Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

This method allows individuals without electronics expertise to build instruments such as this fluorimeter for isothermal nucleic acid amplification and detection, which is critical for molecular diagnostics. The main advantage of this technique is that the system can be completely assembled from commercially available materials and open source software at a low cost. This fluorimeter can be used with multiple isothermal amplification methods.

This is important because isothermal amplification methods are increasingly used to detect a wide range of infectious and inherited diseases. To assemble the optical housing, place a 3/16-inch long 4-40 threaded insert into the hole on top of the optics enclosure bottom STL piece and place a 1/4-inch long 4-40 threaded insert into all of the other holes of the piece. Insert the center test board, into the top cavity of the housing with the five pins facing toward the top and closest to the center axis of the device and secure the test board with a 3/16-inch long 4-40 screw.

Place one of the 20-millimeter focal length lenses in the section below the center test board with the convex side facing toward the bottom of the device and away from the test board. To create the first configuration, place the long pass filter, into the next section below the 20-millimeter focal length lens. To create the second configuration, place two yellow emission filter foils into the section below the lens.

To create the first configuration, place the dichroic mirror into the diagonal section near the center of the encasement while observing the filter orientation specified by the manufacturer. To create the second configuration, place the beam splitter into the diagonal section. Place a second 20-millimeter focal length lens into the section below the dichroic mirror or beam splitter depending on configuration, with the convex side pointing toward the top of the device.

To create the first configuration, place the excitation filter in the section to the right of the dichroic mirror, making sure the arrow points toward the dichroic mirror. To create the second configuration, place one blue excitation filter foil into the section to the right of the beam splitter. Place the 15-millimeter focal length lens to the right of the excitation filter with the convex side facing the dichroic mirror.

And place an LED into the remaining section of the print with the LED facing toward the dichroic mirror or beam splitter, depending on the configuration. Make sure that the two wires leading from the LED are inserted into the recess channels so that the print will close tightly. And repeat the setup for the other side of the 3D printing piece.

Then place the extruded portions of the top half of the encasement into the recessed grooves of the bottom half of the encasement to close the empty side of the piece with the optical components. And secure the parts together with 3/8-inch long 4-40 screws. To assemble the electronics and touch screen, connect the two mini breadboards and place the microcontroller into one of the breadboards, ensuring that the micro-USB port of the microcontroller faces outward.

To connect the LED modulation, connect the CTL pin of the LED+driver to a digital pin of the microcontroller. And the LED-pin of the LED driver to a GND pin of the microcontroller. Remove the plastic covers on the back of the breadboards and press the adhesive backing of the breadboards to the 3D printed part to attach the combined breadboards to the inside of the back portion of the LCD screen holder STL printed piece.

Secure the LCD screen holder with the assembled breadboards inside the optical enclosure with 1-inch long 4-40 screws. To connect the LED power supply, connect the LED-positive pin of the LED driver to the positive wire of the first LED. And connect the negative wire of the first LED to the positive wire of the second LED on the breadboard.

Connect the negative wire of the second LED to the LED-pin of the LED driver. To connect the LED power supply, use a barrel jack to 2-pin adapter to connect the positive and negative wires of the 10-volt power supply to the VIN+and VIN-pins of the LED driver, respectively. To connect the sensor test board power supply and data transfer, use a 4-pin female to male jumper wire to connect the SCK, SDA, VDUT, and GND pins on the light-to-digital sensor test boards to the mini breadboard through the gap in the top right of the LCD holder print.

On the breadboard, confirm that the 3.3 volt pin of the microcontroller and the VDUT pin of both test boards, the GND pin of the microcontroller, and GND pin of both test boards, the analog 4-pin of the microcontroller, and the SDA pin of both test boards, and the analog 5-pin of the microcontroller, and the SCK pin of both test boards are all connected. Use four M2.5 screws to secure the single board computer onto the LCD screen holder, with the HDMI and power adapter ports of the single board computer facing upward and the single board computer centered on the 3D printed part. Then connect the touch screen display to the single board computer according to the touch screen instructions.

And connect the HDMI port of the single board computer to the HDMI port of the touch screen. To record the real-time fluorescence data, after the heat block has been turned on and has reached the appropriate temperature, power on the single board computer and use a micro-USB to USB cable to connect the single board computer to the microcontroller. Open the provided Python script on the touch screen and change the measurement time.

Change the variable output file path to the name of the data file the program generates. And change the serial port variables to the desired values. Place two PCR tubes containing the reactions to be monitored into the heat block.

And place the fluorimeter onto the heat block with the PCR tubes centered between the four pegs extruding from each optical channel. After confirming that the 3D printed fluorimeter is attached, plug in the power supply adapter for the LEDs and start the Python program. A graphical user interface will appear on the LCD screen for measuring the real-time fluorescence in both PCR tubes.

At the end of the experiment, view the measurements and the output data files saved in the user-defined location. Once assembled, the fluorimeter performance can be validated by measuring the fluorescence from a dilution series of FITC dye. In this representative analysis, both channels of the fluorimeter showed a linear response across the desired range.

Shown here are the baseline-subtracted fluorescence of the recombinase polymerase amplification positive and the negative control reactions of a standard commercial kit measured on the second configuration of the fluorimeter. Real-time fluorescence measurements of a custom reverse transcription loop-mediated isothermal amplification reaction for SARS-COVID-2 RNA on the first configuration of the fluorimeter shows that amplification occurs as expected over a clinically relevant range of RNA copy numbers. At a time when global supply chains are very stressed, open source pieces of equipment like this fluorimeter can help us to reduce some of the health inequities that are associated with the pandemic.