Technical field
[0001] The present invention relates to a PCR apparatus for nucleic acid amplification.
Background art
[0002] The polymerase chain reaction (PCR) is widely used by research professionals around
the world to amplify small strands of DNA. Typically, PCR is performed using automated
thermal cyclers that alternately heat and cool numerous small tubes containing the
PCR reaction mixture. Since the invention of the PCR, numerous designs for thermocycling
devices have been developed in an effort to increase the speed of nucleic acid amplification.
The need for rapid methods and devices for examining genetic material became particularly
apparent during the pandemic. One way to increase the speed of PCR reaction is to
use thermocyclic plate (2) made of material that allows the temperature to increase
and drop as fast as possible. In the analysis of various temperature characteristics
of materials suitable for ultra-fast temperature change, a graphite was selected -
a material with high thermal conductivity and low specific heat capacity. When comparing
graphite with aluminum, it was found that graphite thermocyclic plate (2) of the same
shape (equal volume) requires about 40% less energy compared to aluminum thermocyclic
plate (2). Graphite allows for higher heating/cooling rates compared to commonly used
aluminum. The closest prior art document
CN203921614U uses a thermocyclic plate (2) comprising thin graphite elements between layers of
other materials. The apparatus described herein uses a thermocyclic plate (2) made
entirely of graphite allowing faster temperature changes.
Brief description of drawings
[0003] Fig. 1 3D representation of the apparatus for nucleic acid amplification.
Detailed description
[0004] The device consists of four main modules:
- 1. Power supply module.
- 2. Temperature module.
- 3. Fluorescence excitation-detection module.
- 4. Control module.
[0005] The power supply module consists of a high-current battery and an external power
supply / charging unit. The PCR device can operate on both internal battery power
and an external power supply. The internal battery is equipped with a protection module
that controls the charging / discharging currents, balances the charging of the cells,
protects against excessive discharge. The external power supply / charging unit can
generate up to 22A at 16.8 V. The external operation of the power supply is organized
in such a way that the batteries are also charged when the device is operated from
an external power supply. The control module constantly monitors the battery charge
level and displays it on the screen. If the charge level is insufficient for one procedure,
the control module will not start the procedure until the unit is connected to an
external power supply or charged enough to perform at least 1 complete procedure.
[0006] The temperature module consists of a thermocyclic plate (2), a heating / cooling
element and an active heat dissipation system. In the analysis of various temperature
characteristics of materials suitable for ultra-fast temperature change, a material
with high thermal conductivity and low specific heat capacity - graphite - was selected.
When comparing graphite with aluminum, it was found that graphite thermocyclic plate
(2) of the same shape (equal volume) requires about 40% less energy compared to aluminum
thermocyclic plate (2). Graphite allows for higher heating / cooling rates compared
to commonly used aluminum. The plate was provided with 2 wells (5) for 0.2 ml tubes
2 channels for fluorescence excitation and 2 channels for fluorescence collection.
To further reduce thermal inertia, the thermocyclic plate (2) design was optimised
to have high surface area to mass ratio. Depending on the mass of the thermocyclic
plate (2) and the required rate of temperature change for heating / cooling, a 430W
Peltier element was selected. To achieve the highest heat transfer coefficient between
the thermocyclic plate (2), the Peltier element, the passive cooling element and a
metallized thermal paste was used. The active heat dissipation system consists of
an aluminum radiator and an active fan that blows heat away from the radiator. The
active cooling element is selected to dissipate 450W of the heat generated by cooling
the thermocyclic plate (2).
[0007] The most efficient energy transfer conditions of the Peltier element were maintained
to ensure the maximum rate of temperature change. The energy transfer of the damper
element is highest when the temperature difference between the hot and cold sides
is smallest. As a result, the radiator temperature was actively controlled by regulating
the fan speed.
[0008] Evaporation of the solution occurs when the sample is heated. Due to the extremely
small volume of the sample, all of the solution evaporates and amplification does
not occur, although the liquid condenses and drains to the bottom of the tube as it
cools. For this purpose, a higher temperature must be maintained at the top of the
tube to prevent evaporation. Amplification experiments showed that the temperature
difference between the enclosure and the maximum thermocyclic plate (2) should be
in the range of 5-10 °C for optimal amplification efficiency.
[0009] The fluorescence excitation-detection module consists of light sources (1), filters
(3) and detectors (6). The components of a fluorescence excitation detection system
are closely related. Excitation should be selected in the spectral range where dye
absorption is highest and recording in the spectral range where dye fluorescence is
most intense. However, the absorption and fluorescence bands overlap and these areas
of maximum intensities are adjacent to each other at a distance of about 20 to 30
nm, so that with conventional color filters, the excitation light enters the detector
(6), thus reducing the overall sensitivity of the system. Depending on the dyes to
be used, a combination of LED and excitation / detection filters (3) was chosen to
ensure no overlap in the excitation and detection areas and a high sensitivity for
fluorescence detection. The last important component in the fluorescence excitation
detection module is the detector (6). During the test, the volume of the test solution
is very small (-10 ul) and the fluorescence intensity is very low, so the detector
(6) must be extremely sensitive. At the same time, the detector (6) must be fast enough
to record fluorescence at a stable temperature point. A fast, high-sensitivity, PIN-type
photodiode has been selected for this purpose. The photovoltaic diode operating mode
is selected to reduce the noise generated by the dark current. To increase the recording
efficiency of the photodiode, it was mounted behind a thermocyclic plate (2) and the
fluorescence from the tube to the photodiode was "transported" by a fiber optic (4)
system that additionally acts as a spatial filter to reduce the effects of reflections
(Add a detailed light removal filtration scheme). The photodiodes are additionally
actively cooled. For the registration of extremely low currents, the photodiodes were
equipped with FET-type two-stage operational amplifiers, additionally shielding them
from external electromagnetic fields using the Faraday cage principle. In the first
stage, the current of the photodiode is converted into a voltage, and in the second
stage, this voltage is increased.
[0010] The control module consists of 5 parts - heating / cooling element controller, passive
coolant temperature controller, temperature feedback controller, fluorescence controller
and process controller. The requirements for the heating / cooling element controller
are - fast and accurate control of high current in both directions of current flow
with minimal heat loss. A multitransistor current control model was chosen for this.
The passive coolant temperature controller controls the active passive coolant temperature
controller, thus ensuring the most efficient energy transfer of the Peltier element.
The purpose of the temperature feedback controller is to periodically record the thermocyclic
plate (2) temperature at high speed and high accuracy and transmit the information
to the process controller. The process controller has an Integrated ADC converter
that digitizes the incoming signal and transmits it for further processing. The fluorescence
controller consists of two modules - the excitation source controller and the detection
module controller. The excitation source controller must be stabilized to ensure uniform
excitation intensity as operating conditions change. The controller of the detection
module must ensure the amplification of the weak fluorescence signal and efficient
noise filtering. One of the most complex components of a device is the process controller.
It must be able to receive information from different input devices, process digital
and analog signals, control different types of controllers, and present the processed
information to output devices. A program and a set of algorithms were written for
parameter registration, analysis and process control. Fuzzy logic algorithm was used
to control the temperature of the thermocyclic plate (2). The algorithm compares the
measured actual thermocyclic plate (2) temperature with the set one, which depends
on the selected test type. The difference between the measured and set temperature
is multiplied by a fixed constant and converted to time. This time is used to generate
a modulation control signal (PWM) for the Peltier module controller.
[0011] The temperature in the thermocycler is controlled by recording the temperature of
the thermocyclic plate (2). Meanwhile, the contact of the tube with the thermocyclic
plate (2) and a certain coefficient of temperature transfer to the solution results
in temperature differences between the thermocyclic plate (2) and the solution. The
temperature in the tube is - 2-5 degrees different from the temperature of the thermocyclic
plate (2) in the apparatus. After about 2-3 seconds, the temperature in the tube reaches
the set value. Taking into account these temperature differences, the temperature
control algorithm was adjusted - the temperature of the plate is raised to such values
(experimental and modeling data) that the solution in the cuvette reaches the temperature
specified in the procedure.
[0012] Principle of operation. The instrument is turned on and the test type is selected
(procedure type with pre-recorded specific number of cycles, temperature settings,
fluorescence recording settings). One type of study is manual, i.e. it is possible
to fully select all parameters according to the user's needs. The battery charge is
estimated and in the event of an insufficient charge level, the selected procedure
is notified to the user, the procedure is not allowed to run on battery power only,
and the procedure is suggested to be performed when connected to an external power
source. If the initial conditions are met, the procedure is initiated. The instrument
asks you to add a sample. After inserting the sample, the instrument checks that the
tube is inserted and after closing the lid, asks you to confirm with the button on
the display that we are starting the amplification procedure. The appliance rolls
up or the lid is properly (completely) closed and the hood starts to heat up. The
hood is heated to maximum capacity. When the enclosure temperature reaches 80% of
the default temperature, the heating of the main thermoblock is switched on. When
both the hood and the main thermoblock reach the set temperatures, a cyclic process
is started, the parameters of which are pre-programmed. The instrument control is
programmed so that the time and temperature parameters of the cycle can be changed
within the ranges specified in the instrument characteristics, and fluorescence recording
is possible at any stage of the cycle. This provides opportunities to perform unique
experiments in the study of the mechanisms of dye intercalation into a DNA molecule.
The display always shows the number of cycles and, depending on the settings, a curve
of the fluorescence intensity and the fluorescence value of the current cycle. The
data is written to the memory chip in real time. After the procedure (all cycles have
been completed), the temperature maintenance devices are switched off. The screen
informs you of the end of the procedure, analyzes the data, and displays the result
- the answer to the clinical question / task. If necessary, the next procedure can
be started immediately by selecting the start new procedure button on the device screen.
When this option is selected, you are again asked to select the type of procedure
you want to perform and the sequence of steps described above is initiated.
1. An apparatus for nucleic acids amplification, comprising:
a power supply module comprised of a battery and/or an external power supply and/or
charging unit for supplying power to the apparatus;
a fluorescence excitation-detection module comprised of one or more light source (1),
filter (3) and detector (6) for measuring an amount of product formed;
a control module for monitoring charge level; and
a temperature module comprised of a thermocyclic plate (2) and a heating/cooling element;
characterised in that the thermocyclic plate (2) is made of graphite allowing to achieve higher heating/cooling
speeds for faster polymerase chain reactions and allowing to form reaction products
more quickly.
2. The apparatus of claim 1, wherein graphite thermocyclic plate (2) further comprises
one or more wells (5) configured to hold a sample wherein the wells (5) are configured
to hold one or more 0.2 ml tubes.
3. The apparatus of claim 1, wherein graphite thermocyclic plate (2) comprises one or
more channels for fluorescence excitation and detection.
4. The apparatus of claim 1, wherein the heating/cooling element is Peltier element.
5. The apparatus of claim 1 and 5, wherein the Peltier element is 430W.
6. The apparatus of claim 1, wherein a metallized thermal paste is used between the thermocyclic
plate (2) and a passive cooling element.
7. The apparatus of claim 1, wherein the power is supplied either by an internal battery
or external power supply.
8. The apparatus of claim 1, wherein the control module continuously monitors and displays
charge level.
9. The apparatus of claim 8, wherein the control module does not allow to start PCR reaction
until the apparatus has a sufficient level of charge for at least a single PCR reaction
or is connected to an external power supply.
10. The apparatus according to claim 1, wherein the thermocyclic plate (2) temperature
is controlled using the fuzzy logic algorithm.
11. The apparatus according to claim 1, for use in dye intercalation to DNA molecules.
12. A method of using the apparatus of claim 1 comprising:
collecting a fluid sample;
disposing the fluid sample within one or more wells (5) of the thermocyclic plate
(2);
amplifying DNA using repeated cycles of heating and cooling.
13. The method of claim 13 for use to detect a gene variant of the CYP4F2 enzyme (rs3093135).
14. A thermocyclic plate (2) made of graphite according to any preceding claims that allows
to increase and decrease temperature more quickly allowing for polymerase chain reactions
to occur faster.
15. A fiber optics (4) used for fluorescence excitation and detection light paths to reduce
thermal effects on light source (1) and detectors (6) and to realize light spatial
filtration to increase spectral filtration effectiveness and consequently increase
signal to noise ratio.