Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

Borcherding

et al. 2020

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.

Section: To Demonstrate the Potential Of Time-resolved Flow Cytometrymentioning

confidence: 99%

Section: To Demonstrate the Potential Of Time-resolved Flow Cytometrymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Borcherding

et al. 2020

“…Bioanalytical, diagnostic, and security applications require the fast determination of a steadily increasing number of analytes or events in parallel in a broad variety of detection formats. Among the most popular approaches is the use of multiparametric fluorescence techniques for sample analysis due to their versatility and often straightforward use 1 – 3 . Many fluorescence techniques rely on a toolbox of luminescent labels for encoding and multiplexing 2 – 6 .…”

Section: Introductionmentioning

confidence: 99%

Luminescence lifetime encoding in time-domain flow cytometry

Wittkamp

et al. 2018

Time-resolved flow cytometry represents an alternative to commonly applied spectral or intensity multiplexing in bioanalytics. At present, the vast majority of the reports on this topic focuses on phase-domain techniques and specific applications. In this report, we present a flow cytometry platform with time-resolved detection based on a compact setup and straightforward time-domain measurements utilizing lifetime-encoded beads with lifetimes in the nanosecond range. We provide general assessment of time-domain flow cytometry and discuss the concept of this platform to address achievable resolution limits, data analysis, and requirements on suitable encoding dyes. Experimental data are complemented by numerical calculations on photon count numbers and impact of noise and measurement time on the obtained lifetime values.

“…The exploitation of the luminophore-characteristic luminescence lifetime (LT) as additional encoding parameter could in principle increase the number of accessible codes in flow cytometry analysis, thereby increasing the degree of multiplexing 6,7,14,[16][17][18][19][20][21] . Time-resolved fluorescence measurements in flow cytometry and lifetime encoding have been discussed for decades 8,[22][23][24][25][26][27][28] . The concept of lifetime multiplexing in a flow, however, has not been adopted in routine applications up to now due to the limited measurement time per object resulting in reduced photon count numbers 27,29 .…”

mentioning

confidence: 99%

Tempo-spectral multiplexing in flow cytometry with lifetime detection using QD-encoded polymer beads

Nifontova

et al. 2020

Semiconductor quantum dots (QDs) embedded into polymer microbeads are known to be very attractive emitters for spectral multiplexing and colour encoding. Their luminescence lifetimes or decay kinetics have been, however, rarely exploited as encoding parameter, although they cover time ranges which are not easily accessible with other luminophores. We demonstrate here the potential of QDs made from II/VI semiconductors with luminescence lifetimes of several 10 ns to expand the lifetime range of organic encoding luminophores in multiplexing applications using time-resolved flow cytometry (LT-FCM). For this purpose, two different types of QD-loaded beads were prepared and characterized by photoluminescence measurements on the ensemble level and by single-particle confocal laser scanning microscopy. Subsequently, these lifetime-encoded microbeads were combined with dye-encoded microparticles in systematic studies to demonstrate the potential of these QDs to increase the number of lifetime codes for lifetime multiplexing and combined multiplexing in the time and colour domain (tempo-spectral multiplexing). These studies were done with a recently developed novel luminescence lifetime flow cytometer (LT-FCM setup) operating in the time-domain, that presents an alternative to reports on phase-sensitive lifetime detection in flow cytometry.Flow cytometry (FCM), where either single cells or particles are optically detected in a flow, is an established and widespread multiparametric fluorescence technique used for many routine and research applications in biology, medical diagnostics, and food analysis 1-5 . FCM typically relies on organic luminophores for optical encoding and multiplexing as well as analyte quantification 6-10 . State-of-the-art FCM instruments are designed to detect spectral and/or intensity codes and can distinguish a large number of different colour and intensity codes, e.g. in the case of bead-based assays 5,11-13 . Commonly applied spectral multiplexing approaches utilizing organic luminophores are, however, limited by the spectral overlap of the emission bands of the different encoding dyes which limits the number of distinguishable codes and can require signal compensation and cross talk correction 6,14,15 . The exploitation of the luminophore-characteristic luminescence lifetime (LT) as additional encoding parameter could in principle increase the number of accessible codes in flow cytometry analysis, thereby increasing the degree of multiplexing 6,7,14,[16][17][18][19][20][21] . Time-resolved fluorescence measurements in flow cytometry and lifetime encoding have been discussed for decades 8,[22][23][24][25][26][27][28] . The concept of lifetime multiplexing in a flow, however, has not been adopted in routine applications up to now due to the limited measurement time per object resulting in reduced photon count numbers 27,29 . Moreover, the vast majority of reports on lifetime detection in flow cytometry relies on frequency-domain techniques (phase fluorometry), which used to be less expensive as f...