Orange-Red-Emitting Carbon Dots for Bilirubin Detection and Its Antibacterial Activity Against Escherichia coli and Staphylococcus aureus.

In this study, orange-red-emitting carbon dots (OR-CDs) were prepared from p-phenylenediamine (p-PDA) and urea as starting precursors through the hydrothermal method. The OR-CDs exhibited bright orange-red fluorescence at 618 nm when excited at 480 nm. The obtained OR-CDs exhibited stable photophysical properties under different physiological conditions. The unique photophysical property of OR-CDs were then utilized for fluorometric determination of bilirubin. The fluorometric assay revealed that the fluorescence intensity of OR-CDs is gradually quenched upon the addition of bilirubin (1-20 µM). The mechanism of fluorescence quenching was evaluated by steady-state fluorescence analysis and time-correlated single photon counting measurements. The OR-CDs showed good selectivity and sensitivity toward bilirubin over other common interfering biomolecules. The present fluorometric assay showed a linear response toward bilirubin between 1 and 10 µM with a limit of detection of 4.80 nM. Further, a fluorescence test cotton swab-based detection probe has been successfully developed by incorporating OR-CDs for the point-of-care detection of bilirubin in biofluids. Furthermore, a light-emitting diode light that emits orange-red light was prepared by embedding the OR-CDs within the poly(vinyl alcohol) polymer matrix. Moreover, the antibacterial activity of OR-CDs was tested against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. The antibacterial efficacy of OR-CDs was demonstrated by various mechanisms, such as reactive oxygen species generation, destruction of cell structure, chemical binding to membrane, and surface wrapping. Interestingly, the survival assay against L929 fibroblast cells exhibits favorable biocompatibility and bioimaging.

Smartphone-enabled colorimetric visual quantification of highly hazardous trivalent chromium ions in environmental waters and catalytic reduction of p-nitroaniline by thiol-functionalized gold nanoparticles.

High-efficiency sensing systems for extremely hazardous chromium (Cr(III)) ions are important due to their detrimental effects on human health and the environment. We employed a spectrophotometric method combined with a smartphone (red, green, and blue (RGB) color ratio)-based detection platform to realize the quick, visually quantifiable in situ detection of Cr(III) ions using surface plasmon resonance (SPR)-aided colorimetry. For optical sensing nanoprobes, we synthesized the 2-Mercapto-5-methyl-1,3,4-thiadiazole (MMT)-modified gold nanoparticles (MMT-AuNPs) using a wet chemical method. By way of a coordination reaction, the Cr(III) ions induce the as-prepared MMT-AuNPs to aggregate and subsequently change the SPR wavelength band. The freshly synthesized MMT-AuNPs exhibited a wine-red color. While Cr(III) ions interact with the MMT-AuNPs, the color of the latter evolved from wine red to purple, thus facilitating visual monitoring. The SPR-relevant color change allowed the quantitative sensing of Cr(III) ions in the range of 40-128 nM, with the limit of detection of 6.93 nM when employing the spectrophotometric method and 12.4 nM when using the smartphone RGB color ratio. Furthermore, we developed the spectrophotometric technique that used the smartphone RGB color ratio for on-site analysis of Cr(III) ions in environmental water samples, indicating the possibility of its chemo-sensing applications for portable quantitative detection devices. Additionally, the catalytic performance of the MMT-AuNPs was demonstrated by the reduction of p-nitroaniline in the presence of sodium borohydride. It was interestingly unveiled that the MMT-AuNPs showed outstanding catalytic performance with a catalytic rate constant of 6.31 × 10-3 s-1.

Facile preparation of dihydrolipoic acid-stabilized red-emitting silver nanoclusters as a sensitive fluorometric probe for sulfide ions detection.

In this work, we report a smartphone-integrated paper-based sensor for the determination of sulfide ions (S2-) using water-soluble dihydrolipoic acid stabilized silver nanoclusters (DHLA-AgNCs) as a nanoprobe. The optical properties of red emitting fluorescent DHLA-AgNCs was confirmed by UV-visible, steady state flourometric spectroscopic studies. The HR-TEM analysis revealed that the morphology of DHLA-AgNCs was quasi spherical with a grain size of âˆ¼ 5.2 nm. The DHLA-AgNCs exhibited bright red luminescence with strong emission band centered at 650 nm upon the excitation at 420 nm. The excellent fluorescence property of DHLA-AgNCs was further utilized for fluorometric determination of S2- ions. The DHLA-AgNCs can be effectively quenched by increasing concentration of S2- ions owing to the formation of Ag2S complex. The DHLA-AgNCs probe could detect S2- ions preferentially even in the presence of other possible interfering anions with a limit of detection of 32.71 nM. In addition, the proposed technique was effectively used to detect S2- ions in environmental water samples such as tap and drinking water. The detect S2- ions detection was assay and showed good agree compared with the conventional methylene blue approach and showed comparable results. Moreover, a smartphone-paper-based detection assay was developed using the DHLA-AgNCs probe for highly selective and sensitive determination of S2- ions.

Deciphering the binding site and mechanism of new methylene blue with serum albumins: A multispectroscopic and computational investigation.

Herein, the interaction mechanism of new methylene blue (NMB) with human serum albumin (HSA) and bovine serum albumin (BSA) was carefully investigated both experimentally and conceptually, employing experimental and insilico analysis. The steady-state emission spectral studies showed that the emission intensity of HSA and BSA was quenched significantly by NMB. The findings of the Stern-Volmer and double logarithmic plot revealed that the observed emission quenching process was through a static quenching mechanism and the measured binding constant values (Kb) for HSA-NMB and BSA-NMB are 2.766 and 1.187 × 105 dm3 mol-1 respectively. The time-resolved fluorescence lifetime measurement and UV-vis absorption investigation further verify the complex formation between NMB and HSA/BSA. The assessment of thermodynamic parameters disclosed the binding process was spontaneous driven by hydrogen bonds (H-bond) and van der Waals interactions, which contributed a significant role in the complexation. Moreover, the secondary structural conformation and microenvironment of HSA/BSA were modified in the presence of NMB, as evidenced by circular dichroism and synchronous fluorescence data. Molecular docking study predicted a plausible binding mode of NMB inside the binding pocket of HSA and BSA. These results demonstrated that the stabilized NMB is found at the Subdomain IIA (site I) of both the proteins and the results were correlated well with the competitive binding assay. Additionally, the principal components analysis revealed less variation of docked poses for HSA, while, more dispersed docked poses were observed for the BSA model. This also highlights the effects of docking towards a modeled protein (BSA). Molecular dynamic (MD) simulation based binding free energy (ΔGmmgbsa) estimation obtained at 298, 303, 308 and 313 K, were in good agreement with our experimental (ΔGbind) values.

Cysteamine-decorated gold nanoparticles for plasmon-based colorimetric on-site sensors for detecting cyanide ions using the smart-phone color ratio and for catalytic reduction of 4-nitrophenol.

In this paper, we have reported the cyanide ions (CN-) sensing in environmental water samples using cysteamine-capped gold nanoparticles (Cyst-AuNPs) by spectrophotometric, colorimetric, and smartphone-based RGB color detection. The surface plasmon resonance shift at around 525 nm for the Cyst-AuNPs could be used to detect quantitatively the amounts of CN- with concomitant alteration of their color from wine red to purple visualized by the naked eye. For the first time, the Cyst-AuNPs-based visual sensing of CN- was performed using smartphone-based detection with its detection limit of 159 × 10-9 M, ten times lower than that of the highest tolerance level (2 × 10-6 M) permitted by the world health organization. The Cyst-AuNPs displayed excellent specificity for detecting the concentration of 30 × 10-6 M even amid the presence of other interfering inorganic anions with their concentrations about five times higher than it. Environmental real water samples were used to arrange the three different CN- concentrations for plasmon-based colorimetric detection and smartphone-based method. Additionally, the catalytic performance of Cyst-AuNPs was demonstrated for the fast catalytic conversion of hazardous 4-nitrophenol (selected environmental contaminant) to the analogous amino aromatic compounds. A chemical kinetic study showed the conversion rate to be estimated as 1.65 × 10-2 s-1. Cyst-AuNPs can find an application in colorimetric sensing of CN- while being able to be utilized as a catalytic nanomaterial for ecological remedies associated with health care.

Gold nanoparticle-decorated earth-abundant clay nanotubes as catalyst for the degradation of phenothiazine dyes and reduction of 4-(4-nitrophenyl)morpholine.

In the present work, halloysite nanotubes modified with gold nanoparticles (AuNPs-HNT) are successfully prepared by wet chemical method for the catalytic degradation of phenothiazine dyes (azure B (AZB) and toluidine blue O (TBO)) and also cleaner reduction of 4-(4-nitrophenyl)morpholine (4NM) in the sodium borohydride (NaBH4) media. The catalyst is formulated by modifying the HNT support with a 0.964% metal loading using the HNT supports modified with 3-aminopropyl-trimethoxysilane (APTMS) coupling agent to facilitate the anchoring sites to trap the AuNPs and to prevent their agglomeration/aggregation. The AuNPs-HNT catalyst is investigated for structural and morphological characterization to get insights about the formation of the catalyst for the effective catalytic reduction of dyes and 4NM. The microscopic studies demonstrate that AuNPs (2.75 nm) are decorated on the outer surface of HNT. The as-prepared AuNPs-HNT catalyst demonstrates AZB and TBO dye degradation efficiency up to 96% in 10 and 11 min, respectively, and catalytic reduction of 4NM to 4-morpholinoaniline (MAN) is achieved up to 97% in 11 min, in the presence of NaBH4 without the formation of any by-products. The pseudo-first-order rate constant (K1) value of the AuNPs-HNT catalyst for AZB, TBO, and 4NM were calculated to be 0.0078, 0.0055, and 0.0066 s-1, respectively. Moreover, the synthesized catalyst shows an excellent reusability with stable catalytic reduction for 7 successive cycles for both the dyes and 4NM. A plausible mechanism for the catalytic dye degradation and reduction of 4NM by AuNPs-HNT catalyst is proposed as well. The obtained results clearly indicate the potential of AuNPs-HNT as an efficient catalyst for the removal of dye contaminants from the aquatic environments and cleaner reduction of 4NM to MAN, insinuating future pharmaceutical applications.

Water-soluble luminescent copper nanoclusters as a fluorescent quenching probe for the detection of rutin and quercetin based on the inner filter effect.

In the present study, we proposed a simple, sensitive and selective fluorescence method for the detection of rutin (Rut) and quercetin (Que) based on the inner filter effect (IFE) utilizing water- soluble cysteine-stabilized copper nanoclusters (Cys-CuNCs) as a fluorescent probe. The Cys-CuNCs were successfully synthesized and characterized using UV-visible absorption, emission, Fourier-transform infrared (FT-IR) spectroscopy, fluorescence lifetime, high resolution transmission electron microscopy (HR-TEM) and zeta potential measurements. Cys-CuNCs exhibited bluish-green luminescence under UV light with characteristic emission maxima at 486 nm. Cys-CuNCs was successfully exploited as fluorescent probes for the detection of Rut/Que. The addition of increasing concentrations of Rut/Que led to a gradual decrease in the emission intensity of Cys-CuNCs. The decrease in Cys-CuNC emission intensities were attributed to the strong IFE and static quenching mechanism. The calculated limit of detection values for Rut and Que were as low as 0.021 µM and 0.035 µM, respectively. The Cys-CuNCs sensing probe exhibited excellent selectivity in the presence of other potential interfering compounds. Furthermore, the present method was successfully applied to the analysis of both Rut and Que in biological samples.

Smart phone assisted quinoline-hemicyanine based fluorescent probe for the selective detection of glutathione and the application in living cells.

Quinoline appended hemicyanine 6MIM with strong ICT character was successfully synthesized through simple condensation reaction of 6-methoxy-2-chloro-3-formyl quinoline with 2-benzothiazolinium iodide. The photophysical characteristics of synthesized probe revealed that it would selectively detect glutathione (GSH) when it compared with different amino acids including biothiols and the detection limit is found to be 100 nM. The turn off sensor is due to thiol-halogen SNAr nucleophilic substitution between 6MIM and thiol group in glutathione. More importantly, the biosensor 6MIM was effectively applied in the fluorescence bioimaging of GSH in living cells with low cell toxicity. The colorimetric detectable color change of 6MIM-GSH has been effectively integrated with smartphone assisted RGB color value application with lowest detection value of 120 nM.

Colorimetric determination of cysteamine based on the aggregation of polyvinylpyrrolidone-stabilized silver nanoparticles.

A simple, colorimetric and visual method is described for the determination of cysteamine (CA) using polyvinylpyrrolidone-stabilized silver nanoparticles (PVP-AgNPs) as a colorimetric probe. The sensing method was based on the aggregation of PVP-AgNPs that led to the changes in the color and absorption profile of the probe. The aggregation of PVP-AgNPs in the presence of CA was evidenced by using transmission electron microscopy (TEM), zeta and dynamic light scattering (DLS) measurements. A distinct color transition could be observed with the naked eye from pale yellow color of PVP-AgNPs to purple. PVP-AgNPs probe showed an excellent selectivity towards CA versus other interfering biomolecules, cations and anions. Furthermore, the colorimetric probe had a linear response for CA from 0.1 to 1.0 µM concentration range with the limit of detection (LOD) of 4.9 nM. The prepared probe was successfully utilized for the determination of CA in blood serum as biological samples.

Exploring the binding mechanism between methylene blue and ovalbumin using spectroscopic analyses and computational simulations.

The detailed investigation of methylene blue (MB) dye with ovalbumin (OVA) was examined by multispectroscopic and computational techniques. The experimental results of emission spectral studies displayed that the quenching behaviour of OVA with MB dye is due to static quenching mechanism. Isothermal titration calorimetry experimental results suggested that the binding of MB dye became favoured with the aid of a favourable entropy contribution and negative enthalpy. The absorption and circular dichroism spectral experiments showed that the binding of MB dye prompted conformational modifications to the secondary structure of OVA protein. The computational studies have been used to predict the binding region and the stability of MB in OVA protein.Communicated by Ramaswamy H. Sarma.

Synthesis of novel 1,10-phenanthroline derivatives and it used as probes for sensitive detection of Zn2+ and Cd2+ metal ions - Spectroscopic and theoretical approach.

A novel class of unexpected 1,10-phenanthrolinederivatives were synthesized from 2,3-dihydroacridin-4(1H)-ones with 3-aminonaphthalen-2-carboxylic acid in presence of phosphorus oxychloride at 130°C and simple perceptive emission intensity increasing assay was developed effectively to detect the very low concentrations of Zn2+ and Cd2+ ions. Emission intensity of compounds 3(a-c) directly related to the concentrations of Zn2+ and Cd2+ ions was due to metal chelating enhanced fluorescence (CHEF) effect and also its further validated by fluorescence lifetime measurement. Furthermore, the sensing mechanism for compounds 3(a-c) of Zn2+ and Cd2+ were sustained by theoretical calculations. Computational analysis results reveals that compounds 3(a-c) are more interested in Zn2+ ions than that of Cd2+ ions, while, compound 3c is more interested with Zn2+ and Cd2+ ions than those of the rest of the compounds. In addition, this proposed detection analysis has the direct application for monitoring Zn2+ and Cd2+ concentrations in tap and drinking water samples.

Red emitting human serum albumin templated copper nanoclusters as effective candidates for highly specific biosensing of bilirubin.

In this paper, we report a new type of human serum albumin (HSA) stabilized red emissive copper nanoclusters (HSA-CuNCs) were prepared at room temperature and HSA-CuNCs were applied to identify the bilirubin in human urine and blood serum samples. The emission characteristics of synthesized HSA-CuNCs were pH responsive to that the intensity of emission enhanced quickly with varying the pH range from 12 to 6. Emission spectral signal of HSA-CuNCs was found as reduced well with the raise in the amounts of bilirubin attributed to strong binding attraction leads to the non-fluorescent complex formation of HSA-CuNCs with bilirubin. Due to the strong affinity between the nanoprobe and analyte, the red emissive HSA-CuNCs illustrates more specific for the detection bilirubin over the different potential interfering molecules. Two good linear relationships were distinguished the relative emission intensity of HSA-CuNCs versus bilirubin concentrations range from 1.25 × 10-6 to 7.50 × 10-6 M and 5.00 × 10-6 to 2.875 × 10-5 M with lowest limit of detection was determined as 35.00 × 10-9 M and 145.00 × 10-9 M (S/N = 3), respectively. Furthermore, this methodology was effectively used in the quantification of bilirubin in clinical (real) samples. In addition, this fluorometric method offers cost-effective, easy, highly specific and ultrasensitive optical platform for the determination of bilirubin.

Coumarin based hydrazone as an ICT-based fluorescence chemosensor for the detection of Cu2+ ions and the application in HeLa cells.

We have constructed a new coumarin based fluorescence probe BENZPYR with ICT character through condensation of N, N-diethylamino-3-acetyl coumarin with 2-hydrazinobenzothiazole. The absorbance and fluorescence spectral characteristics of BENZPYR revealed that the chemosensor can specifically detect for Cu2+ ions over other different metal ions and the lowest limit of detection was found in nano molar range. The turn off sensor of BENZPYR is related to chelation enhanced quenching (CHEQ) and intramolecular charge transfer (ICT) processes were serve as excellent fluorescent detection of Cu2+ ions in DMF medium. Fluorescence microscopy experiments revealed that probe BENZPYR may have application as a fluorophore to detect the Cu2+ in living cells. The simulated DFT analysis of electronic and structural properties and also UV-vis absorption spectra are in well accordance with the experimental UV-vis absorption spectra.

Investigation of binding interactions between BSA and [EPMpyr][Sal] through spectroscopy studies, thermophysical and thermodynamic properties.

The intensity of research, probing the interactions between proteins and ionic liquid (IL), has been increasing and parallels the fast-growing applications of ILs in biotechnology. The specific aspects which have attracted the involvement of researchers are stabilization, separation, biochemical and enzymatic reactions of proteins. In this work the synthesis of IL, epoxypropyl and N-methyl substituted 2­oxopyrrolidinium cation with salicylate anion, [EPMpyr][Sal], and its interaction with aqueous BSA{BSA(aq)-[EPMpyr][Sal]}. Measurements of thermophysical properties (density (ρ), and speed of sound (u)) showed that both moderately strong and weak interactions occur on treatment of BSA with that chosen IL. H-bond formation, dipole-dipole interactions and ionic interactions occurring in this system were investigated via thermophysical and thermodynamic properties as well as spectroscopic data. Thermodynamic data (excess molar volume (VmE), isentropic compressibility (ks), deviation in isentropic compressibility (∆ks) and intermolecular free length (Lf)) showed that there were stronger interaction between IL and BSA at higher temperature. The data from all the studies were correlated with Redlich Kister polynomial equation. The blue shift observed in the fluorescent spectra was interpreted to indicate that thetryptophan (Trp) residue of BSA moves to a more hydrophobic environment. It was also observed that the addition of more IL to BSA resulted in denaturation of BSA due to high hydrophobic nature of IL. Circular dichroism studies show that there were significant changes in the fine structure of BSA on interaction with IL. From the FTIR spectra the position of H-bond in the secondary structure of BSA was deduced.

New insights into the binding interaction of food protein ovalbumin with malachite green dye by hybrid spectroscopic and molecular docking analysis.

Communicated by Ramaswamy H. Sarma.

Exploring the interaction of Azure dyes with t-RNA by hybrid spectroscopic and computational approaches and its applications toward human lung cancer cell line.

In the present study, in depth characterization of binding aspects of Azure A (AZA) and Azure B (AZB) with transfer Ribonucleic acid (t-RNA) from Escherichia coli (E.coli) is investigated using spectroscopic techniques. The absorbance and fluorescence properties of these dyes have been remarkably changed upon binding with t-RNA. Significant changes in the absorption maxima of the dyes evidence the t-RNA induced metachromasy and the binding clearly revealed the high affinity of AZA and AZB to t-RNA. Strong emission polarization of the bound dyes and strong energy transfer from the guanine base pairs of t-RNA suggested intercalative binding interaction. The stoichiometry of AZA and AZB with t-RNA complexes are determined by the Benesi-Hildebrand plot from emission data. The negative values of free energy change indicated the involvement of hydrophobic forces and noncovalent interactions in the complexation of both the dyes with t-RNA. The 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in A-549 human lung cancer cell lines reveals that binding of t-RNA reduces the toxicity of AZA and AZB. The utility of the present work explores the potential binding applicability of these dyes to t-RNA for their development as effective therapeutic agents and its target at molecular level for the treatment of diseases like cancer.

Protein-Localized Bright-Red Fluorescent Gold Nanoclusters as Cyanide-Selective Colorimetric and Fluorometric Nanoprobes.

Herein, we describe a bright-red-emitting ovalbumin-protected gold nanoclusters (OVA-AuNCs) that were prepared and applied as a luminescent probe for a simple, rapid, and highly sensitive determination of cyanide ions (CN- ions) based on an emission quenching and colorimetric method. Initially, an intense red-emissive fluorescence of the OVA-AuNCs successfully disappeared upon the addition of CN- ions. The resultant emission-quenching process involved CN- ions etching the OVA-AuNC surface, which produced AuCN2 - complexes in the presence of ambient oxygen. Under optimized experimental conditions, the relative emission intensity is inversely relative to CN- ion concentrations ranging from 5.00 × 10-7 to 75.00 × 10-7 mol/L with a linear correlation coefficient of 0.9932. Furthermore, OVA-AuNC-based optical detection systems on both colorimetric and fluorometric assays were tested, which expose highly sensitive and specific determination of CN- ions, and it is easily visualized by the naked eye (day light and UV light). Because of the distinct Elsner reaction between Au atoms of OVA-AuNCs and CN- ions, the recent nanoprobe offered ultrasensitivity and good selectivity with the lowest limit of detection value of 68.00 × 10-9 mol/L. In addition, this fluorescence "turn-off" CN- ion detection method was executed in real water samples. The demonstrated route of OVA-AuNC preparation is extremely easy and quick, making the proposed selective and sensitive CN- ion sensing assay based on the fluorescence response of the OVA-AuNCs for numerous practical applications.

Fluorescence Sensing Approach for High Specific Detection of 2,4,6-Trinitrophenol Using Bright Cyan Blue Color-Emittive Poly(vinylpyrrolidone)-Supported Copper Nanoclusters as a Fluorophore.

In this paper, we illustrate an efficient, convenient, and simplistic fluorescence technique for the specific identification for nitro explosive 2,4,6-trinitrophenol (TNP) in 100% water medium by bright cyan blue color-emitting poly(vinylpyrrolidone)-supported copper nanoclusters (PVP-CuNCs) as a fluorescence probe. PVP-CuNCs exhibited linear fluorescence quenching response toward the increasing concentration of TNP analyte. Surprisingly, TNP only reduces the emission signal of PVP-CuNCs, whereas various nitro explosives cause very slight reducing emission intensity, validating the good specificity of the PVP-CuNC probe toward TNP. The highest Stern-Volmer quenching constant (K sv) value of 1.03 × 107 dm3 mol-1 and the extremely lowest limit of detection of 81.44 × 10-12 mol dm-3 were achieved solely for TNP in 100% water medium which is astonishing and exclusive for this nanoprobe. The sensing pathway for the high sensitivity of PVP-CuNCs assay to quantify the TNP is expected to combine with the inner filter effect process and static quenching. The static quenching mechanism between TNP and PVP-CuNCs is further verified by fluorescence decay measurements. Furthermore, the developed fluorescence sensing platform is applied for the quantification of a trace amount of TNP in real samples named dam water, sea water, and match stick.

Elucidation of Binding Mechanism of Photodynamic Therapeutic Agent Toluidine Blue O with Chicken Egg White Lysozyme by Spectroscopic and Molecular Dynamics Studies.

The nature of binding mechanism of toluidine blue O (TBO) with chicken egg white lysozyme was studied comprehensively by various spectroscopic and computational methods. Both steady state and time-resolved fluorescence studies unambiguously point to the prevalence of static quenching mechanism in lysozyme-TBO system. Thermodynamic parameters revealed that the association of TBO with lysozyme was a spontaneous process in which hydrophobic and hydrogen bond interactions played a pivotal role in the binding process. The secondary and tertiary conformational changes of lysozyme in the presence of TBO were unraveled using absorption, Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD) techniques. Molecular docking studies of lysozyme-TBO system substantiated the findings of site marker experiment and revealed TBO adjacent to Trp-63 and Trp-108 residues of lysozyme. Molecular dynamics (MD) simulation studies of lysozyme-TBO system indicate a stable and effective complexation of TBO with lysozyme. It is hoped that the results presented here will enable further understanding of TBO toxicity.

Probing the interaction of thionine with human serum albumin by multispectroscopic studies and its in vitro cytotoxic activity toward MCF-7 breast cancer cells.

The studies on protein-dye interactions are important in biological process and it is regarded as vital step in rational drug design. The interaction of thionine (TH) with human serum albumin (HSA) was analyzed using isothermal titration calorimetry (ITC), spectroscopic, and molecular docking technique. The emission spectral titration of HSA with TH revealed the formation of HSA-TH complex via static quenching process. The results obtained from absorption, synchronous emission, circular dichroism, and three-dimensional (3D) emission spectral studies demonstrated that TH induces changes in the microenvironment and secondary structure of HSA. Results from ITC experiments suggested that the binding of TH dye was favored by negative enthalpy and a favorable entropy contribution. Site marker competitive binding experiments revealed that the binding site of TH was located in subdomain IIA (Sudlow site I) of HSA. Molecular docking study further substantiates that TH binds to the hydrophobic cavity of subdomain IIA (Sudlow site I) of HSA. Further, we have studied the cytotoxic activity of TH and TH-HSA complex on breast cancer cell lines (MCF-7) by MTT assay and LDH assay. These studies revealed that TH-HSA complex showed the higher level of cytotoxicity in cancer cells than TH dye-treated MCF-7 cells and the significant adverse effect did not found in the normal HBL-100 cells. Fluorescence microscopy analyses of nuclear fragmentation studies validate the significant reduction of viability of TH-HSA-treated human MCF-7 breast cancer cells through activation of apoptotic-mediated pathways.