Expanded single-color barcoding in microspheres with fluorescence anisotropy for multiplexed biochemical detection

Area of Science:

• Biotechnology
• Materials Science
• Analytical Chemistry

Background:

• Conventional fluorescence barcodes suffer from spectral crosstalk, limiting multiplexing capabilities.
• Fluorescence anisotropy (FA) offers a self-referencing method to distinguish spectrally similar fluorophores.
• Developing new barcoding strategies is crucial for high-throughput analyses.

Purpose of the Study:

• To introduce the first fluorescence anisotropy (FA)-based encoding strategy for polystyrene (PS) microspheres.
• To demonstrate the potential of FA for creating spectrally compatible fluorescent barcodes.
• To apply FA-encoded microspheres for sensitive detection of specific oligonucleotide sequences.

Main Methods:

• Incorporating fluorescent labels with similar emission spectra but different properties into PS microspheres.
• Utilizing Förster Resonance Energy Transfer (FRET) and mixing dyes with distinct intrinsic FA values to generate unique FA signals.
• Developing a custom microscopic FA imaging platform for real-time decoding of FA-encoded microspheres.
• Applying the FA-encoded microspheres for the detection of Bacillus cereus oligonucleotides.

Main Results:

• Successfully encoded polystyrene microspheres using fluorescence anisotropy (FA) for the first time.
• Demonstrated single-color 5-plex FA-encoded microspheres with real-time decoding.
• Achieved successful detection of Bacillus cereus oligonucleotide without spectral crosstalk.
• FA signals were stabilized within PS microspheres due to restricted rotational motion.

Conclusions:

• FA-based encoding provides a robust method to overcome spectral crosstalk in fluorescence barcoding.
• This approach significantly expands coding capacity by utilizing the FA dimension.
• FA-encoded microspheres show great promise for multiplexed, high-throughput chemical and biological analyses.