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Analytical methods require efficient and versatile strategies to measure an increasing number of analytes that can be used in conjunction with established platforms like flow cytometry. Spectral multiplexing suffers from problems such as spectral crosstalk and often requires different excitation light sources increasing instrumentation costs. Thus, the number of distinguishable reporters with intensity-based barcodes is limited. An alternative can be lifetime encoding for discrimination of fluorophores based on their fluorescence decay kinetics.
We report on the suitability of µm-sized polymer particles stained with organic dyes for lifetime encoding. These dyes are excitable at a standard laser diode wavelength and detectable within a single spectral window. For lifetime-based discrimination, these dyes display sufficiently different luminescence decay kinetics. We present the spectroscopic properties of these beads and address challenges like the limited number of detectable photons in a flow for the reliable discrimination. These studies are expected to pave the road to new applications of fluorescence lifetime multiplexing for time-domain flow cytometry.
The steadily increasing number of analytes, especially in bioanalytics and environmental contexts, requires the development of efficient and versatile methods for the simultaneous determination of different parameters within a single run. Additionally, these methods should be applicable in conjunction with established platform technologies like flow cytometry.
Fluorescence techniques have proven to fulfill these requirements. Commonly performed spectral multiplexing utilizing a color code suffers from several problems, such as the sensitivity of fluorescence intensity measurements to fluctuations in excitation light intensity and dye concentration and hence, photobleaching and spectral crosstalk limiting the achievable number of detection channels. Moreover, it typically requires different costly excitation light sources.
The efficient and sensitive detection of pathogenic microorganisms in aqueous environments, such as water used in medical applications, drinking water, and cooling water of industrial plants, requires simple and fast methods suitable for multiplexed detection such as flow cytometry (FCM) with optically encoded carrier beads. For this purpose, we combine fluorescent Cd-free Ag−In−S ternary quantum dots (t-QDs) with fluorescence Lifetimes (LTs) of several hundred nanoseconds and superparamagnetic Fe3O4 nanoparticles (SPIONs) with mesoporous CaCO3 microbeads to a magneto-fluorescent bead platform that can be surface-functionalized with bioligands, such as antibodies. This inorganic bead platform enables immuno-magnetic separation, target enrichment, and target quantification with optical readout. The beads can be detected with steady-state and time-resolved fluorescence microscopy and flow cytometry (FCM). Moreover, they are suited for readout by time gated emission. In the following, the preparation of these magneto-fluorescent CaCO3 beads, their spectroscopic and analytic characterization, and their conjugation with bacteria-specific antibodies are presented as well as proof-of-concept measurements with Legionella pneumophila including cell cultivation and plating experiments for bacteria quantification. Additionally, the possibility to discriminate between the long-lived emission of the LT-encoded capture and carrier CaCO3 beads and the short-lived Emission of the dye-stained bacteria with time-resolved fluorescence techniques and single wavelength excitation is demonstrated.
The need for rapid and high-throughput screening in analytical laboratories has led to significant growth in interest in suspension array technologies (SATs), especially with regard to cytometric assays targeting a low to medium number of analytes. Such SAT or bead-based assays rely on spherical objects that constitute the analytical platform. Usually, functionalized polymer or silica (SiO2) microbeads are used which each have distinct advantages and drawbacks. In this paper, we present a straightforward synthetic route to highly monodisperse SiO2-coated polystyrene core−shell (CS) beads for SAT with controllable architectures from smooth to raspberry- and multilayer-like shells by varying the molecular weight of poly(vinylpyrrolidone) (PVP), which was used as the stabilizer of the cores. The combination of both organic polymer core and a structurally controlled inorganic SiO2 shell in one hybrid particle holds great promises for flexible next-generation design of the spherical platform. The particles were characterized by electron microscopy (SEM, T-SEM, and TEM), thermogravimetry, flow cytometry, and nitrogen adsorption/desorption, offering comprehensive information on the composition, size, structure, and surface area. All particles show ideal cytometric detection patterns and facile handling due to the hybrid structure. The beads are endowed with straightforward modification possibilities through the defined SiO2 shells. We successfully implemented the particles in fluorometric SAT model assays, illustrating the benefits of tailored surface area which is readily available for small-molecule anchoring. Very promising assay performance was shown for DNA hybridization assays with quantification limits down to 8 fmol.