Surface Modification and Immunoassys on COC,
Cross-Flow Microfluid Channels and FRET Molecules
Young Jun Kim, Kwang Hyo Chung, Won Ick Jang,
Hye-Yoon Kim, Moon Youn Jung, Seon-Hee Park
BioMems Team, ETRI
Daejeon, South Korea
junkim@etri.re.kr
Abstract—In an effort to manufacture a high-performance
disposable microfluidic system for applications in medical
diagnostics, a series of studies were carried. Surface
modification on COC, nanoparticle-based immunoassays on
COC, manufacturing cross flow microfluidic system, synthesis
of a pair of molecules for fluorescence resonance energy transfer
(FRET) and preparation of a FRET nanoparticle are presented
in this report. Plastics are advantageous compared with the
conventional hard materials such as glass and silicon, in the
application of microfluidic devices. However most plastics lack
proper functional groups or hydrophilicity, which are essential
requirements for microfluidic devices that need various surface
chemistry. Among the plastic materials, cyclo-olefin copolymer
(COC) has been recently reported to be equipped with more
superior physical and optical properties. However since the
COC is synthesized by copolymerizing ethylene with norbornene
based on Metallocene catalyst, COC is composed only of C and
H with no unsaturation. The saturated hydrocarbon polymer
provides high optical clarity, which is also the cause for
inertness of the COC surface. In an effort to find effective
application as a microfluidic device for diagnostic purpose, we
have generated an effective functional group (-OH) on the
surface of COC by treating the COC with O2 plasma. The
surface was characterized by ESCA and the fluorescence
microscopy. We further proceeded to carry out sandwich-type
immunoassays using fluorescence nanoparticles. A series of
nanoparticle-based immunoassays were processed on a COC
plate and the results were compared with those on a glass plate.
AFP was turned out to be detected as low as a few ng/ml.
Microfluidic channels for cross reactions were manufactured.
Two types of fluidic channels were manufactured; one for
surface modification and the other immunoassays. Also a series
of fluorescence molecules were synthesized to choose an
optimum pair for FRET. The behavior of fluorescence and
FRET of the acceptor and donor dyes were studied. The future
research will concern integration of each component for a
disposable microfluidic chip.
I. INTRODUCTION
Much research has been concentrated on developing a
highly efficient diagnostic system with the advantage of facile
978-1-4244-5335-1/09/$26.00 ©2009 IEEE 1184
H2C H2C
+
metallocene
catalyst
ethylene
norbornene
COC
Figure 1. Synthesis and chemical structure of COC
handling. Recently various types of biochip are being
introduced for commercialization. Biochip is in a sense a
department store of different fields of science and engineering.
Microfluidics, detection system and surface chemistry are the
major components. In order to perform highly efficient
function, not only the integration of each component has to be
accomplished but also each constituting element should be
working effectively. Recently plastic materials are increasing
used compared with the conventional hard materials such as
glass and silicon. Plastic materials are easy to handle, its price
is relatively inexpensive, and manufacturing can be promptly
carried out.
Among them a recently developed COC is especially
drawing much attention due to some advantages over
conventional thermoplastics. Since COC is synthesized by
copolymerizing ethylene and norbornene monomers, the
resultant polymer does not contain any functional group. Since
COC is composed only of C and H atoms with full saturation,
the optical clarity is very high, its surface is resistant to
chemicals and the glass transition temperature can be easily
varied by changing the composition ratio of the two
monomers. These properties are beneficial to biochip
application. However lack of functional groups prevented
immunoassays on COC [1, 2, and 3]. A highly performing
biochip often requires covalent linkage of biomolecules on the
chip surface. However lack of proper functional groups
prevents direct installation of biomolecules through chemical
bonding. In this report we did pretreatment using oxygen
plasma followed by acid. On the oxidized COC surface a
series of surface chemistry was carried out to immobilize a
biomolecule. Also based on the established surface chemistry
a series of immunoassays were carried out resulting in a few
IEEE SENSORS 2009 Conference
 ppm/ml detection of AFP. Fluorescence nanoparticles were (A) (B) (C)
used for the purpose of enhancing detection capability.
Nanoparticles embedded with multiple dyes are advantageous
for analyzing low concentration analyte. The sandwich-type
fluorescence immunoassay with nanoparticles involves
diverse variables to be optimized for maximum result.
Previously we have reported optimum values with regard to Figure 3. Contact angle measurement of the (A) bare COC,
the reaction times fro the incubation times and particle (B) oxidized COC and (C) amine functionalized COC.
concentration.
We show a pair of dye molecules and studied their
fluorescence and FRET behavior. When a fluorescence 14000
spectrum of a donor dye superimposes with Uv-vis spectrum on glass t1=60, t2=120 min [bead] = 0.2 mg/ml
of an acceptor dye, energy may be transferred to the acceptor 12000 on glass t1=60, t2=120
on COC t1=60, t2 =60
dye. The degree of the energy transfer is highly sensitive to on COC t1=60, t2=120
the distance between the dye molecules. Also two different 10000

FL intensity [100 ms]

types of microfluidic channels for cross-flow were
manufactured. The fluidic channels will be used for surface
chemistry and immunoassays on a substrate surface.
II. RESULTS AND DISCUSSION
A. Immunoassays on COC
Sandwich-type immunoassays were carried out on a COC
plate using dye-embedded fluorescence nanoparticles. In order
to process the immunoassays we studied the surface
modification of COC. COC was pretreated with oxygen
plasma. After treating the oxidized COC with acid, 3-
aminopropyltrimethoxysilane was used to provide amine
functionality on the COC surface. Since the plasma treatment
is the first step that provides a surface that makes possible of
surface chemistry, a series of treatment experiments were
carried out in order to find out an optimum conditions for the
treatment by varying the treatment power and time. It was
observed that at least 25 Watts (W) had to be used to prepare
appreciable amount of oxidation on COC even regardless of
the time used in this experiment. The oxidation and amine
functionality was confirmed XPS (figure 2). Contact angles
measured for the COC, plasma-treated COC and amine COC
were 97°, 8° and 47° (figure 3).
32000
10000
Si 2p 30000
8000
28000
counts/s
8000
6000
4000
2000
0
0 20 40 60 80 100
AFP [ng/ml]
Figure 4. Nanoparticle-based immunoassay on a COC plate.
After confirming the success of the surface modification on
COC, anti-AFP was immobilized on COC via reacting
glutaraldehyde on the amine-functionalized COC plate. A
series of sandwich-type immunoassays were carried out using
fluorescence nanoparticles on the COC plate immobilized
with anti-AFP. First predetermined amount of AFP was
incubated with fluorescence nanoparticles functionalized on
their surfaces with anti-AFP. The solution of AFP and anti-
AFP nanoparticle was then reacted onto the anti-AFP surface
of COC. The same reactions were repeated on a glass plate in
N1s order to compare the results with those on a COC plate. As
can be seen from figure 4 as the concentration of AFP
increased the fluorescence intensity increased until 100 ng/ml

6000
counts/s

26000
4000
of AFP. Overall the fluorescence intensities were higher on
2000
24000
the glass than on the COC. A few ng/ml was able to be
22000 detected on COC
0
120 110 100 90 80 420 410 400 390 380
5
1.2x10 binding energy (eV)

O1s
1.2x10
5
binding energy (eV)
C1s
B. Cross-flow microfluidic channels
5
1.0x10
Microfluidic channels for cross-flow were fabricated using
5
1.0x10
4
8.0x10
PDMS. Two different types of PDMS casts were prepared in a
4
8.0x10
counts/s
counts/s

4
6.0x10
6.0x10
4

4.0x10
4
metal mold. Figure 5 shows the mold and the PDMS fluidic
4.0x10
4

2.0x10
4 casts for surface modification and immunoassay. A biochip
2.0x10
4
0.0 will form by placing the PDMS cast on the solid substrate.
550 540 530 520 310 300 290 280 270 260
binding energy (eV) binding energy (eV) C. FRET dyes
Figure 2. XPS of the amine functionalized COC. A pair of a donor (PCDMO) and an acceptor (NCDMO)
for FRET were synthesized in order to study the possibility of
using the dye pair for fluorescence nanoparticle.

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 (a) (b) 30000

28000

PCDMO

fluorescence intensity (a.u.)

26000
emission at 742
(c) excitation at
24000
606
638
22000

20000
Figure 5. Pictures of (a) a mold and its PDMS cast for (b)
surface modification and (c) immunoassay. 18000

16000
The absorption maxima for the donor (PCDMO) were 606 nm,
0 5 10 15 20
638 nm and 668 nm and that for the acceptor (NCDMO) were
dye concentration (mg/ml)
668 nm, 734 nm and 773 nm. The maxima for the
fluorescence spectrum of PCDMO were 674 nm, 703 nm and
744 nm. The fluorescence maxima for NCDMO were 784 nm Figure 7. Fluorescence intensities of the PCDMO at higher
and 819 nm. Since the donor’s fluorescence spectrum concentration range.
superimposes with absorption spectrum of the acceptor
molecule FRET was thought to be possible. Fluorescence
intensities of PCDMO were measured while varying the 24000
concentration. Excitations were carried out at 350 nm, 606 nm,
638 nm and 668 nm while the fluorescence intensities were 21000

measured only at 742 nm (figure 6). Fluorescence intensities

fluorescence intensity (a.u.)

were also measured at higher concentration range (figure 7). It 18000

can be observed that while at lower concentration range which

spans from 0.01 mg/ml to 10 mg/ml, generally the intensities 15000

increased, in the higher concentration range which goes upto

20 mg/ml the quenching stated to be involved at around 10 12000
excitation
mg/ml. Also by mixing the acceptor and donor dyes studied 606 nm
9000
the dependence of the FRET intensity on the concentration of 638 nm
the dye. All through the concentration range experimented, the
6000 PCMDO+NCDMO_
FRET intensity tended to increase (figure 8). The FRETs were fluorescence at 784 nm
observed at 784 nm while excitations were carried out at 606
3000
nm and 638 nm. 0 5 10 15 20
dye concentration (mg/ml)

Figure 8. Dependence of FRET from PCDMA and NCDMO

30000
on concentration.
25000
ACKNOWLEDGMENT
fluorescence intensity (a.u.)

20000 We appreciate the support from the Ministry of

Knowledge Economy (09IC1510) and the help from
15000 PCDMO HanChem in the synthesis of the dye molecules.
emmision at 742 nm
10000 excitation (nm) REFERENCES
350
606 [1] Y. J. Kim, W. I Jang, H. B. Pyo and S. H. Park, "Surface Modification
5000 638 of Cycloolefin Copolymer Based on Covalent Bond," Korea Patent
668 (0805816) (2007)
0 [2] C. Jonsson, M. Aronsson etc. “Silane-dextran Chemistry on Lateral
Flow Polymer Chips For Immunoassays”, Lab Chip, vol. 8, pp 1191
0.1 1 10
2008.
concentration of PCDMO (mg/ml) [3] Kim, H. Y. Kim, C. S. Ah, M. Y. Jung, S.-H. Park. “Some Key Factors
in a Bead-based Fluorescence Immunoassay”, Y. J. Biochip J., vol. 2,
Figure 6. Fluorescence intensities of the PCDMO at lower pp 60, 2008.
concentration range.

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