Fluorescent Compounds Research Articles

Effect of different substituents on the fluorescence properties of precursors of synthetic GFP analogues and a polarity-sensitive lipid droplet probe with AIE properties for imaging cells and zebrafish.

The green fluorescent protein (GFP) is a purely natural specialty protein that has been widely used to design synthetic fluorescent probes. In the present work we designed and synthesized a series of fluorescent compounds akin to GFP precursors by a one-step method, and investigated the luminescence properties of the fluorescent compounds by varying the substituents. We presented the first systematic summary of the photophysical data including extinction coefficients and fluorescence quantum yields for this class of fluorescent dyes. We also carried out density functional theory (DFT) calculations for these dyes to investigate the effect of electronic effects due to different substituents. These studied optical properties may provide a reference for later probe design. More interestingly, we have developed a polarity-sensitive lipid droplet probe T-LD with AIE properties on this basis. The probe exhibited not only favorable pH stability and kinetic stability in terms of optical properties, but also solvent discolouration and polarity-sensitive properties, and was able to label intracellular lipid droplets. We successfully applied the probe for intracellular lipid droplet level monitoring and zebrafish imaging.

FABRICATION OF PLASTIC SCINTILLATOR USING POLYSTYRENE MATRIX BASED

Experiments on making a plastic scintillator with polystyrene as a base material mixed with fluorescent compounds (primary and secondary dopants) have been performed. Primary dopants (PTP) used to produce emission at wavelengths of visible light and secondary dopants (POPOP) to shift the visible wavelengths to wavelengths that could be detected by Photomultiplier Tube (PMT) ) were carried out. Experiments were performed on the melting points condition of these materials, which had previously been subjected to a thermo-mechanical analysis using a Thermogravimetric Analysis-Differential Scanning Calorimetry (TGA-DSC) machine, where the melting point was in the range of 200-240°C. Furthermore, the fabrication was carried out using the extrusion technique, where polystyrene pellets mixed with PTP (1.5% by weight) and POPOP (0.05% by weight) were fed into an extrusion machine which has four hot areas to obtain a thin plate plastic scintillator. The plates were then analyzed with a UV-Vis Spectrophotometer to determine the absorption spectrum and Fluorescence Spectrophotometer to determine the emission spectrum. From the results of the analysis, it was found that the samples that went through scintillation pellets and without the addition of antioxidants had absorption spectrum data of 330 nm and emission spectrum of 421 nm. These values are in accordance with the characteristics of plastic scintillators on the market.

Identification and Characterization of Fluorescent Pseudomonas Producing Active Compounds Controlling Plant Pathogens

Fluorescent pseudomonad is one of the biocontrol agents against plant pathogens. Various compounds reportedly can be produced by fluorescent pseudomonad, including chitinase, β-1,3-glucanase, HCN, siderophore, antibiotics, Indole Acetic Acid (IAA), phosphate solvent compounds, and 2,4-diacetylphloroglucinol (DAPG). In this study, it was identified and characterized six isolates of fluorescent pseudomonad (PfPj1, PfPj2, PfKd7, PfCas, PfCas3, and LAHp2). All isolates were isolated from the rhizosphere of various types of plants. The results showed that six isolates were identical to Pseudomonas aeruginosa (93-94%). All bacterial isolates tested were able to produce siderophore, HCN, and solubilize phosphates. The highest siderophore was produced by isolate PfPj2. Whereas isolate PfKd7 had the highest at HCN production and the ability to dissolve phosphates.

Excitation-emission matrices and PARAFAC in the investigation of the bioactive compound effects from the flavoring process in olive oils

The evolution of fluorescent compounds during the thermal degradation of extra virgin and flavored olive oils were monitored using fluorescence spectroscopy (λex 280–400 nm, λem 300–700 nm), without the need for dilution at varying temperatures. The Parallel Factor Analysis (PARAFAC) was applied to this fluorescence data structured as cubic arrays, with the data showing the presence of five fluorophores in the flavored and extra virgin olive oil, where their evolution is dependent on the temperature. Here we show that the bioactive compounds from the flavoring process within olive oil, such as lemon, chili, pepper, and basil, can improve the olive oil’s overall oxidative stability when compared with the extra virgin samples, with the exception of garlic-flavoring.

Novel Antitumor Agents Based on Fluorescent Benzofurazan Derivatives and Mesoporous Silica

Two novel fluorescent mesoporous silica-based hybrid materials were obtained through the covalent grafting of [4-hydrazinyl-7-nitrobenz-[2,1,3-d]-oxadiazole (NBDH) and N1-(7-nitrobenzo[c][1,2,5]-oxadiazol-4-yl) benzene-1,2-diamine (NBD-PD), respectively, inside the channels of mesoporous silica SBA-15. The presence of fluorescent organic compounds (nitrobenzofurazan derivatives) was confirmed by infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), thermal analysis (TG), and fluorescence spectroscopy. The nitrogen physisorption analysis showed that the nitrobenzofurazan derivatives were distributed uniformly on the internal surface of SBA-15, the immobilization process having a negligible effect on the structure of the support. Their antioxidant activity was studied by measuring the ability to reduce free radicals DPPH (free radical scavenging activity), in order to formulate potential applications of the materials obtained. Cytotoxicity of the newly synthesized materials, SBA-NBDH and SBA-NBD-PD, was evaluated on human B16 melanoma cells. The morphology of these cells, internalization and localization of the investigated materials in melanoma and fibroblast cells were examined through fluorescence imaging. The viability of B16 (3D) spheroids after treatment with SBA-NBDH and SBA-NBD-PD was evaluated using MTS assay. The results showed that both materials induced a selective antiproliferative effect, reducing to various degrees the viability of melanoma cells. The observed effect was enhanced with increasing concentration. SBA-NBD-PD exhibited a higher antitumor effect compared to SBA-NBDH starting with a concentration of 125 µg/mL. In both cases, a significantly more pronounced antiproliferative effect on tumor cells compared to normal cells was observed. The viability of B16 spheroids dropped by 40% after treatment with SBA-NBDH and SBA-NBD-PD at 500 µg/mL concentration, indicating a clear cytotoxic effect of the tested compounds. These results suggest that both newly synthesized biomaterials could be promising antitumor agents for applications in cancer therapy.

Spectroscopic techniques for elucidation of structural changes in temperate cowpea cultivars under germination: A useful tool for quality determination and industrial application

The present study investigated the molecular changes induced by germination in cowpea cultivars by spectroscopic techniques. The ATR FT-IR, Intrinsic fluorescence, and UV spectroscopic techniques revealed that germination shows alteration in cowpea cultivars. The ATR-FTIR showed slight differences in band intensities corresponding to carbohydrates, lipids, and protein regions at 700 to 4000 cm-1 wavenumbers. Starch order structure understanding their functionality showed a reduction of short-range ordered structures in Black cowpea flour and long ordered starch fraction in White cowpea flour under germination. The study quantified the degree of ordered starch and their alteration in amorphous/crystallinity nature concerning germination and cowpea cultivars. The secondary structures revealed a reduction of β-sheets and α-helix content, representing decreased ordered protein structures and their shifting into random coils and turn structures. The germinated flours showed decreased fluorescence intensities with the shift from 347 to 348, reflecting the redshifts due to decreased concentrations of fluorescent compounds during germination in cowpea flour. UV spectrum revealed a similar spectrum with an increase in absorption of cowpea flour under germination. The electrophoresis showed variations in the intensities of high (25 to 225 kDa) and low (19 to 10 kDa) molecular weight proteins in germinated cowpea cultivars than the native one. Overall, this study demonstrated the potential of ATR-FTIR, fluorescence and UV-based spectroscopy as non-destructive techniques to assess the alteration of pulses at the molecular level and structural changes under the germination process.

Cascade reaction-based highly sensitive fluorescent sensing systems applicable for dual-pattern fluorescence visualizing of thiophenol flavors in meat products and condiments

Thiophenols (ArSHs) are widely used as popular flavoring ingredients for making daily dishes. Dissecting the ArSHs contents in common foodstuffs is meaningful in the field of food safety science. Herein, a novel small-molecule sensor 2-(1H-benzo[d]imidazol-2-yl)-3-(2-(2,4-dinitrophenoxy)-4-morpholinophenyl)acrylonitrile (NOSA) has been tailored. The NOSA is able to respond to ArSHs, spontaneously yielding highly green-emissive fluorescent iminocoumarin (I500). This cascade reaction-based strategy is sensitive (limit-of-detection = 2.8 nM), rapid (within 5 min), and selective toward ArSH flavors. Probe NOSA has been applied to the determination of ArSHs in real-life meat products and condiments. Moreover, a far-red fluorescent compound, 2-(7-(diethylamino)-4-(4-(methylthio)styryl)-2H-chromen-2-ylidene)malononitrile (CMMT), has been first combined with NOSA to construct a composite probe NOSA@CMMT for the ratiometric detection of ArSHs (I500/I630). System NOSA@CMMT exhibits a conspicuous fluorescence change from deep-red to light-green. Benefitted from the gorgeous chromatic fluctuation, a smartphone-integrated analysis platform is established for the real-time evaluation of ArSHs level.

Chirality Transfer from Polylactide to Achiral Fluorophore in Hierarchical Crystallization for Realizing Handedness-Tunable and Nonreciprocal Circularly Polarized Luminescence

Realizing chiral crystals from achiral compounds is an important but challenging issue. Here, a kebab-structured chiral supra-crystallization complex is constructed in accomplishing hierarchical chiral crystallization of achiral fluorescent compound (1,8-naphthalic anhydride, NA) employing synthetic chiral macromolecules (polylactide, PLA) as a template. Chirality transfer efficiently takes place from PLA to NA crystal during the co-crystallization process, endowing the initial achiral NA crystal with intense optical activity. More importantly, the obtained chiral supra-crystals show remarkable circularly polarized luminescence (CPL) properties, with the highest luminescence dissymmetric factor up to 5 × 10–2. Moreover, handedness-tunable and nonreciprocal CPL emissions are further realized in one single sample by simply flipping and rotating the supra-crystallization composite film, following a polarization mechanism. The work presents the first polymer-based hierarchical chiral supra-crystal with switchable CPL emissions and provides a simple and universal strategy for constructing nonreciprocal CPL materials.

Riboflavin as One of Possible Components of Keroplatus (Insecta: Diptera: Keroplatidae) Fungus Gnat Bioluminescence

Keroplatus is a genus of fungus gnats family Keroplatidae (Diptera, Bibionomorpha). Larvae of some species emit a constant blue light from the body. The bioluminescence of Keroplatidae is one of the least studied of all terrestrial insects and very few facts are known to date of its biology and biochemistry. Here we report the high level of riboflavin in Keroplatus testaceus larvae, a fluorescent compound that might be relative to its bioluminescent system. We suppose that riboflavin may play a role in Keroplatus spp. bioluminescence.

Detection of ampicillin based on the fluorescence of a biocatalytic oxidation product

Antibiotics used in humans and farmed animals are an essential source of water and soil contamination. Ampicillin is a micropollutant commonly found in water, sludge, food, flora, and fauna. However, the methods used for its detection in environmental samples are often complicated and expensive. Therefore, developing more straightforward strategies to detect well-known target antibiotics is necessary. In this context, enzyme-based detection methods have been demonstrated to be selective, sensitive, rapid, and relatively simple. In this study, a fluorescent byproduct from the ampicillin oxidation using Chloroperoxidase (CPO) enzyme was used as a pointer compound to determine ampicillin concentration in environmental water samples. We oxidized 80% ampicillin for 1h, producing a fluorescent compound with m/z 274.2517. A response surface methodology (RSM) based on a central composite design (CCD) was used to evaluate and optimize the effects of hydrogen peroxide, enzyme concentration, and time as independent variables on the maximum fluorescence signal as the response function. The methodology proposes to build a calibration curve that relates the initial concentration of ampicillin with fluorescence intensity after the reaction with CPO, which helps detect ampicillin in the concentration range from 0.035 to 40 μM, with a limit of detection of 0.026 μM. The application of the method to fortified environmental water samples allowed percentages of recovery from 86 to 140%. The formation of the fluorescent compound was not affected by the presence of salts commonly found in wastewater; however, it was affected by other antibiotics. The proposed methodology was tested in the context of water from water bodies, urban, and WWTP effluents.

A fluorescently labelled quaternary ammonium compound (NBD-DDA) to study resistance mechanisms in bacteria.

Quaternary ammonium compounds (QACs) are widely used as active agents in disinfectants, antiseptics, and preservatives. Despite being in use since the 1940s, there remain multiple open questions regarding their detailed mode-of-action and the mechanisms, including phenotypic heterogeneity, that can make bacteria less susceptible to QACs. To facilitate studies on resistance mechanisms towards QACs, we synthesized a fluorescent quaternary ammonium compound, namely N-dodecyl-N,N-dimethyl-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethyl]azanium-iodide (NBD-DDA). NBD-DDA is readily detected by flow cytometry and fluorescence microscopy with standard GFP/FITC-settings, making it suitable for molecular and single-cell studies. As a proof-of-concept, NBD-DDA was then used to investigate resistance mechanisms which can be heterogeneous among individual bacterial cells. Our results reveal that the antimicrobial activity of NBD-DDA against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa is comparable to that of benzalkonium chloride (BAC), a widely used QAC, and benzyl-dimethyl-dodecylammonium chloride (BAC12), a mono-constituent BAC with alkyl-chain length of 12 and high structural similarity to NBD-DDA. Characteristic time-kill kinetics and increased tolerance of a BAC tolerant E. coli strain against NBD-DDA suggest that the mode of action of NBD-DDA is similar to that of BAC. As revealed by confocal laser scanning microscopy (CLSM), NBD-DDA is preferentially localized to the cell envelope of E. coli, which is a primary target of BAC and other QACs. Leveraging these findings and NBD-DDA's fluorescent properties, we show that reduced cellular accumulation is responsible for the evolved BAC tolerance in the BAC tolerant E. coli strain and that NBD-DDA is subject to efflux mediated by TolC. Overall, NBD-DDA's antimicrobial activity, its fluorescent properties, and its ease of detection render it a powerful tool to study resistance mechanisms of QACs in bacteria and highlight its potential to gain detailed insights into its mode-of-action.

Highly Acidic Conditions Drastically Alter the Chemical Composition and Absorption Coefficient of α-Pinene Secondary Organic Aerosol.

Secondary organic aerosols (SOA), formed through the gas-phase oxidation of volatile organic compounds (VOCs), can reside in the atmosphere for many days. The formation of SOA takes place rapidly within hours after VOC emissions, but SOA can undergo much slower physical and chemical processes throughout their lifetime in the atmosphere. The acidity of atmospheric aerosols spans a wide range, with the most acidic particles having negative pH values, which can promote acid-catalyzed reactions. The goal of this work is to elucidate poorly understood mechanisms and rates of acid-catalyzed aging of mixtures of representative SOA compounds. SOA were generated by the ozonolysis of α-pinene in a continuous flow reactor and then collected using a foil substrate. SOA samples were extracted and aged by exposure to varying concentrations of aqueous H2SO4 for 1-2 days. Chemical analysis of fresh and aged samples was conducted using ultra-performance liquid chromatography coupled with photodiode array spectrophotomety and high-resolution mass spectrometry. In addition, UV-vis spectrophotometry and fluorescence spectrophotometry were used to examine the changes in optical properties before and after aging. We observed that SOA that aged in moderately acidic conditions (pH from 0 to 4) experienced small changes in composition, while SOA that aged in a highly acidic environment (pH from -1 to 0) experienced more dramatic changes in composition, including the formation of compounds containing sulfur. Additionally, at highly acidic conditions, light-absorbing and fluorescent compounds appeared, but their identities could not be ascertained due to their small relative abundance. This study shows that acidity is a major driver of SOA aging, resulting in a large change in the chemical composition and optical properties of aerosols in regions where high concentrations of H2SO4 persist, such as upper troposphere and lower stratosphere.

Selective fluorescent sensors for copper(II) ion from julolidine hydrazone derivatives

Three derivatives of N-acylhydrazone julolidine are synthesized and comparatively investigated for their selectivities as metal ion fluorescent sensors. The compound derived from picolinohydrazide is found to be a turn-on fluorescent sensor for Cu2+ in aqueous DMSO media. The mechanistic investigation suggests that Cu2+ promotes the hydrolysis reaction of picolinohydrazide moiety to generate a highly fluorescent compound julolidine-9-carboxaldehyde which has a maximum emission signal at 420 nm. This probe shows an extraordinary selectivity for Cu2+ over other metal ions with a detection limit of 0.1 ppm. Under the optimal conditions, the determinations of Cu2+ in real water samples are successfully executed.

Synthesis and Biological Characterization of Fluorescent Cyclipostins and Cyclophostin Analogues: New Insights for the Diagnosis of Mycobacterial-Related Diseases.

Patients with cystic fibrosis (CF) have a significantly higher risk of acquiring nontuberculous mycobacteria infections, predominantly due to Mycobacterium abscessus, than the healthy population. Because M. abscessus infections are a major cause of clinical decline and morbidity in CF patients, improving treatment and the detection of this mycobacterium in the context of a polymicrobial culture represents a critical component to better manage patient care. We report here the synthesis of fluorescent Dansyl derivatives of four active cyclipostins and cyclophostin analogues (CyCs) and provide new insights regarding the CyC's lack of activity against Gram-negative and Gram-positive bacteria, and above all into their mode of action against intramacrophagic M. abscessus cells. Our results pointed out that the intracellularly active CyC accumulate in acidic compartments within macrophage cells, that this accumulation appears to be essential for their delivery to mycobacteria-containing phagosomes, and consequently, for their antimicrobial effect against intracellular replicating M. abscessus, and that modification of such intracellular localization via disruption of endolysosomal pH strongly affects the CyC accumulation and efficacy. Moreover, we discovered that these fluorescent compounds could become efficient probes to specifically label mycobacterial species with high sensitivity, including M. abscessus in the presence several other pathogens like Pseudomonas aeruginosa and Staphylococcus aureus. Collectively, all present and previous data emphasized the therapeutic potential of unlabeled CyCs and the attractiveness of the fluorescent CyC as a potential new efficient diagnostic tool to be exploited in future diagnostic developments against mycobacterial-related infections, especially against M. abscessus.

Multi-Resonant Thermally Activated Delayed Fluorescent (MR-TADF) Compounds as Photocatalysts.

Donor-acceptor (D-A) thermally activated delayed fluorescent (TADF) compounds, such as 4CzIPN, have become a widely used sub-class of organic photocatalysts for a plethora of photocatalytic reactions. Multi-resonant TADF (MR-TADF) compounds, a subclass of TADF emitters that are rigid nanographene derivatives, such as DiKTa and Mes3 DiKTa, have to date not been explored as photocatalysts. In this study both DiKTa and Mes3 DiKTa were found to give comparable or better product yield than 4CzIPN in a range of photocatalytic processes that rely upon reductive quenching, oxidative quenching, energy transfer and dual photocatalytic processes. In a model oxidative quench process, DiKTa and Mes3 DiKTa gave increased reaction rates in comparison to 4CzIPN, with DiKTa being of particular interest due to the lower material cost (£0.94/mmol) compared to that of 4CzIPN (£3.26/mmol). These results suggest that DiKTa and Mes3 DiKTa would be excellent additions to any chemist's collection of photocatalysts.

A Smartphone-Based Low-Cost Inverted Laser Fluorescence Microscope for Disease Diagnosis.

Fluorescence microscopy is an important tool for disease diagnosis, often requiring costly optical components, such as fluorescence filter cubes and high-power light sources. Due to its high cost, conventional fluorescence microscopy cannot be fully exploited in low-income settings. Smartphone-based fluorescence microscopy becomes an interesting low-cost alternative, but raises challenges in the optical system. We present the development of a low-cost inverted laser fluorescence microscope that uses a smartphone to visualize the fluorescence image of biological samples. Our fluorescence microscope uses a laser-based simplified optical filter system that provides analog optical filtering capabilities of a fluorescence filter cube. Firstly, we validated our inverted optical filtering by visualizing microbeads labeled with three different fluorescent compounds or fluorophores commonly used for disease diagnosis. Secondly, we validated the disease diagnosis capabilities by comparing the results of our device with those of a commercial fluorescence microscope. We successfully detected and visualized Trypanosoma cruzi parasites, responsible for the Chagas infectious disease and the presence of Antineutrophil cytoplasmic antibodies of the ANCA non-communicable autoimmune disease. The samples were labeled with the fluorescein isothiocyanate (FITC) fluorophore, one of the most commonly used fluorophores for disease diagnosis. Our device provides a 400× magnification and is at least one order of magnitude cheaper than conventional commercial fluorescence microscopes.

Antioxidant Effect of Octopus Byproducts in Canned Horse Mackerel (Trachurus trachurus) Previously Subjected to Different Frozen Storage Times.

The effects on lipid damage in canned horse mackerel (Trachurus trachurus) of a prior frozen storage (-18 °C) period and the presence of an octopus (Octopus vulgaris) cooking juice (OCJ) in the packing medium were investigated. An increase of the frozen storage time favoured an increase (p < 0.05) of free fatty acid (FFA), peroxide, and thiobarbituric acid reactive substance contents and a decrease (p < 0.05) of the phospholipid (PL) value and polyene index. Furthermore, an increased presence of OCJ in the packing medium led to an inhibitory effect (p < 0.05) on fluorescent compound formation as well as to a retention (p < 0.05) of the PL and FFA compounds. Colour determination showed a substantial increase (p < 0.05) of L* and b* values in canned fish with previous frozen storage time. Nevertheless, this increase was partly reduced (p < 0.05) by the OCJ presence in the packing medium. It is concluded that previous holding time has led to an increased lipid oxidation development and loss of beneficial lipid constituents (i.e., PLs and polyunsaturated fatty acids). Remarkably, the presence in the packing medium of preservative compounds (i.e., antioxidants) included in waste juice obtained from octopus processing provided an effective tool for lipid preservation and quality enhancement in canned fish.

Structure–Property Relationship of Triscarbazole-diphenyltriazine-type Blue Thermally Activated Delayed Fluorescent Emitters

9-(4-(4,6-Diphenyl-1,3,5-triazin-2-yl)phenyl)-9′,9″-diphenyl-9H,9′H,9″H-3,3′:6′,3″-tercarbazole (1TCzTrz) and 9,9″-bis(4-(4,6-diphenyl-1,3,5-triazin-2-yl)phenyl)-9′-phenyl-9H,9′H,9″H-3,3′:6′,3″-tercarbazole (13TCzTrz) derived from a triscarbazole donor and a diphenyltriazine acceptor were developed as blue fluorophores to study the structure–property relationship in thermally activated delayed fluorescence compounds. 1TCzTrz had one acceptor unit attached to the triscarbazole donor, whereas 13TCzTrz had two acceptor units attached to the triscarbazole donor. The 1TCzTrz emitter worked efficiently relative to the 13TCzTrz emitter because of the high photoluminescence efficiency. The 1TCzTrz device delivered an external quantum efficiency of 23.4% compared with 19.6% of the 13TCzTrz device. However, the 13TCzTrz device was advantageous to increase horizontal emitting dipole orientation.

Donor-Acceptor Type of Fused-Ring Thermally Activated Delayed Fluorescence Compounds Constructed through an Oxygen-Containing Six-Membered Ring.

Nowadays, thermally activated delayed fluorescence (TADF) compounds with a fused-ring core skeleton are getting increasing research interest because of their use in high-performance organic light-emitting diodes (OLEDs). In this study, TADF compounds featuring a D-A-type fused-ring core skeleton are developed. The challenging compatibility of a planarized D-A arrangement and the TADF property is achieved through linking the D and A moieties with two oxygen atoms within a six-membered ring. Compared with a single-oxygen analogue possessing a flexible skeleton and a twisted D-A arrangement, these fused-ring compounds with higher skeleton rigidity show higher photoluminescence quantum yields and narrower emission spectra in toluene and in doped thin films. Their electroluminescent devices achieve high external quantum efficiencies (up to 19.4%), suggesting the potential of rarely achieved D-A-type fused-ring TADF systems to serve as high-performance emitters of OLEDs.

Transit Time index (TTi) as an adaptation of the humification index to illustrate transit time differences in karst hydrosystems: application to the karst springs of the Fontaine de Vaucluse system (southeastern France)

Abstract. Transit time can be estimated thanks to natural tracers, but few of them are usable in the 0–6-month range. The main purpose of this work is to analyze the potential of the ratio of heavy- to light-weight organic compounds (the humification index (HIX); Ohno, 2002; Zsolnay et al., 1999) as a natural tracer of short transit time (Blondel et al., 2012). Critical analysis of former studies shows that although the link between HIX and transit time seems consistent, the whole methodological approach needs to be consolidated. Natural organic matter fluorescence from 289 groundwater samples from four springs and 10 flow points located in the unsaturated zone of the Vaucluse karst system is characterized by parallel factor analysis (PARAFAC) thanks to the excitation–emission matrix (EEM), thus (i) allowing for the identification of main fluorescent compounds of sampled groundwater and (ii) evidencing the inadequacy of HIX 2D emission windows to characterize groundwater organic matter. We then propose a new humification index called the Transit Time index (TTi) based on the Ohno (2002) formula but using PARAFAC components of heavy and light organic matter from our samples instead of 2D windows. Finally, we evaluate TTi relevance as a transit time tracer by (i) performing a detailed analysis of its dynamics on a selected spring (Millet) and (ii) comparing its mean value over karst springs of the Vaucluse karst system. Principal component analysis (PCA) of TTi and other hydrochemical parameters monitored at Millet spring put in relief the different ranges of transit time associated with the different organic matter compounds. PCA results also provide evidence that TTi can detect a small proportion of fast infiltration water within a mix, while other natural tracers of transit time provide no or less sensitive information. TTi distributions at monitored karst springs are consistent with relative transit times expected for the small-scale, short average transit time systems. TTi thus appears as a relevant qualitative tracer of transit time in the 0–6-month range where existing tracers fail and may remain applicable, even in the case of anthropic contamination thanks to PARAFAC modeling. Transforming it into quantitative information is a challenging task which may be possible thanks to intensive studies of organic matter degradation kinetics in natural waters with the help of radiogenic isotope usage or an artificial tracer test.