International Journal of Biological Macromolecules 235 (2023) 123784

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Review

Microfluidic devices for the detection of disease-specific proteins and other

macromolecules, disease modelling and drug development: A review
S. Amir 1, A. Arathi 1, S. Reshma 1, P.V. Mohanan *
Toxicology Division, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology (Govt. of India), Poojapura, Trivandrum 695 012,
Kerala, India

A R T I C L E I N F O A B S T R A C T

Keywords: Microfluidics is a revolutionary technology that has promising applications in the biomedical field.Integrating
Microfluidics microfluidic technology with the traditional assays unravels the innumerable possibilities for translational
Diagnostics biomedical research. Microfluidics has the potential to build up a novel platform for diagnosis and therapy
Organ-on-a-chip
through precise manipulation of fluids and enhanced throughput functions. The developments in microfluidics-
Disease modelling
based devices for diagnostics have evolved in the last decade and have been established for their rapid, effective,
Drug delivery
accurate and economic advantages. The efficiency and sensitivity of such devices to detect disease-specific
macromolecules like proteins and nucleic acids have made crucial impacts in disease diagnosis. The disease
modelling using microfluidic systems provides a more prominent replication of the in vivo microenvironment
and can be a better alternative for the existing disease models. These models can replicate critical micro­
physiology like the dynamic microenvironment, cellular interactions, and biophysical and biochemical cues.
Microfluidics also provides a promising system for high throughput drug screening and delivery applications.
However, microfluidics-based diagnostics still encounter related challenges in the reliability, real-time moni­
toring and reproducibility that circumvents this technology from being impacted in the healthcare industry. This
review highlights the recent microfluidics developments for modelling and diagnosing common diseases,
including cancer, neurological, cardiovascular, respiratory and autoimmune disorders, and its applications in
drug development.

1. Introduction microfluidic cell culture devices or organ-on-chip (OOC) devices. OOC

The emergence of microfluidics has bought a new approach to
biomedical research. Microfluidics has impactful applications in basic
and translational biomedical research due to the peculiar behaviour of
fluids in microenvironments. Several technologies are employed to
fabricate microchannels and chambers that allow fluid flow even in
ranges of femtoliters (fL) in a tunable manner. This warrants numerous
advantages like miniaturisation, portability, cost-effectiveness, minimal
reagent usage, etc. Even though both conventional 2D and hydrogel-
based 3D cell culture systems have established their worth in biomed­
ical research, they lack some of the basic underlying features of human
tissues. These include several key elements, from the spatial orientation
of cells to mechanical cues acted upon the cells, which directly influence
the development and function of organs. Such features that cannot be
incorporated into conventional systems can be recapitulated in
devices provide a robust platform for culturing different cells separately
under continuous perfusion. The complexity of these devices can vary
from a single cell device to multi-organ chips [1,2]. The fast-track ad­
vancements in microfluidics has initiated novel concepts and de­
velopments in many field of science. Microfluidics, as the name
indicates, is the manipulation at fluids in a micro level system and is
capable of interacting with the micro scale biological environment and
provide insight into various micro level cellular functions. The last
decade has seen an immense development in the microfluidic technol­
ogy especially in the organ on a chip development and it would defi­
nitely grow into a field that cannot be differentiated from the biomedical
and biotechnological industry in the next two decades. This review
discuss, firstly, different detection methods established in microfluidic
devices for different diseases. Then, on different disease models, high­
lighting the recapitulation of pathophysiology in such miniature

* Corresponding author.
E-mail address: mohanpv10@gmail.com (P.V. Mohanan).
1
All authors equally contributed.

https://doi.org/10.1016/j.ijbiomac.2023.123784
Received 23 November 2022; Received in revised form 15 February 2023; Accepted 16 February 2023
Available online 21 February 2023
0141-8130/© 2023 Elsevier B.V. All rights reserved.
S. Amir et al.
devices. There view briefly explains the drug screening and drug de­
livery systems. Reference articles were searched using keywords specific
to each sections and subjected to selection by screening the abstracts.
Fig. 1 summarises the outline of this review.
2. Application of microfluidic devices for diagnostics
Recent advances in the development of microfluidic devices and the
integration of mechanical, electrical and optical elements within the
microchannels have made the miniaturisation of conventional labora­
tory tests a viable alternative for disease detection. The use of such
miniature devices for the detection of disease-specific biomolecules
provides a highly sensitive yet cost-effective diagnostic tool. The
portability, cost-effectiveness, enhanced speed and sensitivity and reli­
ability of microfluidic diagnostic devices make them clinically relevant
point-of-care devices. Integration of reagent reservoirs, reaction cham­
bers, micropumps, microvalves, micromixers, biosensors, sorting
mechanisms and detection modules in a single platform enables high-
throughput and early detection of diseases even in bedridden patients.
From earlier Lab-on-a-chip devices which provided miniaturized
reaction chambers, focused and extensive efforts were made to develop
highly sensitive and accurate devices which can detect disease-specific
biomarkers even in femtomolar quantities [3,4]. There are passive and
active microfluidic devices which use internal (diffusion, inertia,
architecture-induced manipulations etc.) and external (electric, mag­
netic, acoustic, thermal, pressure etc.) forces to influence the flow or
distribution of particles inside the microchannels. This helps in the
detection of biomarkers of different diseases. In addition, active
microfluidic devices sometimes employ the functionalization of con­
ducting channels with detection molecules like antibodies, which can be
sensed with the help of biosensors [241]. Disease-specific proteins and
genetic materials are two of the major macromolecule used in disease
diagnostics, which follows immunodiagnostics and genetic diagnostics
respectively [242]. Other approaches like differences in the mechanical
properties of diseased and healthy cells are also exploited for disease
diagnostics. Microfluidic devices, for example with constriction chan­
nels, can be used for the mechanical characterisation of single cells and
thereby acquiring additional information for diagnosis [5].
Fig. 1. Overview of microfluidic device application in human disease research.
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International Journal of Biological Macromolecules 235 (2023) 123784
2.1. Detection systems
2.1.1. Protein based detection
Genetic disorders arise due to the abnormality in the genetic makeup
of an individual. Protein based detection methods of genetic disorders
follow the identification of the abnormal gene product. Sickle cell ane­
mia is one of the most common genetic diseases caused by a point mu­
tation in the hemoglobin gene. μPAD, a paper-based microfluidic device
is a simple, cost-effective, point-of-care diagnostic test developed by X.
Yang, for differentiating healthy individuals from sickle cell trait car­
riers and sickle cell disease patients. The HbS antigen in the sample
blood, which is combined with a reducing agent and phosphate buffer, is
trapped on the paper substrate and generates patterns of different col­
ours that are visible to the naked eye. They are a low-cost platform since
the test takes 20 min and detects Hb without the use of chemicals or
enzymes [6]. Non-invasive prenatal diagnostics (NIPD) have been used
for a long period to diagnose genetic disorders in the fetus [243]. Using
cell-free fetal DNA fragments circulating in maternal plasma for NIPD
have some inherent drawbacks. This can be overcome by isolating and
analysing fetal nucleated red blood cells (fNRBCs). CD147 is a surface
marker used to detect, isolate and enrich fNRBCs. Using this principle,
An affinity microchip was developed in 2017, to identify fetal chromo­
somal aneuploidies including Trisomy 13 and 21 by isolating fNRBCs
utilising anti-CD147 as the immuno-agent and chitosan/hydroxyapatite
nanoparticles [7]. In this study, the isolation of fNRBCs is based on its
protein biomarker and the detection of the aneuploidies is by nucleic
acid hybridization.
The development of digital microfluidic devices based on electro­
wetting offers a platform for high throughput genetic disease screening
in neonates and it is believed to add value to the existing diagnostics.
SEEKER® is one such digital microfluidic device approved United States
Food and Drug Administration (FDA) that has been commercially used
to screen newborns for lysosomal storage disorders (LSDs). It works
based on fluorometric assays for the five LSD enzymes representing
Pompe, Gaucher, Hunter, Hurler and Fabry diseases [244].
2.1.2. Nucleic acid based detection
Non-invasive prenatal diagnostics (NIPD) are rapid and convenient
ways of detecting congenital disabilities in the fetus. Even though free
fetal cells and cell-free fetal DNA fragments circulate through maternal
S. Amir et al.
plasma they are scarce. The potential of microfluidics enables efficient
isolation of such fetal markers, which can then easily be subjected to on-
chip in situ bioanalyses for the detection of genetic abnormalities. This
can realize the development of highly sensitive and accurate and rapid
NIPDs [8]. One such device, developed by Winter et al. [9], used inertial
microfluidics to isolate fetal trophoblast cells from maternal blood
samples based on the size difference between trophoblast cells and white
blood cells (WBCs) [9]. Once fetal-source cells are isolated, fluorescent
in situ hybridization technique is used to detect trisomy 21.
Real-time PCR is another molecular technique used for identifying
abnormalities in the genetic makeup. Several inherent properties of
microfluidic devices like higher surface-to-volume ratio, owe micro­
fluidic PCR devices advantages over conventional PCR thermocyclers,
like low sample requirements and greater control over the reaction steps
[245]. Kang-Yi Lien developed a three-module microfluidic system that
consists of a genomic DNA extraction module, a PCR module, and an
external optical detection module to detect genetic deletions associated
with α-Thalassemia-1. This integrated rapid detection platform extracts
DNA from saliva samples and collects it onto magnetic microbeads,
which are then concentrated by the on-chip magnetic field. Later PCR on
specific genetic deletions can be performed. Finally, an external optical
module connected to the chip detects the amplification via fluorescence
[246]. Single nucleotide polymorphism (SNP) genotyping is another
marker used to identify different genetic disorders, Hereditary hearing
loss is one such condition. Lu et al. developed a microfluidic device that
can screen for multiple SNPs by distributing the sample into Competitive
Allele Specific PCR chambers demonstrating its practical application in
detecting genetic diseases like hereditary hearing loss [10,11].
2.2. Autoimmune disorders based detection
Autoimmune diseases are conditions in which the immune system
attacks self-molecules. Multiple sclerosis, Grave's disease, Hashimoto
thyroiditis, Myasthenia gravis and Pernicious anemia are some exam­
ples. Conventional diagnoses are supported by laboratory tests that
include analysis of biomarkers like inflammatory factors, autoanti­
bodies, and cytokines, flow cytometry, metabolic profiling and HLA
typing etc [247,248]. The advent of microfluidic technology over con­
ventional testing is that it enables parallel analysis of multiple markers
and sample, which in turn enhance the sensitivity and throughput of the
diagnosis [249]. Even though the presence of autoantibodies is not
conclusive evidence for autoimmune diseases, it can certainly support
the signs and symptoms for diagnosis [247].
In 2008, Matsudaira and their team developed a photoirradiation
method to immobilize autoantigens in microarrays of a microfluidic
device. When the sample serum is added to the device, the antigen-
specific autoantibody binds to the microspotted arrays and is identi­
fied using a peroxidase-conjugated secondary antibody. They associated
a CCD (charged-coupled device) camera with the microfluidic device to
detect the intensity of chemical luminescence produced by the peroxi­
dase [12,13]. POCEMON is another powerful diagnostic tool based on
the genomic microarray of HLA typing (Human Leukocyte antigens),
that integrates microfluidics, microelectronics and microarray, to di­
agnose autoimmune diseases like Rheumatoid arthritis and Multiple
sclerosis [14].
2.3. Identification of cancer
Cancer is one of the most aggressive diseases which can be controlled
and even cured if there is an early diagnosis. Common and conventional
tools for diagnosis are imaging techniques, molecular tests for tumour
markers and tissue biopsies. Biomarkers like Circulating tumour cells,
cell-free miRNAs and exosomes allow cost-effective and rapid moni­
toring of the disease. With the advantages of microfluidic point-of-care
devices like portability, and their ability to integrate highly sensitive
biosensors, these diagnosis platforms are promising tools for the early
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International Journal of Biological Macromolecules 235 (2023) 123784
detection of cancers [250].
2.3.1. miRNA based detection
Cell-free DNAs and cancer-specific miRNAs are the nucleic acid
biomarkers of cancer. Detection of miRNA is mediated by customized
probes, mainly single-stranded nucleic acid probes and hair pin-shaped
probes, adsorbed onto the surface of signal transducers, which can be
nanomaterials like magnetic NPs, gold NPs, silver NPs, or electrodes.
The binding of miRNA to the probe produces optical, electrical or me­
chanical signals [15]. Microfluidic platforms are powerful diagnosis
tools for cancer detection because of their capability to incorporate such
probed signal transducers with comparative ease. Song Guo and their
team succeeded in developing a droplet microfluidic device using
isothermal amplification to detect single-cell miRNA. They employed a
DNA hybridisation chain reaction rather than a polymerase chain re­
action to amplify the target gene concentration which is then detected
by fluorescent signals [16,17].
2.3.2. Tumour antigen based detection
Tumour cells detached from tumour sites are circulated through
blood vessels. These are other markers for the tumour that increases or
decrease based on the tumour intensity. Specific antigens called tumour
antigens expressed on the surface of tumour cells are used as probes to
identify and isolate these circulating tumour cells (CTCs).Epithelial cell
adhesion molecule (EpCAM) is a well-known tumour marker [251].
Exploiting this, Sunitha Nagrath and her team developed a CTCchip to
isolate CTCs from peripheral blood, by designing EpCAM antibody-
coated microposts in the fluid chamber. This highly specific and sensi­
tive device successfully isolated CTCs from blood samples of patients
suffering from colon cancer, prostate cancer, and pancreatic and meta­
static lung cancer [18,19]. Another pertinent work uses a microfluidic
technology called PEDOT NanoVelcro to purify CTCs linked to prostate
cancer. This device is made of poly (3,4-ethylene-dioxythiophene) based
nanomaterial. This glycan-stimulated device enhances the capturing
efficiency and increases the specificity of CTC upon conjugation with an
antibody [20].
2.3.3. Exosomal constituents based detection
Exosomes are 30-150 nm-sized extracellular vesicles that play a vital
role in cell-cell communication and contain mRNAs, proteins, lipids, and
double-stranded DNA. These contents can offer crucial information
about cellular identity and tissue origin. Since exosomes are stable in
body fluids and are present in circulation from the early stages of cancer,
they are used as excellent biomarkers for cancer diagnosis [250]. Exo­
somes from each cancer tissue constitute different contents inside. They
can be isolated using microfluidics based on various methods like im­
mune affinity, size and dynamic microfluidic [21,22].A size-dependent
acoustic nano filter device was created by Kyungheon Lee and his col­
leagues to separate microvesicles. Using the ultrasonic standing
approach, the device offers excellent resolution and separation yield of
microvesicles based on their size and density [23,24]. ExoChip, a
different cutting-edge microfluidic technology, enables the extraction
and measurement of exosomes from serum. The PDMS-based Exochip
device is operated with antibodies against CD63, which is overexpressed
in exosomes. As the exosomes are collected with intact RNA, the tech­
nology enables open array analysis to characterize micro-RNA [25,26].
2.4. Detection of neurodegenerative diseases based on specific proteins
The commonly used approaches to diagnose neurodegenerative
diseases including Alzheimer's and Parkinson's are invasive and costly.
This includes lumbar puncture-based CSF sample collection, cognitive
and neuropsychological testing, and neuroimaging-based methods (MRI
and PET). P-TAU, α-synuclein, and β-amyloids are the main biomarkers
of neurodegenerative diseases [252]. Cristiana Mollinari and co-workers
reported that chemically induced neurons from patients show all the
S. Amir et al.
signs of neurodegenerative diseases like misfolded protein accumulation
and mitochondrial abnormalities. They integrated this chemical
reprogramming strategy with microfluidics for early detection of Alz­
heimer’s and Parkinson's disease using antibodies against β-amyloids
and P-TAU [27,28]. In 2018, Zhenzhu and colleagues created an
attractive disposable microfluidic and electrical sensor that was Morpho
Menelaus-inspired. The technology includes a microfluidic section with
SiO2 nanoparticles that improve fluorescence intensity upon IgG and
AD7c-NTP detection as well as an electronic unit covered with
conductive ink to monitor the resistance change rate after static tremors
in patients with neurological diseases [29]. In order to detect Alz­
heimer's, Ning Xia and her colleagues developed a device in which an­
tibodies specific to amyloid-(1-40) and amyloid-(1-42) are immobilised
on two separate channels and measured using surface plasmon reso­
nance. A streptavidin-conjugated antibody specific for the N-terminus of
amyloid-peptides is used to amplify the signal forms [30].
2.5. Detection of infectious diseases
The most destructive microbial infections in human history have
emerged in the twenty-first century. The COVID-19 outbreak has already
displaced millions of people from their homes. Such conditions force the
scientific community to develop affordable, portable, highly sensitive,
and quick diagnostic tools to stop infectious illnesses. Although protein-
based detection may also be used for microbial diagnosis, nucleic acid-
based testing is the main method.
2.5.1. Nucleic acid based detection
An anti-malaria microfluidic device based on paper was established
by Gaolian Xu and colleagues. First, the microbial DNA is extracted, next
loop-mediated isothermal amplification (LAMP), and finally, array-
based fluorescence is used to identify the DNA amplicon. The device
was able to identify three species of Plasmodium, the malarial parasite,
from the patient's whole blood [31,32]. Brian J. Taylor and his col­
leagues developed another adaptable, reliable, and durable tool to di­
agnose malaria that outperforms microscopy's sensitivity by identifying
2 parasites per μl. Plasmodium falciparum and Plasmodium vivax can be
distinguished by the device, which uses PCR primers as the molecular
targets in a hydrogel mixture. This technology outperforms other LAMP-
equipped devices since primer validation is not as necessary [33]. Chih-
Hung Wang and the team reported on the detection of Methicillin-
resistant Staphylococcus aureus (MRSA) utilizing a magnetic bead-
based microfluidic device. The device has a mechanism for extracting
nucleic acids, and isothermally amplifying the target nucleic acids. The
inner and outer primer incorporated in the device can identify six
distinct MRSA sequences [34].
2.5.2. Protein based detection
Nosocomial infections are healthcare-associated infections and the
major causes are Staphylococcus aureus and Enterococcus. In order to
create a sensitive lab-on-chip platform for the detection of nosocomial
infections, Carla M. Carvalho and colleagues employed magneto-
resistive (MR) sensors and phage receptor binding protein (RBP) as
probes. RBPs, which attach to bacterial cells, are used to operationalize
magnetic nanoparticles. When both pathogens are discovered, even in
low quantities, the device's MR sensors create strong signals [35]. To
detect the H1N1 virus using fluorescence-based techniques, a sensitive
and automated microfluidic device was created. Because aptamers are
inexpensive, simple to synthesize, and stable at various temperatures,
the researchers employed an aptamer-based sandwich assay. The test
can be finished in 30 min with the aid of the aptamer-based assay and
microfluidic support, using the least amount of reagents and labour
[36]. Another microfluidic device, created by Siyuan Wang and col­
leagues, to identify live Salmonella typhimurium, a food-borne disease
pathogen. Anti-Salmonella antibodies coated magnetic nanoparticles in
propidium monoazide (PMA) are used in the device, which also employs
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International Journal of Biological Macromolecules 235 (2023) 123784
two magnetic stirring mixers to stir bacterial samples. Magnetic bacteria
that have been subjected to PMA are separated using a magnetic field in
a separating chamber. In the detection chamber, the DNA is extracted
and amplified using LAMP. The LAMP reaction system continuously
measures turbidity to determine the number of the live pathogen [37].
The TMek is another remarkable device that allows for stage-specific
diagnosis and quantification of malaria. By using the conflict between
magnetic forces and gravity, it isolates contaminated RBCs. The micro­
chip can detect the paramagnetic property of the hemozoin nanocrystals
inside the infected RBC. The duration and amplitude of the electric
impulses produced can be used to determine the infection stage and the
number of infected RBCs [38,39].
2.6. Detection of blood related diseases based on mechanical
characterization of blood cells
Microfluidic devices that exploit mechanical properties of cells for
diagnosis employ mechanical forces to characterize their properties.
These mechanical forces are exerted by means of different constriction
channels, fluid stress, optical stretcher, electro-deformation, electropo­
ration and microfluidic pipette aspiration. When cells from the sample
experience deformation, several parameters like transit time, elonga­
tion, recovery time, pressure drop, deformation index, Young's modulus
and viscosity are measured to characterize and compare between dis­
eases and healthy states [5]. For example, RBCs are known to stiffen and
cytoadhere when infected with malarial parasites [40]and leukocytes of
leukemia patients exhibit a decreased Young's modulus [41].
In case of constriction channels, cells are squeezed into a ‘constricted
channel’ (channel with at least one dimension smaller than that of the
cell radius) using hydraulic pressure difference, to record and analyse
their mechanical parameters. Tsukada et al. in 2001, used a micro­
channel chip to deform diabetic and healthy RBCs and reported that
diabetic RBCs show less deformability than healthy RBCs [42]. In 2019,
Faustino et al. proposed a microfluidic device with hyperbolic
converging microchannel to diagnose end-stage kidney disease (ESKD).
Like Tsukada, they used a high-speed video microscopy system, to ac­
quire velocities and deformability ratios. They reported average 8 % and
14 % less elongation inRBCs of ESKD patients without and with diabetes
respectively, compared to healthy individuals [43].The recovery time of
deformed cells is another parameter that can be used for diagnosis. In
Plasmodium falciparum infected RBCs the stretching is reported to be less
and recovery is twice as fast as healthy RBCs. Amirouche et al.
demonstrated this with an oscillating width channel microfluidic device
[44]. These are proof-of-concept devices that has the potential to be used
for disease diagnosis. While some of these devices needs expert hands to
operate while others are simple to use which has more commercializa­
tion potential. Cancer cells are another type of ‘diseased cells’ that can
be distinguished by means of their mechanical properties. Most extra-
neural cancer cells are softer and deformable than benign counter­
parts, while brain cancer cells, deviate from this observation when
elongation and velocities are taken as evaluation parameters. In this
case, entry time into a constriction channel is considered to be more
sensitive parameter for distinguishing cancerous and healthy glial cells
[45].
Table 1 summarises main kinds of microfluidic devices discussed
above and Fig. 2 provides their schematic representations. Disease
diagnosis using microfluidic technology has indeed promising potential
and many of the devices have been successfully commercialized.
Nevertheless, focused efforts are necessary to exploit their full potential
and its translation to the market.
3. Microfluidic disease models
3.1. Disease modelling - why do we need disease models?
Disease models are biological systems that entirely or in part display
S. Amir et al. International Journal of Biological Macromolecules 235 (2023) 123784

Table 1
Some of the microfluidic platforms used for disease diagnosis.
Type of disorder Reference Disease Macromolecule Fabrication Geometry Chip material Active Method
or
Passive

Genetic disorder [6] Sickle Cell Protein μPADs -Meshworkof Chromatography Passive Red colour intensity
Anemia paper fibres paper profile of diffused
bloodstain pattern is used
to differentiate between
SCD, SCT and healthy
samples
[11] Hereditary DNA Injection 112 reaction Polypropylene Active on-chip PCR and
hearing loss molding chambers that are subsequent fluorescence
divided into four scanning
units, and each unit
has an inlet/outlet (
Fig. 2A)
Autoimmune [12] Autoimmune Protein Single serpentine PDMS and Active Autoantibodies adsorbed
disorders diseases channel on PDMS Polystyrene onto the micro spotted
below micro spotted autoantigens are detected
polystyrene chip ( by the chemical
Fig. 2B) luminescence produced by
secondary antibodies
labeled with horseradish
peroxidase (HRP).
Cancer [17] Breast cancer RNA Soft Flow focusing PDMS Active Isothermal amplification
lithography droplet generator ( of target miRNA-21 and
Fig. 2C) subsequent fluorescence
detection by
photomultiplier (PMT)
detector
[19] Metastatic Protein Deep reactive 78,000 equilateral Silicon Active Target circulating tumour
lung, prostate, ion etching triangular array of cells (CTCs) are captured
pancreatic, (DRIE) micro posts, 100 with antibody (EpCAM)-
breast and mm tall and 100 mm coated micro posts
colon cancer in diameter with an
average 50 mm gap
between micro posts
(Fig. 2D)
[25] Specific cancer Protein Soft ExoChip- 12 PDMS Active Capture specific exosome
lithography laterally placed with coated-antibodies
channel of 73 mm in against CD63, staining and
length and 100 μm quantitating exosome with
in height consisting fluorescent carbocyanine
of eight circular dye(DiO) and standard
chambers, with a plate-reader and
diameter of 5 mm subsequent open array
each, equally spaced miRNA profiling
at a distance 9 mm
from each other
connected through a
shorter 0.75 mm
wide channels (
Fig. 2E)
Neurodegenerative [27,28] Familial Protein Soft Six independent PDMS Passive Neuropathological
disorders Alzheimer's lithography parallel channels, markers like β-Amyloid
Disease and each with an inlet and α-Synuclein
Parkinson's and outlet (Fig. 2F) accumulation, TAU
disease phosphorylation and
mitochondrial
abnormalities detection in
chemically induced
neurons by
immunofluorescence
Infectious disease [39] Malaria Protein Reactive ion 1400 cylindrical Silicon Active Infected red blood cells (i-
etching and magnetic Ni RBCs), based on the
electroplating concentrators and paramagnetic properties of
four equivalent hemozoin nanocrystals are
sensors of coplanar sorted and electrically
gold electrodes on detected in whole blood
top of it, in one wall specimen
of the microfluidic
chamber
[31,32] Malaria DNA Device contained filter paper Passive DNA extraction, loop-
three components, a mediated isothermal
filter paper based amplification (LAMP), and
fluidic device with
(continued on next page)

5
S. Amir et al.
Table 1 (continued )
Type of disorder Reference Disease Macromolecule Fabrication
the pathological processes of human or animal diseases. These models
are then used to study various aspects of diseases like pathology, un­
derlying molecular mechanisms, potential target pathways and mole­
cules for therapeutics, and the safety and efficacy of therapeutic
candidates. Current disease models exist in the form of simple cell cul­
ture systems or various animal models. The species variation and inad­
equate representation of human organs in animal models and 2D cell
cultures are still significant concerns. Mimicking organ-level functions
in these conventional models is often limited, let alone the genetic
complexity of many diseases. Diseases that are manifested through
unique genetic makeup and those involving an interplay of the immune
system like autoimmune disorders and human cancers are so unique that
no representative models can be created [46].
The lack of human-relevant preclinical models often leads to delayed
failure of drugs, or in the case of drugs that succeed in preclinical trials,
often develop efficacy (Hepatitis C vaccine, Tuberculosis MV85a) [47]
and safety (Hu5c8 monoclonal antibodies) issues [48] during clinical
trials. Animal disease models that share human phenotype may differ by
their underlying molecular mechanism and hence may produce false
positive results in therapeutic screening. On the other hand, there could
be cases of early rejection of potentially safe therapeutics due to their
toxicity in animal models [49]. Advanced therapeutic strategies like
CRISPR RNA therapies, monoclonal antibodies etc., and target specific
human biomolecules that might not affect non-human models. All these
factors emphasise the need for alternate human-relevant disease models.
In search of advanced and more relevant models for studying the
inner workings of human organs and their diseases, microfluidics has
become one of the most powerful tools. These compartmentalised cell
culture systems can be precisely designed to replicate a microenviron­
ment and mimic the in vivo niche. Initially, these microfluidic chips or
devices were established for laboratory experiments like chemical
analysis [50] or single cell analysis [51,52], which rapidly grew into
more complex ‘Organ-on-chip’ systems. The wide acceptance and pan
applications of microfluidic chips are because they can act as an in vitro
human micro physiological system mimicking healthy and diseased
states of different organs. Critical features like mechanical forces, dy­
namic flow of blood surrogates, the interplay of immune cells in the
circulation, biochemical cues from surrounding cells and multiple
cellular interfaces can be incorporated into these microfluidic devices.
Additionally, the possibility of constituting biosensors inside the chip for
real-time acquisition of physiological parameters and real-time moni­
toring of cellular behaviour under dynamic flow using non-invasive
probes [53].
However, fluid flow inside microchannels often encounters many
challenges. Microbubble formation inside the channel is one of such
practical hurdles which has to be accounted for even before the fabri­
cation of the device. These microbubbles can impair the function of the
microfluidic device by presenting unpredicted shear stress levels and
undesired channel blocks. The bubbles primarily arise either from the
air-device interface or within the device; from the air space inside the
chip or the flowing liquid. To achieve reliable performance, it is
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International Journal of Biological Macromolecules 235 (2023) 123784
Geometry Chip material Active Method
or
Passive
printed hydrophobic array-based fluorescence
wax, acetate film to detection
seal plastic plate
with 4 glass fiber
spots (diameter 3
mm), forming
chambers for LAMP
reaction and one
glass fiber spot with
diameter 4 mm (
Fig. 2G)
important to trap and remove any kind of bubbles in microfluidic de­
vices. Several on-chip and off-chip, active and passive strategies have
been suggested previously. These strategies are principally based on the
concepts of capillarity, buoyancy or permeation and thermo-capillary
flow [54]. For example, some methods utilize complex microstructures
inside the chip to trap bubbles [55]. Recent studies like Huang et al.,
employ vertically sloped microchannels which passively collect the air
bubbles above the fluid flowing plane. However, these sloped channel
devices need advanced 3D microfabrication techniques like two-photon-
polymerisation (2PP) [56]. The selection of the strategy adopted for the
elimination of bubbles depends upon the flexibility of its design, which
allows the incorporation of the above-mentioned concepts in the device.
The following section discusses how various disease pathologies and
phenotypes are mimicked inside a microfluidic device (Fig. 3).
3.2. Neurodegenerative disorders
The brain and spinal cord are the central nervous system compo­
nents. It is a collection of different types of cells that mediate neural
functions. Neurons, glial cells (astrocytes, microglia, ependymal cells
and oligodendroglia) and vascular cells, along with the extracellular
matrix, interact and accomplish various neural functions like nerve
impulse transduction, immunity, protection from external agents,
angiogenesis and nutrient delivery to neurons [57]. Disruption or dys­
regulation of these vital components or biomolecules within the
responsible pathways leads to neurological disorders [58]. The blood-
brain barrier is a specialised system that protects the brain tissue from
toxic materials from blood flow. It consists of astrocytes, pericytes and
endothelial cells. Disruption of BBB integrity has been linked to the
progression of neurological diseases like stroke and Alzheimer's disease
and contributes to cognitive changes [59–61].
The human brain is highly complex beyond the scope of recapitu­
lation using conventional in vitro and animal models, and this gap limits
the possibility of extensive neurological disease modelling. Microfluidic
devices hope to reduce this gap by providing more complex and tuneable
biomimetic in vitro systems. The challenge due to BBB’s restricted
permeability has drawn many researchers to investigate methods for
construction and integrity assessment of BBB models [62–64]. Primarily,
BBB on-a-chip devices are designed so that the neurovascular unit (NVU)
components are compartmentalised. In 2019, Vatine and their team
reported a BBB on-a-chip, incorporating human iPSC-derived tissue in
an organ-on-chip device. They successfully mimicked a physiologically
relevant BBB TEER measurement, organ level responses to neuro­
inflammatory signals, and permeability of soluble biomarkers [65]. A
combination of induced pluripotent stem cells in such systems can be
used explicitly for research in personalized medicines. Yu et al. intro­
duced a BBB on-a-chip device using primary neurovascular unit cells
from neonatal rats and induced neuroinflammatory conditions using
TNF-α under gravity-assisted flow. The addition of TNF-α compromised
the BBB functionality and was successfully mitigated by the glucocor­
ticoid drug dexamethasone [66]. Recapitulation of such diseased and
S. Amir et al. International Journal of Biological Macromolecules 235 (2023) 123784

Fig. 2. Schematic representations of different microfluidic devices used for disease diagnosis.

healthy BBB not only helps us understand the functional aspects of BBB In a study that explored the molecular mechanisms of Parkinson's
but also opens a more reliable therapeutic screen for neurological disease and other neurodegenerative diseases in the context of synu­
diseases. cleinopathies, two sets of experimental proof-of-concept; A co-culture of

7
S. Amir et al.
Fig. 2. (continued).
H4 neuroglioma cells and alpha-synuclein (a-Syn) producing cells and
co-culture of LPS-activated N9 microglial cells with H4 neuroglioma was
designed in a microfluidic cell culture systems. The former method was
used to assess the diffusion of a-Syn and the latter to monitor the
diffusion of ROS to the H4 cell chamber. This device provides a robust
system for understanding the interaction between different cell pop­
ulations via relevant proteins (a-Syn in PD, Tau in Alzheimer's disease)
diffusion and microglia-mediated neuroinflammatory responses [67].
Progressive loss of dopaminergic neurons is another hallmark of Par­
kinson's disease [68]. Moreno and his team developed a phase-guided
3D microfluidic bioreactor which facilitates the differentiation of
human neuroepithelial stem cells to dopaminergic neurons [69]. This
novel device can be used to model PD and developing personalised
medicines. In another study, a Particulate matter 2.5 polluted 3D
microfluidic human brain model demonstrated neuroinflammatory and
neurodegenerative conditions in the brain. The molecular mechanism
was unraveled due to the interleukin-1β and interferon-γ release by the
infiltrating microglial cells, leading to synaptic deterioration, phos­
phoric tau protein accumulation and neuronal damage [70]. To
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International Journal of Biological Macromolecules 235 (2023) 123784
summarize, recreating the pathophysiological conditions inside the de­
vices and characterisation of the disease phenotypes ensure validation
and robustness of a disease model.
3.3. Cancer models
Cancer is another condition that is highly specific in humans. Cancer
formation, progression and metastasis are critically influenced by the
local cellular and molecular microenvironment [71]. Complex in vitro
and in vivo cancer models have been proposed to study the effectiveness
of anticancer agents. Nevertheless, they fail to replicate the human
tumour microenvironment. Using microfluidic organ-on-chip technol­
ogy for cancer modelling has helped in solving these challenges by
providing tumour culture platforms with human physiologically rele­
vant physical, mechanical and biochemical microenvironment [72] and
the possibility of recreating tissue-tissue interfaces that are crucial for
the recapitulation of organ structure and functions [73,74]. Choi et al.
successfully reconstituted the 3-dimensional organisation and tumour
microenvironment of breast cancer in a microfluidic system. A co-
S. Amir et al. International Journal of Biological Macromolecules 235 (2023) 123784

Fig. 3. Flow of clinical research and various diseases models using microfluidics.

culture of breast tumour spheroids with human mammary ductal promote lung tumour cell adhesion on TNF-α-induced inflammatory
epithelial cells and mammary fibroblast cells was used to replicate the brain microvascular endothelium and extravasation. It was also reported
microarchitecture of breast ductal carcinoma [73]. Such models can be that this could be inverted by Rho/Rho-associated protein serine/thre­
used to understand the role of microenvironments in cancer progression onine kinase (ROCK) inhibitor (Y27632) [81,82].
to invasive forms. Another chip designed to emphasise the influence of
the microenvironment on tumour growth was co-cultured T47D human
3.4. Cardiovascular diseases
breast carcinoma cells and human mammary fibroblasts (HMFs) along
with different combinations of ECM materials. This study revealed that
Cardiovascular networks and associated cells are susceptible to
the co-culture of HMFs in a fibronectin-rich environment enhances
environmental biochemical and mechanical cues. These signals mediate
breast cancer growth compared to monocultures [75].
the development and normal functioning of blood vessels. Endothelial
Angiogenesis is another crucial step in the progression of hyperplasia
cells lining the blood vessels change their characteristics and feature like
to neoplasia. The high nutrient requirements of the growing tumour are
phenotypic adaptations, permeability, angiogenesis, blood vessel
met by this outgrowth of blood capillaries [76]. One typical design
regression etc., all under dynamic conditions. The biophysical parame­
followed in microfluidic devices to study angiogenesis is multiple par­
ters highly influence the basic physiology and pathophysiology of the
allel channels separated by micro pillars, which allow the diffusion of
cardiovascular system and cardiovascular diseases [83,84]. While static
biochemical signals and have space for migration cells in response to
cultures fail to provide the hemodynamic environments, microfluidic
specific signals. Kim et al. observed that the human umbilical vascular
systems can replicate the exact biophysical cues and cardiovascular
endothelial cells (HUVECs) show enhanced growth when co-cultured
architecture.
with malignant human U87MG glioblastoma cells in their microfluidic
Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is heart
device. The microvessels formed from the co-cultures HUVECS showed
disease caused by aggregation of amyloid fibrils, and a TTR valine-122
vigorous branching, apparently due to some factors secreted by the
to isoleucine (TTRV122I) is a predominant hereditary background of
cancer cells [77]. Such studies give insights into the angiogenesis around
this disease. In a study, Williams and his team developed a 2 chamber
tumour cells. The mechanical forces on tumour cells also play a role in
microfluidic device populated with a co-culture of TTRV122I expressing
their progression. For example, It is reported that mechanical actuation
hepatic organoids and human iPSC-derived cardiomyocytes. Through
like breathing motion upon tumour cells can suppress cancer cell growth
microchannels connecting the two chambers, the TTRV122I is taken up by
and invasion and become more resistant to therapeutics. The micro­
the cardiomyocyte to exhibit ATTR-CM features. This device mimics
fluidic organ chip in vitro human orthotropic model populated with non-
other vital elements of ATTR-CM like diastolic impairment and con­
small-cell lung cancer recreated the therapeutic responses to the tyro­
duction system disease [85]. This platform can be used to understand
sine kinase inhibitor [74].
further in-depth molecular interactions, genetic regulations and poten­
Metastasis models are another critical area where possibilities of
tial drug screenings. Modelling myocardial ischemia is another exten­
microfluidics have been explored. Primary tumour cells invade through
sively studied cardiac condition where ischemic conditions are recreated
multiple layers of cells and intravasate to blood vessels or the lymphatic
by culturing cells in hypoxic conditions. Veldhuizen et al. presented
system. Then they extravagate at a distant organ interstitium to establish
myocardial ischemia on-a-chip using 3D cardiac tissue culture in hyp­
a metastatic tumour [78]. The effect of the microenvironment on the
oxic conditions and reported validation extensively by molecular level
process of metastasis of lung cancer cells (H1299 lung adenocarcinoma)
analysis [86].
was studied using a three-channel microfluidic device. Different com­
For many diseases, the accuracy of disease models is a limiting factor
binations of collagen and Matrigel provided the variations in the
in drug development. Myocardial infarction or heart attack primarily
microenvironment. It was concluded that at low concentration, Matrigel
results from occlusion in the arteries by platelet-rich thrombus. Anti-
facilitated migration, and at high concentration, the migration was
thrombotic drugs are used to treat such conditions, but there is still a
slowed down [79]. Wang et al. built a 3D microfluidic cell culture model
high risk of extensive bleeding, so the usage of such medications is
to investigate the formation of invadopodia and the mechanism of lung
limited. Arterial thrombosis-on-a-chip is an alternative system that
cancer metastasis [80]. Similarly, Xu et al. presented a simple micro­
generates occlusive thrombi by quenching downstream coagulation. The
fluidic device to study the biochemical and mechanical parameters that
study validated the device using eptifibatide as a positive control [87].

9
S. Amir et al. International Journal of Biological Macromolecules 235 (2023) 123784

Similarly, a particular type of microfluidic device consisting of micro­
channels with a hump-like structure is used to recreate stenosis [88].
Another stenosis chip modelled point-of-care blood profiling device for
atherosclerosis and other associated vascular diseases were also re­
ported to study vascular inflammation and leukocyte-endothelial
interactions.
3.5. Respiratory disorders
Modelling respiratory system physiology in microfluidic devices has
been approached by recreating alveolar-capillary barriers [89,90], small
airway models [91,92], the mechanical strain of breathing [89], and
cellular interactions in the alveoli [93,94]. Lung-on-a-chip is a well-
studied organ-on-a-chip device that has fairly translated all these
physiological processes and established the microenvironment of the
respiratory system [95].
One of the pioneering works of the lung-on-a-chip disease model was
established by Huh et al.; the pulmonary oedema was replicated by
supplying pro-inflammatory cytokines (IL-2) through the microvascular
channel of the device. The device consists of an alveolar-capillary
interface and two hollow chambers on both sides of the culture chan­
nel to mimic the mechanical breathing motion. They found that the
mechanical forces experienced by the cells during normal breathing
influence the rate of vascular leakage and subsequent pulmonary
oedema. This disease model is also used to identify new potential drug
molecules. In a similar design, another pulmonary infection model of
tuberculosis was developed by Thacker et al., in which mouse lung cells
(alveolar epithelial cells) and immune cells (macrophages) were
cultured. The epithelial cells were infected with Mycobacterium tuber­
culosis, and they found that in the absence of surfactants, the bacteria
grow more rapidly and uncontrollably in both epithelial cells and
macrophages [96].
An alternative approach to mimicking the pathophysiology of ge­
netic disorders like cystic fibrosis (CF) is to populate the microfluidic
device with specific primary human cells. Plebani et al. successfully
recapitulated cystic fibrosis physiology in a CF airway chip. The chip
houses patient-derived cystic fibrosis cells in an air-liquid interface and
microvascular endothelial cells under flow. The features like increased
mucus secretion, cilia density and ciliary beating frequency were
replicated in this device [97]. Such devices provide robust platforms for
pathophysiological studies, therapeutic screening and personalised
medicine. Similarly, another work adopted this method for modelling
idiopathic pulmonary fibrosis by incorporating fibroblasts from IPF
patients and healthy fibroblasts treated with TGF-β1 in their devices.

Table 2
Some of the recent disease models in microfluidic platforms.
Organs Diseases Cells used Pathophysiology on chip References

Lungs Chronic Obstructive Bronchial epithelial (BEAS-2B) Increased expression of microRNA-21 and microRNA-34a by [99]
Pulmonary Disease (COPD) inducing oxidative stress using hydrogen peroxide
Idiopathic Pulmonary HPAEpiC (human pulmonary alveolar epithelial TGF-β treatment [100]
Fibrosis(IPF) cell) and HFL1 (human fetal lung fibroblast)
Pulmonary fibrosis Primary normal human lung fibroblasts and TGF-β1 treatment [101]
human lung small airway epithelial cells
SARS-CoV-2 Human primary microvascular endothelial cells, 64-chip microfluidic plate-based platform with two novel [102]
fibroblasts and pericytes vascularized, lower respiratory tract multi-chip models for the
alveoli and the small airway.
Primary ciliary dyskinesia Human-derived Induced pluripotent stem cells Patient derived iPSCs [103]
(iPSC)
Liver Hepatic fibrosis Hepatocyte TGF-β1 treatment [104]
cell line
(HepG2) and human fibroblasts cell line (Hs68)
Hepatic fibrosis Human hepatic stellate cell line (LX2), Primary 100 Mm alcohol treatment on hepatocytes to induce alcohol [105]
hepatocytes from adult female Lewis rats injury and consequent stellate cell activation
Heart Cardiac fibrosis Human cardio fibroblast and human induced TGF-β1 treatment [106]
pluripotent stem cell-derived cardiomyocytes
(hiPSC-CMs)
Transthyretin (TTR) Human iPSC derived cardiomyocyte (hiPSC-CM) Expressing TTR valine-122 to isoleucine (TTRV122I) on hepatic [107]
amyloid cardiomyopathy and human hepatic cell organoid organoid.
(ATTR-CM)
Myocardial infarction Human blood cells Occlusion was created by controlling the blood flow rate <0.001 [87]
mL/min for 3 min or more.
Atherosclerosis Human blood cells By fabricating stenosis at the porous connecting region by either [108]
reducing the width or increasing the height.
Kawasaki disease Human blood cells Collagen coated Total thrombus-formation analysis system (T- [109]
TAS)
Bone Osteoarthritis Human bone marrow-derived adult Chondrogenesis and osteogenesis of Mesenchymal progenitor [110]
mesenchymal stem cells (MSCs) cells derived from Induced pluripotent stem cells.
Infectious Jaundice Human foreskin fibroblast, lung Supplementing bilirubin via vascular channels of the Multi- [111]
disease adenocarcinoma cells, human foetal hepatocyte, chamber Organ-on-a-chip.
Human umbilical vascular endothelial cell.
Type 2 Diabetes mellitus HepaRG liver spheroids and human pancreatic Developed a permeable cross talk between insulin producing [112]
(T2DM) islet microtissue in human stellate cells. pancreatic islet cells and liver spheroids.
Cancer Human-glioblastoma Human glioblastoma cells Bio printed glioblastoma cells collected from patient. [113]
Renal Carcinoma (RC) Clear cell renal cell carcinoma (ccRCC) with Derived ccRCC from six different donors. [114]
human endothelial cells
Brain Alzheimer's disease (AD) Human microglia SV40 cell line, human neural Seeded human neural progenitor cells with over expression of [115]
progenitor cells and human astrocyte cells. APP and PSEN1 with FAD mutation and microglial cells to induce
neuroinflammation via proinflammatory cytokines
Parkinson's disease (PD) Yeast PD model (Process of PD formation is via Over expressing galactose-inducible promoter to produce [116]
α-synuclein inclusion formation is evolutionarily α-synuclein inclusions.
conserved)
Amyotrophic lateral hESC–derived NSCs, ALS-iPSC–derived NSCs, Treatment of glutamic on human motor unit to induce [117]
sclerosis (ALS) Human iPSC-derived Endothelial cells and excitotoxicity which mimic ALS pathophysiology
Human iPSC-derived skeletal myoblasts

10
S. Amir et al.
This system successfully mimicked disease phenotype like fibrosis-like
transformation in cells. The same platform was also used to model the
neutrophil recruitment phenotype of cystic fibrosis [98]. Table 2 sum­
marises some of the recent disease models in microfluidic platforms.
4. Applications in drug development
The clinical failures of multimillion-dollar invested drugs due to
these inaccurate disease models attracted the drug industry to micro­
fluidics. With the possibility of establishing genetically unique disease
models with organ-on-chip devices and the concept of precision medi­
cine, the pharmaceutical field explored a more effective drug develop­
ment pipeline. The advancements in the field of genomics, chemical
engineering, High-throughput screening methodologies and micro­
fluidics have benefited the pharmaceutical industry by providing more
reliable and relevant systems for testing. High-throughput assay are
conventionally done in microtiter well plates, where the drug potency is
tested by adding it into target cells or target compounds. It takes days to
complete the readout in all wells. With the wide applications of micro­
fluidic two new approaches has been made; droplet microfluidic
approach and single phase microfluidic approach, where the concen­
tration gradient generated in a diffusion controlled manner. Rather than
taking the whole cells for potency screening, a more targeted approach is
to study the effect of the drug on a specific molecular pathway or ac­
tivity. But one of the limitations of this approach is that it does not
reflect interference of other biochemical and mechanical cues from the
cell and also the cellular response to the drug. Cytotoxicity analysis is
another aspect of drug development. From the previous sections it is
well clear that organ-on-chip devices can provide better, faster and cost-
effective models for organ level cytotoxic analyses [118,119].
4.1. Drug screening using microfluidic devices
Drug efficacy testing and toxicity screening are very well established
applications of microfluidics. The advantages of microfluidic systems in
the side of human relevance make these approaches more competent. A
detailed study conducted in 2013 shows promising results that the
efficacious concentrations of epithelial-mesenchymal transmission
(EMT) inhibitor determined using the microfluidic system is more closer
to values obtained from human clinical trials than 2D conventional
cultures. Aref et al. tested 12 drugs ranging from those that are in early
phase of preclinical trail and those that has FDA approval [120]. Similar
observations have been made by other studies like [121,122].One of the
causes of post-clinical trial failure of drugs is the unpredicted adverse
effects in humans. As it is already stated, animal models fail to represent
humans in its entirety, so complications in prediction of toxicities should
be expected. Drug metabolism in liver tissues is another concern that
leads to unexpected toxicities in humans. Rightfully, a focus has been
given on the development of liver-on-chip devices. Studies have been
reported combining the metabolomic approach on liver-on-chip devices.
Coupling specifically the liver-on-chip devices with NMR spectroscopy
could give not only details of metabolic pathways involved in drug re­
sponses but also helps to reduce the noise in in vivo omics analysis
[123]. Heart-on-a-chip device was developed and studies using different
doses of isoproterenol [124]. Similarly, other organ-on-a-chip devices
have been developed for toxicity screening of new drugs [125].
5. Microfluidics for drug delivery
Nanoformulations that are designed with specific size provides
several advantages such as improved drug encapsulation, increased
cellular uptake, controlled and accurate release of drugs and proper
targetability [126]. Encapsulation of active ingredients such as anti­
cancer drugs inside the nanocarriers can improve their stability in vivo
and also enhance blood circulation period [127]. Regardless of these
advantages only limited number of nanoformulations are used for
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International Journal of Biological Macromolecules 235 (2023) 123784
clinical trials because of technical hurdles in formulating several phar­
maceutical materials [128]. Depending on the nature and composition
of drug delivery system (DDS) fabricated the nanostructures are made by
techniques such as emulsification, self –assembly or precipitation
[81,129,130]. Technique frequently used in the formation of drug de­
livery system with controlled size include mixing different solutions, an
amphiphile solution with polyelectrolyte solution. Examples of amphi­
phile solution include liposomes or micelles and that of polyelectrolyte
solution include nucleic acids or cationic polymers [131–133]. As a
result of hydrophobic interactions or electrostatic interactions the DDSs
will either precipitate or self-assemble. Regardless of great potential
exhibited by nanosized DDSs in vitro they displayed slow translation to
applications in clinical sector. This is mainly because of many challenges
faced such as large quantity production, and reproducibility of the sys­
tem [134]. The microfluidic system played a great role to overcome
most of the challenges faced. The advantages of using microfluidic
system to fabricate DDSs include the ability to control mixing, low cost
and modification capability [135]. The microfluidic devices provide a
better platform for preparation of different types of DDSs such as lipo­
somes, polymeric and protein based nanoparticles, and multiple emul­
sions [136–139].
6. Strategies for managing formation of DDSs
Amphiphilic copolymers, polyelectrolytes etc. are polymeric mole­
cules which can form nanostructures by precipitation or either by self-
assembling via changing the quality of the solvent by mixing. The size
as well as size distribution of the particles produced is influenced by
rapid mixing of solvent with the antisolvent. Unlike the methods used
for mixing conventionally which normally require time period of sec­
onds, the utilization of microfluidic devices permits controlled rapid
mixing of solvent and antisolvent within the time scale of milliseconds.
Several microfluidic devices are utilized to prepare lipid nanoparticles
via organic solvent injection methodology. The polarity of the solvent
rapidly increases at liquid-liquid interface due to molecular diffusion
thereby forming lipid nanoparticles. Several studies conducted have
shown that microfluidic device design and dimensions on manipulation
affects the properties of lipid nanoparticles and also molecular diffusion
[140–145]. Different microfluidic devices utilize different designs that
bring changes in mechanism of particle formation.
6.1. Hydrodynamic flow focusing
Hydrodynamic Flow Focusing (HFF) is a mixing technique which is
used for the continuous production of several types of DDSs like lipo­
somes, nanoemulsions as well as polymeric nanoparticles. This method
relies on dispersed phase stream which normally contains lipids or
polymeric materials that flow through microchannel at the centre to
come across streams of continuous phase. The mixing of the two streams
occurs at confined areas which lead to formation of droplets providing
conditions like controlled laminar flow. The diameter of the central
channel can be changed to regulate the mixing time in HFF. Different
HFF designs have been formulated to control properties of nano­
formulation produced. HFF devices are either two dimensional or three
dimensional [146–149]. Hood et al. developed three dimensional HFF
for liposome synthesis. The two dimensional HFF devices contain hori­
zontal channel surfaces at the mixing regions and the three dimensional
approach removed such surfaces. The HFF microchannel design regu­
lates the mixing time needed for complete mixing in such devices [150].
6.2. T-Mixer
One of the earliest and simple type geometric designs of microfluidic
device includes T-junction. In these devices the mixing of Organic sol­
vent with the aqueous phase occurs in a perpendicular basis. In some
designs Y-like shape is formed when two inlets of the microfluidic device
S. Amir et al.
unite at Sharpe angle i.e. <90◦ . Coflowing channels are utilized to form
droplets which can be formed as either water in oil(W/O), or oil in water
(O/W) type or as double oil in water in oil (O/W/O) type. In devices with
Y shape the diffusion rate is the key factor that affects mixing and it
occurs in the surface of main channel at aqueous/solvent interface. T-
mixer design can be utilized for the preparation of different types of
nanoformulations which usually provide a mixing range from laminar to
turbulent regime [151–155]. Gunther et al. proposed a strategy known
as segmented gas liquid flow strategy which has the capability to
enhance the efficiency of mixing two miscible liquids inside the micro­
channels of the microfluidic device [156].
6.3. Multi inlet vortex mixers
Large scale production of the polymeric particles is facilitated by
multi inlet vortex mixers via rapid nanoprecipitation. This system uti­
lizes four fluid streams that involve two aqueous solutions and two
organic solvents. The microfluidic device will have different inlets
through which the solutions enter the mixer and it enables highly effi­
cient mixing of components. On comparison with T-mixers the Multi
inlet vortex mixers exhibit more conversion efficiency and high yield of
the product [157], [158].
6.4. Staggered herringbone and toroidal mixers
To physically stir the fluids the active mixers mainly rely on external
sources. But this is different in case of passive mixers. Passive type
mixers facilitate mixing via different mechanisms such as fluid lamina­
tion, hydrodynamic manipulation, and sequential combing and splitting
in the microfluidic device microchannels. Stroock et al. developed
Staggered Herringbone micromixer which is a creative design of the
microchannel developed as a passive mixer. In comparison to other
types of designs with identical dimensions, this design exhibits negli­
gible flow resistance and greater rate of diffusion. Another adder
advantage of the design is that it can produce DDSs with homogenous
particle size via fast and refined kind of mixing performance [159–163].
Another type of mixers are toroidal mixers where mixing of the fluids are
attained by chaotic advection caused by producing forces such as cen­
trifugal forces inside the microchannels [164].
7. Types of drug delivery systems
7.1. Protein based Nano carriers
Synthetic polymers used in developing DDSs face challenges such as
potential toxicity and lack biodegradability. So researchers tend to uti­
lize natural biomaterials as an alternative method to create different
types of DDSs [165].In order to prepare uniformly sized microparticles
of gelatin Hardin et al. formulated a capillary based microfluidic device.
They act as template for DNA functionalized polystyrene adsorption that
in turn facilitates the release of DNA under several thermal conditions
[166]. Wongpinyochit et al. investigated influences of several micro­
fluidic device parameters such as total flow rate and flow rate ratio on
the particle size and shape of silk fibroin. The silk fibroin particles were
produced via desolvation method with the help of two organic solvents
such as acetone and isopropanol. The high ratio maintained of organic
solvent to aqueous solution in combination with least total flow rate
exhibit a significant higher yield in comparison with other conditions.
The properties such as size of the particle, charge at the surface and
polydispersity can be regulated via changing the organic solvent and
also by regulating total flow rate almost 1-12 mL/min and flow rate ratio
as either 1:1, 3:1, 5:1 [139].
7.2. Double and multiple emulsions
Emulsions are formulated via mixing liquid phases that are
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International Journal of Biological Macromolecules 235 (2023) 123784
immiscible in nature. One of which is referred to as dispersed phase and
other as continuous phase [161,167].Multiple emulsions are one of the
common formulations in delivering the drug which is based on forma­
tion and stabilization of droplets within another droplet. Double emul­
sions are formulated when small droplets are arranged or nested one
inside the other [168]. Conventional emulsification techniques have
several limitations that include variability in structure, lack of consis­
tency in encapsulation efficiency [169].These limitations were over­
come by the researchers via developing an alternative protocol with the
help of microfluidic technology referred to as droplet microfluidics. This
technique helped in the formulation of multiple emulsions for the pro­
duction of particles uniform in nature at a single stretch. β-carotene
loaded liposomes were formed using capillary microfluidic device by
Michelon et al. The liposomes thus produced showed high stability and
manageable size distribution [170].
7.3. Lipid based nanoparticles
The nanoparticles that are lipid based in nature that includes lipo­
somes function as drug delivery system as they have the ability to load
the particles inside the lipid shell [171]. Conventional type lipid based
nanoparticles have several advantages such as biocompatibility and
high loading efficiency but to enhance the clinical applications further
improvements are required [172]. Surface modifications are the com­
mon type strategy used to improve lipid nanoparticle formulations that
in turn increase its stability, time of retention in blood and capability of
delivering drugs to the targeted tissues [173–175]. The principle utilized
in the preparation of lipid nanoparticles via microfluidic platforms is
same as that of some of conventional methodologies used such as
organic solvent injection and hydration [176]. The microfluidic tech­
nique used to form lipid nanoparticles not only produced uniform sized
particles but also decreased the time of production [177]. The study
carried out by Nakamura et al. utilized a herringbone chaotic mixer to
produce Ph-sensitive type lipid nanoparticles which is of 30, 100 and
200 nm for specifically targeting the lymph nodes. The lipid nano­
particles formulated of small size that is of 30 nm showed higher tran­
sitivity to lymph nodes when given subcutaneous injection in mice. The
negatively charged lipid nanoparticles were transported efficiently to
lymph nodes compared to neutral and positive charge particles [178].
7.4. Polymeric and hybrid nanoparticles
Several conventional type methods can be used in preparing polymer
based micro and nanoparticles that includes cross linking, evaporation
of solvent, and mixing of solvent and anti-solvent solutions [179]. As
discussed in case of other types of nanoformulations the polymeric
particles can also be synthesized by microfluidic system by rapid mixing.
The most common type of microfluidic system used to produce poly­
meric particles for the purpose of delivering different types of drugs
include HFF and droplet based methodologies [180].The production of
spherical and nonspherical based microparticles are carried out with the
help of droplet based microfluidics through cross linking of ions to
modify the kinetics of drug release [181]. The β-carotene-Pluronic F127
was fabricated using HFF microfluidic reactor via using rapid nano­
precipitation [182]. Another study conducted utilized a T mixer system
to produce Janus nanoparticles which is comprised of PLGA through
rapid nanoprecipitation [183].Spherical polyester type nanoparticles
were prepared in the study conducted by Hong et al. using a herringbone
mixer. The particles produced using the system has a dual enzymatic
stimuli responsive polyester because of ester bonds and sulfide linkages
which is fabricated by microfluidic system [184].
7.5. Magnetic nanoparticles
Microfluidic chip that offers controllability and automation when
merged with magnetic nanoparticles having unique capabilities have
S. Amir et al. International Journal of Biological Macromolecules 235 (2023) 123784

provided enormous progress in biotechnology in recent times. Such
microfluidic chips act as high throughput and portable devices for
contactless detection and manipulation of molecules such as viruses,
RNAs, DNAs and living cells. The methods adopted to synthesize
microfluidic magnetic nanoparticles are continuous flow based and
droplet-based techniques using microreactors. The continuous flow
concept used in the synthesis of nanoparticles reduce the chance of
coalescence [185]. One of the challenges faced in drug delivery is the
rapid clearance of the nanoparticles in the body. So, nanoparticle drug
delivery in a targeted manner to cells like cancer cells has great promise
for increasing their cellular uptake. Different types of drug releasing
particles are developed by researchers using microfluidic chips [186].
Controlled drug release is enhanced by particles such as multi stimuli
responsive particles. The temperature change or the pH change or both
can be used to release drugs. Capillary based droplet microfluidic device
is used in the formation of such particles encapsulating magnetic
nanoparticles. Under the influence of an external magnetic field the
microfluidic magnetic nanoparticles formed is carried to the desired
sites and the capsules are given site specificity properties [187]. Studies
with non-spherical hydrogel microparticles encapsulating magnetic
nanoparticles have also reported. T- junction droplet based microfluidic
chip is used in the generation of such droplets and the droplets took the
shape of the microchannels in the device. UV photopolymerization is
used to fix the shape of the particles [188]. Another study conducted
used L-junction microfluidic device to synthesize magnetic alginate
microparticles and chitosan-coated magnetic alginate for controlling the
drug release. The microfluidic approach helped in achieving uniform
size distribution, adjustable diameter and tunable magnetism. Here
amoxicillin is used as the model drug, pH sensitive drug release and
particle size effect on drug release was also investigated through this
device. It was proved that the smaller particles show faster release when
compared to bigger materials [189].
Organ on chip technology has the potential to provide an alternative,
cost-effective, efficient and fast platform for different therapeutic ap­
plications, from drug screening, safety and efficacy analysis to drug
delivery systems. Table 3 summarises some of the microfluidic devices
that has application in drug delivery. Different types of DDSs have their
own advantages and disadvantages because of their physical and
biochemical properties, these are listed in Table 4. Selection of suitable
DDSs depends upon their specific application. Exploiting newer tech­
nologies and an interdisciplinary approach has proven to be vital in
translating these application into a larger market space.
8. Challenges in translation of microfluidic devices
As the humankind has witnessed a deadly pandemic over the past
two years, the need for advanced diagnostic procedures has become
highly essential for better healthcare. The pandemic had made the world
realize that more than half of the world population still receive inade­
quate healthcare and the other half need to improve the efficiency of
existing healthcare systems. The conventional lab based diagnostic
procedures are insufficient especially for emerging infections that
spread easily among the population in the blink of an eye. Also the
conventional diagnostics deprive time out of patients which can be a
crucial factor between a live or die situation. The development of sub­
stantial technology and tools for diagnostics and scaling up of these
testing procedures need to be done for providing state of the art health
care facilities especially during an epidemic outbreak that can risk the
life of populations.
Microfluidic systems have been integrated with biology for various
applications including DNA Analysis [229], cell handling [230]and
sorting [231,232], protein based applications [233], chemical analysis
[234], and immunoassays [235]. The emergence and development of
lab-on-a-chip has elevated the application of microfluidics in the field of
biotechnology. The microfluidic based cell culture techniques, espe­
cially the organ-on-a-chip applications, have the ability to enhance the
conventional cell culture systems and provide a wider perception of the
cellular functions that otherwise cannot be studied in a conventional
systems. The physiological and molecular functions that are difficult to
identify with the existing culture techniques using plastic dishes can be
elaborately studied with the dynamic cell culture happening in the
microfluidic organ-on-a chip designs. The organ on a chip system can be
employed to investigate the intra cellular and intercellular processes,
model diseases and develop drugs. However keeping in mind the po­
tential advantages of the microfluidic cell culture systems, lab-on-a chip
or organ on a chip as we call it, there is still need to address the elephant
in the room – the translation of the organ on a chip applications.
The conventional practices of cell culture, studying the cellular

Table 3
Summary of some of the microfluidic devices used for drug delivery applications.
Microfluidic devices Drug delivery systems Loaded materials Application References

Staggered Herring bone micromixer Lipid nanoparticles Doxorubicin Pharmaceutical area [190]
Herring bone micromixer Lipid nanoparticles SiRNA DNA Gene silencing and gene delivery [191–194]
Hydrodynamic flow focusing, Herring bone Liposomes Doxorubicin, Propofol, Gene delivery and gene silencing, tumour therapy [195–199]
micromixer and on-chip micro dialysis Vincristine, irinotecan, DNA application, used for inducing and maintaining
sedation
Hydrodynamic flow focusing, Hydrodynamic Polymeric Paclitaxel, Aminated retinoic Used for tumour therapy and anti-inflammation [200–204]
focusing combined with tesla micromixer nanoparticles acid
Rapid microfluidic mixing Polymeric clindamycin phosphate, Transdermal delivery [205]
nanoparticles tretinoin
Rapid microfluidic mixing Polymeric Docetaxel Drug discovery [206]
nanoparticles
Rapid microfluidic mixing Hybrid nanoparticles Doxorubicin Anticancer drug [207]
Glass capillary microfluidic device Microcapsules Rhodamine B, Insulin Glucose responsive release in a controlled manner [208]
Cross junction channel microfluidic chip Microcapsules Ampicillin, Diclofenae Dual drug carriers [209]
3D-Hydrodynamic flow focusing Polymeric Dexamethasone Enhanced drug loading and controllable particle size [210]
nanoparticles
T-mixer Small-molecule Ibuprofen Controllable size and narrow size distribution [211]
nanoparticles
T-mixer Janus and monophasic Paclitaxel and doxorubicin Multiple payloads and modified drug release profile [212]
nanoparticles
Y-junction Amorphous Cefuroxime axetil Enhanced drug dissolution rate and controllable size [213]
nanoparticles
Y-junction Small-molecule Danazol Enhanced drug dissolution rate and controllable size [214]
nanoparticles
Y-junction Lipid NPs pDNA Uniformly coating GO sheets with lipid layers that is [132]
suitable for gene delivery

13
S. Amir et al.
Table 4
Common advantages and disadvantages of different drug delivery system.
Drug delivery Advantages
systems
Emulsions Capable to encapsulate molecules such as antimicrobials, vitamins,
anti-oxidants which are hydrophilic and lipophilic.
Microfluidic devices can be used to generate droplets which are size
controlled and uniform.
Microfluidic devices used to generate emulsions can precisely
manipulate both size and number of encapsulated droplets via
controlling flowrate.
Lipid based Controlled drug release
nanoparticles Lipid based nano particles such as liposomes are most suitable or
drug delivery due to specific arrangement of lipids that mimic cell
membrane
Ability to transfer both hydrophilic and hydrophobic molecules.
Microfluidic devices are used to generate size controlled, lipid based
nano particles
Less processing time to generate liposomes and remote drug loading
was achieved with the five times greater drug-to-lipid ratio using
microfluidic devices.
Polymeric Greater storage stability
nanoparticles Non- toxic
It is biocompatible and biodegradable
Exhibit potential to deliver both genes macromolecules
Drug release in a controlled manner can be achieved by polymer
modification technique.
Prevents premature degradation by protecting active
pharmaceuticals
Microfluidic technology used in polymeric nano particle synthesis
permit rapid formation of libraries of nano particles which poses
different size, surface charge etc.
Microfluidic polymeric synthesis of nanoparticles has several benefits
that include better morphology, size distribution and size controlled
formation.
Protein based They exhibit enhanced loading capacity of different drugs as they
nanocarriers possess multiple binding sites.
Protein nanoparticles enable targeted delivery and also enhanced
uptake of drugs into brain, macrophages and liver.
Surface modification is possible due to various functional groups
present on the surface of the nanoparticles which in turn enable
targeting drugs to specific site of action.
Drug releases can be sustained for prolonged duration.
interaction and other biotechnological research protocols came into
being after years of research and practices. Removing these traditional
systems and integrating the microfluidic based lab-on-a chip devices for
the healthcare applications like diagnostics and disease modelling could
take another one or two decades and number of experiments. The design
development and fabrication of diagnostics is a very tedious process and
there need to be a lot of factors taken into consideration while defining
these products. The testing, validation and regulation of the microfluidic
based devices that are intended to be used in various biomedical and
healthcare applications would take immense efforts, money and time in
the beginning. The valleys of death associated with the approval of these
devices that incorporates microfluidics and miniature devices need to be
addressed carefully.
9. Need for validation of microfluidic cell biological assays
Microfluidic based lab on a chips have been widely studied for its
application as diagnostic tools for various infections and diseases. The
limitations of current in vitro methods, especially the replication of the
micro physiological interactions, paved way for the introduction of
microfluidic based cellular assays thereby accelerating the relevance of
cell culture methods. Microfluidic systems are capable of controlling the
temporal [236] and spatial parameters [237] that are critical for eluci­
dating the cellular and subcellular level interactions. These parameters
could be used the analysis of even the intricate process of cell division
[238] and cell survival. The evolution of microfluidics since the last
decade has been tremendous especially in the field of lab-on-a-chip and
organ-on-a-chip studies. However apart from the basic research and
14
International Journal of Biological Macromolecules 235 (2023) 123784
Disadvantages Reference
Thermodynamically unstable [215–217]
Low shelf-life
Phase inversion is caused if surfactants are improperly selected.
Large emulation particles are usually formed through microfluidic
technology which are unfavorable for many applications.
Stability issues [216];
Low encapsulation efficiency [218–220]
Some cationic formulation causes cytotoxicity
Microfluidic formulation of liposomes or lipid-based nanoparticles
Purification processes are required for natural polymeric [221–225]
nanoparticles.
National polymeric nanoparticles do not exhibit batch to batch
uniformity.
Protein based nanoparticles from animals cannot exhibit sustained [226–228
drug releases due to high solubility in aqueous environments because
of their hydrophilic nature.
Natural protein polymers are heterogeneous molecules or
heterogeneous mixtures of various sizes and so they exhibit batch to
batch variability.
development that is being occurring, microfluidic based cell biology
assays are not able to come out from the shell due to various reasons.
Microfluidic based diagnostics require the successful integration of the
engineering as well as biology background to obtain relevant results.
Often these integration fail at different levels and the system as a whole
is not yet developed into a fully-fledged application. The validation of
the microfluidic based cell biology assays and other diagnostic are very
important to be used in a large scale among the population rather than
the basic research and this scaling up is hindered by various factors from
the development the commercialisation of the products [239]. There is a
high time need to establish the pipeline from the development to the
feasibility of the microfluidic devices. Data interpretation and the
fundamental physical factors involved in the microfluidic micro envi­
ronment needs to be carefully monitored to elucidate the technology at
its maximum potential (Fig. 4). The many examples of microfluidics
based cellular assays are converging on the analysis of the behaviour of
cells in the microfluidic devices with the incorporation of certain soluble
factors in a controlled gradient. Exploring the conventional systems to
include a microfluidic assays would provide a simplified platform for
diagnostics.
The organ-on-a-chip platforms was intended to recapitulate the
micro physiological systems by combining the cell culture, fluidic sys­
tems and analytical methods to establish new in vitro models that could
closely mimic the in vivo conditions. The major factors that excited the
scientist all over the globe was the efficient and much cheaper tools that
could be developed out of these miniature systems without sparing large
volumes of analytes and other requirements that would otherwise be
needed in large quantities. With this agenda, the scientists, technicians
S. Amir et al.
Fig. 4. Translation of microfluidics.
and the companies researched and developed a number of designs of
organ on a chip that could mimic various cellular functions and could be
successfully used for elucidating the physiology pharmacology and
toxicological aspects. However the need for optimisation and validation
of these miniature devices are far way far in line. To be established
among the existing in vitro assay models, organ on a chip would require
extensive optimisation and validation that could strongly support their
in vitro analysis efficiency [240].
10. Conclusion
Microfluidics based disease modelling and diagnosis has witnessed
an immense growth since the last two decades even though the MEMS
technology was introduced to the scientific community way back in
1960s. The scientific arena from the basic biological research to the
complex biological interactions including disease modelling and drug
delivery is utilizing microfluidics to develop better tools for biomedical
research. The advantages of microfluidics including its precision, highly
ordered, controllable flow systems, and the requirement of smaller re­
agent volumes makes them powerful tools for disease modelling and
diagnostics. The ability of these devices to be miniaturized and portable
establishes added advantages for being the better candidates for the
point of care devices also. The microfluidic devices that are manufac­
tured for disease modelling and diagnostics must be highly sensitive,
specific, robust and provide better results as compared to the conven­
tional systems. Nevertheless, there are many challenges that need to be
addressed while integrating microfluidics to the diagnostic develop­
ment. The scaling, optimisation and validation of the devices need to be
carefully and strictly addressed for establishment of a successful system.
The insufficiency of regulatory quantitative analysis methods deprive
these devices of proper validation and optimisation of the data collected.
There need to be advanced systems for elucidating and validating the
results published from the microfluidic device based assays so that they
compete inch by inch with the traditional disease models and
diagnostics.
Ethical approval
This is a review articles. Appropriate approval/Ethical approval is
taken.
15
International Journal of Biological Macromolecules 235 (2023) 123784
Consent to participate
Consent part from the subject is not applicable for this review article.
Consent or publication
All the authors agreed to submit the manuscript in the journal. The
same has approved by the parent Institute.
Funding
There is no funding from outside agencies. The infrastructure support
is provided by the parent Institute.
CRediT authorship contribution statement
Amir, Arathi and Reshma: Investigation, Methodology, Formal
analysis, Resources, Data collection, Writing -writing, original draft,
Editing.
Mohanan: Conceptualization, Supervision, Project administration,
Funding acquisition, Data analysis, final correction, editing and
approval.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
The authors declared that the research data referred to correctly
cited in the manuscript's reference section.
Acknowledgements
The authors wish to express their thanks to the Director and Head,
Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical
Sciences and Technology (Govt. of India), Trivandrum, Kerala, India, for
their support and for providing the infrastructure/support to carry out
this work. Amir and Reshma thank the CSIR, New Delhi and Arathi
thank the DST, New Delhi for Junior Research Fellowship. Mohanan
S. Amir et al.
thank Department of Science and Technology, Govt. of India, New Delhi
for financial support (DST/TDT/DDP-04/2018(G)).
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