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DOI: 10.1039/C9LC01112A
Journal Name
ARTICLE
AHSWYE Microfabricated electrochemical sensing devices

748YIHURJ4NRGEK HF7B
Fumihiro Sassa,a Gokul Chandra Biswasb and Hiroaki Suzukic
Electrochemistry provides possibilities to realize smart microdevices of the next generation with high functionalities.
Lab on a Chip Accepted Manuscript

Electrodes, which constitute major components of electrochemical devices, can be formed by various microfabrication
Received 00th January 20xx, techniques, and integration of the same (or different) components for that purpose is not difficult. Merging this technique
Accepted 00th January 20xx with microfluidics can further expand the areas of application of the resultant devices. To augment the development of
next generation devices, it will be beneficial to review recent technological trends in this field and clarify the directions
DOI: 10.1039/x0xx00000x
required for moving forward. Even when limiting the discussion to electrochemical microdevices, a variety of useful
Published on 13 February 2020. Downloaded on 2/15/2020 1:20:42 AM.
www.rsc.org/ techniques should be considered. Therefore, in this review, we attempted to provide an overview of all relevant
techniques in this context in the hope that it can provide useful comprehensive information.
Introduction electrochemical principles, e.g., transistor-based devices.

Electrochemical sensing devices comprise a major group in However, we limited the scope of the investigation to ensure
the field of bio/chemical sensing. There are many advantages the range of discussion was not expanded unnecessarily and
to using electrochemistry in sensing. First, a wealth of the paper focuses on the main objective of reviewing
techniques can realize highly sensitive detection with rapid techniques and devices. In our discussion, we also mention
response times. Second, because the major components of useful critical techniques originally developed for devices with
electrochemical devices are electrodes, miniaturization, batch dimensions in the order of mm (or larger) that are not used for
fabrication, multiplexing of the same components, and microdevices.
integration with other components on a single chip can be In completing this review, our impression is that there are
performed. Furthermore, electrochemical devices can be really a wide variety of techniques for electrochemical sensing
operated using simple peripheral instruments with low power devices. For specialists of such devices, there may be a
consumption, and the instruments to operate the devices are discovery of novel useful techniques. Also, the information
also simple and portable. Many successful devices have provided in this review will be useful to know the state-of-the-
already been commercialized. art of this theme. We hope this review will contribute to the
The necessity of highly sensitive rapid detection of chemical development of new electrochemical sensing devices.
and biological analytes in samples of very small volumes is
increasing in applications such as point-of-care testing Basic components and fabrication
(POCT).1-5 To handle solutions of very small volumes, Basic setups of electrochemical devices.
microfluidics provides a means to offer additional functions to We will start by briefly mentioning basic components,
electrochemical sensing devices. Data obtained using such setups, and instruments used in electrochemical devices. For
devices can provide critical chemical/biological information amperometry and voltammetry, a three-electrode system is
such as cell functions. typically used, consisting of the working, reference, and
This review is written with the hope of providing a auxiliary electrodes. Two working electrodes are sometimes
comprehensive view of related techniques and devices that used for purposes such as redox cycling. The three-electrode
could assist in the development of new next-generation micro- system is operated by a potentiostat. A constant potential or
electroanalytical devices. We will mainly focus on devices varying potential of a waveform is applied to the working
reported within the last decade for liquid samples, along with electrode with respect to the reference electrode and current
related techniques such as microfluidics. Depending on the generated by a redox reaction is measured. No current flows
category, there are devices based on electrical rather than through the reference electrode and current flows through the
working and auxiliary electrodes. Coulometry is a variation of
a. Graduate
amperometry. Here, the output is the charge, which is the
School of Information Science and Electrical Engineering, Kyushu
University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395. integral of current.
b. School of Life Sciences, Shahjalal University of Science and Technology, Sylhet-
3114, Bangladesh.
c. Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1
Tennodai, Tsukuba, Ibaraki, 305-8573, Japan.

Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here].
This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

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Fumihiro Sassa is currently an Hiroaki SuzukiDOI:received his B.E.
10.1039/C9LC01112A
Assistant Professor at the and M.E. in applied physics and
department of electronics at his Ph.D. in bioelectronics and
Kyushu University, Japan. He biotechnology from the University
achieved his Ph.D. on of Tokyo, Japan, in 1981, 1983,
microfluidics and electro- and 1993, respectively. In 1983,
chemical sensors in 2011 from he joined Fujitsu Laboratories,
University of Tsukuba, Japan. His Ltd. as a researcher. In 1996, he
research focus is on developing moved to University of Tsukuba as
components and systems of an Associate Professor. Since
Fumihiro Sassa micromechanical systems and 2004, he has been a full-time
Hiroaki Suzuki
microfluidic systems employing professor at the Graduate School

soft microfabrication techniques. of Pure and Applied Sciences,
University of Tsukuba. From
Gokul Chandra Biswas is 2016-2018, he was the president
currently an Associate Professor of the Sensors and Micromachines
at the Genetic Engineering and of the Institute of Electrical
Biotechnology Department at Engineers of Japan (IEEJ).

Shahjalal University of Science Currently he is the president of the
and Technology, Bangladesh. He Japan Society of Chemical
achieved his Ph.D. on Nano- Sensors, a fellow of the Royal
Science and Nano-Technology in Society of Chemistry, and a fellow
2017 from University of Tsukuba, of IEEJ.
Japan. His research focus is on gold, and carbon are often used. These electrodes are usually
Gokul Chandra Biswas the development of simple, low- formed by thin- or thick-film techniques, including photolithography,
cost, and automatic biosensing electron-beam lithography, focused ion beam (FIB) milling, and
systems for bio-medical, screen-printing. Thin-film techniques are indispensable for creating
agricultural, food safety, fine patterns (such as interdigitated electrodes used for redox
toxicological and environmental cycling) and promoting integration. On the other hand, they require
analyses. expensive facilities and cost for fabrication often poses a problem.
When a single sensor or a few sensors is/are sufficient for the
For potentiometry, a basic configuration uses two intended purpose and miniaturization is not strictly required,
electrodes: an indicator electrode to detect an analyte, and a screen-printing along with the use of plastic substrates will be more
reference electrode. The potential of the former is measured cost-effective and a practical choice.
with respect to the latter using an electrometer. The potential Materials other than those mentioned above have also been
changes linearly with respect to the logarithm of the used in attempts to improve performance. Boron-doped diamond
concentration of the analyte, as expressed by the Nernst or (BDD) features a wide potential window and low background
Nikolsky–Eisenman equations. current.6 Microfabricated BDD electrodes have been used in the
In impedimetry, the basic setup may be the three-electrode detection of heavy metal ions,7 detection of dopamine,8 and
system mentioned previously, and changes in impedance screening of NAD(P)-dependent oxidoreductases.9 Depending on
around the working electrode are measured. However, in most the application, it can be preferable to form an insulating layer with
practical sensing devices, a pair of electrodes is often used diamond. Coplanar BDD electrodes of desired patterns insulated by
(including interdigitated electrodes made with the same an insulating diamond can be formed by laser micromachining (Fig.
metal). The impedance between the electrodes is analyzed 1A).10 An all-diamond structure can also be fabricated by patterning
using an impedance analyzer. layers of BDD and an insulating diamond to form a stacked-layer
Thin- and t hick-film electrochemical microdevices, including structure.8
the three-electrode systems and microelectrodes, are already For the formation of carbon electrodes, a unique method,
available from many commercial sources. Accordingly, pyrolysis of a photoresist, has been used 11 and carbon electrodes
numerous electrochemical microdevices are constructed using thus fabricated have been used in some devices reported
these commercially available devices. We did not include these recently.12-17 With this method, photoresist patterns are first
devices in the following discussion to focus on the creation of formed and then converted into carbonaceous materials at a high
new devices rather than the progress in detection chemistry. temperature. Carbon electrodes of fine three-dimensional
structures can be formed by pyrolysis after patterning (Fig. 1B).12-14,
Materials and formation of electrodes 16, 17 Otherwise, polyimide can be used to form a patterned carbon
In electrochemical devices, the most critical components are the layer after resist patterning and dry etching.18 This technique is
electrodes. For the working electrodes, materials such as platinum,
2 | J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx

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Journal Name ARTICLE
effective when relatively thick three-dimensional (3D) electrode to form dual electrodes for redox cycling (Fig. 1D).28 The
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patterns are required. area of 9 mm2 contained one billion nanopores DOI: 10.1039/C9LC01112A
with a lateral
As in the case of the carbon electrodes formed by pyrolysis, distance of ~100 nm and pore radii of less than 20 nm.
three-dimensional structures are needed to form working For the formation of relatively large patterns, screen-printing is
electrodes as well as structures such as reaction chambers and flow widely used. However, it presents the issue of a non-negligible
channels. To this end, 3D-printing techniques have also been used amount of inks being wasted. In this regard, inkjet printing may
recently.19 The technique will be useful in forming flow channels, 3D minimize the consumption of materials to be printed and therefore
electrode structures, and other auxiliary structures such as the waste generated.29 With this method, not only electrodes but
microneedles in the trial stage of development of new devices. also small patterns of biomolecules can be prepared. It also
Electroplating is also useful to form nanostructures of metals such facilitates multiple high-resolution deposition steps of molecules.
as platinum, palladium, and gold additionally on patterned Although screen-printing is a practical technique to mass-
electrodes to improve detection sensitivity.20-23 produce inexpensive disposable electrochemical devices, other
When an array or ensemble of electrodes of the nm scale needs effective techniques have also been proposed. Song et al. used

to be formed over a relatively large area in the order of µm, template filtration to prepare electrodes (Fig. 1E).30 Electrode
nanosphere lithography is convenient.24 In this method, a patterns were cut on a polyvinyl chloride (PVC) tape using a laser
monolayer of close-packed nanospheres is first deposited as a engraving machine, and the tape was attached to a porous
template on a substrate with a layer consisting of electrodes and polyvinylidene fluoride (PVDF) membrane. Then, conductive
insulators. The template is then used to form an insulating layer or nanomaterials were deposited there by filtrating their diluted
a protecting layer on areas not protected by the nanospheres. After solutions. The PVC tape was finally peeled off. With this method,
removing the nanospheres, an array or ensemble of nanoelectrodes the composition and thickness of the deposited film can be
is exposed (Fig. 1C). Depending on the structure, other techniques controlled precisely by simply changing the volume or composition
such as electrochemical deposition or dry etching can also be of the solutions. This technique is useful for the formation of
combined to deposit an additional metal layer or to expose electrode patterns of carbon nanotubes (CNTs) and graphene.
underlying metal layers further.24-27 Otherwise, roll-to-roll (R2R) printing (Fig. 1F) 31, 32 can be a powerful
Porous anodic aluminum oxide has also been employed to technique to improve productivity remarkably.
fabricate high-density nanoelectrodes. The porous oxide features
high-density arrays of uniform and parallel pores. Hüske et al. used Removing liquid from internal structure of sensors
the porous oxide directly formed on a platinum electrode pattern
Fig. 1 Methods to form electrode structures for electrochemical sensing devices. (A) All-diamond device with BDD microelectrodes fabricated by laser ablation,
BDD overgrowth, and polishing. Adapted with permission from ref. [10]. Copyright 2014, American Chemical Society. (B) Formation of carbon mesh electrodes by
pyrolysis of photoresist. Reprinted with permission from ref. [17]. Copyright 2018, Elsevier. (C) Nanopore electrode array formed by nanosphere lithography and
reactive ion etching. (i) Formation of a nanoelectrode array. (ii) SEM image of the nanoelectrode array. Adapted with permission from ref. [27]. Copyright 2016,
American Chemical Society. (D) SEM image of the cross section of nanoporous dual electrodes. Adapted with permission from ref. [28]. Copyright 2014, The
royal society of chemistry. (E) Formation of electrode patterns by filtration. (i, ii) Patterning of CNT electrodes by filtration. (iii-v) PVDF filter with a PVC tape
patterned by laser cutting. Adapted with permission from ref. [30]. Copyright 2018, American Chemical Society. (F) Patterning of electrodes by roll-to-roll
printing. Adapted with permission from ref. [32]. Copyright 2018, American Chemical Society.

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Depending on the device, liquids can be additionally used as and carbon (which are formed simultaneously with the working
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internal components of the sensor. Ion-selective electrodes and auxiliary electrodes) are used. However, this DOI:approach
10.1039/C9LC01112A
does not
reference electrodes are representative examples. The presence of work for potentiometric sensors, and an additional structure to
a liquid does not pose serious problems in macroscopic maintain the concentration of primary potential-determining ions is
conventional electrochemical devices. However, it is an obstacle needed, as noted for commercialized liquid-junction electrodes.
toward achieving miniaturization. To realize reliable miniature reference electrodes for
As ion-selective electrodes, the so-called coated wire type microfabricated electrochemical devices, a direct approach is to
electrodes were often used, in which an ion-selective membrane is miniaturize the conventional liquid-junction Ag/AgCl electrode with
directly formed on an electrode.33 However, a problem posed by an internal filling solution. The potential of the Ag/AgCl electrode
this structure was the mismatch of charge carriers between the ion- depends on the concentration of Cl− ions as expressed by the
selective membrane and the electrode. In the ion-selective Nernst equation. In addition, the potential difference generated at
membrane, the carriers are ions. On the other hand, electrons are the liquid junction can be a cause of measurement error. To avoid
the carriers in electrodes. To resolve this issue, an intermediate issues related to this phenomenon, a concentrated potassium

layer is usually used as a redox mediator.34 Such materials can be chloride (KCl) solution is generally used to maintain the
made of conducting polymers such as poly(3,4- concentration of internal Cl− ions at the same level and to ensure
ethylenedioxythiophene) (PEDOT), polypyrrole, and poly(3- sufficient efflux of internal KCl and minimize the liquid junction
octylthiophene) (POT). Otherwise, the double-layer capacitance at potential. As the total volume of the internal solution decreases,
the membrane–electrode interface of the coated-wire type however, it becomes more challenging to meet these requirements.
electrodes was intentionally increased to eliminate factors that lead However, except for cases wherein a very thin probe is used or the
to undesirable drift of the electrode potential. Carbon materials available space is very limited—as in the case of chips of the sub-
such as graphene, graphene oxide, and CNTs have been used for mm order—the situation is not so dire, because the chip needs to
this purpose.34 Advances toward improved solid-state ion-selective conform to a certain size to ensure easy handling. As the size of the
electrodes have been described well in a recent review by Hu et liquid-junction electrode increases, its incorporation in a device and
al.35 Among the microfabricated ion-selective electrodes reported achieving satisfactory performance becomes easier.
so far, coated-wire type electrodes feature frequently. Although Considering long-term storage, the presence of an internal
previous studies to realize solid-state ion-selective electrodes were solution is problematic as in the case of the ion-selective
not necessarily directed toward miniaturization, the techniques will electrodes mentioned previously. It will be ideal if the
be indispensable in realizing reliable microfabricated ion-selective reference electrode functions in a completely dry state. This is
electrodes, and the use of these techniques should be considered a long-standing issue and many groups have attempted to
seriously. develop the so-called solid-state reference electrodes.36, 37 To
this end, structures similar to those employed in solid-state
Miniaturization and integration of reference electrodes ion-selective electrodes have been attempted to be
Although the reference electrode may be an uninteresting fabricated.38 In providing a reliable reference potential, the
component that does not usually attract attention, it is most difficult step will be to design the outermost part of the
indispensable and its performance directly influences that of reference electrode similar to a liquid junction that is in
the entire electrochemical devices. The required stability and contact with the solution. The interfacial potential difference
precision depend on the mode of operation. In amperometry, between this part and the analyte solution should not change
the potential of the working electrode is usually fixed at the when it is in contact with the solution that contains various
diffusion-limiting region at which the generated current does ions of various concentrations. Reference electrodes that
not depend on electrode potential. In this case, the shift in the provide a stable potential have been fabricated by employing a
reference electrode potential is not serious, and even hydrophobic ionic liquid as the outermost layer to resolve this
polarizable electrodes such as those made of platinum, gold, problem.39-42 However, another issue is the use of the liquid,
and carbon can be suitable alternatives, as observed in the which is complicated to incorporate in microfabricated devices.
case of numerous microfabricated amperometric devices. On Although its use may not be difficult for relatively large
the other hand, in potentiometry, the shift in the reference structures such as those found in paper-based devices,
electrode potential directly affects the reading of the potential breakthroughs are required to enable its use in more
of the indicator electrode and causes a serious error. miniaturized devices.
Therefore, currently, commercially available reference
electrodes (in the order of cm) are used in many miniaturized Merging electrochemical microsensors with
potentiometric devices. microfluidics
As mentioned previously, requirements pertaining to the stability Amperometric and voltammetric devices
of the reference electrode potential are not so strict in Independent electrochemical devices such as screen-printed
amperometric sensing. Therefore, the simple so-called pseudo three-electrode systems and interdigitated electrodes for
reference electrodes (that are in direct contact with an analyte impedance measurement or redox cycling are commercially
solution) are often used under limited conditions. The used available. Accordingly, there are many reports of microsensors
electrodes are not restricted to non-polarizable electrodes such as using these commercialized products. The novelty of these devices
Ag/AgCl, and even polarizable electrodes such as platinum, gold,

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lies mainly in the detection of specific analytes, the method of alternative for exchanging sample and reagent View solutions for
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detection, and applications. We will not deal with them here except affinity sensing. A row of plugs including DOI: 10.1039/C9LC01112A
washing plugs
for cases that contain relevant techniques of note. separated by air can be stored in a single tube or flow channel
Some notable works have attempted to integrate sensing and and can be delivered to the sensing site sequentially.52 Yang et
solution processing. Horak et al. fabricated an amperometric device al. used this technique for amperometric detection of tumor
with functions including reagent delivery for the detection of biomarkers.53
Epstein–Barr virus infection.43 Fragoso et al. fabricated an As mentioned previously, electrochemical techniques are
amperometric device for cancer markers (Fig. 2A).44 Sixteen advantageous in many areas of bio/chemical sensing.
working electrodes were placed in separate chambers along with Nevertheless, depending on the analyte, optical
five reservoirs to store reagents and samples. The reservoirs were measurements may be more suitable. Accordingly, all-around
interfaced by septa and the solutions were moved from the sensing devices can be realized in the form of a hybrid,
reservoirs to the sensing region by applying pressure. Lee et al. including sensors based on other detection principles. Ordeig
fabricated a device for amperometric detection of C-reactive et al. reported a hybrid device with integrated flow channels,

protein (CRP) (Fig. 2B).45 The chip consisted of electrodes screen- an electrochemical cell, microlenses, and air mirrors for
printed on a polyethylene terephthalate (PET) film, flow channels, amperometric and absorbance measurements.54
and a cover layer. Complexes consisting of CRP and alkaline Electrochemical devices also provide a method of placing animal
phosphatase were formed by an antigen–antibody reaction and the or microbial cells directly on the chip and measuring the changes in
product was detected. Lee et al. fabricated a device for the activity. Li et al. fabricated a device with a culture chamber and four
voltammetric detection of four biomarkers including fibrinogen, flow channels located downstream, for the amperometric detection
adiponectin, low-density lipoprotein, and 8-isoprostane.46 The of reactive oxygen and nitrogen species secreted from a population
device had five chambers with three-electrode systems, one of of macrophage.55 Ges et al. fabricated a device to monitor the
which was used as the reference. Transport of solutions was secretion catecholamine from adrenal chromaffin cells (Fig. 2E).56
controlled by pneumatic microvalves. Kimura et al. fabricated a An array of U-shaped structures was formed to trap the cells and
device for the early diagnosis of bovine subclinical mastitis.47 catecholamines were detected by amperometry. Measurement of
Neutrophils in the raw milk of mastitic cows were separated from the respiratory activity of cells is required in many situations such as
fat globules in a double-layer flow channel structure and were drug screening applications. Weltin et al. fabricated a device with a
finally trapped and concentrated in a working electrode consisting cell cultivation chamber and sensors for pH, oxygen, lactate, and
of an array of micropillars with immobilized superoxide dismutase. glucose to monitor the metabolism of human cancer cells.57
Superoxide anions secreted from neutrophils were detected, which Extracellular acidification, respiration of the cells, glucose
were used to estimate the number of neutrophils and the consumption, and lactate production were measured using the
seriousness of the disease. Kim et al. used porous membranes to sensors. Marzioch et al. fabricated a sensing device for oxygen and
control flows in multiple flow channels in a structure fabricated by temperature to check the efficacy of photodynamic therapy.58
stacking plastic layers (Fig. 2C).48 They conducted electrochemical Oxygen consumption of T-47D breast cancer cells was measured
immunoassay for the creatine kinase-myocardial band test. during the therapy. Tanabe et al. fabricated a microfluidic device
Although continuous flows of liquids are predominantly used in with a miniaturized Clark-type oxygen electrode to measure the
microfluidic systems, plug (or droplet)-based microfluidics will be bactericidal activity of neutrophils, which is closely related to
more beneficial in creating devices with higher functionalities. First, mental stress.59 Neutrophils and E. coli cells were co-cultured, and
compared with continuous flows, the volume of each plug can be changes in the respiratory activity of E. coli were measured using
reduced remarkably in such cases. Second, multiple plugs can be the oxygen electrode.
processed in a single flow channel. Third, the mixing of components Three-electrode systems have also been used for amperometry
can be very rapid (<1 s) after merging two plugs and moving the in lab-on-a-disc (LoD) devices. In LoD devices, centrifugal force
merged plug in a flow channel. In many previous devices, plugs of moves solutions from the inlet reservoirs near the center of
aqueous solutions were formed by injecting the solution into rotation to compartments in the periphery, for metering, mixing,
another immiscible carrier liquid using a branched flow reaction, and detection. Nwankire et al. fabricated an LoD device
channel structure, which could be used for various purposes.49, for the quantification of ovarian cancer cells.60 The device
50 However, a serious problem with the application of this contained functions including blood separation, cancer cell
method in electrochemical sensing is that electrode surfaces extraction from the plasma, specific capture, and detection by
may be contaminated by the carrier liquid. This difficulty can impedance measurements. Five different assays could be
be alleviated by separating the plugs by air. Based on plug- performed in parallel. Sanger et al. fabricated an LoD device with
based microfluidics, Itoh et al. fabricated a device to measure eight integrated filtration units for sample pre-treatment and three-
the K-value for the freshness of fish (K-value) (Fig. 2D).51 electrode systems for cell-free detection of p-coumaric acid
Solution plugs were formed, merged to mix components, and produced by E. coli. (Fig. 2F).61 Li et al. developed an LoD device
stopped at two sensing sites using auxiliary structures in a with electrochemical sensing and blood sample separation
single flow channel. The sums of concentrations of ATP-related functions.62 The device was used for the determination of glucose,
compounds to calculate the K-value were measured lactate, and uric acid.
amperometrically using two enzyme electrodes in the flow A problem with LoD devices is determining how to establish
channel. Plug-based solution processing can be a good electrical contact between the components on the disk and external

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instruments. A simple method is to stop the rotation and conduct incorporated in the disc. Paraffin wax valves (containing iron oxide
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measurements in a stationary state. However, this method causes nanoparticles used as heaters) were employed DOI: 10.1039/C9LC01112A
for flow control
delay and may influence time-sensitive experiments. Ideally, it is through melting them by laser radiation. Alternatively, Bauer et al.
better to conduct measurements without stopping the rotation. To coupled a battery-powered bipotentiostat to a disc with electrodes
solve this problem, slip-ring setups have been used.63, 64 Kim et al. to send the signal wirelessly.65 Furthermore, LoDs reported recently
used this setup and integrated functions including the capture of by some groups provide power wirelessly to drive an on-chip
analyte molecules, a bead-based enzyme-linked immunosorbent potentiostat along with wireless signal transmission.66, 67
assay, and amperometric detection of C-reactive proteins.63 Many current microfluidic systems rely on external bulky
Rectangular chips with electrodes fabricated separately were pumps and valve systems. To promote automatic solution

Fig. 2 Electrochemical sensing devices coupled with microfluidic functions. (A) Microfluidic device for electrochemical detection of breast cancer markers.
Adapted with permission from ref. [44]. Copyright 2016, The royal society of chemistry. (B) Device for amperometric detection of CRP. Adapted with permission
from ref. [45]. Copyright 2012, Springer. (C) Multilayered polymeric cartridge. (i) Exploded view, (ii) Cross-section of stacked layers. (iii) Top view of cartridge.
Reprinted with permission from ref. [48]. Copyright 2009, Elsevier. (D) Device to determine freshness of fish (I, ii)) PDMS substrate with flow channel structures
and glass substrate with electrodes. (ii) Complete device. (iii) Magnified view of sensing sites with bypass flow channels. Adapted with permission from ref. [51].
Copyright 2013, American Chemical Society. (E) Microfluidic platform for cell trapping and monitoring of cellular secretion. (i, ii, iii) Chromaffin cells in the traps,
(iv) Visualization of a perfusion flow using food dye. (i) Platinum electrodes. (ii) Cell trap. (iii) Chromaffin cells in cell trap. (iv) Flows disrupted by the clumps. (v)
Clumps of cells stuck between the traps. Reprinted with permission from ref. [56]. Copyright 2013, The royal society of chemistry. (F) Photograph of
electrochemical LoD device and magnified view of a unit containing chambers for filtration and detection for screening genetically modified E. coli cells. Reprinted
with permission from ref. [61]. Copyright 2017, Elsevier. (G) EWOD device with integrated potassium ion-selective electrodes. Reprinted with permission from ref.
[70]. Copyright 2018, Elsevier.

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transport, electrowetting is useful. In electrowetting on formed by a sacrificial aluminum etching technique. The
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dielectric (EWOD), wettability of a hydrophobic dielectric layer selectively detected vanillin vapor. DOI: 10.1039/C9LC01112A
formed on an array of electrodes separated with narrow gaps Lin et al. used an electrolyte–insulator–semiconductor structure
is changed by the application of a voltage, and independent for the detection of glucose, urea, and creatinine.83 Alginate
droplets are moved sequentially. Owing to this feature, the microbeads containing magnetic beads and an enzyme were
technique is often called digital microfluidics. An attractive localized using a step-like obstacle structure in a flow channel and a
feature of EWOD devices is not only the transport of droplets magnetic field. Upon the injection of an analyte solution, enzymatic
but also the capability of processing multiple solutions. reactions caused pH changes, which were measured.
Programmable manipulation enables procedures such as Contrary to the ISFETs, ion-selective electrodes have been
metering, transport, separation, and merging of droplets. In used in various devices, particularly in paper-based and
the initial stage, electrodes used for sensing were formed wearable devices. In many of these applications, sensing areas
within the array of driving electrodes for EWOD on the same in the order of mm have been used and the electrode
bottom plate. Some groups integrated a three-electrode potential could be easily measured. Details will be mentioned

system in such devices.68, 69 Farzbod et al. integrated an array later in corresponding sections.
of potassium ion-selective electrodes (ISEs) at the bottom
plate (Fig. 2G).70 A problem with this layout is that the Impedimetric devices
electrodes are crowded and the electrodes for sensing must be Wang et al. fabricated a device for the detection of
exposed without the hydrophobic dielectric layer, making thrombin.84 Gold interdigitated electrodes were formed in a
fabrication more complicated. To overcome this problem, polydimethylsiloxane (PDMS) flow cell. Magnetic beads
sensing electrodes were subsequently formed on the top modified with aptamers were used to capture, separate, and
ground plate.71-73 Shamsi et al. used such an EWOD device and concentrate the target protein. Daprà et al. reported an all-
magnetic beads conjugated with primary antibodies to detect polymer electrochemical device using a conductive polymer
the thyroid stimulating hormone.72 Rackus et al. formed bilayer to form electrodes.85 The electrode was functionalized
nanostructured gold working electrodes along with reference with aptamer probes to detect ampicillin and kanamycin A by
and auxiliary electrodes to detect the Rubella virus by electrochemical impedance spectroscopy(EIS). Ali et al.
electrochemical sandwich enzyme-linked immunosorbent fabricated a microfluidic device for the detection of low-
assay (ELISA).73 density lipoprotein using bovine serum albumin and
antiapolipoprotein B-functionalized carbon nanotube–nickel
Potentiometric devices oxide nanocomposite.86. Chiriacò et al. fabricated a device to
The ion-sensitive field-effect transistor (ISFET) is a representative detect autoantibodies towards phosphorylated a-enolase for
electrochemical microsensing device. Although many reports the diagnosis of pancreatic ductal adenocarcinoma. The device
related to the ISFET were published until the last decade, they have had two PDMS reaction chambers, each of which had four
been decreasing in number since that time. Since the first report of couples of interdigitated electrodes with immobilized peptides
the ISFET in the 1970s, the technology has been established well corresponding to a portion of the enzyme.87 Lakey et al.
and faced challenges in various possible applications.74 Alternatively, fabricated a microfluidic device with five sets of gold
different field-effect transistor (FET) devices based on materials interdigitated electrodes for impedance measurements to
such as carbon nanotubes or graphene have been actively studied. detect a biomarker (D-dimer) for the diagnosis of deep vein
It is a matter of controversy as to whether the detection principles thrombosis and pulmonary embolism.88 Anti-D-dimer antibody
are electrical or electrochemical. Therefore, the new FET sensors was immobilized at the individual sensing sites following the
are not focused on here. Those who are interested in these devices electrochemical patterning of polypyrrole.
should refer to related reviews published recently.75-77 As well as molecules, the detection, number counting, and
Regarding devices based on ISFETs, Yamada et al. fabricated activity monitoring of animal or bacterial cells have also been
a microfluidic device for pH measurements.78 Two ISFETs and investigated using such techniques. Paredes et al. used an
an Ag/AgCl pseudo reference electrode were used for impedimetric device to monitor the growth of a bacterial
differential measurements. Sharma et al. fabricated a biofilm.89 Etayash et al. conducted real-time, label-free
microfluidic chip with ISFETs for pH and potassium ions.79 detection of Gram-positive bacteria (L. monocytogenes) that
Matsuura et al. fabricated a microfluidic device with an ISFET acted as foodborne pathogens using antimicrobial peptides as
to detect biofilm formation by measuring pH changes in the probes.90 Manczak et al. detected monocytes using
culture medium.80 Andrianova et al. reported a microfluidic immobilized antibodies (Fig. 3B).91 An et al. used a benzoboric
device for the detection of organophosphorus pesticides. A acid-modified gold-plated polymeric substrate with a regular
hydrolytic enzyme was used and pH changes were detected 3D microarray to detect rare circulating tumor cells (CTCs)
using an ISFET.81 As an interesting application of the ISFET, efficiently.92 The number of CTCs was measured by EIS and the
Kuznetsov et al. proposed a bioelectronic nose (Fig. 3A).82 An captured CTCs could be detached by a reversible reaction
ISFET with an immobilized aptamer for vanillin was immersed between benzoboric acid on the 3D microarray and sialic acid
in a solution in a flow channel and was separated from the gas on CTCs. In addition to the orthodox methods, some
phase with a hydrophobic membrane with many holes that interesting methods were also used. Damhorst et al. coupled
acted as a gas-to-liquid extraction interface. The structure was Ion-encapsulating liposomes functionalized with antibodies

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with impedance spectroscopy (Fig. 3C).93 The analyte virus was dielectrophoresis.95-99 When objects such as cells View are
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sandwiched between antibodies on the electrodes and polarizable than the medium, they are draggedDOI:to
10.1039/C9LC01112A
a higher electric
liposomes. The ions in the liposomes were released by heating. field region around the smaller detecting electrodes, which is called
Detection of human immunodeficiency virus (HIV) was positive dielectrophoresis (pDEP). By contrast, when the medium is
attempted using the device. more polarizable than the objects, they are repelled from the
In the detection of DNA by label-free impedance spectroscopy, higher electric field region, which is called negative
hybridization in a stationary solution takes time. Wu et al. dielectrophoresis (nDEP).100 Mansoorifar et al. concentrated and
accelerated hybridization by stirring solutions by an AC confined PC-3 prostate cancer cells within microwells by pDEP for
electroosmosis (ACEO) flow (Fig. 3D).94 When animal or bacterial impedance measurements. 100, 101 The cells were treated with the
cells are analyzed by impedimetry in microwells or microfluidic drug Enzalutamide, and dying cells exhibited different spectra
channels, an asymmetric electrode configuration can be easily compared to those of healthy cells. Depending on the scenario, the
established in the structure. This enables concentration of cells by cells were released from the electrode and were used for other

Fig. 3 Potentiometric and impedimetric devices. (A) Bioelectronic nose created with a field-effect transistor with an immobilized aptamer. Reprinted with
permission from ref. [82]. Copyright 2019, Elsevier. (B) Device used for on-chip impedimetric immuno-detection of subpopulations of cells. Reprinted with
permission from ref. [91 ]. Copyright 2016, Elsevier. (C) Impedimetric sensing of virus that employ the release of encapsulated ions from tagged liposomes.
Adapted with permission from ref. [93]. Copyright 2013, Springer. (D) Vortices induced over electrodes by AC electroosmosis for label-free impedimetric DNA
sensing. Reprinted with permission from ref. [94]. Copyright 2013, Elsevier. (E) Concentration of bacteria by negative and positive dielectrophoresis for
impedance measurement. Reprinted with permission from ref. [104]. Copyright 2013, Elsevier. (F) Steps for seeding cells into multiple chambers. (i) Flow channel
1 is filled with the solution, which do not enter the chambers. (ii) Each chamber is filled with the solution by having the solution withdrawn by applying a negative
pressure from port 2. (iii) The solution in flow channel 1 is removed by applying a pressure. Reproduced with permission from ref. [107]. Copyright 2016, The
royal society of chemistry. (G) Microfluidic device with a solution-processing function for label-free impedimetric detection of DNA hybridization analysis. (i) Chip
with electrodes. (ii) Assembled device with assay channels (green) and horizontal (blue) and vertical (red) channels for valve control. (iii) Layout of flow channels
in the stacked layers. Inset shows actuation of the valve. Reprinted with permission from ref. [108]. Copyright 2015, Elsevier. (H) Integrated automated
multiorgan-on-a-chip and sensing platform consisting of modular components, including electrochemical biosensors. Adapted with permission from ref. [117].
Copyright 2017, National Academy of Sciences.

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purposes. Nakano et al. collected bacteria on microelectrodes by with a sensing module for the measurement of oxygen, glucose,
View Article Online
pDEP, and electropermeabilization was used to disrupt cell and lactate concentrations.114 The module DOI: measured
10.1039/C9LC01112A
oxygen
membranes by applying a pulse-like rectangular wave. Intercellular concentration via phosphorescence quenching by employing
ions released by cell disruption were detected.102 Dastider et al. microparticles loaded with a ruthenium-based dye. Additionally, it
used two sets of gold interdigitated electrodes in a flow channel to measured glucose and lactate concentrations amperometrically.
detect E. coli O157:H7.103 A set of electrodes focused and Shin et al. identified a cardiac injury biomarker, which was
concentrated E. coli by pDEP to direct it to other sets of electrodes essentially the creatine kinase-MB in a heart-on-a-chip; this was
with E. coli-specific antibodies used for detection. Hamada et al. achieved by using an aptamer-based impedimetric
used both pDPE and nDEP (Fig. 3E).104 Bacteria introduced into the immunosensor.115 Variations in the concentration of the dose-
device were first repelled by nDEP force exerted by the first dependent creatine kinase-MB were observed, which were caused
electrode and were captured by pDEP on the second electrode. due to the cardiac damage upon exposure to doxorubicin.
Chuang et al. used multi-layer electrodes to trap and release cells Moreover, to continually monitor the secretion of human albumin
from an array of microcavities and to conduct impedance and glutathione-S-transferase-alpha after administering

measurements.105 The device had an ITO top electrode, a middle acetaminophen, Shin et al. integrated impedimetric immunosensors
electrode on the top surface of the microcavity structure, and in a human liver-on-a-chip.116 The immunosensors could be
electrodes at the bottom of the microcavities. By changing the regenerated repeatedly by using a specially designed microfluidic
combination of electrodes, cells were trapped/released individually module. In a modular organ-on-a-chip, Zhang et al. used
in the microcavities by nDEP for impedance measurement of the electrochemical protein biomarker sensors as well as optical pH, O2,
bladder cancer cell. After the measurement, the trapped cells were and temperature sensors.117 The electrode surfaces were
released by pDEP. functionalized with antibodies for the biomarkers, and were
Devices have also been fabricated aiming toward multiplexed regenerated for repeated measurements. Drug-induced organ
detection. Onishi et al. fabricated an impedimetric device for on- toxicity was examined using dual-organ human liver-and-heart-on-
site determination of the minimum inhibitory concentration of a-chip and liver-cancer-and-heart-on-a-chip model systems (Fig. 3H).
antibiotics.106 They formed an array of eight chambers for Some reviews regarding the integration of bio/chemical sensors
impedance measurements to accommodate antibodies of different into organs-on-chips have been reported.118, 119 However, only a
concentrations (Fig. 3F).107 A sample solution containing bacteria few studies on the in situ monitoring of drug-induced organ toxicity
was introduced into the chambers and the solutions in each have been reported that employ organs-on-chips, and studies on
chamber were separated by removing unnecessary solutions with the integration of sensing functions into these organs-on-chips are
the help of surface tension. The growth of the bacteria in each still at the initial stages. Thus, studies on the incorporation of
chamber was monitored by impedance measurements. Ben-Yoav et microfabricated electrochemical sensors in devices are required;
al. formed 3 × 3 individually addressable reaction chambers for the further, an enhancement in the monitoring performance, including
detection of DNA hybridization by EIS (Fig. 3G).108 Dual-layer precision and multiplexed detection improvement, is highly
programmable valves were formed to control solution transport in necessitated.
flow channels formed of rows and columns. The valves closed the
flow channels by bending their PDMS membrane downward. The Paper-based devices
valves allowed the functionalization of multiple working electrodes Compared with that of other electrochemical devices,
with probe DNAs without cross-contamination or requiring parallel development of paper-based devices has been extensively
incubation or measurements with multiple samples. investigated over the last decade. In contrast to the
complementary metal oxide semiconductor (CMOS) devices
Application to Organs-on-Chips mentioned later, the paper-based devices are ultimately
Presently, 3-D human tissue/organ models known as organs-on- simple, which is really advantageous in realizing inexpensive,
chips, are being developed as platforms for personalized medicine portable, and disposable devices. Since the first report,120
and drug screening.109-112 These organs-on-chips facilitate the rapid, many electrochemical paper-based devices and reviews have
inexpensive, and effective investigation of in vivo drug efficacy and been reported.121-129
toxicity, and it is expected that their application will solve the As long as fabrication is concerned, many devices used existing
ethical problems associated with the use of animals. In this study, techniques such as screen-printing with the only exception that the
the in situ measurement of the concentration of indicator substrates were paper. However, some unique techniques specific
molecules secreted by cells was critical in obtaining the information to paper have also been proposed. Ink-jet printing is particularly
regarding the viability and metabolic activity of organs, and will also suitable for paper and was used to draw electrode patterns.130-132
be beneficial in the analysis of multi-organ interactions. Therefore, Santhiago et al. also used a XEROX printer to print patterns of
integrating a sensing function in organs-on-chips is extremely carbon electrodes.133 Along with wax printing already used for
important. paper to form structures such as flow channels, this is very efficient
Moya et al. integrated three inkjet-printed amperometric in mass-producing devices with electrodes. Otherwise, a pencil or
dissolved oxygen sensors in a liver-on-a-chip,113 and primary human pen is used to draw patterns of electrodes, hydrophobic barriers,
and rat hepatocytes were cultured. Their oxygen consumption was and biomolecules.134-139 The method lets users design their original
evaluated, and the concentration distribution of the dissolved electrode patterns themselves. Soum et al. used a digital plotter to
oxygen was determined. Bavli et al. developed a liver-on-a-chip automate the drawing.140 Alternatively, carbon electrodes were

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formed directly on paper boards by pyrolysis using laser light (Fig. withstood strains up to 500% with a negligible influence onOnline
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4A). The carbon composed of graphene sheets with an enhanced electrochemical performance. Lee at al. DOI: used10.1039/C9LC01112A
chemical vapor
active/geometric area ratio and exhibited high conductivity.141 Zang deposition (CVD)-synthesized graphene doped with gold formed on
et al. also formed conductive molybdenum carbide–graphene serpentine gold electrode patterns.166 The electrode showed
composite electrodes by laser-powered conversion from fibrous improved electrochemical activity over bare graphene.
paper soaked with gelatin-mediated inks containing molybdenum In relation to the abovementioned problem, consistency of
ions.142 As an essentially different approach, Fosdick et al. proposed wearability and electrical contact is another critical point to be
the use of metal wires as electrodes.143 The electrodes can be considered when using wearable devices. The electrodes used for
incorporated into the device structure by simply folding paper. The sensing must be flexible to conform to the non-planar skin surface.
wires can be cleaned and modified with molecules prior to To this end, electrodes that take the form of tattoos are very useful
assembly. (Fig. 5A).167 Jia et al. fabricated a temporary-transfer tattoo
Another specific feature of the paper is that it can be folded and amperometric enzyme biosensor for monitoring of lactate levels in
stacked easily. The technique that accompanies the folding of paper human perspiration (Fig. 5B).168 The technique was also used in

is often called the “origami” technique. The technique has been devices for the monitoring of epidermal pH levels,169 trace metals in
used to form not only device structures 143-146 but also to simplify human perspiration,170 and ammonium ions in sweat.171
analytical procedures and realize user-friendly devices. Unlike Depending on the application, there are some additional
previously mentioned microfluidic devices that mostly rely on requirements for the substrates. For example, when applied to
pumps and valves, the transport of solutions in paper-based devices contact lenses to analyze the components of tears,172-174 the
depends on the wicking of solutions, which makes the sequential substrate (and preferably electrodes) must be transparent. The
procedure that handles multiple solutions difficult. The origami substrate must also fit the shape of the eyeball. In other words, the
procedure is effective in circumventing this difficulty.147-149 Fig. 4B electrodes must be formed on a substrate that is not flat. To realize
shows an example of such a device reported by Ding et al. for the this, a method used in previous studies involved forming the
potentiometric detection of organophosphate pesticides.149 electrodes on a flat substrate and deforming it after that.
They separated zones for an enzyme, an enzyme substrate, Furthermore, biocompatibility of materials in contact with the body
and a sample solution on different tabs. When the device was part is necessary, which entails stricter requirements when the
used, the tabs were folded following a predetermined sequence. devices are used on the eye and in the mouth.
The technique was also used for the detection of pesticides.150 A Considering wearability, it will be more preferable if the sensing
modified design was proposed by Yakoh et al. (Fig. 4C).151 Their devices are formed on cloths or underwear. To this end, attempts
device consisted of folding paper and a movable reagent- have been made to form electrodes on textiles. Choudhary et al.
stored pad. When in use, the device is assembled, a sample is used the textile weaving technique to form wearable devices.175 To
spotted, and a carrier buffer is loaded in its reservoir. The make electrodes, silk yarns were coated with conducting inks and
reagent-stored pad is then pulled to introduce reagents or reagents before being woven. To restrict solutions to limited areas,
switch off fluid flow. In the former case, the buffer elutes the hydrophilic and hydrophobic yarns were also formed appropriately.
reagent from the reagent-stored pad to the channel with Liu et al. fabricated an embroidered electrochemical sensor (Fig.
electrodes of the origami folding paper. The same steps can be 5C).176 Electrodes were made from conductive threads and were
repeated for the other reagents. Finally, amperometric and embroidered onto wearable fabrics.
voltammetric measurements are conducted.
Using hydrophilic patches to efficiently sample sweat
Wearable electrochemical devices When sweat is used for analysis, sampling is considered to be
Wearable devices have been a hot topic over the last several straightforward if perspiration is induced through exercise and/or
years. Their application is mainly directed toward personalized temperature elevation. However, sampling sweat under natural
health, fitness, and conditioning of athletes. Numerous papers perspiration conditions results in issues associated with the
and many reviews have already been published on them, even transportation of analytes to the sensor surface. Therefore, to
on those that involve electrochemical components.38, 152-163 facilitate efficient sampling following natural perspiration, a
Therefore, in this section, we will briefly introduce this class of hydrogel patch was formed on the electrodes that were meant for
devices, focusing on the effort to realize properties that wearable amperometric detection.177, 178 Alternatively, perspiration can be
electrochemical devices offer. intentionally and effectively induced through the transdermal
delivery of a stimulating reagent such as pilocarpine to sweat
Designing devices that meet inherent requirements glands; this was achieved via the iontophoresis process, which
A challenging problem with wearable devices is that the device is induces the flow of ions and molecules owing to the application of a
often subjected to mechanical deformations such as stretching and current across the skin. To deliver pilocarpine via iontophoresis and
twisting. In these situations, strain-enduring electrodes are potentiometrically measure chloride ion concentration, Gonzalo-
required. Bandodkar et al. developed inks to form stretchable Ruiz et al. fabricated a device with screen-printed electrodes and a
electrodes by screen-printing.164, 165 The inks employed CNTs and hydrogel.179 Kim et al. integrated tattoo-like electrodes into
elastomeric polyurethane as a binder.165 hydrogels for the iontophoretic delivery of pilocarpine and the
The electrodes printed using these inks with serpentine patterns amperometric detection of ethanol alongside flexible wireless
electronics,180 whereas Emaminejad et al. integrated a hydrogel

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DOI: 10.1039/C9LC01112A
Fig. 4 Techniques used for paper-based electrochemical sensing devices. (A) Formation of carbon electrodes by laser pyrolysis and cutting. Adapted
with permission from ref. [141]. Copyright 2017, Wiley-VCH Verlag GmbH & Co. KGaA. (B) Folding sequence for a paper-based 3D origami
potentiometric device. Adapted with permission from ref. [149]. Copyright 2016, Wiley-VCH Verlag GmbH & Co. KGaA. (C) Paper-based device that
controls solution transport by folding and sliding of paper for electrode modification and voltammetric determination. Adapted with permission from
ref. [151]. Copyright 2019, American Chemical Society.

layer as well as Na+, Cl−, and glucose sensors alongside Contrary to iontophoresis, reverse iontophoresis renders the
iontophoresis electrodes.181 Acetylcholine, methacholine, and non-invasive sampling of interstitial fluid (ISF) possible. In this
pilocarpine were loaded in the hydrogel; further, iontophoresis was regard, Kim et al. developed a device for the simultaneous and
performed wirelessly, while perspiration was induced at periodic independent sampling and analysis of ISF glucose and sweat-
time intervals without causing skin damage. alcohol.182 This device employs the reverse iontophoretic extraction
of ISF and the iontophoretic delivery of pilocarpine into the skin. For
iontophoresis, the device used tattoo-like electrodes, and for
wireless real-time measurements, screen-printed electrodes
formed on a flexible substrate alongside readout circuits were used.
Use of minimally invasive microneedles

Real-time monitoring of metabolites in physiological fluids is
necessary in fields such as diabetes care and sports medicine. In the
development of wearable devices, body fluids such as sweat, saliva,
and tears are often used. However, when more accurate reliable
data are required, direct analysis of whole blood will be more
preferable. In conducting this, a problem is that sampling through a
needle accompanies pain. As a solution, the application of
minimally invasive microneedles has been tried mainly for the
analysis of ISF.
Coupling the microneedles with electrochemical detection using
microdevices can involve two methods. In one method,
electrochemical detection is conducted in a microfluidic system
with hollow microneedles formed vertically at the end of the flow
channel structure. Analyte solutions are withdrawn into the interior
of the device and detected electrochemically. In the other method,
electrodes are formed with microneedles to fabricate on-body
patch-type devices. The electrodes are directly stuck onto the skin
and electrochemical measurements are conducted.
As a device based on the former, Miller et al. formed a
microneedle on a sensing device for K+ ions by two-photon
polymerization.183 The same group also demonstrated the
amperometric detection of myoglobin and troponin using a similar
device with the microneedle.184 Jina et al. reported a device for
continuous glucose monitoring (Fig. 5D).185 The device had an array
of 200 silicon hollow microneedles with a length of ~300 μm and a
50 μm × 50 μm lumen. A screen-printed 3-electrode system
containing a working electrode with immobilized glucose oxidase
was formed in a sensing chamber for the amperometric detection
of glucose. The sensing chamber was initially filled with a buffer
solution. Glucose diffused from the epidermal layer of the skin
through the microneedle array into the buffer solution passively

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Fig. 5 Techniques used for electrochemical wearable devices. (A) Formation of temporary transfer tattoo. (a) The electrode (red) is patterned on the paper
(orange). (b) The adhesive sheet (blue) with protective coating (maroon) is applied to the paper with the electrodes. (c(i)) The protective sheet is removed. (c(ii))
The tattoo is flipped and applied to the skin (green). (c(iii)) The release agent-coated paper is removed and the electrodes are exposed to the wearer's external
environment. In the case that physiological monitoring is desired, the procedure d(i) - d(iii) is followed. (d(i)) The paper is removed. (d(ii)) The tattoo electrode
patterns are applied to the skin. (c(iii)) The protective coating is removed. Adapted with permission from ref. [167]. Copyright 2012, The royal society of
chemistry. (B) Three-electrode “NE” tattoo for the determination of lactate. Adapted with permission from ref. [168]. Copyright 2013, American Chemical Society.
(C) Embroidered Three-electrode systems formed with carbon-coated conductive thread (for W.E. and A.E.) and Ag/AgCl-coated thread (for R.E.). (i-iii) Pictures of
embroidered electrodes on a textile chip, cotton gauze, and cotton t-shirt. (iv) Magnified image of the part indicated in (iii) on the t-shirt. Adapted with
permission from ref. [176]. Copyright 2016, The royal society of chemistry. (D) Amperometric sensing device with an array of microneedles. (i) SEM image of
device. (ii) Magnified SEM image of microneedles. Adapted with permission from ref. [185]. Copyright 2011, Wiley-VCH Verlag GmbH & Co. KGaA. (E)
Mouthguard biosensor with wireless amperometric circuit board. Reprinted with permission from ref. [191]. Copyright 2015, Elsevier. (F) Mouthguard biosensor
for saliva glucose detection. Reprinted with permission from ref. [193]. Copyright 2015, Elsevier. (G) Active contact lens device for wireless health monitoring.
Adapted with permission from ref. [194]. ©2012, IEEE. (H) Glove biosensor with a sensing finger, immobilized organophosphorus hydrolase, and the
collector/sampling finger. The electrodes are connected via a ring bandage to a portable potentiostat on the back of hand with wireless Bluetooth communication
to a smartphone. Adapted with permission from ref. [196]. Copyright 2017, American Chemical Society.
and was detected on the working electrode. The device could lactate oxidase. The same group also fabricated a device with two
provide clinically accurate results over 72 h. working electrodes for the simultaneous monitoring of lactate and
For direct amperometric measurements, working electrode glucose concentrations.189 The performance of the devices was
structures with microneedles of various designs have been assessed in artificial ISF and in human serum. Microneedles were
proposed. Windmiller et al. proposed a working electrode structure also combined with wearable devices to deliver drugs
constructed with an array of platinum-coated solid microneedles transcutaneously. Lee et al. fabricated a patch consisting of a
covered with another array of hollow microneedles that surround heater, temperature, humidity, glucose, and pH sensors along with
the solid microneedle.186 The microrecesses formed between the polymeric microneedles.166 The device measured glucose
two types of microneedles were used to immobilize enzymes in a concentration in sweat and the microneedles were thermally
poly(o-phenylenediamine) (PPD) layer by electropolymerization. activated to release a drug at high glucose concentration.
Detection of glutamate and glucose was demonstrated using this
working electrode structure. The PPD layer rejected electroactive Wireless signal transmission and power transfer
interferents effectively and the microrecesses protect the enzymes Considering the user-friendliness of wearable devices, wireless
upon insertion into the skin. Caliò et al. formed a working electrode device operation and data transmission will be a must as
structure with polymeric microneedles with an immobilized enzyme demonstrated by many recently reported devices. 166, 173, 180-182, 190-
formed on a flexible substrate.187 The microneedles were formed 192 In addition, wireless energy transfer will be another option
on a gold electrode by photolithography using a photocurable to be considered. Bandodkar et al. fabricated a Na+ ion-selective
mixture containing an enzyme. They demonstrated the detection of tattoo electrode paired with a wireless transceiver for the
glucose and lactic acid. Bollella et al. reported a working electrode monitoring of sweat Na+ ion concentration on the human
structure for the sensing of lactate in dermal ISF.188 Electrodes were epidermis.190 Saliva and tears were also used for the analysis using
formed on a structure of 64 polycarbonate microneedles. Gold and wearable devices. Kim et al. fabricated a mouthguard that can
multiwalled carbon nanotubes were electrodeposited followed by monitor the salivary uric acid level (Fig. 5E).191 Screen-printed
the electropolymerization of methylene blue and immobilization of electrodes with immobilized enzyme along with compact electronic

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circuits including a potentiostat, microcontroller, and a Bluetooth In many of the reported microfluidic systems, View solutions are
Article Online
transceiver were incorporated in the mouthguard. Arakawa et al. transported by the application of pressure DOI:using
10.1039/C9LC01112A
an external
fabricated a device used in the human oral cavity for the monitoring pressure source. To realize this on a chip, bubble-based pumps
of saliva glucose (Fig. 5F).193 The device consisted of a platinum that work by the electrolysis of water are useful. For this
electrode with immobilized glucose oxidase along with a Ag/AgCl purpose, a pair of electrodes such as interdigitated electrodes
electrode on a mouth-guard support with a wireless transmitter. are used to produce oxygen and hydrogen bubbles from both
Liao et al. reported a contact lens device with an electrochemical electrodes, which then applies pressure to the solution to be
glucose sensor, a loop antenna, a communication interface, and transported. Following a pioneering device by a group of
readout circuitry (Fig. 5G).194 The device was powered wirelessly University of Twente,206 many electrolysis-based micropumps
using RF power and transmits information about the tear glucose have been reported for various purposes.26, 207-210 Because the
level wirelessly to an external reader. In relation to this technology, structure is simple, the integration of multiple pumps can be
development of a wireless swallowable capsule with realized easily. Nestler et al. fabricated a polymer-based device
electrochemical sensors for gastrointestinal track investigation has with passive microfluidic parts and on-chip electrolytic pumps

been a long-standing issue. Although the development of such (Fig. 6A).211 Although the movement of solutions is
capsule-type devices is challenging and few experimental attempts unidirectional in these micropumps, bidirectional pumping can
have been made,195 they may widely be used in clinical analyses in also be realized employing the reduction of protons and
the near future. oxidation of hydrogen bubbles on a platinum black electrode
The use of smartphones has also emerged as a new trend to by applying appropriate potentials to the electrode.212 This
display obtained results on the spot. As an example, Mishra et function will be useful to withdraw a sample solution from the
al. reported a glove-based device for defense and food security exterior. In these devices, a flexible diaphragm is sometimes
applications (Fig. 5H).196 Electrodes were printed on a finger with employed to separate the electrolyte solution from the
stress-enduring inks and an enzyme was immobilized for solution to be transported. Unlike cases involving other
biosensing. The device also included a swipe sampling finger and a mechanical pumps, transport of the solution is smooth with
function for real-time wireless data transmission to a smartphone. smaller pulsations. To facilitate solution transport, the
The number of reports related to this topic has been increasing micropump can also be coupled with a hydrophobic valve.213
rapidly over the last few years. However, in most of them, the Because of the easiness of integration, electrolysis-based
use of the smartphone has seen major progress rather than micropumps have been actually integrated and used to aid chemical
the improvement of sensing devices themselves. Those who analyses. In these devices, reservoirs for a sample solution,
are interested in this topic should refer to some recently reagents, and washing buffer solutions were coupled with the
published reviews.197-201 pumps and the solutions were transported to the sensing region
sequentially. Schumacher et al. developed a device with multiple
Electrochemical microfluidic components to pumps and demonstrated the application to the assay of proteins
realize on-chip solution processing and DNAs.214 Sin et al. integrated functions including electrolysis-
Microfluidics is becoming an indispensable critical technique for based pumping, AC electrokinetic sample preparation, and
bio/chemical sensing. However, except for techniques such as those electrochemical sensing for the detection of bacterial 16S rRNA (Fig.
involving paper-based devices, current microfluidic devices usually 6B).215 Chen et al. used electrolysis-based micropumps to conduct
rely on external bulky instruments such as pumps, valves, heaters, on-chip ELISA. Necessary reagents and washing buffer solutions
etc. In particular, when the number of flow channels or reaction were delivered to a reaction chamber by switching the multiple
chambers is large, many tubes and wires must be connected to a pumps sequentially (Fig. 6C).216 Geidel et al. integrated multiple
chip as ironically expressed as “spaghetti syndrome.” Depending on electrolysis-based pumps and a photonic sensor to demonstrate the
the applications and situations, this may be acceptable. However, possibility of immunoassay. 217
there are many other cases in which the realization of fully
integrated microfluidic devices brings benefits in improving the
utility for the users. Electrochemical valves
Many active microfluidic components, particularly micropumps As an effective method for autonomous transport of
and microvalves, have been developed over the last three solutions in microfluidic devices, capillary action is
decades.202-205 Among them, electrochemical microfluidic attractive.218 However, with only the capillary action,
components are suitable in promoting miniaturization and processing solutions in a controlled manner cannot be realized.
integration, because the structure and operation of the This problem can be solved by placing switchable hydrophobic
electrochemical components can be very simple. In addition, valves at appropriate locations of flow channels. Satoh et al.
because major components are electrodes that are often made of developed such valves based on direct electrowetting on a
the same materials used for sensing, there will be no or few
gold electrode.219 The valve consisted of a simple electrode
additional steps required for fabrication even if many sensors and
pattern surrounded by a hydrophobic flow channel ceiling and
the microfluidic components are integrated. Here, we shortly
review some critical techniques in realizing electrochemical walls. A solution transported by capillary action stops there.
micropumps and microvalves along with auxiliary techniques. However, by applying an appropriate potential to the
electrode, the valve opens and the solution passes through the
Electrochemical micropumps valve region and moves by capillary action again. Owing to the
very simple structure, many valves could be formed and

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functioned in a network of flow channels. The valves were View Article Online
used in a device to measure the ammonia metabolism of DOI: 10.1039/C9LC01112A
hepatocytes.220 Biswas et al. reported a similar valve based on
the reductive desorption of a hydrophobic self-assembled
monolayer (SAM) of an alkanethiol from a platinum electrode
was also proposed.221 The abovementioned valves were
opened in a two-electrode configuration by the application of
a potential to the electrode that constitutes the valve. If the
three-electrode system is used for the operation and an ion-
selective electrode is used for the reference electrode, valves
that open sensing the concentration of ions can be realized.
Yamaguchi et al. reported a pH-responsive valve and a pH filter
based on this principle.222

EWOD has also been used to form valves for controlling capillary
action. Chen et al. used electrowetting-based valves to control the
transport of solutions by capillary action (Fig. 6D).223 The valves
were formed by forming a SAM of perfluorodecanethiol on silver
patterns. On-chip detection of Salmonella was conducted using an
integrated three-electrode system by sandwich immunoassay.223

An electrolysis-based valve was also reported. Ezkerra et al.
proposed a bubble-based microvalve with a cantilever.224 The valve
was formed next to a flow channel and moved horizontally. When
bubbles are produced by the electrolysis of water, the valve was
closed. Leakage of gaseous pressure through a thin slit opened
the valve.
Toward automatic operation of microfluidic systems

Although the opening of the abovementioned valves was
controlled using a potentiostat, the potential of the valve
electrode can also be applied by coupling it with a redox
reaction in another control flow channel.225 Watanabe et al.
used a structure with the SAM valve mentioned above formed
in one flow channel and a zinc electrode formed in the other
flow channel (Fig. 6E).226 When the control flow channel is
empty, a solution introduced into the main flow channel stops
at the valve. The valve is opened when an electrolyte solution
introduced into the control flow channel wets the zinc
electrode. Following this, zinc is oxidized and the potential of
the electrode changes in the negative direction. This causes
the opening of the valve and the solution again stars to flow.
By forming such a pair of electrodes in appropriate locations of
flow channels, the valves can be opened in a predetermined
manner.

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pH regulation platinum auxiliary electrode that work as View an Article

actuator
Online
Welch et al reported a device with feedback control of DOI:
electrode to cause electrolysis of water.228 By10.1039/C9LC01112A
only connecting
solution pH.227 The device uses an extended-gate ISFET along the three-electrode system to an ordinary potentiostat, the pH
with a pseudo-reference electrode. The pH was regulated by of a solution can be maintained at a predetermined constant
modifying flows through two input channels using integrated value by a feedback function.
pneumatic microfluidic valves. The pH control can also be Formation of a gradient for the pH or other ions is necessary
implemented electrochemically using a non-standard three- depending on the application. For this purpose, a simple
electrode system consisting of a non-polarizable Ag/AgCl method can be the use of a pair of electrodes formed in a
working electrode, pH-sensitive reference electrode, and a small space or within a flow channel. Krabbenborg et al. used

Fig. 6 Electrochemical microfluidic devices. (A) Microfluidic device with two integrated micropumps connected to a flow channel structure with a T-junction.
Adapted with permission from ref. [211]. Copyright 2010, Springer. (B) Microfluidic device with micropumps that deliver solutions to the sensing unit with a
three-electrode configuration. Adapted with permission from ref. [215]. © 2013 IEEE. (C) Microfluidic device with integrated micropumps for ELISA. (i) Layout for
the electrodes for the micropumps. (ii) Picture of the device and the magnified view of the interdigitated carbon electrodes for a micropump. Adapted with
permission from ref. [216]. Copyright 2014, American Institute of Physics under the Creative Commons Attribution 4.0 International License”. (D) Flow control
using electrowetting-based valves. (i) Integrated microfluidic chip consisting of three valves and a three-electrode system. (ii – v) Valve opening and controlled
transport of dye solutions. Adapted with permission from ref. [223]. Copyright 2015, The royal society of chemistry. (E) Device to automatically open a valve
following the injection of a solution in a control flow channel. (i) A solution to be transported stops at the valve. (ii) When the solution in the control flow channel
wets the zinc electrode, zinc is oxidized causing reductive desorption of the hydrophobic SAM of the valve. As a result, the valve opens. (iii)–(viii) Drawing of a
letter following a determined stroke order using a device with the valve and zinc electrodes formed in appropriate locations. Adapted with permission from ref.
[226]. Copyright 2017, The royal society of chemistry. (F) Droplet-based microfluidic pH regulator. Copyright permission will be obtained in the final stage.
Adapted with permission from ref. [230]. Copyright 2014, The royal society of chemistry.

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interdigitated electrodes to form µm-scale gradients of pH or of magnitude compared with that of the case without View Articleusing
Online
Cu+ ions employing electrolysis of water or redox reactions on DOI: 10.1039/C9LC01112A
silver deposition. In this case, a liquid junction was used to
the pair of electrodes.229 Although pH at the electrodes cannot connect the two flow channels. The efficiency of silver
be controlled precisely with only the pair of electrodes, the deposition could also be further improved by connecting the
non-standard three-electrode system mentioned above can analyte solution and the solution for metal deposition using a
also be used for this purpose.228 metal junction instead of a liquid junction. By using a wire with
Zhou et al. proposed a technique to form droplets with redox-active ends, the potentials for the redox reactions for
specific pH values by microfluidics (Fig. 6F).230 The pH was the analyte and silver deposition could be shifted deliberately.
controlled by the electrolysis of water caused by the For hydrogen peroxide as a model analyte, the achieved
application of current pulses. Produced hydrogen and hydroxyl detection limit was 2.4 nM.235
ions were separated and confined in droplets formed by the
application of pressure pulses. By adjusting the applied current Signal amplification by redox cycling
and pressure pulses, droplets of various pH values could be When multiple microelectrodes are placed in close proximity,

formed on demand. chemical crosstalk by reaction products occurs. Although the
crosstalk has an adverse influence depending on the cases, it
Strategies to improve detection sensitivity and can advantageously be used to enhance the amperometric
detection limit signal of reversible redox pairs in a technique called redox
Application of microelectrodes cycling. To intentionally cause redox cycling, a pair of closely
To improve detection performance, microelectrodes and spaced individually addressable electrodes are used. The
nanoelectrodes are often used231 because of their potential of one electrode (generator) is set at a potential that
advantageous features. Around the micro/nanoelectrodes, is sufficient to oxidize (or reduce) the electroactive analyte and
mass transport is enhanced, which results in a high current the product diffuses to the other electrode (collector). The
density. The influence of ohmic drop can also be reduced potential of the collector is set at a potential that is sufficient
because of the small electrode size. Furthermore, with their to reduce (or oxidize) the product. The products from the
small capacitances, the signal-to-background ratio and the collector again diffuse back to the generator, which suppresses
response time can be improved. Current generated on a single the depletion of reactants near the electrodes and increases
micro/nanoelectrode is very small. Therefore, they are usually the current generated from both electrodes. Redox cycling is
deployed in the form of an array. also effective to minimize the influence of irreversible
A challenging issue for microfabricated electrochemical electroactive interferents such as oxygen and L-ascorbic acid
sensing devices is calibration. This is particularly true for because they only deplete around the generator electrode.
disposable devices designed for single use. In the case of Since the early days of this technique, the electrode
amperometric devices, the output current is influenced by the configuration mostly used for this purpose was the
surface state of the working electrode. Therefore, calibration is interdigitated electrodes.236 To enhance the response by
indispensable if precision of the measurement is required. improving the collection efficiency, the distance between the
Furthermore, without signal enhancement such as redox generator and collector electrodes should be as small as
cycling, amperometric detection becomes difficult as the possible. For this purpose, reducing the inter-electrode spacing
volume of the solution decreases, because of the rapid current of the interdigitated electrodes will be a direct method.237
decrease caused by the depletion of analytes in the vicinity of However, the use of electron beam lithography needs an
the working electrode. With regard to these factors, expensive facility and influences the productivity of the device.
coulometry is more beneficial. In coulometry, the output is the To avoid this problem, Partel et al. proposed a simpler method
charge, which is the integral of the current. Therefore, if the that involved forming nanogaps in the order of sub-100 nm
sample volume is very small and the analyte is exhaustively starting from structures of the micrometer scale.238 As SiO2 is
oxidized or reduced on the working electrode, calibration is deposited, gaps between the structures are narrowed and
not necessary.232 Contrary to amperometry, the output undercuts are formed (Fig. 7A). By only depositing gold there,
increases as time elapses, facilitating measurement. On the interdigitated electrodes with nanogaps are easily obtained
other hand, however, the background charge increases. To with the help of the undercut. With this technique, they
improve the sensitivity and detection limit of coulometry by reported an amplification factor of 116 and a collection factor
enhancing the collection efficiency of the analyte and at the of 99.5%.
same time reducing the background charge, Sassa et al. used a
microelectrode array.233 Even if the total electrode area is the
same, the signal to the background ratio can be improved
using microelectrode arrays with thinner electrodes. To
improve the detection limit further, Ikemoto et al. converted
an analyte into silver using microelectrodes formed in separate
flow channels and measured the amount of deposited silver.234
The detection limit could be improved by more than one order

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DOI: 10.1039/C9LC01112A

Fig. 7 Working electrode structures for redox cycling used in devices reported recently. (A) Formation and SEM image of nanogaps between interdigitated
electrodes. Adapted with permission from ref. [238]. Copyright 2015, American Chemical Society. (B) SEM image of interdigitated electrodes. Adapted with
permission from ref. [18]. Copyright 2014, The Electrochemical Society (C) Structure and principle of operation of the electrochemical device with a nanogap.
Immobilized tyrosinase transforms inactive monophenolic molecules into electrochemically active o-quinones. These molecules subsequently undergo redox
cycling. Adapted with permission from ref. [241]. Copyright 2014 American Chemical Society. (D) Nanoring–disc electrode array. Adapted with permission from
ref. [26]. Copyright 2014, American Chemical Society. (E) Plane-recessed microdisk electrode array. Adapted with permission from ref. [247]. Copyright 2014,
American Chemical Society. (F) Inkjet-printed 3D electrode structure with a nanoporous layer between electrodes. Adapted with permission from ref. [250].
Copyright 2017, The royal society of chemistry.
Another method to improve the performance of the deep nanochannel and detected dopamine in the presence of a
interdigitated electrodes is to make them thick and three- 100-fold excess of ascorbic acid.244 The existence of the
dimensional and use both the top surface and sidewalls of the nanochannel improved the selectivity of detection. They also
electrodes. Interdigitated carbon electrodes formed by the demonstrated the ability to differentiate reversible species with
pyrolysis of SU-8 or polyimide were fabricated for this purpose overlapping cyclic voltammograms by adjusting the ring potential.
(Fig.7B).13, 18 An interesting behavior of the recessed ring-disk nanoelectrode
An effective method to alleviate this problem and achieve array is that additional factors can contribute to signal
appreciable performance is to use a nanogap formed between enhancement. The same group reported large current
electrodes in the direction vertical to the substrate. The enhancements in the absence of a supporting electrolyte using a
nanogap can be formed by stacking layers consisting of recessed ring-disk nanoelectrode array by redox cycling in
electrodes, insulators, and a sacrificial layer, and dissolving the combination with ion enrichment and an unshielded ion
sacrificial layer between the electrodes after the stacked migration.27, 245 A current amplification as large as 2000-fold was
structure is completed. In this configuration, the dimensions of achieved. As a structure similar to the ring–disk and ring–ring
the electrodes themselves can be in the order of µm. electrodes, Kanno et al. also used a structure with a nanogap
Therefore, the structure can be made easily by between electrodes formed at the periphery of a hole by the
photolithography. Devices based on this structure have been sacrificial layer etching.246
reported since the middle of 2000s.239, 240 As an application of To cause redox cycling, potentials are usually applied to the
this technique to biosensing, Rassaei et al. used a nanogap generator and collector electrodes. However, by taking advantage
with electrodes and an enzyme.241 Efficient redox cycling of the feature of bipolar electrodes, redox cycling can also be
within the gap enabled detection of molecules of the order of generated by applying a potential to only one of the pair of
zeptomoles produced in the enzymatic reaction (Fig. 7C). electrodes. Zhu et al. proposed the use of self-induced redox cycling
As seen in the devices that employ the nanogap, the vertically on plane-recessed microelectrodes (Fig. 7E).247 In their devices, an
stacked structure of electrodes and insulators facilitates the array of disc electrodes were formed at the bottom of recesses and
formation of a pair of electrodes with an inter-electrode distance in the other electrode was formed at the top plane of an insulating
the order of nm. As a simpler structure, the electrode structure is layer. When a reductive reaction occurs on the disk electrodes at
completed by simply introducing holes into the stacked layers and the bottom, a reverse oxidation reaction is induced at the edge of
exposing the electrodes. Ring–disk electrode arrays (Fig. 7D) 26 and the upper plane at the entrance of the recesses. The oxidation
ring–ring electrode arrays 242, 243 have also been formed. Although current is compensated at other locations by a cathodic reaction
arrays of similar stacked electrode structures of larger dimensions such as the reduction of oxygen. The recorded currents were almost
have already been reported much earlier, a recent trend has been the same as the case when an oxidative potential was intentionally
to form the electrode structure in an array of nanoholes. Ma et al. applied to the top plane. Similarly, Ma et al. formed disk electrodes
used arrays of recessed ring−disk electrodes formed in a 300-nm- at the bottom of an array of nanopores and a planar macroscopic

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top plate separated by a depth of 100 nm.248 The reaction on the Among devices directed for this purpose, many deal Online
View Article with
top electrode in the nanopores was coupled with the other redox DOI: 10.1039/C9LC01112A
neurotransmitters as target analytes. Real-time detection of
reaction at the distal end of the bipolar electrode that accompanies neurotransmitter release provides a direct insight into the
changes in fluorescence or electrochemiluminescence. Thus, redox mechanisms of synaptic communication. Chuang et al. fabricated a
cycling was induced between the recessed disk and the top device with microelectrodes and interdigitated generator/collector
electrode with approximately 30× current amplification. electrodes for the simultaneous detection of action potential and
Although the above-mentioned devices were essentially dopamine release from PC12 cells.259 Picollo et al. used an array of 4
fabricated by the thin-film process, the necessity of an expensive × 4 electrodes for amperometric and voltammetric detection of
facility a facility may pose a problem. To solve this problem, beads exocytosis from chromaffin cells and adrenal slices.260 Wang et al.
can be used to form a gap between the generator and collector formed an array of platinum microelectrodes in a PDMS chamber to
electrodes.249 Adly et al. used inkjet printing to form the layers of detect dopamine released from PC12 cells (Fig. 8A).261 The cells
electrodes the beads (Fig. 7F).250 Needless to say, the existence of were cultured in the PDMS chamber coated with collagen. The
the beads between the electrodes hinders the mass transport of same group fabricated arrays of individually addressable

redox species and influences the efficiency of redox cycling. Further, microelectrodes coated with collagen formed in an SU-8 microwell
the distance between the electrodes and the production yield are in for the detection of neurotransmitter release from a single PC12
the relation of trade-off. Nevertheless, this technique will be useful cell.262 Imaging of the spatial distribution of exocytosis was also
for realizing the redox cycling function in disposable devices. demonstrated. Yakushenko et al. fabricated a device for parallel
real-time recording of vesicular neurotransmitter release using 64
Application of nanotechnology microelectrodes with a minimum electrode diameter of 3 μm (Fig.

Detection sensitivity of these devices can be improved further by 8B).263
taking full advantage of nanomaterials such as graphene, carbon Kisler et al. fabricated transparent microelectrode arrays for
nanotubes, nanowires, and nanoparticles. A variety of techniques simultaneous amperometric measurements and fluorescence
have been developed for the detection of biomolecules such as imaging (Fig. 8C).264 Fluorescence images of chromaffin cells were
proteins and DNAs to achieve remarkable detection sensitivities. taken by total internal reflection fluorescence microscopy while the
With regard to this, numerous papers and many reviews have been release of catecholamine from single vesicles was measured by
published and details are omitted here.251-257 Although the amperometry. Carabellia et al. demonstrated the simultaneous
excellence of the techniques was mainly demonstrated based on detection of exocytotic events of chromaffin cells upon stimulation
devices of conventional sizes, they could also be incorporated in with KCl using an array of microelectrodes of boron-doped
microfabricated devices. However, a problem is that the materials nanocrystalline diamond.265 Wigström et al. fabricated a
and detection chemistry involved tend to become more microelectrode array consisting of 16 1.2-μm-wide platinum band
complicated, making their application to microfabricated devices electrodes (Fig. 8D).266 Catecholamine released from bovine
more challenging. chromaffin cells by single exocytotic events was detected
amperometrically. As for the detection of exocytosis from cells
Multiplexing of electrochemical sensors using microelectrode arrays (MEAs), a review was published in
Simultaneous multiplexed detection of analytes is required in 2015.267 Readers should refer to this for more information about
applications such as screening, detection of dynamic events of devices other than those introduced here.
As a practically important application, Besant et al. used sensor
neurotransmitters, and detection of proteins or DNAs.258 To realize
high-throughput multiplexed detection or real-time in situ arrays for the measurement of antibiotic susceptibility of bacteria
monitoring of high temporal and special resolutions, arraying of (Fig. 8E).268 They used miniaturized wells to capture, concentrate,
sensors is a natural consequence. Detection using such arrays is and analyze bacteria. The metabolic activity of the bacteria was
often carried out by fluorometry due to its high sensitivity and measured electrochemically using a redox-active molecule.
easiness of arraying of sensors. For this technique, the realization of
large-scale sensor arrays is relatively easy as evidenced by DNA Small arrays of sensors for proteins and DNAs
microarrays. On the other hand, fluorometric sensor arrays also
have shortcomings such as the necessity of expensive and bulky
optical instruments, photobleaching of dyes, cross-talk between
dyes, the influence of background fluorescence, the difficulty of
measurement in turbid solutions, and the necessity of labelling for
non-fluorescent analytes. Therefore, depending on the application
and the size of the array, electrochemical sensor arrays can be a
better alternative. It is clear there are challenges in designing and
using such microarrays. The biggest problem is the operation of
multiplexed sensors and signal acquisition, particularly in large-
scale arrays. In the following section, trials to realize arrays of
sensors of relatively small numbers will be introduced.
Sensor arrays for the monitoring of cell activity

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DOI: 10.1039/C9LC01112A

Fig. 8 Integrated electrochemical devices for analysis of cell activity. (A) Array of 36 addressable microelectrodes for spatial measurements of single vesicle
release. Adapted with permission from ref. [261]. Copyright 2013, American Chemical Society. (B) Device for parallel detection of neurotransmitter release. (i)
Array of 64 microelectrodes. (ii) Simultaneous amperometric recordings of spontaneous exocytotic events obtained using 10 microelectrodes. Adapted with
permission from ref. [263]. Copyright 2013, American Chemical Society. (C) Transparent ITO electrode for simultaneous amperometric detection of exocytosis and
fluorescence microscopy. (i) ITO (top) and gold (bottom) electrodes with four (left) and single (right) electrode geometries. (ii) Correlation of results obtained by
amperometry and total internal reflection fluorescence imaging for a vesicle release event from a chromaffin cell. Adapted with permission from ref. [264].
Copyright 2012, Scientific Research Publishing Inc. under the Creative Commons Attribution 4.0 International License. (D) Device with 16 microelectrodes for
detection of exocytosis detection. (i) Side view of sensing region, where the probe is in touch with a cell. (ii-iii) SEM and microscopy images of the microelectrode
array. Adapted with permission from ref. [266]. Copyright 2016, American Chemical Society. (E) Array of microwells with electrodes for phenotypic profiling of
antibiotic-resistant bacteria. Adapted with permission from ref. [268]. Copyright 2011, The royal society of chemistry. (F) Impedimetric immunosensor array for
the detection of tumor marker proteins. Adapted with permission from ref. [269]. Copyright 2011, The royal society of chemistry. (G) Array of 96 sensing sites
formed on a printed circuit board for screening of an enzyme. Adapted with permission from ref. [270]. Copyright 2013, American Chemical Society. (H) Device
for digital protein detection with an array of wells for stripping analysis of enzyme-induced metallization. Adapted with permission from ref. [271]. Copyright
2016, American Chemical Society.
For cases such as diagnosis of cancer, speciation of infectious reported a device with nine chambers each containing a circular
diseases, and profiling of antibiotic-resistance, detection of multiple gold working electrode surrounded by a platinum auxiliary
marker proteins is required. Arrays of electrochemical sensors can electrode (Fig. 8F).269 Ag/AgCl reference electrodes were inserted
be constructed for this purpose. Unlike DNA chips, a challenge is through a sealing lid. A tumor marker, tenascin C, was detected
the preparation of probe molecules. Therefore, the number of impedimetrically following the formation of protein-antibody
sensors integrated in such devices is limited for the detection of a complexes, peroxidase-catalyzed production of the precipitate, and
single or a small number of target proteins, although the possibility redox reactions of ferri-/ferrocyanide. Abdellaoui et al. fabricated a
of the integration of many sensors was demonstrated. Steude et al. device with 96 wells (Fig. 8G).270 A pair of a carbon working

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electrode and a Ag/AgCl electrode as the auxiliary/reference working electrodes in the order of tens or hundreds,
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Online
electrodes were formed in each well by screen-printing. Screening effective techniques have been proposed.DOI: 10.1039/C9LC01112A
of laccase from the fungus Trametes versicolor was conducted. The
device was also used to identify Laccase inhibitors for the enzyme, Use of matrix configuration for electrodes and flow channels
pH optima, and interfering molecules.270 Wu et al. formed a To mitigate the above-mentioned difficulty, layouts of electrodes,
microelectrode array to detect alkaline phosphatase (ALP) secreted flow channels, and reaction chambers in the form of a matrix have
from liver cancer cells by digital analysis (Fig. 8H).271 The product of been proposed. A group of Tohoku University proposed an
the enzymatic reaction of ALP reduced Ag+ to silver. Stripping electrode layout consisting of an n × n array of a pair of working
analysis was conducted for the deposited silver.271 Luo et al. used a electrodes for redox cycling (Fig. 9A). Electrodes in each row and
sensor array with cross-linked poly(ethylene glycol) for column are connected. When a row and a column are selected and
impedimetric detection of insulin and CRP.272 PEG reduced potentials are applied to the electrodes, only the sensing site
unwanted interaction with proteins effectively. corresponding to the selected row and column is activated.
Kling et al. used a unique mechanism to detect up to eight Therefore, only 2n bonding pads are sufficient for connection to an

antibiotics.273 The detection was based on a natural sensing system external instrument to activate n2 sensing sites. The group
including an operator DNA and a repressor protein, which evolved demonstrated operation of up to 1024 (32 × 32) addressable
in certain bacteria to resist antibiotics. The operator DNAs were sensing sites.281 They used this technique in devices for evaluation
immobilized on the surface of a flow channel and the repressor of embryoid bodies through their alkaline phosphatase activity,246,
proteins were bound to the DNA. In the absence of antibiotics, the 282 detection of alkaline phosphatase secreted by HeLa cells,283 DNA
repressor proteins are tightly bound to the operator DNA. However, assay.30 Kätelhön et al. also used two sets of 16 parallel band
when antibiotics are present, the repressor proteins detach from electrodes formed perpendicularly to each other to cause redox
the operator. Changes in the reaction of enzymes conjugated to the cycling and detect current in a nanocavity with a height of 65 nm.284
repressor proteins were detected by amperometry. Lam et al. used bands of reference and auxiliary electrodes
In realizing multiplexed detection of proteins in real samples, formed along flow channels and bands of working electrodes
detection limits must also be sufficiently low. In many cases, formed orthogonally to the flow channels.285 In the presence of
additional amplification is needed for this purpose. Rusling et solutions, the electrical circuit was complete with selected working,
al. proposed methods to realize very low detection limits. In reference, and auxiliary electrodes. The device was used for the
one method, they used detection antibodies with polymerized detection of pathogenic bacteria and antibiotic-resistance markers.
horseradish peroxidase (HRP) labels for the detection of Zhang et al. further used a technique to separate solutions. They
interleukin-6 (IL-6).274 Otherwise, they used magnetic beads formed a set of gold band pseudo-reference and auxiliary
massively loaded with enzyme labels and secondary antibodies electrodes and another set of gold band working electrodes formed
for protein sensor arrays and have reported very low detection orthogonally to make 400 (20 × 20) sensing sites (Fig. 9B).286 Each
limits.275-277 They further used this technique in a device with site covered by the three-electrode system was covered with a
an array of 32 microelectrodes, which was further multiplexed porous wettable square each separated by hydrophobic barriers.
with an 8-port manifold to allow 256 measurements.278
As for large arrays of DNA sensors, the CMOS technology Application of bipolar electrochemistry
mentioned later will be indispensable. Depending on the Bipolar electrochemistry provides another solution for a
application, small arrays are also used. Pursey et al. fabricated a high number of sensors being required.287-291 In orthodox
multi-sensor chip that detects three specific bladder cancer DNA devices, small rectangular electrodes (bipolar electrodes: BPE)
markers simultaneously.279 Their DNA probe had a hairpin structure are placed in a solution between two driving electrodes (open
with a porphyrin marker at the end, which became BPE). In bipolar devices, the electrodes are operated wirelessly
electrochemically active accompanying hybridization with the and simultaneously without leads for connection to external
target strand through a structural change. Slinker et.al. fabricated instruments. (Fig. 9C) shows the principle of detection for the
an array of 16 DNA-modified electrodes for multiplexed open bipolar system. When a voltage is applied to the driving
detection of DNAs and DNA binding proteins.280 Detection using electrodes, a potential drop is generated in the solution
the device relied on DNA mediated charge transfer. between the driving electrodes. For a sufficiently low ionic
strength, a uniform potential gradient is expected. As a result,
Decreasing number of electrical connections in an interfacial potential difference develops between the
multiplexed sensing devices electrode and the solution, and the potential difference
In applications such as the high-throughput speciation of depends on the location on the electrode and is the largest at
infectious diseases, antibiotic-resistance profiling, and both poles. If the potential difference is sufficiently large at the
screening of cell libraries, large arrays of individually cathodic and anodic poles, reduction and oxidation occur,
addressable sensing components are required. Compared with respectively, in the presence of redox compounds. Because the
optical methods, this is really a challenge with regard to BPEs are electrically isolated from external instruments,
operation and data acquisition, because electrodes may need outputs cannot be obtained in the form of a current. Therefore,
to be connected to external instruments through a large optical methods are alternatively used to obtain the
number of contact pads. For devices with the number of information on redox reactions, and
electrochemiluminescence (ECL), particularly based on the

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Ru(bpy)32+/tripropylamine (TPA) system, is often used. An principle of detection is essentially different. In closed bipolar
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advantage of ECL is that it does not need external light systems, the sum of all the interfacial DOI: 10.1039/C9LC01112A
potential differences
sources. Therefore, the background can be minimized and between the electrodes and the solutions and ohmic drop in
detection can be highly sensitive. Although the Ru(bpy)32+/TPA the solutions is equal to the driving voltage. Therefore, closed
system is predominantly used in bipolar devices, coupling the bipolar devices can be operated with a relatively small applied
anodic or cathodic reactions to an enzymatic reaction needs voltage of approximately 1 V.
consideration. For this purpose, the luminol/H2O2 ECL system The closed BPEs can effectively be used for imaging.
may alternatively be used to couple the reaction with Guerrette et al. used a large array of BPE and separated the
enzymatic reactions that produce H2O2.292 A great advantage analyte solution and the other resazurin solution for imaging
of the open bipolar system is that the structure of each BPE (Fig. 9E).296 The analyte on the anode side was detected by
can be very simple and a large array of BPEs can easily be fluorescence emitted from the reaction product on the
realized. Chow et al. demonstrated the simultaneous cathodic pole. Oja et al. demonstrated two- and three-
operation of 1000 BPEs. 293 (Fig. 9D) dimensional imaging of concentration profiles in diffusion

In open bipolar ECL devices, the background signal from the layers of carbon fiber microelectrodes over an array of BPEs.297
driving electrode poses a problem. In addition, the reactants 3-dimensional imaging was realized by collecting 2-
for the anodic and cathodic reactions must be separated dimensional images at different axial positions. Although these
depending on the situation. To solve these problems, devices are not necessarily fabricated by photolithography or
compartments for the anodic and cathodic reactions have other related microfabrication techniques, they demonstrate a
been separated and solutions have been connected with a promising application of this technique. This technique was
BPE.294, 295 The configuration in which BPEs are in contact with also used for multiplexed detection. Wu et al. formed an array
solutions of different compartments is called a closed bipolar of reactions and sensing channels connected by BPEs for the
system. The open and closed bipolar systems may be multiplex detection of cancer biomarkers (Fig. 9F).298 Aptamers
considered as simple structural variations. However, their or antibodies and an electroactive tag synthesized by doping a
Fig. 9 Techniques to reduce number of electrical connections. (A) Crossbar electrode array in the form of a matrix. Adapted with permission from ref. [281].
Copyright 2011, The royal society of chemistry. (B) Array of individually addressable electrochemical cells with crossbar electrodes. Adapted with permission from
ref. [286]. Copyright 2017, American Chemical Society. (C) Principle of detection of the open bipolar system. (i) Reactions that occur at the anodic and cathodic
poles. (ii) Variation of the potential in the solution and polarizations at the anodic and cathodic poles. Adapted with permission from ref. [289]. Copyright 2013,
Wiley-VCH Verlag GmbH & Co. KGaA. (D) Open bipolar device with a large array of BPEs (i) and ECL observed from the BPEs (ii). Adapted with permission from ref.
[293]. Copyright 2009, American Chemical Society. (E) 2D array of closed bipolar electrodes for fluorescence imaging. Adapted with permission from ref. [296].
Copyright 2013, American Chemical Society. (F) Array of closed bipolar electrodes for the detection of cancer biomarkers. Adapted with permission from ref.
[298]. Copyright 2015, American Chemical Society. (G) Light-addressable potentiometric sensor with a light source coupled with an X-Y stage. Reprinted with
permission from ref. [311]. Copyright 2012, Elsevier.

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large number of thionine molecules in silica nanoparticles can be trade-offs. Furthermore, a light source,Viewa Article focusing
Online
were used for the cathodes.298 Wang et al. reported a DOI: 10.1039/C9LC01112A
optics, and a scanning mechanism are additionally required.
multicolor ECL device based on closed BPE employing the To solve the problem related to signal transduction, some
potential dependence of the emission color of alternative techniques have been used. One such technique
electrochemiluminophores.299 Multicolor (green−yellow−red) used a fixed array of light-emitting diodes (LEDs) or lasers to
ECL was realized using a mixture of [Ru(bpy)3]2+ and [Ir(ppy)3] illuminate multiple regions on the sensing surface.306, 307 The
with TPA as the co-reactant. The device was used for the photocurrent generated at each spot is modulated with the
quantification of prostate-specific antigen (PSA). A PSA frequency of the corresponding LED. The total measured
concentration-dependent silver bridge was made in the gap of photocurrent is a superposition of the local photocurrents. The
the BPEs to modulate the conductivity. The color change photocurrent derived from a single spot can be obtained by a
depended on the concentration of PSA. fast Fourier transform.306 An organic light-emitting diode panel
Open bipolar systems are promising to realize large-scale sensor was also used for high-resolution imaging. Miyamoto et al.
arrays for analytes such as DNAs.300 As a trend, reported devices used this device to demonstrate pH imaging with 135 frames

have been shifting to closed bipolar systems. This may be per second (fps).308 Alternatively, a bundle of optical fibers was
reasonable considering that they can solve the problems of the used to achieve guided light spots flexibly. Miyamoto et al.
open bipolar systems. On the other hand, because of the additional achieved two-dimensional pH imaging in a flow channel at a
structure that separates solutions, the operation of a large array of maximum rate of 100 fps.309 The combination of an optical
BPE that can easily be achieved using the open bipolar systems 293 chopper and microlens array can also realize multi-spot
becomes difficult. A drawback of the BPE devices is that they must interrogation using a single beam.310 The digital micromirror
rely on reporting techniques such as ECL. In this respect, for a device (DMD) used in video projectors is also effective in
limited number of integrated BPEs, the superiority over the increasing the number of light spots. It consists of a large array
conventional three-electrode systems that measure current directly of micromirrors that can be flipped individually, which enables
is not convincing. the rapid production of a large number of light patterns.
Nevertheless, the technique used in closed bipolar systems Wagner et al. employed this technique to readout (Fig. 9G).311
provides a solution for a difficult problem in miniaturizing The LAPS is effective for monitoring the metabolic activities of
electrochemical systems, which often require liquid junctions. living cells. Wagner et al. recorded the metabolic activity of
However, microfabricating good liquid junctions without an ohmic macrophages at 16 measurement spots for the concentration
drop is really challenging. With regard to this, closed bipolar gradients of a drug.312 Shaibani et al. conducted a real-time
systems demonstrate that solutions can also be connected using measurement of cancer cell metabolism and their response to
metal junctions.301, 302 The technique will facilitate designing more anticancer drugs by detecting local changes in the pH as cancer cells
sophisticated electrochemical microsystems. released lactate.313 The same group used pH-sensitive hydrogel
nanofibers for the sensitive coating of LAPS to detect pH changes
Light-addressable electrochemical devices caused as a result of sugar fermentation by E. coli.314 Su et al.
The use of light also solves the problem of selective signal fabricated a device with the LAPS and an impedance sensor for
acquisition. Light-addressable electrochemical devices are simultaneous monitoring of extracellular acidification and growth of
operated based on this method. One of the critical advantages HeLa cells.9 Hu et al. used LAPS coupled with a microfluidic system
of the devices is that only a thin sensing plate is sufficient and to monitor cellular metabolism and drug effect.315 Human breast
no fine patterns as seen in microelectrode arrays are cancer cells MCF-7 were used and real-time monitoring of
necessary. The light-addressable potentiometric sensor (LAPS) extracellular acidification was conducted rapidly and sensitively
is one such device.303-305 The sensing part of the LAPS consists under physiological conditions. Although the light beam was not
of an electrolyte–insulator–semiconductor structure. A change necessarily scanned and only the pH-sensing function was used in
in the potential difference at the electrolyte–insulator these cases, 2D detection was also conducted. Takenaga et al.
interface changes the thickness of the depletion layer in the observed the metabolic activity of Chinese hamster ovary and
semiconductor. When a location is illuminated in this state, tumor cells.307 They used a 4×4 array of infrared LEDs as the light
electron–hole pairs are generated and separated in the source. The pH changes accompanying the addition of glucose and
depletion layer, which then generates photocurrents. The changes in metabolic activity were measured. Wu et al. used LAPS
illuminated locations become independent sensing sites. for the 4×4-spot label-free detection of DNA using a 4×4 array of
Therefore, an array of sensors can be realized with only a infrared LEDs.306 Although the LAPS was developed to detect the pH
simple structure. The LAPS can also be used for chemical in the initial stage, it can be sensitive to other ions and molecules
imaging. As mentioned later, such image sensors can also be such as DNA.304 Du et al. used bioengineered olfactory sensory
realized by the CMOS technology in the form of a large array of neurons to make the LAPS respond to odorant molecules.308
ISFETs integrated with other electronic components. However, The LAPS has an electrolyte–insulator–semiconductor structure
the number and locations of pixels are unchangeable, which and detects changes in the potential difference at the electrolyte–
may cause inconvenience in cases where the location of the insulator interface. In other words, detection of the progress of
molecules or objects is unpredictable. The problem can be redox reactions is not possible due to the presence of an insulator
alleviated in the case of the LAPS. On the other hand, however, layer. However, by removing it and using an appropriate
semiconductor surface, amperometric detection is also possible.316
a large sensing area and the length of time for measurement

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Choudhury et al. used this technique and demonstrated multi-spot The ISFETs directly detected pH changes caused by the release
View Article of
Online
detection of redox reactions on a planar hydrogen-terminated protons during nucleic acid polymerization.DOI:
The10.1039/C9LC01112A
amplification and
silicon electrode modified with well-defined organic monolayers.317 measurement started with the binding of the complementary
An n-type semiconductor can be used for oxidation, whereas a p- primer to its target sequence, and no changes were observed for a
type semiconductor can be used for reduction. They also
mismatch primer.
demonstrated the applicability of their device to the detection of
Devices directed to chemical imaging have also been
DNA hybridization. Seo et al. used hematite (α-Fe2O3) for the anode
for the selective detection of oxidizable biomolecules.318 They developed. Hayasaka et al. fabricated an array of 20 × 20
demonstrated quantitative analysis of dopamine. boron-doped diamond microelectrodes for the real-time
imaging of histamine (Fig 10E).327 The electrodes were formed
Higher-level integration by CMOS technology on a separate substrate and transferred to another CMOS LSI
wafer. Rothe et al. integrated an array of 32 × 32 platinum
In the previous section, techniques for multiplexed
black working electrodes, on-chip reference and auxiliary
detection were introduced. However, as the number of
electrodes, and potentiostats (Fig. 10F).328 The device could

sensors increases to the order of hundreds, thousands, or
detect faradaic currents that were more than 10,000 times
more, the application of these techniques will be difficult. This
smaller than charging currents without a Faraday cage.328
naturally leads to the integration of necessary components
Electrochemical images could be acquired at up to 90 fps.
including sensors and circuits for operation and signal
Bellin et.al. fabricated a device with an array of 1,824
processing by the CMOS processes. By amplifying and digitizing
electrodes multiplexed to 38 parallel output channels for the
signals on the same chip, noise interference originating from

imaging of phenazine metabolites produced from biofilms of
lengthy signal paths can also be avoided, enabling
Pseudomonas aeruginosa.329 They demonstrated potential-
measurements without additional shielding such as that by a
sweep-based electrochemical imaging. Nemeth et al.
Faraday cage. Although CMOS devices are the most
fabricated a high-speed ion-imaging device with 64 x 64 pixels
sophisticated among devices treated in this review, trials to
consisting of an array of ISFETs.330 The device can display the
integrate components other than sensors can be found even in
distribution of protons at approximately 100 fps. The same
the 1980s and some reviews have been published.74, 319, 320
group also reported another imaging device with 16 x 16 pixels
During the last decade, the level of integration of CMOS-based
that operates at 333 fps.331 Sawada et al. reported various
electrochemical sensing devices advanced further and very
charge-coupled device (CCD)-type chemical imaging devices
sophisticated devices that may be called systems-on-a-chip
with an array of pH-ISFETs including one with 128 x 128
have been reported.
pixels.332 (Fig. 10G). They also reported applications in imaging
As examples of multiplexed detection, Kim et al. fabricated a
of the distribution of the concentration of potassium ions or
sensor array consisting of 100 platinum electrodes for the
acetylcholine.
simultaneous amperometric detection of neurotransmitter release
Unlike physical sensors, chemical sensors often contain
from chromaffin cells (Fig. 10A).321 The electrode array was
unstable components such as biomolecules and the Ag/AgCl
operated using integrated potentiostats with time-division
reference electrode. Among them, a troublesome component
multiplexing to limit the number of outputs. Inoue et al. fabricated
to be considered is the reference electrode. As mentioned
an array of 400 measurement points for electrochemical imaging
previously, Ag/AgCl electrodes formed by the thin-film process
and multi-point biosensing (Fig. 10B).322 The device had a light-
are very easily damaged. Therefore, it would imprudent to try
shield structure to reduce noise generated by a photocurrent and a
and integrate them onto the chip. For amperometric devices, a
mode-selection function. Jafari et al. developed a 54-channel device
realistic solution will be to use a pseudo-reference electrode
that conducts fast-scan cyclic voltammetry and calibration for DNA
fabricated from noble metals. For potentiometric devices, this
analysis.323 The device contained 600 time-multiplexed DNA sensors
does not work. In this case, the reference electrode should be
and 54 pH sensors along with an arbitrary waveform generator, a
separated as an independent module. In this case, the size of
memory, a current conveyer, an analog-to-digital converter (ADC),
the module can be comparable with the size of the sensing
and a wireless transmitter. The function of the device was validated
chip and the use of screen-printed thick-film electrodes will
by detecting prostate cancer DNA and the achieved detection limit
alternatively be used.
was 10 aM. Ganguli et al. fabricated an array of 1024 × 1024 ISFETs
with immobilized peptide nucleic acid (PNA) probes for label-free
sensing of nucleic acids (Fig. 10C).324 They detected miRNA Let-7b of
concentrations as low as 1 nM. Rothberg et al. fabricated an array
of 1.2 million ISFET pH sensors for DNA sequencing (Fig. 10D).325
The sequence was determined by sensing ions produced by
template-directed DNA polymerase synthesis. Sequencing of
bacterial genomes was demonstrated using this device. Toumazou
et al. demonstrated label-free real-time amplification and detection
by polymerase chain reaction (PCR) and loop-mediated isothermal
amplification using a device containing 40 ISFETs, 10 temperature
sensors, resistive heaters, signal processing and control circuitry.326

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DOI: 10.1039/C9LC01112A

Fig. 10 Highly integrated electrochemical sensing devices fabricated by CMOS process. (A) 10 × 10 array of working electrodes with in-pixel CMOS potentiostats.
for detection of exocytosis of chromaffin cells. (i) Photograph of the chip. (ii) Magnified views of an element obtained at different focal planes. Reprinted with
permission from ref. [321]. Copyright 2013, Elsevier. (B) Amperometric bioimaging chip. (i) Array of 400 individually addressable electrodes for imaging of
distribution of O2 and H2O2. (ii) Color maps of distribution of current before and after injection of H2O2 and Na2SO3 solution. Adapted with permission from ref.
[322]. Copyright 2015, The royal society of chemistry. (C) 1024 x 1024 ISFET array for label-free microRNA detection. Adapted with permission from ref. [324].
Copyright 2018, Springer. (D) DNA sequencing chip with 1.2 million wells. (i) Structure of a well and principle of sequencing. (ii) An 8” wafer with ~200 chips. (iii)
Picture of a chip. Adapted with permission from ref. [325]. Copyright 2011, Springer Nature Limited (E) Array of boron-doped diamond microelectrodes for
amperometric sensing. Adapted with permission from ref. [327]. ©2009 IEEE (F) High-speed chemical imaging sensor array with 1024 Pt or Pt black working
electrodes. (i) Platinum electrodes with different diameters. (ii–iv) SEM images of Pt and Pt black electrodes. Adapted with permission from ref. [328]. Copyright
2014, American Chemical Society. (G) Images of pH recorded by the device and corresponding optical image when a buffer solution was additionally dropped
onto the sensitive area of the device. (i) Initial state (pH 9.01). (ii) After a solution of pH 4.01 was dropped. (iii) Diffusion of the solution. Adapted with permission
from ref. [332]. Copyright 2014, The IEICE (H) Implantable wireless glucose sensor chip. Reprinted with permission from ref. [333]. Copyright 2019, Elsevier. (I)
Wireless sensing chip for alcohol monitoring injectable using a syringe. Adapted with permission from ref. [334]. ©2009 IEEE (J) CMOS chip for electrochemical
detection embedded in a microfluidic device. Adapted with permission from ref. [335]. Copyright 2013, The royal society of chemistry.
An issue with the highly integrated CMOS devices will be the

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fabrication cost. When a very large number of sensors need to will include devices for environmental monitoring,View implantable
Article Online
be used, the integration of sensors and other circuits is a devices, and devices directed to basicDOI: 10.1039/C9LC01112A
research that use
natural consequence. On the other hand, in applications where animals. Furthermore, the use of smartphones as a platform
the cost for analysis must be low, they will not be competitive for the collection of data is increasing,197, 336-339 which suggests
enough compared with the opposite extreme such as that applicable fields for electrochemical sensing devices will
disposable paper-based devices. While investigating previous expand further.
CMOS-based devices, we found devices with only a few
sensors integrated with operational circuits such as those for
potentiostats. Although these may suggest a step toward more Acknowledgments
sophisticated devices, we wonder if they can be replaced with We thank Dr. Yoshitaka Yoshizumi and Dr. Isa Anshori for their
simpler devices such as the paper-based devices operated support in completing this review. This work was partially
using external instruments. Even in such cases, cost reduction supported by JSPS KAKENHI Grant Number 19H05680.
could be achieved by decreasing the chip size and increasing

the number of chips obtained from a wafer. Mujeeb et al.
developed a CMOS-based implantable chip with a size of only References
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