Fluorescence enhancement via aggregation effect due to microenvironmental alterations in human hemoglobin protein in presence of carbon quantum dots (CQD): Comparative spectroscopic approach.

Abstract CQDs have emerged with outstanding properties as a star member of carbon nanomaterial family and in order to reveal its wide-range of application in biological microenvironment the interactions between human hemoglobin (HHb) and CQD and also with ethylenediamine-functionalized CQD (NCQD) are assessed using several techniques. Firstly, UV–vis absorption spectra of HHb reveal hyperchromic effect in the region of absorbance of tryptophan and tyrosine residues and also hypochromicity of Soret band in presence of CQD and NCQD. Interestingly, steady-state fluorescence spectroscopy reveal distinct fluorescence enhancement of HHb with significant red shift thereby indicating exposures of tryptophan and tyrosine residues to a more hydrophilic environment. However synchronous fluorescence spectra reveal that the microenvironment of tryptophan and tyrosine residues is altered in opposite manner, i.e. exposure of tryptophan residues to a more hydrophilic environment and the tyrosine residues to a more hydrophobic environment. Moreover the fluorescence enhancement is observed to be accompanied by increase in average fluorescence-lifetime and decrease in steady-state anisotropy thus signifying a decrease in restriction of rotational motion. Furthermore tryptophan residues within HHb appear to interact more with CQD compared to NCQD. Thermodynamic parameters as revealed by Isothermal Titration Calorimetry (ITC) demonstrate that electrostatic, hydrogen bonding and hydrophobic interactions are the predominant modes of interactions in presence of CQD. Whereas hydrophobic and hydrogen bonding interactions are the major interacting forces in presence of NCQD with five-site sequential binding as best-fit model in both the cases. Such interactions also appear to be associated with an increase in aggregation of HHb as evident from the measurements by atomic force microscopy (AFM) and dynamic light scattering (DLS) study. Although FT-IR spectra display alteration of amide I band, but the overall secondary structure of HHb seems to be nearly retained even in presence of CQDs, as evident in the CD spectra. These observations thus highlight the potential biomedical application of CQDs in biological microenvironment of human especially as drug-delivery system. Also bimolecular interaction of HHb as a model protein with other nanoparticles at the nano bio-interface has been outlined. Graphical abstract It is apparent that the fluorescence enhancement is accompanied by an increase in average lifetime. Since the lifetime determines the time available for the fluorophore to interact with or diffuse in its environment, thus the tryptophan and tyrosine residues within HHb are probably getting more exposed to the hydrophilic environment in presence of CQD as well as NCQD. Also there are possibilities that the CQD and NCQD can induce motional freedom within HHb as a result of which the tryptophan and tyrosine residues get exposed to the polar environment and such phenomenon in turn may bring about an increase in average lifetime. Unlabelled Image Highlights • Fluorescence enhancement of hemoglobin is viewed with CQD and NCQD • Tyrosine residues getting exposed to hydrophobic environment (blue shift) with CQDs • Tryptophan residues getting exposed to hydrophilic environment (red shift) with CQDs. • ITC measurements reveal the mechanisms of the nature of interactions. • The present results indicate potential applications of CQDs in drug-delivery systems [ABSTRACT FROM AUTHOR]