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Journal of Nanobiotechnology
Open Access
Review
Microfluidics in biotechnology
Richard Barry*
1
and Dimitri Ivanov
2
Address:
1
School of Biological Sciences Royal Holloway, University of London Egham, Surrey TW20 0EX United Kingdom and
2
"Laboratoire de
Physique des Polymres, CP223 Universit Libre de Bruxelles" B-1050 Brussels Belgium
Email: Richard Barry* - [email protected]; Dimitri Ivanov - [email protected]
* Corresponding author
Abstract
Microfluidics enables biotechnological processes to proceed on a scale (microns) at which physical
processes such as osmotic movement, electrophoretic-motility and surface interactions become
enhanced. At the microscale sample volumes and assay times are reduced, and procedural costs
are lowered. The versatility of microfluidic devices allows interfacing with current methods and
technologies. Microfluidics has been applied to DNA analysis methods and shown to accelerate
DNA microarray assay hybridisation times. The linking of microfluidics to protein analysis
techologies, e.g. mass spectrometry, enables picomole amounts of peptide to be analysed within a
controlled micro-environment. The flexibility of microfluidics will facilitate its exploitation in assay
development across multiple biotechnological disciplines.
Background
Current analytical techniques in biotechnology can
potentially benefit from an integrated reduction in scale
through lowered production and operating costs, and via
the specific dynamics of flowing fluids occurring at the
mico-scale, which enable the generation of accurate quan-
titative assays. Microfluidics combines multiple disci-
plines including biotechnology, microtechnology,
physics, and analytical chemistry and has flourished as a
research field. The processes involved in biotechnology
and microfluidics technologies take place on a very small
scale (microns to millimetres) where some physical proc-
esses can become enhanced, e.g. osmotic movement, elec-
trophoretic motility and surface interactions.
Microfluidics technology has essentially taken advantage
of the inherent properties of liquids and gases at the
microscale and combined this with semiconductor tech-
nology in order to build singular devices using a stream-
lined manufacturing process.
Commercial products/technologies
In general, microfluidic devices can offer a number of
advantages over more conventional systems, e.g. their
compact size, disposable nature, increased utility and a
prerequisite for reduced concentrations of sample rea-
gents. Miniaturised assemblies can be designed to per-
form a wide range of tasks that range from detecting
airborne toxins to analysing DNA and protein sequences.
Therefore, microfluidics systems provide a real potential
for improving the efficiency of techniques applied in drug
discovery and diagnostics. In order for microfluidic tech-
nology to interface with, and provide improvements for,
current assaying techniques it needs to be adaptable.
Some commercial microfluidics systems illustrate their
suitability to biotechnological applications.
Typical devices include passive flow systems, such as the
Passive Fluid Control (PFC) micro fluid analysis system
by BioMicro Systems http://www.biomicro.com. PFC
incorporates 'building block-like' components into circuit
designs in order to carry out sample processing, e.g.
Published: 31 March 2004
Journal of Nanobiotechnology 2004, 2:2
Received: 04 December 2003
Accepted: 31 March 2004
This article is available from: http://www.jnanobiotechnology.com/content/2/1/2
2004 Barry and Ivanov; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in
all media for any purpose, provided this notice is preserved along with the article's original URL.
Journal of Nanobiotechnology 2004, 2 http://www.jnanobiotechnology.com/content/2/1/2
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immobilisation, mixing, incubation. Essentially, PFC uti-
lises hydrophobicity and 'passive valves' (a narrowing of
capillaries) to control the movement of small volumes of
fluids (< 1 l) within a network of channels. Incorpora-
tion of active or passive pumps can also be used to control
the movement of fluids in microfluidic systems, e.g.
Nanostream's Snap-n-Flow system http://www.nanos
tream.com. Modules are 'snapped' together to construct a
completely integrated and versatile system. A further setup
by Gyros http://www.gyrosmicro.com has integrated a CD
element with the movement and control of nanolitre vol-
umes. When the CD is set spinning centrifugal forces are
created allowing the device to be used to produce a con-
trolled passage of samples through 'microfabricated units'
on the surface of the CD. This technology can be applied
to sample preparation for maldi-mass spectrometric
analysis.
Microfluidics systems capable of assaying 'unprocessed'
biological samples, e.g. blood, have been developed
therefore eliminating the requirement for sample prepara-
tion, e.g. Micronics http://www.micronics.net. Micronics'
MicroFlow system can be used to extract analytes directly
from whole blood and other particulate suspensions (5
200 l volumes). The system utilises disposable 'lab
cards', e.g. the ActiveH card can be used for sample prep-
aration and isolation whereas the ActiveT card can be
used in immunoassays.
DNA applications
Some specific microfluidic systems have been developed
that are capable of a range of DNA-type analyses. A micro-
fluidic integrated system, which minimises sample
processing and handling, has been developed for PCR
analysis. Here DNA typing is achieved from whole blood
Capillary flow direct PCR analysis Figure 1
Capillary flow direct PCR analysis. Whole blood samples are used for direct PCR analysis. Samples are manipulated within
microfluidic channels.
Syringe
pump
Thermal Cycling -
Reaction Chambers
(Buffers,
Washes
etc.)
Sample
Inlet
manifold
Microfluidic
capillaries
Analysis of
PCR Products
Fluorescent
Scanning +
Analysis
Software
Capillary column -
separation
Waste
Voltage
supply
(Adapted from Zhang et al., 1999)
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samples using capillary microfluidics and capillary array
electrophoresis [1], see Figure 1, whereby blood is used
directly as the sample template for a PCR amplification
analysis.
Microfluidics technology has also illustrated a potential to
be allied with the detection of very low numbers of DNA
molecules, i.e. potentially individual molecules. Foquet et
al. [2] have shown that the construction of fluidic chan-
nels of <1 m enables the detection and relative propor-
tions of mixtures of DNA molecules to be measured. In
addition, using an electrical field to control the flow rates
analysis times of only several milliseconds per DNA mol-
ecule become achievable.
Electrophoretic mobility shift assays for the detection of
DNA-protein interactions have also been carried out in a
microfluidic chip environment [3]. Some of the benefits
achieved are reduced sample volumes, an avoidance of
labelling procedures and decreased analysis times.
The application of DNA microarrays revolutionised the
analysis of gene expression studies. However, the tech-
nique generally relies on passive diffusion of the sample
volume, containing the target analytes, towards the
immobilised probe elements and this can result in long
hybridisation times (normally hours). A method of accel-
erating the hybridisation time for DNA arrays using plas-
tic microfluidic chips, comprising networks of
microfluidics channels plus an integrated pump, have
been developed [4]. It has been shown that 'high initial
hybridisation velocities' can be attained and that equilib-
rium, in terms of bound versus free analyte, is quickly
achieved and so negates the requirement for such long
hybridisation events. The assembly of arrays into micro-
fluidic channels in order to improve the kinetics associ-
ated with hybridisation has also been shown by other
researchers. A low-density array, generated within micro-
fluidics channels, has been used to detect gene fragments
(K-Ras) carrying a point mutation [5]. Again it was found
that microfluidics reduced the hybridisation time in this
assay from hours, i.e. the time required in conventional
static hybridisations, down to less than 1 minute. An alter-
native method of reducing array hybridisation times
based on cavitation microstreaming has also been shown
[6]. Essentially cavitation microstreaming involves the use
of a sound field to induce the vibration of air-bubbles (at
a solid surface) present within a fluid. This ultimately
Microfluidic mass spectrometric protein analysis Figure 2
Microfluidic mass spectrometric protein analysis. Proteins are applied directly to a membrane, desalted and directed by micro-
fluidic channel to mass spectrometric analysis.
Hydrophobic
Membrane
(supported in
micro-channel)
Micro-electrode driving capillary flow
Protein Sample (In-Flow)
Micro-capillary
Proteins de-salted and eluted
from membrane
Electrospray
Ionisation-MS
(Adapted from Lion et al. 2003)
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causes a circulatory flow within the fluid and so mixing
times become reduced from hours to seconds. Hybridisa-
tion signals and kinetics are also reported to increase by
approximately 5-fold.
Protein applications
Microfluidic technology has also been incorporated into
the analysis of proteins / peptides [7,8]. In particular,
microfluidics can be linked with a mass spectrometric
analysis of proteins or peptides. Thus, peptides can be
adsorbed onto hydrophobic membranes, desalted, and
through the use of microfluidics eluted in a controlled
manner to allow the direct mass spectrometric analysis of
picomole amounts of peptides by electrospray ionisation
mass spectrometry procedures [9], Figure 2.
The recently reported combinatorial peptidomics
approach [10] is also perfectly suited for use with inte-
grated microfluidic systems and in principle allows iden-
tification of tryptic peptides directly from the crude
proteolytic digest. Combinatorial peptidomics initially
utilises peptidomics where a protein sample is proteolyti-
cally digested prior to assaying, and combines it with a
combinatorial depletion of the digest (peptide pool) by
chemical cross-linking via amino acid side chains to allow
a subsequent profiling of the resulting sample, Figure 3.
Other protein analysis methods have utilised microfluid-
ics channels linked to membranes imprinted with trypsin.
This allows the amount of protein delivered to the mem-
brane, the reaction temperature within the device and the
reaction time to be directly controlled for optimal diges-
tion [11]. Thus, using microfluidics the sample can be
supplied directly from upstream processing procedures,
e.g. purification products from cell lysates. The peptide
mixture can subsequently be analysed by electrospray ion-
isation mass spectrometry. Therefore, protein identifica-
tion can be achieved in minutes using nanograms of
sample.
The development of protein microarray methods [12-14],
analogous to DNA microarray technologies, for protein /
peptide analysis has the potential to hasten the discovery
of proteins of pharmacological value. As is the case with
DNA microarrays it is important that sample volumes
required for analysis are low, the sensitivity of the assay is
high (particularly for low-abundance proteins), and
hybridisation times are kept to a minimum in order to
Combinatorial peptidomics Figure 3
Combinatorial peptidomics. Sample solubilisation and protein purification are not necessary, since proteolyric digection may be
carried on native cells/tissues (dashed lines). The amino acid filtering (depletion) step may be repeated using combinations of
up to 6 amino acid "filters", i.e. chemically reactive surfaces (e.g. derivatised beads) able to covalently cross-link particular
amino-acids. Chemical depletion reduces the complexity of the peptide pool to a sufficient degree to make it compatible with
direct MS detection.
Protein sample
Protein extraction
and solubilisation
Proteolysis
Peptide pool
Direct MS
analysis
Amino acid-specific binding
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produce an efficient assay. A system incorporating protein
microarrays, fluorescent detection and integrated micro-
fluidics using planar waveguide technology has been
developed [15]. In combination these components enable
quantitative measurements for protein profiling to be car-
ried out with high sensitivity and also require shorter
analysis times than static hybridisations.
Future prospects
Finally, more novel uses for microfluidic technology at a
cellular level include the handling of mammalian
embryos [16], the manipulation of embryos and oocytes
in assisted reproduction [17] and even the isolation of
motile spermatozoa [18]. It is evident that the inherent
flexibility of microfluidic systems will allow them to
permeate and advance the development of assays in mul-
tiple biological, chemical and physical disciplines. Thus,
microfluidics should ultimately reduce the cost of run-
ning assays, decrease procedural times and limit the
required concentration and hands-on manipulation of
samples.
Acknowledgements
RB acknowledges the support from the Research Strategy Fund, Royal Hol-
loway Univesity of London.
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