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Multiplexing Fluorescence Anisotropy Using Frequency Encoding.

Title: Multiplexing Fluorescence Anisotropy Using Frequency Encoding.
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Name(s): Schrell, Adrian M, author
Mukhitov, Nikita, author
Roper, Michael G, author
Type of Resource: text
Genre: Journal Article
Text
Date Issued: 2016-08-16
Physical Form: computer
online resource
Extent: 1 online resource
Language(s): English
Abstract/Description: In this report, a method to multiplex fluorescence anisotropy measurements is described using frequency encoding. As a demonstration of the method, simultaneous competitive immunoassays for insulin and glucagon were performed by measuring the ratio of bound and free Cy5-insulin and FITC-glucagon in the presence of their respective antibodies. A vertically polarized 635 nm laser was pulsed at 73 Hz and used to excite Cy5-insulin, while a vertically polarized 488 nm laser pulsed at 137 Hz excited FITC-glucagon. The total emission was split into parallel and perpendicular polarizations and collected onto separate photomultiplier tubes. The signals from each channel were demodulated using a fast Fourier transform, resolving the contributions from each fluorophore. Anisotropy calculations were carried out using the magnitude of the peaks in the frequency domain. The method produced the expected shape of the calibration curves with limits of detection of 0.6 and 5 nM for insulin and glucagon, respectively. This methodology could readily be expanded to other biological systems and further multiplexed to monitor increased numbers of analytes.
Identifier: FSU_pmch_27440478 (IID), 10.1021/acs.analchem.6b02131 (DOI), PMC4991543 (PMCID), 27440478 (RID), 27440478 (EID)
Grant Number: R01 DK080714
Publication Note: This NIH-funded author manuscript originally appeared in PubMed Central at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991543.
Subject(s): Fluorescence Polarization
Fluorescent Dyes/chemistry
Glucagon/analysis
Immunoassay
Insulin/analysis
Lasers
Persistent Link to This Record: http://purl.flvc.org/fsu/fd/FSU_pmch_27440478
Owner Institution: FSU
Is Part Of: Analytical chemistry.
1520-6882
Issue: iss. 16, vol. 88

Choose the citation style.
Schrell, A. M., Mukhitov, N., & Roper, M. G. (2016). Multiplexing Fluorescence Anisotropy Using Frequency Encoding. Analytical Chemistry. Retrieved from http://purl.flvc.org/fsu/fd/FSU_pmch_27440478