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With the Calibration Kit Spectral Fluorescence Standards BAM-F001BAM-F005, we developed a simple
tool for the characterization of the relative spectral responsivity and the long-term stability of the emission channel of
fluorescence instruments under routine measurement conditions thereby providing the basis for an improved comparability
of fluorescence measurements and eventually standardization. This first set of traceable fluorescence standards,
which links fluorescence measurements to the spectral radiance scale in the spectral range of 300770 nm and has been optimized for spectrofluorometers, can be employed for different measurement geometries and can be adapted to different fluorescence techniques with proper consideration of the underlying measurement principles.
This report summarizes problems associated with the comparability of measurements of photoluminescence and procedures for the characterization of relevant instruments, focusing on physical and chemical fluorescence standards. To provide recommendations on selecting and using such standards, we derive general and scope-specific requirements and quality criteria for suitable devices and materials and briefly address metrological requirements linked to the realization of comparable measurements. Special emphasis is dedicated to liquid and solid chromophore-based fluorescence standards developed or currently tested by us.
The need for the traceable characterization of fluorescence instruments is emphasized from a chemists point of view, focusing on spectral fluorescence standards for the determination of the wavelength- and polarization-dependent relative spectral responsivity and relative spectral irradiance of fluorescence measuring systems, respectively. In a first step, major sources of error of fluorescence measurements and instrument calibration are revealed to underline the importance of this issue and to illustrate advantages and disadvantages of physical and chemical transfer standards for generation of spectral correction curves. Secondly, examples for sets of traceable chemical emission and excitation standards are shown that cover a broad spectral region and simple procedures for the determination of corrected emission spectra with acceptable uncertainties are presented. With proper consideration of the respective measurement principle and geometry, these dye-based characterization procedures can be not only applied to spectrofluorometers but also to other types of fluorescence measuring systems and even to Raman spectrometers.
We present a novel, simple, and fast colorimetric method to quantify the total number of carboxy groups on polymer microparticle and nanoparticle surfaces. This method exploits that small divalent transition metal cations (M2+ = Ni2+, Co2+, Cd2+) are efficiently bound to these surface functional groups, which allows their extraction by a single centrifugation step. Remaining M2+ in the supernatant is subsequently quantified spectrophotometrically after addition of the metal ion indicator pyrocatechol violet, for which Ni2+ was identified to be the most suitable transition metal cation. We demonstrate that the difference between added and detected M2+ is nicely correlated to the number of surface carboxy groups as determined by conductometry, thereby affording a validated measure for the trueness of this procedure. The variation coefficient of ~5% found in reproducibility studies underlines the potential of this novel method that can find conceivable applications for the characterization of different types of poly(carboxylic acid)-functionalized materials, e.g., for quality control by manufacturers of such materials.
The fluorine content of polymer particles labelled with 2,2,2-trifluoroethylamine was reliably quantified with overlapping sensitivity ranges by XPS and solid-state NMR. This provides a first step towards reference materials for the metrological traceability of surface group quantifications. The extension of this concept to fluorescence spectroscopy is illustrated.
We introduce a method to determine the number of accessible functional groups on a polymer microsphere surface based on the interaction between the macrocyclic host cucurbit[7]uril (CB7) and a guest reacted to the microsphere surface. After centrifugation, CB7 in the supernatant is quantified by addition of a fluorescent dye. The difference between added and detected CB7 affords the number of accessible surface functional groups.
The amount of grafted poly(acrylic acid) on poly(methyl methacrylate) micro- and nanoparticles was quantified by conductometry, 13C solid-state NMR, fluorophore labeling, a supramolecular assay based on high-affinity binding of cucurbit[7]uril, and two colorimetric assays based on toluidine blue and nickel complexation by pyrocatechol violet. The methods were thoroughly validated and compared with respect to reproducibility, sensitivity, and ease of use. The results demonstrate that only a small but constant fraction of the surface functional groups is accessible to covalent surface derivatization independently of the total number of surface functional groups, and different contributing factors are discussed that determine the number of probe molecules which can be bound to the polymer surface. The fluorophore labeling approach was modified to exclude artifacts due to fluorescence quenching, but absolute quantum yield measurements still indicate a major uncertainty in routine fluorescence-based surface group quantifications, which is directly relevant for biochemical assays and medical diagnostics. Comparison with results from protein labeling with streptavidin suggests a porous network of poly(acrylic acid) chains on the particle surface, which allows diffusion of small molecules (cutoff between 1.6 and 6.5 nm) into the network.
The development of fluorescence applications in the life and material sciences has proceeded largely without sufficient concern for the measurement uncertainties related to the characterization of fluorescence instruments. In this first part of a two-part series on the state-of-the-art comparability of corrected emission spectra, four National Metrology Institutes active in high-precision steady-state fluorometry performed a first comparison of fluorescence measurement capabilities by evaluating physical transfer standard (PTS)-based and reference material (RM)-based calibration methods. To identify achievable comparability and sources of error in instrument calibration, the emission spectra of three test dyes in the wavelength region from 300 to 770 nm were corrected and compared using both calibration methods. The results, obtained for typical spectrofluorometric (0°/90° transmitting) and colorimetric (45°/0° front-face) measurement geometries, demonstrated a comparability of corrected emission spectra within a relative standard uncertainty of 4.2% for PTS- and 2.4% for RM-based spectral correction when measurements and calibrations were performed under identical conditions. Moreover, the emission spectra of RMs F001 to F005, certified by BAM, Federal Institute for Materials Research and Testing, were confirmed. These RMs were subsequently used for the assessment of the comparability of RM-based corrected emission spectra of field laboratories using common commercial spectrofluorometers and routine measurement conditions in part 2 of this series (subsequent paper in this issue).
In the second part of this two-part series on the state-of-the-art comparability of corrected emission spectra, we have extended this assessment to the broader community of fluorescence spectroscopists by involving 12 field laboratories that were randomly selected on the basis of their fluorescence measuring equipment. These laboratories performed a reference material (RM)-based fluorometer calibration with commercially available spectral fluorescence standards following a standard operating procedure that involved routine measurement conditions and the data evaluation software LINKCORR developed and provided by the Federal Institute for Materials Research and Testing (BAM). This instrument-specific emission correction curve was subsequently used for the determination of the corrected emission spectra of three test dyes, X, QS, and Y, revealing an average accuracy of 6.8% for the corrected emission spectra. This compares well with the relative standard uncertainties of 4.2% for physical standard-based spectral corrections demonstrated in the first part of this study (previous paper in this issue) involving an international group of four expert laboratories. The excellent comparability of the measurements of the field laboratories also demonstrates the effectiveness of RM-based correction procedures.