A Generalizable Fluorescence Sensor Platform for Sample Preparation-Free Protein Detection.

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Biosensor Technology

Background:

• Enzyme-linked immunosorbent assays (ELISAs) provide high sensitivity and specificity for molecular detection.
• However, ELISAs often require extensive sample preparation and multiple reagents, limiting their application in rapid diagnostics.

Purpose of the Study:

• To introduce a generalizable biosensor platform for single-step, sample preparation-free detection of protein analytes.
• To achieve high sensitivity detection in complex biological samples.

Main Methods:

• Development of the NanoFluor system, conjugating Janelia Fluor dyes to nanobodies via HaloTag and a glycine-serine linker.
• Utilizing a fluorescence switch mechanism where dye emission is activated upon nanobody target binding.
• Employing molecular dynamics and QM/MM computational models to elucidate the fluorescence mechanism.

Main Results:

• The NanoFluor system demonstrated picomolar detection limits for diverse protein targets.
• Successful multiplexed detection was achieved in complex samples, including undiluted serum.
• The biosensor design proved versatile and simple, with a clear mechanistic basis for fluorescence change.

Conclusions:

• The NanoFluor biosensor platform enables rapid, sensitive, and sample preparation-free protein detection.
• This technology holds significant potential for point-of-care diagnostics and other molecular detection applications.
• The mechanistic understanding provides a foundation for further biosensor development.