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

Abstract

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 I 2 S 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 highaccuracy temperature management in PCR tubes (±0.1 °C) allowing us to perform highresolution thermal shift assays (TSA) from exemplary biological applications.

Nanomolar Protein Thermal Profiling with Modified Cyanine Dyes

“…To assess the compatibility of the one-step assay with the presence of various chemicals, we determined the T m values of MDH (100 nM) in both the absence and presence of MgCl 2 (2.5 mM), CaCl 2 (1 mM), dithiothreitol (DTT, 10 mM), and glycerol (2.5%) using the one-step peptide-probe assays (Figure S32). The chemicals slightly increased the T m value of MDH (1–2.2 °C) as expected because these chemicals have been reported to increase protein T m values. − The experiments were run under a typical concentration used in assay solutions, demonstrating the compatibility of the one-step peptide-probe for assaying protein stability.…”

Emerging Technologies for Fluorescence-Based Optical Test Strip Readers

“…Correct sensing is very important to quantify the target molecule as the amount of the target analyte in the sample immediately correlates with the produced light intensity [17][18][19]. In some cases, the target analyte concentration in the sample is estimated by comparing the intensity of the fluorescent signal to a reference signal [20]. Additional components such as a microcontroller, a user interface, and software for data analysis and interpretation are included in the design of optical test-strip reader devices [21].…”