Microfluidics for single cell analysis
Huabing Yin1
and Damian Marshall2
Substantial evidence shows that the heterogeneity of individual
cells within a genetically identical population can be critical to
their chance of survival. Methods that use average responses
from a population often mask the difference from individual
cells. To fully understand cell-to-cell variability, a complete
analysis of an individual cell, from its live state to cell lysates, is
essential. Highly sensitive detection of multiple components
and high throughput analysis of a large number of individual
cells remain the key challenges to realise this aim. In this
context, microfluidics and lab-on-a-chip technology have
emerged as the most promising avenue to address these
challenges.In this review, we will focus on the recent
development in microfluidics that are aimed at total single cell
analysis on chip, that is, from an individual live cell to its gene
and proteins. We also discuss the opportunities that
microfluidic based single cell analysis can bring into the drug
discovery process.
Addresses
1
Biomedical Engineering, School of Engineering, University of Glasgow,
Glasgow G12 8LT, UK
2
LGC Ltd, Teddington, UK
Corresponding author: Yin, Huabing (huabing.yin@glasgow.ac.uk)
Current Opinion in Biotechnology 2012, 23:110–119
This review comes from a themed issue on
Analytical biotechnology
Edited by Wei E. Huang and Jizhong Zhou
Available online 29th November 2011
0958-1669/$ – see front matter
# 2011 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2011.11.002
Introduction
Cellular analysis underpins many fields including life
science, diagnostics, the pharmaceutical industry and
renewable energy [1,2]. Conventional cell-based assays
measure the average response from a population of cells,
assuming that an average response is representative of a
typical cell within a population. However, this simplification
can result in a misleading interpretation. For
example, an average of 50% protein expression in a cell
population can represent either a 100% response in half
the cells or a 50% response in all.
A plethora of evidence has shown cellular heterogeneity
commonly exists within an isogenic or clonal population
[3]. This heterogeneity can arise through genetic drift,
differences in cell development or cell cycle status,
differences in cell age due to proliferation and passaging
as well as non-genetic heterogeneity arising from the
inherent stochasticity of cellular processes. Whether in
isolation or caused through a combination of the above
events, cellular heterogeneity can dramatically influence
cellular decision making and cell fate [4], however, this
can be masked by the average response from a population.
One approach to solve this dilemma is to analyse a
population at individual cell level. However, multiple
individual cells are required to obtain statistically meaningful
data, and therefore high throughput analysis is
critical.
A number of single cell analysis methods have been
established. Some conventional examples are listed in
Table 1. Microscopic imaging of a cell is the most obvious
approach and has been well established for wide range of
applications, such as physiological studies, measurements
of gene and protein expression. However, assays on single
cells are difficult to perform. The patch-clamp technique
enables highly sensitive measurement of changes in ion
channels, however it requires high skills to perform and
has limitations when detecting complex changes.
Furthermore, both of these methods are low throughput.
Conventional high throughput tools for single cell
analysis include well established methods such as Flow
Cytometry (and Imaging Flow Cytometry, Fluorescence
Activated Cell Sorting) that can detect, sort and collect
cells with desired properties. However, as data are only
collected at a single time point, these techniques still do
not permit dynamic monitoring of cell response.
Microfluidics has emerged as a powerful enabling technology
for investigating the inherent complexity of cellular
systems [5]. Typical microfluidic channels have
dimension of tens to hundreds of microns that are comparable
to the size of a single cell (10 mm in size and
roughly 1 pL in volume) [5]. At this length scale, the
physical behaviour of fluids is fundamentally different
from that seen in large channels: Laminar flow forms
when two fluid streams come together and mixing of
molecules across their interface only occurs through diffusion.
This phenomenon permits the accurate realisation
of complex molecular trajectories and has been effectively
exploited in many circumstances, such as in gradient
formation [6,7] and in localised stimuli delivery to
subcellular compartments [8].
In addition to the conventional methods already
described, new microtechnology based tools for single
cell analysis have also emerged in the last decade; for
example, optical/magnetic tweezing and the use of
Available online at www.sciencedirect.com
Current Opinion in Biotechnology 2012, 23:110–119 www.sciencedirect.com
patterned substrates (Table 1). However, these can
only address challenges in one or a few aspects of single
cell analysis. However, from an individual live cell to its
gene and proteins. By contrast, microfluidics has been
rapidly developed into a powerful approach capable of
integrating multiple functions for micrototal analysis of
biological systems (mTAS) and single cell analysis
[2,9
].
A rapid increasing body of new discoveries have been
enabled by microfluidics that would not be possible
otherwise. As discussed in this review, whole-genome
molecular haplotyping of single human cells has recently
been achieved using microfluidic technology [10
]. It is
arguable that microfluidics will be a central part of the
next generation sequencing focused towards personal
genomics. Capabilities in other single cell ‘omic’ studies,
including metabolomics, transcriptomics and proteomics
[11
] have also been significantly accelerated partly owing
to the rapid development of microfluidics. This advancement
will enable information on the subtle variability that
exists in biological systems to be revealed, and will have
significant implications for understanding the progression
of a disease as well as potential of transforming system
biology.
As an enabling technology, microfluidics owes its power
to handing small volumes of liquid (nanoliter to picoliter),
streamlining multiple procedures on a single chip,
with scope for parallelization. Recently developed droplet-microfluidics
has also emerged as a new forerunner
for single cell encapsulation and analysis with massive
parallelization. High throughput screening of rare cells to
a drug library has been achieved, providing addition
information on cell heterogeneity response [12
]. The
use of microdroplet confinement has enabled new
insights into the nature of quorum sensing, suggesting
it is a ‘‘cell-autonomous mechanism for diffusion or
efficiency sensing’’ [9
].
In this review, we will discuss various microfluidic applications
for single cell analysis. We focus on recent developments
aimed at total single cell analysis on chip, that is,
from an individual live cell to its gene and proteins. We
recognise the rapidly expanding number of applications
for single cell analysis and do not attempt to cover all
these topics. Instead, we will focus on the opportunities
that microfluidic based single cell analysis can bring into
the drug discovery process.
Single cell analysis on chip: overview
Most cellular processes are not isolated single events, but
are interconnected and hierarchically organised from
molecules to a whole cell. As such, multiparameter
analysis, for example, coupling epigenetic to gene expression
or protein expression to physiological measurement
at the level of single cells would enable a better understanding
of the whole process. To achieve this, a
complete analysis of an individual cell, from its live state
to cell lyses, is essential. We define such a process as
‘‘total analysis of single cells’’ and list it as one of the three
strategies that could be employed for cell analysis on chip
(Figure 1). The three strategies are roughly categorised
by the subject of interest, that is, whole cells or cell lysates
or both, and therefore the associated on chip operations
vary. In the last decade numerous technologies have been
developed for each operation making it impracticable to
list them all [1,2,13,14]. However, a few major approaches
are illustrated for each operation.
Although it appears that ‘‘total analysis of single cells’’ is
just a sequential combination of the other two strategies
(i.e. using whole cell and cell lysates respectively), it is far
from trivial. The total amount of analyte in a single cell is
Microfluidics technology Yin and Marshall 111
Table 1
Comparison of exampled approaches to single cell analysis
Approaches Main applications Key advantage Key disadvantage
Microscopic imaging  Morphological studies
 Gene and protein expressions
 Intracellular communications
Generic and well established Difficult to perform assays
on single cells
Patch clamp  Ion channel studies Very sensitive Limited applications
Flow cytometry  Gene and protein expression
 Population studies
Very high throughput Requires labeled cells
in suspension
Tweezing (e.g.
optical, magnetic)
 Manipulation
 Single cell mechanics
Low force range (pN) Requires complex
optical systems
Patterned substrates  Cell–cell communication studies
 Controlled cell proliferation and
differentiation, guidance
Simple and versatile Requires fabrication
capability
Microfluidics  All of the above, that is, a wide
range of applications from cell
manipulation to total single
cell analysis
Enabling technology
for integrated total
single cell analysis
Has not yet gained
popular acceptance
www.sciencedirect.com Current Opinion in Biotechnology 2012, 23:110–119
very low (N.B. a single somatic cell weighs 500 pg) [15].
Furthermore, the majority of the targets of interest are of
low copy number and among abundant interferences.
These complexities and limitations make the detection
of an analytes in a single living cell highly challenging.
Whether the combination of these operations is even
capable of appropriately sensitive detection is perhaps
the first thing to consider. For example, mass spectrometry
is the major tool for proteomics. However, integration
of mass spectrometry based proteomics and on chip single
cell analysis has yet to be achieved. The challenges for
single cell analysis on chip are substantial but also provide
great opportunities to advance technologies in the field of
microfluidics and its associated applications (see below).
Single cell analysis on chip: challenges and
prospects
A detailed understanding of the complex heterogeneity in
cell populations and its impact on cell behaviour and
biological responses can only be achieved using highly
sensitive methods with resolution at the single cell and
ideally subcellular level (e.g. down to the level of single
molecules). In addition, since the processes occur with
varying time scales (e.g. from seconds to hours), dynamic
control of conditions is essential. However, to achieve
total single cell analysis, the development of high
throughput systems capable of handling and analysing
individual cells is essential. In this section, we highlight
the recent advances towards this goal.
Spatiotemporal single cell manipulation
Several single cell immobilisation methods have been
developed, including microwells and traps. Traps can
function via a range of means including physical geometry,
hydrodynamics, magnetic force, dielectrophoresis and
optical or acoustic tweezers [16,17
,18] (Figure 2). In the
early stages of trap development, enhancement of trapping
efficiency has been a major focus – single cell
trapping efficiency has now reached 97% with optimised
trap architectures [18]. Recent development has seen a
tendency towards cell–cell interaction and long term
measurement of cell activities [18,20,21,22
,23]. Of
special interest is recent work that tracks the lineages
of hundreds of single cells in parallel for detailed study of
the time scales of heterogeneity in a population [22
]
(Figure 2). Via a similar method, the mechanisms governing
hematopoietic stem cell fate decision (e.g. selfrenewal
and differentiation processes) were revealed [23].
Recently, hydrodynamic cell trapping systems have
shown great promise for high throughput handing and
manipulation of single cells that would otherwise be
impractical [17
,19
,24
] (Figure 2). Various designs
of microwell, weir and microjail arrays have been used
to passively trap hundreds of single cells. Controlled
pairing and fusing of different cell types for fusionmediated
reprogramming has also been achieved [19
]
(Figure 2). Successful long term observation of the
response of normal and disordered single cells to drugs
112 Analytical biotechnology
Figure 1
Strategies
(a) Whole cell
(Biological analysis)
- On chip culture
- Immobilisation
- Trapping
- Chemical
- Biological
(e.g cell-cell)
- Physical
- Label-based
(e.g. fluorescence
luminescence)
- Label free
(e.g. impedance,
Raman, AFM)
- μwell
- μFACS
- DEP
- Optical tweezers
- Dynamic flow
B4. DetectionB3. Separation
- CE
- Affinity binding
- qPCR, RT-PCR
- Electrochemical
- ELISA
- ESI-MS, MS
B. Cell lysates
(B2-4)
(b) Cell lysates
(Biochemical analysis)
A. Whole cell
(A1-4)
Total single cell analysis
Cells
(clonal/wild)
- On chip culture
- Immobilisation
- Trapping
- Chemical
- Electrical
- Physical
- Optical
B1. Live cell handling B2. Lysis
A1. Live cell handling A2. Stimulation
A3. inspection
(non-invasive)
A4. Selection
Operations
Current Opinion in Biotechnology
Overview of microfluidic approaches for single cell analysis via three different strategies. The total single cell analysis requires the sequential
combination of strategies A and B. The approaches listed for each operation are not exhaustive. More detail can be found in several excellent reviews
[1,2,13,14]. Abbreviations: CE, Capillary Electrophoresis; ESIMS, Electrospray Ionization Mass Spectrometry; MS, Mass Spectrometry; DEP,
Dielectrophoresis; ELISA, Enzyme linked Immunosorbent Assays; qPCR, quantitative Polymerase Chain Reaction; RT-PCR, Reverse Transcription
Polymerase Chain Reaction; mFACS, Micro Fluorescence Activated Cell Sorting.
Current Opinion in Biotechnology 2012, 23:110–119 www.sciencedirect.com
demonstrates the potential of microfluidics in personalised
diagnostics [24
].
Single living cell detection
Although cell response can be measured quantitatively in
many ways, non-invasive methods (and treatments) are
required for living cell detection. At present fluorescence
techniques remain the most common methods owing to
their established nature and abundance of commercially
available probes. Since cell-to-cell variability is temporally
and spatially dependent [4], high content quantification of
all components involved would be ideal. A single time
pointfluorescencemeasurement,such as flowcytometry,is
well suited for high throughput single component quantification.
Spatiotemporal measurement of protein translocation,
interactions and modification rely on imaging
technologies such as Fluorescence Resonance Energy
Transfer (FRET) and Quantitative Time-Lapse Fluorescence
Microscopy [13,25]. However, the large data rich
files generated by these techniques compromise through-
put.
Recently, an intriguing approach has been developed for
both high content and high throughput detection [26]. It
used a photomultiplier to combine one-dimensional imaging
with microfluidic flowcytometry and demonstrated its
usefulnessinhighcontentscreeningwitha speedofseveral
thousands cells per second. This work, together with the
work from Chung et al. [27] illustrates that integration
between microfluidics, automation and conventional
microscopy could expand the ability of a biological lab
for high resolution and large-scale quantitative exper-
iments.
Development of label free techniques continues. A
powerful method capable of measuring growth rate at
the single cell level was developed using a Suspended
Microchannel Resonator technique coupled with microfluidic
control [28
]. It is envisaged that this method will
contribute to our understanding of many cellular processes
that affect cell growth.
Cell lysate analysis
Substantial developments have been made to integrate
major analytical methods currently used for genomics and
proteomic for single cell analysis [11
]. A few notable
examples that could contribute to future developments
are discussed below.
Gene
Gene expression analysis of single cells has been demonstrated
by various groups [29,30
,31]. For example,
Toriello et al. have developed integrated microfluidic
devices that couple single cell selection and capture,
enzymatic reaction and quantitative detection all on a
single platform [30
] (Figure 3). By quantifying mRNA
from two distinct populations at the single cell level, they
illustrated that stochastic variations in gene expression
and silencing within single cells is masked by bulk
measurements [30
]. Quake and colleagues have pioneered
large-scale gene expression analysis from single
cells. The technology they developed has been exploited
in a wide range of applications, such as a recent work on
the whole-genome molecular haplotyping of single
human metaphase cell [10
] (Figure 3). Indeed, developments
in this field advance rapidly, and many technologies
have started to enter commercial markets.
Examples of companies offering the technology include
Fluidigm Corporation, Oxford Nanopore Technology,
and Genechip Affymetrix. These technologies are massively
expanding the throughput for gene expression
measurements as well our understanding of the differential
gene expression profiles that underlie cell behaviours.
For example, Petriv et al. recently used the
Fluidigm dynamic array system to perform 80,000 RTqPCR
assays to map the expression of micro RNA’s in the
hematopoietic hierarchy and showed using single cells
the major reprogramming events that occurs upon cell
differentiation [32].
Epigenetics
Changes in gene expression within a cell population can
be caused by mechanisms other than the underlying
DNA sequence. Processes such as DNA methylation or
histone deacetylation can activate or silence genes without
genomic alteration. These epigenetic processes are a
major factor in non-Mendelian disease such as Alzheimer’s
and Parkinson’s disease [33] and can influence the
way cells behave in response to drugs [34]. Unlike gene
expression studies the application of single cell
approaches to epigenetic analysis has so far been limited.
However, a recent study by Kantlehner et al. [35] demonstrated
an approach for DNA methylation profiling within
the regulatory CpG islands of single cells for genes which
are aberrantly methylated in several types of cancer.
Their single cell approach was aided by the fact they
could reduce sample volumes down from the standard
0.2–1.5 mL PCR volumes to 5 mL which increased their
experimental success rate. This type of analysis would
therefore lend itself to microfluidic approaches where the
reactions volumes could be reduced even further and it is
probably only a matter of time before these studies are
reported in the literature.
Protein
In comparison to gene expression analysis, measuring
proteins from a single cell represents another level of
challenge [11
,14]. This is due to the extremely low
concentrations of proteins (sometimes less than 1000
copies per cell) and that, unlike DNA or RNA, proteins
cannot be directly amplified. Currently, single cell proteomics
is still in its infancy. Two main development
focuses are prominent, namely, sensitivity enhancement
and improved integration and automation.
Microfluidics technology Yin and Marshall 113
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114 Analytical biotechnology
Figure 2
1 2
1 Load green cells
‘up’
2 Transfer cells
‘down’
3 Load red cells
‘down’
(a) (b)
(c)
t=0 500
Taken from D. Di Carlo et. al, Analytical Chemistry
750 1000 1200 min
off
30 µm
off on21
Pho84 Hsp12 Rps8b
Taken from A. M. Skelly et. al, Nature Methods
(d)
Scale bar: 5µm All scale bars: 10µm
Taken from A.C. Rowat et. al, PNAS
1E1D1C1B
Gd V~
1A
Cells
Live Dead
488nm 555nm
1
Dyes
50µm2 2A 2B 2D
Laser Slit
2C
2E
Taken from E. Brouzes et. al, PNAS
Scale bar: 50 µm
Current Opinion in Biotechnology
Current Opinion in Biotechnology 2012, 23:110–119 www.sciencedirect.com
Innovative combinations of microfluidic devices and optical
microscopy have proven to be an efficient way to cope
with the detection of low number of proteins [36
,37].
The whole process is essential to this, namely single cell
manipulation, separation and detection all in one device.
This not only minimises loss of protein and reduces
contamination but also enables a correlation between
protein expression and the phenotypic stage of a singe
cell at a specific time point (i.e. when it is lysed). A good
example is shown in the work from Huang et al. where
individual b2 adrenergic receptors from a single cell were
successfully counted in a microfluidic channel using
cylindrical optics [36
]. With the aim towards single cell
proteomics, Salehi-Reyhani et al. have developed an
integrated microfluidic antibody capture chip and demonstrated
detection of the human tumour suppressor protein
p53 from a single cell [37].
Despite many advances in fluorescence based technologies,
problems in spectra overlapping and uncertainties
from labeling procedures ultimately restrict the total
number of simultaneous measurements. A promising
alternative is to combine single cell analysis with mass
spectrometry. Currently, full integration has been challenged
by a lack of an effective interface between the
mass spectrometer and miniaturised sample preparation
Microfluidics technology Yin and Marshall 115
(Figure 2 Legend) Single cell manipulation on chip. (A) Cell trapping by hydrodynamic focusing principles. (1) A schematic and (2) A phase contrast
image of an array of high quantity single-cell isolates. Reproduced by permission of the American Chemical Society [17
]. (B) Single cell manipulation
enabling precise control of cell-to-cell contact for cell fusion. (1) to (3) show schematics and corresponding red/green micrographs of a three-step cellloading
protocol. Reproduced by permission of Nature [19
]. (C) Restrained and monitored growth of individual cells allowing interrogation of the
lineage of single cells. (1) Bright field image showing single progenitor cells constrained to grow in a line. (2) Variations of three different protein
expressions in lineages of cells were revealed. Reproduced by permission of the National Academy of Sciences [22
]. (D) Droplet microfluidic for high
throughput single cell manipulation and analysis. A series of schematics (1) and corresponding micrographs (2) showing 5 optimised modules
employed for analysis of cell viability. Reproduced by permission of the National Academy of Sciences [12
].
Figure 3
cell-sorting region
(a)
control channels
amplification region
(c)
(b) 2
Taken from H.C. Fan et. al, Nature biotechnology
3
Sample inlet
3 valve
pump
RTDs
Reactor
Heater
Gold cell
capture pad
Hold chamber
Affinity capture
chamber
Anode
CE separation
channel
1 cm
Taken from N.M. Toriello et. al, PNASTaken from J.S. Mellors et. al, Analytical chemistry
50
heme
α 23+
α 24+
α 22+
α 21+
α 20+
α 19+ α 18+β 22+
β 21+
β 20+ β 19+
β 18+
β 17+
β 16+
40
30
20
10
0
50
40
30
20
IoncountIoncount
10
0
600 650 700 750 800
m/z
850 900 950 1000
600 650 700 750 800
m/z
850 900 950 1000
80 μm
Chromosomes released by
protease digestion
Partitioning of
chromosome suspension
into 48 chambers
Capture of a metaphase cell
1
5. Multiple strand displacement
amplification of
isolated chromosomes
6. Retrieval of amplified DNA for
downstream analyses
100 μm
4.3.2.
1.
Single chromosome
C
SC
B
B
Current Opinion in Biotechnology
Individual cell analysis from a live cell to gene or protein expressions. (A) An integrated microfluidic approach for whole-genome molecular haplotyping
of single cells. (1) An overview image of the device. (2–6) The stages that a cell progresses through during analysis. Reproduced by permission of
Nature [10
]. (B) integration of microfluidic device with ESI-MS for on line single cell analysis. (1) A diagram showing the basic operation. (2) Mass
spectra generated by a normal single cell lysis and (3) by a cell with higher haemoglobin content. Reproduced by permission of the American Chemical
Society [38
]. (C) An integrated device for gene expression analysis from a single cell. The processes of single cell capture, mRNA transcription and
amplification were all integrated on chip. Reproduced by permission of the National Academy of Sciences [30
].
www.sciencedirect.com Current Opinion in Biotechnology 2012, 23:110–119
platform [38
,39] (Figure 3). However, this is starting to
be addressed. For example, Mao et al. [40], have recently
reported the development of a microfluidic system which
allows high throughput nanoelectrospray into the mass
spectrometer providing sensitivity comparable to the
larger volume standard capillary emitters. While this is
not a fully integrated system it does open the possibility
for handling the volumes obtained from single cells and
presenting them to the mass spectrometer. Achieving this
complete integration between the two platforms will be
an important step forward towards single cell proteomics.
Metabolites
The cellular metabolome can provide a highly sensitive
measure of a cellular phenotype with quantification of
intracellular metabolite concentrations forming an integral
part of systems biology and pre-clinical drug toxicity
analysis [41]. Similar to protein analysis, single cell metabolomics
is challenging due to the small quantities of
analytes which are present in single cells, with a typical
1 pL cell volume containing metabolite concentrations in
the low femtomole range [15]. In common with single cell
protein analysis, direct amplification of the analytes cannot
be performed. To date, most metabolite profiling studies
on single cells have used approaches based on molecular
sensors such as FRET [42] which limits the analysis to
specific metabolites and prevents ‘omics’ level detection.
More recently however, mass spectrometry based
approaches have reached the limits of detection required
for single cell metabolomics making the technology suitable
for use in combination with microfluidic systems
[38
,43]. For example, Zenobi’s group developed a microfluidic
device which can position single cells onto a specialisedslideformetabolome
analysis ofADP,ATP, GTP,and
UDP-Glucose by matrix assisted laser desorption ionization
(MALDI) mass spectrometry. Meanwhile, Ramsey’s
group coupled a microfluidic system which incorporated
elements for single cell lysis, a solution electrophoresis
channel, where cellular constituents can be separated, and
an electrosmotic pump to direct the eluted cell components
to the mass spectrometer. These microfluidic
based mass spectrometry systems, while still at the experimental
stage, open up the possibility for multiple simultaneous
quantitative detection of multiple metabolites at
the single cell level and could uncover the subtle metabolite
concentration differences that are currently hidden
due to stochastic variability.
Cells in droplets
In the last few years droplet microfluidics has emerged as
a promising avenue for single cell encapsulation, dynamic
living cell assay, and single cell immunoanalysis
[12
,44,45] (Figure 2). In these cases, microfluidic
devices enable high throughput generation of femtoliter
sized and picoliter sized aqueous droplets in an immiscible
carrier, such as oil. These droplets are effectively
nanolabs that accommodate single cells and host all
subsequent reactions. High throughput cytotoxicity
screening of drugs [12
] (Figure 2), in-droplet cell lysis
and intracellular content analysis have all been illustrated
[46]. An advantage of in-droplet analysis is that the
droplet confines the lysate, preventing its dilution
through diffusion. Currently, most of these analyses are
based on fluorescence and therefore suffer from the
limitations described above. Recently, online Mass spectrometry
of individual microdroplets has been demonstrated
[47
]; this could greatly enhance the sensitivity of
droplet-based single cell analysis.
Microfluidic single cell analysis in drug
discovery
Cell based assays form fundamental practices at various
stages of the drug discovery process. To reduce the cost of
discovery, high throughput screening using robotics and
multi-well plates is the principal tool for pharmaceutical
industry. In general, the average response from a population
in a well (tens or hundreds of cells) is used as the
readout. However, the increasing evidence of heterogeneous
responses from individual cells invites the introduction
of new strategies capable of revealing information
at both the individual and population level. We envisage
that the development of microfluidic single cell analysis is
oneof the most attractive approaches towards this goal, and
illustrate its potential with a few recent examples below.
Concentration profiling
The concentration and time course of a drug (and
multiple drugs) interacting with a cell are essential in
evaluating its efficacy [48]. Laminar flow within microfluidic
devices provides a unique advantage to create
purpose designed concentration profiles (e.g. gradients)
for various applications, such as the study of chemotaxis
[7]. Dependent on the cell type studied, the drug concentration
can be delivered by either flowing over an
adhered cell layer on a substrate or by encapsulation
together with cells in a microdroplet [12
]. In the case
of adhered cells, different gradient generators have been
reported for creating concentration profiles for the
pharmacological screening of voltage-gated human
hERG K+
channels [49] and toxicity tests [50]. Such
systems enable the rapid acquisition of a large amount
of high content data that is essential to draw statistically
meaningful conclusions. It should also be noted that shear
stress can induce similar response to the chemical stimuli
for adherent cells [51] and the high flow rate used for
gradient generation might damage shear sensitive cells
[50]. The droplet-based cytotoxicity screen is of specific
interest for large scale single cell based screening [12
] as
it offers high throughput, precise delivery and powerful
manipulation permitting a multitude of possibilities.
Miniaturisation for future discoveries
In recognition of the challenges that current target-driven
drug discovery faces, there is an ongoing paradigm shift
116 Analytical biotechnology
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towards pathway-driven drug discovery [52]. Multiparameter
phenotype profiling of a compound is essential
to understand the biological pathway but faces many
challenges [52]. In this context, microfluidic technology
will be one of the enabling technologies to address these
challenges, as exemplified in the many new discoveries
that would not be possible by conventional methods
[53,54]. For example, recently, Bao et al. has developed
a microfluidic system that enables precise timing control
with integration of multiple simultaneous experiments.
With this system, tracking responses of individual cells
across multiple stimulations was achieved, leading to the
new finding that the variability in G-protein-Coupled
Signalling is associated with long-lived cell state difference
rather than from stochasticity [54].
Conclusions
The impact of the rapid expansion of high throughput
single cell analysis is evident in its great potential for
numerous applications, including drug discovery, diagnostics,
cancer research, regenerative medicine, system and
synthetic biology, and many others. The implementation
of microfluidic technologies in single cell analysis is one of
the most promising approaches that not only offers infor-
mationrich,highthroughputscreeningbutalsoenablesthe
creation of innovative conditions that are impractical or
impossible by conventional means. The possibilities for
distinguishing the difference between individual cells and
the benefits from miniaturisation (e.g. confinement) have
led to many discoveries in both traditional biopharmaceutical
communities and in emerging fields such as synthetic
biology [9
,52]. Recent research has only just started to
show the full potential of microfluidics in this field.
Major challenges still remain. Full integration of microfluidic
(and the miniaturised platforms) for single cell
manipulation with established analytic methods, such as
mass spectrometry and NMR, traditionally used for high
sensitivity single molecule detection is still some way off.
Developing effective strategies for extracting system
level information the very large data sets generated by
total single cell analysis of population is essential. New
breakthroughs in these areas will enable microfluidics
approaches to transition from an interesting research topic
to routinely used tool for drug screening and diagnostics.
Acknowledgements
HY acknowledge support from the Royal Academy of Engineering, also the
Engineering and Physical Science Research Council (EPSRC). DM
acknowledges support from the UK National Measurement System.
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