Biosensors Integration in Blood−Brain Barrier-on-a-Chip: Emerging
Platform for Monitoring Neurodegenerative Diseases
Moǹica Mir,* Sujey Palma-Florez, Anna Lagunas, Maria José López-Martínez, and Josep Samitier
Cite This: ACS Sens. 2022, 7, 1237−1247 Read Online
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ABSTRACT: Over the most recent decades, the development of
new biological platforms to study disease progression and drug
efficacy has been of great interest due to the high increase in the
rate of neurodegenerative diseases (NDDs). Therefore, blood−
brain barrier (BBB) as an organ-on-a-chip (OoC) platform to
mimic brain-barrier performance could offer a deeper under-
standing of NDDs as well as a very valuable tool for drug
permeability testing for new treatments. A very attractive
improvement of BBB-oC technology is the integration of detection
systems to provide continuous monitoring of biomarkers in real
time and a fully automated analysis of drug permeably, rendering
more efficient platforms for commercialization. In this Perspective,
an overview of the main BBB-oC configurations is introduced and a critical vision of the BBB-oC platforms integrating electronic
read out systems is detailed, indicating the strengths and weaknesses of current devices, proposing the great potential for biosensors
integration in BBB-oC. In this direction, we name potential biomarkers to monitor the evolution of NDDs related to the BBB and/or
drug cytotoxicity using biosensor technology in BBB-oC.
KEYWORDS: organ-on-a-chip (OoC), biosensors, blood−brain barrier (BBB),
transepithelial/transendothelial electrical resistance (TEER), neurodegenerative diseases (NDDs)
In the most recent decades, age-dependent diseases such asneurodegenerative diseases (NDDs) have become more
prevalent, partly because life expectancy has increased.
Unfortunately, the efficacy of pharmacological treatment of
these NDDs has very high preclinical and clinical failure rates
with the worst outcomes observed in Alzheimer’s disease
(AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis
(ALS), and neuromuscular disorders. In the case of AD, it is
estimated that developing a disease-modifying treatment could
take about 13 years and cost more than $5.5 billion. But if even
a regulated success is achieved, the prevalence of the disease
could be halved in just 5 years by the year 2050.1,2 Animal
studies remain the gold standard for preclinical validation of
drugs in pharmaceutical development. However, their results
for predicting success in NDDs clinical assays have been
disappointing as the accuracy and reproducibility of the results
obtained are impaired due to species differences between
animal and human systems. Moreover, the use of animal
models is expensive, time-consuming, and subjected to ethical
constraints. Therefore, efforts are focused on the development
of in vitro platforms that reproduce both the physiological and
pathological scenarios. Organs-on-a-chip (OoC) are micro-
engineered biomimetic systems able to recapitulate key
functions of living organs. They are microfluidic platforms
created with manufacturing methods, used in microchip
technology, which contains a cell culture in perfused
chambers.3 By integrating living cells cultures in these
microfluidic platforms, the most relevant biological and
mechanical properties of minimal organic functional units
can be reproduced. OoC in vitro models are an interesting
alternative due to their ability to reliably reproduce biological
characteristics of in vivo physiological and pathological
conditions with human based cells for the study of disease
progression, drug testing, and drug permeability, among others.
The study of blood−brain barrier (BBB) physiology raises
special interest since it is one of the most extensive and
restrictive developed barriers in the central nervous system,
which acts as a natural guard protecting the brain from the
entrance of neurotoxic agents, drugs, invading pathogens, and
circulating blood cells.4−7 Therefore, BBB is key to the study of
brain-directed drugs to reduce drug development failure and to
study BBB dysfunction that is linked to many NDDs, making
Received: February 14, 2022
Accepted: April 12, 2022
Published: May 13, 2022
Perspectivepubs.acs.org/acssensors
© 2022 The Authors. Published by
American Chemical Society
1237
https://doi.org/10.1021/acssensors.2c00333
ACS Sens. 2022, 7, 1237−1247
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the BBB an interesting in vitro model to be developed in an
OoC. In the reported BBB-oCs, most of the works monitor the
correct evolution of the BBB through microscopy images,
while just a few examples are describing other techniques, such
as electrodes integration in the chip for the automatable read
out. Transendothelial electrical resistance (TEER) is the only
integrated detection technique in BBB-oC used to study the
permeability and cell behavior of the BBB endothelial cell
(EC) layer.31 However, TEER offers very limited information
and its lack of specificity makes it not entirely conclusive and
highly dependent on the environment and experimental
settings displaying highly variable results.
However, there are other technologies that can be integrated
into OoC that can offer many advantages and open new
applications, such as biosensors. They are often applied in
medical diagnosis and other areas and are capable of detecting
almost any type of analyte selectively and sensitively. The
integration of biosensors may bring many advantages on BBB-
oC for reaching an automatized monitoring of a wide range of
analytes and biomarkers as a throughput device for a
personalized study of the diseases or drug testing in NDDs.
In this Perspective, the BBB-oC configurations found in
literature are summarized and TEER sensing and influencing
factors are presented by providing solutions to overcome them.
Moreover, described here is a prospective view of new
perspectives of this technology by integrating biosensors into
BBB-oC to achieve a high-throughput system that could reach
the market for personalized medicine and drug detection in
NND. In this direction, the most relevant biomarkers related
to BBB dysfunction in NDDs are described, as well as the
possibilities offered by different biosensors technologies for
their specific detection.
■ BBB-oC PLATFORMS FOR PHYSIOLOGICAL AND
PATHOLOGICAL MIMICKING OF THE BRAIN
Cell Types Involved in Blood−Brain Barrier-on-a-
Chip. BBB is a complex tubular branched network composed
of an EC barrier, linked by TJs and surrounded in the
parenchymal by pericytes and astrocyte cells. This physical
barrier keeps apart blood from neural tissue regulating the
molecular transport and acts as a metabolic and immunological
barrier.8 BBB-oC technology offers the ability to tune
geometry, mechanical, and biochemical factors to mimic the
human environment in vivo.
There are some examples of BBB-oC found in literature that
show the capability to mimic in vivo environment better than
the standard Transwell assays, a static membrane-based old
technology that is the gold standard of in vitro model for
permeability studies of biological barriers. The introduction of
microfabricated platforms combined with dynamic flow offers a
Figure 1. Pictures showing different approaches for BBB-oC configuration. (A) PMMA layers in stack conformation with TEER system included.
Reproduced with permission from 19. Copyright 2021 Elsevier. (B) Flank configuration consisting of two layers of PDMS using a collagen gel to
mimic the natural extracellular matrix in brain. Reproduced with permission from ref 20. Copyright 2016 The Authors under Creative Commons
Attribution 4.0 International License, published by Springer Nature. (C) PDMS layers in flank position and hydrogel consisting in collagen,
matrigel and hyaluronic acid and its TEER system. Reproduced with permission from ref 22. Copyright 2017 Elsevier. (D) Polycaprolactone/
poly(D,L-lactide-co-glycolide) (PCL/PLGA) microfluidic tubular configuration was made by freeze-coating a 3D-printed sacrificial template.
Reproduced with permission from ref 24. Copyright 2020 Elsevier. (E) Tubular structure microchannel via viscous finger patterning technique
using type I collagen hydrogel and its TEER system included, Reproduced with permission from ref 25. Copyright 2020 Wiley. (F) PDMS devices
used to perform a vasculogenesis model. Reprinted with permission from ref 27. Copyright 2017 The Authors under Creative Commons
Attribution 4.0 International License, published by Springer Nature.
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more realistic design, in which cellular shear stress can be
applied on the EC, mimicking the mechanical stimulation
produced by the blood flow in vivo, and allowing 3D cell
culture through embedding hydrogel with cells inside the
microchannel, which render models closer to in vivo
conditions, increasing the biological relevance. However, cell
characterization through imaging is not completely adapted for
3D structures inspection in the chip since 3D configuration
introduces some technical issues.
Regarding cell type, first BBB-oC models included only one
type of cultured cells: ECs mainly from mouse and mice origin.
However, several studies reported that astrocytes have a key
role in the BBB function because they modulate the protein
expression, endothelium differentiation, and the formation and
maintaining of the TJs.9,10 They also showed a protector/
clearance performance over disrupting substances such as
histamine.11 In the same way, other authors have included
more than two cellular types to mimic the BBB in a more
accurate manner, employing ECs, pericytes, astrocytes,
neurons, and microglia. Remarkably, the inclusion of pericytes
in the system displayed a higher barrier restriction and low
permeability of [14C]-mannitol and [14C]-urea in the BBB-oC.
To include pathological conditions, some authors cocultured
ECs and tumoral cells such as glioblastoma (U87). They tested
the coculture of these two cell types to develop a tool for future
high-throughput screening of different antitumor drugs and
evaluate their efficiency to crossing the BBB. To go toward a
more realistic physiological barrier, recent BBB-oC models
included human primary bone marrow-derived mesenchymal
stem cells (BM-MSCs)12 or even brain microvascular
endothelial cells (BMECs) from human induced pluripotent
stem cells (hiPSCs). More recently, some authors created a 3D
self-organized microvascular model of the human BBB with
hiPSC-ECs and primary pericytes and astrocytes, even hiPSC-
derived BBB microvessels, validating barrier function and EC
behavior. The use of hiPSCs brings important new future
applications for OoC platforms in personalized medicine, as
they can be obtained directly from the patient allowing drug
testing and disease monitoring for each patient. Recent
progress has been performed to generate AD, PD, and
Huntington’s disease models from patient-derived iPSCs.13
Nevertheless, although paving the way to personalized BBB
models, the use of hiPSCs is subjected to the efficacy of the
differentiation process and there is a need for more
standardized protocols.14
Chip Designs for Blood−Brain Barrier-on-a-Chip. To
faithfully reproduce the physiological scenario, BBB-oC
designs are presented in the literature mainly in three different
configurations: stack, flank, and tubular. In the stack or vertical
configuration, two channels are piled up containing a
membrane between them that separates the endothelial and
the neuronal culture. This fabrication method is more complex
because it requires the assembling of the chip components at
micrometric precision but allows the use of different types of
membranes embedded between the two channels. This
membrane separated EC culture in one channel from neuronal
cells in the other channel. As mentioned above, the
intercommunication of neuronal and EC is necessary for
proper TJs development. Most of the published works used a
commercial membrane, but other authors fabricated their own
membranes with a desirable material, thickness, and pore
size.15−18 However, the use of a very thick membrane could
discourage cell−cell interaction and hinder the observation of
cell cultures on the top of the membrane, limited by the optical
working distance of the microscope objective and the
transparency of the membrane. Remarkably, some authors
have incorporated TEER systems in this kind of configuration,
using two PDMS layers enclosing two channels and separated
by a porous polycarbonate (PC) membrane with 0.4 mm pores
and 10 μm thickness. PDMS layers were placed between two
glass slides, and Ag was sputtered for electrodes fabrication.
More recently, integrated electrodes and multifrequency TEER
with machine learning algorithms have been incorporated in
multilayered microfluidic platform using PMMA (Figure
1A).19
To facilitate optical inspection, other authors preferred the
flanked or horizontal distribution where two or three channels,
separated by pillars, are patterned in the same layer. Pillars are
usually distributed at short distance along the middle channel
to set a barrier between the cell cultures. In evolved systems,
3D hydrogel with cultured cells is used to substitute the
commercial membrane (Figure 1B,C).20,22,23 This fabrication
approach is easier to industrialize than other configurations,
which are currently commercialized by companies such as
MIMETAS.21 Usually, a positive electrode at the input and a
negative electrode at the output are used for TEER
measurements in this distribution (Figure 1C). The third
arrangement in BBC-oC is based on tubular structures to
mimic the cylindrical-like brain capillaries geometry based on
the formation of hollow fibers as scaffolds that allows the
tubular shape. Different fabrication methods have been used,
but most of them rely on a sacrificial layer that defines the
cylinder (Figure 1D,E).24,25 Moreover, in the line of a hollow
build up, PDMS is used to hold a wire where researchers gelled
Type I collagen and agarose around it. Then, they remove this
wire, creating a bare channel for better mimicking of human
brain venules.26 In a different way, two-photon lithography is
employed to obtain microtubes and reproduce a biomimetic/
biohybrid BBB model at 1:1 scale.15 For the TEER
determination, electrodes are connected into the lumen and
outside of the BBB of the chip (Figure 1E).25 Another
interesting approach for 3D BBB fabrication on a chip is the
technique mimicking the natural formation and maintenance
of our vasculature, angiogenesis (Figure 1F).27 Some examples
are the works carried out in the Kamm laboratory, among
others, where human umbilical vein endothelial cells
(HUVECs) were used for new EC to sprout and split
perpendicularly to the initial EC channel for the growing of
new secondary vessels with morphology similar as natural
ones.23,25,27
Currently, some examples of BBB-oC are commercially
available and in some cases including integrated TEER read
out. AIM biotech launched a platform that allows 40
simultaneous experiments on a single plate. They provide
protocols for creating 3D cultures seeding different types of
cells in a set of three interconnected channels. Elveflow offers a
complete kit of a microfluidic platform and flow controllers for
OoC experiments, and Alveolix displays a chip to model a wide
range of tissue barriers that allows barrier integrity measure-
ments. Moreover, Emulate Co. provides from shell a BBB-oC
including five human cell types: neurons, astrocytes, pericytes,
microglia, and brain microvascular endothelial cells to mimic
the morphological and functional characteristics of cortical
brain tissue. MIMETAS Co. developed a multi-BBB-oC in a
plate format, named Organoplate. This platform includes high-
throughput TEER measurements for their commercial chip.
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This system allows one to measure up to 40 samples at once in
less than a minute, providing an efficient platform to evaluate
the barrier permeability.21
■ TEER SENSING IN BBB-OC
A very attractive element in OoC is the integration of detection
systems, as it will allow the continuous monitoring of the cells
in the chip in real time. As well, the integration of sensors in
OoC offers fully automated analysis, increasing their
commercial interest.
One of the most important factors in TEER measurements is
correlated with the electrodes position. To perform precise
TEER measurements and minimize noise, the electrodes
should be located close to the cell monolayer in a fixed
distance and position. The measurement of in vivo BBB
permeability in humans is not straightforward, and this value
changes from young to old and from healthy to sick. Several
authors have obtained high TEER values in their BBB-oC
designs, closer to in vivo (1500−8000 Ω·cm2).30,31 However,
most of them present designs of electrodes located far from the
cell monolayer, which means a high-resistance contribution
from the solution. Therefore, other authors proposed the used
of fixed electrodes as Ag/AgCl thin-film electrode in the top
and bottom of the membrane and using a four-probe-method
measurement system, which is based on the use of two
electrodes to transport the current and two others to detect the
voltage.34 Some disadvantages of this strategy are the
specialized cleanroom requirement for the electrode’s
fabrication as well as the lack of visualization of the cell
barrier by microscopy due the electrodes position over the
membrane. To overcome these drawbacks, electrodes wires
inserted into guiding channels at the top and bottom layers
allow visualization of the cell monolayer.
Another relevant factor for a precise TEER determination is
the uniformity of the current density through the cell culture.
For this purpose, properties such as the ratio between the
electrode and membrane area and the shape of the electrode
play a key role in reaching a homogeneous current density28,32
because small electrodes against a large membrane cannot
produce enough current across it; leading to an overestimation
of TEER. A circular electrode design located in the top and
bottom of the cylindrical vertical BBB-oC allowed a relatively
uniform electrical current density across the membrane.35
Moreover, the TEER measurement depends on the
electrode material. It has been observed the influence between
the uses of gold (Au) or indium tin oxide (ITO) electrodes in
the TEER values by impedance spectroscopy. Other
extensively employed material for electrodes in BBB-oC is
Ag/AgCl due to their nonpolarizable properties and has a
lower cost.12,16,21,24,35−39 Unfortunately, solid state Ag/AgCl
electrodes can lead to surface degradation with subsequent
signal drift and cytotoxicity effects.29 To avoid these problems
from Ag/AgCl electrodes, other authors used different
materials such as Pt.33 However, it is important to note that
Pt, in contrast to Ag/AgCl, presents a significant interfacial
resistance of electrodes medium that may bring signal
instability, although it can be eliminated by a four-probes
system.32 Temperature and the ionic composition of the cell
culture medium are also other variables to be considered in all
of the electrochemical measurements such as TEER.29,40
Previous results revealed that the resistance decreased with
increasing temperature and ionic concentration (Figure 2A,B),
showing a decrease in sensitivity of 11-fold for temperature and
5-fold for ion concentration ranges.41 To minimize inaccura-
cies in TEER measurements, room temperature needs to be
stable and changing the medium before recording each of the
Figure 2. Elements that determine TEER values: (A) temperature and (B) ion concentration effect over impedance recorded at 10 kHz.
Reproduced with permission from ref 41. Copyright 2016 Elsevier. (C and D) Increased TEER values of ECs cocultured with other neurovascular
cells compared to alone. Panel C reproduced with permission from ref 25. Copyright 2020 Wiley. (E−H) Increasing of TEER values by shear stress
in a dynamic system over a static control. Panels D and H reproduced with permission from ref 39. Copyright 2020 The Authors under Creative
Commons Attribution 4.0 International License, published by Springer Nature. Panel E reproduced with permission from ref 42. Copyright 2021
The Authors under Creative Commons Attribution 4.0 International License, published by MDPI. Panel F reproduced with permission from ref 22.
Copyright 2017 Elsevier. Panel G reproduced with permission from ref 37. Copyright 2015 The Authors under Creative Commons Attribution 4.0
International License, published by PLOS.
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impedance spectra, using always the same medium in the
measurement.
As well as the TEER measurement setup, the BBB
phenotype used in the barrier is an important factor in
TEER results as it can be critically affected by factors such as
the type of cells included in the barrier, cell differentiation
factors, and the shear stress.43−47 So far most BBB-oC works
showed increasing TEER values when EC was cultured with
other types of neurovascular cells,48 such as pericytes and
astrocytes, due to their supply of promoting factors for the
BBB formation (Figure 2C,D).25,39 Also, previous work where
TEER was monitored over the days of culture, showed
significance difference in values due to the optimal incubation
time for EC to develop TJs properly.21,49 Moreover, several
studies demonstrated that shear stress has a mechanotrans-
ductive effect that up-regulates the TJs expressions and RNA
levels of BBB transporters.50,51 As well, most BBB-oCs with
applied shear stress showed higher electrical resistance (Figure
2E−H).22,37,39,42 Remarkably, it is important to consider the
use of in vivo brain microcapillaries shear force values (5−25
dyn/cm2)45,52,53 to mimic a more reliable BBB environment.
However, until now authors have predominantly used shear
forces below in vivo values, except some who applied a
physiological shear stress about 5.8−20 dyn/cm2.11,33,42,54,55
One of the main reasons why physiological shear cannot be
applied is the limitation to reduce the dimension of the BBB-
oC channel, which is inversely related to shear stress.
■ FUTURE OPORTUNITIES FOR THE INTEGRATION
OF BIOSENSORS IN BBB-oC
TEER values could be influenced by multiple factors and
demonstrate selectivity issues. Biosensors permits one to
increase selectivity and to detect a wide range of analytes to
further expand the applications of BBB-oC by including the
detection of disease-specific analytes for a deeper under-
standing on NDDs progression and drug permeation and
testing its performance in brain-based cells. Despite the great
advantages offered by this technology, biosensors integrated in
BBB-oC have not yet been reported. This section proposes the
most relevant biomarkers to monitor the main NDDs related
with damage to the BBB, as well as analytes to evaluate drug
cytotoxicity (Table 1). The possible designs and configurations
of these sensors are also described, paying special attention to
automatized analysis for the monitoring of disease evolution on
the chip to take BBB-oC technology a step further
In biosensors, optical detection is one of the most widely
used methods to read out, such as surface plasmon resonance
and optical waveguide spectroscopy where the physical
behavior of the sensor surface is changing upon the interaction
with the analyte. Also, fluorescence and colorimetric labeled
antibodies are used in a sandwich format to elucidate the
interaction with the analyte. However, to integrate real-time
continuous monitoring into OoC platforms, the transducer
must be miniaturized at low cost and integrated into
microfluidic channels with compatible fabrication techniques
Table 1. Proposed Analytes and Biosensors for BBB-oC Monitoring
detection focus analytes/biomarkers recommended bipreceptor recommended biosensor
drugs Paclitaxel, Simvastatine, Fluvastatin, ... aptamer Aptabeacon
cytotoxicity LDH and/or glutamate lactate oxidase and glutamate oxidase enzymatic sensor
ions Ca+2, Na+, K+, and/or Fe2+/3+ ionophores ISE
neuro-inflammation markers cytokines, chemokines, CAMS, MMPs aptamer and/or antibody aptabeacon and/or impedance immunosensor
ROS hydrogen peroxide HRP enzymatic sensor
Figure 3. (A) Flank design of a BBB-oC considering two blood channels with a central brain chamber. Detail of channels, where biosensors or array
of sensors has been integrated, and TEER electrodes on each side of the endothelial barrier. (B) Schematic drawing of ion-selective membrane for
potassium detection. Reproduced with permission from ref 58. Copyright 2014 The Authors under Creative Commons Attribution 3.0 Unported
License, published by MCPI. (C) Illustration of the mechanism underlying the detection of H2O2 with HRP-AuNPs. Reproduced with permission
of ref 59. Copyright 2015 The Authors under Creative Commons Attribution 4.0 International License, published by PLOS. (D) Scheme of the
aptabecon for drugs binding-induced change in the electron. Reproduced with permission from ref 60. Copyright 2019 American Chemical Society.
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(Figure 3A). Also, it is desirable that the sensor be label-free
and reagent-free for a continuous monitoring, which is not
always possible in optics read-out systems. In this direction,
electrochemical transducers are attractive because the recorded
signal of the electrochemical reaction does not need translation
into a digital signal, the reading equipment being less expensive
and their microfabrication technology affordable for electro-
chemical sensors integration in microfluidics.56,57
Drugs Analysis. Currently, the most extended use for the
BBB-oC platforms is the study of drug delivery to the brain
through the BBB. The usual method to characterize the drug
transport across the brain barrier is by quantification with
external analysis of the remaining drug in the blood channel,
using reverse-phase high-performance liquid chromatography
(HPLC).61 But this technology is not available in most
laboratories because it is expensive and requires a specialized
analytical chemist. An integrated specific sensor for the drug
may allow the automatization of the process at lower cost.
Most of the drugs used in NDDs are low molecular weight
(∼150−300 Da) organic molecules. Then, the most efficient
bioreceptor in biosensors for these types of analytes are
aptamers, which are a synthetic DNA strain able to fold into
well-defined three-dimensional structures and able to bind with
a corresponding target through molecular recognition.62,63 An
electrochemical aptamer-beacon-based sensor is an excellent
candidate for label-free and reagent-free analyses for integrated
in situ detection.64,65 The binding of the analyte with the redox
labeled aptamer induces a 3D conformational change with a
subsequent modification on the distance of the redox tag with
respect to the electrode modifying the electron transfer kinetics
of the redox tag (Figure 3A). Electrochemical aptabeacon has
been reported for drug monitoring (vancomycin) in plasma
using gold electrodes and a self-assembled monolayer of
thiolated-aptamer methylene blue labeled (Figure 3D).60
Cytotoxicity Monitoring. Besides the monitoring of drug
permeability across the BBB, it is also relevant to analyze the
toxicity generated in the brain vasculature by the medication.
Cytotoxicity of drugs can be monitored by the detection of
secreted markers in the extracellular matrix of the cell culture.
Some authors evaluated the cytotoxicity of ECs on the basis of
lactate dehydrogenase (LDH) by colorimetric quantification or
fluorescence immune staining of the death cells,66 but both
techniques do not measure in real time, detecting late
apoptosis and requiring culture fixation. Integrated biosensors
would offer real-time monitoring and quantification of early
apoptosis in an automatized way. For this specific analysis an
electrochemical enzymatic biosensor for LDH detection can be
used. Enzymatic sensors are based on the very specific lock and
key model between the enzyme and its substrate, producing a
catalytic reaction with exponential product formation. Most of
the enzymatic interactions are associated with a reduction−
oxidation reaction that produces electrons, electrochemical
detection being the best candidate. Therefore, enzymes could
be an attractive bioreceptor to determine by electrochemistry
certain molecules in NDDs, permitting low limits of detection,
high selectivity, label-free sensing, and reusability due to
reversible enzyme−substrate interaction. The integration of an
enzymatic sensor in OoC proceeds by the immobilization on
the electrode of the enzyme associated with a redox mediator,
which helps in the electron transfer of the produced electrodes
in the redox reaction to the electrode to perform amperometric
detection. For LDH detection, an electrochemical enzymatic
sensor functionalized with lactate oxidase and a redox mediator
can be used, such as that reported by Park et al.67 Another real-
time biomarker for cell cytotoxicity analysis is glutamate that
can be affected by neuronal toxicity. Also, in this case
electrochemical enzymatic sensors based on glutamate oxidase
or glutamate dehydrogenase are excellent candidates for this
purpose.68,69
To get the complete picture of an in vitro model of NDD
where the neurovasculature is affected and to analyze the
neuronal toxicity of drugs permeated through the BBB, it is
required to combine the coculture of cells from the cerebral
vasculature with neurons. The progressive degeneration of the
activity of the neurons offers a clear evolution of the NDD,
being relevant to detect it from an early stage. The
physiological function of neurons is to send information to
neighboring neurons over a long distance in the form of
electrical impulses, which involves ions flow (sodium,
potassium, calcium, and chlorine) through ion channels. The
degradation of neurons causes neuronal activity to be reduced,
affecting the flow of ions. Therefore, the change in the
concentration of these ions in the extracellular matrix is an
indicator of the neuron’s dysfunction. Ion-selective electrodes
(ISE) are based on organic molecules (ionophores or ion-
exchange substance) contained in a polymeric matrix as
poly(vinyl chloride) drop cast on the working electrode. Here,
the ions are attracted by the ionophore and the attachment of
the ions on the working electrode generates a potential
difference with respect to the reference electrode (Figure 3B).
This difference can be measured by potentiometry in a
reagent-less and label-free manner.70 The most typical
chemical receptors in sensors for the neuronal involved ions
are valinomycin for potassium detection,71 bis[(12-crown-
4)methyl] 2,2-didodecylmalonate for sodium detection,72 and
bis(2-ethylhexyl) adipate for calcium detection.67 However,
Na+, K+, and Ca2+ monitoring in cell culture has some
difficulties due to the presence of these ions in the cell medium
and so poor signal-to-noise ratio as well as a fast change on the
ion’s concentrations, which limit its usefulness for analyzing
neuronal activity. Even so, some examples of neural analysis
with ISE can be found in the literature, such as the
microelectrode modified with tetraphenylarsonium tetrakis(p-
biphenylyl)borate contained in poly(vinyl chloride) membrane
for calcium detection in the intracellular matrix of giant
neurons.73
Inflammation and Oxidative Stress Biomarkers. One
of the most important pathological hallmarks of many NDDs
contributing to cognitive decline is the increase of the BBB
permeability.74 Vascular damage in NNDs is produced mainly
by neuroinflammation, caused by either pathogens or trauma,
and involves both the vascular and immune systems.75
Secreted inflammatory cytokines, such as IL-1β, IL-6, IL-17,
IFN-γ, and TNF-α, regulate the expression and configuration
of a cell junction’s proteins in the ECs of the BBB, altering the
barrier permeability.76,77 Moreover, these cytokines up-
regulate, individually or synergistically, the expression of pro-
inflammatory chemokines, such as CCL2, CCL3, CCL4,
CCL5, CXCL8, CXCL9, CXCL10, and CX3CL1,78,79 and cell-
adhesion molecules (CAMs), such as ICAM-1, VCAM-1,
ALCAM, MCAM, E-selectin, and P-selectin in the ECs of the
BBB.80,81 These chemokines and CAMs promote leukocytes
adhesion to ECs and facilitate their extravasation across the
BBB aided by matrix metalloproteinases (MMPs).82,83 Several
MMPs such as ICAM-1, VCAM-1, ALCAM, MCAM, E-
selectin, and P-selectin, which are critical in tissue remodeling,
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are also involved in the neuroinflammation process either
acting as signaling molecules in neuroinflammatory pathways
or proteolyzing cerebrovascular basement membrane and TJ
proteins.84 Leukocyte infiltration across the BBB initiates a
series of events mostly leading to demyelination and axonal
loss, thereby causing severe neuronal damage.85 During this
process, there is an active production of reactive oxygen species
(ROS) from activated microglia and macrophages.86,87 The
superoxide anion (O2
−), one of the most abundant of ROS,
can easily react with nitric oxide to generate peroxynitrite
anion (ONOO−), a powerful oxidant for proteins that alters
their normal function.88 These include TJs proteins and
signaling proteins, which can be promoted again an
inflammatory response. Thus, oxidative stress and inflamma-
tion are involved in a kind of self-perpetuating cycle,85
producing multiple biomarkers that warn about the different
stages of this process, which are very interesting to analyze in
the in vitro model to understand the evolution of the disease
on the chip.
Neuroinflammation is a known protective mechanism of the
brain, and it is also a common characteristic in the
pathogenesis of several neurodegenerative diseases such as
AD, PD, ALS, and multiple sclerosis (MS), among others.89
Therefore, the biomarkers of neuroinflammation, which
directly affect the BBB permeability, can be considered as
nonspecific markers of the aforementioned NDDs, being useful
for the study of different diseases models. Most of the
neuroinflammatory biomarkers specified (cytokines, chemo-
kines, CAMs, and MMPs) are proteins.
Biosensors can be integrated in BBB-oC devices, placed in
the outlet of the brain chamber of the OoC, for real-time
monitoring of these biomarkers using aptamers and/or
antibodies for sensing proteins as previously discussed. For
the detection of this interaction, a labeling through a secondary
labeled antibody in a sandwich format is usually required in the
sensor. However, for continuous and real-time monitoring is
required a label-free and reagent-less biosensor. Thus, an
attractive option to include in the OoC is an impedance
spectroscopy electrochemical read out, which has high
sensitivity without requiring labeling.35 This technique permits
one to monitor the evolution of the electrical circuit created on
the sensor surface, which is modified by the interaction
between the antigen−antibody complex. Examples of impedi-
metric immunosensor for diagnosis purpose has been reported
for the neuroinflammatory biomarkers; cytokines,90 chemo-
kines,91 CAMs,92 and MMPs,93 as well as aptabeacons for IFN-
γ cytokine analysis by Liu et al.94
Another important factor in detecting neuroinflammation is
monitoring ROS production. Hydrogen peroxide (H2O2) is
the most stable ROS and, therefore, one of the preferred
targets for ROS analysis. Different enzymes such as HRP,59
and superoxide dismutase95 have been used in the detection of
ROS, more common being the use of HRP enzyme as
bioreceptor (Figure 3C). For ROS monitoring in a BBB-oC,
screen-printed, or inkjet-printed carbon electrodes combined
with an Ag/AgCl or Pt (more expensive but less cytotoxic)
reference electrode can be integrated in the microfluidic outlet
channel of the brain coculture chamber. The carbon electrode
needs to be functionalized with HRP enzyme combined with
redox mediator for the amperometric detection of H2O2.
96
The NDDs share common hallmarks, which bring many
advantages in the technology development since one platform
elaborated for a specific analyte can be useful for the study or
application in different NDDs in OoC.
■ CONCLUSIONS AND FUTURE OUTLOOK
The OoC gives us a closer view of the specific parts and
minimal functions of an organ allowing a detailed simulation
and the study of the mechanical and physiological responses
related to different pathologies. Although great progress has
been made in the field of BBB-oC, these platforms are still at a
very early stage as the developed systems are a very simplified
adaptation of the real physiology of the BBB. Currently, the
cell culture development into the BBB-oC is widely monitored
by fluorescence labeling of specific proteins with a confocal
microscope. This method is time-consuming and costly and
does not permit a real-time monitoring and automatization of
the detection, hindering the access of this technology to the
market. Currently, TEER is the only type of integrated sensor
in BBB-oC that contains all of these advantages. The wide
range of electrode materials, shapes, sizes, and configurations
for TEER measurement was described in this review, advising
the most efficient configuration. However, this type of
measurement brings limited information about the BBB and
suffers from the influence of many variables (temperature,
electrolyte concentration, and others) in the response leading
to conflicting results. With the commented-on difficulties,
examples of BBB-oC have been commercialized. Besides the
generic barrier integrity, new BBB-oC models are expected to
go one step further and allow for the continuous monitoring of
markers of inflammation and cell damage. Therefore, the
integration of biosensor’s technology opens many possibilities
for sensing on this type of device for continuous monitoring of
many different analytes. The continuous control of the
concentration evolution of various molecules inside the cell
culture of the BBB-oC may contribute with relevant
information for a deeper knowledge of the disease correlated
with BBB permeability and drug testing. Moreover, the
pathological similarities between NDDs simplify the technol-
ogy development since the same biosensor can be useful for
the study of different illnesses. The disruption of the BBB is a
consequence in various NDDs, and as mentioned before there
are many different biomarkers evolving that need a closer
study. This Perspective brings a forward-looking vision in the
BBB-oC area to develop multiple fully automated selective
analyzers, describing the most relevant biomarkers in NDDs
correlated with the BBB and the possible biosensors strategies
that could be performed to be integrated into BBB-oC to
measure and monitor these molecules.
As we have already advanced in the Perspective, this
technology should move in the direction of a fully automated
multianalyzer to be used in a wider range of applications. For
this technology to reach a wider market, the BBB-oC must be
designed as an easy-to-use analyzer tool to be used for drug
testing or custom drugs in NDD in the hospital or
pharmaceutical industry. We hope that this work will help to
develop more accurate and reliable TEER measurements in the
future BBB-oC and allow the availability of new representative
BBB models integrating biosensors for the study and real-time
detection of new analytes in the chip.
■ AUTHOR INFORMATION
Corresponding Author
Mo ̀nica Mir − Biomedical Research Networking Center in
Bioengineering, Biomaterials, and Nanomedicine (CIBER-
ACS Sensors pubs.acs.org/acssensors Perspective
https://doi.org/10.1021/acssensors.2c00333
ACS Sens. 2022, 7, 1237−1247
1243
BBN), 28029 Madrid, Spain; Nanobioengineering Group,
Institute for Bioengineering of Catalonia (IBEC), Barcelona
Institute of Science and Technology (BIST), Barcelona
08028, Spain; Department of Electronics and Biomedical
Engineering, University of Barcelona, 08028 Barcelona,
Spain; orcid.org/0000-0002-1490-8373; Email: mmir@
ibecbarcelona.eu, mmir@ub.edu
Authors
Sujey Palma-Florez − Nanobioengineering Group, Institute for
Bioengineering of Catalonia (IBEC), Barcelona Institute of
Science and Technology (BIST), Barcelona 08028, Spain;
Department of Electronics and Biomedical Engineering,
University of Barcelona, 08028 Barcelona, Spain
Anna Lagunas − Biomedical Research Networking Center in
Bioengineering, Biomaterials, and Nanomedicine (CIBER-
BBN), 28029 Madrid, Spain; Nanobioengineering Group,
Institute for Bioengineering of Catalonia (IBEC), Barcelona
Institute of Science and Technology (BIST), Barcelona
08028, Spain; orcid.org/0000-0002-0936-3401
Maria José López-Martínez − Biomedical Research
Networking Center in Bioengineering, Biomaterials, and
Nanomedicine (CIBER-BBN), 28029 Madrid, Spain;
Nanobioengineering Group, Institute for Bioengineering of
Catalonia (IBEC), Barcelona Institute of Science and
Technology (BIST), Barcelona 08028, Spain; Department of
Electronics and Biomedical Engineering, University of
Barcelona, 08028 Barcelona, Spain
Josep Samitier − Biomedical Research Networking Center in
Bioengineering, Biomaterials, and Nanomedicine (CIBER-
BBN), 28029 Madrid, Spain; Nanobioengineering Group,
Institute for Bioengineering of Catalonia (IBEC), Barcelona
Institute of Science and Technology (BIST), Barcelona
08028, Spain; Department of Electronics and Biomedical
Engineering, University of Barcelona, 08028 Barcelona, Spain
Complete contact information is available at:
https://pubs.acs.org/10.1021/acssensors.2c00333
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
CIBER-BBN is an initiative funded by the VI National R&D&I
Plan 2008−2011, Iniciativa Ingenio 2010, Consolider Program,
CIBER Actions and financed by the Instituto de Salud Carlos
III with assistance from the European Regional Development
Fund (Grant CB06/01/0055). The Nanobioengineering group
in the Institute of Bioengineering of Catalonia (IBEC) has
support from the Commission for Universities and Research of
the Department of Innovation, Universities, and Enterprise of
the Generalitat de Catalunya (Grant 2017 SGR 1079), and it is
part of the CERCA Programme/Generalitat de Catalunya.
This work is supported by the Project In vitro micro-
physiological platform to mimic the blood-central nervous system
barriers: Brain and spinal cord applications (RTI2018-097038-
B-C21), funded by the Spanish Ministry of Economy and
Competitiveness under the National Program of R&D. S.P.-F.
is supported by the program for predoctoral contracts for the
training of doctors of the State Training Subprogram for the
Promotion of Talent and its Employability in R+D+I
(L05ACPRE286) by the Spanish Ministry of Science and
Innovation.
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