Lifetime encoding in flow cytometry for bead-based sensing of biomolecular interaction

Abstract

To demonstrate the potential of time-resolved flow cytometry (FCM) for bioanalysis, clinical diagnostics, and optically encoded bead-based assays, we performed a proof-of-principle study to detect biomolecular interactions utilizing fluorescence lifetime (LT)-encoded micron-sized polymer beads bearing target-specific bioligands and a recently developed prototype lifetime flow cytometer (LT-FCM setup). This instrument is equipped with a single excitation light source and different fluorescence detectors, one operated in the photon-counting mode for time-resolved measurements of fluorescence decays and three detectors for conventional intensity measurements in different spectral windows. First, discrimination of bead-bound biomolecules was demonstrated in the time domain exemplarily for two targets, Streptavidin (SAv) and the tumor marker human chorionic gonadotropin (HCG). In a second step, the determination of biomolecule concentration levels was addressed representatively for the inflammation-related biomarker tumor necrosis factor (TNF-α) utilizing fluorescence intensity measurements in a second channel of the LT-FCM instrument. Our results underline the applicability of LT-FCM in the time domain for measurements of biomolecular interactions in suspension assays. In the future, the combination of spectral and LT encoding and multiplexing and the expansion of the time scale from the lower nanosecond range to the longer nanosecond and the microsecond region is expected to provide many distinguishable codes. This enables an increasing degree of multiplexing which could be attractive for high throughput screening applications.

A Review of New High-Throughput Methods Designed for Fluorescence Lifetime Sensing From Cells and Tissues

“…The long-lifetime td-FCM instrument measured the focused cells acoustically, and the throughput was lower compared to conventional cytometry. Similarly, Kage et al [ 69 ] developed a td-FCM system to measure the lifetime values of four types of quantum-dot-doped polymer beads. In this study, it was demonstrated that longer lifetimes of the quantum-dot-doped beads can be discriminated from shorter lifetimes found in dye-based beads by measuring the respective decay by separate tempo-spectral channels.…”

Microchip imaging cytometer: making healthcare available, accessible, and affordable

“…Spectral multiplexing identifies the analyte by its emission spectrum. Chia et al reported optical sectioning fluorescence microscopy using the spectral multiplexing method 89 . As shown in Fig.…”

Toward absolute viability measurements for bacteria

“…Several groups have performed frequency-based lifetime flow cytometry by retrofitting lifetime channels into older cytometers [48] or have integrated flow-cells into a benchtop lifetime system [49]. The vast majority of the work in the literature focuses on lifetime flow cytometry of mammalian cells [50,51] although one article reports changing lifetime of EGFP expressing E. coli as they are phagocytized into macrophages [52]. The promise that lifetime-based viability measurements shows in this work should be transitioned to a lifetime flow cytometer so that the technique may meet its full potential.…”