A low-cost 3D-printable differential scanning fluorometer for protein and RNA melting experiments

Published: 29 November 2021| Version 2 | DOI: 10.17632/73rt8s7pwd.2
Contributor:
Fabian Barthels

Description

Differential scanning fluorimetry (DSF) is a widely used biophysical technique with applications to drug discovery and protein biochemistry. DSF experiments are commonly performed in commercial real-time polymerase chain reaction (qPCR) thermal cyclers or nanoDSF instruments. Here, we report the construction, validation, and example applications of an open-source DSF system for 176 €, which, in addition to protein-DSF experiments, also proved to be a versatile biophysical instrument for less conventional RNA-DSF experiments. Using 3D-printed parts made of polyoxymethylene, we were able to fabricate a thermostable machine chassis for protein-melting experiments. The combination of blue high-power LEDs as the light source and stage light foil as filter components was proven to be a reliable and affordable alternative to conventional optics equipment for the detection of SYPRO Orange or Sybr Gold fluorescence. The ESP32 microcontroller is the core piece of this openDSF instrument, while the in-built I2S interface was found to be a powerful analog-to-digital converter for fast acquisition of fluorescence and temperature data. Airflow heating and inline temperature control by thermistors enabled high-accuracy temperature management in PCR tubes (±0.1 °C) allowing us to perform high-resolution thermal shift assays (TSA) from exemplary biological applications.

Files

Steps to reproduce

AirDiffuser: The air diffuser (POM) divides the heated airflow among the four individual measuring cells and leads to turbulent air mixing and thus to a homogeneous temperature profile. DiodeShield: The diode shield (PETG) is designed to shield the photodiodes from external light or daylight to reduce the noise of the recorded fluorescence signal. FanAdapter: The fan adapter (PETG) converts the rectangular outlet of the Sunon radial fan to a circular fitting of the heating tube. FluorescenceCell: The fluorescence cell (PETG) creates a framework into which the sample holders are mounted. HeatingTube: The heating tube (POM) incorporates the coiled heating wire. Housing: The housing of the circuit boards protects the electronic components during use. LEDHolder: High-power LEDs are glued onto the LED holders and serve to stabilize them. openDSF: Project folder (openDSF.zip) for insertion into an ESP_IDF development environment. Python GUI (openDSF.py) to control the device from the user’s PC. PhotoDiodePCB: Material for the fabrication of printed circuit boards of the photodiode modules. SampleCover: A light and temperature shielding cover (PETG+POM) is placed over the measuring cell, through which the temperature sensors are inserted into the PCR tubes. The heated airflow of the instrument exits through this cover, which is why the contact points to the hot air were protected with thermally robust POM inserts. SampleHolder: The sample holders (POM) are shaped as Laval nozzles to ensure optimal airflow and heat transfer. Due to the pressure loss of the nozzle, homogeneous air distribution is achieved. The sample holders provide holes for the LEDs and photodiodes. TemperatureSensor: The temperature sensors (PETG) hold the individual thermistors and shield the sensitive electrical contacts from moisture and physical contact. WiringDiagram: Circuit diagram overview of the electronic components of the openDSF system created with KiCad Eeschema. 3D-printing profiles: List of 3D-printer settings (.ini) for the different filaments used (PETG and POM).

Institutions

Johannes Gutenberg Universitat Mainz

Categories

Drug Discovery, Biophysical Chemistry, Open Source Product

License