ThermoX

Overview

Antimicrobial Resistance (AMR) represents a growing challenge in medical treatment, complicating the management of bacterial infections and demanding innovative approaches beyond the creation of new antibiotics. One of the greatest challenges with preventing the spread of AMR is accurately diagnosing the type of pathogen that causes an infection. This can result in viral infections being mistaken for bacterial ones, leading to improper antibiotic treatment. As pathogens are continually exposed to unnecessary antibiotics, it drives further AMR development. Thus, highlighting an urgent need for accurate diagnostic tools. Quantitative Polymerase Chain Reaction (qPCR) has emerged as a vital diagnostic tool, instrumental in accurately diagnosing infections. qPCR is a wet lab procedure similar to Polymerase Chain Reaction (PCR), however with qPCR, users can monitor the amplification of target DNA using fluorescent dyes or probes in real time. Yet, many conventional qPCR machines are often costly and inaccessible in low-resource areas, creating significant obstacles for effective disease detection and monitoring (Ramírez AL, 2018). Acknowledging this disparity, Lambert iGEM aims to create a cost-effective qPCR machine utilizing heating components from Gaudi Labs’ PocketPCR along with a 488-nanometer wavelength laser and a photodiode. In turn, providing a low-cost solution that ensures broader access to reliable diagnostic testing, which ultimately aids in the global effort to mitigate AMR.

Design

During the design process of ThermoX, our team divided the project into three key subsystems:

1. Thermocycling (PCR): This subsystem is responsible for the rapid heating and cooling required for precise DNA amplification.
2. Intermediate Mechanism: This subsystem serves as the crucial link connecting the other two subsystems.
3. Real-time fluorescence detection: This subsystem enables the monitoring and quantifying the reaction as it progresses

This approach allowed us to tackle the complex requirements of each subsystem independently while ensuring seamless integration in our final design.

Polymerase Chain Reaction (PCR)

In order to conduct PCR (Polymerase Chain Reaction) in a cost-effective manner, Lambert iGEM iterated upon PocketPCR [GaudiLabs, 2021] – a 99 euro, open-source thermocycler capable of simultaneously characterizing 5 PCR tubes. PocketPCR works as a conventional thermocycler consisting of a heating block, assistive cooling, and PID temperature controller, yet it is sold at a fraction of the size and cost of commercially available thermocyclers. Thus, making it an ideal choice for ThermoX.

Intermediate Mechanism: Electromagnet

In order to quantify the fluorescence in each tube Lambert iGEM designed an intermediate mechanism to transfer the tube from the thermocycling subsystem to the fluorescence quantification subsystem (see Figure 1). The mechanism relies upon an electropermanent magnet sequentially being raised and lowered to individually lift each tube to be quantified then restored to its original position. To accomplish this we attached the magnet to a rack and pinion assembly and fitted each tube with a 3D printed structure that holds a piece of metal. The rack and pinion assembly is a mechanical system that converts rotational energy into linear motion using a circular gear and a gear rack. In the context of our system, by rotating the circular gear clockwise and counterclockwise we can control the vertical position of the magnet. When it is lowered a current is run through the electropermanent magnet generating a magnetic field, transitioning it into its “on” stage. This attracts the metal which is attached to the tube. When the magnet is raised it brings along the tube and positions it for quantification. Following quantification, the tube is lowered and current ceases to flow through the magnet, transitioning the system into its “off” state, breaking the link between the magnet and the tube.

Fluorescence Detection

ThermoX quantifies fluorescence utilizing three parts: a 488 nanometer (nm) laser, photodiode, and filter. In ThermoX, the 488 nm laser is positioned perpendicular to the tubes, emitting light that passes through the tube and excites the dye. The resulting fluorescence passes through the filter and is detected by the photodiode situated behind the tube, creating a linear optical pathway. The photodiode, a semiconductor device that converts light into electrical current, captures the laser output and transmits the resulting fluorescence signal to the Arduino microcontroller, where the intensity of the fluorescence is quantified in relative fluorescence units (RFU). To calibrate the system, we will evaluate varying concentrations of Green Fluorescent Proteins (GFP), creating graphs that correlate GFP concentrations to RFU values. Building on this, we developed a model to translate RFU into DNA concentrations, aiding our hardware team’s efforts. The ThermoX system then efficiently measures each of the 5 tubes in sequence, with the heat block rotating after each quantification. By combining the fluorescence data from ThermoX with our modeling simulations, Lambert iGEM successfully developed a frugal system capable of accurately performing quantitative PCR (qPCR), advancing DNA analysis with an affordable approach.

Parts + Cost Breakdown

Software

To complement ThermoX’s hardware, Lambert iGEM created a software app to facilitate easy and efficient use of the qPCR machines. Existing frugal qPCR devices have complicated, outdated, or inefficient software tools making it challenging for the user to operate the device. ThermoX’s tool features a simplistic design with a user-friendly interface, making it possible for anyone to use the device (see Figure 1).

The app is designed in the language _____, and connects seamlessly to the ThermoX device via Bluetooth. Using an Arduino microcontroller, the software app can send instructions to the device based on user input, including cycle length, temperature, and number of cycles. In order to generate fluorescent curves from qPCR experiments, the microcontroller collects RFU values from the photodiode and converts them into digital values using its Analog-to-Digital (ADC) converter and sends it to a Google Firestore Database via wifi to be displayed in the software. The mobile app shows the fluorescence intensity relied upon by the concentration of the inhA gene as well as PCR data as shown in Figure 2.

Results

To validate the accuracy of ThermoX, Lambert iGEM ran several cycles of PCR and quantified qGRN Master Mix concentrations to prove the laser-photodiode system is functional.

[Graph showing results of ThermoX through several samples of whatever we test with]

To determine its effectiveness compared to commercial thermocyclers and fluorometers, Lambert iGEM also tested the PCR aspect and the quantification section separately, showing the efficiency of a frugal qPCR machine.

[Graph showing results of just the PCR part] [Graph showing results of just the fluorometer part]

Future Implementations & Conclusion

ThermoX shows accurate results in quantifying the qGRN Master Mix, and proves its reliability when compared to commercial qPCR machines.