Fluorophore Fluorescein Research Articles

BPA Endocrine Disruptor Detection at the Cutting Edge: FPIA and ELISA Immunoassays.

BPA is a chemical commonly used in the production of polymer-based materials that can have detrimental effects on the thyroid gland and impact human reproductive health. Various expensive methods, such as liquid and gas chromatography, have been suggested for detecting BPA. The fluorescence polarization immunoassay (FPIA) is an inexpensive and efficient homogeneous mix-and-read method that allows for high-throughput screening. FPIA offers high specificity and sensitivity and can be carried out in a single phase within a timeframe of 20-30 min. In this study, new tracer molecules were designed that linked the fluorescein fluorophore with and without a spacer to the bisphenol A moiety. To assess the influence of the C6 spacer on the sensitivity of an assay based on the respective antibody, hapten-protein conjugates were synthesized and assessed for performance in an ELISA setup, and this resulted in a highly sensitive assay with a detection limit of 0.05 g/L. The lowest limit of detection was reached by employing the spacer derivate in the FPIA and was 1.0 μg/L, working range from 2 to 155 μg/L. The validation of the methods was conducted using actual samples compared to LC-MS/MS, which served as the reference method. The FPIA and ELISA both demonstrated satisfactory concordance.

Comparative Study of Intraoperative Fluorescein and Indocyanine Green Videoangiography for Ruptured Cerebral Aneurysms Clipping: A Single Centre Study of 30 Cases.

Aim This study assesses the application of microscope integrated videoangiography techniques in aneurysm clipping surgery using Indocyanine Green and Fluorescein fluorophores and evaluates merits and demerits of each technique. Materials and Methods Total 30 patients of cerebral aneurysmal clipping were included. Standard microsurgical procedures were done. After clipping, we administered a 25 mg bolus intravenous dose of indocyanine green with microscope focused through the INFRARED 800 camera module, followed by administration of 60 mg bolus intravenous dose of fluorescein with microscope focused through the yellow 560 module and images were assessed. Results The average aneurysm size was 17 mm. In 12 patients (40%), FL-VA allowed better assessment of perforating arteries (seven cases) or distal branches (three cases) or both (two cases), when compared with ICG-VA. In one case of MCA (M1) aneurysm, ICG-VA showed no fluorescent signal in one of the distal trunks whereas FL-VA showed normal signal. In one case of ACOM aneurysm, perforators were missed on ICG-VA but were seen on FL-VA. FL-VA was able to identify inadequate aneurysm clipping in one case. In two patients, FL-VA provided the advantage of real-time manipulation of the vessels to expose the vessels and aneurysms of interest. Fluorescein detected all the perforators that were visible under white light (68/68) whereas ICG was able to detect 56 (82.35%) perforators ( p -value< 0.05). Conclusion Intraoperative ICG and Fluorescein videoangiography recognize inadequate occlusion of aneurysm, decreased flow in branches or perforators. When various study parameters were considered such as ability to assess small size perforators, branching vessels, adequacy of aneurysmal clipping, and useful information on repeat imaging, FL-VA was found superior to ICG-VA.

Fluorescence dequenching assay for the activity of TEV protease

Tobacco etch virus (TEV) protease is a widely used protease for fusion tag cleavage. Despite its widespread usage, an assay to quickly and easily quantify its activity in laboratory settings is still lacking. Thus, researchers may encounter inefficient cleavage of the desired fusion proteins due to poor activity of a given TEV protease preparation. Here, we describe the development and implementation of a fluorescence dequenching-based assay to quantify TEV protease activity and assess kinetic parameters. The peptide substrate used in this assay consists of a C-terminal TAMRA fluorophore, an N-terminal fluorescein fluorophore, and the canonical TEV protease recognition sequence. The assay is based on a reduction of fluorescence quenching of fluorescein upon cleavage by TEV protease. The substrate peptide was studied spectroscopically to assess feasibility and to propose a plausible mechanism of the assay. The assay was optimized and applied to obtain rapid assessments of TEV protease activity in purified samples and crude lysate extracts. The kinetic data obtained from improved TEV protease variants were compared with a traditional SDS-PAGE assay. Finally, the assay was applied to determine the optimum pH for TEV protease. Further, the study found that the assay is a rapid and simple approach to quantify TEV protease activity. The findings of the assay on crude lysate extracts, activity assay of TEV protease variants, and assessment of optimum pH for TEV protease reactions demonstrate the robust utility of the assay.

Detection of ochratoxin A using a "turn-on" fluorescence assay based on guanine quenching of the aptamer.

Ochratoxin A (OTA) is a common mycotoxin with high carcinogenicity; therefore, it is crucial to establish a simple, rapid, and sensitive method for its detection. In this study, we developed a “turn-on” fluorescence assay for detecting OTA based on guanine quenching of the aptamer. The method uses fluorescein (FAM) fluorophore to label the complementary strand of the OTA aptamer, Fc-DNA. In the absence of OTA, the Fc-DNA hybridizes with the aptamer to form a double strand. Due to the occurrence of photo-induced electron transfer (PET), the FAM fluorescence signal is quenched as the FAM on the Fc-DNA approaches the guanine of the aptamer at the 5′ end. When OTA is present, the aptamer binds to it and thus, is unable to hybridize with Fc-DNA to form a double strand; the FAM fluorescence signal is restored as FAM moves away from the guanine of the aptamer. The assay achieved OTA detection at a detection limit of 28.4 nM. The application of the original guanine of the aptamer as the quenching agent helps avoid the complex designing and labeling of the aptamer, which ensures the high affinity of the aptamer for OTA. Meanwhile, this “turn-on” detection mode helps avoid potential false-positive results as in the “turn-off” mode and improves the assay’s sensitivity. Additionally, the method has good selectivity and can be used to detect OTA in traditional Chinese medicine. This method provides a simple, low-cost, and rapid method for OTA detection.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s44211-022-00199-z.

Design, fabrication and testing of 3D printed smartphone-based device for collection of intrinsic fluorescence from human cervix

Fluorescence spectroscopy has the potential to identify discriminatory signatures, crucial for early diagnosis of cervical cancer. We demonstrate here the design, fabrication and testing of a 3D printed smartphone based spectroscopic device. Polarized fluorescence and elastic scattering spectra are captured through the device using a 405 nm laser and a white LED source respectively. The device has been calibrated by comparison of spectra of standard fluorophores (Flavin adenine dinucleotide, fluorescein, rhodamine, and porphyrin) with the corresponding spectra collected from a commercial spectrometer. A few cervical tissue spectra have also been captured for proof of its applicability as a portable, standalone device for the collection of intrinsic fluorescence spectra from human cervix.

Development of Fluorescent Aptasensors Based on G-Quadruplex Quenching Ability for Ochratoxin A and Potassium Ions Detection.

G-quadruplexes have received significant attention in aptasensing due to their structural polymorphisms and unique binding properties. In this work, we exploited the fluorescence-quenching properties of G-quadruplex to develop a simple, fast, and sensitive platform for fluorescence detection of ochratoxin A (OTA) and potassium ions (K+) with a label-free fluorophore and quencher strategy. The quenching ability of G-quadruplex was confirmed during the recognition process after the formation of the G-quadruplex structure and the quenching of the labeled fluorescein fluorophore (FAM). The fluorescence-quenching mechanism was studied by introducing specific ligands of G-quadruplex to enhance the quenching effect, to show that this phenomenon is due to photo-induced electron transfer. The proposed fluorescence sensor based on G-quadruplex quenching showed excellent selectivity with a low detection limit of 0.19 nM and 0.24 µM for OTA and K+, respectively. Moreover, we demonstrated that our detection method enables accurate concentration determination of real samples with the prospect of practical application. Therefore, G-quadruplexes can be excellent candidates as quenchers, and the strategy implemented in the study can be extended to an aptasensor with G-quadruplex.

One-Step Assembly of Fluorescence-Based Cyanide Sensors from Inexpensive, Off-The-Shelf Materials.

We report a simple and versatile approach to assemble sensitive and selective fluorescence “turn-on” sensors for cyanide by combining three off-the-shelf materials; namely fluorescent dye, 1-vinyl imidazole polymer, and cupric chloride. The cyanide-sensing species is a non-fluorescent fluorophore-polymer-Cu2+ complex; which forms as a result of the imidazole polymer’s ability to bind both fluorophore and fluorescence quencher (Cu2+). Cyanide removes Cu2+ from these complexes; thereby “turning-on” sensor fluorescence. These sensors are water-soluble and have a detection limit of ~2.5 μM (CN−) in water. Our ternary complex-based sensing approach also enables facile emission tuning; we demonstrate the convenient, synthesis-free preparation of blue and green-emitting sensors using distyrylbiphenyl and fluorescein fluorophores, respectively. Furthermore; these ternary complexes are easily immobilized using agarose to create cyanide-sensing hydrogels; which are then used in a simple; novel microdiffusion apparatus to achieve interference-free cyanide analysis of aqueous media. The present study provides an inexpensive approach for portable; interference-free cyanide detection.

Efficient Polymer Pendant Approach toward High Stable Organic Fluorophore for Sensing Ultratrace Hg2+ with Improved Biological Compatibility and Cell Permeability.

A convenient and efficient method to eliminate the aggregation effect of organic photoelectric sensing materials and to improve biological compatibility and cell permeability as well was developed by hanging organic fluorophores on a polymer chain, for example, fluorescein fluorophores had been controllably hung on polyacrylamide main chains with a 1:2 stoichiometric ratio by a simple copolymerization strategy. The results showed that introduction of water-soluble bioactive polyacrylamide main chains into fluorescein fluorophores via covalent bonds could effectively improve their optical stability by deteriorating π-π stack and charge-transfer interactions among different fluorophores. More importantly, the resultant materials possessed low toxicity and excellent cell permeability ten times larger than their precursor fluorescein fluorophore, which made it express an especially turn-on fluorescent response to ultratrace Hg2+ both in aqueous and living cells by forming stable 5-member-ring complexes with Hg2+ with a correlation coefficient of 0.997 and a low detection limit of 4.0 × 10-10 mol·L-1. This work provides promising insight into constructing some practical sensing materials for environmentally-friendly biological analyses.

Silica nanoparticles with thermally activated delayed fluorescence for live cell imaging.

Thermally activated delayed fluorescence (TADF) has revolutionized the field of organic light emitting diodes owing to the possibility of harvesting non-emissive triplet states and converting them in emissive singlet states. This mechanism generates a long-lived delayed fluorescence component which can also be used in sensing oxygen concentration, measuring local temperature, or on imaging. Despite this strong potential, only recently TADF has emerged as a powerful tool to develop metal-free long-lived luminescent probes for imaging and sensing. The application of TADF molecules in aqueous and/or biological media requires specific structural features that allow complexation with biomolecules or enable emission in the aggregated state, in order to retain the delayed fluorescence that is characteristic of these compounds. Herein we demonstrate a facile method that maintains the optical properties of solvated dyes by dispersing TADF molecules in nanoparticles. TADF dye-doped silica nanoparticles are prepared using a modified fluorescein fluorophore. However, the strategy can be used with many other TADF dyes. The covalent grafting of the TADF emitter into the inorganic matrix effectively preserves and transfers the optical properties of the free dye into the luminescent nanomaterials. Importantly, the silica matrix is efficient in shielding the dye from solvent polarity effects and increases delayed fluorescence lifetime. The prepared nanoparticles are effectively internalized by human cells, even at low incubation concentrations, localizing primarily in the cytosol, enabling fluorescence microscopy imaging at low dye concentrations.

Ratiometric assay of CARM1 activity using a FRET-based fluorescent probe.

One of the regulatory mechanisms of epigenetic gene expression is the post-translational methylation of arginine residues, which is catalyzed by protein arginine methyltransferases (PRMTs). Abnormal expression of PRMT4/CARM1, one of the PRMTs, is associated with various diseases, including cancers. Here, we designed and synthesized a Förster resonance energy transfer (FRET)-based probe, FRC, which contains coumarin and fluorescein fluorophores at the N-terminus and C-terminus of a peptide containing an arginine residue within an appropriate amino acid sequence to serve as a substrate of CARM1; the two fluorophores act as a FRET donor and a FRET acceptor, respectively. Since trypsin specifically hydrolyzes the arginine residue, but not a monomethylarginine or dimethylarginine residue, CARM1 activity can be evaluated from the change of the coumarin/fluorescein fluorescence ratio of FRC in the presence of trypsin.

Octachloro-fluorescein: Synthesis and photosensitizer performance evaluation

Fluorescence image-guided photodynamic therapy (PDT) receives great attention since it provides both the diagnostic and therapeutic information. Theoretically, fluorescence and the photodynamic performance (singlet oxygen) can be traced back to the same origin, namely the Jablonski energy diagram. Therefore, designing photosensitizers with balanced fluorescence and singlet oxygen generation is essentially important. Heavy atom effect (HAE) is an effective approach for designing highly efficient photosensitizers, but at the sacrifice of fluorescence. Herein, through analysis of the well-known fluorophore fluorescein and the photosensitizer Rose Bengal (RB), a new kind of photosensitizer, octachloro-fluorescein (OCF), was designed and synthesized. Structurally, OCF was obtained through anchoring chlorine atoms (heavier than H but lighter than I) onto all the existing open sites of fluorescein, resting in a 5-fold singlet oxygen quantum yield increase over fluorescein, but a much higher fluorescence quantum yield (Φfl = 0.628) over RB. The photodynamic performance of OCF was characterized both photophysically and photochemically. Besides, the image-guided PDT performance of OCF was further evaluated with HeLa cells, demonstrating that elegant heavy atom substitution of fluorescein may provide an effective way of balancing the fluorescence and singlet oxygen generation.

Ultrasensitive fluorescence detection of Fe3+ ions using fluorescein isothiocyanate functionalized Ag/SiO2/SiO2 core–shell nanocomposites

Fluorescein isothiocyanate (FITC) functionalized Ag/SiO2/SiO2 core–shell (CS) nanoparticles (NPs) and nanorods (NRs) are synthesized by chemical method to perform the fluorescence detection of Fe3+ in aqueous medium. The Fe3+ ion sensing is performed on the basis of metal-enhanced fluorescence and subsequent fluorescence quenching of fluorophore (fluorescein isothiocyanate) in aqueous medium. The intensity of fluorescence of FITC is found to get enhanced with increasing the silica shell thickness. The fluorescence intensity of FITC of spherical Ag/SiO2/SiO2 (FITC) CS NPs (AgSiF50) is amplified up to 2.25-folds whereas, in case of Ag/SiO2/SiO2 (FITC) CS NRs (AgSiFNR), the fluorescence intensity of FITC is amplified up to 3.63-folds compared to that of pure FITC. Owing to the amplified fluorescence of FITC, AgSiF50 and AgSiFNR samples are evaluated for detection of Fe3+ in an aqueous medium and the proposed nanoprobes exhibit good sensitivity towards Fe3+ detection because of strong binding affinity of Fe3+ with isothiocyanate group of FITC. The limit of detections for AgSiF50 and AgSiFNR are found to be 19.4 nM and 0.8 nM, respectively. The BET surface areas of AgSiF50 and AgSiFNR are calculated to be 157.64 m2 g−1 and 193.42 m2 g−1, respectively. The higher sensitivity of AgSiFNR compared to AgSiF50 for Fe3+ ion sensing is accredited to the higher adsorption capacity and higher fluorescence of FITC. This Ag/SiO2/SiO2 (FITC) CS NRs probe is a very simple and efficient sensor for Fe3+ ion detection and thus, using this sample, a very simple Arduino based electronic device is fabricated which can give real-time application of the idea.

Highly Efficient, Chemically Stable, and UV/Blue-Light-Excitable Biluminescent Security Ink to Combat Counterfeiting.

A strategy has been demonstrated to design a biluminescent security ink using Eu(TTA)3Phen (ETP) and fluorescein for protecting the currency and other essential documents, viz., passport, bank check, certificates, etc. against counterfeiting. The biluminescent security ink exhibits strong red and green emission under 367 and 445 nm excitations, respectively. As it is quite challenging to prepare a material that possesses two prominent (green and red) and distinguishable colors upon excitation with two separate light-emitting diode (LED) sources, emitting at different wavelengths, the biluminescent security ink would be hard to counterfeit as compared with the existing luminescent security ink that exhibits single color under UV light exposure. To check its feasibility for security application, the patterns printed out using the biluminescent security ink were kept under a hot and humid atmosphere for 150 days. Also, the ETP and fluorescein fluorophores were exposed to UV light for a prolonged time, which do not show any sign of deterioration in their luminescence intensities. Furthermore, to check their chemical stability, printed patterns were also exposed to chemicals that have potential to wipe out ink, viz., detergent, ethanol, acetone, and sodium hypochlorite (bleach) solution, and it was noticed that it is well stable against these chemicals. Because of the reasons mentioned above and easy availability of 367 and 445 nm LEDs at low cost, authors believe that the application of this biluminescent security ink can trigger the realization of the full potential of this advanced security feature in detecting fake currency.

In vitro and in vivo accumulation of magnetic nanoporous silica nanoparticles on implant materials with different magnetic properties

BackgroundIn orthopedic surgery, implant-associated infections are still a major problem. For the improvement of the selective therapy in the infection area, magnetic nanoparticles as drug carriers are promising when used in combination with magnetizable implants and an externally applied magnetic field. These implants principally increase the strength of the magnetic field resulting in an enhanced accumulation of the drug loaded particles in the target area and therewith a reduction of the needed amount and the risk of undesirable side effects. In the present study magnetic nanoporous silica core–shell nanoparticles, modified with fluorophores (fluorescein isothiocyanate/FITC or rhodamine B isothiocyanate/RITC) and poly(ethylene glycol) (PEG), were used in combination with metallic plates of different magnetic properties and with a magnetic field. In vitro and in vivo experiments were performed to investigate particle accumulation and retention and their biocompatibility.ResultsSpherical magnetic silica core–shell nanoparticles with reproducible superparamagnetic behavior and high porosity were synthesized. Based on in vitro proliferation and viability tests the modification with organic fluorophores and PEG led to highly biocompatible fluorescent particles, and good dispersibility. In a circular tube system martensitic steel 1.4112 showed superior accumulation and retention of the magnetic particles in comparison to ferritic steel 1.4521 and a Ti90Al6V4 control. In vivo tests in a mouse model where the nanoparticles were injected subcutaneously showed the good biocompatibility of the magnetic silica nanoparticles and their accumulation on the surface of a metallic plate, which had been implanted before, and in the surrounding tissue.ConclusionWith their superparamagnetic properties and their high porosity, multifunctional magnetic nanoporous silica nanoparticles are ideal candidates as drug carriers. In combination with their good biocompatibility in vitro, they have ideal properties for an implant directed magnetic drug targeting. Missing adverse clinical and histological effects proved the good biocompatibility in vivo. Accumulation and retention of the nanoparticles could be influenced by the magnetic properties of the implanted plates; a remanent martensitic steel plate significantly improved both values in vitro. Therefore, the use of magnetizable implant materials in combination with the magnetic nanoparticles has promising potential for the selective treatment of implant-associated infections.

Optimization of an Anti-NMDA Receptor Autoantibody Diagnostic Bioassay

Anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is one of the most frequently encountered autoimmune encephalitis. The pathogenesis of both anti-NMDAR encephalitis and schizophrenia involve down-regulation of NMDA receptors. Whether autoantibody-mediated destruction of neuronal NMDA receptors is associated with schizophrenia or first-episode psychosis (FEP) remains unclear, as the current findings from different groups are inconsistent. The main culprits are likely due to heterogeneity of autoantibodies (autoAbs) in a patient's blood or cerebrospinal fluid (CSF), as well as due to limitation of the current detection methods for anti-NMDAR autoAbs. Here, we optimized the current diagnostic method based on the only commercially-available anti-NMDAR test kit. We first increased detection sensitivity by replacing reporter fluorophore fluorescein isothiocyanate (FITC) in the kit with Alexa Fluor 488, which is superior in resisting photobleaching. We also found that using an advanced imaging system could increase the detection limit, compared to using a simple fluorescence microscope. To improve test accuracy, we implemented secondary labeling with a well-characterized mouse anti-NR1 monoclonal antibody (mAb) after immunostaining with a patient's sample. The degree of colocalization between mouse and human antisera in NMDAR-expressing cells served to validate test results to be truly anti-NMDAR positive or false-positive. We also incorporated DNA-specific DAPI to simultaneously differentiate autoAbs targeting the plasma membrane from those targeting cell nuclei or perinuclear compartments. All the technical implementation could be integrated in a general hospital laboratory setting, without the need of specialized expertise or equipment. By sharing our experience, we hope this may help improve sensitivity and accuracy of the mainstream method for anti-NMDAR detection.

Testing Fluorescence Lifetime Standards using Two-Photon Excitation and Time-Domain Instrumentation: Fluorescein, Quinine Sulfate and Green Fluorescent Protein

It is essential for everyone working with experimental science to be certain that their instruments produce reliable results, and for fluorescence lifetime experiments, information about fluorescence lifetime standards is crucial. A large part of the literature on lifetime standards dates back to the 1970s and 1980s, and the use of newer and faster measuring devices may deem these results unreliable. We have tested the three commonly used fluorophores fluorescein, quinine sulfate and green fluorescent protein for their suitability to serve as lifetime standards, especially to be used with two-photon excitation measurements in the time-domain. We measured absorption and emission spectra for the fluorophores to determine optimal wavelengths to use for excitation and detector settings. Fluorescence lifetimes were measured for different concentrations, ranging from 10− 3 − 10− 5 M, as well as for various solvents. Fluorescein was soluble in both ethanol, methanol and sulfuric acid, while quinine sulfate was only soluble in sulfuric acid. Green fluorescent protein was prepared in a commercial Tris-HCl, EDTA solution, and all three fluorophores produced stable lifetime results with low uncertainties. No siginificant variation with concentration was measured for any of the fluorophores, and all showed single-exponential decays. All lifetime measurements were carried out using two-photon excitation and lifetime data was obtained in the time-domain using time-correlated single-photon counting.

Synthesis of novel fluorescently labeled water-soluble fullerenes and their application to its cellar uptake and distribution properties

Fullerene is a well-known carbon nanomaterial, which can be potentially used for drug manufacture or delivery. Despite several successful examples of utilizing fullerene derivatives as drug candidate materials, their low water solubility under physiological conditions negatively affects the cell penetration efficiency after treatment. In this work, we successfully synthesized two fullerene derivatives with covalently attached fluorescein and boron dipyrromethene (BODIPY) fluorophore moieties, which exhibited cellular uptake and intracellular localization. While both fluorophores decreased their fluorescence intensity in the vicinity of fullerene, the cellar uptake of the fluorescein-modified fullerene was detected via fluorescence microscopy observations. Moreover, decreases in the fluorescence intensities of the intact fluorescein and BODIPY species were observed when both fluorophores and fullerene coexisted in aqueous media.

In vitro and in vivo assessment of delivery of hydrophobic molecules and plasmid DNAs with PEO–PPO–PEO polymeric micelles on cornea

The stability and bio-distribution of genes or drug complexes with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO–PPO–PEO, Pluronic F-68) polymeric micelles (PM) are essential for an effective nanosized PM delivery system. We used Förster resonance energy transfer (FRET) pairs with PM and measured the FRET ratio to assess the stability of PM in vitro and in vivo on the cornea. The FRET ratio reached a plateau at 0.8 with 3% PM. Differential scanning calorimetry measurement confirmed the complex formation of FRET pairs with PM. Confocal imaging with the fluorophores fluorescein isothiocyanate isomer I (FITC) and rhodamine B base (RhB) also showed the occurrence of FRET pairs in vitro. The fluorophores were mixed with 3% PM solution or the FITC-labeled PEO–PPO–PEO polymers (FITC-P) were mixed with RhB-labeled plasmids (RhB–DNA). In addition, the in vitro corneal permeation of FRET pair complexes with PM reached a 0.8 FRET ratio. One hour after eye drop administration, FRET pairs colocalized in the cytoplasm, and surrounded and entered the nuclei of cells in the cornea, and the polymers were located in the corneal epithelial layers, as detected through anti-PEG immunohistochemistry. Furthermore, fluorescence colocalization in the cytoplasm and cell nucleus of the corneal epithelium was confirmed in tissues where RhB or RhB–DNA complexed with FITC-P was found to accumulate. We demonstrate that at a concentration of 3%, PM can encapsulate FRET pairs or RhB–DNA and retain their integrity within the cornea 1 h after administration, suggesting the feasibility and stability of PEO–PPO–PEO polymers as a vehicle for drug delivery.

Coupling of Molecular Transition with the Surface Plasmon Resonance of Silver Nanoparticles inside the Restricted Environment of Reverse Micelles.

Interaction of molecular transitions of two fluorophores—fluorescein (FL) and safranin O (SAF)—with the surface plasmon resonance (SPR) of silver nanoparticles (AgNPs) inside a water/sodium dioctylsulfosuccinate (AOT)/n-heptane reverse micelle (RM) has been studied using ultraviolet–visible and fluorescence spectroscopies. Here, we exploit the natural capacity of a RM to act simultaneously as a template for nanoparticle formation and host the fluorophores. The fluorophores and reducing agent were loaded together into the water pool; thereafter, silver salt was added, and subsequently, spectral modification and size evolution were monitored by steady-state and time-resolved optical spectroscopy. In the FL–AgNP composite, the SPR band of AgNPs undergoes a strong red shift. Moreover, significant modifications of both the fluorescence intensity and lifetime of FL were found when AgNPs formed inside the RM core. On the contrary, in the SAF–AgNP composite, no such effect was noticed, and the composite system retains the original optical characteristics of their constituents (i.e., both the position of molecular transitions and SPR maximum remain unchanged). This differential effect has been rationalized by the dissimilar plasmon–fluorophore coupling in the two systems, controlled by a combination of spatial distribution and spectral detuning of the molecular absorption maxima of the dyes (455 and 530 nm for FL and SAF, respectively) from the SPR band maximum (∼400 nm) of AgNPs.

Ratiometric imaging of mitochondrial pH in living cells with a colorimetric fluorescent probe based on fluorescein derivative

Mitochondrial pH plays a pivotal role in the regulation of physiological process. Developing ratiometric fluorescent probes for real-time detection of mitochondrial pH fluctuation is still highly demanded yet challenging. Herein, we present a novel strategy to design a ratiometric probe (FDI) by broadening absorption spectrum (360–700nm) of fluorescein fluorophore. Unsaturated dialkene/indole quaternary ammonium moiety has broadened absorption and emission spectrum of fluorescein fluorophore to provide ratiometric detection by CLSM in dual excitation/dual emission mode, as well as directionally accumulated in mitochondria in living cells. Superior to commercially mitochondrial tracker, Carboxy-SNARF, probe FDI exhibits excellent and fast cell-membrane permeability and mitochondrial-targetability. It has been demonstrated that FDI could permit real-time monitoring of pH alkalization of mitochondria stimulated by chloroquine. Owing to nondestructive process and reversible ratiometric response to pH, image acquisition can be repeated frequently to trace and monitor the time course of mitochondrial pH responses. It is clearly confirmed that FDI would be a promising probe for real-time tracking of mitochondrial pH changes in the biomedical and biological fields.