Loss Of Fluorescence Intensity Research Articles

Ultra-low level detection of biomarker bilirubin by graphene quantum dots and bovine serum albumin enabled turn-on sensing

It is known that concentration of unbound (free) bilirubin (BR) instead of the total bilirubin is the critical determinant of cellular uptake and toxicity of bilirubin. Thus, developing fast, effective and sensitive technique for detecting BR, a unique and important biomarker of liver dysfunction, has a long-standing demand. In this study, attempts were made to explore if biocompatible graphene quantum dots (GQDs) could be employed to probe BR at an ultra-low level in aqueous environment through optical techniques. Thus, understanding the possible interactions involved between GQDs and BR is important and necessary to develop protocol for probing BR. The detailed spectral analysis shows that BR molecule quenches the fluorescence of GQDs which may be attributed to a possible fluorescence resonance energy transfer (FRET) involved between the excited state of GQDs and BR. The micro-calorimetric and circular dichroic (CD) measurements have provided binding and structural information in the process of interactions. It is established that with the help of synthesized GQDs, BR can be detected as low as 0.15 nM selectively and this appears to be the most sensitive amongst other available methods. Interestingly, it is observed that the addition of bovine serum albumin (BSA) facilitates the recovery of the BR-induced loss in fluorescence intensity of GQDs. The adopted strategy demonstrates that GQD may act as a sensitive fluorescent “turn off–on” probe for detection of BR. The proposed optical method based protocol validated with serum samples isolated from blood for rapid detection of BR at ultra-low level may help in developing a suitable sensing device in future.

The complexometric behavior of selected aroyl-S,N-ketene acetals shows that they are more than AIEgens

Using the established synthetic methods, aroyl-S,N-ketene acetals and subsequent bi- and multichromophores can be readily synthesized. Aside from pronounced AIE (aggregation induced emission) properties, these selected examples possess distinct complexometric behavior for various metals purely based on the underlying structural motifs. This affects the fluorescence properties of the materials which can be readily exploited for metal ion detection and for the formation of different metal-aroyl-S,N-ketene acetal complexes that were confirmed by Job plot analysis. In particular, gold(I), iron(III), and ruthenium (III) ions reveal complexation enhanced or quenched emission. For most dyes, weakly coodinating complexes were observed, only in case of a phenanthroline aroyl-S,N-ketene acetal multichromophore, measurements indicate the formation of a strongly coordinating complex. For this multichromophore, the complexation results in a loss of fluorescence intensity whereas for dimethylamino-aroyl-S,N-ketene acetals and bipyridine bichromophores, the observed quantum yield is nearly tripled upon complexation. Even if no stable complexes are formed, changes in absorption and emission properties allow for a simple ion detection.

Mechanistic insights into α-amylase inhibition, binding affinity and structural changes upon interaction with gallic acid

The inhibitory mechanism of gallic acid (GA) on α-amylase was investigated through enzymatic inhibition assays, multi-spectroscopy, Job plot analysis and in-silico molecular docking. The results reveal that GA competitively binds to the active site of α-amylase in a 1:1 stoichiometry, dominantly via four hydrogen bonds and one hydrophobic interaction. The α-amylase-GA complex showed an increased thermal stabilisation, accompanied by a rise in α-helical and β-sheet components, resulting in a partial folding of the protein structure. The compact structure prevents substrate binding and inhibits enzyme activity. The self-quenching ability of GA was considered in the intrinsic fluorescence analysis, revealing a distinct loss in fluorescence intensity with a blueshift in the α-amylase-GA complex spectra. This observation suggests a direct interaction between GA and tryptophan amino acid residues, as the residues move to a more hydrophobic environment. Findings in this study provide insights into the interaction mechanism between GA and α-amylase, which will aid in understanding the inhibitory mechanism of their complex forms found in nature.

Rational design of dual-modalitye peptide-based probe for detection of Cu(II) and l-histidine in 100% aqueous solution and its application for living cells, test strips and smartphone

A new fluorometric and colorimetric dual-modalitye probe L for detection of Cu2+ and l-His was constructed based on peptide skeleton via solid phase peptide synthesis (SPPS) technology. L exhibited highly selective recognition towards Cu2+ through formation of L-Cu2+ ensemble with a 2:1 binding stoichiometry, and the limit of detection (LOD) for Cu2+ was determined to be 7.1 nM. Correspondingly, L-Cu2+ ensemble was investigated by fluorescence technique which clearly established the high selectivity towards l-His based on fluorescence “off–on” response, and the limit of detection (LOD) for l-His was calculated to be approximately 15.3 nM. The selective sensing of Cu2+ was efficiently reversible with l-His, which was no loss of fluorescence intensity in 10 cycles. Notably, the sensing behavior of L towards Cu2+ and l-His detections was shown to be available within the wider pH interval (7.0–12.0). Cells imaging studies have shown that L was easily applied to the continuous imaging of Cu2+ and l-His in living cells. Furthermore, the test strips were also prepared for rapid and sensitive identification of Cu2+ and l-His. Combining smartphones and color changes, the semi-quantitative relationship between the R/B and the concentration of Cu2+ was established, and the limit of detection (LOD) reached up to 0.29 µM.

Low Concentration Ethanol Prevents Oxidative Stress- Induced Cardiac Cells Injury Under HyperglycemicConditions by Inhibiting the SAPK/JNK Signaling Pathway and ROS Generation

The purpose of this study was to determine the effects and mechanisms of ethanol on oxidative stress-induced cardiac H9c2 cells mitochondrial injury under hyperglycemic conditions. Under hyperglycemic conditions, ethanol pretreatment (10-100 μM) prevented H2O2-induced mitochondria swelling, as well as decreased cell viability and Respiratory Control Ratio (RCR) in the H9c2 cells. It also prevented TMRE fluorescence intensity loss and DCF fluorescence intensity increase under hyperglycemic conditions. These effects of ethanol were reversed by the SAPK/JNK agonist, anisomycin. Finally, treatment of H9c2 cells with 33mM glucose significantly enhanced Akt and ERK phosphorylation, which was not affected by ethanol. However, ethanol decreased the phosphorylation of SAPK/JNK under hyperglycemic conditions. Collectively, these findings indicate that under hyperglycemic conditions, that ethanol prevents oxidative stress-induced mitochondrial injury in cardiac H9c2 cells by preventing ROS generation via inhibiting the SAPK/JNK signaling pathway.

BAC transgenic mice to study the expression of P2X2 and P2Y1 receptors

Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y1R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y1R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice.

Trioctylphosphine-Assisted Pre-protection Low-Temperature Solvothermal Synthesis of Highly Stable CsPbBr3/TiO2 Nanocomposites.

Lead halide perovskite quantum dots (PQDs) are reported as a promising branch of perovskites, which have recently emerged as a field in luminescent materials research. However, before the practical applications of PQDs can be realized, the problem of poor stability has not yet been solved. Herein, we propose a trioctylphosphine (TOP)-assisted pre-protection low-temperature solvothermal synthesis of highly stable CsPbBr3/TiO2 nanocomposites. Due to the protection of branched ligands and the lower temperature of shell formation, these TOP-modified CsPbBr3 PQDs are successfully incorporated into a TiO2 monolith without a loss of fluorescence intensity. Because the excellent nature of both parent materials is preserved in CsPbBr3/TiO2 nanocomposites, it is found that the as-prepared CsPbBr3/TiO2 nanocomposites not only display excellent photocatalytic activity but also yield improved PL stability, enabling us to build highly stable white light-emitting diodes and to photodegrade rhodamine B.

Utilising fluorescence spectroscopy and optical microscopy to investigate bitumen long-term ageing

Linking the chemical composition of bitumen to its microstructure is challenge due to its complexity, viscoelasticity and sensitivity to high energy input. Therefore, fluorescence spectroscopy combined with fluorescence microscopy are promising tools, as they are capable to link the materials fluorescence behaviour to its respective microstructural features. Herein, these techniques were coupled with dark-field microscopy to gain insight on the chemical composition of these auto fluorescent microstructural features and their changes during long-term-ageing (LTA). The results show that upon LTA a loss in fluorescence intensity goes in line with a shift to higher emission wavelength. While the overall intensity decreases during ageing the density and number of fluorescent particles increases. Dark-field microscopy confirms that these fluorescent particles show particle light scattering, proving that on the micro-scale bitumen is a non-homogeneous material.

Characterization and Potent Application of Pleurotus floridanus Trypsin Inhibitor (PfTI)

Characterization and in vitro inhibition studies of protease inhibitor from the mushroom Pleurotus floridanus (PfTI) towards the pest Papilio demoleus is studied. The addition of 1 mM Mn2+, Na2+, Ba2+ and Ni 2+ enhanced the PfTI activity. The ICP-atomic emission spectrum showed the presence of Ca2+, Mg2+ and Zn2+ in the PfTI. Surfactants SDS and CTAB at a concentration of 1% reduced the PfTI activity whereas, the nonionic detergents Triton X and Tween 80 increased the activity. The inhibitory activity gradually decreased with increase in concentration of DMSO and H2O2. The activity was increased by dithiothreitol up to a concentration of 80 μM and inactivated at 140 μM. The activity of PMSF modified PfTI was drastically reduced to 0.234 U/mL at 4 mM concentration and similar results were obtained for modification of cysteine by N-Ethylmaleimide at slightly higher concentrations. The complex of trypsin and PfTI showed complete loss in fluorescence intensity at 343 nm compared with control. In vitro inhibition studies of PfTI with midgut proteases isolated from citrus pest P. demoleus with protease activity of 1.236 U was decreased to 0.613 U by 50 μL (0.1 mg/mL) of the inhibitor. Inhibitor was stable up to 0.04 M concentration of HCl.

Effect of Vectashield-induced fluorescence quenching on conventional and super-resolution microscopy

Finding the right combination of a fluorescent dye and a mounting medium is crucial for optimal microscopy of fixed samples. It was recently shown that Vectashield, one of the most commonly used mounting media for conventional microscopy, can also be applied to super-resolution direct stochastic optical reconstruction microscopy (dSTORM). dSTORM utilizes conventional dyes and starts with samples in a fluorescent “ON” state. This helps in identifying structures of interest. Subsequently, labelled samples are induced into blinking, which is necessary for determining the position of single molecules and reconstruction of super-resolution images. This is only possible with certain fluorescent dyes and imaging buffers. One of the most widely used dyes for dSTORM, Alexa Fluor 647 (AF647), blinks in Vectashield. However, after preparing immunocytochemical samples in Vectashield, we noticed that the fluorescence intensity of AF647 is quenched. This is particularly evident for dimmer immunostainings, such as stainings of some components of neuronal cytoskeleton and axonal initial segment. Because structures of interest cannot be identified in quenched samples, loss of fluorescence intensity hinders imaging of AF647 in Vectashield. This has consequences for both conventional and dSTORM imaging. To overcome this, we provide: 1) a quantitative analysis of AF647 intensity in different imaging media, 2) a quantitative analysis of the suitability of Vectashield for dSTORM imaging of high and low-abundance AF647-labelled targets. Furthermore, for the first time, we quantitatively analyse the performance of Alexa Fluor Plus 647, a new variant of AF647-conjugated antibody, in dSTORM imaging.

8-Anilino-1-naphthalenesulfonate (ANS) as a probe for Poly(vinyl alcohol)(PVA) swelling

Poly (vinyl alcohol) (PVA) is a hydrophilic polymer developed for biomedical applications. Swelling properties of PVA gels is very important for its drug delivery applications. There are many ways to study the swelling behaviour. Here we are proposing a fluorescent study by using 8-anilino-1-naphthalenesulfonate (ANS) as an extrinsic fluorescent probe. When ANS incorporated PVA films are allowed to swell in water, there is a loss of fluorescence intensity at 453 nm and there is a new peak at 500 nm. Thus, ANS is proposed as a fluorescent probe for the sensing of hydration of PVA.

A simple fluorescent assay for the discovery of protein-protein interaction inhibitors

Due to the therapeutic potential of targeting protein-protein interactions (PPIs) there is a need for easily executed assays to perform high throughput screening (HTS) of inhibitors. We have developed and optimized an innovative and robust fluorescence-based assay for detecting PPI inhibitors, called FluorIA (Fluorescence-based protein-protein Interaction Assay). Targeting the PPI of RAD52 with replication protein A (RPA) was used as an example, and the FluorIA protocol design, optimization and successful application to HTS of large chemical libraries are described. Here enhanced green fluorescent protein (EGFP)-tagged RAD52 detected the PPI using full-length RPA heterotrimer coated, black microtiter plates and loss in fluorescence intensity identified small molecule inhibitors (SMIs) that displaced the EGFP-tagged RAD52. The FluorIA design and protocol can be adapted and applied to detect PPIs for other protein systems. This should push forward efforts to develop targeted therapeutics against protein complexes in pathological processes.

Highly fluorescent, photostable, and biocompatible silicon theranostic nanoprobes against Staphylococcus aureus infections

Human health is severely threatened by bacterial infections, which fuel urgent demand for the development of novel theranostic agents that are suitable for diagnosing and combating bacterial infections. To meet this goal, we synthesized a new kind of theranostic probes, made of vancomycin (Van)-modified fluorescent silicon nanoparticles (SiNPs-Van). The new probes feature good selectivity for Gram-positive bacterial infections, favorable fluorescence, strong resistance to photobleaching (e.g., 12% loss of fluorescence intensity during 40 min continuous laser excitation in the presence of Staphylococcus aureus (S. aureus)), and good biocompatibility (e.g., cell viability > 95% during 24 h treatment). Because of these benefits, the probes were effective at prolonged (i.e., 8-day) fluorescence tracking of S. aureus infections in vivo, thus providing a visible and accurate way to evaluate treatment efficacy. Moreover, the resultant SiNPs-Van with a minimum inhibitory concentration (MIC) of ca. 0.5 μg/mL exhibited high antibacterial efficiency of 92.5%, superior to that (76.5%) of free Van with higher MIC of ca. 1 μg/mL.

Doxorubicin-loaded silicon nanoparticles impregnated into red blood cells featuring bright fluorescence, strong photostability, and lengthened blood residency

Based on the unique advantages of fluorescent silicon nanoparticles (SiNPs), long circulation red blood cells (RBCs), and anti-cancer drug molecules (i.e., doxorubicin (DOX)), we developed multifunctional DOX-loaded SiNPs impregnated into RBCs. Importantly, the resulting drug delivery systems (DDSs) simultaneously exhibited bright fluorescence coupled with robust photostability (i.e., ∼ 24% loss of fluorescent intensity after 25 min continuous laser irradiation) and significantly lengthened blood residency (i.e., t1/2 = 7.31 ± 0.96 h, 3.9-fold longer than pure DOX-loaded SiNPs). Therefore, this novel DDS featuring multi-functionalities shows high potential for cancer diagnosis and therapy, particularly for tumor imaging and chemotherapy in a synchronous manner.

Hybrid Donor-Acceptor Polymer Particles with Amplified Energy Transfer for Detection and On-Demand Treatment of Breast Cancer.

Judicious combination of semiconducting polymers with alternating electron donor (D) and acceptor (A) segments created hybrid nanoparticles with amplified energy transfer and red-shifted emission, while simultaneously providing photothermal capabilities. Hybrid D-A polymer particles (H-DAPPs) passively localized within orthotopic breast tumors, serving as bright fluorescent beacons. Laser stimulation induced heat generation on par with gold nanorods, resulting in selective destruction of the tumor. H-DAPPs can also undergo multiple thermal treatments, with no loss of fluorescence intensity or photothermal potential. These results indicate that H-DAPPs provide new avenues for the synthesis of hybrid nanoparticles useful in localized detection and treatment of disease.

Synthesis and Characterization of a Fluorescent Water-Borne Polyurethane Based on a Novel Naphthalimide Dye

ABSTRACTA waterborne-polyurethane (WPU) dye, based on the fluorescent dye 4-diamino propane-N-allyl-1,8- naphthalimide (WPU-DAN), was synthesized by attaching 4-diamino propane-N-allyl-1,8- naphthalimide (DAN) onto both ends of the polyurethane (PU) chains according to a prepolymer−ionomer process. The synthetic process was confirmed by Fourier transform infrared spectroscopy (FTIR) and UV-visible absorption. The glass transition temperature, molecular weight and average particle sizes were measured. The glass transition temperatures of WPU and WPU-DAN were 46.6 and 49.8°C, respectively. In addition, the particle size distributions of WPU-DAN and WPU were 140 and 134 nm, respectively. The thermal behaviour of WPU-DAN showed improvement compared to WPU. The fluorescence intensity of WPU-DAN was enhanced more than DAN due to the naphthalimide groups attached to the chains, and the fluorescence intensity of WPU-DAN and DAN were increased by increasing temperature. Moreover, the fluorescence intensity of WPU-DAN emulsion was stable during 30 days and no loss of fluorescence intensity occurred for these days.

The great biotechnological potential of a marine polychaete: An alliance between toxin and natural fluorescence

The great biotechnological potential of a marine polychaete: An alliance between toxin and natural fluorescence

Loss of Tryptophan Fluorescence Correlates With Mechanical Stiffness Following Photo-Crosslinking Treatment of Rabbit Cornea.

A clinical treatment option for keratoconus involves the use of UV-initiated photo-crosslinking with riboflavin to increase corneal stiffness. Our study investigates whether endogenous fluorescence changes following treatment for keratoconus can be correlated to alterations in the stiffness of the cornea, thereby guiding treatment of keratoconus. A total of 78 ex vivo rabbit eyes were treated with either riboflavin-dextran solution plus UV light, dextran solution plus UV light, or riboflavin-dextran solution only for half treatment (2.84 J/cm2), standard treatment (5.28 J/cm2), or prolonged treatment (15.84 J/cm2) times. Fluorescent spectroscopy was performed on all samples before and after treatment. The stress-strain relationship was measured for all samples using a uniaxial tensiometer following treatment. We found a dose-dependent decrease in the 290/340 nm excitation/emission fluorescence pair with increase in corneal stiffening following treatment that was significant (P < 0.01) for both standard (5.28 J/cm2 total fluence) and prolonged treatment (15.84 J/cm2) times. We did not observe a significant change in this excitation/emission pair for the dextran-plus-UV or riboflavin-only treatment groups. Loss of fluorescence intensity at the 290/340 nm excitation/emission pair could offer a noninvasive, in situ measurement for guiding the photo-crosslinking treatment of keratoconus. Larger relative decreases in this pair are significantly correlated with longer treatment times and with increases in stiffness and Young's modulus.

Fluorescent and Photostable Silicon Nanoparticles Sensors for Real-Time and Long-Term Intracellular pH Measurement in Live Cells

Fluorescent sensors suitable for dynamic measurement of intracellular pH (pHi) fluctuations should feature the following properties: feeble cytotoxicity, wide-pH range response, and strong fluorescence coupled with good photostability, which are still remaining scanty to date. Herein, by functionalizing fluorescent silicon nanoparticles (SiNPs) with pH-sensitive dopamine (DA) and pH-insensitive rhodamine B isothiocyanate (RBITC), we present the first demonstration of fluorescent SiNPs-based sensors, simultaneously exhibiting minimal toxicity (cell viability of treated cells remains above 95% during 24-h treatment), sensitive fluorescent response to a broad pH range (∼4-10), and bright fluorescence coupled with robust photostability (∼9% loss of fluorescence intensity after 40 min continuous excitation; in contrast, fluorescence of Lyso-tracker is rapidly quenched in 5 min under the same experiment conditions). Taking advantage of these merits, we further employ the resultant fluorescent SiNPs sensors for the detection of lysosomal pH change mediated by nigericin in live HeLa and MCF-7 cells in long-term (e.g., 30 min) manners. Interestingly, two consecutive stages, i.e., alkalization lag phase and logarithmic growth phase, are observed based on recording the whole process of pH change, offering important information for understanding the dynamic process of pHi fluctuations.

Elucidating the impact of glucosylation on human serum albumin: A multi-technique approach

Early glycation products as well as advance glycation end products are involved in pathogenesis of diabetes. Most of studies carried out on AGEs and their possible role in assessing diabetes complications, whereas only a few were focused to highlight the role of Amadori products. In this study, an attempt has been made to investigate a structural and immunological characterizations of Amadori-albumin upon early glucosylation because albumin undergoes fast glycation under hyperglycaemic condition. Amadori-albumin formation was determined by NBT assay and Amadori adducts in glycated samples were confirmed by LC–MS. Structural alterations in Amadori-albumin were characterized by loss in fluorescence intensity, loss in secondary and tertiary structures, exposure of hydrophobic patches, shifting in Amide bands and increment in hydrodynamic radius. Further, presence to autoantibodies against Amadori-albumin in diabetes patients were confirmed by direct binding ELISA and inhibition ELISA. Immunological studies results showed that autoantibodies present in diabetic patients with and without chronic kidney disease (CKD) showed significant binding with Amadori-albumin in comparison to the native protein. Anti Amadori-albumin antibodies predominantly present in CKD patients compare to without CKD patients. Band shift assay results showed true interaction between Amadori-albumin and autoantibodies present in CKD patients. Glucosylation results showed structural alterations in Amadori-albumin and hence generation of neo-epitopes in HSA molecule. Such modifications rendering the protein highly immunogenic that may be recognized as foreign molecule by immune cells and induced autoantibodies in diabetic patients. These finding signify the role of Amadori-albumin in kidney dysfunction in diabetes and raised level of autoantibodies may be used as biomarker for progression of CKD.