Available online at www.sciencedirect.

com

Microfluidics for single cell analysis

Huabing Yin1 and Damian Marshall2

Substantial evidence shows that the heterogeneity of individual differences in cell age due to proliferation and passaging
cells within a genetically identical population can be critical to as well as non-genetic heterogeneity arising from the
their chance of survival. Methods that use average responses inherent stochasticity of cellular processes. Whether in
from a population often mask the difference from individual isolation or caused through a combination of the above
cells. To fully understand cell-to-cell variability, a complete events, cellular heterogeneity can dramatically influence
analysis of an individual cell, from its live state to cell lysates, is cellular decision making and cell fate [4], however, this
essential. Highly sensitive detection of multiple components can be masked by the average response from a population.
and high throughput analysis of a large number of individual One approach to solve this dilemma is to analyse a
cells remain the key challenges to realise this aim. In this population at individual cell level. However, multiple
context, microfluidics and lab-on-a-chip technology have individual cells are required to obtain statistically mean-
emerged as the most promising avenue to address these ingful data, and therefore high throughput analysis is
challenges.In this review, we will focus on the recent critical.
development in microfluidics that are aimed at total single cell
analysis on chip, that is, from an individual live cell to its gene A number of single cell analysis methods have been
and proteins. We also discuss the opportunities that established. Some conventional examples are listed in
microfluidic based single cell analysis can bring into the drug Table 1. Microscopic imaging of a cell is the most obvious
discovery process. approach and has been well established for wide range of
Addresses applications, such as physiological studies, measurements
1
Biomedical Engineering, School of Engineering, University of Glasgow, of gene and protein expression. However, assays on single
Glasgow G12 8LT, UK cells are difficult to perform. The patch-clamp technique
2
LGC Ltd, Teddington, UK
enables highly sensitive measurement of changes in ion
Corresponding author: Yin, Huabing ([email protected]) channels, however it requires high skills to perform and
has limitations when detecting complex changes.
Furthermore, both of these methods are low throughput.
Current Opinion in Biotechnology 2012, 23:110–119 Conventional high throughput tools for single cell
This review comes from a themed issue on analysis include well established methods such as Flow
Analytical biotechnology Cytometry (and Imaging Flow Cytometry, Fluorescence
Edited by Wei E. Huang and Jizhong Zhou Activated Cell Sorting) that can detect, sort and collect
cells with desired properties. However, as data are only
Available online 29th November 2011
collected at a single time point, these techniques still do
0958-1669/$ – see front matter not permit dynamic monitoring of cell response.
# 2011 Elsevier Ltd. All rights reserved.
Microfluidics has emerged as a powerful enabling tech-
DOI 10.1016/j.copbio.2011.11.002
nology for investigating the inherent complexity of cel-
lular systems [5]. Typical microfluidic channels have
dimension of tens to hundreds of microns that are com-
Introduction parable to the size of a single cell (10 mm in size and
Cellular analysis underpins many fields including life roughly 1 pL in volume) [5]. At this length scale, the
science, diagnostics, the pharmaceutical industry and physical behaviour of fluids is fundamentally different
renewable energy [1,2]. Conventional cell-based assays from that seen in large channels: Laminar flow forms
measure the average response from a population of cells, when two fluid streams come together and mixing of
assuming that an average response is representative of a molecules across their interface only occurs through dif-
typical cell within a population. However, this simplifica- fusion. This phenomenon permits the accurate realisation
tion can result in a misleading interpretation. For of complex molecular trajectories and has been effec-
example, an average of 50% protein expression in a cell tively exploited in many circumstances, such as in gra-
population can represent either a 100% response in half dient formation [6,7] and in localised stimuli delivery to
the cells or a 50% response in all. subcellular compartments [8].

A plethora of evidence has shown cellular heterogeneity In addition to the conventional methods already
commonly exists within an isogenic or clonal population described, new microtechnology based tools for single
[3]. This heterogeneity can arise through genetic drift, cell analysis have also emerged in the last decade; for
differences in cell development or cell cycle status, example, optical/magnetic tweezing and the use of

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 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 Generic and well established Difficult to perform assays
 Gene and protein expressions on single cells
 Intracellular communications
Patch clamp  Ion channel studies Very sensitive Limited applications
Flow cytometry  Gene and protein expression Very high throughput Requires labeled cells
 Population studies in suspension
Tweezing (e.g.  Manipulation Low force range (pN) Requires complex
optical, magnetic)  Single cell mechanics optical systems
Patterned substrates  Cell–cell communication studies Simple and versatile Requires fabrication
 Controlled cell proliferation and capability
differentiation, guidance
Microfluidics  All of the above, that is, a wide Enabling technology Has not yet gained
range of applications from cell for integrated total popular acceptance
manipulation to total single single cell analysis
cell analysis

patterned substrates (Table 1). However, these can it is a ‘‘cell-autonomous mechanism for diffusion or
only address challenges in one or a few aspects of single efficiency sensing’’ [9].
cell analysis. However, from an individual live cell to its
gene and proteins. By contrast, microfluidics has been In this review, we will discuss various microfluidic appli-
rapidly developed into a powerful approach capable of cations for single cell analysis. We focus on recent devel-
integrating multiple functions for micrototal analysis of opments aimed at total single cell analysis on chip, that is,
biological systems (mTAS) and single cell analysis from an individual live cell to its gene and proteins. We
[2,9]. recognise the rapidly expanding number of applications
for single cell analysis and do not attempt to cover all
A rapid increasing body of new discoveries have been these topics. Instead, we will focus on the opportunities
enabled by microfluidics that would not be possible that microfluidic based single cell analysis can bring into
otherwise. As discussed in this review, whole-genome the drug discovery process.
molecular haplotyping of single human cells has recently
been achieved using microfluidic technology [10]. It is Single cell analysis on chip: overview
arguable that microfluidics will be a central part of the Most cellular processes are not isolated single events, but
next generation sequencing focused towards personal are interconnected and hierarchically organised from
genomics. Capabilities in other single cell ‘omic’ studies, molecules to a whole cell. As such, multiparameter
including metabolomics, transcriptomics and proteomics analysis, for example, coupling epigenetic to gene expres-
[11] have also been significantly accelerated partly owing sion or protein expression to physiological measurement
to the rapid development of microfluidics. This advance- at the level of single cells would enable a better un-
ment will enable information on the subtle variability that derstanding of the whole process. To achieve this, a
exists in biological systems to be revealed, and will have complete analysis of an individual cell, from its live state
significant implications for understanding the progression to cell lyses, is essential. We define such a process as
of a disease as well as potential of transforming system ‘‘total analysis of single cells’’ and list it as one of the three
biology. strategies that could be employed for cell analysis on chip
(Figure 1). The three strategies are roughly categorised
As an enabling technology, microfluidics owes its power by the subject of interest, that is, whole cells or cell lysates
to handing small volumes of liquid (nanoliter to picoli- or both, and therefore the associated on chip operations
ter), streamlining multiple procedures on a single chip, vary. In the last decade numerous technologies have been
with scope for parallelization. Recently developed dro- developed for each operation making it impracticable to
plet-microfluidics has also emerged as a new forerunner list them all [1,2,13,14]. However, a few major approaches
for single cell encapsulation and analysis with massive are illustrated for each operation.
parallelization. High throughput screening of rare cells to
a drug library has been achieved, providing addition Although it appears that ‘‘total analysis of single cells’’ is
information on cell heterogeneity response [12]. The just a sequential combination of the other two strategies
use of microdroplet confinement has enabled new (i.e. using whole cell and cell lysates respectively), it is far
insights into the nature of quorum sensing, suggesting from trivial. The total amount of analyte in a single cell is

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112 Analytical biotechnology

Figure 1

Strategies Operations

A3. inspection
(a) Whole cell A1. Live cell handling A2. Stimulation A4. Selection
(non-invasive)
(Biological analysis)
- On chip culture - Chemical - Label-based - μwell
- Immobilisation - Biological (e.g. fluorescence - μFACS
- Trapping (e.g cell-cell) luminescence) - DEP
- Physical - Label free - Optical tweezers
(e.g. impedance, - Dynamic flow
Raman, AFM)

(b) Cell lysates B1. Live cell handling B2. Lysis B3. Separation B4. Detection
(Biochemical analysis) - Chemical - qPCR, RT-PCR
- On chip culture
Cells - Electrical - CE - Electrochemical
- Immobilisation
(clonal/wild) - Physical - Affinity binding - ELISA
- Trapping
- Optical - ESI-MS, MS

A. Whole cell B. Cell lysates

Total single cell analysis
(A1-4) (B2-4)

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.

very low (N.B. a single somatic cell weighs 500 pg) [15]. Spatiotemporal single cell manipulation
Furthermore, the majority of the targets of interest are of Several single cell immobilisation methods have been
low copy number and among abundant interferences. developed, including microwells and traps. Traps can
These complexities and limitations make the detection function via a range of means including physical geome-
of an analytes in a single living cell highly challenging. try, hydrodynamics, magnetic force, dielectrophoresis and
Whether the combination of these operations is even optical or acoustic tweezers [16,17,18] (Figure 2). In the
capable of appropriately sensitive detection is perhaps early stages of trap development, enhancement of trap-
the first thing to consider. For example, mass spectrom- ping efficiency has been a major focus – single cell
etry is the major tool for proteomics. However, integration trapping efficiency has now reached 97% with optimised
of mass spectrometry based proteomics and on chip single trap architectures [18]. Recent development has seen a
cell analysis has yet to be achieved. The challenges for tendency towards cell–cell interaction and long term
single cell analysis on chip are substantial but also provide measurement of cell activities [18,20,21,22,23]. Of
great opportunities to advance technologies in the field of special interest is recent work that tracks the lineages
microfluidics and its associated applications (see below). of hundreds of single cells in parallel for detailed study of
the time scales of heterogeneity in a population [22]
Single cell analysis on chip: challenges and (Figure 2). Via a similar method, the mechanisms govern-
prospects ing hematopoietic stem cell fate decision (e.g. self-
A detailed understanding of the complex heterogeneity in renewal and differentiation processes) were revealed [23].
cell populations and its impact on cell behaviour and
biological responses can only be achieved using highly Recently, hydrodynamic cell trapping systems have
sensitive methods with resolution at the single cell and shown great promise for high throughput handing and
ideally subcellular level (e.g. down to the level of single manipulation of single cells that would otherwise be
molecules). In addition, since the processes occur with impractical [17,19,24] (Figure 2). Various designs
varying time scales (e.g. from seconds to hours), dynamic of microwell, weir and microjail arrays have been used
control of conditions is essential. However, to achieve to passively trap hundreds of single cells. Controlled
total single cell analysis, the development of high pairing and fusing of different cell types for fusion-
throughput systems capable of handling and analysing mediated reprogramming has also been achieved [19]
individual cells is essential. In this section, we highlight (Figure 2). Successful long term observation of the
the recent advances towards this goal. response of normal and disordered single cells to drugs

Current Opinion in Biotechnology 2012, 23:110–119 www.sciencedirect.com

 Microfluidics technology Yin and Marshall 113

demonstrates the potential of microfluidics in personal- illustrated that stochastic variations in gene expression
ised diagnostics [24]. and silencing within single cells is masked by bulk
measurements [30]. Quake and colleagues have pio-
Single living cell detection neered large-scale gene expression analysis from single
Although cell response can be measured quantitatively in cells. The technology they developed has been exploited
many ways, non-invasive methods (and treatments) are in a wide range of applications, such as a recent work on
required for living cell detection. At present fluorescence the whole-genome molecular haplotyping of single
techniques remain the most common methods owing to human metaphase cell [10] (Figure 3). Indeed, devel-
their established nature and abundance of commercially opments in this field advance rapidly, and many tech-
available probes. Since cell-to-cell variability is temporally nologies have started to enter commercial markets.
and spatially dependent [4], high content quantification of Examples of companies offering the technology include
all components involved would be ideal. A single time Fluidigm Corporation, Oxford Nanopore Technology,
point fluorescence measurement, such as flow cytometry, is and Genechip Affymetrix. These technologies are mas-
well suited for high throughput single component quanti- sively expanding the throughput for gene expression
fication. Spatiotemporal measurement of protein translo- measurements as well our understanding of the differ-
cation, interactions and modification rely on imaging ential gene expression profiles that underlie cell beha-
technologies such as Fluorescence Resonance Energy viours. For example, Petriv et al. recently used the
Transfer (FRET) and Quantitative Time-Lapse Fluor- Fluidigm dynamic array system to perform 80,000 RT-
escence Microscopy [13,25]. However, the large data rich qPCR assays to map the expression of micro RNA’s in the
files generated by these techniques compromise through- hematopoietic hierarchy and showed using single cells
put. the major reprogramming events that occurs upon cell
differentiation [32].
Recently, an intriguing approach has been developed for
both high content and high throughput detection [26]. It Epigenetics
used a photomultiplier to combine one-dimensional ima- Changes in gene expression within a cell population can
ging with microfluidic flow cytometry and demonstrated its be caused by mechanisms other than the underlying
usefulness in high content screening with a speed of several DNA sequence. Processes such as DNA methylation or
thousands cells per second. This work, together with the histone deacetylation can activate or silence genes with-
work from Chung et al. [27] illustrates that integration out genomic alteration. These epigenetic processes are a
between microfluidics, automation and conventional major factor in non-Mendelian disease such as Alzhei-
microscopy could expand the ability of a biological lab mer’s and Parkinson’s disease [33] and can influence the
for high resolution and large-scale quantitative exper- way cells behave in response to drugs [34]. Unlike gene
iments. expression studies the application of single cell
approaches to epigenetic analysis has so far been limited.
Development of label free techniques continues. A However, a recent study by Kantlehner et al. [35] demon-
powerful method capable of measuring growth rate at strated an approach for DNA methylation profiling within
the single cell level was developed using a Suspended the regulatory CpG islands of single cells for genes which
Microchannel Resonator technique coupled with micro- are aberrantly methylated in several types of cancer.
fluidic control [28]. It is envisaged that this method will Their single cell approach was aided by the fact they
contribute to our understanding of many cellular pro- could reduce sample volumes down from the standard
cesses that affect cell growth. 0.2–1.5 mL PCR volumes to 5 mL which increased their
experimental success rate. This type of analysis would
Cell lysate analysis therefore lend itself to microfluidic approaches where the
Substantial developments have been made to integrate reactions volumes could be reduced even further and it is
major analytical methods currently used for genomics and probably only a matter of time before these studies are
proteomic for single cell analysis [11]. A few notable reported in the literature.
examples that could contribute to future developments
are discussed below. Protein
In comparison to gene expression analysis, measuring
Gene proteins from a single cell represents another level of
Gene expression analysis of single cells has been demon- challenge [11,14]. This is due to the extremely low
strated by various groups [29,30,31]. For example, concentrations of proteins (sometimes less than 1000
Toriello et al. have developed integrated microfluidic copies per cell) and that, unlike DNA or RNA, proteins
devices that couple single cell selection and capture, cannot be directly amplified. Currently, single cell pro-
enzymatic reaction and quantitative detection all on a teomics is still in its infancy. Two main development
single platform [30] (Figure 3). By quantifying mRNA focuses are prominent, namely, sensitivity enhancement
from two distinct populations at the single cell level, they and improved integration and automation.

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114 Analytical biotechnology

Figure 2

(a) (b)
1 Load green cells 2 Transfer cells 3 Load red cells
1 2 ‘up’ ‘down’ ‘down’

Scale bar: 50 µm

30 µm

Taken from D. Di Carlo et. al, Analytical Chemistry Taken from A. M. Skelly et. al, Nature Methods
(c)
t=0 500 750 1000 1200 min Pho84 Hsp12 Rps8b
1 2 off off on

Scale bar: 5µm All scale bars: 10µm

Taken from A.C. Rowat et. al, PNAS
(d) 1 1A 1B 1C 1D 1E
Cells
Gd V~
Live Dead

488nm 555nm

Dyes

2 2A 50µm 2B 2D

2C

2E Laser Slit

Taken from E. Brouzes et. al, PNAS

Current Opinion in Biotechnology

Current Opinion in Biotechnology 2012, 23:110–119 www.sciencedirect.com

 Microfluidics technology Yin and Marshall 115

Figure 3

(a) 1. (c)
cell-sorting region
Sample inlet
control channels

amplification region 3 valve

pump
RTDs
2. 3. 4. Reactor
Partitioning of
chromosome suspension
into 48 chambers
100 μm Heater
80 μm Single chromosome
Chromosomes released by
Gold cell
Capture of a metaphase cell
protease digestion capture pad
5. Multiple strand displacement 6. Retrieval of amplified DNA for
amplification of downstream analyses
isolated chromosomes Hold chamber
Taken from H.C. Fan et. al, Nature biotechnology
50
Affinity capture
α 22+
(b) 40 α 21+ 2 chamber
α 23+ β 21+β 20+ β 19+
Ion count

30 heme β 18+
α 20+ β 17+
CE separation
20 α 19+ α 18+
1 B α 24+ β 22+ β 16+
10
channel
SC
0
600 650 700 750 800
m/z
850 900 950 1000 Anode
C 50

40 3
Ion count

30

B 20

10 1 cm
0
600 650 700 750 800 850 900 950 1000
m/z
Taken from J.S. Mellors et. al, Analytical chemistry Taken from N.M. Toriello et. al, PNAS
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].

Innovative combinations of microfluidic devices and opti- proteomics, Salehi-Reyhani et al. have developed an
cal microscopy have proven to be an efficient way to cope integrated microfluidic antibody capture chip and demon-
with the detection of low number of proteins [36,37]. strated detection of the human tumour suppressor protein
The whole process is essential to this, namely single cell p53 from a single cell [37].
manipulation, separation and detection all in one device.
This not only minimises loss of protein and reduces Despite many advances in fluorescence based technol-
contamination but also enables a correlation between ogies, problems in spectra overlapping and uncertainties
protein expression and the phenotypic stage of a singe from labeling procedures ultimately restrict the total
cell at a specific time point (i.e. when it is lysed). A good number of simultaneous measurements. A promising
example is shown in the work from Huang et al. where alternative is to combine single cell analysis with mass
individual b2 adrenergic receptors from a single cell were spectrometry. Currently, full integration has been chal-
successfully counted in a microfluidic channel using lenged by a lack of an effective interface between the
cylindrical optics [36]. With the aim towards single cell mass spectrometer and miniaturised sample preparation

(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 cell-
loading 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].

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116 Analytical biotechnology

platform [38,39] (Figure 3). However, this is starting to subsequent reactions. High throughput cytotoxicity
be addressed. For example, Mao et al. [40], have recently screening of drugs [12] (Figure 2), in-droplet cell lysis
reported the development of a microfluidic system which and intracellular content analysis have all been illustrated
allows high throughput nanoelectrospray into the mass [46]. An advantage of in-droplet analysis is that the
spectrometer providing sensitivity comparable to the droplet confines the lysate, preventing its dilution
larger volume standard capillary emitters. While this is through diffusion. Currently, most of these analyses are
not a fully integrated system it does open the possibility based on fluorescence and therefore suffer from the
for handling the volumes obtained from single cells and limitations described above. Recently, online Mass spec-
presenting them to the mass spectrometer. Achieving this trometry of individual microdroplets has been demon-
complete integration between the two platforms will be strated [47]; this could greatly enhance the sensitivity of
an important step forward towards single cell proteomics. droplet-based single cell analysis.

Metabolites Microfluidic single cell analysis in drug

The cellular metabolome can provide a highly sensitive discovery
measure of a cellular phenotype with quantification of Cell based assays form fundamental practices at various
intracellular metabolite concentrations forming an integral stages of the drug discovery process. To reduce the cost of
part of systems biology and pre-clinical drug toxicity discovery, high throughput screening using robotics and
analysis [41]. Similar to protein analysis, single cell meta- multi-well plates is the principal tool for pharmaceutical
bolomics is challenging due to the small quantities of industry. In general, the average response from a popu-
analytes which are present in single cells, with a typical lation in a well (tens or hundreds of cells) is used as the
1 pL cell volume containing metabolite concentrations in readout. However, the increasing evidence of hetero-
the low femtomole range [15]. In common with single cell geneous responses from individual cells invites the intro-
protein analysis, direct amplification of the analytes cannot duction of new strategies capable of revealing information
be performed. To date, most metabolite profiling studies at both the individual and population level. We envisage
on single cells have used approaches based on molecular that the development of microfluidic single cell analysis is
sensors such as FRET [42] which limits the analysis to one of the most attractive approaches towards this goal, and
specific metabolites and prevents ‘omics’ level detection. illustrate its potential with a few recent examples below.
More recently however, mass spectrometry based
approaches have reached the limits of detection required Concentration profiling
for single cell metabolomics making the technology suit- The concentration and time course of a drug (and
able for use in combination with microfluidic systems multiple drugs) interacting with a cell are essential in
[38,43]. For example, Zenobi’s group developed a micro- evaluating its efficacy [48]. Laminar flow within micro-
fluidic device which can position single cells onto a special- fluidic devices provides a unique advantage to create
ised slide for metabolome analysis of ADP, ATP, GTP, and purpose designed concentration profiles (e.g. gradients)
UDP-Glucose by matrix assisted laser desorption ioniz- for various applications, such as the study of chemotaxis
ation (MALDI) mass spectrometry. Meanwhile, Ramsey’s [7]. Dependent on the cell type studied, the drug con-
group coupled a microfluidic system which incorporated centration can be delivered by either flowing over an
elements for single cell lysis, a solution electrophoresis adhered cell layer on a substrate or by encapsulation
channel, where cellular constituents can be separated, and together with cells in a microdroplet [12]. In the case
an electrosmotic pump to direct the eluted cell com- of adhered cells, different gradient generators have been
ponents to the mass spectrometer. These microfluidic reported for creating concentration profiles for the
based mass spectrometry systems, while still at the exper- pharmacological screening of voltage-gated human
imental stage, open up the possibility for multiple simul- hERG K+ channels [49] and toxicity tests [50]. Such
taneous quantitative detection of multiple metabolites at systems enable the rapid acquisition of a large amount
the single cell level and could uncover the subtle metab- of high content data that is essential to draw statistically
olite concentration differences that are currently hidden meaningful conclusions. It should also be noted that shear
due to stochastic variability. stress can induce similar response to the chemical stimuli
for adherent cells [51] and the high flow rate used for
Cells in droplets gradient generation might damage shear sensitive cells
In the last few years droplet microfluidics has emerged as [50]. The droplet-based cytotoxicity screen is of specific
a promising avenue for single cell encapsulation, dynamic interest for large scale single cell based screening [12] as
living cell assay, and single cell immunoanalysis it offers high throughput, precise delivery and powerful
[12,44,45] (Figure 2). In these cases, microfluidic manipulation permitting a multitude of possibilities.
devices enable high throughput generation of femtoliter
sized and picoliter sized aqueous droplets in an immis- Miniaturisation for future discoveries
cible carrier, such as oil. These droplets are effectively In recognition of the challenges that current target-driven
nanolabs that accommodate single cells and host all drug discovery faces, there is an ongoing paradigm shift

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 Microfluidics technology Yin and Marshall 117

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synthetic biology, and many others. The implementation molecules and therefore enables an individual cell to rapidly condition its
of microfluidic technologies in single cell analysis is one of microenvironment. Unique advantages of this approach were sum-
the most promising approaches that not only offers infor- marised, including observations of new function and behaviours of
bacteria (e.g. quorum sensing as cell-autonomous mechanism for diffu-
mation rich, high throughput screening but also enables the sion), increase in detection speed, and enhancement in cultivation of rare
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led to many discoveries in both traditional biopharmaceu- genome molecular haplotyping of single human cells. The authors
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