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Chlorination of pool water and wastewater, in food and pharmaceutical production, as well as in pesticide and paper manufacturing is a routinely used technique. However, the amount of chlorine in water must be strictly adjusted, to ensure enough concentration to kill pathogenic bacteria and viruses, while preventing too high concentrations inducing negative effects on human health. As an indicator, a molecular fluorescent probe based on a BODIPY structure was designed. This indicator exhibits a sensitive and selective fluorescence response upon increasing concentrations of hypochlorite in aqueous solvent mixtures. Real-time analyses became possible after the integration of this fluorescent indicator into newly designed 2D & 3D microfluidic chips incorporating a passive sinusoidal mixer and a micro-hydrocyclone, respectively. A comparison of the two microfluidic systems, including their ability to prevent accumulation or circulation of microbubbles, has shown excellent fluidic behaviour for the micro-hydrocyclone device. This system was distinctly more robust against gas bubbles, showed a higher signal gain and allowed to halve the limit of detection to 0.02 mg L–1. The use of the 3D system to quantify the chlorine content of pool water samples for sensitive and quantitative chlorine monitoring has been demonstrated.
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
A “turn-on” fluorescence sensing system based on a BODIPY-cobaloxime complex for the detection of H2S in liquid and gas phase was developed. To that aim, two cobaloxime complexes bearing an axial pyridyl-BODIPY ligand were initially evaluated as sensitive fluorescent HS− indicators in aqueous solution. The sensing mechanism involves the selective substitution of the BODIPY ligand by the HS− anion at the cobalt center, which is accompanied by a strong fluorescence enhancement. The selection of a complex with an ideal stability and reactivity profile toward HS− relied on the optimal interaction between the cobalt metal-center and two different pyridyl BODIPY ligands. Loading the best performing BODIPY-cobaloxime complex onto a polymeric hydrogel membrane allowed us to study the selectivity of the probe for HS− against different anions and cysteine. Successful detection of H2S by the fluorescent “light-up” membrane was not only accomplished for surface water but could also be demonstrated for relevant H2S concentrations in gas phase.
The reliable identification and quantification of phosphorylated amino acids, peptides and proteins is one of the key challenges in contemporary bioanalytical research, noteworthy, to diagnose and treat diseases at an early developmental stage. Miniaturised sensing devices like microfluidic chips combined with “smart” detection chemistry, simple data assessment, processing and presentation are very attractive for benchtop use in clinical environments.
We developed novel synthetic probes targeting phosphorylated amino acids, based on core-shell microparticles consisting of a silica core coated with a molecularly imprinted polymer (MIP) shell. These “plastic antibodies” are extremely robust, resist denaturing solvents and elevated temperatures, can be reproducibly produced at low cost, and potentially overcome many of the practical problems in current bioanalytical detection strategies. The MIP layer contains a fluorescent probe monomer, binds selectively to phosphorylated tyrosine (pTyr) with a significant imprinting factor higher than 3.5 and responds with a “lighting-up” of its fluorescence accompanied by the development of a strongly red-shifted emission band toward the analyte.
In analogy to our previous work [4], the bead-based ratiometric detection scheme has also been successfully transferred to a microfluidic chip format to demonstrate its applicability to rapid assays. Such a miniaturised device could yield an automated pTyr measurement system in the future. The setup was built by coupling a modular microfluidic system [5] for amino acid functionalisation (Fmoc protection) and a multi-layer PDMS/Teflon/glass microfluidic chip [6] for buffering, extraction (micropillars co-flow extraction) and selective adsorption on the MIP core-shell particles.
A miniaturised optical assembly for low-light fluorescence measurements was also developed. Based on small opto-electronic parts and optical fibres, the emission from the MIP particles upon addition of pTyr concentrations from 0.5 – 200 μM could be monitored in real-time.
The reliable identification and quantification of phosphorylated amino acids, peptides and proteins is one of the key challenges in contemporary bioanalytical research, noteworthy, to diagnose and treat diseases at an early developmental stage1. Miniaturised sensing devices like microfluidic chips combined with “smart” detection chemistry, simple data assessment, processing and presentation are very attractive for benchtop use in clinical environments.
We developed novel synthetic probes targeting phosphorylated amino acids, based on core-shell microparticles consisting of a silica core coated with a molecularly imprinted polymer (MIP) shell. These “plastic antibodies” are extremely robust, resist denaturing solvents and elevated temperatures, can be reproducibly produced at low cost, and potentially overcome many of the practical problems in current bioanalytical detection strategies. The MIP layer contains a fluorescent probe monomer, binds selectively to phosphorylated tyrosine (pY) with a significant imprinting factor higher than 3.5 and responds with a “lighting-up” of its fluorescence accompanied by the development of a strongly red-shifted emission band toward the analyte. In analogy to our previous work4, the bead-based ratiometric detection scheme has also been successfully transferred to a microfluidic chip format to demonstrate its applicability to rapid assays. Such a miniaturised device could yield an automated pY measurement system in the future. The setup was built by coupling a modular microfluidic system5 for amino acid functionalisation (Fmoc protection) and, as shown in Figure 1, a multi-layer PDMS/Teflon/glass microfluidic chip6 for buffering, extraction (micropillars co-flow extraction) and selective adsorption on the MIP core-shell particles.
A miniaturised optical assembly for low-light fluorescence measurements was also developed. Based on small opto-electronic parts and optical fibres, the emission from the MIP particles upon addition of pY concentrations from 0.5-200 μM could be monitored in real-time.
The fluorescence quantum yield (QY) of CdSe dot/CdS rod (DR) nanoparticle ensembles is dependent on the Shell growth and excitation wavelength. We analyze the origin of this dependency by comparing the optical properties of DR ensembles to the results obtained in single-particle experiments. On the Ensemble level, we find that the QY of DRs with shell lengths shorter than 40 nm exhibits no dependence on the excitation wavelength, whereas for DRs with shell lengths longer than 50 nm, the QY significantly decreases for excitation above the CdS band gap. Upon excitation in the CdSe core, the ensemble QY, the fluorescence wavelength, and the fluorescence blinking behavior of individual particles are only dependent on the radial CdS shell thickness and not on the CDs shell length. If the photogenerated excitons can reach the CdSe core region, the fluorescence properties will be dependent only on the surface passivation in close vicinity to the CdSe core. The change in QY upon excitation above the band gap of CdS for longer DRs cannot be explained by nonradiative particles because the ratio of emitting DRs is found to be independent of the DR length. We propose a model after which the decrease in QY for longer CdS shells is due to an increasing fraction of nonradiative exciton recombination within the elongated shell. This is supported by an effective-mass-approximation-based calculation, which suggests an optimum length of DRs of about 40 nm, to combine the benefit of high CdS absorption cross section with a high fluorescence QY.
In all fluorescence-based techniques, the measured signals contain not only sample-related but also instrument-specific contributions, which limit the direct comparison of fluorescence data obtained e.g. on different devices or at different times and often hamper quantification. To rule out instrumentation as major source of variability of emission data, accepted fluorescence standards and procedures for the control of instrument specifications and long-term performance are required. For flow cytometry (FCM), a broad variety of fluorophore-stained polymer beads differing in emission wavelength and intensity is available for the testing of the alignment, sensitivity, and other parameters of FCM. These calibration tools are intended to facilitate the assessment of instrument performance to ensure reliable measurements and to improve the comparability of FCM experiments.
As a step towards an improved comparability of fluorescence data, with special emphasis on spectroscopic methods measuring nano- and micrometer-sized fluorescent objects, we are currently developing a set of fluorescent polystyrene (PS) beads loaded with luminophores from the certified BAM-Kit “Spectral fluorescent standards”, initially developed for the calibration of fluorescence spectrometers. Here, we present first results from studies of these fluorophore-loaded polymer beads. Moreover, new beads are made to supplement this kit by encapsulating near-infrared (NIR)-emissive luminophores in PS beads to cover the UV/VIS, and NIR wavelength range.
These beads are designed for calibration of flow cytometers and other fluorescence imaging systems to meet the increasing demand for reliable and comparable fluorescence data especially in strongly regulated areas like e.g. medical diagnostics.
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.
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.