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Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules

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Multiplexing Fluorescence Anisotropy Using Frequency Encoding.

Adrian M Schrell¹, Nikita Mukhitov¹, Michael G Roper¹

¹Department of Chemistry and Biochemistry, Florida State University , 95 Chieftain Way, Tallahassee, Florida 32306, United States.

Analytical Chemistry

|July 22, 2016

Summary

This summary is machine-generated.

This study introduces frequency encoding for multiplexed fluorescence anisotropy, enabling simultaneous detection of insulin and glucagon immunoassays. This method offers a sensitive approach for multi-analyte biological monitoring.

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Area of Science:

Biophysical Chemistry
Analytical Chemistry
Biotechnology

Background:

Multiplexing fluorescence anisotropy measurements is crucial for simultaneous analysis of multiple analytes.
Existing methods may face limitations in sensitivity and throughput for complex biological samples.
Frequency encoding offers a novel approach to differentiate and quantify multiple fluorophores.

Purpose of the Study:

To describe a novel method for multiplexing fluorescence anisotropy measurements using frequency encoding.
To demonstrate the method's capability by performing simultaneous competitive immunoassays for insulin and glucagon.
To establish the sensitivity and applicability of the frequency-encoded fluorescence anisotropy technique.

Main Methods:

Utilized frequency encoding with distinct laser pulsing frequencies (73 Hz for Cy5-insulin, 137 Hz for FITC-glucagon) for simultaneous excitation.
Measured the ratio of bound and free labeled analytes (insulin and glucagon) in competitive immunoassays.
Employed fast Fourier transform (FFT) for signal demodulation and anisotropy calculation in the frequency domain.

Main Results:

Successfully demonstrated simultaneous competitive immunoassays for insulin and glucagon.
Achieved limits of detection of 0.6 nM for insulin and 5 nM for glucagon.
Observed calibration curves with expected shapes, validating the method's performance.

Conclusions:

Frequency-encoded fluorescence anisotropy is a viable method for multiplexed bioassays.
The technique provides sensitive and simultaneous detection of multiple analytes.
This methodology is adaptable for broader applications in biological systems and increased analyte monitoring.