphosphate (ATP) biosensors,
such as cascades of enzymatic reactions, Kucherenko’s group
developed a biosensor system consisting of two biosensors.
74
In detail, the first one was based on GOx and
was designed to measure glucose concentration, and the other one was
based on GOx and hexokinase and was sensitive toward both glucose
and ATP. On this basis, simultaneous determination of glucose and
ATP concentrations by two independent bioselective elements holds
great promise in novel sensing devices. Reed and co-workers demonstrated
electronic field-effect transistors (FETs) as sensitive devices. An
Al2O3-passivated Si nanowire used to mimic transistor
operation was created for measuring enzyme–substrate interactions
via the monitoring of pH change.
75
The
change in pH can be measured by the nanoribbon in solution in real
time and is reflected in the change in drain current through the device.
Urea in phosphate buffered saline (PBS) and penicillinase in PBS and
urine can be effectively detected, at limits of detection of <200
μM and 0.02 units/mL, respectively. The enzyme kinetics can
also be analyzed to accurately determine the kinetic constant. This
direct, rapid, and label-free detection method can be readily generalized
to many unrelated classes of substrates and enzymes. Schöning
and co-workers reported a LBL nanofilm of polyamidoamine (PAMAM) dendrimer
and CNTs on capacitive electrolyte-insulator-semiconductor (EIS) field-effect
sensors for detecting urea.
76
Through the
optimization of the arrangements of a LBL film and the enzyme urease,
adequate film architecture urease sandwiched between the LBL film
and another CNT layer [EIS-(PAMAM/CNT)-urease-CNT] exhibited a superior
output signal performance and higher sensitivity of about 33 mV/decade
by means of capacitance–voltage (C/V) and dynamic constant-capacitance
measurements. It was determined that the presence of the additional
CNT layer was needed to achieve a urea-based EIS sensor with enhanced
properties.
Development of a novel electrochemical interface
along with functional materials and enzyme immobilization plays a
critical role for the rational design and construction of bioelectronic
devices. Wang and co-workers designed a facile and effective electrochemical
sensing platform for the detection of glucose and urea in one sample
without separation; it was developed using CHIT-rGO/concanavalin A
(Con A) as a sensing layer.
77
In this system,
the CHIT-rGO with a large specific surface area was introduced to
immobilize a large amount of Con A, exhibiting nice pH-switchable
behavior to Fe(CN)6
3–. The change of
resistance to charge transfer or amperometric current in the presence
of GOx or urease resulted from the change of glucose or urea concentration,
thus realizing simultaneous detection of glucose and urea based on
in situ pH-switchable enzyme-catalyzed reactions. Karra and Gorski
studied the nafion-induced current amplification in dehydrogenase-based
biosensors.
78
The fabricated biosensors
were designed by sandwiching the enzyme–CHIT/CNTs film between
an electrode and nafion film. The coating of such biosensors with
nafion resulted in the current increase by up to 1000%, depending
on the enzyme. The increase in the biosensor current was attributed
to the pH-driven increase in the enzyme activity inside the two-film
interface. The combination of the two-film interface with enzyme engineering
to modify enzyme activity–pH profiles can lead to the enzyme-based
biosensor devices with highly amplified current output. In addition,
Liu and co-workers adopted an in-site immobilizing method to embed
GOx in copolymer involving N,N-diethylacrylamide
and methyl acrylic acid.
79
The effect of
environmental stimuli, such as temperature, pH, the identity and concentration
of anions, and the concentration of CO2 in solution on
the voltammetric response of Fc dicarboxylic acid (Fc(COOH)2) at the film electrodes
was investigated. This multiresponsive electrochemical
behavior of the system could be further employed to maximize the electrochemical
oxidation of glucose catalyzed by GOx entrapped in the films with
Fc(COOH)2 as the mediator in solution.
Future efforts
were aimed at further miniaturization and integration
of the electronic interface, further facilitating the development
of advanced electroanalytical devices. For example, Wang and co-workers
describe the first example of real-time noninvasive lactate sensing
in human perspiration during exercise events using a flexible printed
temporary-transfer tattoo electrochemical biosensor.
80
This flexible tattoo lactate sensor consists of a mediated
lactate oxidase (LOx) working electrode, prepared by functionalizing
the surface of the printed tattoo electrode with tetrathiafulvalene
and CNTs, followed by tethering the LOx enzyme, and a biocompatible
CHIT overlayer. The lactate biosensor was used for the electrochemical
detection of sweat lactate, thereby substantiating its utility for
the noninvasive assessment of lactate levels and degree of physical
exertion. Mao and co-workers demonstrated a microfluidic chip-based
online electrochemical system for in vivo continuous and simultaneous
monitoring of glucose, lactate, and ascorbate in rat brain.
8
Taking advantage of single-walled CNTs in facilitating
the electrochemical oxidation of ascorbate and dehydrogenases to selectively
catalyze the oxidation of glucose and lactate, the microfluidic chip-based
online electrochemical system allowed the integration of various detection
units into a small device which is more suitable to establish a multicomponent
analysis system with technical simplicity, near real-time nature,
little crosstalk, and low cost.
Genosensors
In
electrochemical genosensors, single stranded DNA (ssDNA) fragments
are immobilized onto the electrode surface as recognition probes for
capturing the target DNA through hybridization. In the presence of
hybrids, signals are generated via various mechanisms and then detected
electrochemically. According to electrochemical detection principles,
several important factors should be considered for the achievement
of good sensitivity and selectivity in the biodetection. The past
two years has witnessed substantial advances toward the development
of a high performance electrochemical sensing platform for DNA detection.
Recent review articles have focused on new methods and new signal
amplification based on functional nanomaterials and enzymes in the
DNA and RNA assays. Xu and co-workers recently evaluated the methods
related to photoelectrochemical DNA biosensors.
81
This kind of photoelectrochemical DNA biosensor provides
excellent sensitivity due to the separation and the different energy
forms of the excitation source and the detection signal. In the another
review article, the sandwich assay based on the biotechnologies and
nanotechnologies for nucleic acids was also introduced.
82
Wu et al. summarized the latest developments
in the application of nanomaterials as signal amplification elements
in ultrasensitive electrochemical detection of DNA (136 citations).
83
From the point of their unique electrochemical
properties, various nanomaterials with different signal amplification
routes have been reviewed briefly. Meanwhile, some other new methods
and related progress were also investigated in the development of
electrochemical DNA sensors.
84−87
For example, a paper analytical device for quantitative
detection of DNA was reported.
87
Here,
we focus on the recent progresses in device fabrication, DNA probe
design, enzyme-based amplification, nanomaterial-enhanced signal amplification,
and other interesting methods.
Nucleic Acid Assay
Design of DNA Probe
The electrochemical DNA sensing
platform consists of capture probes immobilized on the sensing surface
for capturing targets and signal probes with electrochemical tags
for signal generation. In the nucleic acid assays, good detection
sensitivity can be achieved by optimizing hybridization conditions
and improving hybridization efficiency. High detection specificity
relies on the design of specific probes and the elimination of nonspecific
binding on the sensing surface.
88,89
Under the circumstances,
the design of a novel DNA probe was considered to be an efficient
approach to enhance detection sensitivity. Due to the proper distance
between the nucleobases, the rigid amido bonds, the high flexibility
of the aminoethyl linkers, and intramolecular hydrogen bonding, the
peptide nucleic acid (PNA) probe has great sequence specific affinity
and stability and has received great interest in DNA sensors. On the
basis of the advantage of the PNA–DNA hybridization, a rGO-based
FET biosensor used for ultrasensitive label-free detection of DNA
was reported.
90
A detection limit as low
as 100 fM was achieved, which is 1 order of magnitude lower than that
of the previously reported graphene FET DNA biosensor based on DNA–DNA
hybridization. Fan and co-workers have demonstrated a new generation
of electrochemical DNA sensors for sensitive and specific detection
of microRNA (miRNA).
91
In their design,
the use of a DNA tetrahedron ensures the stem-loop structure in a
well controlled density with improved reactivity. The regulation of
the thermodynamic stability of the stem-loop structure decreases the
background signal and increases the specificity as well. The attached
enzymes bring the electrocatalytic signal to amplify the detection.
The combination of these effects improves the sensitivity of the sensor
and can be applied to other miRNA detection methods. At the same time,
DNA tetrahedral nanostructures containing a partially self-complementary
region with a stem-loop hairpin structure were also innovatively designed.
92
An electrochemical redox label was attached
to the reconfigurable tetrahedron edge in such a way that reconfiguration
of this edge changed the distance between the electrode and Fc in
the presence of target DNA. As mentioned above, the immobilization
of the probe DNA on the surface of the electrode dictates the performance
of the resulting sensor. However, it is very difficult to precisely
control the DNA spatial orientation and position on solid surfaces
via formation of the self-assembled monolayer using thiolated ssDNA
molecules. A bovine serum albumin-monolayer-based probe carrier platform
has been reported to improve the performance in comparison to a conventional
thiolated ssDNA probe self-assembled monolayer-based electrochemical
DNA hybridization biosensor.
93
When combined
with an enzyme-amplification method, a detection limit of 0.5 fM was
achieved with high specificity and was reproducible, which was primarily
attributed to the enhanced spatial positioning range and accessibility
of the probes on this novel platform.
Enzyme-Based Amplification
As is well understood, the
application of redox labels is the simplest way to produce an electrochemical
signal. However, a redox label can only transfer one or a few electrons
to or from the electrode surface. The limitation of the number of
electrons transferred directly affects the sensitivity of the DNA
sandwich assay. High sensitivity is highly desired since the DNA levels
are low in some real problems, such as in pathogen DNA detection and
cancer or infectious disease DNA detection. Enzymes have been successfully
used for detection of analytes providing both recognition and amplification
of the binding event with a detectable readout. In this enzyme-based
amplification system, DNA sensors based on enzymatic catalytic reactions,
such as HRP
94,95
and alkaline phosphatase (ALP),
96,97
were used as a substitute for the redox label, thus providing high,
steady, and reproducible signal amplification.
Besides, loading
HRP onto various nanomaterials has become a promising way to further
amplify the detection signal and achieve a lower detection limit for
the analyte. Our group demonstrated an ultrasensitive electrochemical
DAN sensor amplified by CNTs-based labels for the detection of human
acute lymphocytic leukemia (ALL)-related p185 BCR-ABL fusion transcript.
98
Carboxylated CNTs were functionalized with HRP
and target-specific detection probes to amplify the target hybridization
signal. The activity of captured HRP was monitored by square-wave
voltammetry measuring the electroactive enzymatic product in the presence
of 2-aminophenol and hydrogen peroxide. The use of such labels greatly
amplifies hybridization signals and enables the detection of full-length
p185 BCR-ABL transcripts at subfemtomole levels, which corresponds
to picograms of the target gene. The signal-amplified assay achieved
a detection limit of 83 fM (5 × 10–18 mol in
60 μL) target oligonucleotides and has a 4-order-wide dynamic
range of target concentration. The resulting assay allowed robust
discrimination between the perfect match and a three-base mismatch
sequence. Ju et al. reported that noncovalent π–π
interaction led to a stable monolayer stacking of ferric porphyrin
on both sides of the GO and demonstrated a simple and convenient pathway
to fabricate a universal peroxidase mimic by this GO-based nanocomposite.
99
When combined with the Au NPs–SWCNH modified
electrode, the obtained trace label showed greatly enhanced peroxidase
activity toward o-phenylenediamine (o-PD) oxidization
in the presence of H2O2, which recognizes a
biotinylated molecular beacon for specific electrochemical detection
of DNA down to attomolar levels.
On the other hand, several
novel electrochemical label-free methods
using an enzyme-amplification strategy have been reported. For example,
Gao’s group developed a simple and ultrasensitive label-free
miRNA biosensor, based on hybridized miRNA-templated deposition of
an insulating polymer film and electrochemical impedance spectroscopic
detection.
100
Upon hybridization, the neutral
surface of the biosensor was converted to an anionic state by the
hybridized miRNA strands. The deposition of the insulating polymer
film, poly(3,3-dimethoxybenzidine), was then carried out by the HRP-catalyzed
polymerization of 3,3-dimethoxybenzidine in the presence of H2O2. Such a tool may
open a new paradigm in routine
miRNA analysis with a detection limit of 2.0 fM.
Besides this
postamplification strategy toward signal production
by a hybridization event, there are target recycling and other strategies
via nuclease. They have drawn more and more concerns owing to its
striking improvement for the detection sensitivity toward target analytes.
101−103
Dual signal amplification can be readily realized due to the introduction
of the functional nanomaterials. Ju and co-workers combined circular
strand-displacement polymerization with silver enhancement to achieve
a dual signal amplification.
104
After the
molecular beacon hybridized with the target DNA and opened the loop
part, the opened stem then hybridized with the primer assembled on
Au NPs to initiate polymerization of the DNA strand, which led to
the release of the target. The released target found another molecular
beacon to trigger the polymerization cycle, resulting in the multiplication
of the reporter Au NPs on the sensor surface. Sequentially, the Au
NPs-promoted silver deposition afforded a signal trace for electrochemical
stripping analysis of target DNA. This signal showed high selectivity
and can be performed from 10–16 to 10–12 mol L–1 with a detection limit down to the
subfemtomolar
level.
Gao et al. described another amplification method for
DNA detection
by applying rolling circle amplification (RCA), which created a long
ssDNA product and thus significantly enhanced the electronic responses
of Si nanowire FET.
105
Because of the binding
of an abundance of repeated sequences of RCA products, the fabricated
nanosensor showed high sensitivity due to the enhanced signal-to-noise
ratio (SNR). The biosensor has exhibited SNR > 20 for detection
of
1 fM by employing the RCA amplification method, which exceeds the
reported detection SNR by most previously reported DNA sensors.
Nanomaterial-Enhanced Signal Amplification
Given the
limitation of the enzyme (i.e., poor stability and high cost), great
attention has been paid to developing different kinds of functional
nanomaterials, such as metal NPs, quantum dots (QDs), carbon-based
nanomaterials, magnetic NPs, and polymers to design advanced genosensors.
Because of their biological compatibility, high surface area, chemical
stability, nontoxicity, excellent catalytic activity, and conductivity,
the introduction of nanomaterials has greatly improved the analytical
performance, amplified the detection signal, and stabilized the recognition
probes or biosensing interface. It is well-known that the electrode
materials as the key component are most widely used in electroanalytical
investigations and play an important role in constructing high performance
electrochemical sensing platforms to detect target molecules through
different analytical principles. Jiao and co-workers synthesized the
graphene/poly(xanthurenic acid) nanocomposite via a one-step synchronous
electrochemical method.
106
Due to the synergistic
effect, this graphene-based electrochemical platform showed an intrinsic
advantage in highly sensitive impedimetric detection of DNA. They
also synthesized sulfonated PAni-GO, which can be used as a novel
electrode material to direct electrochemical detection of DNA.
107
A similar strategy was also reported to construct
a label-free electrochemical DNA biosensor based on water-soluble
electroactive dye azophloxine-functionalized graphene.
108
A sandwich-type DNA biosensor based on electrochemical
coreduction synthesis of graphene-three-dimensional nanostructure
gold nanocomposite films was developed with high sensitivity due to
its high active surface area and high conductivity.
109
Other materials such as CHIT–ionic liquid
110
and mercury film/carbon nanotubes
111
as well as biocompatible nanostructured magnesium
oxide-CHIT platform
112
were also used as
advanced electrode materials to design high performance genosensors.
Various nanomaterials, especially Au NPs and carbon nanomaterials,
have been used as excellent carriers for loading numerous signal elements
such as enzymes, oligonucleotides, and redox labels. Yu and co-workers
adopted a hairpin sequence as the capture probe with a restriction
site introduced into its stem segment.
113
As shown in Figure 3, with high efficiency
and high fidelity of EcoRI, the enzymatic cleavage
reaction only occurs on those probes that retain their stem–loop
structure without capturing the target, leading to a reduced background
signal. In contrast, the capture probe is opened by the target hybridization,
deforming the restriction site and forcing the biotin tag away from
the electrode. Au NPs modified with a large number of Fc-signaling
probes are captured on the basis of the biotin–streptavidin
complexation. Furthermore, Fc tags can be dragged in close proximity
to the electrode surface via hybridization between the signaling probes
and the capture probe residues after EcoRI treatment,
facilitating interfacial electron transfer and further enhancing the
signal. This sensor achieves an ultralow detection limit to the zeptomole
region and a wide dynamic range over 7 orders of magnitude. Zhang
and co-workers modified Au NPs with two types of signaling reporter
DNAs; one probe is complementary to the target DNA, while the other
is not. The presence of nonmatched probe reduces the cross-reaction
between target DNA and matched probe on the Au NPs, resulting in increased
sensitivity of the sandwich-type DNA biosensor.
114
Figure 3
Design of the cooperative amplification-based electrochemical sensor
for the zeptomole detection of DNA. Reprinted from ref (113). Copyright 2013 American
Chemical Society.
The newly developed nanomaterials
can act as electroactive tracers
for signal amplification by numerous signal species directly from
themselves. Combined with effective methods for determination of nanotracers,
ultrasensitive electrochemical DNA-based assays have been easily developed.
Liu and co-workers presented a novel strategy for simultaneous detection
of multiple DNA targets based on the use of different encoding metal
ions as tags. Metal ions bound to metallothionein molecules can be
released after hybridization with DNA targets and then detected by
stripping voltammetry.
115
Remarkable electrochemical
properties of QD barcodes were also used as enhancing species to improve
the signal. A novel dendritic QD nanocluster was constructed and used
as versatile electrochemiluminescence (ECL) and electrochemical probes
for the detection of DNA and cancer cells.
116
Dai and co-workers combined the high base-mismatch selectivity of
the ligase chain reaction and the remarkable voltammetric properties
of QD barcodes and provided the feasibility of sensitive multiplexed
miRNA analysis detected by square wave voltammetry.
117
Nanomaterials with enzyme-like characteristics were
also used as
a new method for signal amplification in genosensors. Huang and co-workers
fabricated a sensitive gap-electrical biosensor based on self-catalytic
growth of unmodified Au NPs as conductive bridges for amplifying DNA
hybridization events.
118
In the presence
of target DNA, the obtained dsDNA product cannot adsorb onto the surface
of Au NPs due to the electrostatic interaction, which makes the unmodified
Au NPs exhibit excellent GOx-like catalytic activity. Such catalytic
activity can enlarge the diameters of Au NPs in the glucose and HAuCl4 solution and
result in a connection between most of the Au
NPs and a conductive gold film formation with a dramatically increased
conductance. On the contrary, the catalytic activity sites of Au NPs
are fully blocked by ssDNA due to the noncovalent interaction between
nucleotide bases and Au NPs. It is of great significance to explore
the interaction between functional materials with electrochemical
probes in a genosensor system, which provides wide opportunities to
develop a novel sensing platform. Cui and co-workers found that the
ECL of ruthenium(II) complex functionalized GO (Ru–GO) could
be effectively quenched by Fc–ssDNA absorbed on the Ru–GO
nanosheets. The Ru–GO has good discrimination ability over
ssDNA and dsDNA. The mutant ssDNA target responsible for the drug
resistant tuberculosis can hybridize with Fc–ssDNA and release
Fc–ssDNA from the Ru–GO surface, leading to the recovery
of ECL.
119
Zhang and co-workers demonstrated
that the oxygen groups at the surface of CNTs together with the intrinsic
electron properties of CNTs were the major reason for the suppression
of ECL of Ru(bpy)3
2+.
120
Utilizing this essential quenching mechanism, a new signal-on DNA
hybridization assay has been proposed on the basis of the CNTs modified
electrode, where the ssDNA labeled with Ru(bpy)3
2+ derivatives probe (Ru-ssDNA) at the distal end was covalently attached
onto the CNTs electrode. The quenched ECL signal returns in the case
of the presence of complementary ssDNA. Xu and co-workers constructed
a sensitive DNA biosensor based on ECL resonance energy transfer between
RuSi@Ru(bpy)3
2+ and Au@Ag2S NPs.
121
According to the interaction between Au NPs
and DNA immobilized on an electrode surface, Li and co-workers developed
a novel DNA sensing method based on the ultrahigh charge-transfer
efficiency of Au NPs.
122
Moreover, Bonanni
and co-workers used MoS2 nanoflakes as inherently electroactive
labels to design a DNA sensor based on the differential affinity of
MoS2 nanoflakes toward ssDNA and dsDNA.
123
Other Approaches for Signal Amplification
Zuo and co-workers
demonstrated an ultrasensitive detection platform for miRNA by combining
hybridization chain reaction (HCR) amplification and the tetrahedral
DNA nanostructure probes.
124
Among them,
3D tetrahedral DNA nanostructure was the scaffold to immobilize DNA
recognition probes to increase the reactivity and accessibility, while
DNA nanowire tentacles are used for efficient signal amplification
by capturing multiple catalytic enzymes in a highly ordered way. The
synergetic effect of the DNA tetrahedron and nanowire tentacles has
proven to greatly improve sensitivity for both DNA and miRNA detection.
In fact, most of the reports on genosensors involve multiple magnification
approaches mentioned above.Tang and co-workers combined HCR amplification
with silver nanotags to electrochemically monitor nucleic acids with
high sensitivity.
125
Due to the target-triggered
long-range self-assembled DNA nanostructures and HCR, numerous silver
nanotags were directly immobilized onto the long-range DNA nanostructures
without the need of silver enhancement substrates and bioactive enzymes,
each of which produces a strong electronic signal within the applied
potentials. Under optimal conditions, the target-triggered long-range
DNA nanostructures presented good electrochemical behavior for the
detection of human immunodeficiency virus DNA at a concentration as
low as 0.5 fM.
New Methods for DNA Detection
Nanopore
analysis has
emerged as the simplest single-molecule technique. Various ssDNA with
similar lengths can slide through a-hemolysin (a-HL)-based protein
nanopore at a bias voltage and lead to an indistinguishable signal
for DNA detection. Kang et al. combined the DNA probe technique with
nanopore detection and developed a new nanopore DNA biosensor.
126
The DNA sensor relies on the hybridization
reaction between the short HBV target strand and deliberately designed
DNA probes. It was demonstrated that the target HBV DNA could be detected
with high sensitivity and selectivity. Chang and co-workers demonstrated
an ion-exchange nanomembrane sensor for detection of DNA/RNA using
the charge inversion phenomenon when negatively charged nucleic acids
assemble on the surface of the positively charged membrane.
127
Changes in current–voltage characteristics
were used to identify and quantify targets that hybridize with specific
complementary probes covalently functionalized on the membrane surface.
Furthermore, the fabricated sensor is specific and able to distinguish
two base mismatches in the target sequence as well as capable of capturing
and recording the target sequence from a heterogeneous mixture.
Electrochemical Detection of DNA Damage
DNA damage
occurs frequently in organisms. Some endogenous and exogenous chemicals
have been found to induce structural damages to nuclear DNA by base
oxidation or modification. If unrepaired, these damaged DNA may lead
to gene mutation and even tumor generation. Electrochemical genosensors
are well qualified for the rapid screening of industrial and environmental
chemicals for their potential geno-toxicity.
128−130
Specific types of sensors for the identification and quantification
of DNA damage products such as methylated DNA bases were addressed
in these two years.
DNA aberrant methylation represses gene
transcription, deregulates gene expression, and causes various human
diseases. Hence, detecting the DNA aberrant methylation level benefits
the early diagnosis of some tumors and the epigenetic therapy for
DNA methylation-related diseases. Ai’s group developed a series
of electrochemical methods for DNA methylation detection.
131,132
For example, they constructed a photoelectrochemical immunosensor
to assay DNA methylation, where Bi2S3 nanorods
were used as photoelectric conversion material. In this system, the
methyl-CpG-binding domain (MBD) proteins were captured on the electrode
surface through the specific interaction between MBD protein and symmetrical
cytosine methylation in the CpG region of dsDNA. Then, an antihis
tag antibody was captured to further inhibit the photocurrent and
increase the detection sensitivity through the immunoreactions. This
Bi2S3-based photoelectrochemical sensor possessed
excellent photoelectron property and presented high detection specificity,
even distinguishing single-base mismatched sequences.
132
Xie and co-workers reported the highly
sensitive detection of DNA
methylation, methyltransferase activity, and inhibitor screening based
on DNA-Au NPs signal amplification.
133
DNA
hybrid methylated by DNA adenine methylation MTase can not be cleaved
by MboI endounuclease and loaded more intercalated
MB. It should be noted that MB was employed as an electrochemical
indicator and DNA-modified Au NPs were used as a signal amplification
unit because the DNA strands in this composite have strong adsorption
ability for MB. On the basis of this principle, DNA methylation could
be determined on the basis of the voltammetric signal change of MB.
Immunosensors
Immunoassays are the detection platforms based
on specific antigen–antibody
recognition. They are well established standard biodetection methods
used in clinical laboratories for disease diagnosis, in the food industry
for food safety testing, and in monitoring environmental contamination.
134−138
Among immunosensors, electrochemical immunosensors are attractive
and have received considerable attention because of their great features
of being easily used and economical in mass production; having an
excellent LOD with a small volume of samples; and being a paper-based
simple analytical platform. Two recent review articles summarize the
recent trends of electrochemical immunosensors toward POC diagnostics.
139,140
Voltammetry
and Amperometry-Based Immunoassay
Voltammetry
and amperometry, such as linear sweep, differential pulse, square-wave,
and stripping, are the most widely used electrochemical methods for
immunoassay. Most of the strategies discussed below employ the sandwich
immunoassay approach, in which the target antigen (Ag) is captured
by its specific antibody (Ab1) and detected by labeled
secondary antibody (Ab2).
Biomarkers and Bacteria
Detection
The development of
reliable, cost-effective, powerful detection, and monitoring strategies
for cancer diagnosis is particularly important, due to the disease’s
prevalence, high rates of recurrence, and potential lethality.
141,142
Zhang et al.
143
designed a new anodic-stripping
voltammetric immunoassay protocol for the detection of IgG, by using
CdS QDs LBL assembled hollow microspheres as molecular tags. In a
sandwich-type immunoassay format, subsequent anodic-stripping voltammetric
detection of cadmium released under acidic conditions from the coupled
QDs was conducted at an in situ prepared mercury film electrode. The
new immunoassay is promising for enzyme-free and cost-effective analysis
of low-abundance biomarkers. Singh et al.
144
presented a simple immunosensing scheme in which the incubation
period was minimized without a large increase in the detection limit.
This scheme was based on electrochemical-enzymatic redox cycling using
GOx as an enzyme label, Ru(NH3)6
3+ as a redox mediator, and glucose as an enzyme substrate. Fast electron
mediation of Ru(NH3)6
3+ between the
electrode and the GOx label attached to the electrode allows high
signal amplification. Benefiting from this, the detection limit for
carbonhydrate antigen 125 (CA 125) was slightly higher than 0.1 U/mL.
Ren et al.
145
described an electrochemical
biogate for a highly sensitive homogeneous electrochemical immunoassay
by combining target-induced proximity hybridization with a mesoporous
silica nanoprobe. The electroactive MB was sealed in the inner pores
of MSN with single-stranded DNA. More importantly, the in situ recycling
of the proximate complex could be achieved with nicking endonuclease
Nt.BbvCI to open more DNA biogates for release of more MB, thus amplifying
the electrochemical signal. The proposed assay showed a wide detection
range from 0.002 to 100 ng mL–1 with a detection
limit of 1.3 pg mL–1 for prostate-specific antigen.
Parshetti et al.
146
fabricated an ultrasensitive
sandwich-type amperometric immunosensor for the detection of alpha-fetoprotein
(AFP). Au/CHIT modified GCE and antibody-functionalized dumbbell-like
Au–Fe3O4 heterostructures were used as
the sensing platform and immuno-labels, respectively. The authors
showed that the GCE modified with CHIT produced a high electrochemical
response through the conjugation of more Au–Ab1 and
the dumbbell-like Au–Fe3O4 which served
as a dual-probe to immobilize Ab2 onto Au, as well as to
reduce H2O2 by Fe3O4.
That enhanced signal amplification.
Eletxigerra et al.
147
reported an electrochemical
magnetoimmunosensor for detecting a biomarker of tumor necrosis factor
alpha (TNFα). The sensor was constructed by using magnetic microbeads
and disposable screen-printed carbon electrodes, with the addition
of hydroquinone as the electron transfer mediator and H2O2 as the enzyme substrate.
After a thorough optimization
of the assay, extremely low limits of detection were achieved: 2.0
pg mL–1 (36 fM) and 5.8 pg mL–1 (105 fM) for standard solutions and spiked human serum,
respectively.
Bhimji et al.
148
developed the first
electrochemical enzyme-linked immunosorbent assay to detect human
immunodeficiency virus-1 (HIV-1) and HIV-2 in clinical samples. Excellent
performance relative to a commercial gold standard test was obtained,
which was based on the surface functionalization of SU-8 and oxidation
current of p-aminophenol. Because of the heterogeneous
nature of the assay, there is no interference by electroactive substances
or electrode fouling.
The electrochemical immunoassay has also
been used in other fields
besides cancer biomarkers.
149
Grewal et
al.
150
presented a method for label-free
electrochemical detection of a protein from the enteric pathogen Entamoeba histolytica
using cell-free yeast embedded antibody-like
fragments (yeast-scFv) as novel affinity agents. The key architectural
improvements were made, including: (i) avoiding use of secondary antibodies
and (ii) utilizing yeast-scFv cell membrane fragments.
Tlili
et al.
151
integrated two complementary
detection strategies for the identification and quantification of Escherichia coli
based on bacteriophage T4 as a natural
bioreceptor for living bacteria cells. One involves screening and
viability assays, employing bacteriophage as the recognition element
in label-free electrochemical impedance spectroscopy. The other approach
is a confirmation by loop-mediated isothermal amplification to amplify
specifically the E. coli Tuf gene after lysis of
the bound E. coli cells, followed by detection using
linear sweep voltammetry. In another study,
152
biotinylated full antibody-based immunosensors have been optimized
to enable the specific detection of pathogenic bacteria S.
pyogenes in human saliva. Electrodeposited polytyramine was
used as a base layer for the conjugation of biotinyl antibodies via
a biotin-Neutr Avidin bridge. The impedance-based electrochemical
immunosensor showed a linear response (100 cells/10 μL to 105
cells/10 μL) against S. pyogenes in cumulative
incubation and 100 cells/10 μL to 104 cells/10 μL in single-shot
incubation.
Our group
153
proposed
a new approach
for detecting/screening OPs poisons by simultaneously providing the
results of dual biomarkers of both enzyme inhibition and enzyme adducts
(Figure 4). Simultaneously, AChE enzyme activity
of postexposure is also determined. The high detection sensitivity
stems from enrichment of the electroactive product, thiocholine, on
the surface of Fe3O4/Au nanocomposites followed
by electrochemical oxidative desorption of thiocholine. In another
work, we
154
presented the first report
on the development of the Fe3O4 at TiO2 magnetic NPs-based disposable electrochemical
immunosensor with
quantum dot-linked antibodies for sensitive and selective detection
of the OP-butyrylcholinesterase adduct in human plasma. Fe3O4 at TiO2 magnetic NPs
not only selectively
capture phosphorylated adduct by metal chelation but also directly
separate it from biological matrices by simply exerting an external
magnetic field.
Figure 4
(A) Schematic illustration of the preparation of core–shell
Fe3O4/Au magnetic nanocomposites and the electrochemical
oxidative desorption process of thiocholine. (B) Schematic illustration
of the measurement of AChE activity via the reactivation from an OP-exposed
sample. (C) Parallel measurement of AChE activity in a postexposure
sample with and without reactivation. Reprinted from ref (153). Copyright 2013 American
Chemical Society.
Two new electrode functionalization
strategies were developed by
Prieto-Simón et al.
155
The first
strategy relied on hydrazide-phenyl diazonium salts that were electrografted
onto the gold electrode surface. The second strategy involved the
use of mono- and dithiolated self-assembled monolayers carrying hydrazide
functional groups. The immunosensors based on either a direct assay
using electrochemical impedance spectroscopy or a sandwich-assay using
differential pulse voltammetry for MS2 phage detection
were investigated. Their results showed that both immobilization protocols
efficiently controlled the orientation of antibodies and the permeability
of the electroactive species in solution, resulting in strategies
that can be easily tailored to prepare highly sensitive electrochemical
immunosensors.
Amaya-González et al.
156
reported
a highly sensitive approach for gluten analysis using aptamers as
specific receptors, with the successful selection of aptamers for
these water insoluble prolamins that was achieved choosing the immunodominant
apolar peptide from α2-gliadin as a target for selection. The
excellent features of the biosensors make the proposed method a valuable
tool for gluten detection in foods.
Graphene
The combination
of nanomaterials and immunosensors
shows great potential for monitoring biomolecules and sensitive detection
of target analytes. Nanomaterials can be used as carriers to load
signal markers or directly as signal reporters for sensitively detecting
biomarkers, and they can accelerate electron transfer when they are
used as functional materials on electrode surfaces.
157,158
Nowadays, nanomaterials such as carbon materials,
159,160
colloidal nanocrystals (e.g., magnetic NPs, metal NPs, QDs), and
other functional materials
161−164
are being developed to increase the sensitivity
of the electrochemical detection of targets.
Graphene plays
an important role in recent trends for immunosensors fabrication.
Immobilization of the bioactive species is crucial for proper detection.
Graphene offers an easy way to protect and stabilize these species.
In addition to other nanomaterials, graphene-based immunosensors have
exhibited good analytical characteristics and shown great promise
for clinical applications. Recently, Wu et al.
165
introduced different approaches for the fabrication of
graphene and the preparation of graphene-modified electrodes for electrochemical
sensors (119 citations). Zhu et al.
166
provided
an overview of electrochemical biosensing with graphene related materials
and discussed the role of graphene in different sensing protocols
(123 citations). For recent examples, Lin et al.
167
developed an electrochemical immunosensor for the detection
of a cancer biomarker protein in serum at a low concentration with
excellent signal-to-noise ratio. This reusable biosensor utilized
a magnetic GO-modified gold electrode as the detection substrate.
The fabrication method allows for reproducibility, ease of production
and use, and storage stability enabling potential clinical use for
rapid vascular endothelial growth factor detection. Gao et al.
168
constructed a sensitive sandwich-type immunosensor
for detecting alpha-fetoprotein. Poly(diallyldimethylammonium chloride)
(PDDA) protected PB/Au NPs/IL functionalized rGO (IL-rGO-Au-PDDA-PB)
nanocomposite was fabricated and used as a signal amplification label
to enhance the electrochemical response. Lu et al.
169
reported a novel, label-free, and inherent electroactive
redox biosensor based on ultrathin Au–Pt nanowire-decorated
thionine/rGO (AuPtNWs/THI/rGO). The AuPt NWs/THI/rGO composites not
only favor the immobilization of antibody but also facilitate the
electron transfer. It is found that the resultant AuPtNWs/THI/rGO
composites can be designed to act as a sensitive label-free electrochemical
immunosensor for the detection of carcinoembryonic-antigen.
Moreover, in recent years, the study of the graphene-based derivative,
such as heteroatom-doped graphene,
170
GO,
171
and graphene nanoribbon,
172
has been popular and extensive, particularly with respect
to electrochemical applications. Zhu’s group
173
reported an approach to use the exceptional properties
of the NG-based composite for the fabrication of an ultrasensitive
electrochemical immunosensor based on a signal amplification strategy
for the detection of matrix metalloproteinase-2. The design of the
immunosensor also involved a polydopamine functionalized GO hybrid
conjugated to horseradish peroxidase-secondary antibodies by covalent
bonds as a multilabeled and biocompatible probe to increase the electrochemical
response.
Other Nanomaterials
The biomolecule–nanomaterials
hybrid system has excellent prospects for interfacing biological recognition
events with electronic signal transduction so as to design a new generation
of bioelectronic devices with high sensitivity.
161−164
Dendrimers have good conductivity and large potential windows, which
are electrochemically important and make them novel solvent (electrolyte)
systems, holding great promise in many studies of electrochemical
biosensing.
174
Recently, a highly sensitive
electrochemical immunosensor based on dendrimers/Au NPs as a sensor
platform and MWCNT-supported multiple bienzymes (Ab2/MWCNT/GOx/HRP)
as labels was developed by Jeong’s group for the detection
of carcinoembryonic-antigen (CEA) The linear dynamic range of the
proposed immunosensor covered a five-order wide concentration range
in which CEA detection can be made without dilution. The LOD of the
proposed CEA immunosensor was much lower than that of the conventional
ELISA method.
175
Li et al.
176
synthesized a novel ionic liquid, 4-amino-1-(3-mercapto-propyl)-pyridine
hexafluorophosphate (AMPPH), which was used as a functional monomer
to fabricate AMPPH-modified Au NPs (AMPPH-AuNPs) via a one-pot synthesis
method. Rabbit antihuman IgG was immobilized onto the nanointerface
based on AMPPH-Au NPs and used for human IgG immuosensing. The results
indicate that AMPPH-Au NPs improved the immunosensing performance.
Lou et al.
177
proposed a novel competitive
electrochemical immunosensor by combining the electrochemical reduced
GO-AuPd NPs platform with a Ag NPs functionalized polystyrene bionanolabel
for the sensitive detection of human interleukin-6. In the meantime,
they also introduced an electrically heated carbon electrode in the
detection procedure of the immunosensor and further improved the sensitivity.
An ultrasensitive immunoassay method based on the electrochemical
measurement of PAni, which was catalytically produced by an HRP-functionalized
Au NPs (HRP-Au NPs) probe at an immunosensor was developed by Lai
et al. After performing a sandwich immunoreaction, the quantitatively
captured HRP-Au NP nanoprobes could catalyze oxidation of aniline
to produce electroactive PAni on the immunosensor surface. The electrochemical
measurement of PAni enabled a novel detection strategy for the HRP-based
immunoassay.
178
Electrochemiluminescence
In the past two years, ECL
has received much attention and become an important detection method.
179,180
Wang et al.
181
synthesized a novel functionalized
material using surface-decorated fullerene to encapsulate hollow and
porous palladium nanocages. The resultant functionalized material
has a high specific surface area, good electrocatalytic ability, and
efficient photocatalytic activity and has been used to construct an
ECL immunosensor for the detection of Streptococcus suis Serotype 2. A wide linear
detection range of 0.1 pg mL–1 to 100 ng mL–1 is acquired with a relatively low
detection limit of 33.3 fg mL–1. Qi et al.
182
reported an ultrasensitive ECL peptide-based
method for the determination of cardiac troponin I, incorporating
amplification of signal reagent-encapsulated liposome. The principle
was based on the idea of encompassing heavy labels in larger carriers
and on polyvalent binding motifs, employing the Ru1-encapsulated liposome
peptides and the magnetic capture peptides. Ju’s group
183
prepared a hemin functionalized graphene sheet
via the noncovalent assembly of hemin on nitrogen-doped graphene that
acts as an oxygen reduction catalyst to produce sensitive ECL quenching
of quantum dots through the annihilation of dissolved oxygen, the
ECL coreactant, by its electrocatalytic reduction. With the use of
the catalyst with high loading of hemin as a signal tag of the secondary
antibody, a novel ultrasensitive immunoassay for the carcinoembryonic
antigen detection was demonstrated.
Photoelectrochemistry
Photoelectrochemistry is a newly
developed analytical method and now attracts substantial research
scrutiny in various fields.
81,184,185
Tian et al.
186
developed a photoelectrochemical
immunosensor by incorporating graphene quantum dots (GQDs) and highly
oriented silicon nanowires (SiNWs) for the determination of microcystin-LR
in water samples. The GQDs/SiNWs heterostructure was employed for
signal transduction and a biocompatible nanoscaffold for antibody
immobilization. Zhang et al.
187
developed
a new photoelectrochemical biosensor for ultrasensitive detection
of monoclonal antibodies anti-Tn, which was used against the breast
tumor-associated carbohydrate antigen Tn. The detection sensitivity
of 1.0 × 10–13 g/mL was achieved. It should
be noted that the CdSe QDs acted as both photosensitizer and an alternative
multivalent form for carbohydrate antigen with high binding affinity.
This design would facilitate testing for disease-related sugar markers
as well as evaluate the immunogenic properties of carbohydrate vaccine
candidates.
Other Papers of Interest
To achieve
clinical or POC
use, multiplexed electrochemical target detection has been investigated
intensively in the last two years.
188−191
Rusling
192
reviewed multiplexed electrochemical protein detection
and that of translation to personalized cancer diagnostics (60 citiations).
Wu et al.
193
reported the combination
of a signal amplification strategy with a microfluidic paper-based
analytical device for the quantitative analysis of four kinds of cancer
biomarkers as model analytes, namely, alpha-fetoprotein, carcinoembryonic
antigen, cancer antigen 125, and carbohydrate antigen 153. Signal
amplification was achieved through graphene modification of the immunodevice
surface to accelerate the electron transfer and also the use of silica
NPs as a tracing tag to label the signal antibodies. Using the horseradish
peroxidase-O-phenylenediamine-H2O2 electrochemical
detection system, the potential clinical applicability of this biosensor
was demonstrated in the detection of four candidate cancer biomarkers
in serum samples from cancer patients.
Yang et al.
194
reported the design of
a low-cost, portable intelligent microscale electrochemical device
that can automatically deliver multiple reagents in a controlled manner.
The successful adaption of a bubble-based cartridge to the screen
printed electrode system leads to automatic and rapid sample delivery
at the electrode surface in one step with minimal user intervention.
They have performed sensitive and selective detections of several
biological targets, including tumor biomarkers and H1N1 split influenza
vaccine.
Inorganic–organic (poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate))
heterostructured light-emitting diodes (LEDs) based on ZnO nanorods
were presented by Zhang et al. As a proof-of-concept, an advanced
multiplexed photoelectrochemical immunosensor array was fabricated
using the prepared LEDs as an excitation light source, and excellent
performance for the detection of three different cancer biomarkers
was achieved.
195
Kong et al.
196
proposed a branched electrode
platform for label-free, reagentless, and simultaneous tumor markers
detection based on MB and PB, two different redox substrates. The
branched sensing electrode fabricated via photolithography on an ITO
electrode consists of two separate circular areas (2 mm in diameter,
named as W1 and W2), which joined to a rectangular area for the electrical
contact. By equipping each branched sensing pad with different redox
active substrates and different antibodies, the sensor can simultaneously
respond to multiple targets in the sample.
Liu et al.
197
reported on a protocol
for a simultaneous competitive immunoassay for tetracycline (TC) and
chloramphenicol (CAP) on the same sensing interface. Monoclonal anti-TC
and anti-CAP antibodies were conjugated onto CdS and PbS nanoclusters,
respectively. Cd (II) and Pb (II) ions were released from the surface
of the corresponding nanoclusters by treatment with acid and detected
by square wave anodic stripping voltammetry.
There has been
substantial progress in the development of electrochemical
immunosensors; however, a major challenge for electrochemical sensors
is still simultaneous detection of multiple targets in complex biological
samples with reliability, portability, low cost, rapid response, and
excellent selectivity and sensitivity. Hence, the integration of electrochemical
immunoassays into a disposable format will have great potential in
the applications of clinical diagnostics, particularly for POC.
Cytosensors
Considering the vital role of cells in life
science and human health,
cell-related bioassaying has become a hot research topic within the
past decades.
198−203
Thanks to the development of nanotechnology, many kinds of novel
nanomaterials have emerged to anchor recognition units such as antibody,
aptamer, and receptors which can specifically and effectively capture
cells, particularly cancer cells through binding the abnormal and
overexpressed components such as proteins, glycans, and receptors
on the surfaces of cancer cells based on “target-binding”
technology. Developing highly sensitive cytosensors will have a great
impact in health care. Notably, electrochemical cytosensing approaches
play a more and more important role in the analysis and detection
of target cells due to the inherent advantages such as miniaturization,
easy operation, rapid response, satisfied sensitivity, high selectivity,
affordability, and real-time and nondestructive analysis.
204−208
Recent advances on electrochemical cytosensors have been investigated
in the detection of cell type and number, cellular physiological parameters,
and crucial molecules on the cell surface or intracellular, pharmaceutical
evaluation and screening. Different electrochemical methods have been
used in the investigation, including common electrochemical methods,
the ECL method, and the photoelectrochemical method in label-free
or sandwich assays.
209−213
Label-Free
Cytosensing
For label-free electrochemical
cytosensing, the key is to fabricate a biocompatible recognition interface
onto the electrode, coupled with highly sensitive read-out electrical
signal to report the cell-recognition events with the help of biofunctionalized
nanomaterials. Jia’s group reported an ultrasensitive electrochemical
cytosensor for quantification of carcinoembryonic antigen (CEA)-positive
BXPC-3 cells.
214
3D architecture Au@BSA
microspheres were prepared via a convenient and “green”
synthesis route. These microspheres were employed to develop the sensing
layer with the conjugation of monoclonal anti-CEA antibody (anti-CEA).
With its excellent conductivity, stability, and biocompatibility,
the 3D architectural Au@BSA microspheres were used to develop a label-free
cytosensor. Highly specific detection of BXPC-3 cells with a broader
detection range and a detection limit of 18 cells mL–1 has been achieved. Small molecules
are also used for specific recognition
of cells. An effective cytosensor using carboxymethyl CHIT-functionalized
graphene (CMG-G) has been reported.
209
The
electrochemical cytosensor was fabricated and functionalized with
biocompatible CMC-G and a small molecule, folic acid. Electrochemical
impedance spectroscopy (EIS) detection results have showed that the
detection limit for HL-60 cells was achieved at 500 cells per mL.
In addition, aptamers can be useful molecular probes for cell detection.
Quantitative determination of human colon cancer DLD-1 cells were
performed by an electrochemical aptasensor.
215
An effective biosensing interface between the MUC-1 aptamer anchored
on biocompatible carbon nanospheres and Mucin 1 glycoprotein overexpressed
on DLD-1 cells was investigated. Carbon nanospheres not only accelerated
electron transfer but also supplied a highly stable matrix for the
efficient immobilization of target MUC-1 aptamer, considerably amplifying
the electrochemical signals and resulting in sensitive detection performance
toward DLD-1 cells. In order to increase the selectivity and sensitivity
of cytosensors, Peng’s group employed a dual-aptamer recognition
strategy for highly sensitive and specific detection of MEAR cells.
216
Two types of cell-specific aptamers, ss-TLC1c
and ds-TLS11a, offered a unique nanobiointerface for cancer cell detection.
The developed electrochemical cytosensor showed great reliable performance
with satisfied sensitivity and specificity in detecting single MEAR
cancer cells in 109 blood cells from a WBC sample. Real-time
intracellular sensing is of great significance for advancing fundamental
biological and clinical science. Competition strategy is another choice
adopted for developing electrochemical cytosensors. It has been reported
that ultrasensitive electrochemical detection of leukemia cells achieved
a detection limit down to 10 cells.
217
The
aptamer–Fe3O4 MNP/cDNA–Au NPs
nanoprobes were employed to complete the competitive binding with
leukemia cells. Due to a stronger binding affinity between the aptamer
and targeting leukemia cells, the Fe3O4 MNP-bound
aptamers preferred to form the aptamer–targeting cell complex
and then dissociated cDNA–AuNPs nanoconjugates in the presence
of CCRF-CEM cells. Thereafter, the residual Au NPs on the nanoprobes
will perform Au NP-catalyzed silver deposition for amplified signal
read-out. Pioneer work has been done by presenting a direct interface
of vertically aligned single-walled carbon nanotubes (VASWCNTs) with
eukaryotic cells (RAW 264.7 mouse macrophage cell line) for electrochemical
study in an intracellular environment.
218
Combining the features of excellent high aspect ratios, efficient
electron transport, and superior electrical conductivity, VASWCNTs
were anchored onto an ITO substrate and then further wrapped by DNA,
which will enter mouse macrophage cells through endocytosis. This
allows real time monitoring intracellular events. Owing to the advantage
of multiplexing ability, high through-output performance, and low
reagent consumption, microfluidic devices were used to study the on-site
real-time assay of the proliferation and apoptosis of HeLa cells.
219
By virtue of its controllability and
low background, ECL detection was widely used in cytosensing. A label-free
ECL cytosensor was developed for specific determination of early apoptosis.
220
Assembled l-cysteine-capped CdS-QDs/PAni
nanofibers (PAni-NF) were used for the stable and high loading immobilization
of Annexin V, which recognize apoptotic cells through the interaction
between Annexin V and PS exposed on the cell membrane during the cell
apoptosis process. That resulted in a steric effect on the interaction
between sensor and coreactants. More captured cells would lead to
a lower ECL signal. A therapeutical effect could be evaluated by quantifying
an early apoptotic HepG2 cell induced by resveratrol. The results
show that the label-free ECL cytosensor holds a great potential for
rapid detection of cell apoptosis and drug screening. The photoelectrochemical
assay is a new and promising analytical approach for cytosensing.
Low-toxic Ag2S quantum dots were studied in the photoelectrochemical
detecton of cancer cells.
213
As MCF-7 cells
overexpress sialic acid (SA) on their membrane, boronic acid units
were anchored onto a Ag2S quantum dots-based photoelectric
interface that captures MCF-7 cells by virtue of the interaction between
boronic acid and the terminal SA moiety on the membranes of MCF-7
cells. The photocurrent decreased significantly after capturing MCF-7
cells. Because of the diffusion of the sacrificial electron donor
to the surface of the electrode, the electron transfer after the immobilization
of the cell on the electrode surface was blocked and resulted in the
decrease of photocurrent.
Sandwich Cytosensing
The performance
of sandwich cytosensing
relies on the recognition probes specifically capturing the target
cells and the signal probes specifically binding to captured target
cells. Therefore, the key to fabricate high sensitive cytosensors
is to develop high-performance recognition probes and signal probes,
particularly, with the aid of emerging nanomaterials. Figure 5 illustrated a sandwich
electrochemical cytosensor
enhanced by robust nonenzymatic hybrid nanoelectrocatalysts.
205
A kind of Fe3O4@ Ag–Pd
bimetallic nanocage core–satellite hybrid NP was found possessing
significantly more robust electrocatalytic activities than the enzymatic
peroxidase/H2O2 system for signal amplification
in electrochemical cytosensing. The developed electrochemical cytosensor
achieved detection limits of ∼4 MCF-7 and ∼5 T47D cells
in a 1 mL sample. Cho’s group reported an integrated multifunctional
platform based on biotin-doped conducting polymer nanowires for cell
electrochemical sensing.
221
Conductive
disulfide-biotin doped polypyrrole nanowires with a well-ordered three-dimensional
structure effectively immobilize streptavidin (SA) on the surface,
and then, biotin labeled monoclonal antibodies were attached to SA.
Circulating tumor cells (CTCs) were captured by antibodies on the
sensing surface. HRP/antibody-conjugated NPs were employed as signal
probes for quantification of CTCs. The detection range of CTCs is
10 to 1 × 104 cells, and a detection limit as low
as 10 cells was reported. Zhu’s group further reported a multiplex
electrochemical cytosensing platform to simultaneously detect and
classify both acute myeloid leukemia cells (AML) and acute lymphocytic
leukemia cells (ALL).
222
In order to enhance
the specificity of biointerection between recognition element and
targeting cell, a multivalent recognition strategy has been proposed.
207
Poly(amidomine) dendrimer modified rGO offered
a multivalent recognition interface for the immobilization of Concanavalin
A (Con A), that significantly enhanced the cell capture efficiency
and improved the sensitivity of the cytosensing for cell surface glycan.
Two different aptamer-functionalized graphene-Au NPs were integrated
as a recognition probe for capturing two different targeting cells.
SBA-15 loaded with two kinds of redox-tags, HRP and cell-targeting
aptamers, were used as probes for specifically capturing the targeting
cells, amplifying the electrochemical signals, and retaining distinguishable
signals for multiplex cytosensing. An ECL cytosensor was configured
for dynamic evaluation of cell surface N-glycan expression.
223
Due to the specific recognition of Con A with
mannose and the core trimannoside fragment of N-glycan, Con A as cell
recognition element was immobilized onto carboxylic group functionalized
MWCNTs. Con A was doped onto Au NP-modified Ru(bpy)3
2+-loaded silica nanoprobe serving as a signal probe for capturing
mannose and N-glycan expressed on the K562 cell surface. There is
an urgency to explore a portable and disposable device for cost-effective
cytosensing. Yu’s group made good contributions on developing
lab-on-paper techniques for sensitive electrochemical or ECL detection
of cells.
224,225
The constructed microfluidic
paper-based portable analytical devices (μ-PADs or lab-on-paper)
showed great promise in the detection of cells or monitoring cell-related
activities, such as drug screening and other biological research.
Figure 5
Schematic
illustration of the fabrication of Fe3O4@Ag–Pd
hybrid NPs (A) and the cytosensor assembly process
(B). Reprinted from ref (205). Copyright 2014 American Chemical Society.
Conclusions
Inherent sensitivity,
simplicity, speed, and cost benefits continue
to be strong driving forces for the development of electrochemical
sensors and biosensors. In this Review, we have summarized remarkable
advances in the development of novel ultrasensitive electrochemical
assays based on nanomaterials and nanostructures.
There have
been thousands of sensor papers published during the
past two years, where electrochemical sensors represent the most rapidly
growing class. Compared to other methods, such as spectroscopy and
chromatography, the electrochemical measurements are much cheaper
and simpler and easier to miniaturize, which makes them more suitable
for POC detection, particularly for delivering benefits for resource-limited
areas in both developed and developing countries. Besides that, a
wide variety of strategies are used to improve the efficacy of sensing.
Signal amplification for detection to utilize NPs as carriers or tracers,
catalysts, and electronic conductors and produce a synergic effect
among catalytic activity, conductivity, and biocompatibility has been
achieved.
New developments in nanotechnology and material science
as well
as in custom engineering of biorecognition components have advanced
the progress of useful and reliable electrochemical sensors and biosensors.
The materials and biomaterials with rich nanostructures not only improve
the electronic properties and increase the effective electrode surface
for transferring electrochemical signal but also produce detectable
signals for indirect detection of targets. Thus, the resulting methods
possess high sensitivity and good specificity. The synergy of multifunctional
materials, recognition elements, and electrochemical methods is improving
the selectivity, stability, and reproducibility, thus promoting the
development of sensors for assays and bioassays.
Significant
advances have been made in several areas related to
the design and application of electrochemical sensors and biosensors.
However, there is still much effort needed to implement these ultrasensitive
sensors and biosensors for real-world applications. The integration
of electrochemical sensors into (paper-based) microfluidic formats
with the incorporation of unique materials for detection needs to
be extensively explored in the future. The development of these systems
would also lead to significant advantages compared to the current
analytic systems, in terms of simplicity, speediness, cost, and automation.
We envision that a sensing device that can simultaneously monitor
the levels of cell, DNA, RNA, protein, and small molecule-related
markers in a single miniaturized and user-friendly format will offer
the promise of practical applications.