Enzymatically mediated fluorescent copper nanocluster generation for tyramine determination.

This work details the enzymatic generation of fluorescence nanomaterials and the use of this optical signal as the analytical parameter for the quantification of the substrate. More specifically, fluorescent copper nanoclusters have been obtained during the enzymatic reaction of tyramine oxidase and tyramine in the presence of Cu(II); the fluorescence intensity being proportional to the concentration of tyramine. The nanoclusters obtained show fluorescence at 445 nm by being excited at 320 nm and have been characterized by TEM, EDX, and XPS. The formation mechanism has also been studied, suggesting that under the optimal conditions (0.1 M MES buffer and pH = 6), the formation of the nanoclusters is due to the reducing properties of the product of the enzymatic reaction (p-hydroxybenzaldehyde) in MES buffer. The method shows a linear relationship with the concentration of tyramine in the range from 1.0·10-5 to 2.5·10-4 M, a RSD of 3% (n = 5) and a LOD of 6.3·10-6 M. The method has been applied to the determination of tyramine in sausage with good results.

Portable colorimetric enzymatic disposable biosensor for histamine and simultaneous histamine/tyramine determination using a smartphone.

Tyramine oxidase (TAO), peroxidase (HRP), and Amplex Red (AR) have been immobilized on cellulose to obtain disposable biosensors for the determination of histamine. During the enzymatic reaction, AR is oxidized and a pink spot is obtained. Using a smartphone and measuring the G (green) color coordinate, histamine can be determined in the presence of other biogenic amines (putrescine and cadaverine) in concentrations ranging from 2·10-5 M to 5·10-4 M with a 7.5·10-6 M limit of detection (LoD). Despite tyramine interference, experimental conditions are provided which allow rapid and simple histamine and simultaneous histamine/tyramine (semi)quantitative determination in mixtures. Finally, tyramine and histamine were determined in a tuna extract with good results (compared to the reference HPLC-MS method). The methodology can also be applied in solution allowing histamine (and simultaneous histamine/tyramine) determination with a lower LoD (1.8·10-7 M) and a similar selectivity.

In situ enzymatic generation of Au/Pt nanoparticles as an analytical photometric system: proof of concept determination of tyramine.

In situ enzymatic generation of bimetallic nanoparticles, mainly Au/Pt, overcomes the drawbacks (continuous absorbance drift, modest LOQ, and long-time reaction) observed when AuNP alone are produced. In this study, Au/Pt nanoparticles have been characterized by EDS, XPS, and HRTEM images using the enzymatic determination of tyramine with tyramine oxidase (TAO) as a model. Under experimental conditions, the Au/Pt NPs show an absorption maximum at 580 nm which can be related to the concentration of tyramine in the range 1.0 × 10-6M to 2.5 × 10-4M with a RSD of 3.4% (n = 5, using 5 × 10-6M tyramine). The Au/Pt system enables low LOQ (1.0 × 10-6 M), high reduction of the absorbance drift, and a significant shortening of the reaction time (i.e., from 30 to 2 min for a [tyramine] = 1 × 10-4M); additionally, a better selectivity is also obtained. The method has been applied to tyramine determination in cured cheese and no significant differences were obtained compared to a reference method (HRP:TMB). The effect of Pt(II) seems to involve the previous reduction of Au(III) to Au(I) and NP generation from this oxidation state. Finally, a three-step (nucleation-growth-aggregation) kinetic model for the generation of NPs is proposed; this has enabled us to obtain a mathematical equation which explains the experimentally observed variation of the absorbance with time.

Tectomer-Mediated Optical Nanosensors for Tyramine Determination.

The development of optical sensors for in situ testing has become of great interest in the rapid diagnostics industry. We report here the development of simple, low-cost optical nanosensors for the semi-quantitative detection or naked-eye detection of tyramine (a biogenic amine whose production is commonly associated with food spoilage) when coupled to Au(III)/tectomer films deposited on polylactic acid (PLA) supports. Tectomers are two-dimensional oligoglycine self-assemblies, whose terminal amino groups enable both the immobilization of Au(III) and its adhesion to PLA. Upon exposure to tyramine, a non-enzymatic redox reaction takes place in which Au(III) in the tectomer matrix is reduced by tyramine to gold nanoparticles, whose reddish-purple color depends on the tyramine concentration and can be identified by measuring the RGB coordinates (Red-Green-Blue coordinates) using a smartphone color recognition app. Moreover, a more accurate quantification of tyramine in the range from 0.048 to 10 µM could be performed by measuring the reflectance of the sensing layers and the absorbance of the characteristic 550 nm plasmon band of the gold nanoparticles. The relative standard deviation (RSD) of the method was 4.2% (n = 5) with a limit of detection (LOD) of 0.014 µM. A remarkable selectivity was achieved for tyramine detection in the presence of other biogenic amines, especially histamine. This methodology, based on the optical properties of Au(III)/tectomer hybrid coatings, is promising for its application in food quality control and smart food packaging.

Gold nanoparticle formation as an indicator of enzymatic methods: colorimetric L-phenylalanine determination.

An enzymatic-colorimetric method has been developed based on the reaction between L-phenylalanine (L-Phe) and the L-amino acid oxidase (LAAO) in the presence of Au(III), which has led to the formation of gold nanoparticles. The intensity of the localized surface plasmon resonance (LSPR) band of the generated nanoparticles (550 nm) can be related to the concentration of L-Phe in the sample. The mechanism of the LAAO-L-Phe enzyme reaction in the presence of Au(III) has been studied through the evaluation and optimization of experimental conditions. These studies have reinforced the hypothesis that the catalytic center of the enzyme helps the Au(III) reduction and, thanks to the protein, the Au0 form is stabilized as gold nanoparticles (AuNPs). In the calibration study, a sigmoidal relationship between the concentration of the substrate and the LSPR of the nanoparticles was observed. The linearization of the signal has allowed the determination of L-Phe in the range from 17 to 500 µM with an RSD% (150 µM) of 4.8% (n = 3). The method is free of other amino acid interference normally found in blood plasma. These highly competitive results open the possibility of further development of a rapid method for L-Phe determination based on colorimetry.

Electronic Communication in Binuclear Osmium- and Iridium-Polyhydrides.

Reactions of polyhydrides OsH6(PiPr3)2 (1) and IrH5(PiPr3)2 (2) with rollover cyclometalated hydride complexes have been investigated in order to explore the influence of a metal center on the MHn unit of the other in mixed valence binuclear polyhydrides. Hexahydride 1 activates an ortho-CH bond of the heterocyclic moiety of the trihydride metal-ligand compounds OsH3{κ2-C,N-[C5RH2N-py]}(PiPr3)2 (R = H (3), Me (4), Ph (5)). Reactions of 3 and 4 lead to the hexahydrides (PiPr3)2H3Os{µ-[κ2-C,N-[C5RH2N-C5H3N]-N,C-κ2]}OsH3(PiPr3)2 (R = H (6), Me (7)), whereas 5 gives the pentahydride (PiPr3)2H3Os{µ-[κ2-C,N-[C5H3N-C5(C6H4)H2N]-C,N,C-κ3]}OsH2(PiPr3)2 (8). Pentahydride 2 promotes C-H bond activation of 3 and the iridium-dihydride IrH2{κ2-C,N-[C5H3N-py]}(PiPr3)2 (9) to afford the heterobinuclear pentahydride (PiPr3)2H3Os{µ-[κ2-C,N-[C5H3N-C5H3N]-N,C-κ2]}IrH2(PiPr3)2 (10) and the homobinuclear tetrahydride (PiPr3)2H2Ir{µ-[κ2-C,N-[C5H3N-C5H3N]-N,C-κ2]}IrH2(PiPr3)2 (11), respectively. Complexes 6-8 and 11 display HOMO delocalization throughout the metal-heterocycle-metal skeleton. Their sequential oxidation generates mono- and diradicals, which exhibit intervalence charge transfer transitions. This notable ability allows the tuning of the strength of the hydrogen-hydrogen and metal-hydrogen interactions within the MHn units.

Smartphone-interrogated test supports for the enzymatic determination of putrescine and cadaverine in food.

Diamino-oxidase (DAO), horseradish peroxidase (HRP), and tetramethylbenzidine (TMB) have been immobilized into cellulose to obtain circular cellulose test supports (CCTSs) for the determination of cadaverine (Cad) and putrescine (Put). During the enzymatic reaction, TMB is oxidized and a blue spot is obtained. This color (RGB coordinates) is measured with a smartphone and a commercial application. The highest sensitivity is provided by the component R and a linear response is observed for low biogenic amine (BA) concentrations, but a second-order polynomial response better fits the experimental results for a wider concentration range. This has been successfully explained with a model developed to explain the RGB values obtained in this type of analytical system. Optimization studies enable CCTSs to be obtained for Put and Cad determination, which could be used (kept at 4 °C) for at least 45 days if a stabilizer (StabilCoat™ or StabilGuard™) is added during its synthesis. In these conditions, the R coordinate follows the model up to at least 4 × 10-4 M Put and/or Cad (both analytes give the same response). The method permits the Put and Cad determination from 5 × 10-5 M up to 4 × 10-4 M (RSD = 3%, n = 3). The CCTSs have been applied to Put + Cad determination in a tuna sample without any interference by other biogenic amines. The concentration found statistically agrees with that obtained using a HPLC-MS-validated method. Graphical abstract.

Colorimetric-enzymatic determination of tyramine by generation of gold nanoparticles.

In this paper, it has been demonstrated that Au(III) is able to act instead of O2 in the oxidase enzymatic reaction, so that it becomes reduced to purple gold nanoparticles (AuNPs). The plasmon band (at 540 nm) can be used as the analytical signal. Tyramine has been determined using its enzymatic reaction with tyramine oxidase (TAO). The kinetic of the AuNP formation has been studied in the light of both the Avrami equation for crystallization and the Finke-Watsy mechanism for AuNP nucleation and growth. The effects of the Au(III), TAO and tyramine concentrations on the corresponding kinetic constants have been investigated. Working at room temperature, under optimal conditions (phosphate buffer pH 7.0, TAO 0.5 U.mL-1 Au(III) 1 mM), the linear response ranges from 2.5 × 10-5 M to 3.3 × 10-4 M Tyramine (5.6% RSD) and the LOD is 2.9 × 10-6 M. Under these conditions, the signal is measured after 30 min reaction (to obtain the highest sensitivity), but this time can be significantly reduced by increasing the temperature (the reaction is finished after 4 min when working at 50 °C). The method has been applied to tyramine determination in a cheese sample with good results. The new scheme proposed in this paper can be extended, in principle, to other enzymatic methods based on oxidase enzymes. Graphical abstractTyramine is determined by measuring the plasmon band of the gold nanoparticles formed during its enzymatic reaction with Tyramine oxidase. Moreover, a mathematical model has been developed to explain the formation of the gold nanoparticles during the reaction.

Gold nanoclusters as a quenchable fluorescent probe for sensing oxygen at high temperatures.

Gold nanoclusters (AuNCs) capped with lipoic acid (LA) or templated with bovine serum albumin (BSA) are shown to be viable fluorescent probes for oxygen (O2) which acts as a collisional quencher. Quenching of fluorescence, with its lifetimes in the order of 123 ± 9 ns (LA) and 153 ± 15 ns (BSA) (in aqueous solution), is best measured at excitation/emission wavelengths of 400/680 nm and 375/650 nm respectively. It follows the Stern-Volmer model, whose quenching constants (Ksv) and quenching efficiencies (γ) are 1400 M-1 and 0.52 for AuNC@LA and 4479 M-1 and 0.90 for AuNC@BSA. The probes were immobilized on a silica support and tested for response to O2 in gas phase using a commercial instrument. The effect of temperature on the fluorescence of AuNC@LA was studied in the range from 30 to 210 °C. Fluorescence intensity slightly decreases with temperature in the first heating cycle but remains constant in further cycles. The AuNC@LA were studied for their response to O2 in the temperature range from 30 to 100 °C, and even at 100 °C they respond to O2, with a Ksv that slightly drops with increasing temperature. Measuring in gas phase at 100 °C, the sensor has a detection limit of 3% (V/V) of O2 at a signal-to-noise ratio of 3. Graphical Abstract Gold-nanoclusters (AuNCs) fluorescence intensity (λexc = 400 nm, λem = 680 nm) remains constant from 30 to 210 °C and is quenched by O2 following a collisional mechanism. The Stern-Volmer constant (Ksv) slightly changes from 25 °C to 100 °C (at least).

Enzymatic methods for choline-containing water soluble phospholipids based on fluorescence of choline oxidase: Application to lyso-PAF.

In this paper we present methods to determine water soluble phospholipids containing choline (wCh-PL). The analytes were hydrolyzed by the enzyme phospholipase D and the choline formed was oxidized by the enzyme Choline Oxidase (ChOx); the fluorescence changes of the ChOx are followed during the enzymatic reaction, avoiding the necessity of an indicating step. Both reactions (hydrolysis and oxidation) can be combined in two different ways: 1) a two-step process (TSP) in which the hydrolysis reaction takes place during an incubation time and then the oxidation reaction is carried out, the analytical signal being provided by the intrinsic fluorescence of ChOx due to tryptophan; 2) a one-step process (OSP) in which both enzymatic reactions are carried out simultaneously in the same test; in this case the analytical signal is provided by the ChOx extrinsic fluorescence due to a fluorescent probe (Ru (II) chelate) linked to the enzyme (ChOx-RuC). The analytical capabilities of these methods were studied using 1,2-dioctanoyl-sn-glycero-3-phosphocholine (C8PC), a water soluble short alkyl chain Ch-PL as a substrate, and 1-O-hexadecyl-sn-glyceryl-3-phosphorylcholine (lyso-PAF). The analytical features of merit for both analytes using both methods were obtained. The TSP gave a 10-fold sensitivity and lower quantification limit (1.0*10-5 M for lyso-PAF), but OSP reduced the determination time and permitted to use the same enzyme aliquot for several measurements. Both methods gave similar precision (RSD 7%, n = 5). The TSP was applied to the determination of C8PC and lyso-PAF in spiked synthetic serum matrix using the standard addition method. The application of this methodology to PLD activity determination is also discussed.

Fluorescence detection by intensity change based sensors: a theoretical model.

According to Fluorescence Detection by Intensity Changes (FDIC) the fluorescence intensity of many fluorophores depends on the non-covalent (specific and/or non-specific) interactions these fluorophores would be able to establish with the solvent and, more interestingly, with other surrounding molecules. This latter effect is the basis of FDIC for analytical purposes. In this paper, a preliminary study of FDIC applications using a fluorophore supported in a solid medium (sensor film) is presented. First, a mathematical model relating the analyte concentration with the immobilized fluorophore fluorescence is deduced. The model includes all the different mechanisms explaining this relationship: index of refraction or dielectric constant modification, scattering coefficient alteration and sensor film volume increase. Then, the very first experimental results are presented, using different fluorophores and solid supports. The best results were obtained using polyacrylamide (PAA) polymers and coralyne as the fluorophore. This sensor film is applied for albumin and polyethylenglycol determination and the results are compared with those obtained using coralyne in solution. Albumin quenches the coralyne fluorescence in both cases (solution and film), while PEG quenches coralyne fluorescence in films but increases it in solution. These results suggest that the outstanding fluorescence change mechanism is sensor films is the film volume increases, which is different than those observed in solution.

Reagentless fluorescent biosensors based on proteins for continuous monitoring systems.

There is a lack of commercially available efficient and autonomous systems capable of continuous monitoring of (bio)chemical data for clinical, environmental, food, or industrial samples. The weakest link in the design of these systems is the (bio)chemical receptor (bCR). The bCR should have transducer ability, the recognition event should be a single reaction, and the bCR should be easily regenerated. Transport proteins and enzymes are well placed as bCR for optical continuous monitoring systems (OCMS). In this paper we review quantitative aspects and the main transducer strategies which have been developed for transport proteins, using periplasmic binding proteins (linking an environmentally sensitive fluorophore or FRET between two fluorophores) and concanavalin A (competitive reversible assays) as representative examples. Efficient immobilization systems and implementation in OCMS are also reviewed. Some kinds of enzymes can fulfil the necessary requirements to be appropriate bCR. Strategies using flavoenzymes chemically modified with fluorophores can be successfully implemented in OCMS and they are, in our opinion, the most appropriate option.

Selective generation of gold nanostructures mediated by flavo-enzymes to develop optical biosensors.

This paper explores, for the first time, the use of flavo-enzymes for the enzymatic generation of gold nanomaterials. It has been demonstrated that when the oxidation of glucose by GOx is carried out in the presence of Au(III), the in-situ formation of gold nanomaterials is observed. Moreover, depending on the experimental conditions, either nanoparticles (AuNPs) or nanoclusters (AuNCs) are better observed, whose spectroscopical properties can be related to the concentration of glucose. Working at pH 6, only AuNCs with fluorescence at 420 nm (λex=335 nm) are obtained (linear relationship from 6.0·10-5 M to 1.5·10-3 M glucose). However, when the enzymatic reaction is performed at pH 8, AuNPs (λmax=580 nm) are also obtained (linear relationship from 5.5·10-4 M to 2.0·10-3 M glucose). Mathematical equations describing the variation of fluorescence and absorbance values during the reaction have been proposed. The results obtained suggest that AuNCs are formed using GOx as nucleation seeds. Since AuNPs belong to the branched-type, it is suggested that they are obtained by AuNC coalescence. From these models the AuNP molar absorptivity per atom was obtained (2.0(±0.3)·103 M-1cm-1). Finally, the method has been applied to the determination of glucose in orange juice and human plasma samples. Comparing the results with the GOx/HRP/TMB method and a commercial glucometer, no significant differences (P=0.05) are obtained.

Solving Color Reproducibility between Digital Devices: A Robust Approach of Smartphones Color Management for Chemical (Bio)Sensors.

In the past twelve years, digital image colorimetry (DIC) on smartphones has acquired great importance as an alternative to the most common analytical techniques. This analysis method is based on fast, low-cost, and easily-accessible technology, which can provide quantitative information about an analyte through the color changes of a digital image. Despite the fact that DIC is very widespread, it is not exempt from a series of problems that are not fully resolved yet, such as variability of the measurements between smartphones, image format in which color information is stored, power distribution of the illuminant used for the measurements, among others. This article proposes a methodology for the standardization and correction of these problems using self-developed software, together with the use of a 3D printed light box. This methodology is applied to three different colorimetric analyses using different types and brands of smartphones, proving that comparable measurements between devices can be achieved. As color can be related to many target analytes, establishing this measurement methodology can lead to new control analysis applicable to diverse sectors such as alimentary, industrial, agrarian, or sanitary.

Direct minimally invasive enzymatic determination of tyramine in cheese using digital imaging.

An enzymatic method for the direct (without pretreatment) minimally invasive tyramine determination in cheese is proposed. Colorimetric test strips containing tyramine oxidase (TAO), peroxidase and 3,3',5,5'-tetramethylbenzidine (Q-TAO), allow tyramine determination through the RGB chromatic coordinates of the observed blue colour (LOD = 2.6·10-6 M, LOQ = 8.7·10-6 M, RSD% (n = 5; 1.8·10-4 M) = 3.2%). The strips are inserted in the sample for 2 min and then the RGB coordinates are measured using a smartphone. Previously, these Q-TAO strips have been also optimized for tyramine determination in cheese extract. To do that, a spectrophotometric method in solution for tyramine determination in cheese extracts has been developed, which included an in-depth study of the indicating reaction; this study has allowed to gain new information about the spectroscopic properties of different TMB species and, which it is more important, to detect cross-reactions between TAO and TMB species. A mathematical model has also been developed which relate the RGB signals obtained with the tyramine concentrations, the instrumental characteristics of the smartphone and the spectroscopic properties of the absorbing product of the enzymatic reaction.

Globotriaosylceramide-related biomarkers of fabry disease identified in plasma by high-performance thin-layer chromatography - densitometry- mass spectrometry.

Identification of 19 molecular species of globotriaosylceramides (Gb3) in extracts from a Fabry's plasma patient and a healthy control was performed by High-Performance Thin-Layer Chromatography (HPTLC)-densitometry and online coupling to Mass Spectrometry (MS). Separation was carried out on LiChrospher plates using Automated Multiple Development (AMD). Densitometry was performed on twin plates by combining detection in the visible at 550 nm, through previous on-plate orcinol derivatization, and by Ultraviolet 190 nm, using a non-impregnated plate. The latter was directly coupled to an ion-trap mass spectrometer through an automated elution-based interface. Gb3 molecular species, which were identified by HPTLC- Electrospray Mass Spectrometry (+)-MS and confirmed by MS/MS or HPTLC-Atmospheric Pressure Chemical Ionization Mass Spectrometry (+)-MS, are: five isoforms of saturated Gb3; seven isoforms of methylated Gb3; and seven species with two additional double bonds. Twelve of these species were previously reported as biomarkers of Fabry's lysosomal disorder using a Liquid Chromatography-MS-based method, and the other seven are structurally similar, closely related to them. Saturated Gb3 isoforms migrated on LiChrospher plate in one of the separated peaks corresponding to the migration zone of ceramide trihexosides standard. Instead, methylated and unsaturated Gb3 species co-migrated with sphingomyelin species. Ion intensity ESI-MS profiles show that saturated Gb3 species in Fabry's plasma were in higher concentration than in control sample. Before applying the Thin-Layer Chromatography (TLC)-MS interface on HPTLC separated peaks, its positioning precision was first studied using ceramide tri-hexosides as model compound. This provided information on Gb3 peak broadening and splitting during its migration.

CCD detectors for molecular absorption spectrophotometry. A theoretical and experimental study on characteristics and performance.

Uncertainty in charge-coupled devices (CCDs) as UV-vis spectrophotometric detectors is studied here considering that it highly affects the limit of detection of analytical methods. Opposite to photomultiplier-type detectors (PMDs) and diode-array detectors (DADs), where uncertainty is mainly dependent on the photonic signal (shot noise), in CCD detectors uncertainty may come from both independent and dependent effects upon the photonic signal. Shot noise is specially important for high photonic signals in the detector (those for low absorbances) while the uncertainty that is independent of the signal is specially important for low photonic signals in the detector (those for high absorbances). That is, the main source of uncertainty is different depending on the value of the experimental measurement. On the other hand, temperature does not practically affect absorbance measurements, though it is very important for emission measurements (fluorescence, Raman, scattering, etc.). Mathematical equations for uncertainty are proposed with excellent fittings to the experimental data. The equation parameters can be experimentally determined from non-linear regression analysis and used to characterize spectrometers or to test their performance. In order to help buyers and users, some recommendations are finally given considering, among others, cooling, slit, attenuator or fiber optic assemblies.

Selective peracetic acid determination in the presence of hydrogen peroxide using a label free enzymatic method based on catalase.

Peracetic acid (PAA) is selectively determined in the presence of hydrogen peroxide (H(2)O(2)) by using the self-indicating UV-Vis molecular absorption properties of catalase. The PAA reacts with the protein giving an intermediate (Cat-I) which is reduced back by the amino acid core surrounding the heme group. Since the original form of the enzyme and the Cat-I have different UV-Vis absorption properties, the absorbance changes can be used for PAA determination. The H(2)O(2)/catalase reaction is extremely fast so that neither Cat-I compound nor kinetic interferences are observed. The method permits PAA determination in the 5 × 10(-7) to 1.5 × 10(-5) M range, the reproducibility being between 1% and 10%. Using this method, PAA has been successfully determined in water samples treated with commercial PAA/H(2)O(2) biocides. A theoretical study has also been carried out for obtaining a mathematical model able to analytically describe the process.

Analytical possibilities of Putrescine and Cadaverine enzymatic colorimetric determination in tuna based on diamine oxidase: A critical study of the use of ABTS.

The joint determination of putrescine (Put) and cadaverine (Cad) in the presence of other biogenic amines is studied using their enzymatic reaction with diamine oxidase (DAO). Three alternative methods are studied based on the intrinsic colorimetric properties of DAO or horseradish peroxidase (HRP), and the use of 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) colorimetric reagent, respectively. In this last case an in-depth study is carried out in order to explain and solve some drawbacks usually associated with the use of this reagent (especially interferences, interaction with enzymes and instability), and to propose new analytical methodologies which this reagent allows to achieve (transient signal and the use of the violet species). Finally, the method has been applied to Put + Cad determination in a tuna sample without interference of other biogenic amines. The result has been compared with that obtained using a method based on HPLC-MS, which has allowed the new methodology to be validated.

The environmental effect on the fluorescence intensity in solution. An analytical model.

In this paper a mathematical model describing the non-specific interactions of the medium surrounding a fluorophore on its fluorescence intensity is proposed. The model, which has been developed for quantitative analytical applications, is based on the following general ideas: (1) the medium affects the fluorescence quantum yield across the non-radiative decay constant (k(nr)); (2) the k(nr) can be simplified to the singlet-to-triplet intersystem crossing (k(ISC)) constants; (3) k(ISC) follows the energy gap law and then depends on the singlet and triplet energy difference, and (4) the medium, due to solvation, changes the energy of both excited levels (singlet and triplet), then the constants and finally the fluorescence intensity. In our model, the strength of the fluorophore solvation by the solvent (represented by its refraction index, n, dielectric constant, epsilon, and electric charge) changes the singlet (excited)-to-fundamental and the singlet-to-triplet energy gaps, thus the k(ISC) and k(IC) (internal conversion constant) values and in consequence the fluorescence quantum yield. The final model relates the fluorescence intensity (F) with the solvent dielectric constant and refraction index. Finally, the model is particularized for the case of a medium composed of a solvent and a solute, obtaining an F-to-solute concentration relationship and enabling this fact to be used for analytical applications. The very first experimental data are shown demonstrating the fulfilment of this model.