Steady-state Fluorescence Intensity Research Articles

Fit-free analysis of fluorescence lifetime imaging data using chemometrics approach for rapid and nondestructive wood species classification

Abstract Conventional fluorescence spectroscopy has been suggested as a valuable tool for classifying wood species rapidly and non-destructively. However, because it is challenging to conduct absolute emission intensity measurements, fluorescence analysis statistics are difficult to obtain. In this study, another dimension of fluorescence, that is, fluorescence lifetime, was further evaluated to address this issue. A time-resolved fluorescence spectroscopic measurement system was first designed, mainly using a streak camera, picosecond pulsed laser at 403 nm, and a spectroscope, to collect the fluorescence time-delay (FTD) profiles and steady-state fluorescence intensity (FI) spectra simultaneously from 15 wood species. For data analysis, principal component analysis was used to “compress” the mean-centered FTD and FI spectra. Then, support vector machine classification analysis was utilized to train the wood species classification model based on their principal component scores. To avoid overfitting, ten-fold cross-validation was used to train the calibration model using 70 % of the total samples, and the remaining 30 % hold-out validation was used to test its reproducibility. The cross-validation accuracies were 100 % (5 softwoods) and 96 % (10 hardwoods), with test-validation accuracies of 96 % and 89 %.

Understanding Conformational Changes in Human Serum Albumin and Its Interactions with Gold Nanorods: Do Flexible Regions Play a Role in Corona Formation?

The importance of protein-nanoparticle (NP) conjugates for biomedical applications has seen an exponential growth in the past few years. The protein corona formation on NPs with human serum albumin (HSA), being the most abundant protein in blood serum, has become one of the most studied protein analyses under NP-protein interactions as HSA is readily adsorbed on the surface of the NPs. Understanding the fate of the NPs in physiological media along with the change in biological responses due to the formation of the protein corona thus becomes important. We analyzed the HSA protein corona formation on gold nanorods (AuNRs) through different spectroscopic studies in addition to the effects of change in the protein concentration on the protein-NP interactions. Different imaging techniques such as high-resolution transmission electron microscopy, field emission scanning electron microscopy, and atomic force microscopy were used to determine the morphology and the dimensions of the nanorods and the protein-nanorod conjugates. Fourier-transform infrared data showed a reduction in the α-helix content and an increase in β-sheet content for the HSA-AuNR conjugate compared to the native protein. A decrease in steady-state fluorescence intensity occurred with instant addition of AuNR to HSA showing better and efficient quenching of Trp fluorescence for the lower concentration of protein. Time-correlated single photon counting results showed greater energy transfer efficiency and faster decay rate for higher concentrations of proteins. The circular dichroism study gives insight into the secondary structural changes due to unfolding, and a greater change was observed for lower concentrations of protein due to a thermodynamically stable protein corona formation. Surface-enhanced Raman spectroscopy (SERS) indicated the presence of aromatic residues such as Phe, Tyr, and Cys that appear to be close to the surface of the AuNRs in addition to hydrophobic interactions between AuNR and the protein. The disordered and flexible regions mapped onto HSA (PDB: 1AO6), predicted by the intrinsically disordered region predictors, point toward the interactions of similar residues with the nanorods observed from SERS and fluorescence studies. These studies could provide a clearer understanding of the interactions between HSA and AuNRs for possible biological applications.

Photophysical Behaviour of Novel Quaternary Pyrenoyl Salts and Their Sensitivity Towards Bile Salt Micellization.

The present work describes the synthesis and characterization of pyrene derivatives, N-(1-Pyrenoylmethyl) pyridinium bromide (PM-PB) and N-(1-Pyrenoylmethyl)-N,N,N-triethylammonium bromide (PM-TAB). The photophysical behavior of these molecules has been studied in various protic and aprotic solvents. Using steady state fluorescence intensity, fluorescence anisotropy and dynamic fluorescence lifetime studies, the sensitivity of these molecules towards the micellization process of bile salts has been monitored. These derivatives have been effectively used in estimating critical micellar concentration (CMC) of bile salt, sodium deoxycholate (NaDC).

Photophysical Behavior of Plant Flavonols Galangin, Kaempferol, Quercetin, and Myricetin in Homogeneous Media and the DMPC Model Membrane: Unveiling the Influence of the B-Ring Hydroxylation of Flavonols.

Flavonols have been studied extensively because of their interesting biological activities and excited-state intramolecular proton transfer (ESIPT) behavior. Galangin, kaempferol, quercetin, and myricetin are structurally related flavonols that differ only in the number of B-ring hydroxyl substituents. In this work, we have carried out a detailed study on the photophysical behavior of these structurally related flavonols in various solvents and a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) small unilamellar vesicles (SUVs) model membrane. We observed that these flavonols exist in different forms in the ground and excited states depending on the nature of the solvent. The weak intrinsic fluorescence of these flavonols gets enhanced in hydrogen-bond-accepting and alcoholic solvents. The phototautomer fluorescence intensity of these flavonols increases significantly in the DMPC membrane compared to water, suggesting ESIPT activation via binding interaction between flavonols and the membrane. According to our findings, both the number of B-ring hydroxy groups and membrane fluidity affect the flavonol binding with the membrane. The steady-state fluorescence intensity, steady-state anisotropy, fluorescence lifetime, and fluorescence anisotropy decay of flavonols were sensitive towards the temperature-induced DMPC membrane phase change. A quenching study has been performed to investigate the location and distribution of flavonols in the DMPC SUVs. Moreover, the antioxidant potential of flavonols in DMPC SUVs has been examined using the DPPH scavenging method. Our results reveal that the B-ring hydroxy groups significantly affect the photophysics, binding affinity, location, distribution, and DPPH scavenging activity of polyhydroxy-flavonols in the DMPC SUVs.

Ex vivo hypercellular parathyroid gland differentiation using dynamic optical contrast imaging (DOCI).

Primary hyperparathyroidism, often caused by a single adenoma (80-85%) or four-gland hyperplasia (10-15%), can lead to elevated parathyroid hormone (PTH) levels and resultant hypercalcemia. Surgical excision of offending lesions is the standard of care, as the removal of pathologic adenomas reduces PTH and calcium values to baseline. The small size, variable location, and indistinct external features of parathyroid glands can make their identification quite challenging intraoperatively. Our group has developed the dynamic optical contrast imaging (DOCI) technique, a novel realization of dynamic temporally dependent measurements of tissue autofluorescence. In this study, we evaluated the efficacy of using the DOCI technique and normalized steady-state fluorescence intensity data for differentiating types of human parathyroid and thyroid tissues. We demonstrate that the DOCI technique has the capability to distinguish normal parathyroid tissue from diseased parathyroid glands as well as from adjacent healthy thyroid and adipose tissue across 8 different spectral channels between 405nm-600nm (p<0.05). Patient tissue DOCI data was further analyzed with a logistic regression classifier trained across the 8 spectral channels. After computer training, the computer-aided identification was able to accurately locate hypercellular parathyroid tissue with 100% sensitivity and 98.8% specificity within the captured DOCI image.

Curcumin-Induced Membrane Property Changes in DMPC Multilamellar Vesicles and the Effects of Membrane-Destabilizing Molecules on Curcumin-Loaded Multilamellar Vesicles.

Curcumin (CUR) is the major bioactive component of turmeric (Curcuma longa), commonly used as a spice and traditional medicine in India. CUR possesses a wide range of pharmacological benefits, including antioxidant, anticarcinogenic, antimutagenic, anti-inflammatory, anti-Alzheimer, and anti-Parkinson effects. The CUR-membrane interaction is believed to be the reason for such biological activity of CUR. Several research groups have modeled the interaction of CUR with artificial model lipid membranes using various techniques such as nuclear magnetic resonance (NMR), small-angle X-ray scattering (SAXS), and differential scanning calorimetry (DSC). However, the mechanism of its action is still unclear. A fluorescent-probe-based technique could be advantageous to study the CUR-lipid membrane interaction due to its sensitivity toward the local environment and its multiparametric nature. In this work, we have used the intrinsic fluorescence properties of CUR to investigate CUR-induced physical property changes in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilamellar vesicles (MLVs) at various CUR concentrations. By rationalizing the results of steady-state fluorescence intensity, fluorescence anisotropy, temperature-dependent fluorescence intensity, temperature-dependent fluorescence anisotropy, and quenching experiments, we have proposed a model showing concentration-dependent effects of CUR on the DMPC bilayer membrane. We suggest that at low concentrations (≤1 mol %), CUR is homogeneously distributed in the DMPC bilayer membrane in both the solid gel (SG) and liquid crystalline (LC) phases. At high concentrations (>1 mol %), CUR molecules form segregated domains that fluidize both membrane phases. However, the CUR-induced fluidization is less pronounced in the LC phase as some CUR molecules from the domain partition into the bilayer core. Further, the effects of membrane-destabilizing molecules such as bile salts, capsaicin (CAP), and piperine (PIP) on CUR-loaded DMPC multilamellar vesicles were studied. Our work also shows that CUR has a stabilizing effect on the DMPC membrane at high concentrations.

Physicochemical study of dietary fiber methylcellulose and human intestinal bile salt micellar aggregates

The cellulose ether and bile salt mixed systems have a potential to alter the lipid digestion. The basic understanding of the aggregation process, interaction, and physicochemical properties of cellulose ether and bile salt is an essential topic of study. In this work, the interaction between simple dietary fiber methylcellulose (MC) and bile salt sodium deoxycholate (NaDC) has been studied using UV-Vis absorption, steady-state fluorescence intensity, fluorescence quenching, conductivity, light scattering, and zeta potential measurements. Fluorescence properties of hydrophobic probe pyrene were used to find the critical aggregation concentration (CAC), critical micellar concentration (CMC), and aggregation number (Nagg) of the MC/NaDC system. Using conventional conductivity method CAC, CMC, and thermodynamic parameters of micellization i.e. standard Gibbs free energy (∆Gmic0), standard entropy (∆Smic0), and standard enthalpy (∆Hmic0), was calculated to know the information regarding the nature of micellization of MC/NaDC mixed system. The size distribution and zeta potential study give an insight into MC/NaDC interaction before and after the CAC point. The strong hydrophobic interaction between cellulose ether MC and bile salt NaDC shows that this mixed system may have further implications in lipid digestion.

Binding of Calcium and Magnesium Ions to Terrestrial Chromophoric Dissolved Organic Matter (CDOM): A Combination of Steady-State and Time-Resolved Fluorescence Study

Revealing the binding properties of calcium ion (Ca2+) and magnesium ion (Mg2+) to terrestrial chromophoric dissolved organic matter (CDOM) facilities understanding the effect of natural water components on the photophysics of dissolved organic matter. Steady-state and time-resolved fluorescence spectrometry, and dynamic light scattering were applied to investigate the fluorescence quenching process of CDOM by Ca2+ and Mg2+. Due to a remarkable decrease of the steady-state fluorescence intensity and a slight decrease of fluorescence lifetime, the fluorescence quenching of CDOM by cations mainly occurred through a static process. The fluorescence quenching was profound under longer excitation and emission wavelengths. The binding constant (K, L/mol) for Ca2+ to CDOM ranged from 4.29 to 5.09 (lgK), which was approximately one order of magnitude higher than that of Mg2+ to CDOM (3.86 to 4.56). Nevertheless, the efficiency of CDOM fluorescence quenching by Ca2+, Mg2+ was much lower than that by Cu2+. Fluorescence decay became faster in the presence of a high concentration of Ca2+ (&gt;20 mg/L) and Mg2+ (&gt;50 mg/L). In the presence of these two metal ions, particularly for Ca2+, the lifetime of CDOM excited states shifted to the relatively small value side, indicating fluorescence quenching of CDOM mainly occurred through the interaction of Ca2+/Mg2+ with relatively long-lived fluorophores.

Highly Sensitive Optical Sensor for Selective Detection of Fluoride Level in Drinking Water: Methodology to Fabrication of Prototype Device

Excess consumption of fluoride (F–) through drinking water and its detrimental effects on human health have been a serious global concern. Herein, we have developed a facile and highly sensitive spectroscopic technique for selective detection of F– in aqueous media using aluminum phthalocyanine chloride (AlPc-Cl) as a sensor. The absorbance and steady-state fluorescence intensity of AlPc-Cl has been found to decrease in the presence of F– which has been used as a marker for the determination of F– concentration in water. The structural change in AlPc-Cl after addition of F– has been thoroughly studied by using ¹⁹F nuclear magnetic resonance spectroscopy. Our detailed steady-state and time-resolved fluorescence studies reveal that the quenching mechanism is static in nature. The response of the sensor is found to be linear over the F– concentration regime from 0 to 6 ppm with a detection limit of 0.05 ppm. Additionally, it shows an excellent selectivity as well as an insignificant change in sensitivity, even in the presence of interfering iron and aluminum ions. Based on the detailed photophysical study, we have further developed a low cost and portable prototype device that shows an excellent sensitivity with the detection limit of 0.10 ppm.

Antioxidant activity of vitamin E enhanced by cyclodextrin inclusion complex

PurposeThe study aims to find potential method to enhance the stability and antioxidant activity of vitamins.Design/methodology/approachVitamin E (VE) and β-cyclodextrin (CD) were used to prepare the VE-β-CD inclusion complex via a solution method. The yielded VE-β-CD obtained from optimal experimental conditions was characterized. The mechanism of complex formation and the properties of the complex was investigated by steady-state and time-resolved fluorescence techniques. The antioxidant activity of VE-β-CD was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and compared with pure VE.FindingsThree experimental factors – reaction temperature (T), molar ratio of VE to β-CD (rm) and inclusion time (t) – were studied for the preparation of VE-β-CD. At experimental conditions of T = 60°C, rm = 1:3 and t = 3 h, a maximum inclusion rate of VE (78.62%) was obtained. The Fourier-transform infrared spectroscopy (FTIR) shows that VE molecule was physically encapsulated by β-CD in the yielded product. Subsequentially, the steady-state fluorescence intensity of VE was found to increase upon encapsulation by β-CD due to the hindrance of fluorescence quenching of VE molecules inside the hydrophobic cavity of β-CD, and an inclusion constant K was determined to equal 3.58 × 103 L/mol from the fluorescence results. The fluorescent observation was confirmed by time-resolved fluorescence experiment as the lifetime of VE increased with an increase in β-CD concentration.Originality/valueThe ability of removing radicals by VE was significantly enhanced after the formation of inclusion complex with β-CD.

A Fluorescence Study of the Interaction of Anticancer Drug Molecule Doxorubicin Hydrochloride in Pluronic P123 and F127 Micelles.

Drug delivery systems for the sustained and target delivery of doxorubicin to tumor cells are a topic of interest due to the efficacy of the doxorubicin in cancer treatment. The use of polymers such as Pluronic is being studied widely for the formulation of doxorubicin hydrochloride. However, the basic understanding of the physicochemical properties of pluronic micelles in presence of doxorubicin hydrochloride is a very essential topic of study. Doxorubicin hydrochloride is fluorescent; this helped us to study its sensitivity towards the Pluronic microenvironment using the fluorescence technique. In this work, the interaction and place of location of doxorubicin hydrochloride in Pluronic F127 and P123 micelles has been studied extensively using steady-state fluorescence intensity, dynamic fluorescence lifetime, quenching studies, dynamic light scattering, and zeta potential measurements, at different Pluronic concentrations. Using a fluorescence quenching experiment, doxorubicin hydrochloride was found to reside near the hydrophilic PEO corona region of the Pluronic micelles. For both the Pluronic, in the concentration range of study, the micellar size was found to be below 30nm; this may have a greater advantage for various applications.

Amplified fluorescence of Mg2+ selective red-light emitting carbon dot in water and direct evaluation of creatine kinase activity

Carbon quantum dot/carbon dot (CD) exhibiting sustained photoluminescence at longer wavelengths in aqueous solution is difficult to prepare, but has enormous potential in biomedical applications. For the first time, we report the magnesium(II) selective fluorescence enhancement of a red-light emitting anthrarufin and boric acid-derived CD in aqueous solution for direct evaluation of creatine kinase (CK) enzyme activity. The CD displayed visually detectable, intense red fluorescence only in the presence of magnesium ion (Mg2+) at physiological pH value when irradiated with an ultraviolet (UV) source. Concurrently, a significant increase in steady-state fluorescence intensity and fluorescence lifetime was documented. A time-dependent density functional theory (TD-DFT) analysis displayed a bathochromic shift in UV-visible (vis) absorption, and increased oscillator strength of transition resulting from the selective chelation of Mg2+ with β-hydroxy keto functionality on the surface of the CD. The CD-Mg2+ assembly was subsequently used to conceptualize the detection of CK directly through the exploration of the differential binding affinity of Mg2+ with adenosine triphosphate (ATP), adenosine diphophate (ADP), and CD that is otherwise not possible with commercially available kits as of today. Thus, the report delineated here usher grandeur potential of CD for biological explorations related to Mg2+ or ATP sensing and monitoring of Mg2+-dependent enzymatic activity through a clear understanding of the chemistry.

PH induced reorganization of protein-protein interface in liposome encapsulated Ferritin at air/fluid and fluid/solid interfaces

This study reports the pH-dependent assembly - disassembly of the iron storage cage protein ferritin (HSFr), in solution and enclosed in Giant vesicles (GUVs) of DOPC at air/buffer and fluid/solid interface. The surface pressure-area isotherms of films of lipids with the protein at air/buffer interface indicate that there is a disassembly ↔ assembly for pH shift from 7.0 to 2.0 back to 7.0, with a corresponding decrease ↔ increase in the average area/lipid. This disassembly and reassembly processes of ferritin-apoferritin show a pseudo reversible nature. Time resolved fluorescence of the protein in GUVs of mixed lipids of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (NBDPE) with DOPC revealed that the confined protein shows longer lifetimes than the free molecules. CD spectroscopy used to study the conformational preference of protein in the GUVs shows that localization of the protein does not introduce drastic changes in the structure of the vesicles which is confirmed by the fluorescence anisotropy values. The differences seen in the steady state fluorescence intensities and the lifetimes showing the differing encapsulation efficiency of the protein at the 2 pHs are in agreement with our air/buffer interface results. Viscoelastic behaviour of GUVs with the protein from Quartz Crystal Microbalance with dissipation, showed clearly the differences in the protein molecular assembly processes for pH 2.0 and 7.0. Flow cytometry (FC) analysis of the GUVs at pH = 2.0 showed that 18% of total GUVs have HSFr, while at pH 7.0, it is much less (around 7%). This difference could be due to the protein being in the monomeric unfolded state at pH 2.0 with good mobility than at pH 7.0 and therefore can be located easily inside the GUVs. This approach has applications in protein engineering strategies.

Dual effect of non-ionic detergent Triton X-100 on insulin amyloid formation.

Atomic force microscopy, Thioflavin T (ThT) fluorescence assay, circular dichroism spectroscopy, differential scanning calorimetry, and molecular modeling techniques have been employed to investigate the amyloid aggregation of insulin in the presence of non-ionic detergent, Triton X-100 (TX-100). In contrast to recently described inhibition of lysozyme amyloid formation by non-ionic detergents (Siposova, 2017), the amyloid aggregation of insulin in the presence of sub-micellar TX-100 concentration exhibits two dissimilar phases. The first, inhibition phase, is observed at the protein to detergent molar ratio of 1:0.1 to 1:1. During this phase, the insulin amyloid fibril formation is inhibited by TX-100 up to ∼60%. The second, "morphological" phase, is observed at increasing detergent concentration, corresponding to protein:detergent molar ratio of ∼1:1 - 1:10. Under these conditions a significant increase of the steady-state ThT fluorescence intensities and a dramatically changed morphology of the insulin fibrils were observed. Increasing of the detergent concentration above the CMC led to complete inhibition of amyloidogenesis. Analysis of the experimental and molecular modeling results suggests an existence of up to six TX-100 binding sites within dimer of insulin with different binding energy. The physiological relevance of the results is discussed.

(Invited) A Frequency Domain Optofluidics Dissolved Oxygen Sensor

Concentration of Dissolved oxygen (DO) is an important indicator of water quality. Fluorescence quenching is a commonly used optical method to measure DO. Steady-state fluorescence intensity measurements are easy to be influenced by acquisition artifacts, which makes determination of absolution DO values difficult. We developed a miniaturized, optofluidics frequency-domain DO sensor that measures fluorescence quenching using fluorescence lifetime changes of Ruthenium (Ru(dpp)3Cl2) rather than intensity. Dissolved oxygen is quantified by the phase shift between excitation modulation and emission modulation through process. Compact, low cost components such as diode lasers and photodiodes have been used for the potential of scalable distributed sensing applications. The sensor is evaluated using water samples with different oxygen concentrations. Experimental results show that the lifetime measured in frequency domain corresponds well to dissolved oxygen concentration. Long-term experimental demonstrates that the proposed sensor remains stable in the presence of photobleaching; since the measured phase shift is not affected by reduction of the fluorescence intensity. The sensitivity of the DO measurement is also enhanced through the total internal reflection design.

Molecular insight into the inclusion of the dietary plant flavonol fisetin and its chromophore within a chemically modified γ-cyclodextrin: Multi-spectroscopic, molecular docking and solubility studies

We explored the encapsulation of dietary plant flavonols fisetin and its chromophore 3-hydroxyflavone, within 2-hydroxypropyl-γ-cyclodextrin (HPγ-CDx) nano-cavity in aqueous solution using multi-spectroscopic approaches and molecular docking. Upon addition of HPγ-CDx, dramatic changes occur in the intrinsic ‘two color’ fluorescence behavior of the fluorophores. This is manifested by significant increase in the steady state fluorescence intensities, anisotropies, average fluorescence lifetimes and rotational correlation times. Furthermore, in the CDx environment, intrinsically achiral flavonols exhibit prominent induced circular dichroism bands. These findings indicate that the flavonol molecules spontaneously enter the relatively hydrophobic, chiral environment of the HPγ-CDx nano-cavities. Molecular docking computations corroborate the spectroscopic findings, and predict selectivity in orientation of the encapsulated flavonols. HPγ-CDx inclusion increases the aqueous solubility of individual flavonols ∼100–1000 times. The present study demonstrates that the hydroxypropyl substituent in γ-CDx controls the inclusion mode of the flavonols, leading to their enhanced solubilization and altered spectral signatures.

Excited-State Proton Transfer of Phenol Cyanine Picolinium Photoacid.

Steady-state and time-resolved fluorescence techniques as well as quantum-mechanical calculations were used to study the photophysics and photochemistry of a newly synthesized photoacid—the phenol cyanine picolinium salt. We found that the nonradiative rate constant knr of the excited protonated form of the photoacid is larger than that of the excited-state proton transfer (ESPT) to the solvent, kESPT. We estimate that the quantum efficiency of the ESPT process is about 0.16. The nonradiative process is explained by a partial trans–cis isomerization reaction, which leads to the formation of a “dark” excited state that can cross to the ground state by nonadiabatic coupling. Moreover, the ESPT process is coupled to the photo-isomerization reaction, as this latter reaction enhances the photoacidity of the studied compound, as a result of photoinduced charge transfer. To prevent trans–cis isomerization of the cyanine bridge, we conducted experiments of PCyP adsorbed on cellulose in the presence of water. We found that the steady-state fluorescence intensity increased by about a factor of 50 and the lifetime of the ROH band increased by the same factor. The fluorescence intensity of the RO– band with respect to that of the ROH band was the same as in aqueous solution. This explains why inhibiting the photo-isomerization reaction by adsorbing the PCyP on cellulose does not lead to a higher ESPT rate.

Molecular and Thermodynamic Studies on DNA Triplex Formed in the Promoter Region of HMGB1 Gene as a Selective Target for Anticancer Drugs.

HMGB1 (High Mobility Group Box-1) is a very versatile highly abundant architectural protein that plays multiple roles in human health and diseases. Under physiological condition it serves as an amazing assortment of roles in different compartments of cell. The reported high expression of HMGB1 in almost all types of human cancers and inflammatory diseases makes it a critical molecular therapeutic target. In the present study, we have mobilized a proximal twenty one base pair nucleotide (21RY) which is in the promoter region (-55 to-75) of hmgb1 gene and targeted it with triplex forming oligonucleotide (TFO) in combination with two widely used chemotherapeutic drugs, actinomycin (ACT) and adriamycin (ADM). The interaction of actinomycin and adriamycin to 21R*R•Y DNA triplex was studied using UV melting profiles, CD spectroscopy, spectrofluorimetry and Isothermal titration calorimetry. The 21R*R•Y formation was confirmed from biphasic thermal melting profiles, continuous variation method, analysis of CD marker band and thermodynamic parameters. The binding of ADM and ACT to 21R*R•Y was characterized by hypochromic and bathochromic shift in their respective absorption spectrum, quenching (ADM) and enhanced fluorescence (ACT) of steady-state fluorescence intensity, perturbation in the circular dichroic spectrum and change in thermal melting temperatures. The ITC profile and Scatchard plot analysis indicate non-cooperative and higher binding affinity of these drugs to 21R*R•Y compared to their corresponding duplexes. Therefore, combining these chemotherapeutic drugs with triplex forming oligonucleotide may offer new diagnostic and therapeutic options in targeting a gene of interest more specifically with fewer side effects. This study shows that ACT and ADM effectively recognize 21R*R•Y triplex DNA formed on the hmgb1 promoter region.

Photon Antibunching Reveals Static and Dynamic Quenching Interaction of Tryptophan with Atto-655.

Fluorescence correlation spectroscopy (FCS) of photoinduced electron transfer (PET) between the dye Atto-655 and the amino acid tryptophan has been extensively used for studying fast conformational dynamics of small disordered peptides and proteins. However, a precise understanding of the quenching mechanism and its exact rates that would explain ensemble as well as single-molecule spectroscopy results is still lacking. In this contribution, a general unified model for intermolecular PET between Atto-655 and tryptophan is developed, which involves ground-state complex formation, quenching sphere of action, and dynamic quenching at the single-molecule level. We present measurements of fluorescence antibunching, fluorescence lifetime, and steady-state fluorescence intensity and absorbance and demonstrate that our model is capable to describe all results in a global and coherent manner.

Mesoporous silica for drug delivery: Interactions with model fluorescent lipid vesicles and live cells

Formulated mesoporous silica nanoparticle (MSN) systems offer the best possible drug delivery system through the release of drug molecules from the accessible pores. In the present investigation, steady state and time resolved fluorescence techniques along with the fluorescence imaging were applied to investigate the interactions of dye loaded MSN with fluorescent unilamellar vesicles and live cells. Here 1,2-dimyristoyl-sn-glycero-3-phospocholine (DMPC) was used to prepare Small Unilamellar Vesicles (SUVs) as the model membrane with fluorescent 1,6-diphenyl-1,3,5-hexatriene (DPH) molecule incorporated inside the lipid bilayer. The interaction of DPH incorporated DMPC membrane with Fluorescein loaded MSN lead to the release of Fluorescein (Fl) dye from the interior pores of MSN systems. The extent of release of Fl and spatial distribution of the DPH molecule has been explored by monitoring steady-state fluorescence intensity and fluorescence lifetime at physiological condition. To investigate the fate of drug molecule released from MSN, fluorescence anisotropy has been used. The drug delivery efficiency of the MSN as a carrier for doxorubicin (DOX), a fluorescent chemotherapeutic drug, has also been investigated at physiological conditions. The study gives a definite confirmation for high uptake and steady release of DOX in primary oral mucosal non-keratinized squamous cells in comparison to naked DOX treatment.