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Photoluminescence techniques are amongst the most widely used tools in the material and life sciences, with new and exciting applications continuously emerging, due to their many advantages like comparative ease of use, unique sensitivity, non-invasive character, and potential for multiplexing, remote sensing, and miniaturization. Drawbacks are , however, signals, that contain unwanted wavelength- and polarization contributions from instrument-dependent effects, which are time-dependent due to the aging of instrument components, and difficulties to measure absolute fluorescence intensities. Thus, there is a considerable need for standards for intensity, spectral, and temporal fluorescence quantities to meet the increasing need for instrument performance validation and global trends to harmonize physicochemical measurements. In this respect, instrument calibration strategies together with different types of fluorescence standards are presented as well as design concepts for robust, easy-to-use, and format-adaptable fluorescence standards useable for the determination of different fluorescence parameters and a broad variety of fluorescence techniques.
The surface chemistry / functionalization of nanomaterials and microparticles largely controls the stability of these materials as well as their solubility and subsequent biofunctionalization and their interactions with biological systems. Moreover, in the case of some nanomaterials like semiconductor quantum dots or lanthanide-based upconversion nanocrystals, the ligand shell strongly affects their optical properties, e.g., via passivation of surface states and traps that favor luminescence quenching or the protection of surface atoms from quenching water molecules. This renders analytical methods for the quantification of surface groups like functionalities very important. Targets of broad interest are here amino, carboxyl, alkine and maleimide groups used for common bioconjugation reactions and typical ligands like thiols and polyethylene glycol (PEG) molecules of varying length, used for the tuning of material hydrophilicity and biocompatibility, minimization of unspecific interactions, prevention of biofouling, and enhancement of blood circulation times as well as surface-bound biomolecules like streptavidin or other biomolecules relevant e.g., for diagnostic assays. Here, we focus on simple optical methods relying on standard laboratory instrumentation, validated by method comparison and/or mass balances and present examples for their use for the characterization of different types of nanomaterials and microparticles.
Luminescence-based detection methods, ranging from fluorescence spectroscopy for photophysical and mechanistic studies over sensing applications, chromatographic separation techniques and the microarray technology with fluorescence detection to fluorescence microscopy, flow cytometry, single molecule spectroscopy, and molecular imaging to integrating sphere spectroscopy, are among the most widely used methods in the life and material sciences. This is due to e.g., their unique sensitivity enabling the detection of single molecules, potential for multiplexing, ease of combination with spatial resolution, and suitability for remote sensing. Many of these advantages are closely linked to the choice of suitable molecular and nanoscale fluorescent reporters, typically required for signal generation. This includes organic dyes without and with sensor function, fluorophore-encoded polymeric and silica nanoparticles as well as nanocrystalline systems like semiconductor quantum dots and upconversion phosphors, emitting in the visible (vis), near-infrared (NIR), and IR (infrared). Current challenges present the environment sensitivity of most fluorophores, rendering fluorescence spectra, measured intensities/fluorescence quantum yields, and fluorescence decay kinetics matrix-dependent, and instrument-specific distortions of measured fluorescence signals that need to be considered for quantification and comparability of data, particularly fluorescence spectra.
Here, current applications of luminescence-based methods and different types of reporters will be presented. In this context, suitable spectroscopic tools for the characteri-zation of the optical properties of fluorescent reporters and fluorophore-encoded microparticles, analytical tools for the determination of the surface chemistry of different types of particles, and different multiplexing strategies will be discussed.
The surface modification of nanometer- and micrometer-sized particles with polyethylene glycol (PEG) ligands of varying length is a very common strategy to tune their hydrophilicity and biocompatibility, minimize unspecific interactions, improve biofunctionalization efficiencies, and enhance blood circulation times. Nevertheless, simple methods for the quantification of PEG ligands are rare. This is similarly true for the spectroscopic characterization of lanthanide-doped upconverting nanoparticles (UCNPs), novel near infrared (NIR)-excitable nonlinear fluorescence reporters for bioanalysis and theranostics, providing background-free multiple narrow emission bands in the visible and NIR, excellent photostability, and long luminescence lifetimes.
We present here a simple method for the determination of the number of PEG ligands on nanomaterials and screening methods for the fast identification of nonradiative deaction pathways in UCNPs.
Fluorescent particles like nm- and m-sized polymeric beads doped or labeled with different types of fluorophores and nanocrystalline systems like quantum dots and upconversion phosphors emitting in the visible (vis), near-infrared (NIR), and IR (infrared) region are of increasing importance as fluorescent reporters for bioanalysis and medical diagnostics. The assessment and comparison of material performance and the development of rational design strategies for improved systems requires suitable spectroscopic tools for the determination of signal-relevant optical properties and analytical tools for the determination of the number of surface groups, ligands, biomolecules and /or fluorophores per bead. In this respect, suitable spectroscopic tools for the characterization of the optical properties of such materials like photoluminescence quantum yields and brightness values and the determination of their surface chemistry are introduced. This includes integrating sphere setups for absolute measurements of fluorescence quantum yields of liquid and solid, transparent and scattering materials in the wavelength region of 350 nm to 1600 nm at varying excitation power densities for the study of multi-photon processes and simple optical assays, validated by comparison with established analytical techniques relying on different detection principles. Here, different examples for the optical and analytical characterization of different types of nanoscale reporters are presented.
There is an increasing interest in optical reporters like semiconductor quantum dots and upconversion nanocrystals with emission > 800 nm for bioanalysis, medical diagnostics, and safety barcodes. Prerequisites for the comparison of material performance, the mechanistic understanding of nonradiative decay channels, and the rational design of new nanomaterials with improved properties are reliable fluorescence measurements and validated methods for the assessment of their surface chemistry. The latter is of special relevance for nanocrystalline emitters, where surface states and the accessibility of emissive states by quenchers largely control accomplishable photoluminescence quantum yields and hence, signal sizes and detection sensitivities from the reporter side. Here, we present the design of integrating sphere setups for the excitation power density-dependent absolute measurement of emission spectra and photoluminescence quantum yields in the wavelength region of 350 to 1600 nm and results from spectroscopic studies of semiconductor quantum dots and upconversion nanocrystals of different size and surface chemistries in various environments. Subsequently, examples for simple approaches to surface group and ligand analysis are presented.
FLiMFlow – Recent achievements in flow cytometry with lifetime detection and lifetime-encoded beads
(2016)
Flow cytometry is a widespread technique in biological research and clinical applications. Two different directions are currently of importance in development of new methods in this field. Whereas analyses in research become increasingly complex and require a growing number of simultaneously detectable codes and fluorescent labels, also low-cost methods and portable devices are needed in routine application.
Lifetime encoding could present an alternative to common spectral multiplexing. On the one hand, it addresses the need for additional codes by combining spectral and lifetime multiplexing. On the other hand, using only lifetime encoding could help to reduce instrument costs by keeping the number of excitation sources and detectors low.
Here, we report on our recent progress in employing dye-stained lifetime-encoded polymer microparticles as a model system for lifetime encoding in flow cytometry. The discrimination of two lifetime codes was achieved with two bead sets. Moreover, the simultaneous detection of a spectrally different ligand fluorescence signal could be demonstrated.
2,4-Dichlorophenoxyacetic acid (2,4-D) is one important and well-known herbicide that is widely used in agriculture because of its advantages to regulate plant growth. However, the use of large quantities of the treated plants as animal feed leads to residues in meat, milk and eggs. Furthermore, the herbicide can drain away and contaminate ground and drinking water. The ingestion of 2,4-D-contaminated food and water causes damage to the inner organs of humans and animals, e.g., the kidneys and the liver.
Analytical assays based on molecularly imprinted polymers (MIPs) have emerged as a valuable tool in the field of environmental analysis due to the low production costs, stability, format adaptability and the possibility to imprint and thus their ability to recognize a wide variety of target analytes. With regard to optical sensing technologies, however, MIPs have only been used in considerably few applications, especially in fluorescence sensors. This limitation is basically due to the fact that the incorporation of a fluorescently responding moiety into a polymer matrix is challenging. One way to overcome this limitation is to use tailor-made fluorescent indicator monomers for direct transfer of the binding event into an optical signal and coat the MIP via reversible addition-fragmentation chain transfer (RAFT) polymerization as a thin layer onto the surface of silica nanoparticles as primary sensing element.
Here, we present the response behavior of the fluorescent MIP sensor particles in terms of sensitivity for 2,4-D detection (the so-called imprinting factor), discrimination ability against structurally similar compounds and performance in a phase-transfer assay (PTA) on chip, i.e., the implementation of the assay into a microfluidic chip environment, offering a novel simple and rapid way for the detection of herbicides.
2,4-Dichlorophenoxyacetic acid (2,4-D) is one important and well-known herbicide that is widely used in agriculture because of its advantages to regulate plant growth. However, the use of large quantities of the treated plants as animal feed leads to residues in meat, milk and eggs. Furthermore, the herbicide can drain away and contaminate ground and drinking water. The ingestion of 2,4-D-contaminated food and water causes damage to the inner organs of humans and animals, e.g., the kidneys and the liver.
Analytical assays based on molecularly imprinted polymers (MIPs) have emerged as a valuable tool in the field of environmental analysis due to the low production costs, stability, format adaptability and the possibility to imprint and thus their ability to recognize a wide variety of target analytes.5 With regard to optical sensing technologies, however, MIPs have only been used in considerably few applications, especially in fluorescence sensors. This limitation is basically due to the fact that the incorporation of a fluorescently responding moiety into a polymer matrix is challenging. One way to overcome this limitation is to use tailor-made fluorescent indicator monomers for direct transfer of the binding event into an optical signal and coat the MIP via reversible addition-fragmentation chain transfer (RAFT) polymerization as a thin layer onto the surface of silica nanoparticles as primary sensing element.
Here, we present the response behavior of the fluorescent MIP sensor particles in terms of sensitivity for 2,4-D detection (the so-called imprinting factor), discrimination ability against structurally similar compounds and performance in a phase-transfer assay (PTA) on chip, i.e., the implementation of the assay into a microfluidic chip environment, offering a novel simple and rapid way for the detection of herbicides.
Nanocrystalline fluorophores like semiconductor quantum dots and rods and recently also lanthanide-based upconversion phosphors with emission in the visible (vis), near-infrared (NIR), and IR (infrared) region are increasingly being used in bioimaging studies and fluorescence assays as well as in photovoltaics and solid state lighting. The assessment and comparison of material performance as well as the development of rational design strategies for improved systems require spectroscopic tools, which enable the determination of the signal-relevant optical properties like photoluminescence quantum yields and brightness values. In the case of nonlinear fluorescence as shown by upconversion materials, such measurements must be also performed as function of excitation power density. In this work, we report on methods for the absolute determination of the photoluminescence quantum yield and brightness of fluorescent particles in dispersion and as powders based on integrating sphere spectroscopy and underline the importance of such measurements for the understanding of the photophysics of such nanocrystals.
The characterization of the optical properties of photoluminescent systems, that scatter, like dispersions of nanoparticles with sizes exceeding about 25 nm or solid nanophosphors is of increasing importance for many applications in the life and material sciences. Examples present nanoscale optical reporters and dye-doped microparticles for bioimaging, fluorescence assays or DNA sequencing as well as nanocrystalline emitters like semiconductor quantum dots and rods or lanthanide-based nanophosphors embedded into solid matrices for solid state lighting, display technologies, or barcoding/security applications. The assessment and comparison of material performance as well as the development of rational design strategies for improved systems require spectroscopic tools, which enable the determination of the signal-relevant optical properties like photoluminescence quantum yields and brightness values.
This encouraged us to built up an integrating sphere setup enabling absolute measurements of photoluminescence spectra and quantum yields of transparent and scattering photoluminescent dispersions and solid samples in different measurement geometries, i.e., direct and indirect illumination and the combination of both geometries and perform first measurements with selected emitters. Here, the design of this setup is presented and first recommendations concerning suitable measurement geometries are given.
Integrating fluorescent probes with sensing matrices presents a major challenge because usually, when confined in a rather rigid matrix, fluorophores tend to behave completely different than for instance in the molecular state in solution. The lecture reviews the major strategies that have been devised recently to circumvent such issues with special focus on the works carried out in this field by BAM’s Chemical and Optical Sensing Division. Moreover, it will be shown that by using certain strategies not only can the response behavior be retained but synergistic effects can even endow the hybrid with a much better performance than the probe molecule alone.
Macrocycle-containing fluorescent probes continue to be one of the most popular classes of indicator molecules for the sensitive optical detection of ionic inorganic analytes, in particular metal ions, since the first integration of crown ether building blocks into chromophoric π systems more than 30 years ago. However, whereas a large multitude of such probes have been described for operation in organic or mixed aqueous environments, the step to realistic analytical media such as water samples, aqueous food extracts or body fluids is still scarce. On one hand, this is due to considerably low complex stability constants for instance for alkali and alkaline-earth metal ions. On the other hand, many classes of organic dyes that show favourable spectroscopic properties and ion-induced responses in organic solvents are not soluble in neat aqueous media and/or exhibit only significantly quenched fluorescence, even if transition metal ions are binding strongly to the receptor unit. A way to circumvent such problems in a rather simple manner is the steric incorporation of fluorescent probes into mesoporous silica nanomaterials. The local polarity in such pores, whether nascent or modified appropriately with functional silanes, resembles much more a quasi-organic environment while at the same time allowing for free diffusion of water and its cargo.
Two examples of powerful hybrid signalling systems will be presented, utilizing charge transfer-type indicator molecules that show inferior sensing properties in their molecular state. One example discusses mercury(II) determination over an extended concentration range and the other discusses silver(I) and mercury(II) discrimination simply on the basis of photophysical effects retained in the hybrid materials.
Since more than 20 years, optical spectroscopic techniques, in particular fluorescence-based methods, are on the rise in many different areas of chemical and biochemical analysis, with no end being in sight.1,2 Advances in miniaturization and remote applications on one hand and ground-breaking developments in microscopy and laser-based high-throughput instrumentation on the other hand have fuelled these developments substantially.3,4 At the core of utmost of these applications however is not only the instrument, but a small entity that is able to absorb and emit photons and thus to report on the actual (bio)chemistry that is going on in a particular sample of interest.5,6 Besides intense research on various types of luminescent particles (e.g., quantum dots and carbon dots) and proteins, dye chemistry has thus seen its revival and the number of publications dealing with the design, synthesis and application of new fluorescent dyes as probes, stains, labels or indicators is still continuing to grow.
Among the various classes of dyes available as bright fluorophores for a wavelength range that is compatible with many (bio)analytical applications and the respective instrumentation, in particular pyrrolic dyes that do not belong to the classical porphyrins or phthalocyanines have received strong attention recently. Starting perhaps with the revival of the traditional boron-dipyrromethene laser dye ca. 15 years ago,7 these so-called BODIPYs have developed into a colourful spectrum of different derivatives.8 However, the interest in expanding the range of pyrrole-containing π-systems beyond BODIPYs has also constantly increased and has brought about several other pyrrole-based ring systems such as diketopyrrolopyrroles9 or dipyrrolonaphthyridinediones which possess a favourable brightness and other interesting properties. The present contribution will give a critical overview of the field, pinpointing advantages and prospects as well as discussing potential aspects of improvement with an emphasis on the chemical sensing and the longer wavelength range.
Fluorophore labeled proteins and antibodies, referred to also as targeted optical probes, present a promising strategy for a variety of applications from fundamental cell-based biological studies to in vivo diagnostics and image guided surgeries in humans. In this respect, design strategies for the preparation of such conjugates from different dyes including analyte-responsive fluorophores are presented as well as their analytical and spectroscopic characterization employing Absorption spectroscopy and steady state and time-resolved fluorometry. Special emphasis is dedicated to the influence of dye hydrophilicity and labeling density on the optical properties and binding behavior of these dye-bioconjugates including their performance in in vitro and in vivo bioimaging studies.