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Sensors and Actuators B 202 (2014) 6066

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Sensors and Actuators B: Chemical
j our nal homepage: www. el sevi er . com/ l ocat e/ snb
Rapid prototyping of multifunctional microuidic cartridges for
electrochemical biosensing platforms
Jitae Kim
, Yong Shin
, Simon Song
, Joohyung Lee
, Jungkyu Kim
Department of Mechanical Engineering & Institute of Nano Science and Technology, Hanyang University, Seoul, Republic of Korea
Institute of Microelectronics, A*STAR (Agency for Science, Technology and Research), 11 Science Park Road, Singapore Science Park II,
Singapore 117685, Singapore
Department of Mechanical Engineering, Texas Tech University, Lubbock, TX 79406, USA
a r t i c l e i n f o
Article history:
Received 27 January 2014
Received in revised form26 March 2014
Accepted 5 May 2014
Available online 21 May 2014
Rapid prototyping
Porous membrane
One-shot valve
On-chip chemical storage
Microuidic electrochemical immunoassay
Biomarker screening
a b s t r a c t
A multifunctional microuidic cartridge for electrochemical biosensing (CEB) was developed with
a cleanroom-free rapid prototyping technique. A seven-layered CEB platform including a gold (Au)
electrode substrate was fabricated by alternating patterned thin plastic and adhesive lms. To provide
conformal bonding between layers, pressure sensitive adhesive (PSA) tape was adopted to fabricate a leak-
free, porous membranes embedded CEBplatform compatible with relatively high ow rates. Inaddition,
the embedded porous membranes provide multiple functions in the CEB device, including (1) control-
ling uid ow, (2) storing a dried detergent, and (3) trapping liquid waste. To demonstrate the utility of
the CEB, we performed an electrochemical-based immunoassay to detect various concentrations of Cre-
atine Kinase (CK)-Myocardial Band (MB). During the immunoassay, the membranes controlled the ow
path to minimize a carryover between the assay steps and released the stored dried-reagent to remove
non-specically bound detection antibodies. With a linear sweep voltammetry (LSV) electrochemical
sensing technique, a limit of detection of 0.25 ng mL
was achieved. The multilayered prototyping tech-
nique enables rapid and low-cost fabrication of multifunctional microuidic electrochemical devices with
single-use sample processing components.
2014 Elsevier B.V. All rights reserved.
1. Introduction
Miniaturized electrochemical sensors have been broadly
exploredfor rapidandaccuratedetectionof chemical andbiological
agents [15]. Integration of this technology with microuidic sys-
tems enables simple, portableandautomatedbiosensingplatforms.
These benets make microuidic electrochemical (EC) devices an
attractive technology for point-of-care diagnostics and infectious
diseases monitoring in resource-limited settings.
To make the microuidic EC devices more practical, a sig-
nicant effort has been made to develop rapid prototyping
techniques [610]. Soft lithography has been extensively used
due to simplicity, low cost and versatility [7]. To fabricate
the microuidic EC device with the soft-lithography, bonding
between a polydimethylsiloxane (PDMS) channel layer and an

Corresponding author at: Department of Mechanical Engineering, Department

of Internal Medicine, Texas Tech University, Lubbock, TX 79409, USA.
Tel.: +1 806 834 6106.
E-mail address: (J. Kim).
These authors contributed equally to this work.
electrode-deposited substrate was performed simply by oxygen
plasma treatment [11] or physical clamping [12]. This is possible
due to the elastomeric property of PDMS which allows conformal
contact with rigid substrates such as silicon and glass. Recently,
other polymeric materials like polycarbonate (PC) [1315], poly-
methyl-meta-acrylate (PMMA) [16,17], cyclic olen copolymer
(COC) [1820] and polyimide [21] have been used as alternatives
due to undesirable properties of PDMS such as poor chemical com-
patibility, gas permeability and innate hydrophobicity. Although
those polymers are superior to PDMS in terms of native proper-
ties, fabrication, integration and packaging procedures with those
polymers can be more difcult. For example, bonding processes
typically involve the use of high temperature or solvents, resulting
in compromised integrity and resolution of microstructures. As a
result, bonding of an electrode-patterned rigid polymer to another
rigid polymer remains challenging due to leakage.
Bartholomeusz et al. [22] developed a rapid prototyping tech-
nique using xurography, where a cutting plotter was used to cut
microstructures on various adhesive lms (pressure sensitive and
thermal activated adhesive lms). Patterned adhesive lms were
laminated for fabricating multilayered microuidic devices rapidly
and inexpensively. By combining plastic microuidic fabrication
0925-4005/ 2014 Elsevier B.V. All rights reserved.
J. Kimet al. / Sensors and Actuators B 202 (2014) 6066 61
methods with xurography, challenges such as the uid leakage and
electrical cross-talk issues were successfully addressed.
Membrane-integrated microuidic chips [2330] have been
studied for enhanced functionality and mass transport control.
Although porous membranes are used in a wide range of indus-
trial applications, their usage in microuidic systems has been
limited to ltration [23,31] and chemical reactors [32] due to the
lack of simple and reliable fabrication techniques. Yager et al. [33]
presented a microuidic ow-through membrane immunoassay
with dry-stored reagents in a membrane. The use of functionalized
membranes enabled the simplication of assay operation. How-
ever, on-chip integration of multiple membranes, each responsible
for a different function, remains unexplored.
Inthis study, we report a simple rapid prototyping of a multilay-
ered microuidic cartridge for electrochemical biosensor (CEB)
with integrated Au electrodes. By using layer-by-layer assem-
bly, the patterned plastic lms and double-sided PSA tapes were
alternately bonded to each other to fabricate a disposable, multi-
functional CEB at room temperature. Three porous membranes
were integrated into the CEB during the assembly process to uti-
lizefor air owstopper, reagent storageandliquidwasteabsorbent.
To demonstrate the utility of the CEB, we performed a quan-
titative immunoassay for CK-MB, a cardiac biomarker used for
diagnosis of anacutemyocardial infraction(AMI). Thedevelopment
of multilayer electrochemical biosensor from the rapid prototype
technique, multifunctional membranes andsimpleelectrochemical
detections will be able to facilitate advanced point-of-care device
for various biomarker screenings.
2. Experimental
2.1. Fabrication of CEB platform
Fig. 1 presents a schematic for rapid prototyping of the CEB
(85mm54mm1.8mm) with 3-electrode conguration which
was made of 7 patterned layers, comprised of an Au-coated PET
lm(L1), double-sided tape (L2, L4, L6), an acrylic sheet (L3), and a
PET lm(L5, L7).
To fabricate the Au electrodes on PET lm (L1), 100nm thick
Au was deposited by sputtering process through a shadow mask
containing the patterns for electrodes and alignment holes. The
Au-deposited lm (L1) was then treated with plasma to remove
carbon residue on the Au surfaces, and the Au electrodes fur-
ther underwent an antibody immobilization process (see below
in immunoassay protocol section). The double-sided PSAs (ARseal
90880, Adhesives Research), with two release liners laminated on
both sides, were patterned with a cutting plotter (FC2250-120VC,
Graphtec Inc., USA) to create L2/L4/L6 layer. The acrylic sheet was
cut with a CO
laser (VLS 3.60, Universal Laser Systems) to form
uidic reservoirs in L3. Three passes of the laser beam(75% Power,
20% Speed and 1000 PPI) were applied to obtain smooth cutting
edges and near-vertical sidewalls. The laser-cut acrylic sheet was
cleaned in an ultrasonic water bath for 10min, dried with N
and then annealed on a hot plate maintained at 85

C for 90min.
This annealingstepwas includedtoavoidstress cracks intheacrylic
Once all the layers are ready, assembly began with bonding of
the bottomPET lm(L1) and the rst PSA (L2). In the rst step, cut-
out regions (i.e., channels and reservoirs) of the PSAwere peeled off
from the bottom release liner. An application tape, typically one-
sided adhesive backed lm, was applied to the peeled-off side and
squeezed to hold the pattern in place. Then the release liner was
carefully detached, leaving the engraved pattern on the applica-
tion tape pattern transfer [22]. The application tape bearing the
pattern (L2) was placed on a custom-made aligner with 4 press-
tted dowel pins. Each layer has 4 alignment holes at the corners,
and each hole matches the corresponding dowel pin in its position
for accurate alignment. The PET lm (L1) incorporating immobi-
lized antibody was adhesively bonded to the PSA (L2) held on the
aligner using a hand roller. To integrate the acrylic layer (L3) with
the PET-PSA assembly (L1/L2), the assembly was put back on the
aligner withits adhesive side facing upfollowing the removal of the
top release liner along with the application tape. The acrylic sheet
was mated with the assembly held on the aligner to form a PET-
PSA-acrylic assembly (L1/L2/L3). A laser-cut absorbent pad (M3,
790mthick, pure cellulose bers, 133, Pall) was attached to an
adhesive region of the assembly (L1/L2/L3). Next, a pattern trans-
ferred fromthe second PSA (L4) was bonded to the PET-PSA-acrylic
assembly (L1/L2/L3) in the same way as described above (i.e., bond-
ingof L1andL2). The layer-by-layer assemblyprocess was repeated
Fig. 1. Fabrication process of a multilayered, polymeric microuidic cartridge. (A) Exploded view of all patterned layers. Alternating layers of thin plastic lm/sheet (blue)
and double-sided tape (yellow). (B) Schematic cross-section of all layers in the stack. Liquid is drawn into an absorbent-embedded waste reservoir by vacuum when the
tape is pierced. (C) A photograph (top view) of a 3-D microuidic cartridge (85mm54mm1.8mm) constructed by layer-by-layer assembly. M1, M2 and M3 are the rst,
second and third membrane, respectively. C, R, and Wrepresent the counter, reference and working electrode, respectively. The sense electrodes monitor the position of a
liquid meniscus.
62 J. Kimet al. / Sensors and Actuators B 202 (2014) 6066
Table 1
Description of each layer regarding material, thickness, function, and patterning
tool. DST represents the double-sided tape.
Material Thickness (m) Function Pattering tool
Layer 7 PET 125 Lid Cutting plotter
Layer 6 DST 130 Channel Cutting plotter
Layer 5 PET 125 Support Cutting plotter
Layer 4 DST 130 Via hole Cutting plotter
Layer 3 Acrylic 1000 Reservoir CO
Layer 2 DST 130 Channel Cutting plotter
Layer 1 PET 125 Electrode Cutting plotter
until the top PET layer (L7) was integrated into a whole assembly
(L1 through L7) as described in Fig. 1B. The whole assembly was
then compressed multiple times using a desktop cold laminator
to ensure sufcient bonding strength as well as a tight seal of the
integrated Au electrodes. Table 1 shows the summary of detailed
information on each layer.
2.2. Integration of porous membranes
Tween-20 detergent is routinely added to phosphate-buffered
saline (PBS) or tris-buffered saline (TBS) wash buffer to minimize
the background noise from non-specic binding in bioassays. To
simplifytheon-chipassayprocedure, weembeddedaporous mem-
brane containing dried Tween-20 in a CEB. In this way, we could
minimize the complexity of device fabrication by eliminating the
need for liquid buffer storage in the CEB. At rst, a porous mem-
brane (419mthick, spun bonded polyester, 6613, Pall) was cut
into 3-mm and 5-mm circles. The 3-mm disk membranes were
immersed in a tube containing 50% Tween-20 (P9416, Sigma) for
1h. The wetted membranes were removed fromthe tube and dried
in a 50

C oven for 4h to prepare detergent-modied membranes.

Similarly, the 5-mm disk membranes were treated with bovine
serumalbumin (BSA) (A2153, Sigma). The BSA-treated membrane
(M1) andthe Tween-20treatedmembrane (M2) were theninserted
into the corresponding grooves formed by three stacked layers
(L4/L5/L6) and tightly xed in place when the top layer (L7) was
capped. Note that nominal thickness of the membrane was chosen
to be slightly higher than the depth of the groove to create tight t.
This conguration allowed liquid to block air owwhen trapped in
the rst membrane (M1) andtopass throughthe secondmembrane
(M2) for reconstitution of the detergent.
2.3. Surface treatment and immunoassay preparation
Toimmobilizecaptureantibodies ontheworkingelectrode, 6L
of monoclonal anti-CK-MB antibody at 10gmL
CK-MB 7501 SPRN-2, Medix Biochemica) diluted in a PBS buffer
(10mM PBS, pH 7.4 (Sigma)), was dispensed onto the electrode
and incubated at 4

C for 2h in a perti-dish along with a wet tissue

to prevent evaporation of the antibody solution. After the incuba-
tion, the electrode was washed 3 times with a TBST buffer (20mM
TBS, pH 7.4 (T5912, Sigma) and 10mg mL
Tween-20 (P9416,
Sigma)). Then, TBS buffer containing 1% BSA was dropped on all
three electrodes to block the surface. After 1h of incubation at
roomtemperature, the electrodes were washed 3 times with TBST
and dried with N
gas. Once the immobilization steps were n-
ished, the PET lm(L1) was stored at 4

C until used. Labeling of a

detection antibody with alkaline phosphatase (ALP) was done by
using an Alkaline Phosphatase Labeling Kit-SH(LK-13-10, Dojindo,
SouthKorea). We followedthe kit procedure to conjugate 100g of
detection anti-CK-MB antibody (Anti-human CK-MB 7502 SPRN-5,
Medix Biochemica). The conjugate solution was diluted 100 times
to be added in samples. CK-MB antigen (30-1081, Fitzgerald) was
spikedand diluted to nal concentrations of 1, 5, 10 and 25ngmL
in TBS buffer. Each concentration of the CK-MB antigen was mixed
with the ALP-labeled detection antibody at a volume ratio of 1:10
and incubated at 37

C for 30min to execute the rst immunore-

action. The electrochemical substrate solution was prepared in a
mixture of 40mM 4-aminophenyl phosphate (pAPP, A-292, Gold-
bio), 1mM MgCl
(M2393, Sigma) and100mM NaCl (S3014, Sigma)
in 100mM diethyl amine (DEA, pH 9.8, 31589, Sigma).
2.4. Immunoassay for CK-MB detection
Once all the solutions were prepared, an on-chip sandwich-type
immunoassay for CK-MB detection was performed using the CEB
platform. Serially diluted CK-MB antigens (1, 5, 10 and 25ngmL
werepreparedinTBSbuffer. Beforestartingtheon-chipimmunoas-
say, a mixture of 45L of various concentrations of CK-MBantigens
and 5L of the ALP-conjugated anti-CK-MB antibodies were incu-
bated in a water bath maintained at 37

C for 30min. A 200L

volume of substrate solution was pre-loaded in the substrate reser-
voir (2.0mm width1.2mm height), and pierceable tape was then
attached to seal the valves opening (see Fig. 1B). This type of valve
(normally closed) prevents a liquid plug in a conduit frommoving
away fromone side (valves position) when a negative pressure is
applied on the other side. The valve can be opened by simply per-
forating the tape, resulting in the trapped air being balanced with
an ambient pressure.
A 50L sample containing an antigen-antibody-enzyme com-
plexwas loadedtothesampleport. Thesamplespontaneouslylled
the sample reservoir (0.5mm width1.2mm height) by capillary
wicking and stopped at the rst membrane (M1). Upon applica-
tion of a negative pressure via the vacuumport, the sample began
to ow into the main channel (400m width130m height)
past the membrane to reach the absorbent pad (M3) embedded
in the waste reservoir. The ow was maintained at approximately
0.21L s
for 4min (Fig. 2A). Note that the pre-loaded substrate
solution was kept in the substrate reservoir by the normally closed
valve during the sample injection. After the sample injection step,
the wet membrane (M1) stops air owfromleaking intothe sample
Next, a wash step was initiated by perforating the tape with
a sharp needle. The substrate solution was drawn into the main
channel and reconstituted the dried Tween-20 in the second mem-
brane (M2). Subsequently, air owfromthe positive pressure port
was supplied to the junction, thereby generating air bubbles in a
streamof the substrate solution containing the detergent (Fig. 2B).
Such a segmented ow [35] is known to assist in washing on the
electrode surface due to meniscus force and internal recirculation
owinduced within liquid segments. By the time the washing step
was nished, air bubble generation was ended by switching off the
positive pressure port (Fig. 2C). In the meantime, the substrate con-
tinued owing at a owrate of 5.0L s
for 15s. This is to bring a
fresh volume of the substrate, containing little or no Tween-20,
over the electrode region. Lastly, the solution was stopped and
held immobile during the enzymatic reaction by turning off all
the pumps and then venting the remaining pressure in the CEB
(Fig. 2D). After a 5-min enzymatic reaction, linear sweep voltam-
metry(LSV) signals weremeasuredwithaParstat 2273potentiostat
(Princeton Applied Research) in the range of 0.2 to 0.3V at a scan
rate of 0.1Vs
3. Results and discussion
To evaluate sealing performance of the CEB device, we per-
formed leakage tests for liquid and air in the microuidic channels.
Fig. 3A represents the magnied top view of a uidic channel
(the middle region denoted by PET) which is overlapped with Au
J. Kimet al. / Sensors and Actuators B 202 (2014) 6066 63
Fig. 2. Images of a microuidic cartridge during immunoassay operation. The state of pressure (positive, upper port) and vacuum(negative, lower port) pumps for each step
is denoted. (A) Sample transport to the antibody derivatized working electrode (denoted by W). Sample (red arrow) ows through the main channel for immunoreaction
by turning on the negative pressure while substrate solution is held in the reservoir (orange dotted line). (B) Electrode washing and addition of substrate. Air bubbles are
generated at the junction by applying a positive pressure, and non-specically bound target are removed by a meniscus force (blue and orange arrows). (C) By switching off
the positive pressure, air bubbles are removed, and a fresh substrate solution was delivered over the electrodes (orange arrow). (D) By switching off all the pressure sources,
the substrate solution is held on the electrodes during enzymatic reaction and electrochemical detection (orange dotted line).
Fig. 3. Leak-proof sealing of integrated electrodes using a double-sided tape. (A) A magnied viewof an Au electrode-deposited PET lmcrossed by a uidic channel (400m
width 130mheight) formed by cutting a double-sided tape. (B) A red liquid dye owing in the uidic channel which is overlapped with three electrodes (100nmthick)
at a owrate of 9.1L s
(scale bar, 500m).
electrodes on the PET lm(L1). To performa liquid leak test, a red-
dyed liquid was owed through the uidic channel and drained
into a bottle which was connected to the waste reservoir (with no
absorbent pad in this test). The vacuum-driven ow was visually
checked for leakage while the ow rate was gradually increased.
Therewas novisibleleakagearoundtheuidic channel at owrates
upto9.1L s
as showninFig. 3B. Inaddition, we conducteda pre-
liminary test for gas leakage. To detect air leaks out of the CEB,
a syringe pump was set to slowly increase air pressure inside the
CEB that was immersed in water. No air bubbles were observed
up to 20psi. This result demonstrates that the PSA-based bonding
technique is suitable for leak-proof sealing of metal electrodes over
rigid substrates.
For diverse functionality of integrated membranes, we utilized
the rst membrane (M1) as a valve. As depicted in Fig. 4A, a liquid
solution continuously ows to the main channel past the M1 by a
vacuumpump until the solution fromthe reservoir is all dissipated
and captured by the membrane. At this time, a positive pressure is
applied from the right side of the main channel to allow the solu-
tion to be separated fromthe membrane. When M1 was wetted by
the sample solution, it functions as a valve to prevent air inside the
channel frombeing discharged to the reservoir via the membrane.
As shown in Fig. 4B, air can only ow along the main channel but
cannot ow in the direction blocked by the liquid and the mem-
brane. In a similar manner, the third membrane (M3) was used
for isolating the waste solutions from both the main channel and
64 J. Kimet al. / Sensors and Actuators B 202 (2014) 6066
Fig. 4. Operation of the rst membrane (M1) for valve function. (A) A liquid (dotted arrow) passes through the membrane and moves to the main channel by a vacuumpump.
The valve in its open state is used in the sample transport step (red arrow). (B) When the liquid in the sample reservoir is all dissipated, a pressure pump applies a force to
separate the liquid fromthe main channel. The valve in its closed state blocks air ow(blue arrow) fromleaking into the sample reservoir in the air bubble generation step.
the vacuumport. This function will be very useful for the safety of
operators, particularly in screening for contagious diseases.
In addition, the second membrane (M2) was used as a dried
chemical storage. Once the membrane is wetted with a buffer solu-
tion, we can simply reconstitute to obtain the desired buffer. In
the CEB, the substrate solution was exploited for wash (modi-
ed usage) as well as enzymatic reaction (original usage), greatly
simplifying an immunoassay design. For the washing step, by pass-
ing the solution through a Tween-20 dried membrane, the solution
was modiedto 1%Tween-20 buffer to remove the non-specically
bound protein on the electrodes. After reconstituting the dried
Tween-20, the unmodied substrate solution was delivered over
the electrodes for enzymatic reaction. With this simplied forma-
tion, wash performance during the immunoassay was evaluated by
comparing non-specic background signals associated with two
types of membranes (i.e., in the presence vs. absence of Tween-
20). We used 50L of sample containing CK-MB antibody-enzyme
conjugate and 100L of substrate solution (out of total 200L)
with or without 1% Tween-20 in the wash test. The rst 100L
was supplied at different owrates of 0.71, 1.43 and 5.0L s
, fol-
lowed by the second (unmodied) 100L with a xed ow rate
of 5.0L s
. No air bubbles were produced in this procedure in
order to only test the role of Tween-20 rehydrated fromthe mem-
brane to washing efciency. As shown in Fig. 5, the CEB utilizing
the Tween-20 coated membrane yielded a lower background sig-
nal thanone without Tween-20, demonstrating that the membrane
with Tween-20 enhanced washing efciency.
To demonstrate the assay capability of the CEB, we performed
an electrochemical immunoassay for 5 different target concen-
trations (1, 5, 10, 25ngmL
including a negative control). As
described above, a 50L sample containing an antigen-ALP labeled
detection antibody was applied to the CEBs sample port, and
immunoreactionoccurredduringsample ow. Then, 200L of sub-
strate solution was constantly pumped at a ow rate of 5.0L s
for washandhaltedfor a 5-minenzymatic reaction. Fig. 6Apresents
the current responses at the working electrode plotted against
the applied potential in LSV. Faradic current contribution was
Fig. 5. Comparison of background currents (with no target antigen) for wash
performance in the sandwich electrochemical immunoassay. 100L samples of
electrochemical substrate solution with (modied) vs. without (unmodied) 1%
Tween-20 were applied at different ow rates of 0.71, 1.43 and 5.0L s
for the
rst wash. Subsequently, another 100L of unmodied substrate solution was sup-
plied for both second wash and enzymatic reaction. The error bar represents the
standard deviation fromthree independent measurements.
extracted at a xed potential of 0.15V to give a peak anodic cur-
rent. Fig. 6B exhibits the calibration curve of the peak anodic
current versus the concentration of CK-MB. The current responses
changed log-linearly as the analyte concentration increased, which
indicates that the on-chip electrochemical scheme could be used
for quantitative detection in immunoassays. From a series of the
immunoassays, we achieved a limit of detection of 0.25ngmL
theCK-MBassay. ElevatedCK-MBlevel has beencorrelatedwiththe
incidence of myocardial damage, and regular screening of CK-MB
levels has been suggested for the early detection of acute myocar-
dial infarction (AMI) [36]. Although there is no clear threshold
CK-MB value that is denitively diagnostic for AMI, 10ngmL
has been proposed as an acceptable detection limit for early AMI
J. Kimet al. / Sensors and Actuators B 202 (2014) 6066 65
Fig. 6. (A) Current responses obtained fromon-cartridge immunoassay for CK-MB at different concentrations of 25, 10, 5, 1 and 0ngmL
. Linear sweep voltammetry was
run in the range of 0.2 to 0.3V at a scan rate of 0.1Vs
. The immunoassay was processed on the microuidic cartridge with Tween-20 modied membrane. A 200L
volume of substrate solution was constantly supplied at 5.0L s
during wash step. (B) Calibration plot of the anodic peak current vs. the input concentration of CK-MB (1,
5, 10 and 25ngmL
) including a control. The background current (with no target) was 0.110.014A. The test was repeated three times for each concentration.
screening assays [37]. Thus, the performance of the CEB platform
for CK-MB assay is within clinical norms.
4. Conclusion
The development of microuidic systems has promoted
advances in miniaturization and automation of molecular diagnos-
tic testing, however high fabrication costs are often a barrier to
commercializationand/or adaptationtoresource poor settings. The
CEB including a passive valve, reagent storage and electrochemi-
cal sensors was fabricated by alternately stacking thin plastic lms
and double-sided PSA tapes. Our rapid fabrication technique elim-
inates the need for specialized cleanroom equipment and would
provide a miniaturized, affordable platformfor point-of-care (POC)
testing. In addition, the use of pierceable tape and porous mem-
branes in CEB enables programmable control of uid owduring
the performance of an assay. This capability eliminates the need for
mechanical microvalves in the design, thereby signicantly reduc-
ing the amount of off-chip control equipment. The process that is
automated by our CEB platformcan be useful for a broad range of
targets that employ similar immunoassay protocols. In this work,
we demonstrated an on-chip electrochemical immunoassay for
CK-MB and achieved clinically relevant sensitivity. The rapid fab-
rication technique that we present enables the design, fabrication,
and testing of newdevices within a single day.
This research was supported by Basic Science Research Pro-
gram through the National Research Foundation of Korea (NRF)
funded by the Ministry of Education, Science and Technology
(2012R1A6A1029029). J. Kim would like to thank Texas Tech
University for nancial support of this project through newinves-
tigator start-up funding. The authors would also like to thank Dr.
Jensen for fruitful scientic discussions.
[1] J. Kim, J. Elsnab, C. Gehrke, J. Li, B.K. Gale, Microuidic integrated multi-walled
carbon nanotube (MWCNT) sensor for electrochemical nucleic acid concentra-
tion measurement, Sens. Actuator B 185 (2013) 370376.
[2] Y. Sameenoi, K. Koehler, J. Shapiro, K. Boonsong, Y. Sun, J. Collett Jr., et al.,
Microuidic electrochemical sensor for on-line monitoring of aerosol oxidative
activity, J. Am. Chem. Soc. 134 (2012) 1056210568.
[3] J. Wang, Electrochemical biosensors: towards point-of-care cancer diagnostics,
Biosens. Bioelectron. 21 (2006) 18871892.
[4] J.W. Choi, K.W. Oh, J.H. Thomas, W.R. Heineman, H.B. Halsall, J.H. Nevin,
et al., An integrated microuidic biochemical detection system for protein
analysis with magnetic bead-based sampling capabilities, Lab Chip 2 (2002)
[5] A. Bange, H.B. Halsall, W.R. Heineman, Microuidic immunosensor systems,
Biosens. Bioelectron. 20 (2005) 24882503.
[6] T.W. de Haas, H. Fadaei, D. Sinton, Laminated thin-lmTeon chips for petro-
chemical applications, Lab Chip 12 (2012) 42364239.
[7] D.C. Duffy, J.C. McDonald, O.J. Schueller, G.M. Whitesides, Rapid prototyp-
ing of microuidic systems in poly(dimethylsiloxane), Anal. Chem. 70 (1998)
[8] Y. Lu, W. Shi, L. Jiang, J. Qin, B. Lin, Rapid prototyping of paper-based microu-
idics with wax for low-cost, portable bioassay, Electrophoresis 30 (2009)
[9] E. Sollier, C. Murray, P. Maoddi, D. Di Carlo, Rapid prototyping polymers
for microuidic devices and high pressure injections, Lab Chip 11 (2011)
[10] T. Yasukawa, A. Glidle, M. Nomura, J.M. Cooper, Fabrication of robust 2-D and
3-D microuidic networks for lab-on-a-chip bioassays, J. Microelectromech.
Syst. 14 (2005) 839846.
[11] A. Plecis, Y. Chen, Fabrication of microuidic devices based on
glassPDMSglass technology, Microelectron. Eng. 84 (2007) 12651269.
[12] A. Yamaguchi, P. Jin, H. Tsuchiyama, T. Masuda, K. Sun, S. Matsuo, et al., Rapid
fabrication of electrochemical enzyme sensor chip using polydimethylsiloxane
microuidic channel, Anal. Chim. Acta 468 (2002) 143152.
[13] Z. Chen, J. Wang, S. Qian, H.H. Bau, Thermally-actuated, phase change ow
control for microuidic systems, Lab Chip 5 (2005) 12771285.
[14] J. Kim, D. Byun, M.G. Mauk, H.H. Bau, A disposable, self-contained PCR chip, Lab
Chip 9 (2009) 606612.
[15] J. Yang, Y. Liu, C.B. Rauch, R.L. Stevens, R.H. Liu, R. Lenigk, et al., High sensitivity
PCR assay in plastic micro reactors, Lab Chip 2 (2002) 179187.
[16] L. Yao, B. Liu, T. Chen, S. Liu, T. Zuo, Micro ow-through PCR in a PMMA chip
fabricated by KrF excimer laser, Biomed. Microdev. 7 (2005) 253257.
[17] W. Zhang, S. Lin, C. Wang, J. Hu, C. Li, Z. Zhuang, et al., PMMA/PDMS valves and
pumps for disposable microuidics, Lab Chip 9 (2009) 30883094.
[18] Q. Pu, O. Oyesanya, B. Thompson, S. Liu, J.C. Alvarez, On-chipmicropatterning of
plastic (cylic olen copolymer, COC) microuidic channels for the fabrication
of biomolecule microarrays using photografting methods, Langmuir 23 (2007)
[19] J. Steigert, S. Haeberle, T. Brenner, C. Muller, C.P. Steinert, P. Koltay, et al., Rapid
prototyping of microuidic chips in COC, J. Micromech. Microeng. 17 (2007)
[20] R. Novak, N. Ranu, R.A. Mathies, Rapid fabrication of nickel molds for
prototyping embossed plastic microuidic devices, Lab Chip 13 (2013)
[21] B.C. Giordano, J. Ferrance, S. Swedberg, A.F. Huhmer, J.P. Landers, Polymerase
chain reaction in polymeric microchips: DNA amplication in less than 240s,
Anal. Biochem. 291 (2001) 124132.
[22] D.A. Bartholomeusz, R.W. Boutte, J.D. Andrade, Xurography rapid prototyping
of microstructures using a cutting plotter, J. Microelectromech. Syst. 14 (2005)
[23] J. Kim, B.K. Gale, Quantitative and qualitative analysis of a microuidic DNA
extractionsystemusing a nanoporous AlOx membrane, Lab Chip8 (2008) 1516.
[24] J. Kim, M. Mauk, D. Chen, X. Qiu, J. Kim, B. Gale, et al., A PCR reactor with an
integrated alumina membrane for nucleic acid isolation, Analyst 135 (2010)
[25] J. Kim, R. Surapaneni, B.K. Gale, Rapidprototyping of microuidic systems using
a PDMS/polymer tape composite, Lab Chip 9 (2009) 1290.
66 J. Kimet al. / Sensors and Actuators B 202 (2014) 6066
[26] B.H. Chueh, D. Huh, C.R. Kyrtsos, T. Houssin, N. Futai, S. Takayama, Leakage-free
bonding of porous membranes into layered microuidic array systems, Anal.
Chem. 79 (2007) 35043508.
[27] P. Guo, E.W. Hall, R. Schirhagl, H. Mukaibo, C.R. Martin, R.N. Zare, Microuidic
capture and release of bacteria in a conical nanopore array, Lab Chip 12 (2012)
[28] F. Saharil, F. Forsberg, Y. Liu, P. Bettotti, N. Kumar, F. Niklaus, et al., Dry adhesive
bonding of nanoporous inorganic membranes to microuidic devices using the
OSTE(+) dual-cure polymer, J. Micromech. Microeng. 23 (2013) 025021.
[29] F. Tan, P.H.M. Leung, Z.-b. Liu, Y. Zhang, L. Xiao, W. Ye, et al., A PDMS microu-
idic impedance immunosensor for E. coli O157:H7 and Staphylococcus aureus
detection via antibody-immobilized nanoporous membrane, Sens. Actuator B
159 (2011) 328335.
[30] J. Yu, Z. Liu, Q. Liu, K.T. Yuen, A.F.T. Mak, M. Yang, et al., A polyethylene glycol
(PEG) microuidic chip with nanostructures for bacteria rapid patterning and
detection, Sens. Actuator A 154 (2009) 288294.
[31] H.B. Wei, B.H. Chueh, H.L. Wu, E.W. Hall, C.W. Li, R. Schirhagl, et al., Particle
sorting using a porous membrane in a microuidic device, Lab Chip 11 (2011)
[32] D.S. Peterson, T. Rohr, F. Svec, J.M.J. Frechet, Enzymatic microreactor-on-a-
chip: proteinmappingusingtrypsinimmobilizedonporous polymer monoliths
molded in channels of microuidic devices, Anal. Chem. 74 (2002) 40814088.
[33] D.Y. Stevens, C.R. Petri, J.L. Osborn, P. Spicar-Mihalic, K.G. McKenzie, P. Yager,
Enabling a microuidic immunoassay for the developing world by integration
of on-card dry reagent storage, Lab Chip 8 (2008) 20382045.
[34] H. Klank, J.P. Kutter, O. Geschke, CO
-laser micromachining and back-end
processing for rapid productionof PMMA-based microuidic systems, Lab Chip
2 (2002) 242246.
[35] V. Linder, S.K. Sia, G.M. Whitesides, Reagent-loaded cartridges for valveless and
automated uid delivery in microuidic devices, Anal. Chem. 77 (2005) 6471.
[36] D. Chan, L.L. Ng, Biomarkers in acute myocardial infarction, BMC Med. 8 (2010)
[37] A. Qureshi, Y. Gurbuz, J.H. Niazi, Biosensors for cardiac biomarkers detection:
a review, Sens. Actuator B 171 (2012) 6276.
Jitae Kim received a Ph.D. in mechanical & aerospace engineering from the Uni-
versity of California, Irvine (2006). His B.S. and M.S. degrees, both in mechanical
engineering, are fromHanyang University in South Korea (1999) and the University
of Southern California (2001), respectively. He is currently a research professor at
the Institute of Nano Science &Technology (INST), Hanyang University. His interests
andresearchincludedroplet-basedmicrouidics, point-of-care(POC) immunoassay
and microuidic nucleic acid testing (NAT).
Yong Shin received the M.S. degree in Cancer Biology fromSeoul National Univer-
sity, South Korea in 2005 and Ph.D. degree in Molecular Neuro-Biology from Max
Planck Institute of Experimental Medicine and Georg-August-University Goettin-
gen, Germany in 2008. He currently joined Institute of Microelectronics, A*STAR as
a Scientist, Singapore. His research is nowfocused on the development of molecular
diagnostic platform based on optical biophotonics for detection of disease related
Simon Song received his B.S. (1995) in Mechanical Engineering Department,
Hanyang University, Seoul, Korea and M.S. (1997) and Ph.D. (2002) in Mechan-
ical Engineering Department, Stanford University, USA. He joined faculties at
Hanyang University since 2004 and has interest in the development of microu-
idic chips for chemical sensing or sensor synthesis on a microchip as well as
ow visualization technology using medical equipment like magnetic resonance
Joohyung Lee received both B.S. (2009) and M.S. (2011) degrees in Biomedical Engi-
neering fromYonsei University, South Korea. He is currently a Ph.D. student in the
biomedical micro/nano device (BMND) lab at the Texas Tech University, USA. His
research activities are now focused on developing microengineered biomimetic
organ-on-a-chip and chemical/biochemical analysis platforms for environmental
monitoring and biomarker screening.
Jungkyu (Jay) Kim is Assistant Professor, Department of Mechanical Engi-
neering at Texas Tech University. His research expertise includes lab-on-a
chip devices that require a variety of microuidic components for com-
plex chemical/biomedical assays. His work has involved the development of
microuidic control system, microuidic sample processing, nucleic acid sam-
ple preparation and on-chip amplication, protein microarray, CNT biosensor
and immunomagnetic detection. He has authored and co-authored more than
50 peer-reviewed journal and conference publications, 1 book chapters, and 6
patents issued or pending in the area of microuidics, biosensor and cell/tissue