Biomedical Microdevices 7:3, 253–257, 2005


C 2005 Springer Science + Business Media, Inc. Manufactured in The Netherlands.
Micro Flow-through PCR in a PMMA Chip Fabricated
by KrF Excimer Laser∗
Abstract. As the third PCR technology, micro flow-through PCR
chip can amplify DNA specifically in an exponential fashion in vitro.
Nowadays many academies in the world have successfully amplified
DNA using their own-made flow-through PCR chip. In this paper,
the ablation principle of PMMA at 248 nm excimer laser was stud-
ied, then a PMMA based flow-through PCR chip with 20 cycles was
fabricated by excimer laser at 19 kv and 18 mm/min. The chip was
bonded together with another cover chip at 105◦ C, 160N and 20 min-
utes. In the end, it was integrated with electrical thermal thin films
and Pt100 temperature sensors. The temperature controllers was
built standard PID digital temperature controller, the temperature
control precision was ±0.2◦ C. The temperature grads between the
three temperature zones were 16.5 and 22.2◦ C respectively, the gaps
between the temperature zones could realize heat insulation.
Key Words. excimer laser, PCR, chip, hot bond
1. Introduction
PCR was a perfect DNA amplification technology in an
exponential in vitro. Which became one of the important
technologies for gene identification and gene diagnostic
during the last decade. Micro flow-through PCR was the
third PCR technology. According to Table 1, It’s advan-
tage relative to the conventional thermocycler device and
the micro chamber PCR was that it could realize a very
fast and continuous PCR and can amplify various DNA
templates of different sources and properties.
In 1998, the first micro flow-through PCR was invited
by Martin et al., 1998. Nowadays several institutes in
the world have also finished DNA amplification through
the micro flow-through PCR in silicon or glass chip. In
Schneegaß et al. (2001), a house keeping gene with 379
base pairs and a zinc finger protein relevant in human
pathogenesis with 700 base pairs were amplified suc-
cessfully by the IPHT lab through a micro flow-through
PCR in a silicon chip. The typical residence time inside
this 25 cycles system amounted to less then half an
hour. In this year, the whitehead lab (Chiou et al., 2001)
amplified 500 pairs template through 30 cycles in 23
min with 78% amplification efficiency by a closed-cycle
capillary PCR machine. In 2002, the department of
ecosystem engineering of Tokushima University (Suna
et al., 2002) completed an amplification of a 450 bp
segment of Escherichia coli HB101 through a Quartz
Liying Yao, Baoan Liu, Tao Chen, Shibing Liu, and
Tiechuan Zuo
Beijing Institute of Technology, Beijing, China
E-mail: lyyao@emails.bjut.edu.cn, liubaoan@emails.bjut.edu.cn,
chentao@bjut.edu.cn, liushibing@bjut.edu.cn, nclt@bjut.edu.cn
glass micro flow-through PCR chip. An ITO thin film
was deposited on the etched chip and as a thermal source,
the temperature distribution on the ITO thin film was
uniform (within ± 2◦ C) under two flow rates (56 and 152
nl/min). In this year, the semiconductor optoelectronic
lab (Zhang et al., 2002) of Zhejiang university realized
PCR reaction using a silicon–glass-bonding chip.
Although the continuous flow-through PCR has
achieved great progress during the last 6 years, it has
the following disadvantages:
1 most PCR adopted sili-
con or glass as the substrates, so the fabrication progress
of the standard micromachining techniques was complex
and expensive.
2 the thermal source and the temperature
was integrated together with the micro flow-through PCR
chip that increased the cost. In this paper, a cheap PMMA
was chosen as the micro flow-through PCR substrate, and
the excimer laser with 248 nm wavelength was used as the
fabrication method. A cheap electrical thermal thin film
was adopted as the heat source, and it was integrated with
the Pt100 temperature sensor.
2. Experimental
2.1. Construction of the PCR chip system
The PCR chip system consistsed of two parts. The one
was the reaction channel etched into a PMMA chip and
bonded with a cover chip. and the other was the tem-
perature control system integrated with electrical thermal
thin films and Pt100 temperature sensors. Figure 1 was
the schematic of the micro flow-through PCR device. The
yellow zones were the electrical thermal thin films. Three
Pt100 temperature sensors were glued at the bottoms of
three yellow zones. The white zones between the two yel-
low zones were thermal gaps, these were used to insulate
the individual temperature zones from each other.
The chip was designed with channel loops for 20 am-
plification cycles. The outer dimensions of the chip was
40 mm × 63 mm. The chip was fabricated by the ex-
cimer laser with 248 nm wavelength. The channels and the
∗ National Natural Science Foundation of China (Grant No.50175002),
and Beijing (Grant No. 3031001).
253
254 Yao et al.

Table 1. Comparison of three types PCR devices tion at the emission wavelength of KrF excimer laser, so it
through
could be effectively ablated through the laser fabrication
PCR Type Conventional Micro chamber Micro flow system (Figure 3). In this paper, the ablated rule and prin-
ciple of PMMA by KrF excimer laser was researched, then
Reactor types tube Static chamber Micro channel the channel was ablated under proper laser parameters.
Reactor volume Several Several 10 Several to
(µl) Several Several to
One circle time 5 near 1 Less than 1
(Min) 2.2.1. Ablated rule and principle of PMMA. The ab-
Amplification Very good good good lated rule and principle of PMMA was associated with
efficiency
For various
its absorption at 248 nm, so the transmittance of PMMA
√ √ √ was measured in Figure 4. Its transmittance was near zero
DNA template
when the wavelength was less than 300 nm. The absorption
coefficient α of PMMA at 248 nm was about 2.48 µm−1 , so
thermal gaps were etched into the PMMA chip (Figure 2) PMMA can be easily ablated by 248 nm excimer laser. In
using laser direct-write method. The chip was made up of this experiment, a Lamda physik LPX305iF type excimer
an inlet, an outlet, the denaturation zone (94◦ C), the primer laser was used. Its emission wavelength was 248 nm, its
extension zone(72◦ C) and the annealing zone (55◦ C). the maximum pulse energy was 1.2 J, its maximum reple-
chip channel had a width of 0.1 mm, a depth of 0.05 mm, tion frequency was 50 Hz, its facular size was 36 mm ×
a length of 1324 mm, and a total volume of 6.62 µl. 16 mm,its area was 5.76 cm2 , an objective lens with 60 mm
focus was adopted. The objective distance was 660 mm,
and the image distance was 66 mm. A square pattern (side
2.2. Fabrication = 1 mm) was adopted, the side size of the pattern on
PMMA was an important substrate material for biochip workpiece was 104 µm×104 µm so the area of the pat-
(Becker and Locascio, 2002). It had significant absorp- tern on workpiece was 1.08 × 10−4 cm2 . According to the

Fig. 1. The schematic of the micro flow-through PCR device.

Fig. 2. The top view of the micro flow-through PCR chip with fluidic channel layout for 20 PCR-cycles and the individual temperature zone insulated
by thermal gap.
 Micro Flow through PCR in a PMMA Chip Fabricated by KrF Excimer Laser 255

Fig. 3.1. Shock absorber 2 assistant gas 3 laser inlet 4 attenuator 5 Eye fly 6 He-Ne laser 7 collect lens 8 mask 9 fiber lamp 10 objective lens 11 CCD
camera 12 ocular 13 workpieces 14 XYZ control table 15 PC 16 workpiece monitor 17 control box.

Fig. 4. The transmittance curve of PMMA from 190 nm to 1100 nm. Fig. 5. The albation curve of PMMA at different fluence.

functions: F/F ∗ = S ∗ /S, F ∗ = E ∗ /S ∗ . (F is the fluence In the equation(1), dphoto was the photo degradation
at workpiece, S is the area of pattern on workpiece, F ∗ etch rate per pulse , α was the absorption coefficient, Fth
is the light source fluence, S ∗ is the area of the original was the threshold fluence.
facular size, E ∗ is the source light energy.), the Table 2
2 Thermal degradation model (Srinivasan, 1986).

gives the pattern depth in PMMA at different F ∗ and F,
and the ablated principle of PMMA at 248 nm excimer
laser was described in Figure 5.
The ablated rule and principle of the PMMA by excimer
laser was complex. There were several theory models.

[7]
Photo degradation model . When the F was bigger than
Fth and was during some fluence range, the relationship
between etch rate and F followed Beer’ law:
When the F was large enough, the relationship between
etch rate and F followed the nether equation:
dthermal = k0 exp(E ∗ /RT ) (2)
1
In the equation (2), dthermal was the thermal degradation
etch rate per pulse, k0 was the experiment thermal etch rate
coeffient, E ∗ was the active energy, R was the gas constant,
T was the average temperature of the radiated area.

3 SSB model (Srinivasan, 1986). During the whole

dphoto = α −1 ln F/Fth (1) fluence range, the relationship between etch rate and F

Table 2. The ablated rule of PMMA at different voltage of KrF excimer laser

Voltage (Kv) 16 17 18 19 20 21 22 23

E (J) 0.295 0.390 0.486 0.560 0.700 0.830 0.954 1.000

F* (J/cm2 ) 0.051 0.067 0.084 0.097 0.121 0.144 0.165 0.173
F (J/cm2 ) 6.273 8.241 10.332 11.931 14.883 17.712 20.295 21.279
Depth (µm/pulse) 3.248 3.105 3.092 3.113 3.016 2.898 2.896 2.893
256 Yao et al.
Fig. 6. (a) 3D image of circle pattern in PMMA at 23 Kv (b) the pattern section in PMMA at 23 kv (c) roughness of the bottom surface at 23 Kv.
included two parts: photo degradation and thermal degra-
dation.
dtotal = dphoto + dthermal

 

1 F E∗ F
= ln + k0 exp − ln
α Fth Fα Fth
The three model and the experiment’s curves were
given in Figure 5. According to reference (Srinivasan
et al., 1986), when the PMMA interacted with 248 nm
excimer laser, the constants in Equation (1)–(3) were:
α = 2.48 µm−1 , Fth = 0.1 J/cm2 , k0 = 7.7 µm, E ∗ /α =
1.3 J/cm2 . According to Figure 5, when the F was big-
ger than 2 J/cm2 , photo degradation dominated the etch
process. With the increase of F, the thermal degradation
held the etch process, the electrons in excited state ab-
sorbed the energy of incidence photons and changed the
energy to irradiated area vibration heat. Which led to the
temperature of the irradiated area rising sharply and bulk
expanding and forming explosion. The explosion removed
the surface material and induced temperature decreasing
quickly. In this experiment, the etch process was dom-
inated by thermal degradation. When the F was larger
than 20 J/cm2 , the photo degradation would dominated
the etch process again. The slope changed greatly when
the F was less than 6 J/cm2 . Although the experiment curve
overlapped some part of SSB model curve, we found that
it didn’t meet with SSB model ultimately, because the
visible plume absorbed some incidence fluence and de-
creased the F. We also found that the experiment curve
met the plume model curve relative to the Photo, thermal
and SSB modal curve. The plume model (Dyer 2003) was
expressed as the follow:
dplume = µ/(α · µ p ) ln[1 + µ p /µ(F/FT − 1)]
In the equation (4), µ = α/ρ and µ p = α p /ρ p were
mass absorption coeffients for the solid polymer and
plume, respectively. The etch rate per pulse was depen-
dent on whether the plume was less (µ p < µ) or more
(µ p > µ) absorbing than the solid. The detailed infor-
Table 3. The excimer laser parameters for fabrication of PCR chip
Velocity of X Repetition Etch
Laser Voltage F and Y axis frequency rate
parameters (Kv) (mJ/cm2 ) (mm/Min) (Hz) (µm/Pulse)
19 11.931 18 30 3.1
(3)
mation can be found in reference (Dyer, 2003). Figure 6
showed the 3D image and the bottom surface of circle
pattern in PMMA at 23Kv. The roughness sq (sq is root-
mean-square deviation) of the bottom Surface was less
than 0.5 µm. Through the research, a PMMA based flow-
through PCR chip with 20 cycles was fabricated by ex-
cimer laser at19kv and 18 mm/min. the laser parameters
were shown as Table 3.
3. Bonding
The micro flow-through chip with 20 amplification cycles
was bonded together with a cover chip using hot press fit-
tings. The bonding devices included two parts: a DH-101-
OS type electricalthermal constant temperature deciccator
and a spring depressor. The deciccator could offer a con-
stant temperature condition, and the depressor could pro-
vide a constant pressure. In order to bond the micro flow-
through chip with the cover chip together, the influences
of temperature, constant temperature time and pressure
were discussed. Through experiment, the most optimized
bond conditions were: 105◦ C,160 N and 20 minutes. The
seal degree of the bonded PCR chip was checked by KFC
molecular pump, the results were shown in Figure 7. In
Figure 7, (a) was the bonded microchannel section with
width 104 µm and depth 56 µm; (b) was the bottom sur-
(4)
face of the bonded microchannel, the surface presented a
wave shape. Because the excimer laser was pulse laser,
that led to the wave surface.(c) was the bonded 20-cycle
PCR chip, it had good seal character and could endured
0.85 Mpa pressure. It could meet the pressure requirement
(0.1 Mpa) for PCR reaction.
 Micro Flow through PCR in a PMMA Chip Fabricated by KrF Excimer Laser
Fig. 7. (a) The bonded microchannel section (b) the bottom surface of the bonded microchannel (c) the bonded 20-cycle PCR chip.
Fig. 8. The temperature rise curves of the three different temperature
zones on the copper flake.
Fig. 9. The rmographic image showing the three different temperature
zones on the copper flake.
4. Temperature Analysis
The practical temperature control system(TCS) included
three temperature zones. There was a copper flake between
the PCR chip and the electrical thermal thin films. It was
257
used to uniform the three different temperature zones of
the chip. The temperature rise curves of the three zones
were shown in Figure 8, they all attached constant tem-
perature in 5 minutes, the speed of temperature rise was
23◦ C/Min. The surface temperature field distribution of
the three zones was imaged using an infrared camera (Fi-
ure 9). The temperature grads between the three temper-
ature zones were 16.5◦ C and 22.2◦ C respectively in Fig-
ure 9 from the top to down. The temperature wave in the
three temperature zones was less than ±0.2◦ C. So the TCS
could meet the requirement for PCR reaction.
5. Conclusions
In this paper, the ablation principle of PMMA was dis-
cussed. Then a new type PMMA based flow-through PCR
chip was fabricated by 248 nm excimer laser. It could be
bonded together with another cover chip at 105◦ C,160N
and 20 minutes.It was integrated with electrical thermal
thin films and could realize heat insulation between the
three temperature zones. As a perfect DNA amplification
technology, we hope it could realize commercial early
through our efforts.
References
M.U. Kopp, A.J. de Mello, A. Manz, et al., Science 280, 1046(1998).
I. Schneegaß, R. Bräutigamb, and J. M. Köhlera, Lab on chip 1, 42
(2001).
J. Chiou, P. Matsudaira, Ain Sonin, et al., Anal. Chem 73, 2018(2001).
K. Suna, A. Yamaguchi, Y. Ishida, et al., Sensors and Actuators B84,
283(2002).
Q. Zhang, W. Wang et al., Sensors and Actuators B82, 75 (2002).
H. Becker and L.E. Locascio, Talanta 56, 267 (2002).
V. Srinivasan, M.A. Smritic, and S.V. Babu, J. Appl. Phys 59, 3860
(1986).
P.E.Dyer, Appl.Phys.A77,167(2003)