Three-Dimensional FRET Multiplexing for DNA Quantification with Attomolar Detection Limits

The Journal of Physical Chemistry Letters
Xue QiuNiko Hildebrandt

Abstract

Photoluminescence (PL) multiplexing usually relies on spectral or temporal separation. A combination into higher-order multiplexing for biosensing is extremely challenging because the PL intensity is required for target quantification at very low concentrations and the interplay of color, lifetime, and intensity must be carefully adapted. Here, we demonstrate time-gated Förster resonance energy transfer (TG-FRET) from a long-lifetime Tb complex to Cy3.5 and Cy5.5 dyes for spectrotemporal multiplexing of four different DNA targets in the same sample by single-color excitation and two-color detection. We used rolling circle amplification (RCA) for high specificity and sensitivity and for placing Tb donors and dye acceptors at controlled distances within the amplified DNA concatemers. This precise distance tuning led to target-specific PL decays of the FRET pairs and simple, separation-free, and higher-order multiplexed quantification of DNA. The RCA-FRET DNA assay could distinguish very homologous target sequences and provided limits of detection down to 40 zeptomoles (300 aM).

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Citations

Aug 8, 2018·Angewandte Chemie·Chi ChenNiko Hildebrandt
Nov 15, 2018·Scientific Reports·Daniel KageUte Resch-Genger
Dec 14, 2018·Chemical Science·Xue QiuNiko Hildebrandt
Jul 25, 2019·Mikrochimica Acta·Zeki Semih PehlivanMeral Yüce
Nov 12, 2020·Scientific Reports·Daniel KageUte Resch-Genger
Feb 5, 2021·Chemical Society Reviews·Thomas JetValérie Taly
Aug 3, 2021·Analytical Biochemistry·Mahsa ImaniNosratollah Zarghami
Jul 1, 2020·ACS Sensors·Yannick Rondelez, Guillaume Gines
Aug 29, 2019·ACS Applied Materials & Interfaces·Jae Chul ParkYoon Sung Nam
Oct 20, 2018·Analytical Chemistry·Diego Mendez-GonzalezTero Soukka
Jul 7, 2020·Journal of the American Chemical Society·Daniel KovacsK Eszter Borbas
Jun 17, 2020·The Journal of Physical Chemistry Letters·Pradyut RoyPramod P Pillai
Jun 30, 2019·Journal of the American Chemical Society·Chi ChenNiko Hildebrandt
Oct 15, 2021·Basic and Clinical Andrology·Anne-Sophie NeyroudCélia Ravel

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