Project Report

- By Debjit De Sarkar
INDEX
 Acknowledgement
 Introduction – PCR process and Thermocycler
 Brief Overview
 Aim
 Pre-Requisite
 Preparation of Heating element
 Connection Diagram
 Actual Setup
 Modelling of system in COMSOL
 Final model
 Physics Applied
 Results
 Conclusion
 Brief overview of market survey
 Basic Outline
 Heating block
 Suggested Heating Systems
 Heat exchangers
 Top Lid Heater
 Control Strategies
 Basic Coding plan
 Future Plan
 References
Acknowledgement

I would like to express my special thanks of gratitude to my teacher Dr

Santanu Talukder who gave me the golden opportunity to do this
wonderful project on the topic Top Lid Heater for PCR and my friends
Saleem and Anshuman who were working with me on the same project.
This project was a great experience and I learned many things during the
project duration.
Lastly, I would like to thank my parents and friends whose constant
supports were important for me to complete my project successfully.
Introduction
What is Polymerase Chain Reaction(PCR)?
Polymerase chain reaction (PCR), a technique used to make numerous
copies of a specific segment of DNA quickly and accurately. The
polymerase chain reaction enables investigators to obtain the large
quantities of DNA that are required for various experiments and
procedures in molecular biology, forensic
analysis, evolutionary biology, and medical diagnostics.

This diagram shows the three-step process of the polymerase chain reaction.

What is a thermocycler?

Thermocyclers, or thermal cyclers, are instruments used to amplify DNA and

RNA samples by the polymerase chain reaction. The thermocycler raises and
lowers the temperature of the samples in a holding block in discrete, pre-
programmed steps, allowing for denaturation and reannealing of samples with
various reagents. Amplified genetic material can be used in many downstream
applications such as cloning, sequencing, expression analysis, and genotyping .
Function of Top-Lid Heater
Heated lids are another potentially important feature for a thermal
cycler. Heated lids allow a sample to be heated more efficiently—
minimizing formation of nonspecific annealing and primer dimers. This
function also prevents water condensation on the lid and evaporation
of your samples.

Aim of the project

In this project we will be designing and simulating a hot plate system
similar to top lid heating system used in P.C.R. machine which can be
heated up to 105 ◦ C in a controlled manner. This should act as a
replacement for the lid heater of a PCR machine.
Also we aim to create a basic design for an actual thermocycler or PCR
machine by doing market survey for determining the parts required for
the machine.

Brief Overview
We will first make a COMSOL simulation of the top lid heater which will
according to the work we did during our lab project in the 6 th semester.

In this simulation we will try to understand the heat distribution and dissipation
throughout the aluminium plate and the current required to so.

After this we will try to make a basic design of a thermocycler and do a market
survey to find out the necessary parts for building one.
Pre-Requisite
Basic knowledge about COMSOL software will be required.

Preparation of heating element

Heating element was made by pasting the nichrome wire on an aluminium plate
with the help of high temperature resistant glue (Gorilla glue). The current is
supposed to pass through the nichrome wire which will cause it to heat thereby
heating the whole system.

Connection Diagram
Actual Setup

Modelling of System in COMSOL

The whole system is modelled as:

1. The lid is modelled as an aluminium plate of dimension 10 x 10 x 0.2 cms

with required material specifications.

2. The heating element or mesh is modelled with nichrome material.

 3. The glue is modelled by polyurethane layer of depth 2 mm. We are
considering uniform spread of the glue.

4. The ground or the surface in contact with the lower surface is modelled
with marble with dimensions 20cm x 20cm x 0.8 cm.

Final COMSOL model

Physics Applied
 We used the electromagnetic heating function available in
COMSOL.

 Current node or current source is set up at one end and ground

at another end of the nichrome mesh.

 We applied different currents to find the one which gives

desired result i.e. the temperature of aluminium plate should be
at 105⁰C.

 Also to model the heat losses we’ve taken into account for-
heat loss due to radiation and the heat that is lost due to
 conduction to ground, we’ve assigned the parameters
respectively.

Results

Current = 5 A

Current = 6 A
Current = 7 A

Current = 8 A
Conclusion
We can conclude that on the basis of present selection of materials and
the physics that was added the current required to achieve the desired
temperature for aluminium plate is about 7 A to 8 A and the time required
to reach this temperature is about 20 minutes.

Brief overview of the market survey

 Basic outline of thermal cycler

Heating Block
 This consists of TE (thermoelectric module), heat exchanger, heating stage
and sample block.
 A heating stage (aluminium or copper) present above the TE module .
 Sample block will be present above the heating stage, heat from the TE
module will move through heating stage to the sample block containing
reaction wells.
 We can use thermal insulators along the borders of the heating stage so
that heat from the stage does not escape to the ambient. This in turn will
increase the efficiency and performance of the PCR

Suggested Heating Systems

 Peltier module (preferred)
 Wirewound resistor (has limited power wattage and resistance can change
with increase in temperature, not suitable for better performance)
 Cartridge heater

Heat Exchanger
 Heat sink is used for removing the excess heat dissipated from the heat
source.
 Peltier module with heat sink is a good option.
 One potential challenge that might be faced by us is the difference in the
sizes of the different components. The aluminum block has to match the
area of the peltier and the heat sink also has to have a corresponding
mounting surface area to press against the peltier.
 If it’s bigger than required, that won't be a big issue. If the heatsink is
smaller, we can add one thin aluminum plate between the peltier and the
heatsink.
 While the concept of a heat sink itself is relatively standard it is important
to understand that in order to optimize the system we actually need to
preserve a significant amount of heat intentionally to minimize the
difference between the cool and hot side of the TEC.
 One of the fans on a heat sink can be reversed to allow, when desired,
rapid exhaust of the heat within the heat sink. The entire set of heat sinks
is then insulated and one of the temperature probes/thermocouples can
be inserted to allow for monitoring the heat-sink temperature.
 Some suggested products are mentioned in the ppt.

Top-Lid Heater
 It is the heater that heats the top lid of the PCR  covering the PCR reaction
tubes in the PCR machine. It helps in avoiding the condensation of the
reaction mixture droplets. It is an important part of the PCR system, during
past times mineral oil was used to cover the reaction tubes and heated.

 The top lid must be in such that it presses against the top of the reaction tubes
and seals it off so that vapours from reaction tubes do not escape the PCR
machine. We can use also some other sealing measures to properly seal the top
 lid above the reaction chamber so that no component escapes the temperature
of the top lid is kept high and constant, usually, around 100-105 C high enough
to discourage condensation.

 We can have a fixing frame of Top lid such that we can fix this heating part there,
and use it as an opening Lid of the PCR machine.

 Since the temperature of the Top lid covering the reaction tubes needs to be
constant, we can have its temperature controlled by a microcontroller.

Some suggested Controlling Strategies

 Basic PID control
 Hybrid controller
 Using PWM signal to control peltier
The disadvantage of this method is that it causes rapid thermal cycling
which leads to degradation of PCR.

Temperature Profile to be tracked

Some PID based embedded controllers

Software design of PID based control
 This is from the paper “Design and Development of Embedded System Based
PCR Thermal Cycler”
  ARM 32-bit microcontroller is used for controlling the temperature
profile of the heat block. The system of the designed PCR thermal
cycler has input or output devices LCD and Keypad.
 A UART interface can be used to enable the system PC
communication. Software is developed using Embedded C and Free
RTOS system calls. 
Flowchart of PCR execution
Disadvantages of using PID
 Tuning of a PID controller is usually a compromise between the ramp rate and the
overshoot; therefore, the actual ramp rate will not reach the physical limit of the
control system. 

 Experimental results demonstrate that the hybrid control strategy performs better
than the conventional PID controller both in setpoint response and in steady-state
performance of temperature control, so that the accuracy and efficiency of PCR are
improved accordingly.
Hybrid Control
 The hybrid control strategy combines feedforward, feedback, and Bang-Bang actions to
achieve better setpoint response and steady state behavior.
 Based on the steady state model of the thermocycler, the feedforward action serves as the
model predictive control for each setpoint, while the feedback controller is used as the local
control action to accelerate ramp rates and reduce the tracking errors.

 Experimental results demonstrate that, besides the improvement of the tracking accuracy,
both the rise time and the decay time of each thermal cycle have been reduced significantly
with the hybrid control strategy, compared with the conventional PID. Therefore, improved
efficiency and accuracy of the polymerase chain reaction is achieved. 

Peltier Effect
 As current passes through the TE module in the direction shown in Fig. 1, QL is the
absorbed heat on the cold side, and QH the released heat on the hot side.

 QL equals the Peltier-effect heat  𝛂ITL subtracting the Joulean-effect heat ( ½)I^2 R
and the transfer heat K(TH–TL):

Where 𝛂 is the Seeback coefficient, R is electrical resistance, I is operating current, K is

thermal conductivity.

 The applied voltage V is:

 The input power P is:

 And the thermal power given out of the hot side Q H

Based on the energy balance, a high order nonlinear dynamic model of the TE module system has
been developed in “Huang, B.J.; Duang, C.L. System dynamic model and temperature control of a
thermoelectric cooler. Intl. J. Refrig. 2000,23, 197–207”, which is then linearized to a linear second
order model in the paper using small signal linearization methods. In the paper the system was
identified based on step response tests, in order to simplify the model development.

For better understanding reading of the paper is recommended. 

Hybrid Controller Diagram

 G(s),C(s), and F(s) are transfer functions of the plant, feedback controller, and feedforward
controller, respectively. And, ‘r’ is the reference profile, ‘e’ the servo error, ‘u’ the plant
input, y the plant output, uBB the Bang-Bang action, uFF the feedforward action, and uFB the
feedback action.
 As shown in Fig. 4, the feedforward action plays, not only a role of model predictive control,
but also a role of consistent disturbance rejection to the fluctuation of ambient
temperature.
 The hybrid control strategy functions follow. If the absolute value of servo error e is greater
than a preset value 𝜀, then uBB is switched on, so that a maximum ramp rate is obtained;
otherwise, uBB is switched off, while uFF and uFB are switched on. The control action uis,
therefore:

 The switching threshold 𝜀 is chosen as a compromise between ramp rates and

overshoots. In practice, 𝜀 may be tuned to get high ramp rates with very small or,
even, no overshoot. Moreover, it was found that the changes of 1in a small region
have almost no influence on the control performance.
 Feedback Controller
 The tracking error function to be minimized:

 For accurate tracking, the Laplace transform of the ideal feedforward control action
should be the inverse of the plant transfer function

 However, such ideal feedforward control action is found to become easily saturated,
due to the large gain T/kp (e.g. 52.2/5.64 at 808C).

Figure 6 presents the relationship between the applied voltage and the
corresponding steady-state temperature of the heating block when the heating block
is heated with different voltages from the ambient temperature. To simplify the
design of feedforward action, a linear fitting model is developed. The fitted model
equation is:

where, T is the steady state temperature, u is the applied voltage, e(u) the linear fitting
error function, which is caused by the nonlinearity of the system model. e(u) ¼2.36 at
u¼0. The constant 24.26 is the initial temperature of the heat block at u¼0, i.e., the
ambient temperature.
 The feedback controller is used to accelerate ramp rates, reject disturbances, and
reduce the tracking errors. A two-degree of freedom (2-DOF) PID controller is
adopted, of which the derivative action is only applied to the output y, instead of the
error e, so as to avoid derivative kick due to abrupt changes of the setpoint in the
thermal cycling.
  Because, the integral action of simple PID control at steady-state is approximately
equivalent to the feedforward action integral separation is applied in the
feedforward/PID hybrid control strategy to prevent integral saturation

 In Eqs. (12) and (13), T, Kc, Ti, Td are the sampling period, proportional, integral
and derivative gains, respectively, y(.) the actual output, e(.) the error between the
setpoint and the actual output, 𝛿 =  Min{k, |e(k)|≤M},  |e(k)|≤ 𝜀, k = 1, 2, 3 ... , 0 < M
< 𝜀 and M is the switching threshold for the use of integral separation, which is
tuned by trial and error, 1is the switching threshold defined in Eq. (6). Trapezoidal
integration is used to avoid the effect of sudden changes of setpoint or output. In
addition, a nonlinear adjustment is used to suppress overshoot, [13] which makes
the integral action smaller for larger errors.

Embedded System with Hybrid Control

Graphical Results

This graph explains how accurate different methods are tracking down the
required curve.
The graph demonstrate that the hybrid control strategy performs much
better than the best tuned PID controller
Basic Programming Strategy

This is a very basic programming flowchart, modifications need to be made with increasing
complexity
Future Plan
Here we’ve tried to make simulations of the top lid heater with all the given parameters
and we were able to obtain some results now we have to try implementing them through
actual experiments to find their accuracy and improve our model.

Through the market survey we were able to gather information on the parts that can be
used to make a low budget cost thermocycler. We also have compared various controlling
strategies and find out that hybrid control strategy is a better option than conventional PID
control. Now we need to make a final design by exploring the option we’ve gathered for
both hardware and software to make a actual thermocycler.

Conclusion
This project provided great understanding regarding the simulations and modelling device
environments. It also provided us an opportunity to use and get familiar with COMSOL
software and the concept of modelling a system.

Also through the market survey I came to know about various devices and controlling
algorithms. Also I got to read many papers on this topic which I find fascinating. Overall this
summer project was very informative and provided opportunity to explore my area of
interest.

References
 https://www.coleparmer.com/blog/2013/08/29/lab-equipment-issues-to-
consider-in-pcr/
 https://www.electronicscomp.com/tec1-12706-thermoelectric-peltier-cooler-
generator-module-india
 https://www.mouser.in/ProductDetail/Vishay-Dale/RH050150R0FC02/?
qs=oFi4%2FQsgZXYdykWTBf8Cwn8R2%2FshRA70
 https://robu.in/product/12v-40w-ceramic-cartridge-heater/
 https://handling-solutions.eppendorf.com/sample-
handling/amplification/productivity/detailview-productivity/news/peltiers-in-pcr-
matter/
 https://cen.acs.org/articles/92/web/2014/09/Computers-Heat-Sink-Used-
Slash.html
 https://forum.arduino.cc/t/peltier-cooler-box-controlled-by-pid/522067
 http://www.iosrjournals.org/iosr-jeee/Papers/Vol14%20Issue%203/Series-
3/G1403034553.pdf
 https://www.researchgate.net/publication/6522763_Temperature_Control_for_P
CR_Thermocyclers_Based_on_Peltier-Effect_Thermoelectric
 https://www.researchgate.net/publication/259328615_Hybrid_Control_Strategy_
for_Thermal_Cycling_of_PCR_Based_on_a_Microcomputer
 https://online-shop.eppendorf.co.in/IN-en/PCR-44553/Cyclers-
44554/Mastercycler-nexus-PF-14698.html