Chemical Engineering & Processing: Process Intensification 189 (2023) 109394

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Chemical Engineering and Processing - Process

Intensification
journal homepage: www.elsevier.com/locate/cep

Development of a disposable and easy-to-fabricate microfluidic PCR device

for DNA amplification
Hirad Mashouf a, Bahram Talebjedi b, Nishat Tasnim a, Maia Tan b, Sahar Alousi b,
Sepideh Pakpour b, Mina Hoorfar a, *
a
Department of Mechanical Engineering, University of Victoria, Victoria, Canada
b
School of Engineering, University of British Columbia Okanagan Campus, Kelowna, Canada

A R T I C L E I N F O A B S T R A C T

Keywords: The production of microfluidic PCR devices within lab settings in a straightforward process and large numbers is
Microfluidic PCR limited due to complex methods of fabrication and high cost of development. In this study, we developed a
Lambda (λ) DNA amplification unique disposable, low-cost, and easy-to-fabricate microfluidic PCR chip, comprised of a passive micromixer,
Simple fabrication
reaction well, microheater, and a dynamic temperature control system. In addition to being compact, this device
Low-cost PCR chip
has the advantages of microfluidic PCR systems, such as low power and sample consumption, as well as being
disposable microfluidic device
disposable and easy to fabricate. The disposable part of the chip is easy to fabricate and eliminates the run-to-run
carryover contamination while its integration with a reusable heating system reduces the cost of producing a
micro PCR device. The geometry of the micromixer and the pattern of microheater electrodes are analyzed
through experiments to achieve comparable amplification to a conventional thermocycler. The temperature
control system is designed to monitor the temperature within the reaction well with ±1 ◦ C precision. Finally, the
reliability and repeatability of the microfluidic device were verified by amplifying lambda (λ) DNA. The pro­
posed device demonstrated high-quality amplification comparable to the conventional PCR and is an adaptable
and low-cost on-chip PCR device for reliable molecular diagnostics.

1. Introduction introduced to make use of microfluidics in the fields of biomedicine and

Infectious disease detection is quite challenging in the early stages
since the pathogenic genetic material is present at low concentrations in
biological samples. Polymerase chain reaction (PCR) based molecular
diagnostics enable fast and accurate detection of infectious disease
agents [1–3]. To carry out the PCR reaction, the mixture undergoes
thermocycling process consisting of periodic heating and cooling be­
tween constant setpoints specified by a particular PCR protocol. Ther­
mocyclers control the dwell time and temperature of each step according
to the protocol. To develop accurate point-of-care PCR-based molecular
diagnostics, it is necessary to manufacture compact, low-cost, and
easy-to-operate thermocyclers [4,5].
Conventional thermal cyclers are bulky, expensive, energy-intensive
and require sample preparation [6,7]. Microfluidics PCR devices offer
several significant advantages over traditional PCR devices including a
high surface to volume ratio, reduction in the quantity of reactants
consumed, and high controllability [8–11]. The development of micro­
fluidic PCR equipment began since the idea of a lab-on-a-chip was
chemistry. With microfluidics PCR devices, PCR reactions can be per­
formed with much smaller reaction volumes, saving reagent costs and
reducing waste [12,13]. As a result of their smaller reaction volumes and
their ability to heat and cool small volumes of liquid rapidly, micro­
fluidic PCR devices can achieve faster reaction times. It is possible to
improve the efficiency of PCR reactions with microfluidics devices by
controlling the reaction conditions more precisely and by minimizing
the effects of thermal gradients and diffusion. One of the main advan­
tages of the suggested device over previously reported ones is its capa­
bility in large scale production without needing the industrial
techniques and equipment. As an attempt in production of micro PCR
devices with a low power consumption and resource settings, Karprou
et al. [14] introduced a fast, continuous-flow μPCR device with inte­
grated resistive microheaters. The device was fabricated using
PCB-compatible, industrial materials and processes, all on the same PCB
substrate. In another study, Moschou et al. [15], introduced a DNA
amplification featuring the integrated heating elements and was fabri­
cated on a commercially available thin polymeric substrate (Pyralux®

* Corresponding author.

https://doi.org/10.1016/j.cep.2023.109394
Received 28 January 2023; Received in revised form 4 April 2023; Accepted 21 April 2023
Available online 22 April 2023
0255-2701/© 2023 Elsevier B.V. All rights reserved.
H. Mashouf et al.
Polyimide). The suggested chip had a low power consumption and fast
amplification rates due to the small thermal mass of the chip and the low
thermal diffusivity of the polymeric substrate where the heating ele­
ments are located. The proposed device can be produced in lab settings
in large numbers without complex fabrication procedures.
Microfluidic PCR devices can be categorized into two groups: space
domain and time domain PCR devices [16,17]. In space domain PCR, the
sample is circulated through various predetermined temperature zones
[18–20]. Conversely, samples are injected and kept still in a reaction
chamber while a heating system cycles the temperature in time domain
PCR devices [21–25]. Controlling the sample in space domain PCR is
challenging due to the constant movement of the sample, requiring
frequent protocol adjustments. In contrast, time domain PCR allows for
straightforward adjustments to dwelling time and temperature set­
points. Furthermore, conducting PCR using different volumes of sample
is much simpler to achieve in time domain devices. Additionally, space
domain PCR requires more equipment and complex channel designs,
making it more expensive and challenging to implement. Due to its
multilayered design, our device is simple and low-cost, requiring no
complex channel designs or microvalves. If the device were to be clas­
sified as a space domain PCR, it would likely require microvalves.
Nevertheless, there are still concerns about the cost and complexity of
the fabrication process that make these chips inaccessible for large-scale
PCR testing [26–29].
Although a wide variety of highly sophisticated chips are increas­
ingly being demonstrated, few are or can be adopted by the market and
reach the end-users, which could revolutionize healthcare, biology, and
chemistry. Commercial manufacturing technology lacks a strong foun­
dation for large-scale production [30]. To develop low-cost and
low-power microfluidic PCR devices, the temperature control unit of
must have low fabrication cost and complexity. Previous studies have
reported different types of external heat sources such as
temperature-controlled blocks [31], Peltier elements [32], and infrared
lamps [33]. These methods have considerable heat capacity, high power
consumption, increase the size of the device and need bulky and
expensive external components, which are not suitable for developing a
low-cost and compact microfluidic PCR device. Nonetheless, thin film
resistance heaters achieve fast heating/cooling rates and effective power
consumption because they have low thermal masses and can be fabri­
cated near the reaction chamber. Platinum [34–36], gold [37,38],
Aluminum [39], indium tin oxide (ITO) [40], and several thin film
heaters have been patterned on microfluidic chips for PCR applications.
Thin film Platinum has been widely used as a heater due to its high
temperature coefficient of resistivity and linear behavior, while the low
resistance of gold makes it most suitable to be utilized as electrical leads
[41]. In microfluidic chips, it is possible to combine the heater and
sensor into a single component. Linear behavior and high sensitivity are
desired characteristics in temperature sensors. Platinum’s stability and
high resistance allows it to be simultaneously used as thin film for
heating and temperature sensing in microfluidic devices. In these chips,
sensors are usually narrow and small, with higher resistance compared
to the thin film heater to have a fast thermal response [41–43]. Never­
theless, platinum is expensive, and its complicated and dangerous
fabrication procedure involving the lift-off process [44] and wet etching
in toxic aqua regia [45] complicates its incorporation in microfluidic
PCR devices. In a study conducted by Kaprou et al. [46], the authors
introduced a static μPCR devices with resistive microheaters integrated
on PCBs as miniaturized thermocyclers for efficient DNA amplification.
The performance of their device was compared to that of conventional
thermocyclers in terms of amplification efficiency, power consumption,
and duration. The results showed that PCB-based miniaturized ther­
mocycling achieves faster DNA amplification with significantly smaller
power consumption while exhibiting similar efficiency to conventional
thermocyclers. Simulations are used to guide the design of such devices
and propose methods for further improvement of their performance.
Our proposed device employs gold electrodes etched on glass as a
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Chemical Engineering and Processing - Process Intensification 189 (2023) 109394
micro heater unit. The designed temperature control system with NTC
thermistor in this study can perform PCR at any temperature inside the
ordinary working range of PCR protocols. To further reduce energy re­
quirements, the design employs a passive micromixer with channel ge­
ometry that relies on molecular diffusion to efficiently mix PCR
reagents. To prevent washing the reaction chamber after each use or
disposing the chip as whole, our developed microfluidic device is
composed of a reusable and a disposable section. The disposable section
is replaced with a new one upon completion of each PCR reaction. All
components are integrated in a layered design with the aim to reduce the
fabrication cost and eliminate the chance of sample-to-sample cross-
contamination. The temperature control unit is programmed to main­
tain temperature with less than 1 ◦ C error inside common working range
of PCR protocols. The performance of the microfluidic device is verified
by amplifying lambda DNA (λ-DNA) samples.
2. Methodology
2.1. Working principle
The proposed compact and easy-to-fabricate microfluidic PCR device
is developed by integrating several components to create separate
disposable and reusable elements. Fig. 1b illustrates an exploded view of
the microchip comprising a micromixer, reaction chamber well, cover­
slip, microheater, and the temperature control system. Thin film resistor
acting as the microheater electrode is patterned on a glass substrate
while the temperature sensor is attached to backside of it forming the
reusable part of the device. Polydimethylsiloxane (PDMS) is the material
used for creating micromixer and the reaction well. PDMS is a well-
known biocompatible material for biological applications and has
been commonly used for the fabrication of reaction chambers. It has
good light permeability, adheres firmly to flat surfaces, and is suitable
for mass production [47,48]. These two components are fabricated via
soft photolithography and then bonded onto a coverslip.
Assembling the disposable part on a coverslip eliminates sample-to-
sample contamination while channels and reaction chambers in micro­
fluidic devices, which are non-disposable are often washed upon PCR
completion. Therefore, reagents or washing liquids may be left in
channels or absorbed by PDMS due to their porous structure leading to
distortion in the next amplification process results. In this study, it is
proposed to bond the disposable part on a coverslip. If the disposable
part made of PDMS were bonded directly to the glass slide (on which
electrodes are deposited), the whole device would have to be discarded
after each test since it is not possible to separate the PDMS block from
the glass slide. Even after separation, the electrodes would be impacted
and unlikely to function. There must be a biologically clean layer to
insulate the PCR mixture from contacting electrodes directly. The elec­
trodes could be coated with SiO2 and then bonded with PDMS, but that
would increase the fabrication cost.
The advantage of having a separate PDMS block for reaction well and
bonding the micromixer block on top of it is cutting out the need for a
microvalve. Integrating microvalves into PCR microfluidic devices
makes the fabrication process much more challenging, and supple­
mentary equipment, such as an air compressor, is required for the valves
to operate leading to increase in size and cost of the device. Further­
more, since microvalves do not always perform ideally, some of the PCR
mixture might flow back to the channels during the thermocycling
process, resulting in wasting the mixture and lowering the quality of
amplification.
In the proposed microfluidic chip, the target DNA sample and the
master mix solution are first mixed inside the micromixer and streamed
directly into the reaction well. The reaction chamber well is located
above the microheater which carries out the thermocycling process
while the controller unit monitors the temperature inside the well dur­
ing the PCR reaction. After PCR reaction is completed, the PCR product
is analyzed by gel electrophoresis and the disposable part is replaced
H. Mashouf et al.
Fig. 1. The microfluidic device layout for PCR amplification (a) Schematic of the integrated device, and (b) Exploded view of the microchip presenting inte­
gral components.
with a new one.
2.1.1. Thermocycler design
Temperature cycling is performed by heating and cooling the PCR
mixture according to a specific protocol. The thin film microheater
carries out the thermocycling process by Joule heating method. The
temperature gradient is formed inside the PCR reaction well as electric
current passes through the thin film resistive electrode located beneath
it. Achieving uniform temperature distribution over a designated area is
the prime concern in developing microheaters. The pattern design of the
electrode plays a crucial role in addressing this concern, and studies
have been made to find the efficient heater layout. Geometries with
meandering narrow line electrodes are generally incorporated since they
reduce the power consumption [37,49].
Several designs were tested in this study aiming to provide temper­
ature uniformity with ±1 ◦ C precision across a circular area that houses
the PCR well. An infrared imager was used to investigate temperature
uniformity. The designs also focused on providing fast thermal response
times while lowering the power required to heat the electrodes. In order
to accomplish these goals, the size of the electrodes and the distance
between them were gradually modified by conducting several experi­
ments until satisfactory results were achieved.
Fig. 2 shows the microheaters fabricated via photolithography by
patterning Au on glass substrate. The edges of thin-film heaters always
show lower temperatures compared to the middle region because of low
ambient temperature. One approach that has been implemented to
address this issue is to increase the resistance in the corners of the heater
[41]. However, this idea complicates the design of the pattern and its
fabrication as well. Besides working on the pattern of the electrodes and
their dimensions, microheaters cover a larger area than the reaction well
to compensate for heat loss at the edges. In all proposed designs, the
heating region can be fitted into a square with a side length of 1 cm. The
detailed dimensions can be found in Fig. S1.
Fig. 2. Fabricated microheaters by patterning gold on glass substrate. (a) Design 1, (b) Design 2, and (c) Design 3.
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Chemical Engineering and Processing - Process Intensification 189 (2023) 109394
2.1.2. Temperature control unit
The cycling protocol in PCR reaction includes three stages: main­
taining a steady temperature, cooling, and heating which are automat­
ically mediated with the use of a microcontroller. Schematic of the
temperature control unit is illustrated in Fig. S2 of supplementary
document. The microheater is connected to the power supply using
crocodile connections (B&K Precision Corporation, Model 1672)
through the microcontroller. The power supply operates on constant
voltage mode while the controller monitors the time of thermocycling
stages and temperature inside the reaction well. The programmable
controller provides tuned voltage to the heater based on the feedback
from the sensor as the temperature set points are altered according to the
algorithm.
Integrated thin film sensors were not an appropriate option for the
proposed device. Among temperature sensors, thermistors can offer the
required precision and stability while being affordable, compact, and
compatible with microcontrollers. A 10KΩ thermistor (B3950 NTC,
Adafruit) with a negative temperature coefficient is used as the tem­
perature sensor in the micro device. It is crucial to verify that the
measured temperature correlates with the actual temperature distribu­
tion of the PCR mixture in order to ensure an accurate and repeatable
amplification. The sensor was thermally calibrated to report the tem­
perature inside the well. The calibration of the temperature sensing unit
was performed by comparing the temperatures read by two thermistors,
one embedded inside the reaction well, and another attached to the glass
slide. In order to achieve the minimum temperature difference between
the two sensors (less than 0.1 ◦ C), two readings were compared for
temperatures ranging from 40 ◦ C to 98 ◦ C with intervals of 2 ◦ C, and the
sensor attached to the glass slide was adjusted accordingly. The method
of calibration is discussed in supplementary document.
2.1.3. Micromixer design
Mixing in the microscale often occurs as a result of molecular
diffusion process. The low molecule diffusivities would make fluid
mixing extremely challenging and the process of mixing through
H. Mashouf et al.
diffusion would take an excessive amount of time and require an
extremely lengthy channel [50,51]. Therefore, increasing the contact
area between the fluids to be mixed is necessary to hasten the process
and reduce the mixing length. Several micromixers were fabricated and
analyzed to find a convenient geometry for our application. Folding,
stretching, and splitting of the flow streams facilitate mixing at low
Reynolds numbers which can be generated by disturbing the flow with
creating curved channels or embedding obstacles throughout the
channel. Fig. 3 presents the structure of two micromixer with their
detailed dimensions. These designs were selected and underwent mod­
ifications to improve mixing quality by inserting sharp edges and
grooves in channel walls. The channel length is around 80 mm and 60
mm for design 1 and design 2, respectively.
In this study, the mixing index is calculated by comparing the stan­
dard deviation of the pixel intensity of the desired point to that in the
entirely unmixed section (inlet) [52]. To determine the standard devi­
ation, two fluid streams were flowed through the mixer. DI water was
injected from one inlet and a colorful stream from another one. The
experimental apparatus for evaluating mixing quality is shown in
Fig. 4a. ECHO Revolution microscope took RGB Images from the desired
region inside the micromixer channel. These images were then con­
verted to greyscale values via homemade code in computer software.
These values range from 0 to 255, corresponding to the intensity of each
pixel in the original image. The values were then normalized according
to the maximum intensity, resulting in normalized values Xi which
range from 0 to 1. The mixing index is then expressed by:
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
∑N
1 2
σ i=1 (Xi − 〈X〉)
(1)
N
MI = 1 − = 1 − √̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
∑N ̅
σ0 1 2
i=1 (X0i − 〈X0 〉)
N developed in SU-8 developer for around 8 min, washed with isopropanol
Where, 〈X〉 denotes the average value over the normalized values,
and N is the number of data. Standard deviation at the non-mixing
section (σ 0 ) would be equal to 0.5 if the flow rates are similar at the
inlet, implying completely segregated streams. This specific formula was
picked over the others because studies have demonstrated that it is least
impacted by lighting [53]. Fig. 4b demonstrates the process of calcu­
lating the mixing index.
2.2. Fabrication
The components of the microfluidic device are fabricated separately
and then assembled together. To fabricate the microheater, glass slides
were first deposited with gold in the sputtering machine. The thickness
of gold is 1000 Å, and 100 Å chrome acts as the adhesion layer. Positive
photoresist (MICROPOSIT™ S1813™ G2) was spin-coated on the slide
at a spin speed of 500 rpm for 10 s and a spread speed of 3000 rpm for
20 s. The glass slide was then soft-baked at 115 ◦ C for 2 min. For
Fig. 3. Micromixer structures. (a) Design 1, and (b) Design 2.
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Chemical Engineering and Processing - Process Intensification 189 (2023) 109394
transferring the heater pattern, the mask layout was designed using a
CAD program, and UV exposure was carried out for 8 s under a wave­
length of 365 nm and 20 mW/cm2 (Model 204IR, OAI). Then, the
photoresist was developed for 45 s in developer (MICROPOSIT® MF®-
319). To perform the wet etching process, the glass slide with patterned
photoresist was immersed for 30 s into a gold etchant (TFA gold etchant,
Transene). To remove the unwanted chrome layer, the glass was
immersed in chrome etchant (TFE chromium etchant, Transene) for 10 s.
The residual photoresist was then stripped by MICROPOSIT™
REMOVER 1165. At the final stage, the glass was rinsed with water and
isopropanol (IPA) and dried with nitrogen gas.
The steps for fabricating reaction wells are as follows: 10:1 wt ratio of
PDMS pre-polymer and curing agent (SYLGARD™ 184, Dow Inc., MI,
USA) was mixed. The mixture was poured into a mold and degassed for
5 min, followed by baking for 2 h at 80 ◦ C in the oven to create PDMS
sheets. These sheets with a thickness of 3 mm were then cut down into
small blocks of 2 × 1.5 cm. Biopsy punches were used to make holes
throughout the block to form the reaction well. The temperature uni­
formity analysis is conducted to choose the appropriate well diameter.
Negative photoresist SU-8 2075 (MicroChem, MA, USA) was used to
fabricate the master for replica molding of the micromixer. A 3-inch
silicon wafer was first cleaned using isopropanol (IPA). The photore­
sist was spin-coated on the wafer at a speed of 500 rpm for 10 s followed
by a speed of 2100 rpm for 30 s to create approximately 100 µm chan­
nels. The wafer was then put onto a hot plate for 5 minutes at 65 ◦ C and
15 min at 95 ◦ C to perform the soft baking process. The mixer pattern
was exposed onto the wafer by UV light (365 nm and 20 mW/cm2) with
a mask aligner for 13 s. Post-exposure baking processes were performed
at 65 ◦ C and 95 ◦ C for 4 and 10 min, respectively. Then, the wafer was
(IPA), and dried with nitrogen gas. For the last step of fabricating the
mold master, the wafer underwent hard baking process for 5 min at
150 ◦ C. After molds are prepared, micromixer PDMS blocks with 2 mm
thickness are created by implementing similar procedure as reaction
well blocks were fabricated.
To fabricate the disposable part, fluidic access points were punched.
Then, reaction well block and micromixer block were exposed to oxygen
plasma for 20 s (Plasma Etch, PE-50, USA), brought into contact with
each other, and incubated at 80 ◦ C for 2 h to form an irreversible bond.
The final stage of assembling the disposable part is bonding the block of
the reaction well and micromixer to a coverslip (22 × 22 mm, Fish­
erbrand™) using oxygen plasma.
2.3. Sample preparation
The PCR reagents were purchased from Integrated DNA technolo­
gies. The PCR master mix consists of 2 µL Deoxynucleoside triphosphate
(dNTP), 2μL 10X PCR buffer, 1 μL MgCl2, 10.8 μL nuclease-free water,
H. Mashouf et al.
Fig. 4. The experimental procedure for evaluating mixing efficiency (a) Experimental setup and fabricated micromixer structures, and (b) Mixing index calcula­
tion steps.
and 0.4 μL Taq gold (AmpliTaq Gold™ DNA Polymerase). Λ-phage DNA
(Thermo Scientific™, USA) with concentration of 475.2 ng/μL was used
as template. The forward and reverse primers used were 5′ - ATG CCA
CGT AAG CGA AAC A − 3′ , and 5′ - GCA TAA ACG AAG CAG TCG AGT
− 3′ , respectively, each with 0.4 μL volume. The master mix (17 µL) was
automatically mixed with of 100-fold diluted λ-phage DNA (3 µL)
through the micromixer and directly dispensed into the reaction well of
the disposable block. To prevent evaporation, the sample was covered
with 10 μL of mineral oil.
The PCR thermal cycling was applied under the following protocol:
First, to activate the Taq polymerase predenaturation was done at 95 ◦ C
for 5 min. Then, PCR cycle was repeated 40 times consisted of: dena­
turation of DNA template at 95 ◦ C for 15 s, annealing of primer at 54 ◦ C
for 20 s, and extension of the DNA at 72 ◦ C for 20 s. Final step was
elongation at 72 ◦ C for 1 min. Upon PCR completion, the amplified
product was analyzed by gel electrophoresis in a 2% agarose gel Invi­
trogen, and evaluated compared to the positive control band obtained
by a conventional thermocycler (T100 Thermal Cycler, Bio-Rad, USA).
Rnase-free water was used for the negative control as well. The resulting
DNA fragments size was measured by GeneRuler 100 bp DNA Ladder
(SM0241, Thermo Scientific™, USA).
Fig. 5. Spatial thermal uniformity comparison of microheater designs over a circular region using IR imaging at 95 ◦ C. (a) design 1, (b) design 2, and (c) design 3.
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Chemical Engineering and Processing - Process Intensification 189 (2023) 109394
3. Components performance
3.1. Microheater
3.1.1. Spatial thermal uniformity
The microheaters are designed to create uniform heating and fast
thermal response with low power consumption. Temperature uniformity
in an area can be defined as where the temperature difference between
set point temperatures is less than 1 and/ or 2 ◦ C [54]. The spatial
thermal uniformity of the proposed designs was verified by an infrared
imaging system, and their performance was compared over circular
areas. As gold directly reflects infrared light from its surroundings, an
image taken with the resistor facing the IR camera would show a heat
map of the surroundings rather than the actual temperature of the
electrodes [37]. Therefore, images were taken from the backside of the
glass patterned with heater electrodes. Fig. 5 illustrates the images taken
by IR camera (FLIR Flip 4) from microheaters at the temperature of
95 ◦ C (denaturing temperature) where Area1 is a circle of 4 mm in
diameter.
The pattern of heating maps in design 1 and design 3 are almost fully
symmetric unlike rectangular-shaped heating map of design 2.
Numerous small nodes located at small distances from each other pre­
vents large heat dissipations in design 2, resulting in lowest temperature
deviation in Area 1 from the set point and not allowing the temperature
to drop significantly compared to other two designs. Design 1 reported
the most deviation due to the high thermal dissipation from its edges,
whereas in design 3 the center of the heater is surrounded by electrodes
H. Mashouf et al.
to avoid heat dissipation. With the help of these electrodes, design 3
showed an average temperature of 94.9 ◦ C over measured points inside
Area 1. Evidently, as the circular area being studied gets bigger, the
average and minimum values drop.
Changing the voltage from 1 V to 1.8 V increased the temperature in
design 2 from 54 ◦ C to 95 ◦ C. Although this might sound pretty satisfying
for a heater, it might not work well for a thermocycler. A small alteration
in voltage drastically changes the spatial temperature, making temper­
ature control challenging. Moreover, fast heating ramps do not allow
sufficient thermal distribution inside PCR mixture, impacting the quality
of amplification. In contrast, design 1 requires around 14 V to reach
95 ◦ C implying notable power consumption although showing adequate
thermal uniformity. Design 3 needed around 8.1 V and less than 2 Watts
power for maintaining the temperature at 95 ◦ C making it suitable to be
integrated into a microfluidic device. Same performance analysis on
various circular regions and different temperatures almost presented
similar results on spatial thermal uniformity. Considering these expla­
nations, design 3 appeared to be the best fit for the microheater pattern.
Design 3 suggested similar heating map patterns with average tem­
peratures of 72.1 ◦ C and 54.1 ◦ C at extending and annealing tempera­
tures, respectively which is highly satisfying. The deviation of maximum
and minimum temperatures from the desired set point intensifies as the
temperature rises. This happens since heat dissipation to the surround­
ings becomes easier and larger as the temperature difference with the
ambient increases. The IR images of design 3 at 72 ◦ C and 54 ◦ C, can be
found in Fig. S4 of the supplementary document.
3.1.2. Reaction well diameter
Fast thermal distribution and temperature uniformity inside the re­
action well are key parameters in successful PCR process. As the diam­
eter gets smaller, temperature uniformity increases across the diameter,
but uniform thermal distribution along the well’s height might not
occur. Measuring temperature inside the reaction well at different
heights would be inaccurate due to the load effect of the external ther­
mometer. Therefore, it is tried to achieve a satisfactory compromise
between height and temperature uniformity across the circular area of
the corresponding diameter. Fig. S5 illustrates the reaction chamber
PDMS block, its height, and reaction well diameter punched through it.
Well diameters and corresponding mixture heights can be found in
Table 1 of supplementary document as well.
Fig. 6. Well diameter analysis according to the selected microheater (design 3), (a) IR image at 95 ◦ C annotated with different well diameters, (b) Uniform area
percentage inside different well diameters with ±1 ◦ C accuracy, and (c) Cross-section temperature distribution along a horizontal line at 95 ◦ C.
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Chemical Engineering and Processing - Process Intensification 189 (2023) 109394
The temperature uniformity was analyzed inside three circular areas
with diameters of 3.5, 4.5, and 5.5 mm at 95 ◦ C since highest deviation
from desired setpoint happened at this temperature. These diameters are
illustrated in Fig. 6a with D1, D2, and D3 annotations, respectively. We
considered the span of ±1 ◦ C temperature difference as uniform. The
uniformity is then reported as the percentage of measured points in the
circular region which are inside this temperature span. The temperature
uniformity results for each diameter are illustrated in Fig. 6b. D3 shows
unfavorable results with temperature uniformity of 68% at 95 ◦ C. D1, on
the other hand, suggests superior performance by 100% uniformity at all
temperatures. D2 suggests uniformity of 93.1% which is pretty high as
well, and it is not affected by temperature alterations. Nonetheless, the
height of PCR mixture corresponding to D1 would be equal to 2.08 mm,
66% higher than the height for D2. Furthermore, the total height of the
reaction well block will be affected as well since it must occupy mineral
oil which will result in higher consumption of material (PDMS). How­
ever, we decided to choose the final well diameter of 4 mm to have
improved temperature uniformity. Also, the height difference between
D2 and a diameter of 4 mm is not that significant compared to D1.
Table S1 reports the mixture heights corresponding to each reaction well
diameter.
The plot presented in Fig. 6c shows the temperature along a hori­
zontal line crossing through the heated region to better understand
temperature fluctuations inside the circle of D2 at a temperature of
95 ◦ C. The maximum temperature is 95.8 ◦ C, as illustrated before in
Fig. 5c. Large drops in the plot are probably due to the regions which are
not covered by the heater electrodes. The temperature decreases by
moving from the center of the heater toward the edges. The rate of drop
in temperature apparently increases as we get closer to the edges of the
heater.
3.2. Dynamic temperature control unit
For successful PCR amplification, the temperature of the PCR
mixture must be carefully controlled and regulated. PCR reagents are
highly sensitive to temperature and the quality of amplification will be
reflected if the control unit fails to maintain the temperature of the
mixture at specific set points according to the protocol with the lowest
possible fluctuations. The control system in this work is programmed to
operate in the temperature range of common PCR protocols. Sufficient
H. Mashouf et al.
number of tests were conducted to analyze the performance of the
control system.
54 ◦ C and 72 ◦ C are common PCR temperatures for annealing and
extending. Fig. 7a and 7b show the heating performance starting from
these temperatures to two arbitrary setpoints followed by maintaining
the temperature for around 15 s. According to the plots, the temperature
fluctuations after reaching the desired set point is lower than 1 ◦ C, which
can be regarded as uniform. Although microheater design and well
diameter were selected in a way to facilitate thermal uniformity inside
the well, the control system must also allow sufficient time for the re­
action well to reach uniform thermal distribution. To ensure the tem­
perature uniformity of the sample inside the reaction well, a
temperature transition time is required for the set temperature to be
reached inside the well to ensure that the sample reaches the exact
temperature as the sensor is recording. To ensure this smooth transition
of the temperature, the controlling system dynamically adjusts the
heating and cooling rate by considering the aforementioned criteria.
Considering all the limitations, the applied heating rates ranged be­
tween 2 and 2.6 ◦ C/s. As a result of a more uniform temperature dis­
tribution inside the well at lower temperatures, higher heating rates are
observed in the well at lower temperatures. In addition, as the difference
between the two set points narrows, the heating rate decreases.
Fig. 8a and b display the cooling performance of the device in two
separate plots. The set points for each plot are different. Here, the per­
formance of the device is compared to when fans are employed as well.
Two fans (Delta Electronics, AUC0512, 20.2 CFM) were placed at the
bottom of the microheater to facilitate cooling. The average cooling
rates are smaller than the heating rates. It is apparent that fan installa­
tion results in an increase in the cooling rate. The maximum cooling
rates were observed in the absence of a fan at 2.1 ◦ C/s, and with a fan at
1.7 ◦ C/s. The protocol chosen for this particular application involved
denaturation and annealing stages. Considering the cooling rate differ­
ence between using and not using fans we decided against including it in
our device. Utilizing fans decreases cycle time by around 5 s which is not
that significant. However, it would be an extra component that not only
increases the size of the whole microfluidic device but also increases its
power consumption.
Fig. 7. Heating and temperature maintaining performance. (a) Heating from 54 ◦ C to 72 ◦ C and 90 ◦ C, (b) Heating from 72 ◦ C to 85 ◦ C and 95 ◦ C.
7
Chemical Engineering and Processing - Process Intensification 189 (2023) 109394
3.3. Micromixer
At low Reynolds numbers, molecular diffusion prevails in the mixing
mechanism of two streams. Therefore, the time the working fluid stays
in the micromixer influences the mixing quality. The volume ratio of the
target DNA to master mix is 3:17. Different methods can be implemented
to enhance the mixing efficiency of micromixers such as increasing the
length and height of the microchannels, changing the type and design of
the micromixer, or increasing the hydrophilicity of PDMS by Oxygen
plasma treatment or surface coating. Efforts were made to enhance the
efficiency of the process by implementing methods that require fewer
equipment and cost-efficient fabrication steps. Experiments were con­
ducted on micromixer structures to find out the appropriate design,
channel height, and flow rate for sufficient mixing efficiency. It is more
practical to report the amplification results in mixing efficiency ranges
rather than a minimum value for mixing index required for successful
amplification. We found that for efficiencies below 0.85, no amplifica­
tion occurs, or only unspecific bands appear. For efficiencies between
0.85 and 0.9, the results were not repetitive, and the brightness of bands
were not always similar to the positive control, while in efficiencies
above 0.9, results were satisfying. E-gel electrophoresis test results can
be found in Fig. S6 of supplementary document.
Fig. 9a presents the effect of channel height on mixing efficiency at
total flow rate of 20 µL.min− 1. Experiments conducted at other total flow
rates between 0.5 and 50 µL.min− 1 followed the same trend. Increasing
channel height will improve the mixing quality since the contact area
between the two streams and the fluid-fluid interaction time increases;
hence the diffusion rate intensifies as well. Channel height of 40 µm
suggested poor mixing quality. Significant improvement in mixing
quality was observed by increasing channel height from 40 µm to 100
µm although the improvement is not notable from 100 µm to 180 µm.
From the practical view, channels with high heights are difficult to
fabricate, and the molds are more susceptible to getting deformed due to
their high aspect ratio. Therefore, channel height of 100 µm is consid­
ered for the micromixers.
The adverse effect of increasing the flow rate at a constant length is
expected since the working fluids will have less time to diffuse and are
driven toward the outlet without having sufficient time to interact. The
bar chart in Fig. 9b presents the effect of total flow rate on mixing ef­
ficiency. Both designs suggested high efficiencies while total flow rate of
H. Mashouf et al. Chemical Engineering and Processing - Process Intensification 189 (2023) 109394

Fig. 8. Comparison of cooling and temperature maintaining performance of the microheater by utilizing fans (a) Cooling from 95 ◦ C to 54 ◦ C, and (b) Cooling from
70 ◦ C to 50 ◦ C.

Fig. 9. Micromixers mixing performance comparison. (a) Mixing efficiency at different channel heights and total flow rate of 20 µL.min− 1 (b) Mixing efficiency at
different total flow rates and channel height of 100 µm, and (c) Mixing efficiency at channel height of 100 µm and total flow rate of 10 µL.min− 1 with respect to
channel length.

20 µL.min− 1 showed poor performance in comparison to other flow
rates. In addition to poor performance, a high flow rate might damage
the bonding between the micromixer PDMS channel and reaction
chamber PDMS block, resulting in the leakage of the PCR mixture and
impeding the PCR reaction. Handling low target DNA volume (3 µL) can
also be difficult. Therefore, it was decided not to choose a total flow rate
of 20 µL.min− 1 and higher. Both total flow rates of 10 and 4 µL.min− 1
suggested satisfactory results. However, mixing at total flow rate of 4 µL.
min− 1 and lower takes too much time. Since the mixing efficiency of
total flow rate at 10 µL.min− 1 is sufficient for our application, we
decided to choose this flow rate over flow rate of 4 µL.min− 1. Overall,
design 1 displayed better performance over design 2 in terms of final
mixing indices. This can be explained with the chaotic flow regime
created by the high number of grooves across its channel walls.
Although design 1 achieved better mixing efficiency, design 2 is compact
and more convenient for a small disposable device. Unlike design 2,

8
H. Mashouf et al.
design 1 occupies a larger space making its production costly as it re­
quires more material.
It should be noted that the mixing channel of design 1 is around 20
mm longer than design 2. According to the plot in Fig. 9c, they show a
very close mixing index at a distance of 60 mm from the inlet, which is
the total channel length of design 2. Design 1 reaches high efficiencies
much faster than design 2 due to its special obstacle-based geometry.
However, it was not possible to make design 1 as compact as design 2,
even by reducing its length. The most challenging issue, however, was
the formation of bubbles in several regions around the grooves, which
can impact the mixing quality or even block the channel, which did not
happen in experiments using design 2.
Since total flow rate is decided to be 10 µL.min− 1, the flow rates at
the inlets should match with volume ratio of PCR mixtures. Therefore,
the flow rate at one of the inlets would be 1.5 µL.min− 1 and 8.5 µL.min− 1
in the other one. Fig. 10a demonstrate microscope images at different
regions throughout the selected micromixer (design 2) under this flow
rate and channel height of 100 µm. The continuous growth in mixing
quality along the channel is noticeable after the working fluids pass
through each loop. Plots illustrating mixing performance at three
different heights and three different total flow rates are illustrated in
Fig. 10b to better understand the mixing indices. The regions used to
calculate mixing indices have identical distances from each other and
correspond to the microscope images in Fig. 10a. The growth rate of
mixing quality decreases as the mixing efficiency rises to high values.
Mixing efficiency of 0.8 is surpassed after the flow reaches halfway
through the mixer at selected characteristics, and the final efficiency is
equal to 94.17%.
4. Integrated device performance
The developed compact microfluidic chip is shown in Fig. 11a. The
heating component of the chip can be reused for future amplifications,
while the disposable section is simply replaced with a new one after each
PCR cycle is finished. The experimental setup during PCR reaction is
shown in Fig. 11b. After the protocol is finished, the amplified mixture is
recovered for the post PCR process using E-gel electrophoresis.
The PCR reaction is performed following a protocol consisting of 40
cycles. The performance of the developed microfluidic device is pre­
sented in Fig. 12a. The temperature was meticulously maintained at PCR
step set points with a deviation of less than 1 ◦ C/s. The whole
Fig. 10. Micromixer performance. (a) Microscope images throughout design 2 at total flow rate of 10 µL.min−
design 2 at different total flow rates and channel heights.
9
Chemical Engineering and Processing - Process Intensification 189 (2023) 109394
amplification process takes around 70 min, which is lower than con­
ventional PCRs.
The amplification performance of the microfluidic PCR device was
verified by a conventional PCR referred to as λ DNA positive control.
Fig. 12b presents the final results visualized by E-gel electrophoresis.
Lane M is the 100-bp ladder DNA marker. Signal in Lane 1 is obtained by
using the conventional PCR machine for positive control, and lane 5
represents negative control. Bands in lanes 2, 3, and 4 are the PCR
products obtained by different microfluidic devices. To ensure that
contamination is prevented, and PCR amplification results are repro­
ducible, experimental tests were conducted on microfluidic devices with
different disposable and reusable parts. The results demonstrate that this
system can generate the correct amplified PCR base pair and that
degradation does not occur throughout the process. The brightness of
bands can be used to qualitatively assess the efficiency of amplification.
Since no significant difference in the PCR band intensities was observed,
it can be interpreted that the proposed device can perform the amplifi­
cation with high efficiency.
5. Conclusion
In this study, a disposable, compact, low-cost and easy-to-fabricate
microfluidic device is designed to amplify λ-DNA. The disposable
feature of the device eliminates the run-to-run carryover contamination,
while its integration with reusable heating system reduces fabrication
costs. The need for integrating a microvalve to isolate the PCR mixture
from flowing back to micromixer is eliminated by the layered design of
the disposable part. The microheater is fabricated by patterning gold
electrodes on glass substrate and carries out the thermocycling process.
Around 95% of the area housing the reaction well have temperature
uniformity with thermal variation of <1 ◦ C at protocol setpoints (54 ◦ C,
72 ◦ C, 95 ◦ C). The microcontroller unit can maintain the temperature
with less than 1 ◦ C error inside common working range of PCR protocols.
The developed passive micromixer suggested mixing efficiency of
94.17% at channel height of 100 µm and total flow rate of 10 µL.min− 1.
The integrated device proposed high-quality amplification results
comparable to the conventional PCR. Additionally, the studies on the
reproducibility of the device shows its superior performance in gener­
ating consistent and reliable results for different amplification protocols.
Further study can be focused on employing multiple microheaters on a
single chip in order to increase the throughput and utilization of the
1
and channel height of 100 µm, and (b) Performance of
H. Mashouf et al. Chemical Engineering and Processing - Process Intensification 189 (2023) 109394

Fig. 11. Integrated microfluidic device. (a) Fabricated microfluidic chip, and (b) Experimental setup for conducting PCR reaction.

Fig. 12. Integrated device performance. (a) 15 PCR cycles using the developed microfluidic device, and (b) E-gel results of the developed microfluidic device
compared to conventional PCR.

suggested device for the amplification of the target gene from an ani­ Investigation, Validation. Sepideh Pakpour: Conceptualization, Re­
mal’s total DNA. sources, Funding acquisition. Mina Hoorfar: Conceptualization, Su­
pervision, Funding acquisition, Resources.
Statement of novelty and significance
Declaration of Competing Interest
Rapid heat transfer, and low sample and energy consumption are
advantages of microfluidic PCR devices. However, they are unsuitable The authors declare that they have no known competing financial
for mass production and portable use. Our proposed compact device interests or personal relationships that could have appeared to influence
comprises several components integrated in a layered design aiming to the work reported in this paper.
reduce the fabrication cost and eliminate the chance of sample-to-
sample cross-contamination. The microheater and control system form Data availability
the reusable parts of the chip while the reaction well and micromixer are
disposable. The disposable element is easy to fabricate and replaced Data will be made available on request.
with a new one upon completion of PCR reaction. The performance of
the device is verified by amplifying lambda DNA samples.
Acknowledgment
CRediT authorship contribution statement
This work was supported by the Natural Science and Engineering
Hirad Mashouf: Investigation, Data curation, Writing – original Research Council (NSERC) of Canada under the Discovery Grant (Grant
draft, Writing – review & editing. Bahram Talebjedi: Conceptualiza­ no. 341873).
tion, Supervision, Investigation, Methodology, Software, Writing – re­
view & editing. Nishat Tasnim: Writing – review & editing, Project
administration. Maia Tan: Investigation, Validation. Sahar Alousi:

10
H. Mashouf et al.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.cep.2023.109394.
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