Journal of Modern Optics, 2014
Vol. 61, No. 2, 85–101, http://dx.doi.org/10.1080/09500340.2013.873496
Optical detection strategies for centrifugal microfluidic platforms
Damien King, Mary O’Sullivan and Jens Ducrée*
Biomedical Diagnostics Institute, National Centre for Sensor Research, School of Physical Sciences, Dublin City University,
Glasnevin, Dublin 9, Ireland
Downloaded by [Dublin City University] at 06:14 24 August 2014
(Received 10 September 2013; accepted 4 December 2013)
Centrifugal microfluidic systems have become one of the principal platforms for implementing bioanalytical assays, most
notably for biomedical point-of-care diagnostics. These so-called ‘lab-on-a-disc’ systems primarily utilise the rotationally
controlled centrifugal field in combination with capillary forces to automate a range of laboratory unit operations (LUOs)
for sample preparation, such as metering, aliquoting, mixing and extraction for biofluids as well as sorting, isolation and
counting of bioparticles. These centrifugal microfluidic LUOs have been regularly surveyed in the literature. However,
even though absolutely essential to provide true sample-to-answer functionality of lab-on-a-disc platforms, systematic
examination of associated, often optical, read-out technologies has been so far neglected. This review focusses on the
history and state-of-the-art of optical read-out strategies for centrifugal microfluidic platforms, arising (commercial)
application potential and future opportunities.
Keywords: bioanalytical assay; centrifugal microfluidic platform; optical read-out strategy
1. Introduction
1.1. Background
Centrifugal microfluidic platforms have been a focus of
academic and industrial research efforts over the past 20
years. By exploiting the microfluidic liquid handling
capabilities and coupling them with optical detection
modules, a wide range of bioanalytical assays have been
adapted in a ‘sample-to-answer’ fashion on these
‘lab-on-a-disc’ (LoaD) systems.
Centrifugal liquid handling has proven to offer many
intrinsic advantages compared to typical pump-driven
lab-on-a-chip systems. On the one hand, the centrifugal
force is directly controlled by the rotational frequency of
system-innate spindle motor which can be dynamically
varied over more than three orders of magnitude. As it
scales with the square of the spinning frequency, the
resulting centrifugal volume force and flow speeds cover
a significantly wider range than can be achieved with
pressures which can practically be applied to common,
pneumatically or electrophoretically pumped polymer
microfluidic chips. Furthermore, the centrifugal force can
typically be set high enough to virtually rule out other, in
some situations parasitic forces such as surface tension
and capillarity. In addition, a flow-free, mere sedimentation akin to the simple particle hydrodynamics in conventional centrifuge tubes is possible. Due to the inertia of
the disc, the spinning motion is also self-stabilising, thus
eliminating jittering observed on pressure driven pumping
methods. Another advantage of the lab-on-a-disc system
*Corresponding author. Email: [email protected]
© 2014 Taylor & Francis
is the independence of the centrifugal force field from
parameters such as viscosity, conductivity, pH and surface
tension. This way the lab-on-a-disc platform can provide
comparatively robust liquid handling across a substantial
spread of fluidic properties for biofluids such blood,
urine, and mucus as well as assay reagents, solvents and
potential additives.
The rotational actuation, which is typically provided
by a conventional, low-cost spindle motor, also removes
the requirement of external periphery such as pumps and
their associated microfluidic interconnects and ‘world-tochip’ interfacing. Moreover, the modular nature of the
centrifugal platform completely separates the electronic
based driving instrumentation and conventional optical
readout units from the microfluidic sample and reagents
under test. The sample and/or reagents are exclusively
handled by the micro-structured, typically disposable
polymer test disc. These features are particularly beneficial for biological applications since the biofluids under
test can be processed in an encapsulated system, thus
minimising their potential contamination as well as their
biohazard.
By exploiting advancements made in the area of
microfabrication, numerous research groups have
enhanced the liquid handling capabilities of the centrifugal system by utilising geometrical control over capillary
action and hydrodynamic flow resistance in their designs.
While the concept of centrifugal analysers have been
established towards the end of the 1960s, foundational
work on microfluidic systems was initially reported by
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D. King et al.
Schembri et al. [1,2] in the early 1990s (leading to the
company Abaxis Inc. (California, USA) [3]), followed
by Madou and Kellogg in 1998 [4] within the activities
of a company later acquired by Tecan (Mannedorf,
Switzerland) [5] and, quite in parallel, by Gyros
Microlabs (Uppsala, Sweden) [6]. Since these pioneering
commercial ventures, numerous academic and other
corporate research groups have developed microfluidic
liquid handling operations such as volume metering,
valving methods (capillary, hydrophobic, siphoning,
sacrificial, etc.), mixing and routing onto their centrifugal
systems. Madou et al. [7], Ducrée et al. [8] and Gorkin
et al. [9] have provided comprehensive reviews on the
area of centrifugal microfluidic devices. Burger et al.
[10,11] have specifically surveyed centrifugal platforms
for cell handling and analysis applications.
Apart from advantages on behalf of liquid handling,
the centrifugal platform also displays several intrinsic
advantages in terms of optical detection compared to
conventional lab-on-a-chip devices. The actuation and
the detector as well as all moving parts can be entirely
delegated to a robust macroscopic unit which resembles
the “pickup” unit of a conventional optical disc drive
(ODD), e.g. a compact disc (CD) or a digital versatile
disc (DVD) player. The microfluidic lab-on-a-disc device
thus constitutes a merely passive (optical as well as fluidic) module. Also, the mechanical interface between the
polymer disc and the optical detection module is usually
made up by a simple, central clamp holder. Other than in
ODD technology, the detection may also be performed
by conventional optical equipment with the disc either at
rest or by rotating at a sufficiently low frequency.
In summary, the centrifugal platform bears great
potential for comprehensive process integration and
automation of liquid handling and optical detection on a
conceptually simple, compact, robust and widely autonomous instrument for a wide range of bioanalytical assay
formats. Compared to optical detection technologies for
stationary microfluidic lab-on-a-chip systems, the specific
characteristic of for centrifugal systems is the rotation of
the disc substrate with respect to the optical detection
system. In particular for deployment as full-fledged
biomedical sample-to-answer devices in point-of-care
settings, high-performance, yet cost-efficient and portable
detection systems are needed.
In this review, we will survey for the first time
various optical detection concepts that have been
developed for rotational lab-on-a-disc platforms. These
technologies can be coarsely categorised as static, e.g.
stop-and-go mode, and dynamic ‘on-the-fly’ read-out
during rotation. A further distinction can be made
between local ‘0D’ probing, 1D scanning and 2D imaging systems. For static readout, in principle most
schemes available to conventional lab-on-a-chip systems
can be implemented. However, manufacturing-process
related geometrical and mechanical tolerances associated
of the clamping and the typically poor accuracy of
stop-and-go azimuthal positioning of spindle motors
tends to compromise optical alignment. For detection
during rotation, either linear (azimuthal) scanning by a
(laser) beam [12], sometimes fully or partially compatible to the standards defined by the ODD industry
[3,6,13,14], or (bright-field) stroboscopic imaging
approaches [15] are most frequently pursued. Moreover,
imaging-based surface-plasmon-resonance (SPR) detection has been successfully implemented [16].
1.2. Optical detection for centrifugal microfluidic
platforms
Due to its high sensitivity, specificity and the wide array
of available sensing techniques, optical detection has
been the mainstay of microfluidics research. While other
techniques such as electrochemical and impedance based
detection systems may show some benefits in terms of
cost, portability and ease of alignment over conventional
optical systems, the on-chip integration of smaller optical
components may circumvent these issues. The miniaturisation of light sources (LEDs, laser diodes, etc.) and
optical detectors (photodiodes, CCD and CMOS chips)
allows for the elimination of a number of cumbersome
alignment steps with ‘off-chip’ light sources and detectors. The development of highly sensitive and miniaturised optical components may leverage further integration
and simplification of optofluidic sensing schemes.
Techniques such as interferometry, absorbance and
fluorescence, Raman spectroscopy, holography and
contact imaging are some of the most common optical
detection schemes which can be used in conjunction with
microfluidic systems [17–20]. Within this review article,
the compatibility of the above mentioned optical detection techniques with the centrifugal microfluidic platform
will be examined.
The centrifugal microfluidic platform presents numerous advantages and challenges for implementing and
integrating optical detection schemes. A rotational actuation scheme allows for periodic and essentially jitter free
motion (which is self-stabilised by inertia of the rotor).
A rotational setup also has an intrinsic 1D (annular)
scanning capability and can be extended to a 2D
scanning system by the addition of a simple linear stage
moving in the radial direction (as employed by ODDs).
The challenges include azimuthal alignment (stopping of
the spindle motor at a pre-defined position), Z-axis
alignment and depth of focus, short optical path length
in the vertical direction due to the shallow nature of the
common disc substrate, wobble during rotation and the
lack of a permanent mechanical interface, e.g. optical
connectors, between the rotating disc and the optical
detection system (and in some cases even between
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sub-components such as the light source and the optical
detector). In the following sections, numerous optical
detection systems will be discussed which exploit the
inherent features of the centrifugal microfluidic platform;
they simultaneously provide elegant methods to overcome the issues of optical detection measurements on
lab-on-a-disc systems.
Centrifugal microfluidic platforms can be broadly
divided into two main categories. The first are ‘lab-on-adisc systems’ where microfluidic LUOs such as valving,
metering etc. are carried out on-disc [21–23] and
on-rotor/on-disc based optical detection systems are used.
The optical detection methods most strongly associated
with these systems include:
absorbance
label-free detection
label-based detection.
The second category is the ‘Bio-CD’, which is a term
that has been coined to refer to platforms which utilise
the immobilisation of biological samples on a CD- or
DVD-style reflective surface. As surveyed in a 2009
review by Nolte [24], the main method of optical detection associated with Bio-CD platforms is spinning disc
interferometry. Bio-CD platforms also utilise ODD technology as a common method for readout via the ODD
pick-up head. Yu et al. have presented comprehensive
reviews on the use of ODD technology in conjunction
with Bio-CD systems [25,26].
The above mentioned optical detection systems and
ODD-based technology will be outlined in the following
sections. A summary of on-disc optical detection
methods is presented in Table 1 and a summary of
Bio-CD optical detection methods, along with the use of
ODD technology is presented in Table 2.
2. Challenges for optical detection systems on
centrifugal microfluidic platforms
As indicated in the introduction section, due to unavoidable manufacturing tolerances affecting their planarity,
spinning discs tend to wobble, thus impairing the
accuracy of optical alignment. There are two commonly
adopted approaches taken to compensate to stabilise
optical readout from the spinning disc.
The first method uses a closed-loop feedback system
with voice-coil magnetic actuators to dynamically
position the laser head above the fluctuating position of
the disc-based detection zone [27]. The shallow depth of
focus can be related to strong refraction occurring at the
abrupt change of the refractive indices at the disc
surface. This highly dynamic active tracking is achieved
using light-weight plastic lenses to minimise the inertia
of the voice-coil actuators and a pair of detectors. As the
87
disc wobbles, the position of the laser head continuously
adjusts in a closed-loop fashion to the signals obtained
from the detector. Such active tracking method is implemented by ODD read heads where the laser spot size at
the track needs to be reduced into the micron range in
order to enable read-out of high-density data. This strong
focussing at the disc surface implies a very shallow
depth of focus, so active tracking to limit wobbling
induced read-out errors.
The alternate approach to the optical tracking method
outlined previously is to utilise a passive system. Such a
system can mechanically planarise the disc through a
suitable holder in conjunction with an optical system
based on low numerical aperture lenses, thus extending
the length and the depth of focus. In this passive system
no tracking of the disc is required and which significantly simplifies the optical setup [24]. However, while
the resulting spot sizes can be much larger, e.g. tens of
microns, which would be unacceptable for high data
density ODD technologies; however, this optical scheme
is still well suitable for the typically greater feature sizes
of bioanalytical applications where the extended spot
may even help to average out artefacts and inhomogeneities [24].
Another issue is the lack of a direct mechanical
interface, e.g. opto-mechanical connectors, between the
rotating disc the optical detection system. This can be
addressed by free space optical components such as
lenses, mirrors and cameras which can detect optical
signals or image the rotating disc surface without the
need for a direct opto-mechanical connection [28–30].
Numerous applications have also been reported to
improve azimuthal alignment by placing reference
markers on the disc under test [21–23]. The issue of
vertical alignment has been addressed by adjustable
positioning stages for the detector to obtain optimal
focussing [31,32].
3. Disc-based optical detection systems
3.1. Label-free optical detection systems
Label-free optical detection principles can be categorised
into absorbance, scattering and total internal reflection
(TIR). These systems commonly utilise a 1D (annular)
scanning approach as outlined in the following section.
3.1.1. Absorbance-based optical detection systems
The absorbance (or optical density), as defined by the
logarithm of the ratio of the intensities of incident and
transmitted light. A wide range of optical components,
low-cost systems such as paired LEDs and photodiodes,
and more expensive components such as laser diodes and
spectrometers (either in a free space optics or fibre
coupled arrangement) has proven to be suitable for
2D imaging
2D imaging
Lab on a disc
Centrifugal test
stand
Spinit ®
Lab on a disc
system
SAF reader
Portable ELISA
reader
Centrifugal test
stand
PEDD
1D scanning
1D scanning
Piccolo Xpress ®
GyroLab ®
Workstation
1D scanning
1D scanning
1D scanning
2D imaging
1D scanning
1D scanning
System type
Scattering via
enhanced TIR
Fluorescence
Imaging and
fluoresce
Absorbance
Surface Plasmon
Resonance
Absorbance
Enhanced
absorbance
via TIR guidance
Imaging
Absorbance
Fluorescence
Detection method
Water quality
monitoring
Closed loop
process control
Blood monitoring
Cell handing
ELISA
Microfluidic
operations
Blood Analysis
Blood analysis
Automated protein
and antigen
capture
Blood monitoring
Application area
On disc based optical detection methods and application areas.
System
Table 1.
No
Yes
Yes
No
No
Yes
Yes
Yes
Yes
No
Label free
detection
No
No
No
No
Samsung
Biosurfit
No
No
Abaxis Inc.
Gyros AB
Commercially
available
High
High
Medium
Medium
High
High
Medium
High
High
High
High
Medium
Low
High
Medium Medium
Low
High
Medium High
Medium Medium
Medium Low
Medium High
Medium
High
Low
High
Low
Low
Medium
Medium
Low
Medium
[44]
[40]
[37]
[31,32]
[16,45]
[16]
[15]
[12]
[3]
[6]
Ruggedness Maintenance Reference
Medium High
High
Medium
Sensitivity Cost
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D. King et al.
Interferometry
Combined interferometry
and absorbance
Combined interferometry
and fluorescence
Modified ODD
Standard ODD
Standard ODD
2D scanning
2D scanning
2D scanning
2D imaging
2D scanning
2D scanning
2D scanning
Bio-CD
Bio-CD
Bio-CD
Bio-CD
Bio-CD
Modified ODD
Modified ODD
2D imaging
Discipher
Platform
Bio-CD
Bio-CD
Detection method
System type
Immunoassays
Cell counting
Cantilever based
biomolecule detection
ELISA
Protein detection
Cell counting and
imaging
Protein detection
Picometrology
Application area
Bio-CD based optical detection methods and application areas.
System
Table 2.
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Label free
detection
No
No
No
No
No
No
No
Ling Vitae
Commercially
available
High
High
High
High
High
High
High
High
High
Low
Low
Low
Low
High
High
High
High
High Low
Low
Low
Low
Low
High
High
High
Low
[74]
[63]
[64]
[66]
[61]
[51]
[60]
[13,62]
Ruggedness Maintenance Reference
High Low
High Low
Low
Sensitivity Cost
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D. King et al.
centrifugal microfluidic platforms. An overview of
absorbance-based optical detection methods is presented
in Table 1.
While absorbance measurements are highly
compatible with ‘on-the-fly’ detection on a rotor, a major
challenge to overcome constitutes the short (vertical)
optical path length due to the typical shallowness of the
disc substrate. According to the Beer–Lambert law, any
decrease in the optical path length reduces the signal
level of the detection system. Grumann et al. introduced
the concept of on-disc optical beam guidance by total
internal reflection (TIR) [12,33] to significantly extend
the optical path length Iopt on a flat monolithic polymer
disc compared to direct incidence (Figure 1). This technique has been widely used in a range of absorbancebased assays such as blood alcohol [34], blood glucose
[12] and haemoglobin [35] with integrated upstream
plasma extraction from whole blood [36].
Czugala et al. [37] have reported a wireless paired
emitter detector diode device (PEDD) for optical water
quality monitoring on a lab-on-a-disc. The centrifugal
microfluidic platform is based on an ionogel sensing area
combined with a very low-cost optical sensor. It is
applied for quantitative pH and qualitative turbidity
monitoring of water samples at the point-of-need, e.g.
for monitoring environmentally sensitive areas. The
PEDD detector consists of two pairs of surface mounted
LEDs (for reference and sensing regions), placed above
and below the sensing area of the disc (Figure 2). One
LED in each pair acts as the light source while the other
is reverse biased to act as a detector [38]. The PEDD
method has shown excellent sensitivity and signal-tonoise ratio when compared to the more commonly
employed method of pairing an LED and a photodiode
[39]. Given its low-cost optical components along with
its portable and rugged setup, the PEDD device also
lends itself as a biomedical point-of-care in resourcepoor settings.
Nwankire et al. [40] have demonstrated full
integration of sample preparation with read-out of a
multi-parameter liver assay panel (LAP) on a portable
centrifugal microfluidic analysis system (CMAS).
Dissolvable-film based centrifugo-pneumatic valves
provide advanced flow control for plasma extraction (from
finger-prick of blood), metering and aliquoting into separate reaction chambers for colorimetric quantification during rotation using a PEDD detection system. The device
completes the 5-parameter LAP within 20 minutes while
using a tenth of the reagent volumes, thus saving significant time, labour and costs compared to alternative technologies. Due to its comprehensive automation, autonomy
from infrastructure and minimum requirements on operator skills the CMAS system has been successfully tested
in resource-poor settings such as sub-Saharan Africa.
3.1.2. Scattering-based optical detection systems
Scattering-based optical detection systems have also been
implemented on centrifugal microfluidic platforms.
Optical detection methods based on elastic scattering
methods, such as Rayleigh and Mie scattering [41], as
well as inelastic scattering with generation of molecular
vibrations such as surface enhanced Raman spectroscopy
(SERS) [42–44] and surface plasmon resonance (SPR)
[16] have been reported.
Similar to absorbance, scattering based detection
systems can readily be devised for ‘on-the-fly’ detection
using low-cost illumination and detection components.
The main disadvantage is that the resultant measurement
signals can be complex in nature (in particular from
non-linear scattering based systems) and may require
significant post-processing and classification. The
Figure 1. Concept of on disc optical beam guidance by total internal reflection [33]. (© The Association for Laboratory
Automation). (The colour version of this figure is included in the online version of the journal.)
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Journal of Modern Optics
91
Figure 2. (a) Schematic of the electronic circuit used for the paired emitter detector diode (PEDD) system. (b) Prototype of the
PEDD-based centrifugal microfluidic platform [37] (© The Royal Society of Chemistry). (The colour version of this figure is included
in the online version of the journal.)
components required for non-linear scattering detection
tend to be also far more costly than their linear based
counterparts.
Kim et al. have demonstrated the adoption of a CD
optical pick-up technology for Raman micro-spectroscopy by utilising both the focusing and the 2D (lateral)
scanning capabilities of the ODD pickup [42]. Resolution was then characterised by scanning polystyrene
micro-spheres and obtaining Raman images of the structures under test. Choi et al. have shown that signal
amplification of SERS can be realised by pre-concentration on an opto-fluidic disc for effective, label-free
environmental and bio-molecular detection [43]. Cho and
Lee have reported integrated microfluidic SERS
spectroscopy on a disc for high-throughput quantitative
biomedical applications [44]. A SERS probe combined
with a protein concentrator monitored the protein
secretion dynamics as a function of trapped cell density,
accumulation time, and the induced centrifugal force.
Biosurfit (Lisbon, Portugal) have developed a
centrifugal microfluidic platform based on label-free SPR
detection for point-of-care applications [16]. SPR biosensors are used to probe refractive index changes that
occur in the immediate vicinity of a thin metal film
surface. The changes in the refractive index results from
the binding between an analyte in solution and its ligand
immobilised on the sensor surface.
3.1.3. TIR-based meniscus detection
By expanding upon the previous works reported by
Grumann et al. [12] and Steigert et al. [33], which
utilised enhanced TIR at disc-based structures, Hoffmann
et al. [41] presented a novel optical technique for the
resolved localisation of liquid–gas interfaces on centrifugal microfluidic platforms. The setup consists of a line
laser and a linear image sensor array mounted in a
stationary instrument. Their system can locate menisci
during rotation at frequencies up to 30 Hz, for instance to
measure liquid volumes with a high precision, flow rates
and viscosities. Also the presence of often unwanted gas
bubbles can be detected. A systematic summary of both
scattering- and TIR-based optical detection systems is
also included in Table 1.
3.2. Label-based optical detection systems
Enzyme-linked immunosorbent assays (ELISAs) in well
plates are still by far the most popular immunoassay
format over any decades and are read out by previously
described absorbance measurements. ELISA systems
have also been implemented on centrifugal microfluidic
platforms. Lee et al. from Samsung have reported a
portable, fully automated lab-on-a-disc based ELISA
system to test infectious diseases from whole blood [45].
A complete centrifugal spin stand test platform was
developed which included thermal management of the
enclosed test system, a laser diode to perform ferrowax
valve actuation and an optical detection module made up
of two sets of matched LEDs and photodiodes to
perform absorbance measurements at 430 nm and
630 nm, respectively (Figure 3).
For enhanced sensitivity, labelling by fluorescent
bioconjugates of antibody or antigen has emerged.
However, these fluorescent immunoassays require more
complex read-out equipment for excitation and detection.
Yet, cost and size of associated components are
continuously falling to make this contact free detection
method also attractive for the rotating lab-on-a-disc
platforms. Compared to label-free methods, common
sandwich immunoassay formats require a secondary
(labelled) ntibody and at least one additional wash after
exposure of the analyte, thus extending the liquid
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D. King et al.
Figure 3. Portable, lab-on-a-disc based ELISA system with an absorbance optical detection system [45]. (a) Photograph and (b)
schematic of the blood analyser system. A detector module is installed to perform absorbance detection. (c) Top and bottom plate of
the disc before UV bonding. (d) UV bonded disc image. (e) Photograph of the bonded disc with ferrowax valve formation. (f)
Schematic and microscope image showing the side view of the disc mixing chamber. (© The Royal Society of Chemistry). (The
colour version of this figure is included in the online version of the journal.)
handling protocol and requiring further reagent storage /
supply.
Gyros AB (Uppsala, Sweden) have developed a
platform which performs automated immunoassay
processing utilising laser induced fluorescence (LiF) [6].
This ‘GyroLab’ workstation features a liquid-handling
robot for pipetting from a standard well plate to the disc,
a centrifugal platform which carries out the assay and an
optical detection system employing LiF to fully automate
the entire assay protocol between sample preparation to
detection [6].
Puckett et al. have evaluated the effectiveness of
incorporating a protein-based assay onto the centrifugal
microfluidic platform [28]. The authors present a classselective, homogeneous assay for the detection of phenothiazine antidepressant drugs. The fluorescence detection
utilised a spectrophotometer with a fibre optical coupler.
This optical fibre was positioned perpendicular to the
disc in the centre of the detection reservoir at a distance
of ~2 mm and remained stationary throughout the experiment. The excitation wavelength was set at 488 nm, and
the emission monochromator was set at 508 nm (with a
slit width of 5 nm for both) for optimum detection of
green fluorescent protein (GFP). Nagai et al. have
reported a lab-on-a-disc platform for measurement of
biomarkers for mental stress [29]. Upon completion of
the disc rotation, fluorescence was measured using an
imaging scanner and analysed using imaging analysis
software. Riegger et al. have reported on read-out
concepts for multiplexed bead-based fluorescence
immunoassays on centrifugal microfluidic platforms [30].
Nwankire et al. report a centrifugal microfluidic
lab-on-a-disc system for at-line monitoring of human
immunoglobulin G (hIgG) in a typical bioprocess
environment [46]. The novelty of this device is the
combination of a heterogeneous sandwich immunoassay
on a serial-siphon enabled microfluidic disc with automated sequential reagent delivery and surface-confined
supercritical angle fluorescence (SAF) based detection
selectively collects the fluorescence emanating from
surface-bound emitters. The optical detection system
comprises of a focussing lens for excitation with a laser
beam at 635 nm (5-nW laser diode) and a spherical ring
lens structure at the bottom. The fluorescent emission is
transmitted through the spherical sector at the bottom of
the SAF chip, thus avoiding total internal reflection
(TIR), and is redirected by an elliptical mirror towards a
photomultiplier tube (PMT) (Figure 4). Compared to
conventional fluorescence detection setups, the SAF
scheme constitutes a simpler and cost-efficient hardware
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93
Figure 4. (a) Optical paths of the SAF detection system showing the collection of supercritical angle emission. (b) 3D rendering of
the SAF prototype reader and its key components. Inset: Photograph of the prototype system (with and without the housing unit)
including laptop for control and the centrifugal microfluidic platform [46]. (© Elsevier). (The colour version of this figure is included
in the online version of the journal.)
system. A systemised summary of label-based optical
detection systems is presented in Table 1.
4. Image capturing on centrifugal microfluidic
platforms
Grumann et al. developed a high-end experimental setup
for microscopic image capturing on discs spinning [15].
By utilising a microscope-mounted CCD camera
permitting short exposure times (~100 ns) to minimise
smearing, thus achieving resolutions of 4.5 μm at rest
and 10 μm while spinning at 150 Hz. Image capture was
controlled by a real-time PC board which sends delayed
trigger signals to the CCD camera and to a stroboscopic
illumination device upon receiving the zero-crossing
signal of the rotating disc (Figure 5). The common delay
of the trigger signals is electronically adjusted according
to the spinning frequency to appreciably improve the
stability of the captured image sequences.
Imaging of the entire disc area can be enabled by
placing the imaging detector on a linear stage. This
centrifugal ‘test stand’ is mainly devised for development
of centrifugal microfluidic systems in development labs
where its rather large footprint and the high costs are
acceptable. Yet, as cost-efficiency and performance of
opto-electronic components such as the camera have
dramatically increased over recent years, test stands with
significantly cheaper components and even without
stroboscope illumination have demonstrated very reasonable performance for the majority applications in various
labs. For absorbance or luminescence-based detection,
associated optical equipment has been added to these test
stands [21–23,47–51].
Burger et al. have demonstrated a centrifugal
microfluidic platform for the highly efficient manipulation
and analysis of bioparticles, e.g. for bead-based immunoassays [31]. The platform uses an array of geometrical Vcup barriers to trap particles using stopped-flow sedimentation under highly reproducible hydrodynamic conditions. The very high capture efficiency of nearly 100%,
paired with the capability to establish sharply peaked, single-occupancy distributions, enables a novel, digital readout mode for colour multiplexed, particle-based assays
with low-complexity instrumentation.
Burger et al. have also reported a technology which
integrates fluorescence-based detection and manipulation
of individual particles with the previously reported,
highly efficient, array-based particle trapping in scalematched V-cups under stagnant flow conditions [32].
The system demonstrates the capture and manipulation
via optical tweezers of microbeads and HL 60 cells,
along with bright field imaging (beads and cells) and
fluorescent measurements (labelled cells).
Based on a similar imaging system for rotational
platforms, Chen et al. presented a means of detecting
circulating endothelial cells (CECs) from peripheral
blood mononuclear cells (PBMCs) via a microfluidic
disc, with a model cell system of human umbilical
vein endothelial cells (HUVECs) in PBMCs [47]. Hattori and Yashuda have demonstrated the efficacy of a
microfluidic medium exchange method for single cells
using the passive centrifugal force of a rotating microfluidic-chip based platform (Figure 6) [48]. Kubo et al.
have reported a method for trapping and staining single cells in the micro-chambers of a disc shaped cell
separation device [49]. Lee et al. have also demonstrated a single cell trapping method by utilising a lab
on a disc setup [50]. A microfluidic method to construct an array on a disc for staining assays has been
shown by Chen et al. [51].
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Figure 5. The experimental setup and operating principles of the stroboscopic ‘test stand’ for the development of centrifugal
microfluidic platform [15]. (© American Institute of Physics).
Figure 6. Centrifugal test stand for microfluidic medium exchange [48]. (a) System setup; (b) microfluidic chip design; (c) and (d)
collection chamber of double Y-shape microfluidic pathway. (© MDPI).
The centrifugal test stand (Figure 5) platform [15] has
found numerous applications in the area of imaging complex microfluidic operations (such as valving, metering)
[21–23]. Godino et al. presented a new integration and
automation concept for a range of bioassays, leveraged
by cascading a centrifugal pneumatic valving scheme to
move several liquids through shared channel segments
[21]. Nwankire et al. demonstrated all key fluidic steps
for the full integration and automation of a 7-step homogeneous absorbance assay for quantifying nitrate and
nitrite levels in whole blood within about 15 minutes
[23]. A summary of image capturing systems for
centrifugal microfluidic platforms is presented in Table 1.
5. Bio-CD based optical detection systems
The term ‘Bio-CD’ has been coined to refer to
centrifugal microfluidic platforms which utilise the
immobilisation of biological samples on a reflective CD
surface. The immobilised disc is then optically interrogated using either 1D scanning spinning disc interferometry [52–62] or 2D scanning optical readout on the
analogy of ODD technology [25,26,63–75]. Interferometry based optical detection systems for the Bio-CD platform and the use of ODD pick up based readout is
summarised in Table 2.
5.1. Spinning disc interferometry
A comprehensive review by Nolte details the different
classes of spinning disc interferometry, Bio-CD optical
detection, Bio-CD quadrature classes and the main
Bio-CD application areas [24]. While it has been demonstrated that spinning disc interferometry is a highly sensitive and reliable detection method [24,57,59], the
associated optical set-up is bulky and expensive when
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compared to other, more integrated and miniaturised
detection schemes.
Zhao et al. created a Bio-CD based on backscattering
interferometry by patterning reference protein spots
beside the target spots on λ/4 wavelength thick layers of
SiO2, on a dielectric coated glass substrate [52]. When a
Gaussian beam from a laser is incident on the Bio-CD,
small phase differences occur in the reflected light, in a
technique akin to the operation of a conventional CD
with lands and pits. These differences in the signal beam
lead to linear and highly sensitive changes in the interference intensity (Figure 7).
Varma et al. demonstrated gold lands that were
patterned radially in a spoke-wheel fashion onto a 3-inch
silicon wafer or onto a 2-inch dielectric mirror disk [53].
Antibodies were deposited onto the gold ridges using
PDMS stamps, creating selective binding sites for the
target protein (Figure 8). The optical set-up for this
Bio-CD example is very similar to that of Zhao et al.
[54].
Additional methods of interferometric detection of
protein on rotating microfluidic platforms include
phase-contrast configurations that detect local changes in
protein density [55], in-line configurations that detect the
direct protein and disc surface topology [50], and
Figure 7. Detection principle and optical setup of phase
contrast Bio-CD [52]. (a) Principle of differential phase contrast
detection. (b) A schematic of the far-field optical detection
system. (© American Association for Clinical Chemistry).
95
approaches using adaptive beam mixers [56]. All of the
Bio-CD quadrature classes incorporate high stability as a
fundamental and intrinsic component of the detection
[24,57,59]. By using a common-path configuration [53]
in which both the signal and the reference waves are
generated from the same location on the disc and share
the same path to the detector, interferometry based
optical detection systems also show great promise for
multi-analyte detection, particularly when combined with
centrifugally driven microfluidic channels for the protein
patterning steps [60].
Other Bio-CD concepts utilise a combination of
interferometry with other previously described detection
methods. For example, interferometric scanning of a
rotating disc can include absorption as an imaginary
component of a refractive index because detection of
common-path interferometry is an intensity-based
measurement, similar to absorbance based detection [61].
While it is clearly a separate phenomenon, fluorescence
can be combined with interferometric detection when the
disc to be monitored is optimised for both detection
wavelengths to maximise emission [62]. Wang et al.
showed that in addition to the amplitude and phase interferometry channels; there can be other channels, such as
for fluorescence detection or light scattering. Light
scattering and fluorescence share a common detection
configuration with the angle of view far from the specular reflection of the interferometry channel (Figure 9).
Figure 8. Illustration of the radial gold ridge pattern on the
Bio-CD [54]. The gold ridges are illuminated by a focussed
Gaussian beam, such that 50% of the intensity falls on the
substrate and 50% falls on the gold microstructure (© Elsevier).
(The colour version of this figure is included in the online
version of the journal.)
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5.2. Optical disc drive (ODD) pick-up based readout
ODDs represent promising optical readout systems for
centrifugal microfluidic platforms performing chemical
sensing and bio-sensing applications [25,26]. Their
primary advantages encompass the large installation base
as nowadays almost every manufactured computer
contains an ODD, the low price due to high-volume
production and high data resolution with even sub-micron
features.
ODD technology for digital data storage has implemented a 2D scanning mode enabled by the combination
a linear translation stage in the radial direction and the
rotor in the annular scanning in the perpendicular direction, thus enabling, in principle, a 2D scan of the entire
disc. Intrinsic challenges such as the only loosely defined
distance and planarity and rotationally induced periodic
‘wobble’ of the focal plane have been elegantly solved
using a dynamically adjustable lens, disc-based guide
structures (tracks) and software [25–27].
With some additions such as optical detectors and
lenses along with modifications to the CD or DVD
surface, an ODD’s functionality can also be extended to
a 2D imaging system similar to a laser scanning microscope [63,64]. This section will survey optical readout
methods based on standard ‘off-the-shelf’ ODDs along
with systems which are based around modified ODDs.
5.2.1. Standard ODD based optical readout
In order to utilise conventional laser read heads
(‘pickups’) on ODDs in the detection of biological
information, substrates need to be compliant to common
ODD data storage standards such as CDs or DVDs.
Numerous methods have been reported to chemically
functionalise the surfaces of conventional CDs or DVDs,
without adversely affecting their optical and mechanical
properties. These methods have been covered in great
detail in the review articles presented by Nolte [24] and
Yu et al. [25,26].
Imaad et al. have shown a centrifugal microfluidic
platform for microparticle and cell counting using a standard ODD and a disc derived from a standard CD [65].
This five-layer disc consists of a PDMS microfluidic
layer, a thin polycarbonate layer, a photosensitive dye
layer or data layer, a metallic reflective layer and a
plastic protective layer (Figure 10). Bioparticles such as
cells are introduced into the microfluidic channel and
will interfere with the converging laser beam in the
optical pickup, thus causing reading errors in the
encoded digital data previously burned on the dye layer.
The data errors are detected, analysed and correlated
with the number of particles in the microfluidic channel.
La Clair and Burkart presented a method to screen
the interaction between ligands immobilised on standard
CDs and biomolecules [66]. The data on the CD was
then read out in an ODD and the binding of the
biomolecules was assessed by the error rate induced by
the scattering of the laser beam. Bosco et al. have
demonstrated a centrifugal microfluidic platform for
label-free detection of biomolecules based on cantilever
chips [67]. The deflection of the biofunctionalised
cantilevers resulting from binding of analyte molecules
was recorded by an ODD pick up head.
Li et al. demonstrated a digital signal readout
protocol for screening disc based bioassays with standard
ODDs. Three different types of biochemical recognition
Figure 9. Experimental layout using the 488-nm line from an Argon laser incident at 30° and focused on the Bio-CD [62]. The
interferometric signal is detected in the reflected light, while the fluorescence signal is collected by a lens above the disk. The
oblique-incidence design spatially separates the two detection channels (© Optical Society of America).
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97
Figure 10. Cross-sectional view and optical operating principles of a digital microfluidic CD using standard ODD detection and
readout [65]. (© The Royal Society of Chemistry). (The colour version of this figure is included in the online version of the journal.)
reactions (biotin-streptavidin binding, DNA hybridisation
and protein–protein interaction) were performed directly
on a CD in a line array format [68]. By applying a
screen-printing process, Potyrailo et al. demonstrated a
laboratory-scale automated production of sensing films
with an average thickness of ~10 μm [69]. This
technique was then used to deposit colorimetric calciumsensitive sensor films onto a DVD which were exposed
to water with different concentrations of Ca2+, and these
concentrations were then quantified in an ODD [70].
Chen et al. used a double-spiral setup to generate
384 × 384 hybridisation arrays on a standard CD [71].
5.2.2. Modified ODD based optical readout
The 2D scanning functionality outlined in the previous
section may be expanded by further modifications to the
standard ODD. Ramachandraiah et al. modified a
standard ODD as well as standard DVD medium to convert the ODD to a 2D laser scanning microscope [63];
another photodiode is added to the ODD to detect
transmitted and forward-scattered light through the disc
(Figure 11). Furthermore, the DVD is replaced with a
disposable, multilayer, semi-transparent polymer disc that
contains microfluidic channels in addition to the standard, 0.74-μm pitch spiral track. The inner walls of the
fluidic channels are functionalised to specifically capture
the targeted bioparticles.
Samples of blood (or another liquid of interest) are
then pumped into the channels and the ODD is switched
on. The additional photodiode records the transmission
of the ODD’s 658-nm semiconductor laser through the
spinning disc, eventually providing a two-dimensional
image featuring the bound particles which is saved onto
a hard drive. To date, the system has successfully imaged
polymer beads of various sizes (1 μm, 2.8 μm and 5 μm)
suspended in solution, as well as blood-borne CD4+
cells which are an important marker for the immune status of patients with HIV infections (Figure 12). This
technology (both the modified ODD and DVDs) has also
become commercially available as the ‘Discipher’ platform developed by Ling Vitae (Husoysund, Norway)
[13].
Lange et al. converted an ODD to a laser scanning
microscope to monitor the light reflection from gold
nanoparticle-stained immunoassays, which were contact
printed on CD surfaces [64]. With the addition of a
second laser-based detector attached on the upper part
of an ODD, Alexandre et al. fabricated a double-sided
reader to examine both numeric and genomic information on Bio-CDs [72]. Barathur et al. reported that a
modified ODD can measure signals from DNA microarrays with embedded microfluidic channels on a disc
[73]. Banuls et al. detected DNA hybridisation spots
and immunoassays located on the top side of a CD by
installing an additional photodiode in the ODD to monitor the light transmitted through the CD [74]. Morais
et al. developed an ELISA for atrazine on a polycarbonate CD, where absorption of the immuno-reagents
took place on the CD surface. The various probes were
spotted manually on the disc surface. Due to the
absence of microfluidic function, the end user also
needs to perform all the liquid handling steps involved.
Quantitative optical detection was performed using a
standard ODD with an additional integrated planar
photodiode to measure the transmittance through the
surface of the disc [75].
6. Commercial devices
Recently, Chin et al. have comprehensively reviewed on
the commercialisation of microfluidic lab-on-a-chip
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Figure 11. Modified ODD constituting a laser scanning microscope [13,63]. (a) Overview of the DVD laser scanning microscope
system. (b) Operating principle of the ODD laser scanning microscope. (© The Royal Society of Chemistry). (The colour version of
this figure is included in the online version of the journal.)
Figure 12. DVD laser scanning microscope image of polymer beads of 1 μm, 2.8 μm and 5 μm diameter (scale bar 20 μm) [13,63].
(© The Royal Society of Chemistry). (The colour version of this figure is included in the online version of the journal.)
devices for point-of-care applications in general [76].
Since the early 1990s, numerous commercial avenues
have been explored for centrifugal microfluidic lab-on-adisc platforms with integrated optical detection
techniques.
In the mid-1990s, Abaxis Inc. (California, USA) [12]
launched the Piccolo xpress® portable blood analyser
based on colorimetric absorbance measurements for
medical diagnostics. In the meantime they have
established a wide range of disc panels for human and
veterinary diagnostics. Gyros AB (Uppsala, Sweden)
[11] have developed the GyroLab workstation which
performs automated immunoassay processing resorting to
LiF based detection. Samsung (Seoul, South Korea) [13]
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have successfully implemented an ELISA assay system
on their proprietary lab-on-a-disc platform. Roche
Diagnostics (Basel Switzerland) offer fluorescent based
optical readout systems for centrifugal microfluidic
platforms [77].
Various start-up companies have also entered the
market. Quadraspec (Indiana, USA) [78] have developed
the Bio-CD system based on spinning disc interferometry
for multiplexed, label-free assays for protein analysis.
Biosurfit (Lisbon, Portugal) [16] launched a centrifugal
microfluidic platform based on label-free SPR imaging
for point-of-care diagnostics. Radisens Diagnostics
(Cork, Ireland) [79] develop a miniaturised centrifugal
microfluidic platform for point-of-care applications which
utilises fluorescence, absorbance and (elastic) scattering.
They have reached an agreement on licensing microfluidics patent portfolio and from Tecan (Mannedorf,
Switzerland) [5].
7. Summary and conclusion
Centrifugal microfluidic platforms have shown to the
ability to integrate and automate even rather complex
liquid handling protocols of bioanalytical assays in a
high-performance but still robust and user-friendly
fashion. Yet, a full-fledged point-of-care with true
sample-to-answer fashion also requires suitable detection
technologies. Amongst the broad scope of technologies
implemented in the lab-on-a-chip world, contact-free
optical detection has been most commonly used for
lab-on-a-disc systems. On the one hand these schemes
can be categorised in 1D scanning and 2D imaging
devices based on direct, i.e. analogue signals or resorting
to optical configurations for digital data encoding and
tracking derived from ODD technology. On the other
hand, one can distinguish between signal generation
schemes such as elastic (e.g. absorbance, interferometry,
TIR, forward and side scatter) and inelastic scattering
(e.g. SPR, Raman, FTIR), and luminescence (e.g. fluorescence, chemiluminescence) which require different
optical setups and components (Tables 1 and 2). The
schemes might be label-free or require tagging with a
direct or indirect optical signal generator. From an
application point of view, one can also differentiate
between surface-bound heterogeneous as well as
homogeneous assay formats that have been implemented
on the lab-on-a-disc platform.
While there is a rapidly growing number of academic
groups entering the scene, lab-on-a-disc technologies
have been pioneered and are still strongly impacted by
(historic and present) corporate players such as Abaxis
Inc., Gyros AB, Gamera/Tecan, Burstein Technologies,
LifeBits, Bayer, Boehringer Ingelheim and Roche
Diagnostics) and more recently founded companies (Ling
Vitae, Biosurfit and Radisens Diagnostics).
99
Apart from plasma-based molecular biomarkers, we
expect a continued growth in research and development in the areas of blood cell counting, e.g. CD4
counts in HIV diagnostics, detection of circulating
tumour cells (CTCs) and complete blood counts. The
unique combination of low-cost equipment akin to a
conventional CD drive interfacing with a cheap, mass
manufacturable, disposable disc incorporating all
reagents for fully integrated sample preparation and
detection will pave the route towards commercial success in the point-of-care market.
Funding
This work was supported by the Science Foundation Ireland
[grant number: 10/CE/B1821]; the ERDF; the LiPhos project
[80] funded by the European Commission [grant number:
317916].
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