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Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

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Lifetime-based multiplexed detection of viral RNA using fluorogenic aptamers.

Yuan-I Chen¹, Yu-An Kuo¹, Yujie He¹

¹Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, USA.

Biorxiv : the Preprint Server for Biology

|April 27, 2026

Summary

This summary is machine-generated.

This study introduces a new method using DNA fluorogenic aptamers (FAPs) with distinct fluorescence lifetimes for multiplexed viral RNA detection, overcoming cross-reactivity and spectral overlap issues for improved diagnostics.

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Area of Science:

Molecular Biology
Biotechnology
Analytical Chemistry

Background:

Fluorogenic aptamers (FAPs) are promising for nucleic acid sensing but limited by cross-reactivity and spectral overlap in multiplexed applications.
Existing FAP technologies struggle to differentiate multiple targets simultaneously due to similar emission spectra.

Purpose of the Study:

To develop a fluorescence-lifetime-based multiplexed detection strategy for viral nucleic acids using engineered DNA fluorogenic aptamers (FAPs).
To overcome limitations of cross-reactivity and spectral overlap in FAP-based multiplexed sensing.
To engineer FAP variants with distinct fluorescence lifetimes for robust target discrimination.

Main Methods:

Developed FAP-FLIM-NGS (fluorogenic aptamer-based fluorescence lifetime imaging microscopy on next-generation sequencing chips) for high-throughput screening of FAP variants.
Screened approximately 10^4 Lettuce/TO1-biotin complexes on an Illumina MiSeq flow cell to identify variants with distinct fluorescence lifetimes.
Designed split Lettuce probes targeting viral RNA fragments from SARS-CoV-2, MERS-CoV, and influenza A for multiplexed detection assays.

Main Results:

Identified three Lettuce variants with distinct fluorescence lifetimes: smC14T (6.0 ns), dmA5T/C14T (5.2 ns), and dmA5T/T22A (4.4 ns).
Demonstrated robust discrimination of individual and mixed viral RNA targets using phasor plot analysis based on fluorescence lifetimes.
Successfully overcame challenges of cross-reactivity and spectral overlap in multiplexed viral RNA detection.

Conclusions:

Established a generalizable strategy for engineering FAPs with customized photophysical properties, specifically distinct fluorescence lifetimes.
The fluorescence-lifetime-based multiplexed detection strategy offers a robust approach for next-generation diagnostics and molecular sensing.
This method enables precise differentiation of multiple nucleic acid targets, paving the way for advanced biosensing technologies.