Accepted Manuscript

Title: Biochemical analysis on microfluidic chips

Author: Jing Wu, Ziyi He, Qiushui Chen, Jin-Ming Lin

PII: S0165-9936(15)30145-X
DOI: http://dx.doi.org/doi: 10.1016/j.trac.2016.03.013
Reference: TRAC 14704

To appear in: Trends in Analytical Chemistry

Please cite this article as: Jing Wu, Ziyi He, Qiushui Chen, Jin-Ming Lin, Biochemical analysis
on microfluidic chips, Trends in Analytical Chemistry (2016), http://dx.doi.org/doi:
10.1016/j.trac.2016.03.013.

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 Biochemical Analysis on Microfluidic Chips
Jing Wua, Ziyi Heb, Qiushui Chenb, Jin-Ming Linb*
a
School of Science, China University of Geosciences (Beijing), Beijing 100083,
China
b
Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and
Instrumentation, Tsinghua University, Beijing 100084, China

Highlights
 Biochemical analysis plays a crucial role in understanding mechanism of life
activities and giving biological insights into life process.
 Microfluidic chip shows great promise in biochemical analysis field.
 Recent advances in integrated technologies on microfluidic chip for
biochemical analysis have been reviewed.
 New paradigms of biochemical analysis on microfluidic platforms have been
summarized.

Abstract
Biochemical analysis is crucial in understanding mechanism of life activities and
giving biological insights into life process. With inherent merits in flexible design,
microscale of operation, good incorporation with other techniques for manipulation
and detection, microfluidic chip has introduced new paradigms in biochemical
analysis field. Considering the explosive development of microfluidics over the past
decades, in this review, we summarized recent advances in biochemical analysis on
microfluidic platforms. Highlight was put on the integrated technologies such as
optical, electrical, acoustic and magnetic techniques. Focus also was given on relative
applications in biomimetics, drug screening, biomolecular detection, single cell and
stem cell analysis.
Keywords: biochemical analysis; microfluidic chip; optical detector; electronic
methods; acoustic wave; magnetic operation; chip-MS platform; biomimetics; drug

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discovery; biomolecular analysis; single cell; stem cell.

1. Introduction
In native cellular microenvironment, cells are subject to complex biochemical cues
that vary in temporal and spatial scales [1]. These cues can be divided into soluble and
insoluble signaling molecules, including chemokines [2], gradients of cytokines [3],
growth factor secretion from neighboring cells [4], and even biophysical interactions
with the extracellular matrix (ECM) [5]. The autocrine and paracrine signals are
sensed by cells in different ways and regulate cell physiology temporally and spatially.
Meanwhile, cells exert some factors to alter their surrounding microenvironment [6,
7]. Probing the biochemical processes could govern cell behavior and show great
current and potential impact on the bio-analytical chemistry researches. However,
most in vitro cell-based biochemical experiments are performed in two-dimensional
(2D) manner that cells are cultured onto plastic surfaces and treated by coating them
with various solutions [8, 9]. The standard 2D conditions poorly mimic the cellular
microenvironment and are absent of three-dimensional (3D) cues.
Fortunately, microsystems bring unprecedented opportunities for giving analytical
insights into cells and tissues [1, 10-12]. Microfluidic chips enable cells reside in a
more physiologically relevant context and present cells with biochemical cues in a
controllable and reproducible way [13-15]. 3D cell culture on microfluidic chips
makes it possible to retain native tissue-specific functions of cells and recapitulate
tissue-tissue interfaces, spatiotemporal chemical gradients [16, 17]. Various artificial
organ systems have been particularly constructed on microfluidic platforms to
reconstitute the critical organ functions [18, 19]. By integrating with biochemical
analysis techniques, microdevices show great promise for basic biomedical and
pharmaceutical researches. Over the past decade, the microfluidic chips have been
recognized as ideal platforms for biochemical assays which have been widely
expanded to tissue engineering, drug screening, biomolecular detection, single cell
and stem cell analysis (Fig. 1).

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 Fig. 1
In this Review, we describe methods and techniques of biochemical analysis on a
micrometer scale and discuss the application of these microtechnologies in biological
sample analysis. We further present a critical survey of recent advances and future
trends of biochemical analysis in microsystems. Emphasis is placed on works aimed
at gaining biological insights, as well as on efforts to realize advanced biochemical
analysis on chips.

2. Biochemical analysis techniques on chips

Almost every conventional analytical tool in biochemistry labs has an equivalent
microfabricated counterpart on chips. With tremendous advances, microfluidics could
integrate technologies in various fields to perform rapid and reproducible analysis on
small sample volumes as well as eliminate the labor-intensive and potentially
error-prone laboratory manipulations. Here, we highlight several kinds of analytical
methods applied in microfluidic chips in recent years.
2.1 Optical detector
Due to the ubiquitous optical instrumentation in laboratories, quantitative detections
of samples by optical techniques become common in microsystems. Conventional
optical detection technologies, such as absorbance, fluorescence, infrared (IR) and
surface plasmon resonance (SPR), have all been applied in microfluidic chips. Single
mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse
IgG antibodies were co-compartmentalized in droplets to generate a distinguishable
fluorescence signal enabling intracellular, cell-surface or secreted protein screening
(Fig. 2) [20]. The production rate of fluorescent proteins in bacteria was monitored on
microfluidic chip revealing a long period of constant protein production in stationary
phase [21]. Microscopic imaging of whole aging process of budding yeast was
successfully conducted on a microfluidic dissection platform integrated with arrays of
soft elastomer-based micropads [22]. Spatially resolved IR spectroscopy as an
analytical method possessing characteristic of label-free and nondestructive detection
can provide spatiotemporal information on functional groups of biomolecules through

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observing their characteristic vibrational modes.
Fig. 2
With the help of spatially resolved IR spectroscopy, Holman et al. [23] monitored the
spatiotemporal chemical changes in living PC12 cells and found that the cells at the
boundary and center of the colony tended to produce more glycogen and glycoprotein
on an open-channel membrane device. The device composed of a gold-coated porous
membrane between a feeding channel and a viewing chamber to allow long-term
continuous IR measurement of live, adherent mammalian cells. On-chip protein
microarray was fabricated by in vitro transcription and translation to be implemented
for SPR imaging in both clinical and research applications [24]. Cytosolic glucose
levels in yeast were analyzed using genetically encoded Förster resonance energy
transfer (FRET) sensors. Glucose-induced conformational change in the bacterial
periplasmic glucose/galactose binding protein MglB was detected by monitoring
FRET signals between two fluorescent protein variants [25]. Optical method was even
used to pattern different types of cells at single-cell patterning precision. The
propagating light signals of the patterned cells could be detected in real-time giving
promise for the detection of transduction signals among patterned cells [26].
All the above conventional detection units which also called “off-chip” strategies are
separated from the chip platform. While optofluidic is an “on-chip” mode in which
the optics are integrated with or fabricated together with the fluidic functional units
and the optical property of the integrated optics can be tuned by fluidic technology.
All the relevant units, including light sources, filters, mirrors, waveguides, lenses and
detectors, are integrated on the chip. Kim et al. [27] conducted a single-molecule
optofluidic analysis on a microfluidic mixing device which integrated valves and
pumps to accurately accomplish titration of biomolecules with picoliter resolution. An
optical fiber-based, on-chip detection unit with a droplet-based microfluidic unit was
integrated onto a single-layer, optofluidic device for real-time, high-throughput,
quantitative analysis of droplet contents. A detection throughput of 2000 droplets per
second, a detection limit of 20 nM and an excellent reproducibility were achieved on
the device [28]. A random gold nanoisland substrate was used for laser-controlled
4

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optofluidic manipulation based on plasmonic heating. Microfluidic flow guiding,
valving and mixing were firstly realized within the same functional substrate [29].
The more functional units are integrated on the chip, the degree of miniaturization of
the microdevice is higher. With the advance of manufacturing technologies, full
integration of optical elements onto microfluidic chip is becoming feasible.
2.2 Electronic methods
Electrical analysis of biological samples on microfluidic platforms shows prominent
advantages that rich expertise on recording and processing of electronic signals,
readily available technologies for miniaturization and integration, and possibility for
label-free and non-invasive detection [30]. By giving information on cell membrane
capacitance and resistance and cytoplasm conductivity and permittivity, impedance
spectroscopy has been developed as a robust tool for characterization of biological
cells. Its application has been expanded to areas of drug screening [31-34], cellular
biology [35], neuroscience [36-38] and disease diagnosis [39, 40]. Automated
quantification of ovarian cancer cells from whole blood was reported on an
electrochemical Lab-on-a-Disc platform by detecting impedance [41]. The
combination of peptides with electrodes opens a robust, evolutionary avenue in
studies of the interactions between bacteria and antimicrobial peptides (AMPs).
Mannoor et al. [42] reported selective and sensitive detection of infectious agents via
impedance spectroscopy based on hybridization of the AMP magainin I with
functionalized microcapacitive electrode arrays. These devices with electronic
read-out may work as portable pathogen detectors (Fig. 3). Flow impedance
measurement of cells on microfluidic devices provides several advantages over
conventional techniques, such as high sensitivity, saving reagent and sample,
integration of reference measurement electrodes, the use of sheath flow or
dielectrophoretic forces for cell centering and the possibility of online implementation
of cell sorting [43].
Fig. 3
Dielectrophoresis (DEP)-based methods also serve as powerful strategies for
continuous-flow cell sorting on chip. Renaud et al. [44-46] developed
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equilibrium-based continuous cell sorters based on DEP. Applying opposite negative
DEP force fields from electrodes defines a position of equilibrium for the dielectric
particles placed in these fields. In addition to the equilibrium-based sorting device,
some novel cell sorting microdevices based on DEP have been reported such as
Raman-activated cell sorting [47], label-free stem cell sorting [48, 49], DEP viable
cell sorting [50, 51] and electrodeless DEP [52, 53]. Single cells were captured and
repulsed by DEP forces generated by a bipolar electrode. Both faradaic ion
enrichment and depletion zones generated by bipolar electrodes can be exploited to
shape and extend the electric field gradients that are responsible for DEP force. DEP
technologies for manipulating biological cells also show many distinct merits over
other cell-handling techniques including label-free selectivity, cost-saving device
components, and amenability to single-cell and array-based applications [54].
Currently, many novel analytical approaches based on electronic detection at high
speed have been developed on chips. Western blotting assay was conducted in a single
glass microchannel under purely electronic control. Western blotting on microfluidic
platform is an effort trying to reach the long-sought bioanalytical goal in the life
sciences that developing rapid and quantitative analysis methods [55]. Protein
biomarkers in complex biological samples were detected by multiplexed
immunoarrays on microfluidic devices [56-58]. Electrodes even were printed on
paper-based microfluidic device to achieve some biological and chemical applications
such as cancer cell detection [59], enzyme-linked immunosorbent assay (ELISA) [60].
All of them are the results of the technological advances in microfluidics,
microelectronics and electrophysiology.
2.3 Acoustic wave
The introduction of surface acoustic wave (SAW) technology onto lab-on-a-chip
platforms has opened a new frontier in microfluidics. Numerous advantages are
provided by SAW microfluidics: compact and inexpensive devices and accessories,
fast fluid actuation, contact-free particle manipulation, and compatibility with other
microfluidic components [61]. According to the property of acoustic wave, SAW
microfluidics are divided into two types: travelling SAW (TSAW) and standing SAW
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(SSAW). Ultrafast particle and cell concentration are essential to the success of
subsequent analytical procedures and the development of miniaturized biological and
chemical sensors. TSAW devices were used to excite a MHz-order acoustic wave that
propagated into a microlitre droplet to facilitate spatial partitioning between two
different sized suspended microparticles [62]. A novel microfluidic cell sorter
operating in continuous flow at high sorting rates was successfully used to direct
HaCaT cells, fibroblasts from mice and MV3 melanoma cells by acoustic streaming
excited by TSAW [63]. TSAW also allows fast drop formation and real time control of
drop size at quick response times over a wide range [64]. A sophisticated fluid
manipulation tool based on TSAW was demonstrated to carry out the complex
sequence of fluidic manipulations to detect the rodent malaria parasite in blood [65].
Acoustic fields were used to produce the required rotational vortices to mechanically
lyse both the red blood cells and the parasitic cells presented in a drop of blood (Fig.
4a).
Fig. 4
In contrast to TSAW-based devices harnessing the acoustic streaming, SSAW-based
devices use the primary acoustic radiation forces acting on particles via the
surrounding fluid. On-chip functional blocks for sample preprocessing are
indispensable elements for fully portable micrototal analysis systems. Pretreatment of
whole-blood samples [66] and cell washing [67] were successfully conducted on the
SSAW-based microfluidic device. Similar to optical tweezers, “acoustic tweezers”
based on SSAW was provided to trap and manipulate single cells and entire organisms
in a single-layer microfluidic chip (Fig. 4b). The acoustic tweezers utilize the wide
resonance band of chirped interdigital transducers to achieve real-time control of a
SSAW field which enables flexible manipulation of most known microparticles. The
tweezers give promise as powerful tools for many disciplines of science and
engineering [68]. Supported lipid bilayers (SLBs) are crucial in cellular biology
because they regulate the intracellular and intercellular movement of ions, proteins
and other molecules [69]. As a result, SLBs work as a critically important tool in
studying the physical properties of biological membranes and cells. SSAW were used
7

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to generate lateral standing waves leading to a lateral modulation in lipid
concentration. This pattern formation was demonstrated to be reversible and brought
no effect on the integrity of the lipid bilayer [70]. Arrays of molecules, such as DNA
[71] and proteins [72], were bounded to SLBs to enable on-chip screening and
binding assays. Titled-angle SSAW (ta-SSAW)-based microfluidic device was capable
of high-throughput separation of circulating tumor cells (CTCs) from peripheral blood
samples obtained from cancer patients (Fig. 4c). In the fields of cancer research,
diagnostics, drug efficacy assessment, and therapeutics, this method will become an
invaluable supplement tool owning to its simple design, label-free automated
operation and capability to isolate rare CTCs in a viable state [73]. Ta-SSAW also was
introduced to microfluidic channel for improving the efficiency and sensitivity of
acoustic separation techniques. Human breast cancer cells were effectively separated
from non-malignant leukocytes, while preserving the integrity [74]. An organized cell
co-culture platform was constructed by sequentially patterning different types of cells
in the SSAW field. Cell migration dynamics of epithelial cancer cells was real-time
monitored on this platform when they were co-cultured with endothelial cells [75].
SAW-based microfluidics has come a long way in the past decade. Its applications
have been spanned the full spectrum of lab functions.
2.4 Magnetic operation
Utilization of magnetic particles has a combination of advantages that competing
techniques lack: a large surface-to-volume ratio, convenient bio-functionalization,
manipulation by magnetic fields, simplified extraction and buffer replacement steps.
Manipulation of magnetic particles by magnetic fields is compatible with system
miniaturization on account of the following reasons: (1) the tendency of explorations
toward miniaturization has a solid basis that availability of magnetic particles and
corresponding assay reagents; (2) the magnetic fields are strong close to the field
generators; (3) magnetic field gradients are large in the vicinity of structures with high
curvature; (4) only short distances need to be travelled in miniaturized devices [76].
Magnetic particles have been applied in mixing fluids, selective capturing,
concentrating, transferring, labeling specific analytes, and probing biophysical
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properties of analytes.
Rapid mixing of fluids or solutions is a necessary step in most point-of-care testing
devices and has been a topic of everlasting interest in the microfluidic field. Magnetic
field was used to assemble an artificial biomimetic cilia as long chains of spherical
superparamagnetic particles and to actuate the cilia in a simple nonreciprocal manner
resulting in a fluid flow [77]. A flow system consisting of a water-based ferrofluid and
a mixture of DI water and glycerol was mixed by the interaction between a uniform
magnetic field and a magnetic fluid in a microfluidic chamber [78]. The controlled
rotation and torque was generated by uniaxial magnetic microactuators formed by two
bound superparamagnetic particles in a fluid. This demonstration opened a range of
possibilities in lab-on-a-chip applications, such as the actuation of single molecules,
fluid mixing in microfluidic chambers and novel cluster-based assays [79].
High surface-to-volume ratio and many bio-functionalization options are beneficial
for magnetic particles to capture, concentrate, transfer and label analyte in biological
samples. A novel microfluidic platform using “phase-transfer magnetophoresis”
enabled continuous DNA extraction from bacterial Escherichia coli which was
cultured with superparamagnetic bead suspension, lysis and binding buffers (Fig. 5a).
These DNA carrying beads were subsequently transported across the interfaces
between co-flowing laminar streams of sample mixture, washing and elution buffer
actuating by applying a time-varying magnetic field generated by a rotating
permanent magnet [80]. Magneto-capillary valve technology also was reported to be a
flexible approach for integrating purification and enrichment of nucleic acids and
proteins [81]. Transportation of magnetic beads between multiple microfluidic
chambers was even realized to be automated on a centrifugal microfluidic cartridge
“LabDisk” [82]. A concept of multidimensional magnetic and optical barcoding of
droplets based on a magnetiofluidic platform was presented. The platform comprised
multiple functional blocks: encoding area, encoded droplet pool and magnetic
decoding area (Fig. 5b). The platform paved the way for the development of novel
non-optical encoding schemes for multiplexed droplet-based biological assays [83].
Fig. 5
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After the capture, concentration, transferring and labeling of target analytes, further
processing is accurate and specific detection. Concentration of avidin added to the
solution could be quantitatively determinated by monitoring changes in the amplitude
of the optical transmittance of the solution which was caused by self-assembled
chains of functionalized superparamagnetic beads rotating in the solution (Fig. 5c).
This protocol offered a rapid, highly sensitive, inexpensive and homogeneous means
of detecting biorecognition processes [84]. Magnetic particle-scanning for on-chip
ultrasensitive immunodetection also was described. A magnetic particle carrying a
low number of antigens was transported by hydrodynamic forces and was subjected to
a pattern of substrate-bound small magnetic particles functionalized with antibodies
[85]. An electromagnetically actuated platform was demonstrated to perform nucleic
acid extraction from whole blood followed by real-time polymerase chain reaction
(PCR) detection of KRAS oncogene [86]. Isolation and detection of CTCs from blood
samples was reported using a system that integrated a microchip with
immunomagnetics, high-throughput fluidics and size-based filtration [87]. Concerning
the magnetic particles, it is foreseen that particle-based assays will benefit from the
ongoing optimization of particles regarding their surface bio-functionalization,
surface smoothness and their size and magnetization uniformity [88].
2.5 Chip-MS platform
Mass spectrometry (MS) is an important analytical tool which can provide both
qualitative and quantitative information about a specific analyte and identify its
chemical structure. Gas chromatography (GC) [89, 90], liquid chromatography (LC)
[91, 92] and capillary electrophoresis (CE) [93-95] have been coupled with MS and
studied for a long time. All of the technologies have made great contributions in
separation and detection of molecules. However, MS-based technology with higher
throughput, more rapid and convenient analysis, and lower sample consumption is
still in great demand to meet the new challenges in biochemical analysis. In 1997,
Karger [96] and Ramsey [97] provided the concept of microchip-MS for modern
biological analysis. The new method is superior to other detection methods coupling
with chips in giving information for characterization of chemical structure [98]. Since
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then, a number of microfluidic chip-MS studies have been reported.
A series of analytical methods relative to microchip-MS have been developed in our
group, which show their potential as an integrated platform for cell-based biochemical
analysis. C30 beads were used to imitate cells to be introduced into a hole on a simple
microchip. The combination of microfluidic device with electrospray ionization
quadrupole time-of-flight mass spectrometer (ESI-Q-TOF-MS) was successfully
detected a series of herbicides on a single C30 bead [99]. Then, the ESI-Q-TOF-MS
coupled microchip platform was employed to monitor carnosine-protection process
against A42-induced glutamate released from PC12 cells [100]. We detected
multiple cell metabolites on the microchip coupled with ESI-Q-TOF-MS. The
metabolism of vitamin E in human lung epithelial A549 cells was successfully studied
by online microchip-MS platform with high sensitivity and short analysis time. By
integrating solid-phase extraction (SPE) microcolumns, cell culture chambers with
microchips, all the steps of cell culture, metabolism generation, sample pretreatment
and detection were conducted on the chip-MS platform [101]. Multiple gradient
generator also was integrated onto the chip-MS platform. The absorption of
methotrexate and its effects on HepG2 and Caco-2 cells were investigated on the
combination system [102]. Curcumin was used as a model drug to detect drug
permeability in Caco-2 monolayers on a membrane-based microfluidic device
coupled with MS [103]. In order to improve the stability, sensitivity and repeatability
of quantitative analysis on the chip-MS system, we developed a stable isotope
labeling assisted microfluidic chip ESI-MS (SIL-chip-ESI-MS) (Fig. 6). A
dual-isotopic labeling was presented for effective qualitative analysis of multiplex
metabolites. Despite complex biological matrixes, three coeluting pairs of
isotopomers could be easily recognized and identified by SIL-chip-ESI-MS [104]. We
even extended the application of chip-MS platform to study cell-to-cell
communication. The inhibition on growth hormone secretion from GH3 cells by
dopamine released from PC12 cells was investigated and demonstrated [105]. A
“Surface Tension Plug” was presented on a microchip to control the communication
between the 293 and the L-02 cells. Signaling molecules epinephrine and glucose
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were successfully detected using ESI-Q-TOF-MS [106]. An automatic and online
microfluidic chip-MS system was established to quantify noncovalent protein-protein
interactions [107].
Fig. 6
Recently, novel chip-MS platforms have been developed by researchers. An integrated
microfluidic CE-ESI device was demonstrated to separate intact monoclonal antibody
charge variants with online MS identification [108]. An electrochemical chip was
coupled online to MS or LC-MS to generate phase I and phase II drug metabolites and
to demonstrate protein modification by reactive metabolites. This new screening
method gave the potential of this electrochemical chip as a complementary tool for a
variety of drug metabolism studies in the early stages of drug discovery [109]. A
multi-layer microfluidic device was developed for characterization of drug
metabolism in human liver microsomes by coupled with ESI-Q-TOF-MS [110]. A
method combining microfluidics and a miniature MS was applied to quantify abuse
drugs in urine. Cocaine, benzoylecgonine and codeine were quantified from four
samples in less than 15 min using the new method [111]. A Swan-shaped probe
coupled with ESI-MS was designed and applied for high-throughput and
nanoliter-scale analysis of biological samples in both a microfluidic droplet array and
a multiwall plate. The Swan probe had two sections: a U-shaped section with a
micrometer-sized hole for sampling and a tapered tip for sample electrospray
ionization. 256 droplets were analyzed within 90 min with a peak height relative
standard deviation (RSD) of 12.6% [112]. Efforts also were put on miniaturization of
microchip-based MS using SU-8 as a material [113]. All this demonstrate that the
microfluidic chip-MS platform is developing to be a potential useful tool for
biochemical researches [114, 115].

3. Applications
By integrating various technologies from other fields, microfluidic device as a
powerful analytical technique is increasingly being used in various biochemical
researches, such as biomimetics, drug screening, biomolecular analysis and cell

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analysis. Some recent examples of these applications have been reviewed here.
3.1 Biomimetics
Cell culture on microscale platforms better mimics the in vivo cellular
microenvironment than conventional, whereas macroscale systems enabling
physiologically realistic in vitro tissue models to be constructed. Biomimetic
hydrogels are the preferred material for microfluidic chip fabrication. Recently, the
effects of microenvironmental parameters on cell fate were demonstrated in 3D model
by incorporating multiple cell types in microgels which having defined and tunable
mechanical and biochemical properties [116]. CTCs were encapsulated on-chip in a
biomimetic hydrogel matrix and their clonal 3D spheroid growth potential was
assayed by microscopy over one week. The possibility to clonally expand a subset of
captured CTCs in a near-physiological in vitro model powerfully exploit CTC-chip
application and ultimately should improve prediction of treatment responses and
disease progression [117]. Tethered protein gradients were patterned on the surface of
soft synthetic hydrogels in any user-defined shape to mimic the biophysical
characteristics of the natural extracellular milieu and validated to have a certain
impact on single-cell migration [118-120]. Probably, in vitro human skin models are
the most developed and understood engineered constructs due to the clinic and
cosmetics industry requirements [121]. By using a microfluidics-based in vitro skin
wound healing model, it was revealed that the bottom side of the bacterial cellulose
film could better promote the migration of cells to facilitate wound healing through
MTT assay [122]. The microenvironment of bone marrow contains a complex set of
cellular, chemical, structural and physical cues to maintain the viability and function
of the hematopoietic system. In vitro hematopoiesis models always fail to demonstrate
the complex microenvironment and functions of living bone marrow. A method for
fabricating “bone marrow-on-a-chip” allowed in vitro culturing of live marrow with a
functional hematopoietic niche. Hematopoietic stem and progenitor cells could be
retained in the engineering bone marrow for at least 1 week [123]. We developed a
microfluidic 3D culture device to imitate the diffusion process between blood vessels
and tissues. On this microfluidic device, QD cytotoxicity was evaluated by
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determining cell apoptosis, intracellular reactive oxygen species and glutathione with
specific fluorescence probes. The degree of QD cytotoxicity was demonstrated to be
relative to the diffusion distance [124].
Microfluidic devices also serve as novel tools for recapitulating complex organ
functions to improve the biochemical analysis on chips. A biomimetic microsystem of
human lung was fabricated by reconstituting the critical functional alveolar- capillary
interface (Fig. 7a). Complex integrated organ-level responses to bacteria and
inflammatory cytokines were reproduced on this bioinspired microdevice [19]. In
human body, liver is a central nexus integrating metabolic and immunologic
homeostasis. Also, it is the direct or indirect target of most molecular therapeutics
[125]. We integrated bioreactors containing poly(ethylene) glycol hydrogel
encapsulated human liver microsomes onto the microfluidic device to imitate drug
metabolism in human liver and its cytotoxicity on cells. The products of
5’-diphosphate-glucuronosyltransferase (UGT) metabolism of acetaminophen (AP)
were injected into the cell culture chamber for cytotoxicity assay and simultaneously
detected online with ESI-Q-TOF-MS [126]. Alzheimer’s disease (AD) classified as
one of the most costly brain disease imposes an enormous burden on society. A 3D
brain-on-a-chip with an interstitial level of flow was developed as an in vitro model of
AD to meet the growing need for understanding of etiology and faster development of
treatment strategies (Fig. 7b) [127]. The microvasculature is an extensive organ that
supports metabolic activity and mediates the interaction between blood and tissues.
Engineering living microvascular networks in 3D tissue scaffolds developed by Zhang
et al. provided a platform for studying complex vascular phenomena (Fig. 7c). The
angiogenic activities and nonthrombotic nature of the vascular endothelium and its
transition to a prothrombotic state during an inflammatory response were
demonstrated [128]. Metastasis of cancer cells from a primary tumor to secondary loci
is the main cause for cancer-related mortality. A microfluidic 3D in vitro model was
constructed to analyze human breast cancer cell extravasation into bone- and
muscle-mimicking microenvironments through a microvascular network
concentrically wrapped with mural cells and unveil the underlying mechanisms of
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cancer metastases [129]. In vivo-like oriented neural networks between superior
cervical ganglion (SCG) neurons and their effector smooth muscle cells (SMC) were
modeled in a microfluidic device which allowed amperometric detection of individual
neurotransmitter release events inside functional SCG-SMC synapse with carbon fiber
nanoelectrodes as well as recording of postsynaptic potential using glass nanopipette
electrodes [130]. Further advances in the field of organ-on-a-chip platforms can
revolutionize some biochemical analysis process such as drug screening, genetic and
protein analysis. The final goal is to develop a body-on-a-chip platform for systemic
biochemistry analysis.
Fig. 7
3.2 Drug discovery
Bringing a new drug to market is a complex, lengthy and expensive process. An
adequate cell-based assay to efficiently screen and validate potential drug candidates
is crucial in the initial stage of drug discovery [131]. Microfluidic platforms provide
several unique advantages for drug screening. Physical and chemical properties of
drug carriers can be easily and effectively modified by tuning the flow rate and
geometries of microchannels on microfluidic devices. Batches of carriers can be
fabricated with minimal effort and with little variation. Tissue or organ models can be
mimicked in microfluidic systems to be used as in vitro drug screening tools [132].
A robust microfluidic approach increased the number of data points in dose-response
analysis to approximately 10 000 per compound (Fig. 8a). The increased number of
data points resulted in highly precise and reproducible IC50 values [133]. A
microfluidic device featuring an electrokinetic size and mobility trap was formed to
overcome the bottleneck that extraction of target analytes from biological samples.
Ampicillin levels in blood were analyzed within 5 min and a linear response over the
range of 2.5-20 g mL-1 on this device [134]. Concentration gradients play essential
roles in drug screening because they can affect various cell behaviors. A novel
gradient generator was developed to achieve long range and linear chemical gradients
with a dynamic control function. This gradient generator overcome the disadvantages
of the “Christmas tree” design that lacking dynamic control of gradient profile and
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limited length of the gradient. The effect of deoxycycline on the viability of PC-9 lung
cancer cells was tested on this device [135]. Anti-angiogenesis drugs suppress tumor
growth by disrupting oxygen and nutrient supplied from blood to the tumor. Kim et al.
[136] described a method for screening and quantifying the vascular endothelial
growth factor (VEGF)-induced chemotactic response on human umbilical vein
endothelial cells (HUVEC) cultured with different concentrations of bortezomib, a
selective 26S proteasome inhibitor. A 3D microfluidic cell array which reconstructed a
3D tumor microenvironment with cancer cells and microvascular endothelial cells
was used to predict anticancer drug responses in human tumors [137]. Cell gradient
also is a key factor in cytotoxicity assay. We developed a microfluidic cell density
gradient generator composed of eight parallel channels which were surrounded by 1-8
microwells. Series of cell density gradients were successfully generated on this chip
and QD cytotoxicity exhibited obvious cell density-dependence [138].
Fig. 8
The development of microengineered models of the functional units of human organs
or tissues could improve the effectiveness of preclinical predictions of human drug
responses [139-141]. Shuler et al. [142] designed and developed a microfluidic
platform that allowed for long-term maintenance of full thickness human skin
equivalents which were comprised of both the epidermal and dermal compartments.
The toxic effects of doxorubicin on skin cells and structure was examined on this
platform. Our group developed an in vitro liver model to imitate and detect prodrug
metabolism (Fig. 8b). Capecitabine (CAP) was selected as a model compound
because it needed to be metabolized into active intermediate in the liver and then
transformed into final effective drug in tumor cells. This process was realized and
confirmed within this in vitro liver model. The intermediate product was successfully
detected by MS and the anti-tumor effect of the active metabolite was observed
through cell viability assays of MCF-7 cells [143]. Hepatic organoids could be also
formed from co-cultures of HepG2 and NIH-3T3 cells embedded in hydrogel matrices
on a digital microfluidic device. The organoids exhibited better mimics of in vivo liver
tissue than comparable 2D cell culture systems. The effects of a dilution series of
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acetaminophen on apoptosis and necrosis were analyzed on this device [144]. A
higher throughput “heart on a chip” which recapitulated the laminar architecture of
the heart ventricle was presented. The microdevice was employed to test the positive
inotropic effect of isoproterenol on cardiac contractility at dosages ranging from 1 nM
to 100 M [145]. An in vitro microfluidic absorption, distribution, metabolism and
excretion (ADME) profiling system was established to study systemic absorption of
drugs in the small intestine, metabolism by the liver as well as excretion by the kidney.
Dose systemic toxicity testing of drug candidates had been lasted for over 28 days in
the microsystem [146]. Achievements in the organ-on-a-chip field exploit exciting
new avenues for drug discovery and development. The tremendous potential that
organ-on-a-chip technology holds will bring a broad impact on drug discovery and
give a surge in its future progress.
3.3 Biomolecular analysis
3.3.1 Genetic analysis
So far, microsystem for DNA and RNA measurement is one of the most developed
analytical microfluidic field. PCR and other techniques for sample amplification are
currently powerful and popular technique for sensitive genetic analysis. Due to the
high surface-to-volume ratio inside microchannels or microscale reactors, microscale
PCR has been realized and demonstrated to be rapid. A digital PCR using aqueous
droplets in oil as microreaction vessels for individual DNA molecules known as
droplet digital PCR was conducted on a microfluidic chip [147]. Single-cell PCR
gene-expression analysis was implemented on microfluidic chip to dissect the cellular
composition of primary human normal colon and colon cancer epithelia. It was
showed that different gene-expression programs linked to multilineage differentiation
were strongly associated with patient survival [148]. We also established a method
based on PCR coupled with microchip electrophoresis to analyze BCR-ABL fusion
gene [149] and mini-short tandem repeat loci [150]. A crucial step in the genetic assay
process is the isolation of the nucleic acids. Purification of the nucleic acids from raw
samples could enhance the device sensitivity and pave the way for downstream
processes [151]. An array of surface-adhering droplets was reported to facilitate the
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transportation of magnetic purification beads for parallel mRNA extraction through
individual buffer solutions without solid structures [152]. However, the device in Fig.
9a executing all steps of single-cell processing, including cell capture, cell lysis,
reverse transcription and quantitative PCR made it possible to skip nucleic acid
isolation [153].
Fig. 9
Hybridization arrays, real-time probes and electrophoretic sizing for analysis are
always contained on microchips. A microfluidic chip with 3 × 3 arrayed
electrochemical sensors for the analysis of DNA hybridization events was presented.
The electrochemical signal given by the microfluidic chip was validated and
demonstrated to be repeatable [154]. A label-free DNA sensing platform was
developed with low-voltage electrolyte-gated transistors. A floating-gate electrode
functionalized with ssDNA whose potential was determined by both capacitive
coupling with a primary, addressable gate electrode and the presence of adsorbed
molecules. When DNA was hybridized at the floating gate, it offset the primary gate
voltage. The offset was related to the number density of dsDNA molecules [155]. A
temperature jump (T-jump) setup enabled heating and cooling of aqueous solutions by
~ 50 ℃ on a 1-s time scale. The setup was comprised by a thermally conductive
sapphire substrate with light-absorptive nano-coating, a microfluidic device and a
rapidly switched moderate-power IR laser with the laser beam focused on the
nano-coating. The setup could be used to probe folding and unfolding dynamics of
DNA hairpins after direct and inverse temperature jumps [156]. Chip-base analysis of
mRNA levels in enriched tumor exosomes obtained from blood even could monitor
the level change during treatment. A microfluidic platform termed immune-magnetic
exosome RNA (iMER) analysis was presented in Fig. 9b which integrated
immunomagnetic selection, RNA collection and real-time PCR into the format of a
single chip [157]. Zero-mode waveguides (ZMWs) are attractive technology for
single-molecule imaging. Conical lens-based dark-field fluorescence microscopy was
combined with a ZMW/microfluidic chip and revealed weak DNA-protein and
protein-protein interactions found with T4 replisomal proteins [158]. “DNA
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dumbbells”— single molecules of DNA with a microscopic bead attached at each end
were generated on a microfluidic device. This design facilitated the examination of
the role of 3D DNA conformation and dynamics in protein-DNA interactions [159].
The advantage of microfluidic technology that high-throughput features more
prominently in genetic analysis. A million-membered metagenomic library was
screened in microfluidic picolitre droplet compartments [160]. Ohler et. al [161]
developed a high-throughput microfluidic device in which 64 Arabidopsis thaliana
seedlings could be grown and their roots were imaged by confocal microscopy over
several days without manual intervention. They tracked hundreds of roots to capture
detailed expression patterns of 12 transgenic reporter lines under different conditions.
The integration of microfluidic selection with high-throughput DNA sequencing
technology was described for rapid and efficient discovery of nucleic acid aptamers.
The copy number and enrichment-fold of more than 10 million individual sequences
were tracked by this method through multiple selection rounds [162]. Huang et. al
[163] prepared 94 libraries consisting of single mouse embryonic cells and technical
replicates of extracted RNA through implementing microfluidic technology in
single-cell RNA sequencing. They also quantified variation between and within
different types of mouse embryonic cells. The advantages of microfluidic approaches
that enhanced measurement precision, detection sensitivity and high throughput were
throughly validated in the single-cell transcriptome analysis. Integration of multiple
instruments has been a major trend in fabricating microfludic chips for biochemical
analysis. An efficient microfluidic single-cell analysis chip in combination with
high-resolution time-lapse microscopy was developed to firstly determine the yeast
replicative lifespan in a high-throughput manner. This chip gave a promise for the
usefulness of yeast replicative lifespan assays as a broad genetic screening platform
for research on aging [164].

3.3.2 Protein analysis

The separation of required protein analytes from raw biosamples is of critical
importance in modern proteomics. An on-chip all SiNW filtering, selective separation,
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desalting, and preconcentration platform for target proteins analysis was developed.
The SiNW forest with ultralarge binding surface area was modified with antibody to
separate required protein, followed by the release of these proteins in a controlled
liquid media. The released proteins were detected by supersensitive SiNW-based
field-effect transistor arrays fabricated on the same chip [165]. A robust
chemomechanical sorting system also was designed to catch and release target
biomolecules from a solution mixture. Target-specific, reversible binding sites
attached to microscopic fins were the main part of the system. The microscopic fins
were embedded in a responsive hydrogel that moved the cargo between two
chemically distinct environments. Thrombin was separated as an example on this
platform by synchronizing the pH-dependent binding strength of a thrombin-specific
aptamer with volume changes of the pH-responsive hydrogel in a biphasic
microfluidic regime (Fig. 10) [166]. The evolution of enzyme catalyst was performed
on a microfluidic device by caged in gel-shell beads. These hydrogel beads were
surrounded with a polyelectrolyte shell that enclosed an enzyme, its encoding DNA
and the fluorescent reaction product [167]. The activity of millions of glycosidase
sequence variants was mapped by Abate et. al through combining droplet microfluidic
screening with next-generation DNA sequencing. Microfluidic-based deep mutational
scanning provided a comprehensive and unbiased view to dissect enzyme function
[168]. Otten et al. [169] described a microfluidic platform for on-chip expression,
covalent surface attachment and measurement of single-molecule protein mechanical
properties. A dockerin tag on each protein molecule allowed thousands of pulling
cycles performed by a single cohesion-modified cantilever. We also reported some
microchips for protein analysis. An integrated microfluidic device was developed for
real-time analysis of secreted protein VEGF165 by aptamer-functionalized
microchannels. Besides that, investigation of various oxygen concentrations and
distances effect on cell migration was conducted on this platform [170]. An
aptamer-modified microchip for ultrasensitive detection of thrombin using rolling
circle amplification and G-quadruplex DNAzyme was developed in our lab [171]. We
also assayed multiplex proteins based on tunable aptamer by microchip
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electrophoresis. Different lengths of aptamers modulated the electrophoretic mobility
of proteins allowing the protein to be effectively separated on the microchip. Under
optimum conditions, good linearities of quantitative assays of three kinds of proteins
(PDGF-BB, VEGF165 and thrombin) were achieved with a dynamic range of
5.00-150.0 nM [172]. ELISA which measures protein abundance using bulky
fluorescent scanners is the current gold standard method for protein biomarker
detection. ELISA is widely used in biochemical analysis with high specificity and
sensitivity but lacks of portability. Carrying out ELISA on microfluidic platform can
eliminate this disadvantage. A digital microfluidic platform present by Javanmard et.
al making use of simple electronics was translated into a portable handheld device and
could be adapted to assay different types of proteomic biomarkers [173]. Micro
open-sandwich ELISA allowed rapid and noncompetitive detection of a small
biomarker peptide [174]. A novel microfluidic ELISA microplate with standard
SBS-configured 96-well microplate architecture was introduced as the next generation
assay platform for unparalleled assay performances [175]. An integrated microfluidic
solid-phase ELISA platform was developed for rapid and ultrasensitive detection of
proteins with a wide dynamic range. Two key novelties existed in this design that an
integrated microwell-patterned assay chamber which could be pneumatically actuated
to reduce the volume of chemifluorescent reaction and monolithic integration of
on-chip pumps to allow programmable fluid delivery and effective mixing [176].
ELISA was even performed in a 96-microzone plate fabricated in paper [177].
Interaction between biomacromolecules plays a significant role in revealing the
underlying mechanisms of cellular activities. A unique continuous-flow laminar mixer
based on microfluidic dual-hydrodynamic focusing was investigated to characterize
the kinetics of DNA-protein interactions. The interaction kinetics of G-quadruplex
and single-stranded DNA binding protein was successfully interrogated by this device
[178]. Biomolecular assay on microfluidic chip can give fundamental biology
investigations to decipher mechanisms of some in vivo biochemical processes.
Fig. 10
3.4 Cell analysis
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3.4.1 Single cells
The capability of integrated microfluidics to accurately manipulate, handle and
analyze very small volumes has opened up new opportunities for analysis of
intracellular constituent at single-cell level. Compared to conventional biological
assays that statistically analyze the average response from a large population of cells,
single-cell assay can tell the differences between individual cells allowing more
precise understanding of single-cell behavior. The challenging in studying single cells
is requiring hundreds or thousands of isolated single cells. Numerous
microfluidic-based techniques such as microwell arrays [179, 180], DEP [181, 182],
microscale physical trap arrays [183] and microdroplets [184, 185] have been
developed and successfully utilized to capture single cells. Using fluid dynamics
simulations in combination with particle image velocimetry to systematically
optimize trap architectures, Kobel et al. [186] enhanced single cell trapping efficiency
up to 97%. A U-shaped hydrodynamic trap was incorporated into a two-layer
microfluidic device platform for single cell capture, culture and clonal expansion.
Both adherent and non-adherent cells were trapped with high efficiency and in a high
throughput manner [187]. An optical micro-tweezers was integrated into a hybrid
polymethylmethacrylate (PMMA)-glass microfluidic circuit to trap a single cell and
excite its Raman and fluorescence response, thus making monitoring the single cell
reaction to different stimuli to be possible [188]. Single-cell encapsulation rate was
improved by Liu et. al through integrating droplet generation with
fluorescence-activated droplet sorting which isolated droplets containing one cell
[189]. Our group fabricated rounded bottom microwell arrays in PDMS by moulding
a monolayer of ordered polystyrene (PS) microspheres. PS microspheres were
self-assembled on a glass slide and partially melted at 240 ℃ to be used as master to
generate microwell arrays. Single cells can be retained in the microwells with high
efficiency [190]. We also designed a novel microfluidic platform integrated with
cell-recognizable aptamer-encoded microwells to isolate single tumor cells with
satisfied single-cell occupancy and unique bioselectivity. The single-cell capture
efficiency was significantly enhanced from 0.5% to 88.2% through the introduction of
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the aptamer into the microwells with optimized size [191]. After isolation,
microfluidic approaches are found to be well suited to resolve a variety of single-cell
analytics including signal response, growth dynamics, gene expression measurements
and protein analysis. A chemical signal generator based on hydrodynamic gating
permitted resolving temporal dynamics of single cells [192]. A microfluidic
hydrodynamic trapping system was designed with arrays of bypass structures for
trapping individual cells and developed as a digital single-cell assay system to
monitor cellular dynamics under drug treatment over time [193]. A high-throughput
single-cell multidrug resistance analysis device was developed to examine both the
chemo-sensitizing effect and the cytotoxity of the potential chemo-sensitizing agents
[194]. Five clinically established chemotherapeutic reagents were conveniently
assayed on this system. Large-scale, highly parallel single-cell transcriptomics was
realized in tiny droplets in which single cells were captured along with sets of
barcoded primer beads. Transcriptionally distinct cell populations in mouse retinal
tissue were revealed by this analytical method [195] . Similar method also was used to
enable single-cell transcriptomics of a large number of cells in a heterogeneous
population. Population structure, gene expression relationships, and the heterogeneous
onset of differentiation in mouse embryonic stem cells were uncovered depending on
this development [196]. Single-cell genomic damage which was generally identified
with a “comet assay” was demonstrated to be analyzed in an ultrahigh-throughput
approach with an agarose-based microfluidic chip. Up to 10 000 individual cells were
simultaneously detected with better reproducibility [197]. Picoliter-sized droplets
were present for compartmentalized whole genome amplification to minimize
amplification bias and contaminants showing potential as an efficient tool for
effective amplification of low-input DNA for single-cell genomics [198]. Voldman et
al. [199] presented a microfluidic device to trap and properly pair thousands of cells.
The device was compatible with both chemical and electrical fusion protocols.
Recently, they further applied this device to investigate immune response by pairing
lymphocytes (Fig. 11a). Using this platform, they characterized early activation
dynamics of CD8 T cells and investigated the extent of heterogeneity in T-cell
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activation and the correlation of multiple readouts [200]. A microfluidic migration
platform to simultaneously analyze four qualitative migration patterns:
chemoattraction, -repulsion, -kinesis and -inhibition was designed and validated using
single-cell quantitative matrics of direction, speed, persistence and fraction of cells
responding (Fig. 11b) [201]. We designed a microfluidic system consisted of a PDMS
microwell array for single-cell arrangement. Depending on this platform, we found
that QD cytotoxicity was dependent on cellular uptake in various cell cycle phases
because the accumulation and dilution of QDs happened in single cell cycles. The
rank of QD cytotoxicity in different cell cycle phases was G2/M > S > G0/G1 [202].
A single-cell analysis pipeline for studies of signaling dynamics was described.
Microfluidic cell culture, precise stimulation of cells with signaling molecules or
drugs, live-cell microscopy, computerized cell tracking, on-chip staining of key
proteins and subsequent retrieval of cells for high-throughput gene expression
analysis were incorporated into this pipeline. This pipeline represented a major step
forward in dynamic single-cell analysis because it enhanced experimental precision
and throughput by orders of magnitude and introduced much-desired new capabilities
in cell and fluid handling [203]. A microchip was designed to quantify the levels of a
dozen cytoplasmic and membrane proteins from single cells. Single-cell analysis
uniquely revealed single-cell heterogeneity and different types and strengths of
protein-protein interactions. This platform helped to comprehensively understand
altered signal transduction networks in tumor cells and gave insight into the effect of
targeted therapies on protein signaling networks [204]. A microscope slide supporting
a 30-m-thick photoactive polyacrylamide gel was developed to conduct ~103
concurrent single-cell western blots in ~4 h. This method was applied to monitor
single-cell differentiation of rat neural stem cells and responses to mitogen
stimulation [205]. Liu et. al [206] proposed a novel single-cell chemical proteomics
strategy to profile low-abundance membrane proteins in single cells. They further
applied this method to investigate single primary cells and observed significant
heterogeneity among individual mouse cerebellar granule neurons. A complete cycle
of single cell analysis was achieved on a lab-on-a-chip that combines
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micro/nano-fabricated features with a Convex Lens-Induced Confinement device. All
the steps of cell trapping, cell isolation, lysis, protein digestion, genomic DNA
extraction and on-chip genomic DNA linearization were included [207]. A novel
microfluidic single-cell culture method was present by Buckanovich et. al to directly
observe the differentiation capacity of four heterogeneous ovarian cancer cell
populations. The data suggested a hierarchical differentiation pattern in which bone
morphologenetic protein 2 acted as a feedback mechanism promoting ovarian cancer
stem-like cell expansion and suppressing progenitor proliferation [208]. Microfluidics
will work as a necessary single-cell analytical technique in biotechnological process
and strain characterization.
Fig. 11
3.4.2 Stem cells
Stem cells are one kind of cells that capable of potentially differentiating into specific
tissue types and continuously self-renewaling through replication. Thus, they can
offer a consistent supply of physiologically relevant cells from validated
pathogen-free sources that differentiate into mature somatic cells [209]. Stem cell
analysis is an emerging field of great significance in healing damaged tissues and
replacing non-functional organs. The combination of microfabrication-based
technologies with stem cell analysis shows advantages in spatially and temporally
controlling cell growth and stimuli by combining surfaces that mimic complex
biochemistry and geometry properties of the ECM to maintain a homogeneous culture
of undifferentiated cells and direct stem cell differentiation reliably. Occhetta et al.
[210] designed and validated a microfluidic platform to culture 3D micromasses of
human bone marrow-derived mesenchymal stromal cells under continuous flow
perfusion. Defined concentrations of morphogens were delivered to specific culture
units yielding more uniform cell response throughout the 3D structures of perfused
micromasses and a 34-fold higher percentage of proliferating cells at day 7. A
microfluidic platform containing thousands of nanoliter-scale chambers suitable for
live-cell imaging studies was used for single hematopoietic stem cell proliferation
analysis (Fig. 11c). The automated medium exchange of this system made it possible
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to define when Steel factor stimulation was first required by adult hematopoietic stem
cells (HSCs) in vitro as the point of exit from quiescence [211]. A large array of 2D
no-flow chambers was designed to allow the monitoring of single HSCs for several
days. Live human HSCs could be stained with fluorescent primary antibodies to
reveal their stage in the hematopoiesis differentiation pathway. Human HSCs’ growth
rate, polarization and migration were correlated to their differentiation stage on this
chip [212]. We proposed a novel PDMS porous membrane-assembled 3D microdevice
for feeder-separated co-culture of mouse embryonic stem (mES) cells (Fig. 11d). Pure
mES cells of good pluripotency were separated from mouse embryonic fibroblasts
with easy and high efficiency [213].
Niches are complex microenvironments that regulate adult stem cell fate in vivo by
molecular interactions [214] and are critical to maintain stem cells in their multipotent
and often quiescent state. Upon activation, inches influence the decision of single
cells to self-renew or to undergo differentiation into the various lineages of a tissue.
Microfluidics is ideally suitable to give well-defined chemical and physical stimuli to
stem cells, so artificial niches are suitable constructed on microfluidic platform to
investigate biological behavior of stem cells. Lutolf’s group have made a great
contribution in this field. They applied microfabrication and micropatterning
technologies into dissecting the complex molecular interplay of stem cells and their
niches [215]. Hydrogel engineering was combined with droplet microfluidic
technology to form cell-type-specific artificial extracellular matrix in high-throughput.
The robust expansion or differentiation of stem cells was stimulated via display of
specific niche components on the microcarrier surface [216]. Artificial niche
microarrays in which individual microwells was functionalized with combinations of
proteins were reported to test the effect of cell-cell interactions on adipogenic
differentiation of adherent human mesenchymal stem cells (MSCs) and the effect of
substrate stiffness on osteogenic MSC differentiation. Artificial niches supporting
extensive self-renewal of nonadherent mouse neural stem cells also were identified
[217]. Single-cell analysis in microwell arrays revealed a highly heterogeneous
division behavior of HSCs [218]. Recently, droplet-based microfluidics has grown to
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be a promising strategy to build in vitro stem cell niches in a miniaturized and highly
precise fashion for stem cell biology [219]. A microfluidic gradient generator was
used to study brain-derived neurotrophic factor (BDNF)-enhanced neural stem cell
(NSC) chemotaxis which was critical for NSC migration during brain development
and neural tissue regeneration [220]. Osteogenic differentiated factors were
encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres which were
patterned into grooves by Teflon chips. These factors induced the osteogenic
differentiation of adipose-derived stem cells. The results demonstrated that a
combination of chemical and topographical cues was more effective to direct the
osteogenic commitment of stem cells than either was alone [221]. Cell-secreted
factors were demonstrated to be necessary to maintain self-renewal of mES cells on a
microfluidic perfusion device [222]. Another major advantage of microfluidic
technology is integrating electrical, optical, magnetic and acoustic sensors into a
single device to give observation on stem cells. Kang et al. [223] presented a
microfluidic device that coupled on-chip culture of adherent cells and transfection by
localized electroporation. Differentiation of NSCs and transfection of postmitotic
neurons with a green fluorescent protein plasmid were demonstrated on this device. A
radio frequency identification-based sensor platform was designed by integrating
sensitive elements onto glass substrate comprised of two comb-shaped interdigitated
gold electrodes covering an area of 1.8 mm × 2 mm. The platform was adopted to
characterize cultivation and differentiation of human bone marrow-derived
multipotent stem cells over periods of up to several days and weeks [224]. Osteogenic
differentiation of MSCs was evaluated on a SPR-based system. A high correlation
between the duration of osteogenic induction and the difference in refractive angle
shift with coefficient was demonstrated [225]. In the light of an increasing demand of
stem cells for cytotoxicity assay, disease modeling, drug screening and cell-based
therapies, there is an increasing necessity for advanced in vitro stem cell analysis
systems. Advances in biomaterials and microtechnology for stem cell analysis even
make it possible to offer mechanistic insight into organogenesis and unveil powerful
new models for drug discovery, as well as strategies for tissue regeneration in the
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clinic [226-228].

4. Conclusions and outlook

Owning to the inherent advances in device design, fabrication and flexible integration
with other microscale techniques, microfluidics has experienced an explosive growth
during the past decades and become a major analytical technology with enlarging
application fields. As a technique enabling precise manipulation of fluid at the
micrometer scale, microfluidics has paved a revolutionary way for time-saving and
cost-saving detection in sophisticated biochemical analysis. Not only traditional
biochemical operations were miniaturized onto the microfluidic device, but also new
platforms for generating unconventional strategies to overcome the challenges in
biochemical analysis were provided.
For improving microfluidic applications in biochemical analysis, there are still some
challenges have to be addressed: (1) Highly integrated microdevices coupled with
large and multifunctional instruments such as MS, nuclear magnetic resonance
spectrometer have to be designed. Although we have explored in the field of chip
coupled with MS, fully-integrated chip-MS platforms need to be further developed.
Besides that, how to miniaturize these large instruments to make them adjustable to be
coupled with chip still demand prompt development. (2) Automated manipulation of
microfluidic chips desire more convenient sample pretreatment and high-throughput
analysis. This promotion will advance microfluidics to the upper level in biochemical
analysis. (3) Real-time analysis of multiple targets of in vivo biochemical process in
situ is a crucial area in microfluidics. With the development of online microfluidic
chip-based biochemical analysis, it is hoped that portable and inexpensive
microfluidic devices will play a great role in point-of-care diagnosis and drug
discovery in the future.

Acknowledgements
This work was supported by the National Natural Science Foundation of China (No.
21405143, 21435002, 21227006) and the Fundamental Research Funds for the
Central Universities (No. 2-9-2014-023).
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Corresponding Author
*E-mail: jmlin@mail.tsinghua.edu.cn.

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Figure captions:
Fig. 1. Biochemical analysis integrated on microfluidic chip.

Fig. 2. Principle of single mouse hybridoma cell analysis and sorting depending on fluorescent

signals (Reprinted with permission from ref. [20]. Copyright 2013 Nature America, Inc.).

Fig. 3. AMP-based electrical detection of bacteria. (a) Schematic of AMPs immobilized on an

interdigitated microelectrode array. (b) Magnified image of theAMP magainin I in helical form,

modified with a terminal cysteine residue, and with clearly defined hydrophobic and hydrophilic

faces. (c) Detection of bacteriais achieved via binding of target cells to the immobilized AMPs. (d)

Image of the microfluidic chip (Reprinted with permission from ref. [42]. Copyright 2010

National Academy of Sciences.).

Fig. 4. SAW-based microfluidic devices. (a) A TSAW-based microfluidic tool detect the rodent

malaria parasite in blood (Reprinted with permission from ref. [65]. Copyright 2012 National

Academy of Sciences.). (b) Device structure and working mechanism of the acoustic tweezers

(Reprinted with permission from ref. [68].Copyright 2012 National Academy of Sciences.). (c)

Schematic illustration and image of the high-throughput ta-SSAW device for cancer cell

separation (Reprinted with permission from ref. [73]. Copyright 2015 National Academy of

Sciences.).

Fig. 5. Magnetic-based microfluidic technologies. (a) Photograph of the chip that was used for

continuous DNA extraction using phase-transfer magnetophoresis (Reprinted with permission

from ref. [80]. Copyright 2010 The Royal Society of Chemistry.). (b) Conceptual image of a

lab-on-a-chip droplet-based magnetofluidic platform which is composed of several functional

blocks (Reprinted with permission from ref. [83]. Copyright 2015 The Royal Society of

Chemistry.). (c) Schematic illustration of the process of magneto-optical biomolecular detection

by rotating super paramagnetic bead chains in solution (Reprinted with permission from ref. [84].

Copyright 2010 American Chemical Society.).

Fig. 6. Schematic diagram of the SIL-chip-ESI-MS system (Reprinted with permission from ref.

[104]. Copyright 2012 American Chemical Society.).

Fig. 7. Biomimetics on microfluidic chips. (a) Biologically inspired design of a human breathing

lung-on-a-chip microdevice (Reprinted with permission from ref. [19]. Copyright 2010 by the

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American Association for the Advancement of Science.). (b) Schematic diagram of a 3D

brain-on-a-chip with an interstitial level of flow (Reprinted with permission from ref. [127].

Copyright 2015 The Royal Society of Chemistry.). (c) Schematic cross-sectional view of a

microvasculature model (Reprinted with permission from ref. [128]. Copyright 2012 National

Academy of Sciences.).

Fig. 8. Drug discovery on microfluidic devices. (a) The microfluidic screening system (Reprinted

with permission from ref. [133]. Copyright 2012 National Academy of Sciences.). (b) Schematic

of the microfluidic device and cell co-culture process (Reprinted with permission from ref. [143].

Copyright 2015 Elsevier B.V.).

Fig. 9. Genetic analysis on microfluidic chip. (a) Design and operation of the microfluidic device

for single-cell gene expression analysis (Reprinted with permission from ref. [153]. Copyright

2011 National Academy of Sciences.). (b) The immunomagnetic exosomal RNA (iMER) platform

(Reprinted with permission from ref. [157]. Copyright 2015 Macmillan Publishers Limited.).

Fig. 10. Scheme depicting a cross-sectional view of the design of the chemomechanically

modulated biomolecule catch-and-release system (Reprinted with permission from ref. [166].

Copyright 2015 Macmillan Publishers Limited.).

Fig. 11. Cell analysis on microfluidic chips. (a) Microfluidic device for immune cell pairing

(Reprinted with permission from ref. [200]. Copyright 2015 Macmillan Publishers Limited.). (b)

Measuring attraction, repulsion, kinesis and inhibition during leukocyte responses to chemical

gradients (Reprinted with permission from ref. [201]. Copyright 2014 Macmillan Publishers

Limited.). (c) Iso-osmotic perfusion microfluidic cell culture array (Reprinted with permission

from ref. [211]. Copyright 2011 Nature America, Inc.). (d) Schematic of PDMS porous

membrane-assembled microfluidic feeder-separated co-culture system (Reprinted with permission

from ref. [213]. Copyright 2013 Nature Publishing Group, a division of Macmillan Publishers

Limited.).

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