In-lab synthesized turn-off fluorescence sensor for estimation of Gemigliptin and Rosuvastatin polypill appraised by Spider diagram, AGREE and whiteness metrics.

Gemigliptin-Rosuvastatin single-pill combination is a promising therapeutic tool in the effective control of hyperglycemia and hypercholesterolemia. Organic sensors with high quantum yields have profoundly significant applications in the pharmaceutical industry, such as routine quality control of marketed formulations. Herein, the fluorescence sensor, 2-Morpholino-4,6-dimethyl nicotinonitrile 3, (λex; 226 nm, λem; 406 nm), was synthesized with a fluorescence quantum yield of 56.86% and fully characterized in our laboratory. This sensor showed high efficiency for the determination of Gemigliptin (GEM) and Rosuvastatin (RSV) traces through their stoichiometric interactions and simultaneously fractionated by selective solvation. The interaction between the stated analytes and sensor 3 was a quenching effect. Various experimental parameters and the turn-off mechanism were addressed. The adopted approach fulfilled the ICH validation criteria and showed linear satisfactory ranges, 0.2-2 and 0.1-1 µg/mL for GEM and RSV, respectively with nano-limits of detection less than 30 ng/mL for both analytes. The synthesized sensor has been successfully applied for GEM and RSV co-assessment in their synthetic polypill with excellent % recoveries of 98.83 ± 0.86 and 100.19 ± 0.64, respectively. No statistically significant difference between the results of the proposed and reported spectrophotometric methods in terms of the F- and t-tests. Ecological and whiteness appraisals of the proposed study were conducted via three novel approaches: the Greenness Index via Spider Diagram, the Analytical Greenness Metric, and the Red-Green-Blue 12 model. The aforementioned metrics proved the superiority of the adopted approach over the previously published one regarding eco-friendliness and sustainability. Our devised fluorimetric turn-off sensing method showed high sensitivity, selectivity, feasibility, and rapidity with minimal cost and environmental burden over other sophisticated techniques, making it reliable in quality control labs.

Chip-Based Spectrofluorimetric Determination of Iodine in a Multi-Syringe Flow Platform with and without In-Line Digestion-Application to Salt, Pharmaceuticals, and Algae Samples.

In this work, a flow-based spectrofluorimetric method for iodine determination was developed. The system consisted of a miniaturized chip-based flow manifold for solutions handling and with integrated spectrofluorimetric detection. A multi-syringe module was used as a liquid driver. Iodide was quantified from its catalytic effect on the redox reaction between Ce(IV) and As(III), based on the Sandell-Kolthoff reaction. The method was applied for the determination of iodine in salt, pharmaceuticals, supplement pills, and seaweed samples without off-line pre-treatment. An in-line oxidation process, aided by UV radiation, was implemented to analyse some samples (supplement pills and seaweed samples) to eliminate interferences and release iodine from organo-iodine compounds. This feature, combined with the fluorometric reaction, makes this method simpler, faster, and more sensitive than the classic approach of the Sandell-Kolthoff reaction. The method allowed iodine to be determined within a range of 0.20-4.0 µmol L-1, with or without the in-line UV digestion, with a limit of detection of 0.028 µmol L-1 and 0.025 µmol L-1, respectively.

Study of Methionine Choline Deficient Diet-Induced Steatosis in Mice Using Endogenous Fluorescence Spectroscopy.

Non-alcoholic fatty liver disease is a highly prevalent condition worldwide that increases the risk to develop liver fibrosis, cirrhosis, and hepatocellular carcinoma. Thus, it is imperative to develop novel diagnostic tools that together with liver biopsy help to differentiate mild and advanced degrees of steatosis. Ex-vivo liver samples were collected from mice fed a methionine-choline deficient diet for two or eight weeks, and from a control group. The degree of hepatic steatosis was histologically evaluated, and fat content was assessed by Oil-Red O staining. On the other hand, fluorescence spectroscopy was used for the assessment of the steatosis progression. Fluorescence spectra were recorded at excitation wavelengths of 330, 365, 385, 405, and 415 nm by establishing surface contact of the fiber optic probe with the liver specimens. A multi-variate statistical approach based on principal component analysis followed by quadratic discriminant analysis was applied to spectral data to obtain classifiers able to distinguish mild and moderate stages of steatosis at the different excitation wavelengths. Receiver Operating Characteristic (ROC) curves were computed to compare classifier's performances for each one of the five excitation wavelengths and steatosis stages. Optimal sensitivity and specificity were calculated from the corresponding ROC curves using the Youden index. Intensity in the endogenous fluorescence spectra at the given wavelengths progressively increased according to the time of exposure to diet. The area under the curve of the spectra was able to discriminate control liver samples from those with steatosis and differentiate among the time of exposure to the diet for most of the used excitation wavelengths. High specificities and sensitivities were obtained for every case; however, fluorescence spectra obtained by exciting with 405 nm yielded the best results distinguishing between the mentioned classes with a total classification error of 1.5% and optimal sensitivities and specificities better than 98.6% and 99.3%, respectively.

Yellow-emissive carbon dots with a large Stokes shift are viable fluorescent probes for detection and cellular imaging of silver ions and glutathione.

Yellow-emissive carbon dots (Y-CDs) were prepared by a solvothermal method using anhydrous citric acid and 2,3-phenazinediamine as the starting materials. The Y-CDs display a 24% fluorescence quantum yield, a 188-nm Stokes' shift and excellent stability. They are shown here to be excellent fluorescent probes for the determination of Ag(I) ion and glutathione (GSH). If exposed to Ag(I) ions, they are bound by the carboxy groups of the Y-CDs, and this causes quenching of fluorescence (with excitation/emission maxima at 380/568 nm) via a static quenching mechanism. This effect was used to design a fluorometric assay for Ag(I). The quenched fluorescence of the Y-CDs can be restored by adding GSH due to the high affinity of GSH for Ag(I). The calibration plot for Ag(I) is linear in the 1-4 µM Ag(I) concentration range, and the limit of detection is 31 nM. The respective values for GSH are 5-32 µM, and 76 nM, respectively. The method was applied to the detection of Ag(I) in spiked environmental water samples and gave recoveries ranging from 93 to 107%. It was also applied to the determination of GSH in tomatoes and purple grapes and gave satisfactory recoveries. The Y-CDs display low cytotoxicity and were successfully used to image Ag(I) and GSH in H1299 cells. Graphical abstract Schematic presentation of the mechanism of yellow fluorescent CDs for the detection of Ag+ and glutathione.

High-performance organized media-enhanced spectrofluorimetric determination of pirimiphos-methyl in maize.

A novel method for the determination of pirimiphos-methyl (PMM) in maize grains by fluorescence spectroscopy and three-way calibration was developed. The formation of supramolecular complexes and their effect on the luminescence properties of PMM were studied. A solvent extraction step followed by solid phase extraction for sample clean-up was optimized. A chemometric approach consisting in PARAFAC as second-order data processing tool and piecewise direct standardization (PDS) for reducing the complexity of the calibration process was developed to overcome the matrix effect. This strategy allowed dealing with the matrix effect while reducing the number of samples to be processed and, consequently, the solvent consumption and the total analysis time. Finally, three-way calibration was applied to predict the PMM concentration in unknown samples. The mean recovery was 115% and the limits of detection and quantitation were in the order of the parts per trillion, i.e. 6 and 20 ng g-1, respectively.

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement. In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

Nonlinear relation between concentration and fluorescence emission of protoporphyrin IX in calibrated phantoms.

5-ALA-induced protoporphyrin IX (PpIX) has shown its relevance in medical assisting techniques, notably in the detection of glioma (brain tumors). Validation of instruments on phantoms is mandatory and a standardization procedure has recently been proposed. This procedure yields phantoms recipes to realize a linear relationship between PpIX concentration and fluorescence emission intensity. The present study puts forward phantoms where this linear relationship cannot be used. We propose a model that considers two states of PpIX, corresponding to two different aggregates of PpIX, with fluorescence spectra peaking at 634 and 620 nm, respectively. We characterize the influence of these two states on PpIX fluorescence emission spectra in phantoms with steady concentration of PpIX and various microenvironment parameters (surfactant, Intralipid or bovine blood concentration, and pH). We show that, with fixed PpIX concentration, a modification of the microenvironment induces a variation of the emitted spectrum, notably a shift in its central wavelength. We show that this modification reveals a variation of proportions of the two states. This establishes phantom microenvironment regimes where the usual single state model is biased while a linear combination of the two spectra enables accurate recovering of any measured spectra.

Determination of intracellular pH in phytoplankton using the fluorescent probe, SNARF, with detection by fluorescence spectroscopy.

The maintenance of pH homeostasis is critical for a variety of cellular metabolic processes. Although ocean acidification is likely to influence cellular metabolism and energy balance, the degree to which intracellular pH in phytoplankton differs from the external environment under varying environmental pH levels is not well characterized. While there are numerous existing methods for the determination of intracellular pH in the form of single peak emission (e.g., BCECF) and radioisotopic (e.g., 14C-DMO) indicators for use with phytoplankton, the fluorescent pH indicator seminaphtharhodafluor (SNARF) has not been established as a robust method for measuring in vivo pH in phytoplankton. SNARF has superior accuracy and sensitivity since it exhibits dual emission peaks from a single excitation wavelength and the ratio of the two are related to pH. The use of a ratio limiting variations in fluorescence due to dye loading, photobleaching, and instrument variation; moreover, like other fluorescence-based assays, it does not require the specialized equipment and permits that radioisotopic methods do. As a first step, we tested the performance of SNARF for measuring intracellular pH in vivo in a number of phytoplankton taxa. SNARF detection was accomplished using fluorescence spectroscopy (FS) and laser scanning microscopy (LSM). Since SNARF fluorescence is activated by cleavage of an ester group from the core fluorophore by non-specific esterases, we measured esterase activity using fluorescein diacetate (FDA) to characterize variability in esterase activity among phytoplankton taxa, with a view towards its influence on assay performance. Esterase activity cell volume; however, there was no indication that enzyme specificity and differences in individual esterase profiles adversely affected SNARF performance in phytoplankton. Assays of intracellular pH using SNARF were comparable to those made with 14C-labeled DMO, an accepted standard method. Thus, SNARF provides robust measurements of intracellular pH in phytoplankton, constituting a useful tool in investigations of the effects of ocean acidification and fluctuations in environmental pH on cellular physiology.

Time to Get Serious about Measurement in Synthetic Biology.

For synthetic biology to mature, composition of devices into functional systems must become routine. This requires widespread adoption of comparable and replicable units of measurement. Interlaboratory studies organized through the International Genetically Engineered Machine (iGEM) competition show that fluorescence can be calibrated with simple, low-cost protocols, so fluorescence should no longer be published without units.

Random initialisation of the excitation-emission matrix fluorescence spectral variables in constraint fashion for subsequent multivariate curve resolution alternating least square analysis on a peculiarly designed calibration set: Simultaneous sensing of nine polycyclic aromatic hydrocarbons in water samples.

Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic and mutagenic in nature therefore their sensing in water sample is an important analytical task. In the present work, a novel approach that is based on the random initialisation of the excitation-emission matrix fluorescence (EEMF) spectral variables in constraint fashion for subsequent multivariate curve resolution alternating least Square (MCR-ALS) analysis is introduced for simultaneously sensing the complex dilute aqueous mixture of PAHs. The usefulness of the proposed analytical approach is successfully demonstrated by applying it intentionally on a calibration set that is peculiar in many senses. The peculiarity mainly arises because the designed (i) the calibration set consist of nine PAHS having significant spectral overlap, (ii) the concentration of each PAH in different samples are kept constant and (iii) any two samples differ only in the presence and absence of the PAHs. The proposed approach is found to make precise and accurate estimation of each of the nine PAHs without involving any pre-separation. In summary, the proposed approach provides a simple and cost-effective procedure for simultaneous sensing of several PAHs in water samples. The proposed approach could be very useful in developing countries.

Validation of Fluorescence Spectroscopy to Detect Adulteration of Edible Oil in Extra Virgin Olive Oil (EVOO) by Applying Chemometrics.

Due to high price and nutritional values of extra virgin olive oil (EVOO), it is vulnerable to adulteration internationally. Refined oil or other vegetable oils are commonly blended with EVOO and to unmask such fraud, quick, and reliable technique needs to be standardized and developed. Therefore, in this study, adulteration of edible oil (sunflower oil) is made with pure EVOO and analyzed using fluorescence spectroscopy (excitation wavelength at 350 nm) in conjunction with principal component analysis (PCA) and partial least squares (PLS) regression. Fluorescent spectra contain fingerprints of chlorophyll and carotenoids that are characteristics of EVOO and differentiated it from sunflower oil. A broad intense hump corresponding to conjugated hydroperoxides is seen in sunflower oil in the range of 441-489 nm with the maximum at 469 nm whereas pure EVOO has low intensity doublet peaks in this region at 441 nm and 469 nm. Visible changes in spectra are observed in adulterated EVOO by increasing the concentration of sunflower oil, with an increase in doublet peak and correspondingly decrease in chlorophyll peak intensity. Principal component analysis showed a distinct clustering of adulterated samples of different concentrations. Subsequently, the PLS regression model was best fitted over the complete data set on the basis of coefficient of determination (R2), standard error of calibration (SEC), and standard error of prediction (SEP) of values 0.99, 0.617, and 0.623 respectively. In addition to adulterant, test samples and imported commercial brands of EVOO were also used for prediction and validation of the models. Fluorescence spectroscopy combined with chemometrics showed its robustness to identify and quantify the specified adulterant in pure EVOO.

Determination of enantiomeric excess of some amino acids by second-order calibration of kinetic-fluorescence data.

In this investigation a new non-separative kinetic-spectroflourimetric method is proposed for the determination of lysine (lys), leucine (leu) and phenylalanine (phe) enantiomers as their o-phthaldialdehyde (OPA) derivatives in the presence of an optically active chiral thiol compound, 1-mercapto-2-propanol (MP). At ambient temperature and in the borate buffer media of pH 9.6, MP, OPA, as highly selective fluorogenic reagents, and amino acid (AA) enantiomers reacts with each other to yield two fluorescent diasteriomers of D and L-AA with maximum difference in fluorescence intensity at about 450 nm. To achieve information from the small spectral changes, the data are analyzed by Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) method. Linear calibration curves are achieved to distinct D and L-lys, leu and phe in different mole ratios by applying appropriate constraints in MCR-ALS procedures. This is the first application of MCR-ALS in determination of enantiomeric excess (ee) using OPA/MP adduct as chiral reagent, which benefits from direct time dependent-fluorescence spectral analysis and does not require prior separation of chiral analytes. Both the cross-validated correlation coefficient (Q2) and root mean squares error of prediction (RMSEP) indicated satisfactory prediction ability of this method.

A simple and economical spectrofluorimetric alternative for Al routine analysis in seafood.

A simple and economical spectrofluorimetric alternative for aluminium determination in bivalve mollusks based on the fluorescent blue-green colour complex between Al(III) and salicylaldehyde picolinoylhydrazone (SAPH) has been studied. The factors that are most likely to affect were optimized with a Box-Behnken design. Optimum conditions were: pH 6.6, 0.9 mol L-1 acetic acid/acetate buffer, 3.0 mmol L-1 SAPH, and 50% ethanol. Detection and quantitation limits were found to be 2.7 µg L-1 and 9.1 µg L-1, respectively. The upper limit of application was assessed through the limit of linearity which was set as 300 µg L-1. Intra-day repeatability and inter-day repeatability were evaluated showing an excellent precision for the fluorescence method (both < 5%). The method was sensitive enough for the satisfactory determination of aluminium in several bivalve mollusk samples both fresh and canned seafood. The results showed that commercial fresh wild products presented the smallest Al concentration (6-27 mg per 100 g dry weight), while bivalves preserved in cans the concentration was considerably higher (75 mg per 100 g dry weight). Thus, differences between Al concentration related to processing were identified. The study shows a simple, cost-effective and reliable tool for routine aluminium determination in seafood for food quality control.

Molecular interactions between warfarin and human (HSA) or bovine (BSA) serum albumin evaluated by isothermal titration calorimetry (ITC), fluorescence spectrometry (FS) and frontal analysis capillary electrophoresis (FA/CE).

Interaction thermodynamics between warfarin, a very popular anticoagulant, and Sudlow I binding site of human (HSA) or bovine (BSA) serum albumin have been examined in strictly controlled experimental conditions (HEPES buffer 50 mM, pH 7.4 and 25 °C) by means of isothermal titration calorimetry (ITC), fluorescence spectrometry (FS) and frontal analysis capillary electrophoresis (FA/CE). Each technique is based on measurements of a different property of the biochemical system, and then the results allow a critical discussion about the suitability of each approach to estimate the drug-protein binding parameters. The strongest interaction step is properly evaluated by the three assayed approaches being the derived binding constants strongly consistent: from 4 × 104 to 7 × 104 for HSA and from 0.8 × 105 to 1.2 × 105 for BSA. Binding enthalpy variations also show consistent results: -5.4 and -5.6 Kcal/mol for HSA and -4.3 and -3.7 Kcal/mol for BSA, as measured by ITC and FS, respectively. Further high order interaction events for both albumins are detected only by FA/CE.

Transfer rates of aflatoxins from herbal medicines to decoctions determined by an optimized high-performance liquid chromatography with fluorescence detection method.

OBJECTIVES: This study aimed to explore the transfer rates of aflatoxins from several contaminated herbal medicines by fungi to their decoctions. METHODS: Five types of commonly used herbal medicines including Lilii Bulbus, Hordei Fructus Germinatus, Nelumbinis Semen, Polygalae Radix and Bombyx Batryticatus were selected as the examples. Raw herbal medicine samples were treated by ultrasonication-assisted extraction with 70% methanol and immunoaffinity column clean-up, and the decoctions were prepared following the commonly used boiling method with water for 2 h. Then, the optimized high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method was validated for the quantitative analysis of four aflatoxins (AFG2 , AFG1 , AFB2 and AFB1 ) after postcolumn photochemical derivatization, which was proved to be reliable and sensitive. KEY FINDINGS: Aflatoxins were detected to be transferred from the herbal medicines to decoctions with significantly different transfer rates in the five types of herbal medicines. Quietly high transfer rates of 7.26-115.36% for AFG2 , 4.37-26.37% for AFB1 and 9.64-47.68% for AFB2 were obtained. AFB1 as the most toxic aflatoxin expressed the lowest transfer rate, but still exhibited high amount in the samples. CONCLUSIONS: Therefore, the monitoring of aflatoxins in herbal medicines and their decoctions is in great urgency to ensure the security of consumers taking decoctions.

The diagnostic value of the native fluorescence visualization device for early detection of premalignant/malignant lesions of the oral cavity.

PURPOSE: The present study aimed to determine the diagnostic value of a native fluorescence visualization device in the identification of oral malignant/dysplastic lesions. METHODS: This study involved 45 patients who had oral lesions that were suspected to be malignant, potentially malignant, or benign. The patients visited the Oral Medicine Department of the Mashhad Dental School. The sensitivity, specificity, positive and negative predictive values, and likelihood ratio of this device were determined. RESULTS: The histopathological assessment of samples showed 9 cases of oral squamous cell carcinoma and 12 lesions with dysplasia. Ten samples of dysplastic lesions and all malignant lesions appeared dark or red/orange when examined with the native fluorescence visualization device. In 90% of the dysplastic/malignant lesions, the label-free fluorescence results were positive. The sensitivity, specificity, and positive and negative predictive values of this device were 90%, 15%, 40%, and 71%, respectively. CONCLUSIONS: The native fluorescence visualization device can be used in specialized centers as an adjunctive device to increase the sensitivity of a clinical examination, but is not capable of distinguishing benign lesions from malignant and dysplastic ones due to its low specificity.

Method Verification Requirements for an Advanced Imaging System for Microbial Plate Count Enumeration.

The Growth Direct™ System that automates the incubation and reading of membrane filtration microbial counts on soybean-casein digest, Sabouraud dextrose, and R2A agar differs only from the traditional method in that micro-colonies on the membrane are counted using an advanced imaging system up to 50% earlier in the incubation. Based on the recommendations in USP <1223> Validation of New Microbiological Testing Methods, the system may be implemented in a microbiology laboratory after simple method verification and not a full method validation.LAY ABSTRACT: The Growth Direct™ System that automates the incubation and reading of microbial counts on membranes on solid agar differs only from the traditional method in that micro-colonies on the membrane are counted using an advanced imaging system up to 50% earlier in the incubation time. Based on the recommendations in USP <1223> Validation of New Microbiological Testing Methods, the system may be implemented in a microbiology laboratory after simple method verification and not a full method validation.

Self-calibrated fluorescent probe resembled as an indicator of the lysosomal phosphatase pertaining to the cancer cells.

A self-calibrated fluorescent probe Lyso-Phos has synthesized followed by a straightforward synthetic pathway. Lyso-Phos acts as an indicator for lysosomal phosphatase. Its photophysical property including cellular imaging was described. Lyso-Phos showed ratiometric UV-Vis- absorption changes from λabs 370nm to λabs 450nm in the presence of alkaline phosphatase (ALP). On the other hand, fluorescence intensity λem 560nm of Lyso-Phos has increased around 45-fold in the presence of ALP. The probe Lyso-Phos was found to be highly chemoselective toward the phosphatase compared with other ubiquitous entities in cellular milieu. The non-toxic nature of the Lyso-phos has accounted by observing higher cell viability in prostate cancer- LnCap, fibrosarcoma HT1080 and normal mouse embryo fibroblast NIH3T3 cells. Further, the probe Lyso-Phos was utilized for tracking of cellular phosphatase in live-cells. Lyso-Phos enabled to track cellular phosphatase by the extent of fluorescence labeling of LnCap cells which showed reasonable uptake efficiency in the presence of Lyso-Phos as indicated by the intracellular fluorescence. The phosphoester bond in the probe was cleaved by intracellular alkaline phosphatase leading to turn on fluorescence of the fluorescent probe Lyso-Phos. Finally, cellular colocalization with Lyso-Tracker empowered our speculation that Lyso-Phos can track endogenous phosphatase in the lysosomes. Altogether these findings suggest that Lyso-Phos would be powerful probe to detect phosphates in cancer cells.

Fluorescence Spectroscopy Application in the Express-evaluation of the Gastrointestinal Microflora.

Possible application is studied for fluorescence spectroscopy in the express-evaluation of the gastrointestinal microflora. This diagnostic approach is feasible because of the difference in fluorescent spectra from different microorganisms, with anaerobic microorganisms exhibiting the most pronounced fluorescence. It was found that fluorescence strength raised with an increase in the bacteria Bacteroides and bifid bacteria, whereas it decreased with an increase in Escherichia coli. The bacteria Bacteroides and bifid bacteria are the most typical representatives of anaerobic bacteria, whereas the bacteria Escherichia coli arc the typical representatives of aerobic bacteria. According to the literature, Bacteroides and bifid bacteria reveal an intense fluorescence. Bearing in mind that these genera of bacteria play an important role in the gastrointestinal microbiocenosis, fluorescence spectroscopy can be used as a diagnostic means for gastrointestinal dysbacteriosis.

Fluorescent rare earth solutions as intrinsic wavelength standards for protein fluorescence spectroscopy.

Trivalent Gd, Tm, and Dy solutions can be used as intrinsic excitation and emission standards to validate the UV and violet-blue wavelength accuracy of a spectrofluorimeter. Europium extends the range into the red. To attain sufficient sensitivity, these luminescent rare earth ions require deuterated reagents or carbonate complexation, which allow the use of ordinary water and thus preparation in virtually any laboratory. Such solutions are particularly valuable as system suitability standards (SST) for protein fluorescence spectroscopy to detect red shifts of the intrinsic fluorescence maximum in stability and storage studies.