1914

IEEE SENSORS JOURNAL, VOL. 12, NO. 6, JUNE 2012

Glass-Based Continuous-Flow PCR Chip With a

Portable Control System for DNA Amplification
Ning Xue, Student Member, IEEE, and Weiping Yan

Abstract A glass-based continuous-flow polymerase chain

reaction (PCR) chip has been designed and fabricated. The device
consists of a glass microfluidic channel, three NiCr heaters, and
three Ni thermometers on the silicon substrate. An intelligent
temperature-control circuit system has been designed to achieve
desirable temperature control (95, 72, and 55 C) at the three
temperature zones of the PCR chip. Simulation underneath the
microfluidic channel using the finite element method shows that
the temperature distribution through the three temperature zones
are relatively uniform. A mixture of DNA samples for PCR was
allowed to flow through the microfluidic channel under different
flow rates. The amplified sample of the target DNA obtained
from the PCR chip was then separated by electrophoresis and
was analyzed using an ultraviolet analyzer. The result indicates
that DNA amplification can be achieved and that its amplification
factor depends greatly on the injection rate of the sample. The
optimum sample-flow rate is 0.6 l/min.
Index Terms DNA amplification, microelectromechanical
systems (MEMS), Ni thermometer, NiCr heater, polymerase chain
reaction (PCR) chip, temperature control system.

I. I NTRODUCTION

ECENTLY, miniaturization of analytical systems for

application in biomedicine and clinic diagnosis have
received much attention due to its advantages such as low
reagents consumption, short reaction time, and low cost. The
purpose of lab-on-a-chip system is to integrate the functions
of biosample preconditioning, chemical reaction, separation of
samples and detection of the constituents into a single chip.
Thus, research related to a polymerase chain reaction (PCR)
chip is a vital part for lab-on-a-chip system [1][5]. During
the PCR reaction process, DNA molecules are amplified in
the presence of an enzyme, respective primes and dNTPs
in three alternate temperature zones, consisting of a hightemperature denaturation zone (95 C), a low-temperature
annealing zone (5060 C), and a medium-temperature extension zone (72 C). Usually, PCR chips microfabricated in
two structures can carry out the amplification reaction: a
micro-chamber PCR chip and a continuous-flow PCR chip.

Manuscript received November 27, 2011; accepted December 20, 2011.

Date of publication January 3, 2012; date of current version April 25, 2012.
The associate editor coordinating the review of this paper and approving it
for publication was Prof. Elliot R. Brown.
N. Xue is with the Department of Electrical Engineering, University
of Texas at Dallas, Richardson, TX 75080 USA (e-mail:
nxx083000@utdallas.edu).
W. Yan is with the School of Electronic Science and Technology,
Dalian University of Technology, Dalian 116024, China (e-mail:
yanwp@utdallas.edu).
Color versions of one or more of the figures in this paper are available
online at http://ieeexplore.ieee.org.
Digital Object Identifier 10.1109/JSEN.2011.2182047

In micro-chamber PCR chip, DNA mixtures are reacted and

DNAs are amplified in a micro-chamber made of materials
such as silicon, glass and polymers. In the continuous-flow
PCR chip, under the action of a certain force, the PCR
mixtures flow through the serpentine channel maintained at
three different temperatures. Commonly, the channel of this
chip is designed for 1530 amplification cycles. Each cycle is
expected to finish one DNA-molecules-amplification process.
Compared to microchamber PCR chip, a continuous-flow PCR
chip has the advantage of short reaction time, and rapid and
steady temperature control. In addition, it can be integrated
with capillary electrophoresis (CE) and yield PCR-CE chips.
Table 1 lists the comparison among three types of PCR chips
in terms of the required sample volume, reaction time, cost,
and integration to other lab-on-a-chip systems [6]-[10].
Glass, poly(dimethylsiloxane) (PDMS), and silicon are
three common materials used to compose the PCR chips. Kim
developed a PDMS-based microchamber PCR chip, where
one temperature cycle was set to 70 seconds [11]. However
very few papers have reported a continuous-flow PCR chip
made of PDMS because the latters high hydrophobic nature
can impede the flow of samples in the microchannel. Silicon
is a common material used in both microelectromechanical
systems, popularly known as MEMS, and the semiconductor
field. Hence, many previous microfabricated PCR chips have
used silicon as their structural or channel material. However,
silicon has certain undesirable properties such as opacity
and least bio-compatibility which have limited its further
development as the material of choice for lab-on-a-chip
systems. Glass is an ideal material to compose lab-on-a-chip
system, because it is rigid, transparent, bio-compatible and
hydrophilic [1], [5], [12].
We introduce a glass-channel PCR chip in this study.
Based on the simulation using the finite element analysis
(FEM), a continuous-flow PCR chip has been designed and
fabricated, including a micro-reaction glass channel, heaters,
and thermometers on a silicon substrate. The three constanttemperature zones were controlled by a portable circuit system.
II. E XPERIMENT
A. Design
The microfluidic circulation channel was etched on a sodalime glass substrate. Another glass lid with the microfluidic
channel was covered on the glass substrate. Three heaters and
thermometers were fabricated on an oxidized silicon substrate.
Finally, the glass substrate and silicon chip were bonded to
form the PCR chip, as shown in Fig. 1.

1530437X/$31.00 2012 IEEE

XUE AND YAN : GLASS-BASED CONTINUOUS-FLOW PCR CHIP WITH A PORTABLE CONTROL SYSTEM FOR DNA AMPLIFICATION

TABLE I
C OMPARISON A MONG T HREE T YPES OF PCR D EVICES

1915

2.8 cm

2.7 mm
4 mm

Minimum
required
sample
volume

Reaction
time
(30 cycles)

Cost
of
device

Integration
with other
micro-chips

Conventional
PCR machine

20 l

30-60 min

High

No

Microfabricated
continuous-flow
PCR chip

< 10 l

2-30 min

Low

Easy

Microchamber
PCR chip

10-50 l

15-30 min

Low

Difficult

1 cm

NiCr heater
Fig. 3.

Ni thermometer

Schematic layout of a heater and a thermometer.

Not to scale
Cover-glass

(a)

(b)

(c)

(d)

Substrate-glass

Thermometers and heaters

Fig. 1.

Schematic view of cross-section of the PCR chip.

2.5 cm
DNA denaturation zone
1 cm

DNA extension zone

1 cm

1 cm
DNA annealing zone
Sample inlet
Fig. 2.

Sample outlet

Micro-circulation channel of continuous-flow PCR chip.

To obtain high DNA-amplification efficiency, the duration

of denaturation and annealing should be shorter, whereas the
extension time should be longer. As a result, in our continuousflow PCR chip, different lengths of the microreaction channel
were designed in each of the temperature zones. In addition,
to ensure DNA strands fully unfastened, the DNA should be
preheated before the DNA amplification starts. This process
can be achieved by extending the length of the denaturation
channel before the first circle.
A micro-channel with 20 cycles was designed in the PCR
chip (Fig. 2). The length ratio of the channels with three
different temperature zones is 2:2:5, and the total length of
the microchannel is approximately 1.9 m. The width and
depth of the microchannel are 120 and 30 m, respectively.
Moreover, to have a smooth liquid flow, the bending area of
the channel was designed in an arc. The three temperature
zones are marked in Fig. 2.
As a heater, a NiCr thin film has relatively high heating
efficiency and good stability, whereas a Ni film is used
as the material for temperature sensor because of its high

Fig. 4. Thermal distribution analysis of continuous-flow PCR chip. Thicknesses of glass are (a) 0.6 mm, (b) 1.4 mm, and (c) 2.2 mm. (d) Cross-sectional
thermal distribution of 1.4-mm-thick glass.

temperature coefficient of resistance (TCR) [13]. Three heaters

were designed in a serpentine shape to enlarge the heating
area and increase the resistance. To maintain the accuracy of
temperature measurement, three thermometers were placed at
the center of each heater. The design of one pair of heater and
thermometer is shown in Fig. 3, in which the trace width of
the NiCr heater is 400 m and the gap between two traces of
the heater is 300 m; similarly, the trace width is 75 m and
gap is 100 m for the Ni thermometer.
B. FEM Simulation
In our PCR chip, the heaters and thermometers are underneath the glass substrate, thus a uniform thermal distribution on the surface of the channel is the main concern.
Thermal distributions of the glass substrate with different
thicknesses (0.6, 1.4 and 2.2 mm) were studied using the
ANSYS software. Three different temperatures (95, 72, and
55 C) were applied at the three bottom zones of the glass
substrate. The simulated thermal-distribution profiles on the
surface of the channel are depicted in Fig. 4 (a through c).
Nonuniformity of thermal distribution for the 2.2-mm-thick
glass obstructs the performance of the PCR process; whereas
0.6 and 1.4-mm-thick glasses are suitable for use because of
the observation of uniform thermal distribution on the surface
of the channel. Moreover, a temperature difference exists
between the channel and the heater. A cross-section view of
the thermal distribution across a 1.4-mm-thick glass substrate
is shown in Fig. 4d, showing that an average difference of
2 C exists between the heater and the surface of channel.

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IEEE SENSORS JOURNAL, VOL. 12, NO. 6, JUNE 2012

Mask
Photoresist
Cr
Glass

SiN
SiO2
Silicon

(a)
(f)
NiCr Ni NiCr

(b)
(g)
(c)
Glass lid
Glass substrate

(d)
Glass lid
Glass substrate

(e)

(h)

Not to scale

Fig. 5.
Fabrication process of continuous-flow PCR chip. (a)(e) Glass
fabrication: (a) Cr patterning, (b) Cr etching, (c) glass etching, (d) protection
layers removal, (e) thermal bonding of two glasses to form sealed channel.
(f)(g) Silicon fabrication: (f) SiN deposition, (g) Ni thermometer and NiCr
heater patterning. (h) Bonding of glass and silicon.

Photograph of packaged PCR chip.

Amplifier and
A/D
LCD display

95 C

700
600
Temperature (C)

Fig. 7.

Micro
processer

72 C

500
55 C

Keypad control

400
PE coupler
and amplifier

100

Fig. 8.

Diagram of temperature-detection-control system of PCR chip.

0
0

Fig. 6.

100

200

300 400 500

Time (min)

600

700

Temperature curve for glass thermal bonding.

Accordingly, this temperature difference must be considered

in the design of the temperature control system. We chose
1.4-mm-thick glass because it is a commercially available
glass product and has good uniformity of thermal distribution
in the channel.

silicon wafer that is used as the electrical isolation layer.

Subsequently, thin films of NiCr and Ni thin films were
deposited and patterned as the heater and the thermometer,
respectively. Finally, the three pieces of heaters and
thermometers were attached to the bottom of the glass
substrate by thermally conductive glue. The photograph of
a packaged fabrication device is shown in Fig. 7, where the
pads of the heaters and thermometers are connected to the
pins on the printer circuit boards.

C. Fabrication

D. Temperature-Control System

A 1.4-mm-thick glass, coated with 145-nm-thick Cr /

570-nm-thick photoresist, was used to fabricate the
micro-circulation channel. The fabrication process of
the micro-circulation channel is shown in Fig. 5. First, the
photoresist was patterned, followed by Cr-etching. A 30-mthick glass channel was then anisotropically etched using
HF:HNO3 :H2 O = 5 ml:10 ml:80 ml, with an etching rate of
0.5 m/min. Another unprocessed glass was used as the lid;
3-mm-diameter inlet and outlet holes were drilled through the
glass lid using an ultrasonic driller. To seal the microfludic
channel, the two glass plate were placed in contact with each
other with a heavy load on top and were thermally treated in
a programmable air convection oven. And the temperaturecontrol curve is shown in Fig. 6. Uniform firm bonding can
be achieved after 34 cycles of temperature control.
For the silicon fabrication process, a 170-nm-thick layer
of silicon nitride was deposited on a 2-m-thick oxidized

The hardware temperature-control system was designed to

implement the function of temperature sensing and temperature control. To achieve rapid and stable constant-temperature
conditions (95, 72, and 55 C) at the various temperature
zones, a proportional-integral-differential (PID) algorithm was
adopted to calculate the heating time, while the analysis of
the complete dataset was accomplished in the microprocessor.
The diagram of the hardware system is presented in Fig. 8,
and it mainly includes temperature data-collection module
(Wheatstone bridge, voltage amplifier, and analog-to-digital
converter) to acquire temperature information from the thermometers, 8052 microprocessor, power output-driving module
(photoelectric coupler and power amplifier) that turns on/off
the heaters voltage, keypad control, liquid crystal display
(LCD) display, and RS232 communication module. Finally,
this control circuit was connected to the personal computer
(PC) interface for real-time temperature monitoring.

XUE AND YAN : GLASS-BASED CONTINUOUS-FLOW PCR CHIP WITH A PORTABLE CONTROL SYSTEM FOR DNA AMPLIFICATION

1917

140
Data window

T/C
120
110
100
90
80
70
60
50
40
30
20
10
00

Temperature (C)

120
100
80

5
Time/min

60
40

Fig. 11. PCR chip testing setup with control circuit and PC interface control.

20
0

10

Fig. 9.

Resistance (k)

95 C
72 C
55 C

20

30
Voltage (V)

40

50

Curve of voltage-temperature of NiCr heater.

0.70
0.68
0.66
0.64
0.62
0.60
0.58
0.56
0.54
0.52
0.50
0.48

Resistance 1
Resistance 2
Resistance 1 36 h later
Resistance 2 36 h later

Fig. 12. Electrophoresis diagram of amplified DNA samples under different

flow rates (a) 7.6 l/min, (b) 3.1 l/min, (c) 1.2 l/min, (d) 0.6 l/min.
(Bars in column 1 presents the standard plant DNA, bars in columns 2 and 3
presents the PCR results by conventional PCR machine and microfabricated
continuous flow PCR chip, respectively.)

20

40

60
80
Temperature (C)

100

Fig. 10. Resistance response of Ni thin film as a function of temperature

and time.

III. R ESULTS AND D ISCUSSION

A. Characterization of Heater and Thermometer
An excellent heater should maintain a stable resistance even
under changing of temperatures. Lower power input to obtain
a high temperature is also preferable. We have proved that the
resistance of the thin film of NiCr alloy can remain constant
even under conditions of variable temperatures [13] - [14]. The
curve of voltage-temperature of the fabricated NiCr heater in
the PCR chip is shown in Fig. 9. Within the voltage range
of 545 V, the corresponding temperature range is 2595 C.
Because the highest temperature is 95 C for the PCR chip,
the fabricated NiCr film can satisfy the requirement as a heater
in a PCR chip.
The sensitivity of the thermometer corresponds to the slop
of the resistance-temperature curve. From Fig. 10, the resistance of the Ni thin film has a linear temperature response,
and its TCR is 4.0103/C.
B. System Setup
The system setup is presented in Fig. 11. Saline (0.9 %
NaCl) solution was injected into the inlet of the microchannel
using a syringe pump, and the flow rate was set to 1 l/min.
The temperature values of the three zones (55, 72, and 95 C)
were preset through the PC program. As the program started,

the preset temperature data were sent to the microprocessor

for analysis and the heating intervals of the three heaters
were calculated from the PID control program. In Fig. 11,
the window of real-time monitoring from the PC program
shows that the response time of the heater was approximately
20 seconds. The control circuit can maintain the temperature
sufficiently stable, with a small variation of 1 C during the
sample flow.
C. In Vitro Testing
To verify the feasibility of DNA amplification in our
continuous-flow PCR chip, 2 L of plant DNA, 5 L of forward primer, 5 L of reverse primer, 8 L of dNTP, 10 L of
phosphate buffer solution, 0.5L of TaKaRa EX Taq enzyme
and 69.5L of distilled water were mixed to prepare a DNA
mixture sample. The sample flowed through the fabricated
PCR chip, and the temperatures were controlled at constant
levels. Meanwhile, the above DNA mixture sample was amplified 30 cycles in a commercial PCR thermal cycler machine
(TP-600, Dalian, China), as a control. After the PCR process,
both the DNA analytes were subjected to electrophoresis under
an electric field of 2 V/cm in an electrophoresis apparatus (30
III, Beijing Liuyi Instrument Factory, China) to separate the
DNA molecules in the mixture. Finally, an ultraviolet analyzer
(JY02S, Junyi-Dongfang Electrophoresis Equipment, China)
was used to obtain the electrophoresis diagram. The results of
DNA amplification under different sample flow rates compared
with the DNA amplification result from a conventional PCR
machine are shown in Fig. 12. In Fig. 12, Column 1 shows the
different standard plant DNA samples; Column 2 contains the
DNA-separation result from the commercial PCR machine,

1918

IEEE SENSORS JOURNAL, VOL. 12, NO. 6, JUNE 2012

and Column 3 presents the DNA-amplification result obtained

from our PCR chip. It verifies that our microfabricated
continuous-flow PCR system can accomplish amplification
of the DNA samples. Furthermore, the figure shows that a
slower sample flow rate can enhance the efficiency of DNA
amplification. The optimum flow rate is 0.6 l/min.
IV. C ONCLUSION
Based on FEM analysis of the thermal distribution over the
surface of a microfluidic channel, a glass-based continuousflow PCR chip was successfully designed and fabricated.
A portable temperature-control/detection circuit was also
designed based on a 8052 microprocessor. The temperature
variation was only 1 C during sample injection. Finally, the
DNA-amplification performance of the fabricated PCR chip
was established. The sample of DNA mixture flows through
the PCR chip at different flow rates. The result verifies the
feasibility of DNA amplification by our continuous-flow PCR
device, which is of great importance for the future study of a
PCR-CE system.

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Ning Xue (S11) received the B.S. and M.S. degrees in electrical engineering
from the Dalian University of Technology, Dalian, China, in 2005 and
2008 respectively. He has worked in the bioMEMS microfluidc devices for
his master research. He is currently pursuing the Ph.D. degree with the
Department of Electrical Engineering, University of Texas, Dallas.
He joined Micro-Nano Devices and Systems Laboratory, Microelectromechanical Systems (MEMS) Research Group, University of Texas at Dallas,
Richardson, TX, in 2009. His current research interests include MEMS fabrication, microfluidic device, bioMEMS for wireless implantable applications,
pressure sensors, and radio frequency (RF) MEMS of on-chip inductor and
capacitor.

Weiping Yan received the M.S. degree in physics and devices of semiconductors from Jilin University, Changchun, China, in 1975 and 1989, respectively.
She was with the Department of Electronic Science, Jilin University, from
1975 to 1990. She has been with the Department of Electronic Engineering,
Dalian University, Dalian, China, since 1990. Her current research interests
include the fabrication of the integrated biochip, design, and fabrication of
semiconductor sensors.