Surface and Optical Properties of PVP Stabilized PHINPs
The histogram of particle size distribution of PVP stabilized PHINPs presented in Fig. 1 shows narrower particle size distribution in the range from 50-100 nm and the average particle size is 77.5 nm. Fig. 2 shows FESEM images of air dried film of aqueous suspension of PHINPs in different stabilizers and film prepared without stabilizer. It is seen that the image of the film prepared in PVP stabilizer reveals spherical monodispersed particles of average size 150 nm. However morphology of PHINPs prepared using other stabilizers seen irregular in SEM images. The observed larger size of the nanoparticle spheres in FESEM analysis as compared to the value obtained from DLS technique is due to agglomeration of nanoparticles which forms large size spheres during evaporation of water from aqueous suspension in the process of the preparation of thin film on silicon wafers.
The zeta potential distribution of PVP stabilized PHINPs shown in Fig. 3 gives -24.9 mV zeta potential due to electron rich ‘O’ atoms of PVP capping. The hydrophilic pyrrolidone part acted as head group while the hydrophobic -[CH2-CH] n part is the tail. During the formation of nanosuspension the PHI molecules nucleate and aggregate to form dimeric species in aqueous medium. The absorption spectrum of nano suspension and that of dilute solution of PHI in acetone presented in Fig. 4 shows blue shift indicating formation of H-type of aggregates due to lateral π-stacking effect between neighboring PHI molecules in suspension [16-17]. However the fluorescence spectrum (D) of nanosuspension in Fig. 5, peaking at 406 nm is seen enhanced very strongly in comparison with the weak structured fluorescence of dilute solution of PHI appeared in the wavelength region of 400-500 nm (spectrum B). The broad, pronounced fluorescence spectrum of nanosuspension of PHI is also shifted towards blue of the fluorescence spectrum of its dilute solution. In addition Fig. 5, shows a large stoke shift of 7116 cm-1 between the excitation spectrum (C) and fluorescence spectrum (D) of nanosuspension of PHI. On the contrary the stoke shift value of 3932 cm-1 estimated from excitation spectrum (A) and fluorescence spectrum (B) of dilute solution of PHI in acetone is small. These observations suggest that the fluorescence of nanosuspension is aggregation induced enhanced emission (AIEE). The results of pH dependence of fluorescence intensity presented in Fig. 6 show maxima at pH 7.5.
Recognition Test of Nanosuspension of PHI for HSA
The fluorescence spectra produced using 315 nm excitation wavelength of nanosuspension of PHI in presence of various biologically important molecules such as HSA, bovine albumin serum (BSA), D-penicillamine, guanine, sucrose, glucose, cyanocobalmine, hemoglobin, vit.B-1, glutathione each of 1.8 µM concentration are given in Fig. 7. The careful observation of the spectra reveals that the fluorescence of nano suspension of PHI is enhanced significantly by HSA as compared to enhancement induced by BSA, while other biomolecules decreases the fluorescence of nanosuspension. In addition to this the bar diagram in Fig. 8 indicating effect of fluorescence change estimated as, (‘F0’-is the intrinsic fluorescence of nanosuspension and ‘F’ is the fluorescence in presence of biomolecules) supports to the observation of recognition of HSA by suspended nanoparticles of PHI. The blue bar in Fig. 8 shows enhancement effect by HSA and BSA. The enhancement produced by HSA is significantly large in comparison with BSA. The orange bar seen in the same figure reveals that the fluorescence enhancement induced by HSA is not affected even in presence of coexisting substances. In addition to these the comparison of the fluorescence spectrum of nanosuspension containing HSA with the pure spectrum without HSA shown a blue shift of about 2035cm-1 while BSA does not produced any spectral shift. This observation led to consider binding between PHI nanoaggregate and HSA molecule only.
Studies on Binding Interactions between HSA and PHI Nanoparticles
The gradually increasing amounts of HSA solution in the concentration range 0.1-1.8 µM were added to definite amount of nanosuspension of PHI buffered at pH 7.5 taken in the different test tubes (ESI, Table S2). Fluorescence spectra of the nanosuspension monitored at excitation wavelength of 315 nm in presence of HSA and without HSA are shown in Fig. 9. From the spectra it is seen that the fluorescence intensity of PHI nanosuspension not only increases significantly but also the wavelength of maximum emission shifts from 406 nm to 375 nm as concentrations of HSA increases.
The fluorescence enhancement and observed wavelength shift effect are discussed on the basis of complex formation between PVP capped PHI nanosuspension through the electron rich ‘O’ atoms of PVP with HSA molecule. It is thought that PVP may acts as receptor between PHINPs and HSA. This proposed binding interaction mechanism further supported by the double logarithmic plot [18].The binding constant ‘K’ and binding sites ‘n’ are estimated from the double logarithmic plot Log10 (F0-F)/F Vs Log [HSA] as shown in Fig. 10. This plot is based on equation 1 given below.
![](https://myfiles.space/user_files/58853_cdc0f79cc190fa60/58853_custom_files/img1618252381.png)
where F0 and F are the fluorescence intensities of PHINPs in absence and presence of HSA concentration. The value of K obtained from the plot is 7.030 × 105 Lit. mol-1 indicates good stability of complex while the value of binding site (n) nearly equal to 1 indicates availability of one possible binding site on the surface of suspended nanoparticles.
Fluorescence lifetime determinations of nanosuspension of PHI in presence of HSA and without HSA are found in harmony with the proposed mechanism of molecular interaction leading to formation of complex. Fig. 11 shows the decay profile of PHINPs in absence and in presence of HSA solution. The life time 7.29 ns (Fig. 11[A]) of suspension without HSA found to increase in order of 8.02 ns, 8.52 ns and 10.61 ns (Fig. 11[B-D]) when concentration of HSA solution was 0.3 μM, 0.4 μM and 1.0 μM respectively. The molecular interactions and complexation lead to prolonged fluorescence lifetime [19].Thus increase in lifetime of suspension with addition of HSA suggests stabilization of PVP-PHINP-HSA complex. The schematic representation of fluorescence enhancement of PHINPs upon interaction with HSA is graphically presented in scheme 2. The average particle size and zeta potential measurements further confirm complex formation between PHI nanoparticles and HSA. The zeta potential of as prepared nanosuspension -24.9 mV seen in Fig. 12 decreases to -16.7 mV and to -10.6 mV when concentration of HSA increased from 0.5 μM and to 1.0 μM, while at the same time the particle size seen increasing from 77.5 nm to 250 nm and to 481.1 nm respectively. The increase in particle size and decrease in zeta potential led to consider adsorption of HSA over the negatively charged surface of suspended particles [20]. Thus the observed fluorescence enhancement is attributed to the possible interaction with negatively charged oxygen atom of PVP with HSA as presented in scheme 2.
Exploration of mechanism of binding between PVP capped PHINPs and HSA by absorption spectroscopy
The absorption spectra of the PHINPs in presence of different amounts of HSA showed in Fig. S3 reveals the red shift in wavelength of maximum absorbance as indicating complex formation between PVP capped PHINPs and HSA. The PVP acts as receptor between them for enhancing the rate of charge transfer in complex formation.
Molecular Docking Studies
To provide further, the deeper insight into the interaction of HSA with PHINPs, a molecular docking technique is used. The experiment of docking studies carried out between human serum albumin (2bx8.pdb) and PVP-N-PHI is shown in Fig. 13. The three-dimensional structure of human serum albumin (2bx8.pdb) was extracted from RCSB PDB. Further the three dimensional structure of PVP-N-PHI was built by SPARTAN ver 6.0.1 Softwareand minimized by using HartreeFock (HF) method in the SPARTAN ver 6.0.1 Software [21-22]. After completion of minimization the PVP-PHINPs structure with less energy were used for the molecular docking with human serum albumin (2bx8.pdb). Finally, the docked complex of PVP-N-PHI with human serum albumin (2bx8.pdb) was analyzed by CHIMERA.The best conformation with least binding energy of 5.02KJ/mol was visualized as presented in Fig. 13.
The best possible solution obtained by the molecular docking study suggests that PVP capped PHI nanoparticle is able to interact with HSA by means of hydrogen bonding. The amino acid residues of human serum albumin (2bx8.pdb) such as TYR-150, GLN-196, HIS -242, LEU-238, ARG-257 are having strong hydrogen bonding interactions with the PVP-PHINPs. These interactions are predicted to occur at distances required for effective H-bonding ranging from 1.837Å to 2.774 Å (Table 1). Thus the formation of PVP-PHINP-HSA complex through hydrogen bonding interactions is strengthened by molecular docking analysis.
Application of Nanosuspension of PHI for the Detection of HSA in Biological sample
A calibration curve as shown in Fig. 14 is constructed by plotting fluorescence intensity (∆F) increase as a function of concentration of standard solution of HSA added in the known amount suspension of PHINPs. The calibration graph in Fig.14 is linear over the range of concentration of HSA from 0.0 µM to 1.8 µM. The correlation coefficient of the plot is 0.9902. The limit of detection calculated by using equation 2 is 0.032287µM [23].
![](https://myfiles.space/user_files/58853_cdc0f79cc190fa60/58853_custom_files/img1618252396.png)
Where,‘s’ is the standard deviation of ‘y’ intercept of the regression line, ‘K’ is the slope of the calibration graph. An analytical method is developed for the quantification of HSA in the serum samples collected from the Health Centre of Shivaji University, Kolhapur. The blood samples were allowed to clot at room temperature for 15-30 minutes and then the clot was removed by centrifuging at 2000 rpm for 10 minutes in refrigerated centrifuge. The resulting supernatant liquid is serum. Serum consists of antibodies, antigens, electrolytes, hormones and proteins [24].An appropriate amount of the serum samples were spiked with HSA to prepare two synthetic samples of concentration 0.3 µM and 0.7 µM, and diluted by distilled water so that fluorescence intensity of samplesolutions is in the working range of calibration curve. The results of analysis shown in Table 2 indicate that the amount of HSA found by proposed method is in close agreement with amount added with good percent recovery.