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PAPER www.rsc.org/loc | Lab on a Chip

Integrated microfluidic platform for the electrochemical detection of breast

cancer markers in patient serum samples
Alex Fragoso,*a Daniel Latta,b Noemi Laboria,a Frithjof von Germar,b Thomas E. Hansen-Hagge,b
Wolfgang Kemmner,c Claudia G€ artner,d Richard Klemm,d Klaus S. Dreseb and Ciara K. O’Sullivan*ae
Received 10th September 2010, Accepted 9th November 2010
DOI: 10.1039/c0lc00398k

A microsystem integrating electrochemical detection for the simultaneous detection of protein markers
of breast cancer is reported. The microfluidic platform was realized by high precision milling of
Published on 01 December 2010 on http://pubs.rsc.org | doi:10.1039/C0LC00398K

polycarbonate sheets and features two well distinguishable sections: a detection zone incorporating the
electrode arrays and the fluid storage part. The detection area is divided into separate microfluidic
chambers addressing selected electrodes for the measurement of samples and calibrators. The fluidic
storage part of the platform consists of five reservoirs to store the reagents and sample, which are
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interfaced by septa. These reservoirs have the appropriate volume to run a single assay per cartridge
and are manually filled. The liquids from the reservoirs are actuated by applying a positive air pressure
(i.e. via a programmable syringe pump) through the septa and are driven to the detection zone via two
turning valves. The application of the realised platform in the individual and simultaneous
electrochemical detection of proteic cancer markers with very low detection limits are demonstrated.
The microsystem has also been validated using real patient serum samples and excellent correlation
with ELISA results obtained.

Introduction processing can also be easily miniaturised and integrated onto

the same platform by carefully designing application specific
Recent advances in the fabrication of microfluidic platforms integrated circuits.7
initiated during the late 90s have facilitated the realisation of There are some examples in the literature of electrochemical
micro total analysis systems as they enable the miniaturization, immunoassays on microchip platforms. Strategies for electrical
integration and automation of (bio)chemical assays.1 These decoupling have been implemented for conventional capillary
platforms combine a series of fluidic unit operations (transport, electrophoresis with electrochemical detection (CE-EC) that
metering, mixing, incubation, detection, etc.)2 within a device avoids the inherent difficulties in isolating both separation and
with interconnected elements, preferably in a single packaged detection fields, allowing the integration of CE-EC in a photo-
support manufactured using cheap polymeric materials.3 ablated polymer microdevice with electroosmotic driven flow.8
The integration of miniaturised fluidic handling and delivery Wang et al. have reported on a microfluidic device integrating
systems with chemical and biochemical sensors provides applied pre-column reactions of alkaline phosphatase-labeled antibody
scientists with powerful tools for in-field measurements away (anti-mouse IgG) with the antigen (mouse IgG), followed by
from central laboratories.4,5 Amongst the various classes of electrophoretic separation of the free antibody and antibody–
elements able to transduce a chemical or biochemical event into antigen complex and amperometric postcolumn detection.9 On-
a measurable signal, electrochemical platforms undoubtedly chip separation of free and bound antigens has also been
present the most promising advantages. Electrodes of all type, achieved based on differences in isoelectric point using a multi-
sizes and geometries can easily be integrated within a microfluidic channeled column coated with a cation-exchange resin.10 In this
platform and provide excellent sensitivity and versatility in case, off-chip detection was based on the amperometric response
comparison to other transduction techniques based on, for of a ferrocene-labeled antibody which avoids the use of enzy-
example, optical or mass sensing.6 Furthermore, the associated matic substrates enabling the detection of histamine released in
electronics used to drive the electrochemical detection and signal whole blood within 2 min.
A more advanced microfluidic device was developed by Ko
a
Nanobiotechnology and Bioanalysis Group, Departament d’Enginyeria and co-workers based on a poly(dimethyl siloxane) (PDMS)
Quımica, Universitat Rovira i Virgili, Avinguda Pa€ısos Catalans, 26, substrate molded by polymer casting bonded to a poly-
43007 Tarragona, Spain. E-mail: [email protected]; ciara.osullivan@
(methylmethacrylate) (PMMA) bottom substrate fabricated by
urv.cat; Fax: +34-977-889621; Tel: +34-977-558579
b
Institut f€
ur Mikrotechnik Mainz, Carl Zeiss Strasse 18-20, 55129 Mainz, hot embossing.11 Two inlet ports and an air vent are opened
Germany through the PDMS top substrate and the fluidic control was
c
Research Group Surgical Oncology, Charit e Universit€
atsmedizin Berlin, implemented by designing the microfluidic channels with various
Campus Buch, Lindenberger Weg 80, 13125 Berlin, Germany geometries of width, depth, and shape. The system was used for
d
Microfluidic ChipShop GmbH, Carl Zeiss Promenade 10, 07745 Jena,
Germany
the amperometric detection of model analytes such as strepta-
e
Institucio Catalana de Recerca i Estudis Avançats, Passeig Lluıs vidin and anti-ferritin antibodies carried out on gold electrodes
Companys 23, 08010 Barcelona, Spain integrated onto the PMMA bottom substrate. Alternatively, the

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screen printing technique can also be used to pattern the elec- 10% of women with early localized breast cancer and in about
trodes on the microfluidic platform.12 70% of those with metastatic breast cancer.20 CA15-3 may also be
Cancer is one of the main causes of mortality worldwide, the elevated in healthy people and in individuals with other cancers,
cure for which has not yet been achieved and early diagnosis is conditions, or diseases, such as colorectal cancer, lung cancer,
critical to avoid a fatal outcome. Certain proteins can signal the cirrhosis, hepatitis, and benign breast disease. Finally, PSA is
presence or recurrence of cancer although only a few serum a 30 kDa single-chain glycoprotein expressed predominantly by
markers are truly specific for a disease in a defined organ. In human prostate. It is usually present in the blood at low
cancer, the multi-factorial nature of oncogenesis and the concentrations (<4 ng mL1) in healthy males and because of the
heterogeneity in oncogenic pathways make it unlikely that correlation of PSA concentration with tumour volume and tissue
a single biomarker will detect all cancers with high specificity and specificity, its use as a tumour marker for prostate cancer has
sensitivity. Multiplex analysis of panels of markers has thus the flourished over the past decade.21,22
potential to provide information about patient prognosis and
Published on 01 December 2010 on http://pubs.rsc.org | doi:10.1039/C0LC00398K

response to therapy that cannot be obtained by analysis of single Experimental

markers. Hence, quantitative identification of biomarkers in
a mixture without separation and at clinically relevant concen- Reagents
trations is a crucial requirement for the development of more
Dithiol 22-(3,5-bis((6-mercaptohexyl)oxy)phenyl)-3,6,9,12,15,18,21-
effective and simpler diagnostic devices. Arrayed tests enable the
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heptaoxadocosanoic acid N-hydroxysuccinimide ester (DT2-

use of pattern recognition approaches to assess disease changes,
NHS) was purchased from SensoPath Technologies (Bozeman,
which are important in disease diagnostics and monitoring.13
MT). Stock solutions (1 mM) were prepared in acetonitrile,
In the present paper, we report on the development of
purged with argon and kept at 20  C when not in use. Carci-
a packaged amperometric immunosensor for the detection
noembryonic antigen (CEA) and prostate specific antigen (PSA)
of cancer markers in real samples based on monolayers of
were purchased from Scipac Ltd (Kent, UK). Monoclonal anti-
a carboxylic acid terminated dithiol14,15 supporting a monoclonal
CEA, anti-CA15-3 and anti-PSA antibodies were kindly
capture antibody that recognizes the target antigen and is
provided by Fujirebio Diagnostics AB (G€ oteborg, Sweden). The
detected by an in-house prepared peroxidase–antibody reporter
affinity column Freezyme Conjugation purification kit and N-
conjugate. The microfluidic platform has been engineered to
succinimidyl-S-acetylthioacetate (SATA) were purchased from
contain two separated areas: a detection zone incorporating an
Pierce.
array of 16 working electrodes and a fluid storage section. The
detection zone is divided into separate microfluidic chambers
addressing selected electrodes for the measurement of samples Preparation of antibody–HRP conjugates
and calibrators. The fluidic storage part of the platform consists One milligram of the corresponding monoclonal antibody was
of five reservoirs to store the reagents and sample, which are dissolved in 0.1 M MES, 0.15 M NaCl, pH 7.2, to obtain a final
interfaced by septa and actuated by applying a positive air volume of 1500 mL. A 10 mL aliquot of a solution containing 1
pressure via a programmable syringe pump. In comparison with mg of SATA was then dissolved in 100 mL of dried DMSO and
other existing microfluidic methods such as CE-based micro- added to the antibody solution and the mixture was gently stirred
chips,16 this device has the advantage of integrating a series of for 30 minutes at room temperature, protected from light. A
microfluidic operations in a single platform, namely, reagent deacetylation solution (100 mL) consisting of 0.5 M hydroxyl-
storage and transport, metering, incubation and detection. In amine hydrochloride in 0.15 mM NaCl, pH 7.2, was added to the
addition, the design has been conceived in such a way that if the SATA–antibody mixture and allowed to react for 2 hours at
fluidic operations are fully automated, the assay only requires room temperature, again protected from light. Subsequently,
the end-user intervention of application of sample, highlighting 1 mg of maleimide activated HRP was added and the solution
the simplicity of its operation. was incubated for 90 minutes at 37  C. To deactivate the
For this purpose, we have chosen a panel of clinically relevant maleimide groups and avoid further non-desired reactions, an
tumour biomarkers, including the carcinoembryonic antigen aliquot of 2-mercaptoethanol was added to the solution to a final
(CEA), cancer antigen 15-3 (CA15-3) and prostate specific concentration of 0.15 mM and stirring continued for 15 minutes.
antigen (PSA) as model systems. CEA is present in about 95% of Purification of the resulting conjugate was carried out with the
all colon tumours and 50% of breast tumours, and is also asso- metal chelate affinity chromatography Freezyme conjugation
ciated with ovarian carcinoma, lung cancer and others.17 purification kit. Finally, the pure conjugate was concentrated by
Therefore, it can either be part of a panel of cancer markers for passing through a molecular weight cut-off filter of 50 kDa at
different cancers or, more importantly, can also be used as an 7500 rpm for 5–10 minutes and the conjugates were washed with
independent prognostic factor. The normal CEA levels in healthy Milli-Q water.
adults lie in the range 3–5 ng mL1, although some benign
diseases can increase these levels up to 10 ng mL1.18 Cancer
Fabrication of the electrode arrays and microfluidic cell
antigen 15-3 is a protein that is produced by normal breast cells.
In many patients with cancerous breast tumors, there is an The lithographic process employed to fabricate the electrode
increased production of CA15-3, which is shed by the tumor cells arrays was achieved as follows (Scheme 1): a circular borofloat
and enters the bloodstream, making it useful as a tumor marker glass wafer (f ¼ 12 cm) was fully covered with 5 mm of LOR5214
to follow the course of the cancer.19 CA15-3 levels above the lift-off resist, later UV patterned to reveal the reference electrode
clinically relevant threshold (30 U mL1) are present in about areas and corresponding connections. Ten nanometres of

626 | Lab Chip, 2011, 11, 625–631 This journal is ª The Royal Society of Chemistry 2011
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Published on 01 December 2010 on http://pubs.rsc.org | doi:10.1039/C0LC00398K
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Scheme 2 Fabrication process for the microfluidic platform: (a) poly-

carbonate block; (b) septa; (c) electrode array; and (d) valves.

(75.5  25.5  5 mm, Scheme 2). The channels (1 mm depth),

reservoirs and valve holes were realised by high-precision milling
and sealed with a layer of a pressure sensitive adhesive. Valve
bodies were fabricated by injection moulding and integrated on
the chip by means of a metallic holder.

Electrochemical measurements
The working electrodes were spotted with 5 mL of a stock solu-
tion of DT2-NHS for 3 hours followed by rinsing with copious
amounts of acetonitrile. The capture antibodies were covalently
immobilized on NHS-activated SAM by spotting with 5 mL of
a 0.5 mg mL1 solution of antibody in 10 mM acetate buffer, pH
Scheme 1 (a) Fabrication process for the electrode array; (b) top view of
5.0, for one hour at 37  C. The remaining NHS active ester sites
the electrodes arrangement: CE: counter electrode. were blocked with 1.0 M ethanolamine, pH 8.5, for 30 minutes at
37  C. The modified arrays were then assembled in the micro-
fluidic cells by means of a double-sided adhesive gasket.
Antigens were diluted in phosphate buffer, pH 7.2, to the
titanium, used as a glass/metal adhesion promoter, were sput-
desired concentration, while real serum samples were applied
tered followed by a 150 nm thick silver layer. The patterned
without any dilution for 2 minutes at room temperature. After
substrate was fully coated a second time with the same lift-off
rinsing with PBS, a 1 mg mL1 aliquot of the prepared HRP-
resist and UV patterned to reveal working and counter electrode
labelled antibody was added and left to incubate to form
areas and their corresponding connections, and finally coated
a sandwich immunocomplex for 2 minutes. The amperometric
with a 10 nm thick titanium layer followed by a 150 nm thick
measurements were carried out by first recording the background
gold layer. The remaining photoresist was finally removed with
response at 0.2 V in PBS followed by injection of a mixture of
acetone, revealing the planar gold and silver patterns. Since the
1 mM hydroquinone/1 mM H2O2 in PBS, pH 6.
connection lines between the electrodes and their connection
points have to cross the fluidic channels, an insulation of the chip
was required. Thus, an SU8 negative photoresist was applied to Results and discussion
the whole surface of the chip and was only partially removed at
Microfluidic platform design and fabrication
the electrode and connection areas. This ensured that only elec-
trode would be later exposed to the sample fluids. In order to The microfluidic platform was realized by high precision milling
extend storage lifetime by reducing contamination of the elec- of polycarbonate sheets, which offers flexibility and rapid turn
trode surfaces, the patterned wafers were coated with a 5 mm over of the desired designs. It contains two well distinguishable
protection layer of AZ6632, which can be easily removed by sections: a detection zone incorporating the electrode arrays and
rinsing the arrays with acetone, isopropanol and water immedi- the fluid storage part (Fig. 1).
ately prior to use. The detection zone is divided into separate microfluidic
The microfluidic cell was fabricated in a polycarbonate chambers addressing selected electrodes for the measurement of
substrate and diced to have the format of a microscope slide samples and calibrators depending on the assay to be realised.

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Fig. 1 Microfluidic platform components: (1) washing buffer reservoir,

(2) substrate reservoir, (3) secondary antibody reservoir, (4) sample
reservoir, (5) calibrator reservoir, (6) waste reservoir, (7) valve A, (8)
valve B, and (9) detection zone with integrated electrode array.
Fig. 2 Valve positions in the microfluidic platform.
Published on 01 December 2010 on http://pubs.rsc.org | doi:10.1039/C0LC00398K

The electrode arrays were fabricated using photolithographic

design. However, care should be taken to avoid the use of very
deposition technologies in order to realize three-electrode cells
high air pressure to drive the liquids to avoid the generation of air
comprising of gold counter and working electrodes as well as
bubbles inside the channels.
silver reference electrode. They consist of 16 independently
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Fig. 3 shows a flow diagram of the sandwich assay protocol

addressable gold working electrodes (1  1 mm2) in a 4  4
employed in this work for the detection of proteic cancer
arrangement placed between a silver pseudo reference (0.2 
markers. The first step is a rehydration of the sensor surface,
1 mm2) and a gold counter electrode of the same size in order to
followed by the successive incubations of sample and HRP-
create 16 planar electrochemical cells. The working principle is
labelled secondary antibody for a given period of time (see next
similar to any classical three-electrode cell in which the potential
section). Detection is carried out amperometrically after injec-
at the working electrode is applied versus the reference electrode,
tion of the substrate mixture. Although the integrated micro-
most commonly made of silver, and the counter electrode (made
fluidic platform has been devised to be disposable, the chip can
of an inert material, in this case gold) serves to close the elec-
be disassembled following measurement and most parts can be
trochemical circuit. All reference and counter electrodes were
re-used.
short circuited at their connection points to realise an array of 16
working electrodes surrounded by common reference and
counter electrodes. The array also features an equalized length to
the connection pads from the working electrodes to the On-chip optimisation of assay time
connector in order to avoid differences in electrical resistance
between these two points. The electrode array is integrated on the Incubation times are one of the bottlenecks in assay development
fluidic chip using a double-sided medical grade adhesive foil of since it not only influences the overall assay time but also the
50 mm thickness previously laser machined to generate micro- analytical performance of the immunosensor and the time
channel structures of 1 mm width and is connected via pogopin required for the complete reaction between the target and the
connectors to an external multichannel potentiostat. immobilized biorecognition probe.15 For the optimisation of the
The fluidic storage part of the platform consists of five reser- incubation times, electrode arrays modified with the capture
voirs to store the reagents and sample, which are interfaced by antibody were integrated in the microsystem and the target and
rubber septa plugged into the injection ports. These reservoirs reporter conjugate were injected into the system and incubated
have the appropriate volume to run a single assay per cartridge for variable times. Fig. 4 shows the influence of incubation time
and are manually filled. A waste reservoir is placed at the top left on the amperometric response of the system in the presence of
corner of the chip to collect the liquids from the different reser- 100 ng mL1 CEA. As can be seen, the optimum combination of
voirs during the assay. incubation times was 2 minutes for each sample and reporter
conjugate. These very fast incubation times contrast greatly with
Operation of the microfluidic platform those commonly used in ELISA (30–60 minutes) and, as has been
previously shown, highlight the importance of the use of
The liquids from the reservoirs are actuated by applying a posi- microfluidic systems in a speeding-up of analytical processes.23,24
tive air pressure (i.e. via a programmable syringe pump) through A similar result was obtained with the other markers, allowing
the septa and are driven to the detection zone via two turning the possibility of a fast multiplexed assay.
valves (Fig. 2). Valve A has 6 positions and serves to select one of
the five reservoirs from which the liquid is pumped or a venting
position used to empty the microfluidic channels and connected
directly with a waste reservoir. The second valve selects to which
chamber in the detection zone the selected liquid will be driven,
i.e. calibrator or sample chambers and has two positions. For
example, the combination of positions A1–B1 will drive the
washing buffer to the calibrator channel. No liquid cross-
contamination is observed due to the efficiency of the sealing of
the valves, in spite of the apparent complexity of the fluidic Fig. 3 Assay structure indicating the reservoirs employed in each step.

628 | Lab Chip, 2011, 11, 625–631 This journal is ª The Royal Society of Chemistry 2011
 View Online
Published on 01 December 2010 on http://pubs.rsc.org | doi:10.1039/C0LC00398K

Fig. 4 Amperometric responses obtained for the detection of CEA at

different sample and reported antibody conjugation times.
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Proof of principle of individual detection of protein markers

The approach for multiplexed electrochemical detection consists

in the immobilisation of several marker probes on an electrode
array, each electrode being functionalised with a particular probe
selective to the desired target. The immobilization of antibodies
against CEA, CA15-3 and PSA was achieved via crosslinking to
a bipodal dithiol chemisorbed on gold electrodes.14
Multiplexed detection requires that the sensor is, in the first
place, able to detect each individual marker individually with
good sensitivity. Fig. 5 shows the calibration curves obtained for
each marker. The amperomeric response increased linearly with
antigen concentration in the studied range with a LOD ¼ 0.2 ng
mL1 for CEA, 5.2 U mL1 for CA15-3 and 2 ng mL1 for PSA.
All these values are well below the commonly accepted concen-
tration thresholds used in clinical diagnosis for these
markers,17,19,21 which demonstrates the sensitivity of the devel-
oped electrochemical assays. The markedly higher currents
obtained with PSA is due to its lower molecular weight (34 kDa)
as compared with CEA and CA15-3 (200 and 450 kDa, respec-
tively), which increases the number of captured antigen mole-
cules on the surface and thus generates a higher electrochemical
signal after interaction with the labelled reporter conjugate.

Reproducibility studies
Fig. 5 Calibration plots obtained for the amperometric detection of
In order to assess the reproducibility of the on-chip electro- CEA (a), PSA (b) and CA15-3 (c).
chemical detection of cancer markers using the developed
microfluidic cell, electrode arrays were modified with the usual
and anti-PSA in alternating electrode spots. An antigen mixture
surface chemistry and the same concentration of CEA was
composed of CEA (50 ng mL1) and PSA (20 ng mL1) was then
applied on all electrodes. Fig. 6 shows the amperometric
applied to the sensor surface and the response obtained in the 16
responses obtained in the detection of 100 ng mL1 of CEA. The
channels was measured amperometrically (Fig. 7). As can be
average current value obtained was (391  27) nA, with
seen, the signal obtained in the respective channels is very
a residual standard deviation (RSD) of 7% (n ¼ 16). At lower
reproducible, with RSD lower than 8% for both CEA and PSA,
CEA concentrations (10 ng mL1) the average signal was (89 
and the ability to differentiate both signals and detect CEA and
10) nA with RSD ¼ 11% (n ¼ 16), indicating an excellent degree
PSA simultaneously is clearly demonstrated.
of reproducibility of the microfluidic platform.

Multiplexed electrochemical detection of cancer markers Real sample analysis

To test the possibility of simultaneous detection of several The developed immunosensor was applied to the detection of
protein markers, electrode arrays were modified with anti-CEA CEA in real samples using serum samples from cancer patients.

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patients with an excellent correlation between the CEA levels

obtained with the immunosensor and ELISA. Therefore, the
microfluidic platform not only functions with high accuracy and
sensitivity but also matches the performance of an established
technique such as ELISA with the advantage of vastly reduced
assay time and reagent consumption.

Conclusions
In this work, we demonstrate the versatility of a microsystem
integrating electrochemical detection in the individual and
simultaneous detection of breast cancer markers in real samples.
The microfluidic platform was realized by high precision milling
Published on 01 December 2010 on http://pubs.rsc.org | doi:10.1039/C0LC00398K

of polycarbonate sheets and features two well distinguishable

sections: a detection zone incorporating the electrode arrays and
the fluid storage part. The detection zone is divided into separate
microfluidic chambers addressing selected electrodes for the
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Fig. 6 Current values obtained in the amperometric detection of 100 ng measurement of samples and calibrators. The fluidic storage part
mL1 CEA using the microfluidic platform. of the platform consists of five reservoirs to store the reagents
and sample, which are interfaced by septa. These reservoirs have
the appropriate volume to run a single assay per cartridge and
are manually filled. The liquids from the reservoirs are actuated
by applying a positive air pressure (i.e. via a programmable
syringe pump) through the septa and are driven to the detection
zone via two turning valves. The application of realised platform
in the individual and simultaneous electrochemical detection of
proteic cancer markers with very low detection limits is demon-
strated. The microsystem has also been validated using real
patient serum samples and excellent correlation with ELISA
results obtained.
This device has the advantage of integrating the microfluidic
operations required to execute on-chip electrochemical immu-
noassays in a single platform—namely, reagent storage and
transport, metering, incubation and detection. In addition, the
design has been conceived in such a way that with fully auto-
mated fluidic operations, the assay only requires the introduction
of microlitre amounts of sample by the end user, thus facilitating
Fig. 7 Amperometric responses obtained for the simultaneous detection its operation and vastly reducing the reagent consumption and
of CEA (50 ng mL1) and PSA (20 ng mL1) using the microfluidic assay time.
platform. The results presented here on the successful integration of
fluidic storage of assay reagents with electrochemical detection
Samples were taken and divided into two aliquots, one for are thus in the direction of achieving low cost disposable
ELISA and one for immunosensor detection. As can be seen cartridges amenable for point of care applications. Advances in
from Table 1, samples 1–4 showed values lower than 10 ng mL1 microfabrication and packaging necessarily need to meet with
of CEA, which is the commonly accepted cut-off value used in the specifications of stable reagents for storage25 with the
clinical diagnosis.17 These samples are from patients under objective of achieving a fully disposable detection platform.
cancer remission and can be thus considered as normal values.
Therefore, the device can differentiate between normal and ill Acknowledgements
The authors thank the EU SmartHEALTH project (FP6-2004-
Table 1 CEA levels (in ng mL1, duplicate measurements) measured in
IST-NMP-2-016817) for financial support and Fujirebio Diag-
serum samples with the developed immunosensor and ELISA
nostics AB for providing the monoclonal antibodies. A.F. thanks
Sample Immunosensor ELISA Ministerio de Ciencia e Innovacion, Spain, for a ‘‘Ram on y
Cajal’’ Research Professorship.
1 4.4  1.7 3.6  0.7
2 5.8  1.1 6.9  0.4
3 6.8  1.4 5.9  0.6 References
4 7.7  0.7 8.9  1.1
5 20.1  2.9 22.5  1.1 1 J. West, M. Becker, S. Tombrink and A. Manz, Anal. Chem., 2008, 80,
6 43.6  2.7 41.5  3.3 4403.
2 S. Haeberle and R. Zengerle, Lab Chip, 2007, 7, 1094.

630 | Lab Chip, 2011, 11, 625–631 This journal is ª The Royal Society of Chemistry 2011
 View Online

3 H. Becker and C. G€ artner, Electrophoresis, 2000, 21, 12. 15 H. Nassef, M. C. Bermudo Redondo, P. Ciclitira, H. J. Ellis,
4 A. J. Tudos, G. A. J. Besselink and R. B. M. Schasfoort, Lab Chip, A. Fragoso and C. K. O’Sullivan, Anal. Chem., 2008, 80, 9265.
2001, 1, 83. 16 S. F. Y. Li and L. J. Kricka, Clin. Chem. (Washington, DC, U. S.),
5 A. Bange, H. B. Halsall and W. R. Heineman, Biosens. Bioelectron., 2006, 52, 37.
2005, 20, 2488. 17 K. Bremer, S. Micus and G. Bremer, Eur. J. Cancer, 1995, 31, S262.
6 R. Polsky, J. C. Harper, D. R. Wheeler and S. M. Brozik, 18 Z. Mujagic, H. Mujagic and B. Prnjavorac, Med. Arh., 2004, 58, 23.
Electroanalysis, 2008, 20, 671. 19 S. Ali, K. Leitzel, V. M. Chinchilli, L. Engle, L. Demers,
7 E. Nebling, T. Grunwald, J. Albers, P. Schafer and R. Hintsche, Anal. L. H. A. Harvey, W. Carney, J. W. Allard and A. Lipton, Clin.
Chem., 2004, 76, 689. Chem. (Washington, DC, U. S.), 2002, 48, 1314.
8 J. Rossier, R. Ferrigno and H. H. Girault, J. Electroanal. Chem., 20 B. De La Lande, K. Hacene, J. L. Floiras, N. Alatrakchi and
2000, 492, 15. M. F. Pichon, Int. J. Biol. Markers, 2002, 17, 231.
9 J. Wang, A. Ib ~ ez, M. P. Chatrathi and A. Escarpa, Anal. Chem.,
an 21 U. H. Stenman, J. Leinonen, W. Zhang and P. Finne, Semin. Cancer
2001, 73, 5323. Biol., 1999, 9, 83.
10 T. Lim, H. Ohta and T. Matsunaga, Anal. Chem., 2003, 75, 3316. 22 A. Healy, C. J. Hayes, P. Leonard, L. McKenna and R. O’Kennedy,
11 J. S. Ko, H. C. Yoon, H. Yang, H. Pyo, K. Chung, S. Kim and Trends Biotechnol., 2007, 25, 125.
Y. Kim, Lab Chip, 2003, 3, 106. 23 N. Lion, F. Reymond, H. H. Girault and J. S. Rossier, Curr. Top.
Published on 01 December 2010 on http://pubs.rsc.org | doi:10.1039/C0LC00398K

12 H. Dong, C. Li, Y. Zhang, X. Cao and Y. Gan, Lab Chip, 2007, 7, Biotechnol., 2004, 15, 31.
1752. 24 S. C. Jacobson, C. T. Culbertson, J. E. Daler and J. M. Ramsey, Anal.
13 I. E. Tothill, Semin. Cell Dev. Biol., 2009, 20, 55–62. Chem., 1998, 70, 3476.
14 A. Fragoso, N. Laboria, D. Latta and C. K. O’Sullivan, Anal. Chem., 25 A. Fragoso, N. Laboria, C. K. O’Sullivan, Anal. Lett., DOI: 10.1080/
2008, 80, 2556. 00032719.2010.539732.
Downloaded by University of Toronto on 05 November 2012

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