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Guaranteeing safety and security of citizens requires a significant effort and innovative tools from national and international agencies and governments, especially when it comes to the field of explosives detection. The need to detect Improvised Explosive Devices (IEDs) and Home-made Explosives (HMEs) at a point of suspicion, has grown rapidly due to the ease with which the precursors can be obtained and the reagents synthesised. The limited availability of immunoanalytical tools for HME detection presents an opportunity for the development of new devices, which enable a rapid detection and recognise the target analyte with high specificity and sensitivity. In this work, we introduce an optical biosensor for highly specific and sensitive HME detection. The immunoassay system is placed in a hydrogel environment permeable to the analyte and transparent to light interrogating the fluorescently labelled antibodies. The readout of the immunoanalytical system is realized with Supercritical Angle Fluorescence (SAF), an advanced microscopy technique. To accomplish this, we made use of recent, commercial high resolution (< 22 µm) Liquid Crystal Display 3D printers to fabricate a parabolic optical element with high refractive index (RI>1.5) and transmission values (>90%) from photo-resin. Aiming at a new generation of sensors, which not only can meet the requirements of trace detection, but can also be used for substance identification, the combination of immunoanalytical recognition with SAF detection offers a modularity and versatility that is principally well suitable for the measurements of target analytes at trace levels.
Optical measurements of scattering luminescent materials dispersed in liquid and solid matrices and luminescent powders play an important role in fundamental research and industry. Typical examples are luminescent nano- and microparticles and phosphors of different composition in different matrices or incorporated into ceramics with applications in energy conversion, solid-state lighting, medical diagnostics, and security barcoding. The key parameter for the performance of these materials is the photoluminescence quantum yield QY, i.e., the number of emitted photons per number of absorbed photons. QY of transparent luminophore solutions can be determined relatively to a fluorescence quantum yield standard of known QY. Such standards are meanwhile available as certified reference materials.[1] The determination of QY of scattering liquid and solid samples like dispersions of luminescent nanoparticles, solid phosphors, and optoceramics requires, however, absolute measurements with an integrating sphere setup. Although the importance of reliable absolute QY measurements has been recognized, no interlaboratory comparisons (ILCs) on measurement uncertainties and the identification of typical sources of uncertainty have been yet reported. Also, no scattering reference materials with known QY are available.
We present here the results of a first ILC of 3 laboratories from academia and industry performed to identify and quantify sources of uncertainty of absolute QY measurements of scattering samples. Thereby, two types of commercial stand-alone integrating sphere setups with different illumination and detection geometries were utilized for measuring QY of transparent and scattering dye solutions and solid phosphors. As representative and industrially relevant solid and scattering samples, YAG:Ce optoceramics of varying surface roughness were chosen, applied, e.g., as converter materials for blue light emitting diodes. Special emphasis was dedicated to the influence of the measurement geometry, the optical properties of the blank, utilized to determine the number of photons of the incident excitation light absorbed by the sample, and the sample-specific surface roughness. While matching QY values could be obtained for transparent dye solutions and scattering dispersions, here using a blank with scattering properties closely matching those of the sample, QY measurements of optoceramic samples with different blanks revealed substantial differences, with the blank's optical
properties accounting for measurement uncertainties of more than 20 %. Based upon the ILC results, we recommend non-absorbing blank materials with a high reflectivity (>95 %) such as a 2 mm-thick PTFE target placed on the sample holder which reveals a near-Lambertian light scattering behavior, yielding a homogeneous light distribution within the integrating sphere.
Ensuring the purity of air and water is essential for the overall well-being of life on earth and the sustainability of the planet's diverse ecosystems. To achieve the goal of zero pollution, as outlined in the 2020 European Green Deal by the European Commission,[1] significant efforts are in progress. A key aspect of this commitment involves advancing more efficient and economically viable methods for treating wastewater. This includes the systematic monitoring of harmful pollutants such as heavy metals, microplastics, pesticides, and pharmaceuticals.
One example is the presence of the anti-inflammatory drug diclofenac in water systems, primarily originating from its use as a gel or lotion for joint pain treatment. Diclofenac contamination in surface waters has been detected at approximately 10 μg L-1 (0.03 μM)[2] which is not solely due to widespread usage but also because of the drug's resistance to microbial degradation. Conventional wastewater treatment plants (WWTPs), which rely on biodegradation, sludge sorption, ozone oxidation, and powdered activated carbon treatment, struggle to efficiently remove diclofenac from wastewater.[3],[4] For instance, to enable WWTPs to efficiently monitor and optimize their processes, it would be advantageous to develop on-site detection and extraction methods for persistent pharmaceutical residues in aqueous samples.
In this work, a sol-gel process was used to prepare Nile blue-doped silica nanoparticles (dSiO2-NPs) with a diameter of ca. 30 nm that were further functionalized to enable reversible-addition-fragmentation chain-transfer (RAFT) polymerization. To achieve fluorescence detection, a fluorescent monomer was used as a probe for diclofenac in ethyl acetate, generating stable complexes through hydrogen bond formation. The diclofenac/fluorescent monomer complexes were imprinted into thin molecularly imprinted polymer (MIP) shells on the surface of the dSiO2-NPs. Thus, the MIP binding behaviour could be easily evaluated by fluorescence titrations to monitor the spectral changes upon addition of the analyte. Doping the core substrate with Nile blue generates effective dual fluorescent signal transduction. This approach does not solely depend on a single fluorescence emission band in response to analyte recognition. Instead, it enables the fluorescent core to function as an internal reference, minimizing analyte-independent factors such as background fluorescence, instrumental fluctuation, and operational parameters.[5] Rebinding studies showed that the MIP particles have excellent selectivity towards the imprinted template and good discrimination against the competitor ibuprofen, with a discrimination factor of 2.5. Additionally, the limit of detection was determined to be 0.6 μM. Thus, with further optimization of the MIP, there is potential for the development of a MIP-based biphasic extract-&-detect fluorescence assay for simple, sensitive and specific sensing of diclofenac in aqueous samples down to the required concentrations of 0.03 μM.
Fluorescent labels have strongly contributed to many advancements in bioanalysis, molecular biology, molecular imaging, and medical diagnostics. Despite a large toolbox of molecular and nanoscale fluorophores to choose from, there is still a need for brighter labels, e.g., for flow cytometry and fluorescence microscopy, that are preferably of molecular nature. This requires versatile concepts for fluorophore multimerization, which involves the shielding of dyes from other chromophores and possible quenchers in their neighborhood. In addition, to increase the number of readout parameters for fluorescence microscopy and eventually also flow cytometry, control and tuning of the labels’ fluorescence lifetimes is desired. Searching for bright multi-chromophoric or multimeric labels, we developed PEGylated dyes bearing functional groups for their bioconjugation and explored their spectroscopic properties and photostability in comparison to those of the respective monomeric dyes for two exemplarily chosen fluorophores excitable at 488 nm. Subsequently, these dyes were conjugated with anti-CD4 and anti-CD8 immunoglobulins to obtain fluorescent conjugates suitable for the labeling of cells and beads. Finally, the suitability of these novel labels for fluorescence lifetime imaging and target discrimination based upon lifetime measurements was assessed. Based upon the results of our spectroscopic studies including measurements of fluorescence quantum yields (QY) and fluorescence decay kinetics we could demonstrate the absence of significant dye-dye interactions and self-quenching in these multimeric labels. Moreover, in a first fluorescence lifetime imaging (FLIM) study, we could show the future potential of this multimerization concept for lifetime discrimination and multiplexing.
Scattering luminescent materials dispersed in liquid and solid matrices and luminescent powders are increasingly relevant for fundamental research and industry. Examples are luminescent nano- and microparticles and phosphors of different compositions in various matrices or incorporated into ceramics with applications in energy conversion, solid-state lighting, medical diagnostics, and security barcoding. The key parameter to characterize the performance of these materials is the photoluminescence/fluorescence quantum yield (Φf), i.e., the number of emitted photons per number of absorbed photons. To identify and quantify the sources of uncertainty of absolute measurements of Φf of scattering samples, the first interlaboratory comparison (ILC) of three laboratories from academia and industry was performed by following identical measurement protocols. Thereby, two types ofcommercial stand-alone integrating sphere setups with different illumination and detection geometries were utilized for measuring the Φf of transparent and scattering dye solutions and solid phosphors, namely, YAG:Ce optoceramics of varying surface roughness, used as converter materials for blue light emitting diodes. Special emphasis was dedicated to the influence of the measurement geometry, the optical properties of the blank utilized to determine the number of photons of the incident excitation light absorbed by the sample, and the sample-specific surface roughness. While the Φf values of the liquid samples matched between instruments, Φf measurements of the optoceramics with different blanks revealed substantial differences. The ILC results underline the importance of the measurement geometry, sample position, and blank for reliable Φf data of scattering the YAG:Ce optoceramics, with the blank’s optical properties accounting for uncertainties exceeding 20%.
This contribution reports the development of a polymerizable BODIPY-type fluorescent probe targeting small-molecule carboxylates for incorporation into molecularly imprinted polymers (MIPs). The design of the probe crosslinker includes a urea recognition site p-conjugated to the 3-position of the BODIPY core and two methacrylate moieties. Titration experiments with a carboxylate-expressing antibiotic, levofloxacin (LEVO), showed a blue shift of the absorption band as well as a broadening and decrease in emission, attributed to hydrogen bonding between the probe’s urea group and the carboxylate group of the antibiotic. Using this probe crosslinker, core–shell particles with a silica core and a thin MIP shell were prepared for the detection of LEVO. The MIP exhibited highly selective recognition of LEVO, with an imprinting factor of 18.1 compared to the non-imprinted polymer. Transmission electron microscopy confirmed the core–shell structure and spectroscopic studies revealed that the receptor’s positioning leads to a unique perturbation of the polymethinic character of the BODIPY chromophore, entailing the favourable responses. These features are fully preserved in the MIP, whereas no such response was observed for competitors such as ampicillin. The sensory particles allowed to detect LEVO down to submicromolar concentrations in dioxane. We have developed here for the first time a BODIPY probe for organic carboxylates and incorporated it into polymers using the imprinting technique, paving the way for BODIPY-type fluorescent MIP sensors.
Heterogeneous sandwich immunoassays are widely used for biomarker detection in bioanalysis and medical diagnostics. The high analyte sensitivity of the current “gold standard” enzyme-linked immunosorbent assay (ELISA) originates from the signal-generating enzymatic amplification step, yielding a high number of optically detectable reporter molecules. For future point-of-care testing (POCT) and point-of-need applications, there is an increasing interest in more simple detection strategies that circumvent time-consuming and temperature-dependent enzymatic reactions. A common concept to aim for detection limits comparable to those of enzymatic amplification reactions is the usage of polymer nanoparticles (NP) stained with a large number of chromophores. We explored different simple NP-based signal amplification strategies for heterogeneous sandwich immunoassays
that rely on an extraction-triggered release step of different types of optically detectable reporters. Therefore, streptavidinfunctionalized polystyrene particles (PSP) are utilized as carriers for (i) the fluorescent dye coumarin 153 (C153) and (ii) hemin (hem) molecules catalyzing the luminol reaction enabling chemiluminescence (CL) detection. Additionally, (iii) NP labeling with hemin-based microperoxidase MP11 was assessed. For each amplification approach, the PSP was first systematically optimized regarding size, loading concentration, and surface chemistry. Then, for an immunoassay for the inflammation marker C- eactive protein (CRP), the analyte sensitivity achievable with optimized PSP
ystems was compared with the established ELISA concept for photometric and CL detection. Careful optimization led to a limit of detection (LOD) of 0.1 ng/mL for MP11-labeled PSP and CL detection, performing similarly well to a photometric ELISA (0.13 ng/mL), which demonstrates the huge potential of our novel assay concept.
SWIR luminescent nanomaterials – key chemical parameters for bright probes for in vivo bioimaging
(2024)
A current challenge for studying physio-pathological phenomena and diseaserelated processes in living organisms with non-invasive optical bioimaging is the development of bright optical reporters that enable deep tissue penetration, a high detection sensitivity, and a high spatial and temporal resolution. The focus of this project are nanomaterials, which absorb and emit in the shortwave infrared (SWIR) between ~900–2500 nm where scattering, absorption, and autofluorescence of the tissue are strongly reduced compared to the visible and NIR.
Antibacterial photodynamic therapy (a‐PDT) has emerged as a promising non‐invasive therapeutic modality that utilizes the combination of a photosensitive agent, molecular oxygen, and excitation light to generate reactive oxygen species (ROS), demonstrating remarkable activity against multidrug‐resistant bacterial infections. However, the effective use of conventional photosensitizers is significantly limited by a number of their shortcomings, namely, poor water solubility and low selectivity. Herein, we present a novel biocompatible water‐soluble nanocomposite based on hydrophobic tetraphenylporphyrin (TPP) molecules and hydrophilic ternary AgInS2/ZnS quantum dots incorporated into a chitosan matrix as an improved photosensitizer for a‐PDT. We demonstrated that TPP molecules could be successfully transferred into chitosan solution while remaining primarily in the form of monomers, which are capable of singlet oxygen generation. We performed a detailed analysis of the Förster resonance energy transfer (FRET) between quantum dots and TPP molecules within the nanocomposite and proposed the mechanism of the singlet oxygen efficiency enhancement via FRET.
The development of new fluorescent organic probes effective in the NIR-II region is currently a fast-growing field and represents a challenge in the domain of medical imaging. In this study, we have designed and synthesized an innovative series of aza-boron dipyrromethenes emitting in the NIR-II region. We have investigated the effect of different water-solubilizing groups not only on the photophysical properties of the compounds but also on their in vitro and in vivo performance after bioconjugation to the antibody trastuzumab. Remarkably, we discovered that the most lipophilic compound unexpectedly displayed the most favorable in vivo properties after bioconjugation. This underlines the profound influence that the fluorophore functionalization approach can have on the efficiency of the resulting imaging agent.
Surface functionalized silica nanoparticles (SiO2-NP) gained great interest in the life and material sciences, as they can be used e.g. as drug carriers, fluorescent sensors, and multimodal labels in bioanalytical assays and imaging applications. They are highly stable, are easily produced and modified on a large scale at low cost and can be labeled or stained with a multitude of sensor dyes. These dye modified particle conjugates have several advantages as compared to conventional molecular probes like enhanced brightness, ease of designing ratiometric systems by combining analyte-sensitive and inert reference dyes, and increased photostability. Moreover, stained nanoparticles can enable the use of hydrophobic dyes in aqueous environments.
Here we present our work on multicolored sensors for the measurement of pH, oxygen and saccharides utilizing amorphous SiO2 NPs.
In the focus of division Biophotonics are the design, preparation, analytical and spectroscopic characterization, and application of molecular and nanoscale
functional materials, particularly materials with a photoluminescence in the visible, near infrared (NIR) and short-wave infrared (SWIR). This includes optical reporters for bioimaging and sensing, security and authentication barcodes, and materials for solid state lighting, energy conversion, and photovoltaics. For the identification of optimum particle structures quantitative spectroscopic studies are performed under application-relevant conditions, focusing on the key performance parameter photoluminescence quantum yield. In addition, simple, cost-efficient, and standardizable strategies for quantifying functional groups on the surface of nano- and microparticles are developed, here with a focus on optical assays and electrochemical titration methods, cross-validated by more advanced methods such as quantitative NMR. In addition, reference materials and reference products are developed for optical methods, particularly luminescence techniques, and for analytical methods utilized for the characterization of nanomaterials.
Photoluminescence Quantum Yields of Luminescent Nanocrystals and Particles in the UV/vis/NIR/SWIR
(2023)
The rational design of functional luminescent materials such as semiconductor quantum dots and lanthanide-based upconversion nanoparticles, all photophysical and mechanistic studies, and the comparison of different emitters require accurate and quantitative photoluminescence measurements. Particularly the reliable determination of the key performance parameter photoluminescence quantum yield (f), the number of emitted per absorbed photons, and the brightness are of special importance for luminescence applications in the life and material sciences and nano(bio)photonics.[1] In this context, examples for absolute measurements of the photoluminescence quantum yields of UV/vis/NIR/SWIR emissive semiconductor quantum dots and rods, made from different materials, and spectrally shifting lanthanide upconversion nanocrystals with different surface chemistries in transparent matrices are presented including excitation wavelength and power density dependent studies utilizing integration sphere spectroscopy.[2,3] In addition, procedures for the absolute determination of the photoluminescence quantum yields of scattering dispersions of larger size quantum rods and differently sized inorganic particles have been developed as well as procedures for the characterization of solid luminescent nanomaterials such as different perovskites and YAG:Cer converter materials.[4] Thereby, challenges and pitfalls of f measurements in different wavelength regions including the SWIR and material-specific effects related to certain emitter classes are addressed, achievable uncertainties are quantified, and relative and absolute measurements of photoluminescence quantum yield measurements are compared to underline limitations of the former approach. Finally, a set of novel UV/vis/NIR quantum yield standards is presented including their certification with a complete uncertainty budget.[5]
Surface-functionalized polymer beads encoded with molecular luminophores and nanocrystalline emitters such as semiconductor nanocrystals, often referred to as quantum dots (QDs), or magnetic nanoparticles are broadly used in the life sciences as reporters and carrier beads. Many of these applications require a profound knowledge of the chemical nature and total number of their surface functional groups (FGs), that control bead charge, colloidal stability, hydrophobicity, and the interaction with the environment and biological systems. For bioanalytical applications, also the number of groups accessible for the subsequent functionalization with, e.g., biomolecules or targeting ligands is relevant. In this study, we explore the influence of QD encoding on the amount of carboxylic acid (COOH) surface FGs of 2 μm polystyrene microparticles (PSMPs). This is done for frequently employed oleic acid and oleylamine stabilized, luminescent core/shell CdSe QDs and two commonly used encoding procedures. This included QD addition during bead formation by a thermally induced polymerization reaction and a post synthetic swelling procedure. The accessible number of COOH groups on the surface of QD-encoded and pristine beads was quantified by two colorimetric assays, utilizing differently sized reporters and electrostatic and covalent interactions. The results were compared to the total number of FGs obtained by a conductometric titration and Fourier transform infrared spectroscopy (FTIR). In addition, a comparison of the impact of QD and dye encoding on the bead surface chemistry was performed. Our results demonstrate the influence of QD encoding and the QD-encoding strategy on the number of surface FG that is ascribed to an interaction of the QDs with the carboxylic acid groups on the bead surface. These findings are of considerable relevance for applications of nanoparticle-encoded beads and safe-by-design concepts for nanomaterials.
Current challenges and objectives for non-invasive optical bioimaging are deep tissue penetration, high detection sensitivity, high spatial and temporal resolution, and fast data acquisition. A promising spectral window to tackle these challenges is the short-wave infrared (SWIR) ranging from 900 nm to 1700 nm where scattering, absorption, and autofluorescence of biological components are strongly reduced compared to the visible/NIR. At present, the best performing SWIR contrast agents are based on nanomaterials containing toxic heavy-metal ions like cadmium or lead, which raises great concerns for biological applications. Promising heavy-metal free nanoscale candidates are gold nanoclusters (AuNCs) and Ag2S nanoparticles (NPs). The photoluminescence (PL) of both types of nanomaterials is very sensitive to their size, composition of their surface ligand shell, and element composition, which provides an elegant handle to fine-tune their absorption and emission features and boost thereby the size of the signals recorded in bioimaging studies.
Aiming for the development of SWIR contrast agents with optimum performance, we dived deeper into the photophysical processes occurring in these nanomaterials, thereby exploring in depth how the environment, surface ligand composition, and the incorporation of transition metals influence the optical properties of AuNCs and Ag2S NPs. We observed a strong enhancement of the SWIR emission of AuNCs upon exposure to different local environments (in solution, polymer, and in the solid state). Addition of metal ions such as Zn2+ to Ag2S based NPs led to a strong PL enhancement, yielding PL quantum yields of about 10% and thus making them highly suitable for non-invasive deep imaging of vascular networks and 3D fluid flow mapping.
The rational design of next generation molecular and nanoscale reporters and the comparison of different emitter classes require the determination of the fluorometric key performance parameter fluorescence quantum yield (Φf), i.e., the number of emitted photons per number of absorbed photons. Main prerequisites for reliable Φf measurements, which are for transparent luminophore solutions commonly done relative to a reference, i.e., a fluorescence quantum yield standard of known Φf, are reliable and validated instrument calibration procedures to consider wavelength-, polarization-, and time-dependent instrument specific signal contributions, and sufficiently well characterized fluorescence quantum yield standards. As the standard’s Φf value directly contributes to the calculation of the sample’s Φf, its accuracy presents one of the main sources of uncertainty of relative Φf measurements. To close this gap, we developed a first set of 12 fluorescence quantum yield standards, which absorb and emit in the wavelength region of 330−1000 nm and absolutely determined their Φf values with two independently calibrated integrating sphere setups.
Criteria for standard selection and the configuration of these novel fluorescence reference materials are given, and the certification procedure is presented including homogeneity and stability studies and the calculation of complete uncertainty budgets for the certified Φf values. The ultimate goal is to provide the community of fluorescence users with available reference materials as a basis for an improved comparability and reliability of quantum yield data since the measurement of this spectroscopic key property is an essential part of the characterization of any new emitter.
In recent years, the demand for reliable, versatile, fluorescent pH and oxygen sensors has increased rapidly in many biomedical applications since these analytes are important indicators of cell function or certain diseases. Therefore, sensor particles are needed that are small enough to penetrate cells, non-toxic, and allow for close-up optical monitoring. When developing such sensor systems, one must consider the pH and oxygen range detectable by the sensor dye and the matrix material of the used carrier particles. Here, we present the development of pH- and oxygen-responsive polymeric beads functionalized with fluorescent dyad molecules that consist of an analyte-responsive fluorophore and an analyte-inert dye.
Photodynamic therapy (PDT) used for treating cancer relies on the generation of highly reactive oxygen species, for example, singlet oxygen 1O2, by light-induced excitation of a photosensitizer (PS) in the presence of molecular oxygen, inducing DNA damage in close proximity of the PS. Although many precious metal complexes have been explored as PS for PDT and received clinical approval, only recently, the potential of photoactive complexes of nonnoble metals as PS has been discovered. Using the DNA origami technology that can absolutely quantify DNA strand break cross sections, we assessed the potential of the luminescent transition metal complex [Cr(ddpd)2]3+ (ddpd=N,N’-dimethyl-N,N’-dipyridine-2-ylpyridine-2,6-diamine) to damage DNA in an air-saturated aqueous environment upon UV/Vis illumination. The quantum yield for strand breakage, that is, the ratio of DNA strand breaks to the number of absorbed photons, was determined to 1–4%, indicating efficient transformation of photons into DNA strand breaks by [Cr(ddpd)2]3+.
The core−shell NaYF4:Yb3+/Tm3+@NaYF4:Yb3+ upconversion nanoparticles were successfully prepared by a solvothermal method, and a layer of mesoporous silica (mSiO2) was successfully coated on the periphery of the core−shell nanoparticles to transform their surface from lipophilic to hydrophilic, further expanding their applications in biological tissues. The physical phase, morphology, structure, and fluorescence properties were characterized by X-ray diffraction (XRD), field emission transmission electron microscopy (TEM), Fourier infrared spectroscopy (FT-IR), ζ potential analysis, and fluorescence spectroscopy. It was found that the material has a hexagonal structure with good hydrophilicity and emits intense fluorescence under 980 nm pump laser excitation. The non-contact temperature sensing performance of nanoparticles was evaluated by analyzing the upconversion fluorescence of Tm3+ (1G4 → 3F4 and 3F3 → 3H6) in the temperature range of 284−344 K. The absolute and relative sensitivities were found to be 0.0067 K−1 and 1.08 % K−1, respectively, with high-temperature measurement reliability and good temperature cycling performance. More importantly, its temperature measurement in phosphate-buffered saline (PBS) solution is accurate. In addition, the temperature of the cells can be increased by adjusting the laser power density and laser irradiation time. Therefore, an optical temperature sensing platform was built to realize the application of real-time monitoring of cancer cell temperature and the dual function of photothermal therapy.
Fluorescent semiconductor nanocrystals, also known as quantum dots (QDs), enabled many advancements in biotechnology, photovoltaics, photocatalysis, quantum computing and display devices. The high versatility of this nanomaterial is based on their unique size-tunable photoluminescence properties, which can be adjusted from the visible to the near-infrared range. In contrast to other nanomaterials, QDs made the transition from a laboratory curiosity to the utilization in commercial products, like the QLED television screen or in smartphone displays. The best investigated QDs are composed of heavy metals like cadmium or lead, which is not the best choice in terms of toxicity and environmental pollution. A more promising material is Indium Phosphide (InP), which is also currently used by Samsung, Sony and co. in the QLED displays.
In this contribution, I would like to give you a sneak peek behind the curtains of nanomaterial synthesis and show how this material is produced, how to stabilize their structural properties, and assess their toxicity in environmentally relevant conditions. Furthermore, I would like to present a synthesis method to accomplish the last open challenge in display technology of a blue luminescent LED based on QDs by introducing a new element to the InP QDs.
Luminescence techniques are amongst the most commonly used analytical methods in the life and material sciences due to their high sensitivity and non-destructive and multiparametric character. Photoluminescence signals are, however, affected by wavelength-, polarization-, and time-dependent instrument specific effect and the compound-specific photoluminescence quantum yield. The former hamper the comparability of fluorescence measurements, while the relative determination of the latter requires suitable quantum yield standards with well-known photoluminescence quantum yields (QY). For the simple correction of instrument specific effects in the wavelength region of 300 nm to 950 nm, the set of the five certified spectral fluorescence standards BAM-F001 – BAM-F005, has been extended to the NIR range by including two new fluorescence standards currently under certification. For the reliable and accurate determination of QY which is the key performance parameter for the comparison of different luminophores, we certified a set of 12 quantum yield standards, which absorb and emit in the wavelength range from 300 nm to 1000 nm.
Current trends in materials and life sciences are flanked by the need to push detection limits to single molecules or single cells, enable the characterization of increasingly complex matrices or sophisticated nanostructures, speed up the time of analysis, reduce instrument complexity and costs, and improve the reliability of data. This requires suitable analytical tools such as spectroscopic, separation and imaging techniques, mass spectrometry, and hyphenated techniques as well as sensors and their adaptation to application-specific challenges in the environmental, food, consumer product, health sector, nanotechnology, and bioanalysis. Increasing concerns about health threatening known or emerging pollutants in drinking water, consumer products, and food and about the safety of nanomaterials led to a new awareness of the importance of analytical sciences. Another important driver in this direction is the increasing demand by legislation, particularly in view of the 17 sustainable development goals by the United Nations addressing clean energy, industry, and innovation, sustainable cities, clean water, and responsible consumption and production. In this respect, also the development of analytical methods that enable the characterization of material flows in production processes and support recycling concepts of precious raw materials becomes more and more relevant. In the future, this will provide the basis for greener production in the chemical industry utilizing recycled or sustainable starting materials.
This makes analytical chemistry an essential player in terms of the circular economy helping to increase the sustainability of production processes. In the life sciences sector, products based on proteins, such as therapeutic and diagnostic antibodies, increase in importance. These increasingly biotechnologically produced functional biomolecules pose a high level of complexity of matrix and structural features that can be met only by highly advanced methods for separation, characterization, and detection. In addition, metrological traceability and target definition are still significant challenges for the future, particularly in the life sciences.
However, innovative reference materials as required for the health and food sector and the characterization of advanced materials can only be developed when suitable analytical protocols are available. The so-called reproducibility crisis in sciences underlines the importance of improved measures of quality control for all kinds of measurements and material characterization. This calls for thorough method validation concepts, suitable reference materials, and regular interlaboratory comparisons of measurements as well as better training of scientists in analytical sciences.
The important contribution of analytical sciences to these developments is highlighted by a broad collection of research papers, trend articles, and critical reviews from these different application fields. Special emphasis is dedicated to often-overlooked quality assurance and reference materials.
Current trends in materials and life sciences are flanked by the need to push detection limits to single molecules or single cells, enable the characterization of increasingly complex matrices or sophisticated nanostructures, speed up the time of analysis, reduce instrument complexity and costs, and improve the reliability of data. This requires suitable analytical tools such as spectroscopic, separation and imaging techniques, mass spectrometry, and hyphenated techniques as well as sensors and their adaptation to application-specific challenges in the environmental, food, consumer product, health sector, nanotechnology, and bioanalysis. Increasing concerns about health threatening known or emerging pollutants in drinking water, consumer products, and food and about the safety of nanomaterials led to a new awareness of the importance of analytical sciences. Another important driver in this direction is the increasing demand by legislation, particularly in view of the 17 sustainable development goals by the United Nations addressing clean energy, industry, and innovation, sustainable cities, clean water, and responsible consumption and production. In this respect, also the development of analytical methods that enable the characterization of material flows in production processes and support recycling concepts of precious raw materials becomes more and more relevant. In the future, this will provide the basis for greener production in the chemical industry utilizing recycled or sustainable starting materials.
This makes analytical chemistry an essential player in terms of the circular economy helping to increase the sustainability of production processes. In the life sciences sector, products based on proteins, such as therapeutic and diagnostic antibodies, increase in importance. These increasingly biotechnologically produced functional biomolecules pose a high level of complexity of matrix and structural features that can be met only by highly advanced methods for separation, characterization, and detection. In addition, metrological traceability and target definition are still significant challenges for the future, particularly in the life sciences.
However, innovative reference materials as required for the health and food sector and the characterization of advanced materials can only be developed when suitable analytical protocols are available. The so-called reproducibility crisis in sciences underlines the importance of improved measures of quality control for all kinds of measurements and material characterization. This calls for thorough method validation concepts, suitable reference materials, and regular interlaboratory comparisons of measurements as well as better training of scientists in analytical sciences.
The important contribution of analytical sciences to these developments is highlighted by a broad collection of research papers, trend articles, and critical reviews from these different application fields. Special emphasis is dedicated to often-overlooked quality assurance and reference materials.
pH presents one of the most important analytes in the life and material sciences, indicating, e.g., diseases and corrosion processes. This includes the optical monitoring of pH in living cells for studying cellular internalization pathways, such as phagocytosis, endocytosis, and receptor mediated internalization with the aid of molecular and nanoscale fluorescent sensors. Nanoparticle (NP)-based sensors, that are labelled or stained with a multitude of sensor dyes, have several advantages as compared to conventional molecular probes like enhanced brightness, i.e., amplified signals, ease of designing ratiometric systems by combining analyte-sensitive and inert reference dyes, and increased photostability. Moreover, this can enable the use of hydrophobic dyes in aqueous environments. Versatile templates and carriers for the fabrication of nanosensors by staining and/or labelling with different fluorophores and sensor molecules are surface-functionalized particles like silica (SiO2) and polystyrene (PS) particles. Here we present a platform of blue-red-green fluorescent pH nanosensors for the measurement of acidic, neutral, and basic pH utilizing both types of matrices and two spectrally distinguishable sensor dyes with an integrated reference dye and demonstrate its applicability for cellular studies.
Herein, we report three crystals of styryl quinoline derivatives. All these three crystals are mechanically flexible crystals, isostructural and also all of them comply with the common underlying features for elas- tic flexibility like absence of slip plane, criss-cross packing arrangement of neighbouring tapes, presence of weak and dispersive interactions such as halogen bonds, hydrogen bonds etc. The interactions facilitate easy movement of molecules under application of pressure thereby imparting elasticity. Further, the crys- tals were found to be blue light emitting making them promising candidates for optical waveguides. The optical properties were combined with flexibility by using crystal engineering approach towards achiev- ing various applications such as flexible OLEDs, optical waveguides, flexible optoelectronics etc.
NaYF4:Yb3+/Tm3+@NaGdF4:Nd3+/Yb3+ upconversion nanoparticles (UCNPs) were prepared using a solvothermal method, and the effects of key factors such as the content of sensitiser Nd 3+ and Yb3+ on their luminescence properties were investigated. The nanoparticles are homogeneous in size and well dispersed. Under 808 nm excitation, it can produce strong upconversion fluorescence. At the same time, the nanoparticles have good temperature-sensing properties at the thermally coupled energy levels of 700 nm and 646 nm for Tm3+. Using its fluorescence intensity ratio (FIR), accurate temperature measurements can be performed, and it has been found that it exhibits different temperature sensing properties in low and high-temperature regions. The maximum relative sensitivity was found to be 0.88% K-1 and 1.89% K-1 for the lowtemperature region of 285 K-345 K and the high-temperature region of 345 K-495 K. The nanoparticles were applied to the internal temperature measurement of lithium batteries and the actual high-temperature environment, respectively, and were found to have good temperature measurementt performance.
A new “green” and mild synthesis of highly stable microcrystalline Cs2AgxNa1-xBiyIn1-yCl6 (CANBIC) perovskites under ambient conditions was developed that is scalable to the multi-gram production. Under UV illumination, the CANBIC perovskites emit intense broadband photoluminescence (PL) with a quantum yield (QY) of 92% observed for x = 0.35 and y = 0.01-0.02. The combination of strong UV absorbance and broadband visible emission, high PL QY, and long PL lifetimes of up to 1.4 μs, along with an outstanding stability makes these CANBICs a promising material class for many optical applications.
A first tricolor fluorescent pH nanosensor is presented, which was rationally designed from biocompatible carboxylated polystyrene nanoparticles and two analyte-responsive molecular fluorophores. Its fabrication involved particle staining with a blue-red-emissive dyad, consisting of a rhodamine moiety responsive to acidic pH values and a pH-inert quinoline fluorophore, followed by the covalent attachment of a fluorescein dye to the particle surface that signals neutral and basic pH values with a green fluorescence. These sensor particles change their fluorescence from blue to red and green, depending on the pH and excitation wavelength, and enable ratiometric pH measurements in the pH range of 3.0−9.0. The localization of the different sensor dyes in the particle core and at the particle surface was confirmed with fluorescence microscopy utilizing analogously prepared polystyrene microparticles. To show the application potential of these polystyrene-based multicolor sensor particles, fluorescence microscopy studies with a human A549 cell line were performed, which revealed the cellular uptake of the pH nanosensor and the differently colored emissions in different cell organelles, that is, compartments of the endosomal-lysosomal pathway. Our results demonstrate the underexplored potential of biocompatible polystyrene particles for multicolor and multianalyte sensing and bioimaging utilizing hydrophobic and/or hydrophilic stimuli-responsive luminophores.
Atomically precise gold nanoclusters are a fascinating class of nanomaterials that exhibit molecule-like properties and have outstanding photoluminescence (PL). Their ultrasmall size, molecular chemistry, and biocompatibility make them extremely appealing for selective biomolecule labeling in investigations of biological mechanisms at the cellular and anatomical levels. In this work, we report a simple route to incorporate a preformed Au25 nanocluster into a model bovine serum albumin (BSA) protein. A new approach combining small-angle X-ray scattering and molecular modeling provides a clear localization of a single Au25 within the protein to a cysteine residue on the gold nanocluster surface.
Attaching Au25 to BSA strikingly modifies the PL properties with enhancement and a redshift in the second near-infrared (NIR-II) window. This study paves the way to conrol the design of selective sensitive probes in biomolecules through a ligand-based strategy to enable the optical detection of biomolecules in a cellular environment by live imaging.
Biofilms are ubiquitous in nature and in the man-made environment. Given their harmful effects on human health, an in-depth understanding of biofilms and the monitoring of their formation and growth are important. Particularly relevant for many metabolic processes and survival strategies of biofilms is their extracellular pH. However, most conventional techniques are not suited for minimally invasive pH measurements of living biofilms. Here, a fluorescent nanosensor is presented for ratiometric measurements of pH in biofilms in the range of pH 4.5–9.5 using confocal laser scanning microscopy. The nanosensor consists of biocompatible polystyrene nanoparticles loaded with pH-inert dye Nile Red and is surface functionalized with a pH-responsive fluorescein dye. Its performance was validated by fluorometrically monitoring the time-dependent changes in pH in E. coli biofilms after glucose inoculation at 37 °C and 4 °C. This revealed a temperature-dependent decrease in pH over a 4-h period caused by the acidifying glucose metabolism of E. coli. These studies demonstrate the applicability of this nanosensor to characterize the chemical microenvironment in biofilms with fluorescence methods.
Flexible organic crystals (elastic and plastic) are important materials for optical waveguides, tunable optoelectronic devices, and photonic integrated circuits. Here, we present highly elastic organic crystals of a Schiff base, 1-((E)-(2,5-dichlorophenylimino)methyl)naphthalen-2-ol (1), and an azine molecule, 2,4-dibromo-6-((E)-((E)-(2,6-dichlorobenzylidene)hydrazono)methyl)phenol (2). These microcrystals are highly flexible under external mechanical force, both in the macroscopic and the microscopic regimes. The mechanical flexibility of these crystals arises as a result of weak and dispersive C−H⋅⋅⋅Cl, Cl⋅⋅⋅Cl, Br⋅⋅⋅Br, and π⋅⋅⋅π stacking interactions. Singly and doubly-bent geometries were achieved from their straight shape by a micromechanical approach using the AFM cantilever tip. Crystals of molecules 1 and 2 display a bright-green and red fluorescence (FL), respectively, and selective reabsorption of a part of their FL band. Crystals 1 and 2 exhibit optical-path-dependent low loss emissions at the termini of crystal in their straight and even in extremely bent geometries. Interestingly, the excitation position-dependent optical modes appear in both linear and bent waveguides of crystals 1 and 2, confirming their light-trapping ability.
Functionalized nanomaterials (NM) with their unique size-dependent properties are of increasing relevance for current and future developments in various fields such as medical and pharmaceutical industry, computing, electronics or food and consumer products. The performance and safety of NM are determined by the sum of their intrinsic physicochemical properties.1 Besides other key parameters, the particle surface chemistry, which is largely controlled by the chemical nature and density of functional groups and ligands, must be considered for a better performance, stability, and processibility of NM, as well as their interaction with the environment. Thus, particle standards with well-designed surfaces and methods for functional group quantification can foster the sustainable development of functional and safe(r) NM.2 Here we provide a brief overview of the ongoing research in division Biophotonics to design tailored amorphous silica reference particles with bioanalytically relevant functional groups and ligands, for the development of standardized and validated surface functional group quantification methods.
Bifunctional fluorescent molecular oxoanion probes based on the benzoxadiazole (BD) chromophore are described which integrate a thiourea binding motif and a polymerizable 2-aminoethyl methacrylate unit in the 4,7-positions of the BD core. Concerted charge transfer in this electron donor-acceptor-donor architecture endows the dyes with strongly Stokes shifted (up to >250 nm) absorption and fluorescence. Binding of electron-rich carboxylate guests at the thiourea receptor leads to further analyte-induced red-shifts of the emission, shifting the fluorescence maximum of the complexes to ≥700 nm. Association constants for acetate are ranging from 1–5×105 M−1 in acetonitrile. Integration of one of the fluorescent probes through its polymerizable moiety into molecularly imprinted polymers (MIPs) grafted from the surface of submicron silica cores yielded fluorescent MIP-coated particle probes for the selective detection of antibiotics containing aliphatic carboxylate groups such as enoxacin (ENOX) at micromolar concentrations in highly polar solvents like acetonitrile.
Recent progress has shown that using wavelengths between 1,000 and 2,000 nm, referred to as the shortwave-infrared or near-infrared (NIR)-II range, can enable high-resolution in vivo imaging at depths not possible with conventional optical wavelengths.
However, few bioconjugatable probes of the type that have proven invaluable for multiplexed imaging in the visible and NIR range are available for imaging these wavelengths. Using rational design, we have generated persulfonated indocyanine dyes with absorbance maxima at 872 and 1,072 nm through catechol-ring and aryl-ring fusion, respectively, onto the nonamethine scaffold. Multiplexed two-color and three-color in vivo imaging using monoclonal antibody and dextran conjugates in several tumor models illustrate the benefits of concurrent labeling of the tumor and healthy surrounding tissue and lymphatics.
These efforts are enabled by complementary advances in a custom-built NIR/shortwave-infrared imaging setup and software package for multicolor real-time imaging.
Introduction. Comparing different emitter classes and rationally designing the next generation of molecular and nanoscale probes for bioimaging applications require accurate and quantitative methods for the measurement of the key parameter photoluminescence quantum yield f.1 f equals the number of emitted per number of absorbed photons. This is particularly relevant for increasingly used fluorescence imaging in the short wave-infrared region (SWIR) ≥ 900 nm providing deeper penetration depths, a better image resolution, and an improved signal-to-noise or tumor-to-background ratio.2, 3 However, spectroscopic measurements in the SWIR are more challenging and require specific calibrations and standards.
The concept of aggregation-induced emission represents a means to rationalise photoluminescence of usually nonfluorescent excimers in solid-state materials. In this publication, we study the photophysical properties of selected diaminodicyanoquinone (DADQ) derivatives in the solid state using a combined approach of experiment and theory. DADQs are a class of high-dipole organic chromophores promising for applications in non-linear optics and light-harvesting devices. Among the compounds investigated, we find both aggregation-induced emission and aggregation-caused quenching effects rationalised by calculated energy transfer rates. Analysis of fluorescence spectra and lifetime measurements provide the interesting result that (at least) two emissive species seem to contribute to the photophysical properties of DADQs. The main emission peak is notably broadened in the long-wavelength limit and exhibits a blue-shifted shoulder. We employ high-level quantum-chemical methods to validate a molecular approach to a solid-state problem and show that the complex emission features of DADQs can be attributed to a combination of H-type aggregates, monomers, and crystal structure defects.
Aroyl-S,N-ketene acetal-based bichromophores can be readily synthesized in a consecutive three-component synthesis in good to excellent yields by condensation of aroyl chlorides and an N-(p-bromobenzyl) 2-methyl benzothiazolium salt followed by a Suzuki coupling, yielding a library of 31 bichromophoric fluorophores with substitution patterntunable emission properties. Varying both chromophores enables different communication pathways between the chromophores, exploiting aggregation-induced emission (AIE) and energy transfer (ET) properties, and thus, furnishing aggregation-based fluorescence switches. Possible applications range from fluorometric analysis of alcoholic beverages to pH sensors.
Lanthanide-based upconversion nanoparticles (UCNPs) like hexagonal 𝛽-NaYF4 UCNPs doped with Yb3+ and Er3+, which efficiently convert 976 nm light to ultraviolet, visible, and near infrared photons, offer new strategies for luminescence-based sensing, barcoding, and Imaging. Their upconversion (UC) luminescence (UCL) features like UCL intensity, quantum yield, relative spectral distribution / UCL luminescence color, and luminescence decay kinetics are, however, strongly influenced by particle size, dopant ion concentration, particle architecture, surface chemistry including presence and thickness of surface passivation and shielding shells, microenvironment/presence of quenchers with high energy vibrations, and excitation power density (P).
We present here a comprehensive study of the influence of excitation power density on the UCL features of different types of UCNPs, focusing on Yb3+ and Er3+ co-doped NaYF4 core-only and core-shell nanostructures with different sizes and doping ion concentration, which underlines the importance of P-dependent optimum dopant concentrations for UCNP performance and the potential of P-tuning of UCL.
Molecular aggregation alters the optical properties of a system as fluorescence may be activated or quenched. This is usually described within the well-established framework of H- and J-aggregates. While H-aggregates show nonfluorescent blueshifted absorption bands with respect to the isolated monomer, Jaggregates are fluorescent displaying a redshifted peak. In this publication, we employ a combined approach of experiment and theory to study the complex aggregation features and photophysical properties of diaminodicyanoquinone derivatives, which show unusual and puzzling nonfluorescent redshifted Absorption bands upon aggregation. Our theoretical analysis demonstrates that stable aggregates do not account for the experimental observations.
Instead, we propose an unprecedented mechanism involving metastable dimeric species formed from stable dimers to generate nonfluorescent J-aggregates. These results represent a novel kind of aggregation-induced optical effect and may have Broad implications for the photophysics of dye aggregates.
In this work, we describe the synthesis, structural and photophysical characterization of four novel Pd(II) and Pt(II) complexes bearing tetradentate luminophoric ligands with high photoluminescence quantum yields (FL) and long excited state lifetimes (s) at room temperature, where the results were interpreted by means of DFT calculations. Incorporation of fluorine atoms into the tetradentate ligand favors aggregation and thereby, a shortened average distance between the metal centers, which provides accessibility to metal–metal-to-ligand charge-transfer (3MMLCT) excimers acting as red-shifted Energy traps if compared with the monomeric entities. This supramolecular approach provides an elegant way to enable room-temperature phosphorescence from Pd(II) complexes, which are otherwise quenched by a thermal population of dissociative states due to a lower ligand field splitting. Encapsulation of These complexes in 100 nm-sized aminated polystyrene nanoparticles enables concentration-controlled aggregation-enhanced dual emission. This phenomenon facilitates the tunability of the absorption and emission colors while providing a rigidified environment supporting an enhanced FL up to about 80% and extended s exceeding 100 ms. Additionally, these nanoarrays constitute rare examples for selfreferenced oxygen reporters, since the phosphorescence of the aggregates is insensitive to external influences, whereas the monomeric species drop in luminescence lifetime and intensity with increasing triplet molecular dioxygen concentrations (diffusion-controlled quenching).
The use of inorganic lanthanide-doped upconversion nanoparticles (UCNP) in bioimaging and cellular studies requires biocompatible particles. One possible cause of UCNP toxicity is the release of potentially harmful fluoride and lanthanide ions as revealed by dilution studies in aqueous environments, particularly under high dilution conditions. To address this issue, suitable surface coatings preventing such effects in combination with fast screening methods suited for online monitoring and in situ analyses are desired.
Here we present systematic studies of differently sized β-NaYF4:Yb,Er UCNP stabilized with different surface coatings and hydrophilic ligands varying in binding strength to the particle surface in various aqueous environments at different temperatures and UCNP concentrations. The concentration of the fluoride and lanthanide ions released upon particle dissolution was quantified electrochemically with a fluoride ion-sensitive electrode and inductively coupled plasma optical emission spectrometry (ICP-OES) and monitored fluorometrically, thereby exploiting the sensitivity of the upconversion luminescence to changes in size and surface chemistry. Moreover, changes in surface chemistry were determined with X-Ray photoelectron spectroscopy (XPS). Based upon our results, we could derive optimum screening parameters for UCNP stability studies and determine conditions and coating procedures and ligands for enhancing UCNP stability in aqueous environments.
We demonstrate the potential of time-resolved luminescence spectroscopy for the straightforward assessment and in situ monitoring of the stability of upconversion nanocrystals (UCNPs). Therefore, we prepared hexagonal NaYF4:Yb3+,Er3+ UCNPs with various coatings with a focus on phosphonate ligands of different valency, using different ligand exchange procedures, and studied their dissolution behaviour in phosphate-buffered saline (PBS) dispersions at 20 °C and 37 °C with various analytical methods. The amount of the released UCNPs constituting fluoride ions was quantified by potentiometry using a Fluoride ion-sensitive electrode and particle disintegration was confirmed by transmission electron microscopy studies of the differently aged UCNPs. In parallel, the luminescence features of the UCNPs were measured with special emphasis on the lifetime of the sensitizer emission to demonstrate its suitability as Screening parameter for UCNP stability and changes in particle composition. The excellent correlation between the changes in luminescence lifetime and fluoride concentration highlights the potential of our luminescence lifetime method for UCNP stability screening and thereby indirect monitoring of the release of potentially hazardous fluoride ions during uptake and dissolution in biological systems. Additionally, the developed in situ optical method was used to distinguish the dissolution dynamics of differently sized and differently coated UCNPs.
The absorption and emission properties of organic dyes are generally tuned by altering the substitution pattern. However, tuning the fluorescence lifetimes over a range of several 10 ns while barely affecting the spectral features and maintaining a moderate fluorescence quantum yield is challenging. Such properties are required for lifetime multiplexing and barcoding applications. Here, we show how this can be achieved for the class of fluoranthene dyes, which have substitution-dependent lifetimes between 6 and 33 ns for single wavelength excitation and emission. We explore the substitution-dependent emissive properties in the crystalline solid state that would prevent applications.
Furthermore, by analyzing dye mixtures and embedding the dyes in carboxyfunctionalized 8 μm-sized polystyrene particles, the unprecedented potential of these dyes as labels and encoding fluorophores for time-resolved fluorescence detection techniques is demonstrated.
A brief introduction to fluorescence spectroscopy will be provided, ranging from typically measured fluorescence quantities over instrument-specific contributions to measured fluorescence signals to selected applications. In this context, an overview of the photoluminescence properties of molecular and nanoscale luminescence reporters will be given including a brief insight into their photophysics and fluorescence standards designed by division Biophotonics for the calibration and instrument performance validation of fluorescence measuring devices will be presented.
A brief introduction to fluorescence spectroscopy will be provided, ranging from typically measured fluorescence quantities over instrument-specific contributions to measured fluorescence signals to selected applications. In this context, an overview of the photoluminescence properties of molecular and nanoscale luminescence reporters will be given including a brief insight into their photophysics and fluorescence standards designed by division Biophotonics for the calibration and instrument performance validation of fluorescence measuring devices will be presented.
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.
We studied the dissolution behavior of β NaYF4:Yb(20%), Er(2%) UCNP of two different sizes in biologically relevant media i.e., water (neutral pH), phosphate buffered saline (PBS), and Dulbecco’s modified Eagle medium (DMEM) at different temperatures and particle concentrations. Special emphasis was dedicated to assess the influence of different surface functionalizations, particularly the potential of mesoporous and microporous silica shells of different thicknesses for UCNP stabilization and protection. Dissolution was quantified electrochemically using a fluoride ion selective electrode (ISE) and by inductively coupled plasma optical emission spectrometry (ICP OES). In addition, dissolution was monitored fluorometrically. These experiments revealed that a thick microporous silica shell drastically decreased dissolution. Our results also underline the critical influence of the chemical composition of the aqueous environment on UCNP dissolution. In DMEM, we observed the formation of a layer of adsorbed molecules on the UCNP surface that protected the UCNP from dissolution and enhanced their fluorescence. Examination of this layer by X ray photoelectron spectroscopy (XPS) and mass spectrometry (MS) suggested that mainly phenylalanine, lysine, and glucose are adsorbed from DMEM. These findings should be considered in the future for cellular toxicity studies with UCNP and other nanoparticles and the design of new biocompatible surface coatings.
We developed a procedure to prepare luminescent LiYF4:Yb/LiYF4 and LiYF4:Yb,Er/LiYF4 core/shell nanocrystals with a size of approximately 40 nm revealing luminescence decay times of the dopant ions that approach those of high-quality laser crystals of LiYF4:Yb (Yb:YLF) and LiYF4:Yb,Er (Yb,Er:YLF) with identical doping concentrations. As the luminescence decay times of Yb3+ and Er3+ are known to be very sensitive to the presence of quenchers, the long decay times of the core/shell nanocrystals indicate a very low number of defects in the core particles and at the core/shell interfaces. This improvement in the performance was achieved by introducing two important modifications in the commonly used oleic acid based synthesis. First, the shell was prepared via anewly developed method characterized by a very low nucleation rate for particles of pure LiYF4 shell material. Second, anhydrous acetates were used as precursors and additional drying steps were applied to reduce the incorporation of OH− in the crystal lattice, known to quench the emission of Yb3+ ions. Excitation power density (P)-dependent absolute measurements of the upconversion luminescence quantum yield (Φ,UC) of LiYF4:Yb,Er/LiYF4 core/shell particles reveal a maximum value of 1.25% at P of 180 W·cm−2.
Although lower than the values reported for NaYF4:18%Yb,2%Er core/shell nanocrystals with comparable sizes, these Φ, UC values are the highest reported so far for LiYF4:18%Yb,2%Er/LiYF4 nanocrystals without additional dopants. Further improvements May nevertheless be possible by optimizing the dopant concentrations in the LiYF4 nanocrystals.
Tricalcium aluminate (C3A) is found with less than 10% wt. of the total composition; however, during hydration, C3A plays an important role in the early hydration of cement in the presence of gypsum as a set retarder.
The aim of this investigation is to assess the suitability of optical spectroscopy and a dye-based optical probe to monitor early hydration of C3A in the presence of gypsum and hemihydrate. Optical evaluation was performed using steady-state fluorescence and diffuses reflectance spectroscopy (UV-VisDR). Phase characterization during hydration was done with in-situ X-ray diffraction. UV-VisDR with a cyanine dye probe was used to monitor the formation of metastable phases and was employed together with fluorescence spectroscopy, to follow the Aggregation and disaggregation of the dye during hydration. In conclusion, for the first time, a cyanine dye was identified as a feasible and stable probe to monitor C3A hydration changes in the presence of calcium sulfate.
We present a comparative study of the spectroscopic properties of the donor–acceptor–donor substituted dyes triphenylamine-allylidenemalononitrile-julolidine (TMJ) and triphenylamine-allylidenemalononitriletriphenylamine (TMT), bearing one and two propeller-like triphenylamine donor moieties, in solvents of varying polarity and viscosity and in the aggregated and solid state. Our results reveal control of the aggregation-induced spectroscopic changes and the packing motifs of the dye molecules in the solid state by the chemical nature and structure of the second nitrogen-containing donor, i.e., a planar and a rigid julolidine or a twisted triphenyl group. Assuming that the TMT and TMJ aggregates show a comparable arrangement of the molecules to the respective crystals, these different molecular interactions in the solid state are responsible for aggregation induced emission (AIE) in the case of TMT and its absence for TMJ. Moreover, a versatile strategy for the fluorescence enhancement of only weakly emissive AIE dyes is shown, turning these dyes into bright nanoscale fluorescent reporters by using them as stains for preformed polymer particles.
Merocyanine–triarylamine bichromophores are readily synthesized by sequentially Pd-catalyzed insertion alkynylation–Michael–Suzuki four-component reactions. White-light emissive systems form upon aggregation in 1 : 99 and 0.1 : 99.9 vol% CH2Cl2–cyclohexane mixtures, ascribed to aggregation-induced dual emission (AIDE) in combination with partial energy transfer between both chromophore units as supported by spectroscopic studies.
N-Benzyl aroyl-S,N-ketene acetals can be readily synthesized by condensation of aroyl chlorides and N-Benzyl 2-methyl benzothiazolium salts in good to excellent yields, yielding a library of 35 chromophores with bright solid-state emission and aggregation-induced emission characteristics.
Varying the substituent from electron-donating to electronwithdrawing enables the tuning of the solid-state emission Color from deep blue to red.
We report the synthesis and spectroscopic characteristics of two different sets of carbon dots (CDs) formed by hydrothermal reaction between citric acid and polyethylenimine (PEI) or 2,3-diaminopyridine (DAP). Although the formation of amide-based species and the presence of citrazinic acid type derivates assumed to be responsible for a blue emission is confirmed for both CDs by elemental analysis, infrared spectroscopy, and mass spectrometry, a higher abundance of sp2-hybridized nitrogen is observed for DAP-based CDs, which causes a red-shift of the n-π* absorption band relative to the one of PEI-based CDs. These CD Systems possess high photoluminescence quantum yields (QY) of ∼40% and ∼48% at neutral pH, demonstrating a possible tuning of the optical properties by the amine precursor. pH-Dependent spectroscopic studies revealed a drop in QY to < 9% (pH ∼ 1) and < 21% (pH ∼ 12) for both types of CDs under acidic and basic conditions. In contrast, significant differences in the pHdependency of the n-π* transitions are found for both CD types which are ascribed to different (de)protonation sequences of the CD-specific fluorophores and functional groups using Zeta potential analysis.
We present here the design and characterization of a set of spectral calibration beads. These calibration beads are intended for the determination and regular control of the spectral characteristics of fluorescence microscopes and other fluorescence measuring devices for the readout of bead-based assays. This set consists of micrometer-sized polymer beads loaded with dyes from the liquid Calibration Kit Spectral Fluorescence Standards developed and certified by BAM for the wavelength-dependent Determination of the spectral responsivity of fluorescencemeasuring devices like spectrofluorometers. To cover the wavelength Region from 400 to 800 nm, two new near-infrared emissive dyes were included, which were spectroscopically characterized in solution and encapsulated in the beads. The resulting set of beads presents the first step towards a new platform of spectral calibration beads for the determination of the spectral characteristics of fluorescence instruments like fluorescence microscopes, FCM setups, and microtiter plate readers, thereby meeting the increasing demand for reliable and comparable fluorescence data especially in strongly regulated areas, e.g., medical diagnostics. This will eventually provide the basis for standardized calibration procedures for imaging systems as an alternative to microchannel slides containing dye solutions previously reported by us.
The employment of type-I pyrethroids for airplane disinfection in recent years underlines the necessity to develop sensing schemes for the rapid detection of these pesticides directly at the point-of-use. Antibody-gated indicator-releasing materials were thus developed and implemented with test strips for lateral-flow assay-based analysis employing a smartphone for readout. Besides a proper matching of pore sizes and gating macromolecules, the functionalization of both the material's outer surface as well as the strips with PEG chains enhanced system performance. This simple assay allowed for the detection of permethrin as a target molecule at concentrations down to the lower ppb level in less than 5 minutes.
Water-Soluble Aza-BODIPYs: Biocompatible Organic Dyes for High Contrast In Vivo NIR-II Imaging
(2020)
A simple NIR-II emitting water-soluble system has been developed and
applied in vitro and in vivo. In vitro, the fluorophore quickly accumulated in 2D and 3D cell cultures and rapidly reached the tumor in rodents, showing high NIR-II contrast for up to 1 week. This very efficient probe possesses all the qualities necessary for translation to the clinic as well as for the development of NIR-II emitting materials.
We synthesized a generation of water-soluble, atomically precise gold nanoclusters (Au NCs) with anisotropic Surface containing a short dithiol pegylated chain (AuMHA/TDT). The AuMHA/TDT exhibit a high brightness (QY ∼ 6%) in the shortwave infrared (SWIR) spectrum with a detection above 1250 nm.
Furthermore, they show an extended half-life in blood (t1/2ß = 19.54 ± 0.05 h) and a very weak accumulation in organs. We also developed a non-invasive, whole-body vascular imaging system in the SWIR window with high-resolution, benefiting from a series of Monte Carlo image processing. The imaging process enabled to improve contrast by 1 order of magnitude and enhance the spatial Resolution by 59%. After systemic administration of these nanoprobes in mice, we can quantify vessel complexity in depth (>4 mm), allowing to detect very subtle vascular disorders non-invasively in bone morphogenetic protein 9 (Bmp9)-deficient mice. The combination of these anisotropic surface charged Au NCs plus an improved SWIR imaging device allows a precise mapping at high-resolution and an in depth understanding of the organization of the vascular network in live animals.
We report on the temperature- and structural-dependent optical properties and photophysics of a set of boron dipyrromethene (BODIPY) dyes with different substitution patterns of their meso-aryl subunit. Single-crystal Xray diffraction analysis of the compounds enabled a classification of the dyes into a sterically hindered and a unhindered group. The steric hindrance refers to a blocked rotational motion of the aryl subunit around the bond connecting this moiety to the meso-position of the BODIPY core. The energy barriers related to this rotation were simulated by DFT calculations. As follows from the relatively low rotational barrier calculated to about 17 kcal/mol, a free rotation is only possible for sterically unhindered compounds. Rotational barriers of more than 40 kcal/mol determined for the sterically hindered compounds suggest an effective freezing of the rotational motion in These molecules. With the aid of temperature-dependent spectroscopic measurements, we could show that the ability to rotate directly affects the optical properties of our set of BODIPY dyes. This accounts for the strong temperature dependence of the fluorescence of the sterically unhindered compounds which show a drastic decrease in fluorescence quantum yield and a significant shortening in fluorescence lifetime upon heating. The optical properties of the sterically hindered compounds, however, are barely affected by temperature. Our results suggest a nonradiative deactivation of the first excited singlet state of the sterically unhindered compounds caused by a conical intersection of the potential energy surfaces of the Ground and first excited state which is accessible by rotation of the meso-subunit. This is in good agreement with previously reported deactivation mechanisms. In addition, our results suggest the presence of a second nonradiative depopulation pathway of the first excited singlet state which is particularly relevant for the sterically hindered compounds.
Singlet oxygen can severely damage biological tissue, which is exploited in photodynamic therapy (PDT). In PDT, the effective range is limited by the distribution of the photosensitizer (PS) and the illuminated area. However, no distinction is made between healthy and pathological tissue, which can cause undesired damage. This encouraged us to exploit the more acidic pH of cancerous tissue and design pH-controllable singlet oxygen-generating boron-dipyrromethene (BODIPY) dyes. A pH sensitivity of the dyes is achieved by the introduction of an electronically decoupled, photoinduced electron transfer (PET)-capable subunit in meso-position of the BODIPY core. To favor triplet-state formation as required for singlet Oxygen generation, iodine substituents were introduced at the chromophore core. The resulting pH-controlled singlet oxygen-generating dyes with pKa values in the physiological range were subsequently assessed regarding their potential as pH-controlled PS for PDT.
Using HeLa cells, we could successfully demonstrate markedly different pH-dependent cytotoxicities upon illumination.
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.
In summary, we report a highly modular solid TTA-UC system comprising of a crystalline, thermally stable PCN222(Pd) MOF with CA-coated MOF channels and with a DPA annihilator embedded in a solution-like environment in the MOF channels. This solid material displays blue upconverted delayed emission with a luminescence lifetime of 373 us, a threshold value of 329 mW*cm-2 and a triplet–triplet energy transfer efficiency of 82%. This optical application adds another facet to the versatile chemistry of PCN-222 MOFs. The design concept is also applicable to other TTA-UC pairs and enables tuning of the UCL color, for example, by replacing DPA with other dyes as exemplarily shown for 2,5,8,11-tetra-tert-butyl-perylene, that yields UCL at 450 nm. Current work aims to reduce the oxygen sensitivity and to increase the retention of the trapped annihilators in organic environments, for example, by tuning the chain length of the carboxylic acid and by coating the MOF surface. In addition, the TTA-UC efficiency will be further enhanced by reducing the reabsorption of the UC emission caused by Pd(TCPP) and by optimizing the sensitizer/annihilator interface.
We report the synthesis and characterization of carbon nanodots (CDs) with high quantum yield (>50%) and tailored optical absorption as well as emission properties. A well-described protocol with polyethyleneimine (PEI) as amine precursor is used as a reference to a new CD system which is stabilized by aromatic 2,3-diaminopyridine (DAP) molecules instead. The DAP stabilizer is installed in order to red-shift the absorption peak of the n-π* electron transition allowing efficient radiative recombination and light emission. Size, shape, and chemical composition of the samples are determined by (HR)TEM, EDX and FTIR-spectroscopy. Optical parameters are investigated using UV-VIS, PL and QY measurements. Several parameters such as concentration, excitation wavelength and pH are studied. Zeta-potential analysis indicate that pH-induced (de-)protonation processes of functional moieties directly affect the n-π* energy bands. This results in unique pH-dependent absorption and emission characteristics which are discussed on the specific chemical composition of each CD system.
Multiplexed encoding schemes of nano- and micrometer sized particles with fluorescent dyes or quantum dots (QDs) and their optical detection, are of increasing interest for applications in the life sciences, for example in flow cytometry. Almost all strategies utilizing fluorescence focus on spectrally distinguishable emission bands or colors and different intensity levels as fluorescence codes. The fluorescence parameter lifetime has been, however, barely exploited. In this work the goal is to perform multiplexing with encoding fluorophores with different fluorescence lifetimes (LTs). In comparison to the spectral multiplexing strategies this has the advantage, that the different fluorescence LT codes can be measured with the excitation and emission wavelength, thus reducing instrument costs. Moreover, LTs should not depend on emitter concentration. Unlike organic dyes, the LTs of which are typically < 10 ns, the fluorescence LTs of ternary semiconductor QDs that represent a “green” alternative to conventional Cd-containing QDs are in the range of several hundred ns, independent of oxygen concentration, and can be tuned to a certain extent by chemical composition and surface chemistry. This present a time region that can be barely covered by other emitters that have either much shorter or longer lifetimes. In this project, different encoding strategies will be assessed and the encoded particles will be then used for fluorescence assays for the analysis of several targets in parallel. Therefor the encoded particles will be functionalized with different target-specific bioligands and read out with a specifically designed flow cytometer enabling time-resolved fluorescence detection. With this instrument, the particles will be discriminated by their fluorescence LTs In one detection channel while the analytes will be quantified by fluorescence labels in a second channel in the intensity domain.
Multiplexed encoding schemes of nano- and micrometer sized polymer particles with fluorescent dyes or quantum dots (QDs) and their optical detection, are of increasing interest for applications in the life sciences, for example in flow cytometry. Almost all strategies utilizing fluorescence focus on spectrally distinguishable emission bands or colors and different intensity levels as fluorescence codes.
In this work the goal is to perform multiplexing with encoding fluorophores with different fluorescence lifetimes (LTs). In comparison to the spectral multiplexing strategies this has the advantage, that the different fluorescence LT codes can be measured with the same excitation and emission wavelength, thus reducing instrumental costs. Moreover, LTs should not depend on emitter concentration. Unlike organic dyes, the LTs of which are typically <10ns, the fluorescence LTs of ternary semiconductor QDs which represent a “green” alternative to conventional Cd-containing QDs are in the range of several hundred ns, independent of oxygen concentration, and can be tuned to a certain extent by chemical composition and surface chemistry. This presents a time region that can barely be covered by other emitters that have either much shorter or longer lifetimes. In this project, different encoding strategies will be assessed, and the encoded particles will then be used for fluorescence assays for the analysis of several targets in parallel. Therefore, the encoded particles will be functionalized with different target-specific bioligands and read out with a specifically designed flow cytometer enabling time-resolved fluorescence detection. With this instrument, the particles will be discriminated by their fluorescence LTs in one detection channel while the analytes will be quantified by fluorescence labels in a second channel in the intensity domain.
Spectral encoding of cells or particles and the discrimination of multiple spectral codes are a critical process in flow cytometry (FCM). Typical issues in spectral encoding are, e.g., the spectral overlap of codes, or the increasing complexity of instruments . The exploitation of the photoluminescence lifetime (LT) as an encoding parameter could be used to circumvent both of these issues, as it adds another dimension to the parameter space, or, when used as a stand-alone parameter, requiring only one excitation light source and one detector. While LT encoding was considered already decades ago it is still not implemented as a routine technique in FCM yet, mainly due to the challenge of very few photons being available within the limited transition time of a cell or particle through the laser spot.
Recently, we demonstrated LT-FCM based on luminophores with ns LTs in a compact and low-cost flow cytometer. Measurements on polymer microbeads containing luminophores with distinctly different excited state LTs enabled the complete discrimination of three LT codes and five codes in total could be identified.
Now, we have extended our approach towards considerably longer LTs by custom-made polymer microbeads loaded with different ratios of InP/ZnS and AgInS2 quantum dots. The use of these materials significantly expands the usable time range for LT encoding to up to several hundred ns. Our studies demonstrate the possibility to further increase the number of viable LT codes for multiplexing in LT-FCM without the need for extensive hardware modifications.
The comparison of different emitter classes and the rational design of the next generation of molecular and nanoscale reporters require accurate and quantitative photo-luminescence measurements. This is of special importance for all photoluminescence applications in the life and material sciences and nanobiophotonics. In the following, procedures for the determination of the spectroscopic key parameter photoluminescence quantum yield, i.e., the number of emitted per absorbed photons, in the UV/vis/NIR/SWIR are presented including pitfalls and achievable uncertainties and material-specific effects related to certain emitter classes are addressed.
Bioanalytical, diagnostic, and security applications require the fast and sensitive determination of a steadily increasing number of analytes or events in parallel in a broad variety of detection formats.[1,2] Ideal candidates for spectral encoding and multiplexing schemes are luminescent nanocrystals like semiconductor quantum dots (QDs), particularly Cd-containing II/VI QDs with their narrow and symmetric emission bands. With the availability of relatively simple and inexpensive instrumentation for time-resolved fluorescence measurements, similar strategies utilizing the compound-specific parameter fluorescence lifetime or fluorescence decay kinetics become increasingly attractive.[3-5] The potential of different types of QDs like II/VI, III/V and Cd-free ternary QDs such as AgInS (AIS) QDs for lifetime-based encoding and multiplexing has been, however, barely utilized, although the lifetimes of these nanocrystals cover a time windows which is barely accessible with other fluorophores. Here we present a brief insight into the photophysics of AIS QDs and show the potential of dye- and QD-encoded beads for lifetime-based encoding and detection schemes in conjunction with flow cytometry and fluorescence lifetime imaging microscopy
Diaminodicyanoquinones: Fluorescent dyes with high dipole moments and electron-acceptor properties
(2019)
Fluorescent dyes are applied in various fields of research,includingsolarcellsandlight-emittingdevices,andas reporters for assays and bioimaging studies.Fluorescent dyes with an added high dipole moment pave the way to nonlinear optics and polarity sensitivity.Redox activity makes it possible to switch the moleculeQsphotophysical properties.Diaminodicyanoquinone derivatives possess high dipole moments,yet only lowfluorescence quantum yields,and have therefore been neglected as fluorescent dyes.Here we investigate the fluorescencepropertiesofdiaminodicyanoquinonesusingacombined theoretical and experimental approach and derive molecules with afluorescence quantum yield exceeding 90%. The diaminodicyanoquinone core moiety provides chemical versatility and can be integrated into novel molecular architectures with unique photophysical features.
We present a comprehensive investigation of the magnetic and optical properties of an ytterbium complex, which combines two desirable and practical features into a single molecular system. Based upon YbIII Ions that promote near-infrared optical activity and a chemical backbone that is ideal for an in-depth understanding of the magnetic behaviour, we have designed a multifunctional opto-magnetic species that operates as a luminescent thermometer and as a single-molecule magnet (SMM). Our magnetic investigations, in conjunction with ab initio calculations, reveal one of the highest energy barriers reported for an YbIII-based complex. Moreover, we correlate this anisotropic barrier with the Emission spectrum of the compound, wherein we provide a complete assignment of the energetic profile of the complex. Such studies lay the foundation for the design of exciting multi-faceted materials that are able to retain information at the single-molecule level and possess built-in thermal self-monitoring capabilities.
The rational synthesis and use of nanomaterials require the characterization of many different properties, ranging from particle size and size distribution over surface chemistry to more applicationrelevant features like optical, electrochemical, and magnetic properties. In the following, several methods for the characterization of functional groups on nanomaterials, like polymer and silica nanoparticles, semiconductor quantum dots, and lanthanide-based upconversion nanocrystals are presented. Additionally, procedures for the measurement of the key spectroscopic performance parameters of nanomaterials with linear and nonlinear photoluminescence, such as the photoluminescence quantum yield, are presented for the UV/vis/NIR/SWIR.
Structural modularity of polymer frameworks is a key advantage of covalent organic polymers, however, only C, N, O, Si and S have found their way into their building blocks so far. Here, we expand the toolbox available to polymer and materials chemists by one additional nonmetal, phosphorus. Starting with a building block that contains a λ⁵‐phosphinine (C₅P) moiety, we evaluate a number of polymerisation protocols, finally obtaining a π‐conjugated, covalent phosphinine‐based framework (CPF‐1) via Suzuki‐Miyaura coupling. CPF‐1 is a weakly porous polymer glass (72.4 m2 g‐1 N2 BET at 77 K) with green fluorescence (λmax 546 nm) and extremely high thermal stability. The polymer catalyzes hydrogen evolution from water under UV and visible light irradiation without the need for additional co‐catalyst at a rate of 33.3 μmol h‐¹ g‐¹. Our results demonstrate for the first time the incorporation of the phosphinine motif into a complex polymer framework. Phosphinine‐based frameworks show promising electronic and optical properties that might spark future interest in their applications in light‐emitting devices and heterogeneous catalysis.
Nanomaterials are used in many different applications in the material and life sciences. Examples are optical reporters, barcodes, and nanosensors, magnetic and optical contrast agents, and catalysts. Due to their small size and large surface area, there are also concerns about their interaction with and uptake by biological systems. This has initiated an ever increasing number of cyctoxicity studies of nanomaterials of different chemical composition and surface chemistry, but until now, the toxicological results presented by different research groups often do not address or differ regarding a potential genotoxicity of these nanomaterials. This underlines the need for a standardized test procedure to detect genotoxicity.1,2
Aiming at the development of fast, easy to use, and automatable microscopic methods for the determination of the genotoxicity of different types of nanoparticles, we assess the potential of the fluorometric γH2AX assay for this purpose. This assay, which can be run on an automated microscopic detection system, relies on the determination of DNA double strand breaks as a sign for genotoxicity.3 Here, we present first results obtained with broadly used nanomaterials like CdSe/CdS and InP/ZnS quantum dots as well as iron oxide, gold, and polymer particles of different surface chemistry with previously tested colloidal stability. These studies will be also used to establish nanomaterials as positive and negative genotoxicity controls or standards for assay performance validation for users of this fluorometric genotoxicity assay. In the future, after proper validation, this microscopic platform technology will be expanded to other typical toxicity assays.
References. (1) Landsiedel, R.; Kapp, M. D.; Schulz, M.; Wiench, K.; Oesch, F., Reviews in Mutation Research 2009, 681, 241-258. (2) Henriksen-Lacey, M.; Carregal-Romero, S.; Liz-Marzán, L. M., Bioconjugate Chem. 2016, 28, 212-221. (3) Willitzki, A.; Lorenz, S.; Hiemann, R.; Guttek, K.; Goihl, A.; Hartig, R.; Conrad, K.; Feist, E.; Sack, U.; Schierack, P., Cytometry Part A 2013, 83, 1017-1026.
Correlating the photoluminescence (PL) properties of nanomaterials like semiconductor nanocrystals (QDs) and upconversion nanocrystals (UCNPs) assessed in ensemble studies and at the single particle level is increasingly relevant for applications of these nanomaterials in the life sciences like bioimaging studies or their use as reporters in microfluidic assays. Here we present a comparison of the spectroscopic properties of ensembles and single emitters for QDs like II/VI QDs and cadmium-free AIS/ZnS QDs as well as different UCNPs. The overall goal of this study was to derive particle architectures well suited for spectroscopic and microscopic applications.
Relative and Absolute Methods for Measuring Photoluminescence Quantum Yields of UV/vis/NIR Emitters
(2019)
One of the key spectroscopic performance parameters of molecular and particulate emitters is the photoluminescence quantum yield (PL QY) that provides a direct measure for the number of emitted per absorbed photons. This triggered the interest in methods suitable for measuring this property for emitters in various environments in the UV/vis/NIR and above 1000 nm as well as on the ensemble and single emitter level. Moreover, for nonlinear emitters like lanthanide-based upconversion nanocrystals methods including instrumentation for power density-dependent PL QY studies are required.
An overview of the research activities in Division Biophotonics of BAM is given and suitable relative and absolute methods for the deter-mination of PL QY of organic dyes and different types of application-relevant nanomaterials in dispersion and in the solid state are presen-ted. This covers also the design and calibration of integrating sphere setups, achievable uncertainties, and candidates for PL QY reference materials.
Carbon dots have attracted much attention due to their unique optical, chemical and electronic properties enabling a wide range of applications. The properties of carbon dots can be effectively adjusted through modifying their chemical composition. However, a major challenge remains in understanding the core and surface contributions to optical and electronic transitions. Here, three blue luminescent carbon dots with carboxyl, amino and hydroxyl groups were comprehensively characterized by UV-vis absorption and emission spectroscopy, synchrotron-based X-ray spectroscopy, and infrared spectroscopy. The influence of the surface functionality on their fluorescence was probed by pH-dependent photoluminescence measurements. Moreover, the hydrogen bonding interactions between water and the surface groups of carbon dots were characterized by infrared spectroscopy. Our results show that both core and surface electronic states of blue luminescent carbon dots contribute to electronic acceptor levels while the chemical nature of the surface groups determines the hydrogen bonding behavior of the carbon dots. This comprehensive spectroscopic study demonstrates that the surface chemistry has a profound influence on the electronic configuration and surface–water interaction of carbon dots, thus affecting their photoluminescence properties.
Lanthanide-based upconversion nanoparticles (UCNPs) like hexagonal Beta-NaYF4 UCNPs doped with Yb3+ and Er3+, which efficiently convert 976 nm light to ultraviolet, visible, and near infrared photons, offer new strategies for luminescence-based sensing, barcoding, and imaging. The properties of their upconversion (UC) luminescence (UCL) are, however, strongly influenced by particle size, the concentration and spatial arrangement of the dopant ions, surface chemistry including presence and thickness of surface passivation and shielding shells, microenvironment/presence of quenchers with high energy vibrations, and excitation power density (P). We present here a comprehensive study of the influence of UCNP size and particle architecture for Yb3+ and Er3+ co-doped NaYF4 core-only and core-shell nanostructures in the size range of about 5 nm to 50 nm, which underlines the importance of particle synthesis, surface chemistry, and quantitative luminescence measurements for mechanistic insights and the determination of application-relevant matrix- and P-dependent optimum dopand concentrations.
During the last years, there has been a rapid rise in the use of nanomaterials in consumer products. Especially silver nanoparticles are frequently used because of their well-known optical and antimicrobial properties. However, the toxicological studies focusing on silver nanoparticles are controversial, either claiming or denying a specific nano-efffect. To contribute to localizing nanoparticles in toxicological studies and to investigate the interaction of particles with cells, a fluorescent marker is often used to monitor their transport and possible degradation. A major problem, in this context is the issue of binding stability of a fluorescent marker which is attached to the particle.
In order to overcome this problem we provide an investigation of the binding properties of fluorescence-labeled BSA to small silver nanoparticles. Therefore, we synthesized small silver nanoparticles which are stabilized by poly(acrylic acid). The particles are available as reference candidate material and were thoroughly characterized in an earlier study. The ligand was exchanged by fluorescence marked albumin (BSA-FITC). The adsorption of the ligands was monitored by dynamic light scattering (DLS). To verify that the observed effects on the hydrodynamic radius originate from the successful ligand exchange and not from agglomeration or aggregation we used small angle X-ray scattering (SAXS). The fluorescent particles were characterized by UV/Vis and fluorescence spectroscopy. Afterwards, desorption of the ligand BSA-FITC was monitored by fluorescence spectroscopy and the uptake of particles in different in vitro models was studied.
The particles are spherical and show no sign of aggregation after successful ligand exchange. The fluorescence intensity is quenched significantly by the presence of the silver cores as expected, but the remaining fluorescence intensity was high enough to use these particles in biological investigations. Half-life of fluorescence labeling on the particle was 21 d in a highly concentrated solution of non-labeled BSA. Thus, a very high dilution and long incubation times are needed to remove BSA-FITC from the particles. Finally, the fluorescence-labeled silver nanoparticles were used for uptake studies in human liver and intestinal cells, showing a high uptake for HepG2 liver cells and almost no uptake in differentiated intestinal Caco-2 cells. In conclusion, we showed production of fluorescence-marked silver nanoparticles. The fluorescence marker is strongly adsorbed to the silver surface which is crucial for future investigations in biological matrices. This is necessary for a successful investigation of the toxicological potential of silver nanoparticles.
DNA origami nanostructures provide a platform where dye molecules can be arranged with nanoscale accuracy allowing to assemble multiple fluorophores without dye–dye aggregation. Aiming to develop a bright and sensitive ratiometric sensor system, we systematically studied the optical properties of nanoarrays of dyes built on DNA origami platforms using a DNA template that provides a high versatility of label choice at minimum cost. The dyes are arranged at distances, at which they efficiently interact by Förster resonance energy transfer (FRET). To optimize array brightness, the FRET efficiencies between the donor fluorescein (FAM) and the acceptor cyanine 3 were determined for different sizes of the array and for different arrangements of the dye molecules within the array. By utilizing nanoarrays providing optimum FRET efficiency and brightness, we subsequently designed a ratiometric pH nanosensor using coumarin 343 as a pH-inert FRET donor and FAM as a pH-responsive acceptor. Our results indicate that the sensitivity of a ratiometric sensor can be improved simply by arranging the dyes into a well-defined array. The dyes used here can be easily replaced by other analyte-responsive dyes, demonstrating the huge potential of DNA nanotechnology for light harvesting, signal enhancement, and sensing schemes in life sciences.
The increasing interest in molecular and nanoscale emitters with photoluminescence > 800 nm and recently also > 1000 nm for bioanalysis, medical diagnostics, bioimaging, and safety Barcodes requires quantitative spectroscopic studies, which are, however still challenging in this long wavelength region. This is of special relevance for nanocrystalline emitters like semiconductor quantum dots and rods as well as lanthanide-based upconversion and downconversion nanocrystals, 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. Moreover, nonlinear emitters like lanthanide-based upconversion nanocrystals require also power density-dependent studies of their luminescence spectra, quantum yields, and decay kinetics. Here, we present suitable absolute methods and underline the impact of such measurements on a profound mechanistic understanding of the nonradiative deactivation pathways in semiconductor and upconversion nanocrystals of different chemical composition and particle architecture.
Cancer is a leading cause of death worldwide, and its early detection and resultant treatment contributes significantly to patient recovery and survival. Detection is currently based on magnetic resonance imaging and computed tomography, methods that are expensive, while processing of the results is time consuming. There is a need for low-cost cancer-detection techniques that give conclusive results in the shortest time possible. Molecularly imprinted polymers (MIPs) targeting tumor markers on cancerous cells may provide a cheaper solution for cancer detection. Thin MIP layers immobilized on particle platforms are known to give faster response times and increased selectivity in comparison to bulk MIPs. It has been reported that a fluorescent monomer can be incorporated into the MIP layer, allowing for faster detection of the target group, thus significantly shortening the turn-around time for biopsies.
Changes in sialylation patterns of cell surface glycoproteins indicate malignancy. Here, we present the development of MIPs that target sialic acid-terminated glycoproteins (SA MIPs), prepared as a thin layer on a silica nanoparticle platform. A fluorescent monomer is incorporated into the MIP layer, and upon binding of the target group to the specific binding pockets in the MIP, the fluorescence signal is enhanced. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are used for structural characterization. To validate the specificity, fluorescence changes of MIPs in the presence and absence of template are compared to their corresponding non-imprinted polymer particles (NIP). Initial binding experiments with tumor cells using fluorescence microscopy demonstrate that the presented technique shows promise as a cheaper alternative to current detection methods, while allowing for relatively shorter analysis of biopsy results.
Phage display is used to find specific target binding peptides for polypropylene (PP) surfaces. PP is one of the most commonly used plastics in the world. Millions of tons are produced every year. PP binders are of particular interest because so far gluing or printing on PP is challenging due to its low surface energy. A phage display protocol for PP was developed followed by Next Generation DNA Sequencing of the whole phage library. Data analysis of millions of sequences yields promising peptide candidates which were synthesized as PEG conjugates. Fluorescence-based adsorption-elution-experiments show high adsorption on PP for several sequences.
The overall interest in nanotoxicity, triggered by the increasing use of nanomaterials in the material and life sciences, and the synthesis of an ever increasing number of new functional nanoparticles calls for standardized test procedures1,2 and for efficient approaches to screen the potential genotoxicity of these materials. Aiming at the development of fast and easy to use, automated microscopic methods for the determination of the genotoxicity of different types of nanoparticles, we assess the potential of the fluorometric γH2AX assay for this purpose. This assay, which can be run on an automated microscopic detection system, relies on the detection of DNA double strand breaks as a sign for genotoxicity3. Here, we provide first results obtained with broadly used nanomaterials like CdSe/CdS and InP/ZnS quantum dots as well as iron oxide, gold, and polymer particles of different surface chemistry with previously tested colloidal stability and different cell lines like Hep-2 and 8E11 cells, which reveal a dependence of the genotoxicity on the chemical composition as well as the surface chemistry of these nanomaterials. These studies will be also used to establish nanomaterials as positive and negative genotoxicity controls or standards for assay performance validation for users of this fluorometric genotoxicity assay. In the future, after proper validation, this microscopic platform technology will be expanded to other typical toxicity assays.
Quantum dot (QD) based nanomaterials are very promising materials for the fabrication of optoelectronic devices like solar cells, light emitting diodes (LEDs), and photodetectors as well as as reporters for chemo- and biosensing and bioimaging. Many of These applications involve the monitoring of changes in photoluminescence intensity and energy transfer processes which can strongly depend on excitation wavelength or energy. In this work, we analyzed the excitation energy dependence (EED) of the photoluminescence quantum yields (PL QYs) and decay kinetics and the circular dichroism (CD) spectra of CdSe/CdS core/shell QDs with different thicknesses of the surface passivation shell. Our results demonstrate a strong correlation between the spectral position of local maxima observed in the EED of PL QY and the zero-crossing points of the CD profiles. Theoretical analysis of the energy band structure of the QDs with effective mass approximation suggests that these structures could correspond to exciton energy levels. This underlines the potential of CD spectroscopy for the study of electronic energy structure of chiroptically active nanocrystals which reveal quantum confinement effects.
The implementation of fluorescent methods is of outstanding importance in the field of optical chemical sensor Technology and biosciences. Their bioanalytical applications are manifold including fluorescence microscopy, fluorescence in situ hybridization, DNA sequencing, fluorescence-activated cell sorting, immunoassays, analysis of DNA and Protein microarrays, and quantitative PCR, just to name a few examples. Particularly, fluorescence microscopy is a valuable method in the versatile field of biomedical imaging methods which nowadays utilizes different fluorescence Parameters like emission wavelength/Color and lifetime for the discrimination between different targets. Sectional Images are available with confocal microscopes. Tissue, cells or single cellular compartments can be stained and visualized with fluorescent dyes and biomolecules can be selectively labeled with fluorescent dyes to Monitor biomolecular interactions inside cells or at Membrane bound receptors.
On the other hand , fluorophores can act as indicator (or "molecular probe") to visualize intrinsically colorless and non-fluorescent ionic and neutral analytes such as pH, Oxygen (pO2), metal ions, anions, hydrogen peroxide or bioactive small organic molecules such as Sugars or nucleotides. Thereby, their photoluminescent properties (fluorescence or phoporescence intensity, exitation and/or Emission wavelength, emission lifetime or anisotropy) respond to the presence of these species in their immediate Environment. In general, the use of luminescent probes has the advantage that they can be delivered directly into the sample, and detected in a contactless remote mode. By now, these probes are often encapsulated in different types of nanoparticles (NPs) made from (biodegradable) organic polymers, biopolymers or inorganic materials like silica or bound to their surface.
Micelles are of increasing importance as versatile carriers for hydrophobic substances and nanoprobes for a wide range of pharmaceutical, diagnostic, medical, and therapeutic applications. A key Parameter indicating the Formation and stability of micelles is the critical micelle concentration (CMC). In this respect, we determined the CMC of common anionic, cationic, and non-ionic surfactants fluorometrically using different fluorescent probes and fluorescence Parameters for Signal detection and ompared the results with conductometric and surface Tension measurements. Based upon These results, requirements, Advantages, and pitfalls of each methods are discussed. Our study underlines the versatility of fluorometric methods that do not impose specific requirements on surfactants and are especially suited for the quantification of very low CMC values. Conductivity and surface Tension measurements yield smaller uncertainties particularly for high CMC values, yet are more time- and substance consuming and not suitable for every surfactant.
Lanthanide-doped upconversion nanoparticles (UCNPs) are of great interest for biomedical applications. Currently, the applicability of UCNP bionanotechnology is hampered by the generally low luminescence intensity of UCNPs and inefficient energy Transfer from UCNPs to surface-bound chromophores used e.g. for photodynamic therapy or analyte sensing. In this work, we address the low-Efficiency issue by developing versatile core-Shell nanostructures, where high-concentration sensitizers and activators are confined in the core and Shell Region of representative hexagonal NaYF2:Yb,Er UCNPs. After Doping concentration optimization, the sensitizer-rich core is able to harvest/accumulate more excitation energy and generate almost one order of Magnitude higher luminescence intesity than conventional homogeneously doped nanostructures. At the same time, the activator Ions located in the Shell enable a ~6 times more efficient resonant energy Transfer from UCNPs to surface-bound acceptor dye molecules due to the short distance between donor-acceptor pairs. Our work provides new insights into the rational design of UCNPs and will greatly encrease the General applicability of upconversion nanotechnologies.
At the core of luminescence color and lifetime Tuning of rare earth doped upconverting nanoparticles (UCNPs), is the understanding of the Impact of the particle architecture for commonly used sensitizer (S) and activator (A) Ions. In this respect, a series of core@Shell NaYF4 UCNPs doped with Yb3+ and Ho3+ ions are presented here, where the same dopant concentrations are distributed in different particle architectures following the scheme: YbHo core and YbHo@..., ...@YbHo, Yb@Ho, Ho@Yb, YbHo@Yb, and Yb@YbHo core-Shell NPs. As refealed by quantitative steady-state and time-resolved luminescence studies, the relative spatial Distribution of the A and S ions in the UCNPs and their protection from surface quenching has a critical Impact on ther luminescence characteristics. Although the increased amount of Yb3+ Ions boosts UCNP Performance by amplifying the Absorption, the Yb3+ ions can also efficiently dissipate the energy stored in the material through energy Migration to the surface, thereby reducing the Overall energy Transfer Efficiency to the activator ions. The results provide yet another proof that UC Phosphor chemistry combined with materials Engineering through intentional core@shell structures may help to fine-tune the luminescence Features of UCNPs for their specific future applications in biosensing, bioimaging, photovoltaics, and Display technologies.
Due to their unique physical properties, particularly their electronic and luminescent properties, graphene quantum dots (GQDs) are expected to be suitable for a wide range of applications in bioimaging, electro-optical and photonic materials or energy harvesting among others.1 Tuning the surface chemistry provides an efficient approach to modulate the fluorescence and distinct electronic properties of GQDs.2 Nevertheless, the role of surface chemistry on the electronic structure of GQDs remains poorly understood. In this presentation, we will compare systematically the electronic and chemical structures of GQDs functionalized with carboxylic and aminated groups to those of non-functionalized GQDs, combining theoretical and experimental approaches, here various photon-based spectroscopies. First, the electronic structure of GQDs was characterized by soft X-ray absorption (XA) and X-ray emission (XE) spectroscopies, probing unoccupied and occupied electronic states, respectively, at the carbon K edge for the first time. The interpretation of the XA/XE spectra was done based on theoretical calculations. Then, the chemical structure of the GQDs was characterized in situ by ATR-FTIR in water, thereby accounting for the importance of the interface between GQDs and water believed to play a central role in the chemical reactivity and the optical properties. We previously demonstrated that monitoring the OH vibrations of water molecules during exposure to humid air was a powerful method to probe H-bonding environment around carbon nanomaterials.3 For GQDs, clear surface-dependent water adsorption profiles are observed and discussed. Finally, UV/Vis absorption and photoluminescence measurements were done to characterize the optical properties of these GQDs. Our results suggest that the surface chemistry of the GQDs affects significantly their electronic structure and optical properties. These findings will contribute to an improved understanding of the structure–activity relationship of GQDs and other carbon nanomaterials with surface modifications.
There is an increasing interest in optical reporters like semiconductor and lanthanide-based nanocrystals with emission > 800 nm and recently also > 1000 nm for bioanalysis, medical diagnostics, and safety barcodes. Mandatory for the comparison of different emitter classes and the rational design of the next generation of reporters for the short wavelength infrared (SWIR) region are reliable and quantitative photoluminescence measurements in this challenging wavelength region. This is of special relevance for nanocrystalline emitters like semiconductor quantum dots and rods as well as for upconversion and downconversion nanocrystals, where surface states and the accessibility of emissive states by quenchers largely control accomplishable quantum yields and hence, signal sizes and detection sensitivities from the reporter side. Such measurements are currently hampered by the lack of suitable methods and standards for instrument calibration and validation as well as by the lack of quantum yield standards with emission > 800 nm and especially > 1000 nm.
In this respect, we present the design of integrating sphere setups for absolute and excitation power densitydependent measurements of emission spectra and quantum yields in the wavelength region of 650 to 1650 nm
including calibration strategies and first candidates for potential fluorescence standards. Subsequently, the photoluminescence properties of different types of nanocrystals are presented and discussed including absolute photoluminescence measurements of upconversion and down conversion emission in different solvents.
There is an increasing interest in molecular and nanoscale with emission > 800 nm and recently also > 1000 nm for bioanalysis, medical diagnostics, bioimaging, and safety barcodes. Mandatory for the comparison of different emitter classes and the rational design of the next generation of reporters for the short wavelength infrared (SWIR) Region are reliable and quantitative photoluminescence measurements in this challenging wavelength region. This is of special relevance for nanocrystalline emitters like semiconductor quantum dots and rods as well as lanthanide-based upconversion and downconversion nanocrystals, 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. Such measurements are currently hampered by the lack of suitable methods and standards for instrument calibration and validation and quantum yield standards with emission > 800 nm and especially > 1000 nm.
In this respect, we present the design of integrating sphere setups for absolute and excitation power density-dependent measurements of emission spectra and photoluminescence quantum yields in the wavelength Region of 650 to 1650 nm including calibration strategies and first candidates for potential fluorescence standards.
Subsequently, the photoluminescence properties of different types of nanocrystals are presented including the upconversion and downconversion emission of differently sized and surface functionalized lanthanide-doped nanoparticles and photoluminescence quenching effects are quantified.
Upconversion nanoparticles (UCNPs) offer new strategies for luminescence-based sensing and imaging. One of the best studied materials are ..-NaYF4 UCNPs doped with 20 % Yb3+ and 2 % Er3+, which efficiently convert 976 nm light to photons emitted at 540 nm, 655 nm, and 845 nm, respectively, reveal long luminescence lifetimes (> 100 µs), are photostable and chemically inert. Their upconversion (UC) luminescence (UCL) properties are, however, strongly influenced by particle size, surface chemistry, and microenvironment. In addition, the multiphotonic absorption processes responsible for UCL render UCL excitation power density (..) dependent. This makes quantitative UCL measurements as well as the determination of UC quantum yields (.UC) very challenging.
The rational design of brighter UCNPs particle architectures and the interest in identifying optimum particle architectures for FRET-based sensing and imaging schemes, which often rely on core-only UCNPs to minimize donor-acceptor distances, encouraged us to assess the influence of particle size and P on UCL. Here, we present the photophysical properties of a series of hexagonal NaYF4 UCNPs with sizes from 10 to 43 nm with different surface ligands dispersed in organic solvents and water studied by steady state and time-resolved fluorometry as well as quantitatively by integrating sphere spectrometry with P varied over about three orders of magnitude. Our results underline the need for really quantitative luminescence studies for mechanistic insights and the potential of high P to compensate for UCL quenching due to high energy phonons and surface effects.
Comparison of fluorescence measurements performed on different fluorescence instruments, analyte quantification from fluorescence intensities as well as the determination of fluorescence quantum yields require instrument calibration and consideration of the wavelength-dependent instrument-specific quantities spectral photon flux reaching the sample and spectral responsivity. Here, we present guidelines and recommendations for the qualification of fluorescence instruments and introduce suitable chromophore-based reference materials. Moreover, the design concepts of the different BAM fluorescence standards are discussed.
Semiconductor nanocrystals with a spherical (QDs) core and a spherical or a rod-shaped Shell, u.a., so-called Quantum dot-Quantum rods (QDQRs) are increasingly used as fluorescent Reporters or optically active components in the life and material science, e.g., in solid state lightening including Plasma Displays. (1,2) Morever, there is an increasing interest in materials with emission >800 nm for bioanalysis, medical diagnostics, and safety barcodes. Prerequisites for the mechanistic understanding of nonradiativ decay channels needed for the rational design of improved nanomaterials and the comparison of material Performance are reliable fluorescence measurements and validated methods for the assessment of their surface chemistry. (3,4) The latter is of particular importance for nanocrystalline Emitters, where surface states and the accessibility of emissive states by quenchers largely control photoluminescence properties. (5) Here, we present results from systematic spectroscopic studies including absolutely measured photolumunescence Quantum yields of different vissible and NIR emisisve QD and QDQRs Systems of varying particle architecture size and surface chemistries in Dispersion and embedded in salt crystals. (6,7)
In this review we present new concepts and recent progress in the application of semiconductur quantum dots (QD) as labels in two important areas of biology, bioimaging and biosensing. We analyze the biologically relevant properties of QDs focusing on the following topics: QD surface treatment and stability labeling of cellular structures and receptors with QDs, incorporation of QDs in living cells, cytotoxicity of QDs and influence of the biolocical environment on the biological and optical properties of QDs. Initially, we consider utilization of QDs as agants in high-resolution bioimaging techniques that can provide information at the molecular levels. The deverse range of modern live-cell QD-based imaging techniques with resolution far beyond the diffraction limit of light is examined. In each technique, we discuss the pros and cons of QD use and deliberate how QDs can be further engineered to facilitate their application in the respective imaging techniques and to produce significant improvements in resolution. Then we review QD-based point-of-care bioassays, bioprobes, and biosensors designed in different formats ranging from analytic biochemistry assays and ELISA, to novel point-of-care smartphone integrated QD-based biotests. Here, a wide range of QD-based fluorescence bioassays with optical transduction, electrochemiluminescence and photoelectrochemical assays are discussedc. Finally, this review provides an analysis of the prospects of application of QDs in selected important Areas of biology.
Photoluminescence techniques are amongst the most widely used Tools in the life sciences, with new and exciting applications in medical diagnostics and molecular Imaging continuously emerging. Advantages include their comparative ease of use, unique sensitivity, non-invasive character, and potential for Multiplexing, remote sensing, and miniaturization. General drawbacks are, however, signals, that contain unwanted wavelength- and polarization contributions from Instrument-dependent effects, which are also time-dependent due to aging of Instrument-components, and difficulties to measure absolute flourescence entensities. Moreover, scattering Systems require Special measurement geometries and the interest in new optical Reporters with Emission > 1000 nm strategies for reliable measurements in the second diagnostic for the comparison of material Performance and the rational designg of new flourophores with improved properties.
Here, we present strategies to versatile method-adaptable liquid and solid flourescence Standards for different flourescence paramters including traceable Instrument calibration procedures and the design of integrating spere setups for the absolute measurements of emission spectra and Quantum yields in the wavelength Region of 350 to 1600 nm. Examples are multi-Emitter glasses, spectral flourescence Standards, and quantum yield Standards for the UV/vis/NIR.
The quantum yield is a critically important parameter in the development of lanthanide-based upconverting nanoparticles (UCNPs) for use as novel contrast agents in biological imaging and optical reporters in assays. The present work focuses on the influence of the beam Profile in measuring the quantum yield (f) of nonscattering dispersions of nonlinear upconverting probes, by establishing a relation between f and excitation light power density from a rate equation analysis. A resulting 60% correction in the measured f due to the beam profile utilized for excitation underlines the significance of the beam profile in such measurements, and its impact when comparing results from different Setups and groups across the world.
The unparalleled excited-state potential-energy landscape of the chromium(III)-based dye [1]3+ ([Cr(ddpd)2]3+; ddpd=N,N’-dimethyl-N,N’-dipyridin-2-ylpyridin-2,6-diamine) enables a strong dual emission in the near infrared region. The temperature dependence of this dual emission allows the use of [1]3+ as an unprecedented molecular ratiometric thermometer in the 210–373 K temperature range in organic and in aqueous media. Incorporation of [1]3+ in biocompatible nanocarriers, such as 100 nm-sized polystyrene nanoparticles and solutol micelles, provides nanodimensional thermometers operating under physiological conditions.
A set of highly fluorescent, pH-responsive boron dipyrromethene dyes covering the pH range of 5-12 is presented for broad range pH measurements in mixed aqueous-organic median and polymer matrices. Readout in the intensity Domain with low cost and miniaturized Instrumentation utilizes reversible protonation induced switching ON of their initially completely quenched flourescence mediated by photoinduced electron Transfer. All dyes, rationally designed to reveal closely matching Absorption and Emission properties, are accessible via facile two-step reactions in Overall yields of up to 20%. By modifying the Substitution pattern of the meso-Aryl substiuent, the pKa values could be fine-tuned from 6 to 11. Integration of these molecules into polymeric films by a simple mixing procedure yielded reversible and longterm stable pH sensors for naked eye detection.
Lanthanide-doped photon-upconversion nanoparticles (UCNPs) have been the Focus of many Research activities in materials and life sciences in the last 15 years because of their potential to convert light between different spectral regions and their unique photophysical properties. To fully exploit the application potential of These facinating nanomaterials, a number of challenges have to be overcome, such as the low brightness, particularly of small UCNPs, and the reliable quantification of the excitation-power-density-dependent upconversion luminescence. In this series of critical Reviews, recent developments in the design, Synthesis, optical-spectroscopic characterization, and application of UCNPs are presented with Special Focus on bioanalysis and the life sciences. Here we guide the reader from the Synthesis of UCNPs to different concepts to enhance their luminescence, including the required optical-spectroscopic assessment to quantify material Performance; surface modification strategies and bioanalytical applications as well as selected examples of the use of UCNPs as reporters in different Assay formats are addressed in part II. Future Trends and challenges in the field of upconversion are discussed with Special emphasis on UCNP Synthesis and material characterization, particularly quantitative luminescence studies.