Filtern
Erscheinungsjahr
- 2017 (42) (entfernen)
Dokumenttyp
- Zeitschriftenartikel (16)
- Vortrag (16)
- Posterpräsentation (7)
- Buchkapitel (2)
- Beitrag zu einem Tagungsband (1)
Sprache
- Englisch (42)
Schlagworte
- Fluorescence (42) (entfernen)
Eingeladener Vortrag
- nein (16)
Due to the demand of monitoring the water quality regarding emerging pollutants, such as drug residues, selective, high-throughput and multi-target analytical methods must be established. On the one hand, the influence of sewage on natural surface waters must be routinely monitored. On the other hand, estimation of removal efficiencies of pollutants, such as drug residues, is in the focus of industrial and public wastewater treatment. Immunoassays, such as ELISA, offer the possibility to be highly sensitive and selective due to the high target affine and specific recognition of antibodies to target molecules. Batch-wise processing in microtiter plates allow the necessary high-throughput, but only a single analyte can be determined within one measurement.
To overcome these disadvantages, we developed a four-plex micro-bead based flow cytometric assay, which is adaptable for the microtiter plate format. The modular and self-prepared bead support consists of polystyrene-core-silica-shell particles. While the polystyrene core is used for encoding, by introducing different amounts of fluorescent dyes, the silica-shell creates a solid-support for the immunoassay: The target analytes, three drugs, carbamazepine, diclofenac and caffeine and the f ecal marker isolithocholic acid are covalently coupled to amino-groups on the surface via NHS-chemistry. A competitive immunoassay is subsequently conducted in a simple mix-and-read procedure. Finally, we could use SAFIA to assess the influent of treated and untreated waste water on the Douro river estuary in Portugal. The results of the analysis are comparable to ELISA. However, measurements could be carried out in 25% the time of analysis.
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.
We report on a novel and simple approach to surface ligand design of CdSe-based nanocrystals (NCs) with biocompatible, heterobifunctional polyethylene glycol (PEG) molecules. This method provides high transfer yields of the NCs into aqueous media with preservation of the narrow and symmetric emission bands of the initial organic-capped NCs regardless of their interior crystal structure and surface chemistry. The PEG-functionalized NCs show small sizes, high photoluminescence quantum yields of up to 75%, as well as impressive optical and colloidal stability. This universal approach is applied to different fluorescent nanomaterials (CdSe/CdS, CdSe/CdSCdxZn1-xS, and CdSe/CdS/ZnS), extending the great potential of organic-capped NCs for biological applications.
Because ammonia and its reaction products can cause considerable damage to human health and ecosystems, there is a need for reliably operating and reversibly interacting sensor materials to monitor traces of gaseous ammonia in ambient air, which at best can be used on-site for in-the-field measurements. Herein, the development of a sensor material for gaseous ammonia in the lower ppm to ppb range using optical fluorescence as transduction mechanism is presented. A fluorescent dye, which shows reversible fluorescence enhancement in the presence of ammonia is incorporated into a polymer matrix, the latter to ensure the accumulation of ammonia. The sensor material is integrated into a prototype of a miniaturized sensor device, facilitating long-term operation. To calibrate the optical sensor system a gas standard generator, producing standard gas mixtures, is used, leading to a sensitivity down to lower ppm concentrations of ammonia.
Pollution through emission of toxic gases is of utmost environmental concern, raising the interest in developing reliable gas sensors. Exemplarily, ammonia and its conversion products can provoke considerable damage on human health and ecosystems. Hence, there is a need for reliable and reversible sensor materials to monitor traces of gaseous ammonia in ambient air, which at best can be used on-site for field measurements. Although various types of sensors such as potentiometric, amperometric, and biological sensors are available for detecting trace amounts of gases, fluorescent sensors have gained importance due to several advantages such as high sensitivity, possible miniaturization, as well as potential multiplexing. Herein, we present the development of a sensor material for gaseous ammonia in the lower ppm or even ppb range using optical fluorescence as transduction mechanism due to its intrinsically high sensitivity and high spatial resolution.[1] Therefore, a fluorescent dye, which shows reversible fluorescence enhancement in the presence of the analyte was incorporated into a polymer matrix, the latter to ensure the accumulation of ammonia. To calibrate the designed optical sensor system a gas standard generator was used, producing standard gas mixtures, which comply with the metrological traceability for ammonia gas standards in the desired environmentally relevant measurement range.[2] Beside the development of a highly sensitive, selective, and reversible sensor, the integration of such systems into mobile sensor devices is addressed. Therefore, a prototype of a miniaturized hand-held instrument was developed enabling a straightforward and long-term read-out of the measurement signal.
Fluorescence based sensing is a versatile approach for the trace analysis outside of the laboratory, requiring suitable sensor materials and their integration into sensing devices. The versatility of fluorophores as probes, especially in terms of the possibility to tailor their optical as well as their recognition properties by synthetic modifications in a wide range, renders them a superior active component for the preparation of optical sensor devices. Recent works at BAM in this field include, for example, the detection of nerve gas agents, illustrating impressively the aforementioned benefits of fluorophores in optical sensing applications.
In the interdisciplinary approach presented here, we target hazardous gases such as ammonia, benzene, and hydrogen sulfide, next to others, which pose a major threat to human health and environmental safety and for which the availability of a sensitive and reliable detection method is highly desirable.
The dyes presented follow a “turn-on” fluorescence schematic which allows for the selective and sensitive detection of the respective gaseous analyte. The immobilization of the probe in polymeric matrices is then the next step toward the fabrication of a prototype device for molecular sensing.
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.
Ammonia and its reaction products can cause considerable damage of human health and ecosystems, increasing the necessity for reliable and reversible sensors to monitor traces of gaseous ammonia in ambient air directly on-site or in the field. Although various types of gas sensors are available, fluorescence sensors have gained importance due to advantages such as high sensitivity and facile miniaturization.
Here, we present the development of a sensor material for the detection of gaseous ammonia in the lower ppm to ppb range by incorporation of a fluorescent dye, which shows reversible fluorescence modulations as a function of analyte concentration, into a polymer matrix to ensure the accumulation of ammonia. A gas standard generator producing standard gas mixtures, which comply with the metrological traceability in the desired environmentally relevant measurement range, was used to calibrate the optical sensor system. To integrate the sensor material into a mobile device, a prototype of a hand-held instrument was developed, enabling straightforward data acquisition over a long period.
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)
The fluorescence properties of three molecular rotors, related to 4-dimethylamino-4-nitrostilbene (4-DNS), are studied versus different diesel/kerosene blends. In nonviscous solvents, these compounds can populate a twisted intramolecular charge transfer state which deactivates nonradiatively, successfully suppressing fluorescence emission. Solution experiments with diesel/kerosene blends showed a good linear correlation between the fluorescence intensity of the probe molecules and the diesel fraction of the blend. The dyes have been immobilized on paper, retaining their fluorescence behavior, i.e., negligible emission in the presence of nonviscous organic solvents and increasing fluorescence when the environment is increasingly viscous. When the impregnated paper is devised as a test strip, the latter is compatible with a newly designed smartphone reader system, which allows in-the-field measurements. The method can safely detect the presence of kerosene in diesel at ≥7%, which competes favorably with current standard methods for the detection of diesel adulteration.
A 2,3-diaminophenazine bis-urea fluorescent probe monomer (1) was developed. It responds to phenylphosphate and phosphorylated amino acids in a ratiometric fashion with enhanced fluorescence accompanied by the development of a redshifted emission band arising from an excited-state proton transfer (ESPT) process in the hydrogen-bonded probe/analyte complex. The two urea groups of 1 form a cleft-like binding pocket (Kb>10^10 L^2 mol^-2 for 1:2 complex). Imprinting of 1 in presence of ethyl ester- and fluorenylmethyloxycarbonyl (Fmoc)-protected phosphorylated tyrosine (Fmoc-pTyr-OEt) as the template, methacrylamide as co-monomer, and ethyleneglycol dimethacrylate as crosslinker gave few-nanometer-thick molecularly imprinted polymer (MIP) shells on silica core microparticles with excellent selectivity for the template in a buffered biphasic assay. The supramolecular recognition Features were established by spectroscopic and NMR studies. Rational screening of comonomers and cross-linkers allowed to single out the best performing MIP components, giving significant imprinting factors (IF>3.5) while retaining ESPT emission and the ratiometric response in the thin polymer shell. Combination of the bead-based detection scheme with the phase-transfer assay dramatically improved the IF to 15.9, allowing sensitive determination of the analyte directly in aqueous media.
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.
Coumarin-rhodamine hybrids-novel probes for the optical measurements of viscosity and polarity
(2017)
A comprehensive systematic study of Absorption and fluorescence properties in solvents of varying viscosity and polarity of three novel and red-emitting coumarin-rhodamine hybrid derivatives with differences in the rigidity of their substituents is presented. This includes ethanol-polyethylene glycol, toluene-polyethylene glycol, and tolueneparaffin mixtures. Moreover, protonation-induced effects on the spectroscopic properties are studied. A viscosity-induced emission enhancement was observed for all coumarin-rhodamine hybrid derivatives. MCR2 bearing a julolidine donor showed the expected low sensitivity to viscosity whereas MCR3 with its freely rotatable diphenylamino Substituent revealed a particularly pronounced sensitivity to this parameter.
Moreover, MCR2 shows an enhancement in Emission in the open, i.e., protonated form in conjunction with a largely Stokes shift fluorescence in the deep red spectral region. This enables the application of these dyes as viscosity sensors and as far red emitting pH-sensitive probes.
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.
The 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 stage. Small sensing devices like microfluidic chips combined with “smart” detection chemistry and simple data assessment, processing and presentation are attractive in this regard. 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 shell (MIP). These “plastic antibodies” which are extremely robust, resist denaturing solvents and high temperatures, can be reproducibly produced at low cost, can potentially overcome many of the problems in the current detection strategies. The MIP layer, containing a fluorescent probe monomer, responds to phosphorylated amino acids such as phosphorylated tyrosine (pTyr) with a significant imprinting factor, i.e. selectivity, higher than 3.5, and a “lighting-up” of its fluorescence accompanied by the development of a strongly red-shifted emission band. 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 device could yield an automated pTyr measurement system in the future. The setup was built by coupling a PDMS/Teflon/glass microfluidic chip with an optical setup for fluorescence measurements able to extract and monitor pTyr concentration from 0.5–200 μM.
Fluorescent sensory MIP (molecularly imprinted polymer) particles were combined with a droplet-based 3D microfluidic system for the sensitive and selective determination of 2,4-dichlorophenoxyacetic acid (2,4-D) in water samples. 2,4-D being an important and widely used herbicide to regulate plant growth, its extensive use leads to food or ground water contamination. Analytical assays based on MIP have emerged as a valuable tool in the field of environmental analysis thanks to low production costs, stability, format adaptability and their ability to recognize a wide variety of targets, such as the critical neutral organic molecules present in water: herbicides, pesticides, antibiotics, etc.
To accomplish this, a tailor-made fluorescent indicator cross-linker was designed that translates directly the binding event into an enhanced fluorescence signal. This phenoxazinone-type cross-linker was co-polymerized into a thin MIP layer grafted from the surface of silica microparticles. The latter was achieved by integration of the fluorescent core-shell MIP sensor particles into a modular microfluidic platform that allows for an in-line phase-transfer assay, extracting the analyte from aqueous sample droplets into organic phase droplets that contain the sensor particles.
This tool offering a novel simple and rapid way for the detection of herbicides, real-time fluorescence determination of 2,4-D down to 20 nM was realized with the system and applied for the analysis of worlwide surface water samples.
Molecularly imprinted polymers (MIPs) are an established and powerful matrix for the selective enrichment and separation of chemical species, especially of small organic molecules. Because MIPs contain cavities in their matrix that are complementary in size, shape and electronic/electrostatic or hydrogen bonding demand to the imprinted target molecule or template, they are frequently termed “artificial antibodies”. Compared to natural antibodies, they are chemically and physically much more robust. MIP formation proceeds through the polymerization of a mixture of functional monomers and cross-linkers in the presence of the template with subsequent extraction of the latter. While this strategy has been successfully employed for separation MIPs since decades, the development of sensory MIPs has long been limited to the combination of a MIP as enrichment phase with a separate signalling element in a discontinuous fashion, being unsuitable for many sensing applications. Only very recently, the implementation of specifically designed fluorescent probes into MIPs has been successfully accomplished, fluorescence being one of the most versatile, sensitive and easily miniaturizable techniques. This presentation will introduce basic design considerations, challenges, limitations and the potential that lies with such sensor materials on the background of the work carried out in our group.
In this work, a family of pH-responsive fluorescent probes has been designed in a rational manner with the aid of quantum chemistry tools, covering the entire pH range from 0−14. Relying on the boron−dipyrromethene (BODIPY) core, all the probes as well as selected reference dyes display very similar spectroscopic properties with ON−OFF fluorescence switching responses, facilitating optical readout in simple devices used for detection and analysis. Embedding of the probes and reference dyes into hydrogel spots on a plastic strip yielded a test strip that reversibly indicates pH with a considerably small uncertainty of ~0.1 pH units. These strips are not only reusable but, combined with a 3D-printed case that can be attached to a smartphone, the USB port of which drives the integrated LED used for excitation, allows for autonomous operation in on-site or in-the-field applications; the developed Android application software (“app”) further simplifies operation for unskilled users.
Besides the traditional areas of application such as separation and enrichment which made molecularly imprinted polymers (MIPs) very attractive, they have emerged as a valuable detection tool in the field of environmental analysis due to the low production costs, high stability, format adaptability and the possibility to imprint and thus specifically recognize a wide variety of target analytes. Regarding optical sensing, however, MIPs have only been used in considerably few applications, especially in fluorescence sensors, basically because of the challenge to incorporate a fluorescently responding moiety into a polymer matrix. One way to overcome this limitation is the coating of a thin MIP layer onto the surface of silica nanoparticles using tailor-made fluorescent indicator monomers or cross-linkers for direct transfer of the binding event into an optical signal.
Regarding sensors for environmental monitoring, microfluidic devices utilizing optical detection modules are especially appealing because of their versatility in terms of miniaturization and automation. So far, MIPs have only rarely been used in combination with microfluidic sensor devices.
Here, we present the hydrogen bond-mediated optical response of fluorescent MIP sensor particles against a typical small-molecule analyte 2,4-D (2,4-dichlorophen¬oxyacetic acid) which is an important herbicide widely used in agriculture and known to cause adverse health effects when ingested by contaminated water. By combining the sensor particles with droplet-based 3D microfluidics, a microfluidic phase-transfer assay was designed which enables the direct analysis of 2,4-D in river and lake water without sample pre-treatment or clean-up.
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.
In Part II of this Review series on lanthanide-doped photon-upconversion nanoparticles (UCNPs), we present and critically discuss the Performance and suitability of UCNPs as background-free luminescent Reporters in bioimaging and bioanalytical applications. The preparation of a biocompatible nanoparticle surface is an integral step for all life - science-related applications. UCNPs have found their way into a large number of diagnostic platforms, homogeneous and heterogeneous assay formats, and sensor applications. Many bioanalytical detection schemes involve Förster resonance energy transfert (FRET), which is still debated for UCNPs and Needs to be much improved. The Need for dedicated and standardized instruments as well as recent studies on the Dissolution and potential toxicity of UCNPs are addressed. Finally we outline future Trends and challenges in the field of upconversion.
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.
The rational design of functional nanomaterials for optical applications in the material and life sciences requires optical-spectroscopic methods for the quantitative characterization of their signal-relevant optical properties. Additionally, methods for the simple and quantitative analysis of the surface chemistry are desired as the chemical nature and number of the surface groups and ligands can affect the optical features and controls the interaction of these nanomaterials with their environment. Here, we present quantitative photoluminescenvce studies of different types of vis/NIR-emissive nanomaterials like semiconductor quantum dots and upconversion nanocrystals will be presented and their relevance for the mechanistic understanding of nonradiative decay channels and the rational design of new nanomaterials will be underpinned. In this respect, also validation concepts for such measurements and absolute fluorometry will be introduced. In addition, the potential of optical spectroscopy for surface group and ligand analysis surface chemistry will be demonstrated exemplarily for semiconductor quantum dots
Lanthanide-doped photon-upconversion nanoparticles (UCNPs) have been in the focus of many research activities in the material and life sciences over the last 15 years because of their potential to convert light between different spectral regions and their unique photophysical properties. In order to fully exploit the application potential of these fascinating nanomaterials, a number of challenges has to be overcome such as the low brightness particularly of small UCNPs and the reliable quantification of the excitation power density (P)-dependent upconversion luminescence (UCL). Here, the need and requirements on the characterization of the optical properties of UCNPs are discussed with special focus on the reliabiliy and comparability of relative and quantitative luminescence measurements and prerequisites for their standardization.
Multiparametric analyses involving optical techniques like flow cytometry are at the core of studying complex systems in biological research and diagnostic applications. However, for fluorescence-based techniques, the number of reporters distinguishable in spectral multiplexing is limited by spectral overlap and requires a multitude of excitation light sources and detection Systems. Intensity encoding often used for bead assays suffers from problems regarding dye concentration control and excitation light intensity fluctuations. An alternative is luminescence lifetime encoding, particularly to minimize instrument costs.
Here, we report on dye-stained polymer microparticles for lifetime encoding in flow cytometry with different organic dyes. This ranges from studies of the impact of parameters like dye loading concentration and particle diameter on fluorescence decay behavior to the demonstration of lifetime code reading and simultaneous ligand fluorescence signal detection with single-wavelength excitation in a flow.
Dye-stained lifetime-encoded polymer microbeads for application in time-resolved flow cytometry
(2017)
Flow cytometry is a standard analytical tool for biological research and in medical applications. There are different requirements triggering recent device and method development depending on the desired field of application. One trend is governed by the need for an increasing number of simultaneously detectable codes, i.e., fluorescent labels. The other one focuses on cost-effective methods and development of miniaturized, portable devices.
Fluorophore encoding is usually based on spectral encoding. However, this approach is hampered by, e.g., spectral crosstalk. Additionally, the sensitivity of fluorescence intensity measurements to fluctuations in excitation light intensity and dye concentration limits the achievable number of detection channels. Moreover, spectral multiplexing typically requires several costly excitation light sources. Lifetime multiplexing and the discrimination between different encoding fluorophores and carrier beads based on their fluorescence decay kinetics could present an innovative alternative. Encoded beads, i.e., beads with lifetime codes corresponding to the surface chemistry, have been employed to evaluate the feasibility of this approach with a custom designed flow cytometer equipped with a pulsed light source and a fast detector for time-resolved measurements in a flow.
In a first step, we used steady state and time-resolved photoluminescence measurements for the spectroscopic characterization of micrometer-sized dye-stained PMMA beads. Subsequently, the potential use of these microbeads for flow cytometry applications was analyzed with a prototype flow cytometer with lifetime detection.
With our proof-of-concept studies, we could demonstrate that lifetime discrimination and simultaneous readout of a ligand fluorescence signal for analyte quantification is feasible with a set of dye-stained polymer microbeads at single wavelength excitation. These studies are expected to pave the road for new applications of fluorescence lifetime multiplexing in time-domain flow cytometry and bead-based assays in general.
Flow cytometry is a widely used method in biological research and medical diagnostics. Depending on the respective application, two opposing directions of development are currently of interest. On the one hand, there is a need for analyses of growing complexity employing more and more fluorescent labels and codes. On the other hand, cost-effective methods and portable, miniaturized, and robust instruments are desired.
Commonly performed spectral multiplexing utilizing a color code suffers from several problems, such as the sensitivity of fluorescence intensity measurements to fluctuations in excitation light intensity and dye concentration and hence, photobleaching, dye leaking for certain encoding procedures, and spectral crosstalk, limiting the achievable number of detection channels. Moreover, it typically requires several costly excitation light sources. An innovative alternative can be lifetime multiplexing and the discrimination between different encoding fluorophores and carrier beads based on their fluorescence decay kinetics. In order to examine the potential of this approach, dye encoded beads (lifetime encoded surface chemistry) were prepared using several fluorophores from different dye classes and their suitability for lifetime discrimination in a flow was tested in conjunction with a custom designed flow cytometer equipped with a pulsed light source and a fast detector.
In a first step, the spectroscopic properties of micrometer-sized dye-stained PMMA beads were studied by means of steady state and time-resolved photoluminescence measurements. For the performance of studies on the practical use of these microbeads in flow cytometry applications, a custom-built demonstrator model for a flow system was employed.
Our results demonstrated that lifetime discrimination and simultaneous readout of a ligand fluorescence signal for analyte quantification with a set of dye-stained polymer microbeads at single wavelength excitation is feasible. These studies are expected to pave the road for new applications of fluorescence lifetime multiplexing within the framework of time-domain flow cytometry and bead-based assays.
For the application of time-resolved detection in the framework of flow cytometry, suitable luminescence lifetime code carriers are required. Here we report on our achievements concerning strategies to increase the accessible range of lifetime values and to realize continuously tunable lifetimes.
To that end, we investigated polymer (PMMA) microbeads stained with mixtures of dyes exhibiting different fluorescence decay kinetics. At the expense of spectrally varying decay kinetics, it is possible to modify the lifetime by changing the dye concentration ratio.
Moreover, semiconductor quantum dots incorporated into polymer beads were studied as alternative luminophores outperforming organic dyes with respect to long luminescence lifetimes, flexible choice of excitation wavelength and narrow spectral emission width.
Our experiments demonstrate that lifetime adaption with dye mixing is basically feasible and semiconductor quantum dots represent promising candidates for long-lifetime codes.
Lanthanide-doped up-converting nanoparticles (UCNPs) are promising reporters for medical diagnostics and bioimaging. Current limitations present their relative low absorption cross sections and low fluorescence efficiencies, with the latter being affected by particle size, surface chemistry, and microenvironment, particularly water.
Here, we present results from systematic studies of the excitation power density dependent upconversion luminescence spectra, intensities/intensity ratios of the individual emission bands, slope factors, and quantum yields of UCNPs of varying size, dopant concentration, and surface chemistry in different microenvironments as well as the up- and downconversion luminescence decay kinetics of the different emission bands. Moreover, first studies of the energy transfer from UCNPs to surface-bound organic dyes acting as fluorescence acceptors are shown. Based upon these measurements, fluorescence deactivation channels are identified and spectroscopic parameters for the screening of material performance are derived.
Lanthanide-doped up-converting nanoparticles (UCNPs) are promising reporters for medical diagnostics and bioimaging, which are excited in the near infrared (NIR) by multiphoton absorption processes, and show multiple narrow emission bands in the visible (vis) and NIR, long luminescence lifetimes in the μs range, and excellent photostability. Current limitations present their relative low absorption cross sections and low fluorescence efficiencies, with the latter being affected by particle size, surface chemistry, and microenvironment, particularly water.
Here, we present results from systematic studies of the excitation power density dependent upconversion luminescence spectra, intensities/intensity ratios of the individual emission bands, slope factors, and quantum yields of UCNPs of varying size, dopant concentration, and surface chemistry in different microenvironments as well as the up- and downconversion luminescence decay kinetics of the different emission bands. Moreover, first studies of the energy transfer from UCNPs to surface-bound organic dyes acting as fluorescence acceptors are shown. Based upon these measurements, fluorescence deactivation channels are identified and spectroscopic parameters for the screening of material performance are derived.
An ideal sensor system is a combination of a selective receptor, an effective transducer, and a sensitive detector. To utilize molecularly imprinted polymers (MIPs) as responsive recognition phases in sensors, the employment of fluorescent molecules or nanoparticles (NPs) that show prominent changes in their spectroscopic properties after binding of the target molecule in the MIP’s cavity is particularly attractive. Such fluorescent MIPs (fMIPs) act through target-induced quenching, enhancement, or spectral shifts of the fluorescence. This contribution introduces different strategies of incorporation of fluorescent dyes, probes, and NPs into fMIPs. In addition, various sensing mechanisms are reviewed, and depending on the application of the sensor, the different deployable formats, their advantages, drawbacks, and impact will be presented and discussed.