Hydrophobic quercetin encapsulated hemoglobin nanoparticles: formulation and spectroscopic characterization.

Various natural proteins are finding application in drug delivery for their high biodegradability and biocompatibility. Albumins are well explored and now focus is shifting to other proteins like hemoglobin (Hb) with unique structural properties. In the present study Hb is allowed to denature at pH 5.0 and model hydrophobic drug quercetin (Q) is encapsulated via self-assembly and hydrophobic interactions. Fluorimetric titrations record highest binding between Hb and Q at pH 5.0, rendering significant structural changes in Hb as captured in CD spectra. A decrease in fluorescence life time of tryptophan residues from 3.31 ns in Hb to 2.89 ns in presence of Q at pH 5.0; surmises efficient binding of Q at the hydrophobic core housing tryptophan. Peak shifts in Fourier transform infrared spectroscopy spectra of Hb-Q compared to Hb evidence significant interactions between them at pH 5.0. Significant spectral changes in soret band region of Hb on addition of Q at pH 5.0 envisages unfolding of porphyrin ring and binding influence of Q. Efficient formation of Hb-Q nanoparticles (NPs) at pH 5.0 is established by DLS, SEM and TEM.Communicated by Ramaswamy H. Sarma.

Room temperature synthesis of NIR emitting Ag2S nanoparticles through aqueous route and its influence on structural modulation of DNA.

Near infra-red (NIR) light emitting nanomaterials had shown great promise in clinical imaging in view of negligible absorption by skin or tissue of mammalian. Thus, it demands for synthesizing stable NIR emitting nanomaterials in water environment. The present work presents synthesis of biologically acceptable luminescent near-IR emitting silver sulfide nanoparticles through an aqueous route using 2-mercaptoethanol. The prepared as-synthesized Ag2S nanoparticles exhibited bright photoluminescence with quantum yield of ca. 4%. X-ray diffraction (XRD) analysis indicated that the products were monoclinic α-Ag2S. Fourier transform infrared spectral analysis revealed that the stretching vibration at 2560 cm-1 responsible for S-H bond of thiol group disappeared suggesting the conjugation of 2-mercaptoethanol with Ag2S nanoparticles. In view of investigating any possible effect on genetic materials, interactions of the synthesized particles with calf thymus DNA was investigated employing Ethidium bromide (EB) as structural probe. To understand the binding mechanism, the UV-vis absorption, fluorescence and circular dichroism (CD) spectroscopic, as well as DNA melting studies measurements were carried out. The observed results confirm that nanoparticles interact with DNA through groove binding.

Physicochemical Understanding of Protein-Bound Quantum Dot-Based Sensitive Probing of Bilirubin: Validation with Real Samples and Implications of Protein Conformation in Sensing.

Precise and rapid determination of free bilirubin (BR), a key biomarker of pathological conditions of the liver, is important clinical issue. The present study demonstrates that the combination of the strong specific affinic properties of protein, bovine serum albumin (BSA), toward bilirubin and luminescence of well-characterized semiconductor quantum dots (QDs) can offer a simple, fast, and sensitive technique for the determination of free bilirubin through quenching analysis. Here, BSA molecule not only stabilizes the quantum dots in an aqueous environment but also helps bring BR closer to QDs during the interactions of CdSe-BSA QDs with BR. Further, it is revealed through photophysical investigation that the conformation of protein molecule may play an important role in biomolecular sensing considering bilirubin as a model target molecule. The luminescence of CdSe-BSA QDs was highly responsive toward bilirubin, where nearly 90% of emission intensity was quenched on adding only 40 µM bilirubin, suggesting strong interactions involved between synthesized QDs and bilirubin. Solvent polarity dependence on luminescence changes confirms strong electrostatic interaction between the QDs and BR. The applicability of the synthesized quantum dots in sensing bilirubin has been checked in the presence of different possible interfering agents and also with plasma isolated from real blood samples of both normal and hepatitis patients. It was observed that bilirubin as control sample as well as in human serum sample can be optimally measured at pH 7.5, 25 °C. Thus, the proposed strategy being able to measure free BR even at least two orders of magnitude lower than bilirubin level in normal blood may provide a reasonable protocol to determine BR in the pathophysiology of many critical human diseases, like hepatitis and Gilbert's syndrome in the near future.

Interaction of flavonols with human serum albumin: a biophysical study showing structure-activity relationship and enhancement when coated on silver nanoparticles.

Binding affinities of flavonols namely quercetin, myricetin, and kaempferol to human serum albumin (HSA) were determined fluorimetrically and the order was observed to be myricetin > quercetin > kaempferol demonstrating structure-activity relationship. Quercetin-coated silver nanoparticles (AgNPs) show higher binding affinity to HSA compared to free quercetin with binding constants 6.04 × 107 M-1 and 4.2 × 106 M-1, respectively. Using site-specific markers it is concluded that free quercetin and that coated on AgNPs bind at different sites. Significant structural changes in circular dichroism (CD) spectra of HSA were recorded with quercetin-coated AgNPs compared to free quercetin. These results were further substantiated by time-resolved fluorescence spectroscopy where fluorescence life time of the tryptophan residue in HSA-quercetin-coated AgNPs complex decreased to 3.63 ns from 4.22 ns in HSA-quercetin complex. Isothermal calorimetric studies reveal two binding modes for quercetin-coated AgNPs and also higher binding constants compared to free quercetin. These higher binding affinities are attributed to altered properties of quercetin when coated on AgNPs enabling it to reach the binding sites other than site II where free quercetin mainly binds.

Synthesis of Excitation Independent Highly Luminescent Graphene Quantum Dots through Perchloric Acid Oxidation.

We demonstrate a facile liquid phase exfoliation method by only using perchloric acid to synthesize graphene quantum dots (GQDs) having excitation independent strong emission with a quantum yield of about 14%. The proposed simplified synthesis strategy can help in overcoming the limitations of existing aqueous routes which produce GQDs with excitation dependent emission and of low quantum efficiency. Photoluminescence (PL) properties of GQDs have been studied in detail to understand the origin of emission. As-synthesized GQDs show excitation independent photoluminesce (PL) which suggests that the synthesized materials do not have any significant defects. Spectral analysis suggests that the PL emission of the well-defined GQDs originates mainly from the peripheral functional groups conjugated with carbon backbone planes. We also demonstrate a relatively longer PL lifetime (average lifetime of about 10 ns) of the synthesized GQDs determined by time correlated single photon counting (TCSPC) measurement and this high lifetime suggests that the synthesized GQDs may be suitable for biomacromolecular probing. In addition, as-synthesized GQDs interestingly show delayed fluorescence and steady state anisotropy, which make the material an appropriate candidate for application in sensing and bioimaging of cells and organisms.

Aqueous Synthesis of Protein-Encapsulated ZnSe Quantum Dots and Physical Significance of Semiconductor-Induced CuII Ion Sensing.

In view of their promising bio-applicability, we have synthesized water-soluble bovine serum albumin (BSA)-encapsulated ZnSe quantum dots (QDs) with visible emission with longer average luminescence lifetimes of approximately 125 ns at ambient conditions. BSA-ZnSe QDs are shown to be efficient selective copper ion probes in the presence of physiologically important metal ions through luminescence quenching with a high Stern-Volmer constant (3.3×105 m-1 ). The mechanism of sensing has been explained in terms of electron transfer processes and the apparent rate of electron transfer (Ket ) from ZnSe QDs to Cu2+ has been calculated to be 2.8×108  s-1 . It is demonstrated that the negative conduction band potential plays a major role in the feasibility of the electron transfer process, which is reflected in the higher efficacy of ZnSe QDs in sensing copper(II) ions over other group II-VI quantum dots, namely, CdSe, ZnS, or CdS. The results observed with cysteine-capped QDs are almost identical to those with BSA-encapsulated QDs and this presumably negates the possible reason of CuII ion induced quenching ascribed to its binding with surface groups or replacement of metal sites as proposed by several groups previously.

Exploiting the biomimetic and luminescence properties of multivalent dendrimer-semiconductor nanohybrid materials in the ultra-low level determination of folic acid.

In view of the enhanced generation of folate receptors in cancerous cells and diseases linked to the deficiency of folic acid, such as anemia, mental devolution, congenital malformation, etc., the development of a simple method for the ultra-sensitive determination of folic acid remains a long-standing issue for practical applications in medicine and biotechnology. Thus, the proposed luminescence based strategy involving multifunctional poly(amidoamine) (PAMAM) dendrimer encapsulated quantum dots (QDs) as a probe provides a simple, fast and efficient method for the selective determination of folic acid at the nano-molar level. Absorption and Fourier transform infra-red (FTIR) spectroscopy provide evidence of the binding of folic acid with dendrimer amine groups. The emission quenching of dendrimer encapsulated CdS QDs follows a linear Stern-Volmer plot with an exceedingly high value of the Stern-Volmer constant (KSV = 8.4 × 106 M-1) facilitating a higher detection efficiency. Similar quenching analysis with dendrimer-ZnS QDs showed a slightly lower Stern-Volmer constant (KSV = 2.29 × 106 M-1). The lower probing efficiency of the protein or amino acid capping of QDs has been explained through zeta potential measurements. The solvent polarity dependence suggests a charge transfer process responsible for the emission quenching of CdS QDs, which is static in nature as revealed by lifetime measurements. The determination of folic acid at this low level is not affected by possible interfering molecules, such as vitamin C, vitamin B12 and uric acid. Calorimetric measurements showed that the exothermic binding of folic acid with a dendrimer follows enthalpy-entropy compensation. The detailed mechanistic aspect of interactions of folic acid with the QD probe helps in a better understanding of the detection process, which in turn can assist in developing a dendrimer based material for image analysis and drug delivery in folate receptor rich cells.

A comparative evaluation of the activity modulation of flavo and non-flavo enzymes induced by graphene oxide.

Graphene, and its water soluble derivative graphene oxide, has shown great promise in various biomedical applications, such as cancer therapeutics, drug delivery, etc. and in industrial applications such as enzyme immobilization, etc. Thus, modulation of the activities of different classes of enzymes by graphene materials is an important aspect in the formulation of different biological applications. We have demonstrated here how flavin adenine dinucleotide (FAD) moieties protect the binding site from conformational change in the presence of an inhibitor, graphene oxide, and also explore differences in the mode of interactions between flavo and non-flavo enzymes. It was shown that there was a much greater loss of activity with the non-flavo enzyme, l-lactate dehydrogenase (LDH), of ∼74% compared to that with the flavo-enzyme, glucose oxidase (GOX), of ∼45%, in the presence of equal concentrations of GO. Furthermore, GO acts as an enzyme inhibitor and the mode of inhibition is uncompetitive for GOX and competitive for LDH. Circular dichromism measurements showed a 21% decrease in the α helix of GOX and a 31% decrease in the α helix of LDH in the presence of a given concentration of GO (0.5 mg mL-1). There was a slight change in the average emission lifetime of tryptophan in GOX in the presence of GO from 3.2 to 2.6 ns. In contrast, there was no change in the average emission lifetime of tryptophan in LDH in the presence of GO. The extents of fluorescence quenching for GOX and LDH were 39% and 70% upon addition of a certain amount of GO. The present study provides insight into the development of sensors through the immobilization of enzymes and the possible formulation of a multifunctional protein and graphene composite system for various biomedical applications such as bio-sensing, gene and drug delivery, etc.

SERS Enhancement on the Basis of Temperature-Dependent Chemisorption: Microcalorimetric Evidence.

The mechanism of surface-enhanced Raman spectroscopy (SERS) is not very clear in view of the magnitude of the contribution of electromagnetic factor as well as the chemical mechanism. This report presents the extent of adsorption at different temperatures in terms of signal enhancements in SERS employing silver nanoparticles (AgNPs) of various shapes as substrate and dye molecules, crystal violet or Rhodamine 6G, as model Raman probes. Initially, the SERS signal increases with increasing temperature until a maximum intensity is reached, before it gradually decreases with increasing temperature. This trend is independent of the shape of the Raman substrates and probes. However, the temperature at which maximum intensity is obtained may depend upon the nature of the Raman probe. The energetics involved in the chemisorption process between dye molecules and AgNPs were determined through isothermal titration calorimetry and their implications for the observed SERS signals were assessed. The maximum heat change occurred at the temperature at which the maximum signal enhancement in SERS was obtained and the enhanced interaction at optimum temperature was confirmed by absorption spectroscopy.

Synthesis and spectral measurements of sulphonated graphene: some anomalous observations.

The present report demonstrates how a sulphonation process, a key route for synthesizing water soluble graphene, can influence the optical behavior of precursor graphene oxide, intermediate reaction products and sulphonated graphene. We observed that there is constant emission maximum at 500 nm for graphene oxide in the excitation range of 320-450 nm. During sulphonation, sulphonated reduced graphene oxide (rGO-SO3H) is initially formed which has an emission at 358 nm. However, the reduction of oxygen containing groups in rGO-SO3H with hydrazine hydrate leading to the formation of SG caused a shift in the emission to 430 nm, which has been attributed to the extended delocalization of π-electrons involving the phenyl sulphonate group. In the present investigation, we have identified many existing anomalies in the important spectral features of these materials, such as violation of Kasha's rule on fluorescence and pH dependence emission. Furthermore, it has also been shown that proper care is necessary to be taken in monitoring the fluorescence of sulphonated graphene in view of possible interference from the components produced during sulphonation.