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Dye-biomolecule conjugates and NIR-fluorescent particles for targeting of disease-related biomarkers
(2011)
Indispensable for fluorescence imaging are highly specific and sensitive molecular probes that absorb and emit in the near infrared (NIR) spectral region and respond to or target molecular species or processes. Here, we present approaches to targeted fluorescent probes for in vivo imaging in the intensity and lifetime domain exploiting NIR dyes. Screening schemes for the fast identification of suitable fluorophores are derived and design criteria for highly emissive optical probes. In addition, as a signal amplification strategy that enables also the use of hydrophobic NIR fluorophores as fluorescent reporters, first steps towards versatile strategies for the preparation of NIR-fluorescent polymeric particles are presented that can be utilized also for the design of targeted and analyte-responsive probes.
Polystyrene nanoparticles (PS-NPs) were doped with an oxygen-sensitive near-infrared (NIR)-emissive palladium meso-tetraphenylporphyrin and an inert reference dye which are both excitable at 635 nm. The nanosensors were characterized with special emphasis on fundamental parameters such as absolute photoluminescence quantum yield and fluorescence lifetime. The PS-NPs were employed for ratiometric dual-wavelength and lifetime-based photoluminescent oxygen sensing. They were efficiently taken up by cultured murine alveolar macrophages, yielding a characteristic and reversible change in ratiometric response with decreasing oxygen concentration. This correlated with the cellular hypoxic status verified by analysis of hypoxia inducible factor-1α (HIF-1α) accumulation. In addition, the surface of PS-NPs was functionalized with polyethylene glycol (PEG) and the monoclonal antibody herceptin, and their binding to HER2/neu-overexpressing tumor cells was confirmed in vitro. First experiments with tumor-bearing mouse revealed a distinctive ratiometric response within the tumor upon hypoxic condition induced by animal sacrifice. These results demonstrate the potential of these referenced NIR nanosensors for in vitro and in vivo imaging that present a new generation of optical probes for oncology.
Photoluminescence techniques are amongst the most widely used tools in the material and life sciences, with new and exciting applications continuously emerging. Advantages include their comparative ease of use, unique sensitivity, non-invasive character, and potential for multiplexing, remote sensing, and miniaturization. Increasing applications of fluorescence techniques in the life sciences and emerging medical applications of fluorescence microscopic techniques including 1P and 2P fluorescence microscopy combined with fluorescence lifetime imaging (FLIM) in e.g. in vivo eye diagnostics boosted the demand for robust, easy-to-use, and reliable fluorescence standards to ensure the reliability and comparability of fluorescence data. This includes fluorescence standards for the consideration of instrument-specific spectral and intensity distortions of measured signals and instrument performance validation, fluorescence intensity standards for the quantification of measured intensities and for signal referencing, and lately, also fluorescence lifetime standards.
Synthesis and characterisation of highly fluorescent core-shell nanoparticles based on Alexa dyes
(2012)
Current and future developments in the
emerging field of nanobiotechnology are closely
linked to the rational design of novel fluorescent
nanomaterials, e.g. for biosensing and imaging applications.
Here, the synthesis of bright near infrared
(NIR)-emissive nanoparticles based on the grafting
of silica nanoparticles (SNPs) with 3-aminopropyl
triethoxysilane (APTES) followed by covalent attachment
of Alexa dyes and their subsequent shielding by an
additional silica shell are presented. These nanoparticles
were investigated by dynamic light scattering (DLS),
transmission electron microscopy (TEM) and fluorescence
spectroscopy. TEM studies revealed the monodispersity
of the initially prepared and fluorophorelabelled
silica particles and the subsequent formation of
raspberry-like structures after addition of a silica
precursor.Measurements of absolute fluorescence quantum
yields of these scattering particle suspensions with
an integrating sphere setup demonstrated the influence
of dye labelling density-dependent fluorophore aggregation
on the signaling behaviour of such nanoparticles.
The rational design of nano- and micrometer-sized particles with tailor-made optical properties for biological, diagnostic, and photonic applications requires tools to characterize the signal-relevant properties of these typically scattering bead suspensions. This includes methods for the preferably nondestructive quantification of the number of fluorophores per particle and the measurement of absolute fluorescence quantum yields and absorption coefficients of suspensions of fluorescent beads for material performance optimization and comparison. Here, as a first proof-of-concept, we present the first time determination of the number of dye molecules per bead using nondestructive quantitative (19F) NMR spectroscopy and 1000 nm-sized carboxylated polystyrene particles loaded with varying concentrations of the laser dye coumarin 153 containing a CF3 group. Additionally, the signal-relevant optical properties of these dye-loaded particles were determined in aqueous suspension in comparison to the free dye in solvents of different polarity with a custom-built integrating sphere setup that enables spectrally resolved measurements of emission, transmission, and reflectance as well absolute fluorescence quantum yields. These measurements present an important step toward absolute brightness values and quantitative fluorescence analysis with particle systems that can be exploited, for example, for optical imaging techniques and different fluorescence assays as well as for the metrological traceability of fluorescence methods.
Water-dispersible and (bio)functionalizable nanoclays have a considerable potential as inexpensive carriers for organic molecules like drugs and fluorophores. Aiming at simple design strategies for red-emissive optical probes for the life sciences from commercial precursors with minimum synthetic effort, we systematically studied the dye loading behavior and stability of differently functionalized laponites. Here, we present a comprehensive study of the absorption and emission properties of the red emissive hydrophobic and neutral dye Nile Red, a well-known polarity probe, which is almost insoluble and nonemissive in water. Adsorption of this probe onto disk-shaped nanoclays was studied in aqueous dispersion as function of dye concentration, in the absence and presence of the cationic surfactant cetyltrimethylammonium bromide (CTAB) assisting dye loading, and as a function of pH. This laponite loading strategy yields strongly fluorescent nanoclay suspensions with a fluorescence quantum yield of 0.34 at low dye loading concentration. The dye concentration-, CTAB-, and pH-dependent absorption, fluorescence emission, and fluorescence excitation spectra of the Nile-Red6#8211;nanoclay suspensions suggest the formation of several Nile Red species including emissive Nile Red monomers facing a polar environment, nonemissive H-type dimers, and protonated Nile Red molecules that are also nonfluorescent. Formation of all nonemissive Nile Red species could be suppressed by modification of the laponite with CTAB. This underlines the great potential of properly modified and functionalized laponite nanodisks as platform for optical probes with drug delivery capacities, for example, for tumor and therapy imaging. Moreover, comparison of the Nile Red dimer absorption spectra with absorption spectra of previously studied Nile Red aggregates in dendrimer systems and micelles and other literature systems reveals a considerable dependence of the dimer absorption band on microenvironment polarity which has not yet been reported so far for H-type dye aggregates.
The increase in information content from bioassays and bioimaging requires robust and efficient strategies for the detection of multiple analytes or targets in a single measurement, thereby addressing current health and security concerns. For fluorescence techniques, an attractive alternative to commonly performed spectral or color multiplexing presents lifetime multiplexing and the discrimination between different fluorophores based on their fluorescence decay kinetics. This strategy relies on fluorescent labels with sufficiently different lifetimes that are excitable at the same wavelength and detectable within the same spectral window. Here, we report on lifetime multiplexing and discrimination with a set of nanometer-sized particles loaded with near-infrared emissive organic fluorophores chosen to display very similar absorption and emission spectra, yet different fluorescence decay kinetics in suspension. Furthermore, as a first proof-of-concept, we describe bioimaging studies with 3T3 fibroblasts and J774 macrophages, incubated with mixtures of these reporters employing fluorescence lifetime imaging microscopy. These proof-of-concept measurements underline the potential of fluorescent nanoparticle reporters in fluorescence lifetime multiplexing, barcoding, and imaging for cellular studies, cell-based assays, and molecular imaging.
Current optical probes including engineered nanoparticles (NPs) are constructed from near infrared (NIR)-emissive organic dyes with narrow absorption and emission bands and small Stokes shifts prone to aggregation-induced self-quenching. Here, we present the new asymmetric cyanine Itrybe with broad, almost environment-insensitive absorption and emission bands in the diagnostic window, offering a unique flexibility of the choice of excitation and detection wavelengths compared to common NIR dyes. This strongly emissive dye was spectroscopically studied in different solvents and encapsulated into differently sized (15, 25, 100 nm) amino-modified polystyrene NPs (PSNPs) via a one-step staining procedure. As proof-of-concept for its potential for pre-/clinical imaging applications, Itrybe-loaded NPs were surface-functionalized with polyethylene glycol (PEG) and the tumor-targeting antibody Herceptin and their binding specificity to the tumor-specific biomarker HER2 was systematically assessed. Itrybe-loaded NPs display strong fluorescence signals in vitro and in vivo and Herceptin-conjugated NPs bind specifically to HER2 as demonstrated in immunoassays as well as on tumor cells and sections from mouse tumor xenografts in vitro. This demonstrates that our design strategy exploiting broad band-absorbing and -emitting dyes yields versatile and bright NIR probes with a high potential for e.g. the sensitive detection and characterization of tumor development and progression.
In order to develop simple and versatile procedures for the preparation of red emissive particles, various one-step swelling procedures for the loading of fluorophores into nanometer- and micrometer-sized polystyrene particles were systematically assessed. Parameters studied for model dyes from common dye classes include the composition of the swelling medium, dye charge and polarity, dye concentration, and particle surface chemistry. The dye loading procedures were compared based upon the efficiency of dye incorporation, fluorescence intensity, and colloidal stability of the resulting particles as well as the absence of dye leaking as determined by absorption and fluorescence spectroscopy, flow cytometry, and measurements of zeta potentials. In addition, for the first time, the influence of the amount of incorporated dye on the absolute fluorescence quantum yield and brightness of the fluorescent particles was investigated for selected chromophores in differently sized particles using a custom-made calibrated integrating sphere setup. Our results demonstrate the general suitability of these one-step loading procedures for efficient particle staining with neutral, zwitterionic, and charged fluorophores like oxazines, coumarines, squaraines, xanthenes, and cyanines emitting in the visible and near infrared. Dye polarity was identified as a suitable tool to estimate the loading efficiency of fluorophores into these polymer particles.
One of the most active research areas in the life and material sciences is the design and synthesis of fluorescent nano- and micrometre sized particles for applications e.g. as labels, sensor systems, and platforms for fluorescence assays or barcoding materials. The reliable and reproducible fabrication of such particles as well as many applications require accurate, simple, and versatile procedures for the determination of the dye content per particle which affects e.g. the brightness of these materials and their surface charge and thus, colloidal stability. Here, four fast and inexpensive spectroscopic methods for the quantification of the fluorophore content of beads are presented and compared for nanometre- and micrometre sized polystyrene particles loaded or labeled with commercial fluorophores, differing in dye class, charge, and hydrophilicity. This included the determination of the amount of incorporated dye from absorption spectra of bead suspensions, via dissolving of the polymer matrix, via extraction of the polymer matrix, and from the supernatant of the swelling solution or reaction mixture. Method validation was performed with a sulfur-containing dye and elemental analysis. Based upon this method comparison and the accomplishable uncertainties, two reliable strategies for particle characterization and bead process control are identified that can be easily extended to other materials.
Bioanalytical, clinical, and security applications increasingly require simple, efficient, and versatile strategies to measure an ever increasing number of analytes or events in parallel in a broad variety of detection formats as well as in conjunction with chromatographic separation techniques or flow cytometry. An attractive alternative to common optical multiplexing and encoding methods utilizing spectral multiplexing/color encoding and intensity encoding is lifetime multiplexing, which relies on the discrimination between different fluorescent reporters based on their fluorescence decay kinetics. Here, we propose a platform of surface-functionalizable polymeric nanoparticles stained with fluorophores differing in their fluorescence lifetimes as a new multiplexing and encoding approach. Proof-of-concept measurements with different sets of lifetime-encoded polystyrene nanoparticles are presented, obtained via staining of preformed particles with visible (vis)- and near-infrared (NIR)-emissive organic dyes, which display very similar absorption and emission spectra to enable excitation and detection at the same wavelengths, yet sufficiently different fluorescence decay kinetics in suspension, thereby minimizing instrumentation costs. Data analysis was performed with a linear combination approach in the lifetime domain. Our results and first cell experiments with these reporter sets underline the suitability of our multiplexing strategy for the discrimination between and the quantification of different labels. This simple and versatile concept can be extended to all types of fluorophores, thereby expanding the accessible time scale, and can be used, e.g., for the design of labels and targeted probes for fluorescence assays and molecular imaging, cellular imaging studies, and barcoding applications, also in conjunction with spectral and intensity encoding.
The optical properties of semiconductor nanocrystals (SC NCs) are largely controlled by their size and surface chemistry, i.e., the chemical composition and thickness of inorganic passivation shells and the chemical nature and number of surface ligands as well as the strength of their bonds to surface atoms.
The latter is particularly important for CdTe NCs, which – together with alloyed CdₓHg₁₋ₓTe – are the only SC NCs that can be prepared in water in high quality without the need for an additional inorganic passivation shell. Aiming at a better understanding of the role of stabilizing ligands for the control of the application-relevant fluorescence features of SC NCs, we assessed the influence of two of the most commonly used monodentate thiol ligands, thioglycolic acid (TGA) and mercaptopropionic acid (MPA),
on the colloidal stability, photoluminescence (PL) quantum yield (QY), and PL decay behavior of a set of CdTe NC colloids. As an indirect measure for the strength of the coordinative bond of the ligands to SC NC surface atoms, the influence of the pH (pD) and the concentration on the PL properties of these colloids was examined in water and D₂O and compared to the results from previous dilution studies with a set of thiol-capped Cd₁₋ₓHgₓTe SC NCs in D₂O. As a prerequisite for these studies, the number of surface ligands was determined photometrically at different steps of purification after SC NC synthesis with Ellman’s test.
Our results demonstrate ligand control of the pH-dependent PL of these SC NCs, with MPA-stabilized CdTe NCs being less prone to luminescence quenching than TGA-capped ones. For both types of CdTe colloids, ligand desorption is more pronounced in H₂O compared to D₂O, underlining also the role of hydrogen bonding and solvent molecules.
The combination of complementary characterization techniques such as SEM (Scanning Electron Microscopy), T-SEM (Scanning Electron Microscopy in Transmission Mode), EDX (Energy Dispersive X-ray Spectroscopy) and SAM (Scanning Auger Microscopy) has been proven to be a powerful and relatively quick characterization strategy for comprehensive morphological and chemical characterization of individual silica and titania nanoparticles. The selected real life test materials, silica and titania, are listed in the OECD guidance manual as representative examples because they are often used as commercial nanomaterials. Imaging by high resolution SEM and in the transmission mode by T-SEM allows almost simultaneous surface and in-depth inspection of the same particle using the same instrument. EDX and SAM enable the chemical characterization of bulk and surface of individual nanoparticles. The core–shell properties of silica based materials are addressed as well. Titania nominally coated by silane purchased from an industrial source has been found to be inhomogeneous in terms of chemical composition.
To improve the reliability of fluorescence data in the life and material sciences and to enable accreditation of fluorescence techniques, standardization concepts are required that guarantee and improve the comparability of fluorescence measurements. At the core of such concepts are simple and evaluated fluorescence standards for the consideration of instrument-specific spectral and intensity distortions of measured signals and for instrument performance validation (IPV). Similarly in need are fluorescence intensity standards for the quantification from measured intensities and for signal referencing, thereby accounting for excitation light-induced intensity fluctuations. These standards should be preferably certified, especially for use in regulated areas like medical diagnostics. This encouraged us to develop liquid and solid standards for different fluorescence parameters and techniques for use under routine measurement conditions in different formates. Special emphasis was dedicated to the determination and control of the spectral responsivity of detection systems, wavelength accuracy, homogeneity of illumination, and intensity referencing for e.g. spectrofluorometers, fluorescence sensors and confocal laser scanning fluorescence microscopes. Here, we will present design concepts and examples for mono- and multifunctional fluorescence standards that provide traceability to radiometric units and present a first step towards a toolbox of standards.
Increasing the information content from bioassays which requires robust and efficient strategies for the detection of multiple analytes or targets in a single measurement is an important field of research, especially in the context of meeting current security and health concerns. An attractive alternative to spectral multiplexing, which relies on fluorescent labels excitable at the same wavelength, yet sufficiently differing in their emission spectra or color presents lifetime multiplexing. For this purpose, we recently introduced a new strategy based on 'pattern-matching' in the lifetime domain, which was exemplary exploited for the discrimination between organic dyes and quantum dot labels revealing multi-exponential decay kinetics and allowed quantification of these labels. Meanwhile, we have succeeded in extending this lifetime multiplexing approach to nanometer-sized particle labels and probes absorbing and emitting in the visible (vis) and near-infrared (NIR) spectral region. Here, we present a first proof-of-principle of this approach for a pair of NIR-fluorescent particles. Each particle is loaded with a single organic dye chosen to display very similar absorption and emission spectra, yet different fluorescence decay kinetics. Examples for the lifetime-based distinction between pairs of these fluorescent nanoparticles in solution and in cells are presented. The results underline the potential of fluorescenc lifetime multiplexing in life science and bioanalysis.
The widespread use and acceptance of fluorescence techniques especially in regulated areas like medical diagnostics is closely linked to standardization concepts that guarantee and improve the comparability and reliability of fluorescence measurements. At the core of such concepts are dependable fluorescence standards that are preferably certified. The ever rising interest in fluorescence measurements in the near-infrared (NIR) spectral region renders the availability of spectral and intensity standards for this wavelength region increasingly important. This encouraged us to develop approaches to solid NIR standards based upon dye-doped polymers and assess their applicationrelevant properties in comparison to metal ion-doped glasses. The overall goal is here to provide inexpensive, easily fabricated, and robust internal and external calibration tools for a broad variety of fluorescence instruments ranging e.g. from spectrofluorometers over fluorescence microscopes to miniaturized fluorescence sensors.
Simple, fast, and versatile methods for the quantification of thiol groups are of considerable interest not only for protein analysis but also for the characterization of the surface chemistry of nanomaterials stabilized with thiol ligands or bearing thiol groups for the subsequent (bio-) functionalization via maleimide−thiol chemistry. Here, we compare two simple colorimetric assays, the widely used Ellman’s assay performed at alkaline pH and the aldrithiol assay executed at acidic and neutral pH, with respect to their potential for the quantification of thiol groups and thiol ligands on different types of nanoparticles like polystyrene nanoparticles, semiconductor nanocrystals (SC NC), and noble metal particles, and we derive criteria for their use. In order to assess the underlying reaction mechanisms and to obtain stoichiometry factors mandatory for reliable thiol quantification, both methods were studied photometrically and with electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), thereby demonstrating the influence of different thiols on the reaction mechanism. Our results underline the suitability of both methods for the quantification of directly accessible thiol groups or ligands on the surface of 2D- and 3D-supports, here exemplarily polystyrene nanoparticles. Moreover, we could derive strategies for the use of these simple assays for the determination of masked (i.e., not directly accessible) thiol groups like disulfides such as lipoic acid and thiol stabilizing ligands coordinatively bound to Cd and/or Hg surface atoms of II/VI and ternary SC NC and to gold and silver nanoparticles.
Critical review of the determination of photoluminescence quantum yields of luminescent reporters
(2015)
A crucial variable for methodical performance evaluation and comparison of luminescent reporters is the photoluminescence quantum yield (Φ pl). This quantity, defined as the number of emitted photons per number of absorbed photons, is the direct measure of the efficiency of the conversion of absorbed photons into emitted light for small organic dyes, fluorescent proteins, metal–ligand complexes, metal clusters, polymeric nanoparticles, and semiconductor and up-conversion nanocrystals. Φ pl determines the sensitivity for the detection of a specific analyte from the chromophore perspective, together with its molar-absorption coefficient at the excitation wavelength. In this review we discuss different optical and photothermal methods for measuring Φ pl of transparent and scattering systems for the most common classes of luminescent reporters, and critically evaluate their potential and limitations. In addition, reporter-specific effects and sources of uncertainty are addressed. The ultimate objective is to provide users of fluorescence techniques with validated tools for the determination of Φ pl, including a series of Φ pl standards for the ultraviolet, visible, and near-infrared regions, and to enable better judgment of the reliability of literature data.
Aiming at the development of validated protocols
for protein conjugation of nanomaterials and the determination
of protein labeling densities, we systematically assessed
the conjugation of the model protein streptavidin (SAv) to
100-, 500-, and 1000-nm-sized polystyrene and silica nanoparticles
and dye-encoded polymer particles with two
established conjugation chemistries, based upon achievable
coupling efficiencies and labeling densities. Bioconjugation
reactions compared included EDC/sulfo NHS ester chemistry
for direct binding of the SAv to carboxyl groups at the particle
surface and maleimide-thiol chemistry in conjunction with
heterobifunctional PEG linkers and aminated nanoparticles
(NPs). Quantification of the total and functional amounts of
SAv on these nanomaterials and unreacted SAv in solution
was performed with the BCA assay and the biotin–FITC
(BF) titration, relying on different signal generation principles,
which are thus prone to different interferences. Our results
revealed a clear influence of the conjugation chemistry on
the amount of NP crosslinking, yet under optimized reaction
conditions, EDC/sulfo NHS ester chemistry and the attachment
via heterobifunctional PEG linkers led to comparably
efficient SAv coupling and good labeling densities. Particle
size can obviously affect protein labeling densities and
particularly protein functionality, especially for larger
particles. For unstained nanoparticles, direct bioconjugation
seems to be the most efficient strategy, whereas for dyeencoded
nanoparticles, PEG linkers are to be favored for the
prevention of dye–protein interactions which can affect
protein functionality specifically in the case of direct SAv
binding. Moreover, an influence of particle size on achievable
protein labeling densities and protein functionality could be
demonstrated.
We designed and synthesized triphenylamine based and coumarin fused rhodamine hybrid dyes and characterized using 1H, 13C NMR and HR-LCMS analysis. Both the newly synthesized hybrid dyes were found to show red shifted absorption as well as emissions and large Stokes shift (40e68 nm) as compared to the small Stokes shift (25e30 nm) of reported dyes Rhodamine B and 101. Photophysical properties of these dyes were studied in different solvents and according to the solvents acidity or basicity they preferred to remain in their spirocyclic or open form in different ratio. We studied the spirocyclic as well as open form derivatives of these dyes for their viscosity sensitivity in three different mixture of solvents i.e. polar-protic [EtOH-PEG 400], polar-aprotic [toluene-PEG 400] and non-polaraprotic [toluene-paraffin]. They are found to show very high viscosity sensitivity in polar-protic mixture of solvents [EtOH-PEG 400] and hence concluded that both polarity as well as viscosity factor worked together for the higher emission enhancement rather than only viscosity factor. As these dyes showed very high viscosity sensitivity in their spirocyclic as well as open form, they can be utilized as viscosity sensors in visible as well as deep red region. We also correlated our experimental finding theoretically by using Density Functional theory computations.