Chapter-4: Photophysical Properties of Luminol

Transcription

1 Chapter-4: Photophysical Properties of Luminol 4.1. Introduction Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, LH 2 ) is a versatile chemical that shows striking blue chemiluminescence in presence of certain metal ions when treated with appropriate oxidizing agent like hydrogen peroxide. This unique feature of LH 2 is often exploited by forensic investigators to detect trace amount of blood left in the crime scene. LH 2 is also used by the biologists as a cellular assay to detect copper, iron and cyanides etc [ ]. Further, LH 2 enhanced chemiluminescent probes have been used to characterize and quantify the secretion of oxygen by phagocytozing cells [108]. The use of LH 2 chemiluminescence has also been reported recently for facile detection of proteins [109], cancer biomarkers [110], as well as for reactive oxygen species produced by human neutrophils [111]. The ultra-high sensitivity of - time-resolved chemiluminescence behavior of LH 2 can be used to measure OH/O 2 radical species concentration as low as mol dm -3 in water [112]. An important aspect of LH 2 chemiluminescence is its different degrees of sensitivity from one substance to another. LH 2 shows higher sensitivity to animal or human blood, organic tissues and fluids than to other compounds containing metal ions, such as paints, metallic surfaces, household products, or vegetable enzymes. Therefore, the light emitted by LH 2 has different intensities and time duration, depending on the material of contact making it an efficient forensic marker Luminol Fluorescence in Homogeneous Media General spectral behavior: Importance of hydrogen bonding Solution phase spectroscopic properties of LH 2 have drawn enormous interest in recent times due to the biochemical relevance of its photoactivity. Photophysical properties of LH 2 in different solvents and solvent mixtures as well as its interaction with several biological molecules were reported in the literature [ ]. LH 2 exhibits two principle absorption bands in 300 and 350 nm region, whereas, a single broad fluorescence emission appears at 400 nm region. Interestingly, the fluorescence emission peak shows large spectral shift towards longer wavelength in hydrogen bonding solvents. This shift is believed to be due to the stabilization of 68

2 the charge transfer excited state of the intermolecular hydrogen bonded complex of LH 2 with solvent [113]. Figure 4.1 Possible tautomeric structures of luminol (LH 2 ). Numbering scheme used in calculation is also shown. Intermolecular hydrogen bonding and solubility of organic systems is known to play a crucial role in determining the biological activity as well as its application in forensic science [79, ]. In general, formation of intermolecular hydrogen bond between solute and solvent results in a decrease in Gibb s free energy and thus promotes mixing. Hydrogen bonding can occur in different modes, depending on the structure of the solute and solvent. The situation becomes more complicated when a solute molecule possesses multiple hydrogen bonding site and the solvent molecule can act both as a proton donor as well as a proton acceptor. Under this condition, the competition among different molecular species resulting from hydrogen bonding interaction between the solute and the solvent molecules becomes inevitable. LH 2 provides a unique example to study the hydrogen bonding effect because the molecule itself 69

3 can exist in more than one prototropic species (figure 4.1) having multiple hydrogen bonding sites. The keto-amine (I) structure can go to the enol-imine (III) form in single step or through the intermediate structures IIa and/or IIb, respectively. These inter-conversion and associated spectroscopic properties will depend strongly on the relative abundance of several species, as well as their hydrogen bonding mode with the solvent. Furthermore, the efficacy of hydrogen bond formation in the excited state may change due to charge redistribution after excitation. Also, relatively strong and unstructured fluorescence of LH 2 is an additional advantage to use the spectroscopic ruler for quantitative estimation of the effect of different solvent parameters. The chemiluminescence and the fluorescence band of LH 2 in water appear in the same wavelength region ( nm) [115]; so quantitative characterization of this band on different solvent parameters is indispensable. In the following sections, we use steady state spectral properties of LH 2 in a series of pure solvents with varying polarity as well as hydrogen bond donor and acceptor abilities to find quantitative information about their relative contribution. The list of all the solvents and some of the important physical parameters are collected in table 4.1. Furthermore, the effects of specific LH 2 water hydrogen bonding on the solution phase spectral properties were theoretically modeled by using density functional approach. Figure 4.2 Absorption (a), Fluorescence emission (b and c, at exc = 350 and 290 nm, respectively) and excitation (d, mon = 425 nm) spectra of mol dm -3 aqueous solution of LH 2. 70

4 Table 4.1 Solvent parameters No. Solvent f(,n) a E T (30) b c c *c 1. Acetonitrile Ethyl acetate Benzene Tetrahydrofuran ,4-dioxane Toluene DMSO DMF Dichloromethane Butanol Methanol Propanol Pentyl alcohol Water Acetone Isopropanol a Polarity parameter; b Reichardt solvent parameter; c Kamlet-Taft solvent parameters Steady state spectral properties in pure solvents Figure 4.2 shows some representative absorption and emission spectra of LH 2 in aqueous medium and table 4.2 summarizes the steady state spectral behavior of LH 2 in solvents with varying polarity and hydrogen bonding parameters. In homogeneous solvents, LH 2 shows two absorption maxima. One relatively structured high energy peak appear at nm region; whereas the other unstructured, broad low energy absorption in the nm region. However, the emission obtained by exciting at both these absorptions show strongly intense, unstructured and broad spectra ranging from 375 to 520 nm. The excitation spectra corresponding to this emission again show a broad peak at 350 nm. In a recent report [115], 71

5 Vasilescu et. al. proposed an acid-base type of equilibrium to explain the two absorption bands of LH 2 in highly alkaline (ph 12.0) DMSO solution for the formation of corresponding dianion of structure III. This was further confirmed by the concomitant quenching of LH 2 fluorescence and appearance of new broad band at nm with increasing alkali concentration. However, the formation of the dianionic species in the present experimental condition of neutral aqueous LH 2 solution (ph 6.4) can be ruled out. The absence of any new fluorescence band further supports this hypothesis. The origin of the broad absorption in nm region can be assigned as S 1 (π) S 0 (π) transition; whereas, the origin of the high energy absorption at 300 nm may be due to S 2 S 0 excitation. From the relatively large absorption coefficient ( max dm 3 mol -1 cm -1 ) of this band, which is comparable to that of 350 nm absorption ( max dm 3 mol -1 cm -1 ), it can be concluded that this transition is also π* π in nature. The assignment of these absorptions as well as further verification for the long wavelength absorption at nm to be originated from the anionic species, is confirmed from theoretical calculations described in the following sections. Although, the results in table 4.2 do not show any regular variation of steady state spectral properties, careful observation reveal several interesting trends. For example, fluorescence maxima (ν em ) shows appreciable shift in protic solvents along with almost two fold increase in fluorescence quantum yield (φ f ), when compared with their aprotic counterpart. Furthermore, full width at half maxima (FWHM) for both the absorption and emission peaks are much higher in water compared to the other solvents. All these results indicate that consideration of hydrogen bonding interaction is very important to describe LH 2 photophysics, more particularly in aqueous medium Solvatochromism of luminol photophysics: Estimation of contributions from solvent parameters To verify the effect of solvent polarity, several steady state spectral parameters of LH 2 in a variety of solvents mentioned in table 4.1 were plotted against the solvent polarity parameter f(, n). From the results given in figure 4.3, it is clear that the spectroscopic properties of LH 2 do not show any regular solvatochromism behavior on the solvent polarity parameter. This observation points to the existence of specific solute-solvent interactions. As a trial, the empirical solvent polarity scale, E T (30), built with a betaine dye, was used, as it is the most 72

6 popular choice to correlate several solvent dependent spectral properties. The uni-parametric scale depends on both the solvent dielectric properties and hydrogen bonding acidity, but it does not take care of solvent hydrogen bonding acceptor basicity [76]. The specificity of Lewis acid base interactions in E T (30) parameter arise from the negative charge localized on the phenolic oxygen of betaine molecule. As it is seen in figure 4.4 again, there is no linear correlation of either LH 2 absorption/emission energies or Stokes-shift even with this parameter. A break point, mostly influenced by LH 2 emission properties like fluorescence maxima (ν em ) and Stokes shift ( ν ss ), is obtained around E T (30) = 38 kcal mol -1. This clearly indicates that apart from solvent polarity, LH 2 solvatochromism is strongly modulated by both solvent hydrogen bond donor acidity and solvent hydrogen bond acceptor basicity parameters also. Figure 4.3 Variation of absorption ( a ), emission ( em ) energies and Stokes-shift ( ss ) with solvent polarity parameter, f(,n). In-view of this situation, one must look at multi-parametric approach, as devised by Kamlet and Taft and mentioned in equation (3.3), to assess the contribution of different solvent parameters on LH 2 solvatochromism. The s, a and b coefficients in equation (3.3) were all obtained by multiple linear regression analysis and the results are given in tables 4.3 and

7 Table 4.2 Steady state spectral properties of LH 2 in homogeneous solvents a Solvent b abs /cm -1 em /cm -1 exc /cm -1 ss /cm -1 f em /cm -1 exc /cm a Abbreviations used: = absorption, emission and excitation energy; ss = Stokes shift; f = fluorescence quantum yield; = corresponding full width at half maxima (FWHM), b The name of the solvents are listed in table 4.1. Few representative correlation diagrams of the experimental values with those calculated from multiple regression analysis using equation (3.3), are shown in figure 4.5. A close look into the tables reveals several interesting feature for LH 2 solvatochromism: (i) in general, the contributions from a as well as b parameters are significant relative to the s parameter, indicating the importance of solvent hydrogen bonding in LH 2 spectroscopy; (ii) the excited state spectral properties like fluorescence maxima, Stokes-shift, quantum yield etc. are mostly controlled by solvent hydrogen bond acidity function (a parameter), whereas, both a and b contributes almost equally in the absorption property. This indicates an efficient charge localization in LH 2 upon excitation (see DFT calculation results in the following section); (iii) the 74

8 charge localization in the excited state is further confirmed by the negative values of both a and s [121]; (iv) almost two fold increase in fluorescence quantum yield in protic medium (table 4.2) is mainly due to the hydrogen bond acidity of the solvents with a/s 1.5. However, solvents like DMSO with higher hydrogen bonding acceptor (HBA) tendency has very little effect ( 8%) on φ f. For example, the φ f value of LH 2 in water is about 0.79 compared to that in 1,4-dioxane (0.25) and DMSO (0.34). This observation is also in line with our finding that the yield of LH 2 fluorescence decreases substantially in presence water soluble proteins like bovine and human serum albumins and discussed more in detail in the next section. The decreased fluorescence intensity of LH 2 on binding to albumins most likely reflects reduced water access to the chromophore in the bound state. Finally, (v) the large spectral shift in water and other hydroxylic solvents, as observed in table 4.2, is due to the negative value of a parameter and its corresponding larger contribution (table 4.4). In summary, the solvatochromic analysis reveals that in polar protic solvents, like water for example, several spectroscopic species may be present due to the hydrogen bonded donor and acceptor properties of the solvent in the ground state; however, in the excited sate, the main fluorescing species is originated due to the hydrogen bonded complex formation of LH 2 through the solvent hydrogen bonding acidity behavior. Figure 4.4 Variation of absorption ( a ), emission ( em ) energies and Stokes-shift ( ss ) with solvent E T (30) parameter. The solid line guides the eye along the variation of em and ss with the solvent parameters. 75

9 Table 4.3 Regression fit to solvatochromic parameters towards the steady state spectral properties of LH 2. a Property (P) P 0 s a b R 2 SD abs /cm em /cm exc /cm ss / cm f em /cm exc /cm a The regression analysis was done using equation (3.3); R 2 and SD indicate correlation coefficient and standard deviation in the regression analysis, respectively. Figure 4.5 Correlation diagram of LH 2 emission energy ( em ), Stokes-shift ( ss ) and fluorescence quantum yield ( f ) with predicted values from Kamlet-Taft equation. 76

10 Table 4.4 Relative values (in percentage) of the solvatochromic parameters towards LH 2 steady state spectral properties. Property (P) P s (%) P a (%) P b (%) abs em exc ss f em exc Calculation Using Density Functional Theory Energetic of different conformers in the ground state Full geometry optimization of different conformers of LH 2 in isolated condition (structures given in figure 4.1 along with the numbering scheme) as well as with different degree of hydration, was done using B3LYP/ G(d,p) methodology. The fully optimized structures are shown in figure 4.6(a-d). The energy parameters, relative to the most stable structure, are given in table 4.5. It is clear that the conformer IIb is the most stable structure in the isolated, monohydrated as well as in the dihydrated configuration. However, comparison of relative energies indicates that LH 2 most likely exists in dihydrated complex structure represented by IIb-S3. The structure represented by I-S3 is about 7.6 kj mol -1 higher in energy than IIb-S3. It may still be possible that this high energy structure will have relatively less abundance in the solution along with the structure IIb-S3 in the ground state; and more so, in the excited state (see below). However, the existence of all other conformers represented by IIa and III can be neglected to discuss the spectroscopic behavior of LH 2 in water. This is because of their relatively higher energy; they are unlikely to be present in solution mixture. It is to be noted here that only primary hydrogen bonded complex with water was considered to give different complexes like S1, S2, and S3 (figure 4.6). It is obvious that additional solvent molecules will combine to give secondary water cluster around LH 2 and the actual hydrated 77

11 structure is far more complex than what is considered for these calculations. However, we restrict ourselves only in the first hydration layer, as the effect of internal hydrogen bonding (IHB) is expected to diminish very fast from the center of origin. Consequently, it is reasonable to believe that any further addition of water structure will have insignificant contribution toward the relative energy parameters. Table 4.5 Relative energy (kj mol -1 ) of fully optimized structures of different conformers of LH 2 in gas phase calculated at B3LYP/ G(d,p) level. Structure a I IIa IIb III Isolated Monohydrated (S1) Monohydrated (S2) Dihydrated (S3) a See figures 4.6(a-d) for the optimized structures of different conformers with varying degree of hydration. Figure 4.6 (a) Fully optimized structures of I conformer of LH 2 in isolated, monohydrated I-S1 and I-S2, and dihydrated I-S3 states. The geometry optimization was done at B3LYP/ G(d,p) level of calculation. Bond lengths and angles are given in Å and degrees, respectively. 78

12 Figure 4.6 (b) Fully optimized structures of IIa conformer of LH 2. The other details are similar to figure 4.6(a). 79

13 Figure 4.6 (c) Fully optimized structures of IIb conformer of LH 2 in isolated (i), monohydrated IIb- S1 and IIb-S2 (ii & iii, respectively), and dihydrated IIb-S3 (iv) states. The geometry optimization was done at B3LYP/ G(d,p) level of calculation. Bond lengths and angles are given in Å and degrees, respectively. 80

14 Figure 4.6 (d) Fully optimized structures of III conformer of LH 2. The other details are similar to figure 4.6(a) TD-DFT calculation on the excited state The excited states of the two most stable ground state structures of dihydrated LH 2 discussed above, i.e. for I-S3 and IIb-S3, were calculated using TD-DFT procedure. The calculated transition energies and the corresponding oscillator strengths for several singlet excitations within the experimentally observed absorption wavelength range of nm region for both the structures are given in table 4.6. It is noted that the nature of transition as well as its energy and oscillator strength is comparable for both the structures. The calculated S 1 S 0 transition wavelength of 335 nm is in close agreement with the experimentally observed value of 350 nm and the gas phase absorption energy of 354 nm (tables 4.2 and 4.3). The nature of the second lowest energy transition in the experiment cannot be assigned unambiguously 81

15 because of the close energy separation of the next two calculated values (294 and 281 nm, respectively) and their similar oscillator strengths. However, all the associated orbitals those might be involved in excitation with significant contribution in this wavelength range, viz. HOMO-1, HOMO, LUMO and LUMO+1 (table 4.6), are shown to be of π type in nature (figure 4.7). Further, to confirm that no proton dissociated anionic species contributes in this wavelength region, TD-DFT calculation was performed on fully optimized anionic species of conformer IIb. The lowest energy absorption appears at 450 nm region that is in close agreement with the experimentally obtained value of nm reported by Vasilescu et. al. [115]. These authors proposed the existence of proton dissociated dinionic structure of conformer III responsible for this absorption. However, from the energy parameters given in table 4.5, it is clear that the formation of this conformation itself is very unlikely. So, the anionic species responsible for the long wavelength absorption is believed to be originated from conformer IIb. Furthermore, comparison of the nature of HOMO and LUMO in figure 4.7 reveals that on excitation, the electron density is more localized on carbonyl oxygen and imino nitrogen atoms (O12 and N10, respectively in figure 4.1), thereby increasing the basicity at these points significantly. This confirms the importance of hydrogen bond donating ability (acidity) of the solvent to discuss the spectroscopic behavior of LH 2, more particularly in the excited state, as indeed observed from LSER analysis discussed above. Table 4.6 Calculated singlet excited state transitions, associated energies and oscillator strength (f) of I-S3 and IIb-S3. a I-S3 IIb-S3 Singlet Transition Energy /nm f Transition Energy /nm f State 1 H L H L H-1 L H L H-1 L+1 H L H-1 L H L a The frontier orbitals HOMO and LUMO are designated as H and L, respectively. 82

16 Figure 4.7 Frontier orbital diagram for the most stable ground state conformer of LH 2 (structure IIb). The arrow mark in LUMO indicates charge localization on the oxygen and nitrogen atoms on electronic excitation. The calculated energy difference between I and IIb in the ground state is 14.1 kj mol -1. The potential energy surface (PES) (figure 4.8), constructed by intrinsic reaction coordinate (IRC) calculation from the transition state (TS), indicates that the water assisted conversion of I to IIb is associated with a large activation barrier of 55 kj mol -1. So, in ground state, relative abundance of the high energy structure (I-S3) will be very less, both from kinetic and thermodynamic point of view. However, TD-DFT calculation results show that, the relative energy difference between I-S3 and IIb-S3 is very small in the first excited state ( 3.4 kj mol -1 ). Hence, simple Boltzmann distribution predicts approximately 25% population of excited state to exist as I-S3 in solution at room temperature. Therefore, the photochemistry of LH 2 can be considered as an average property of both these structures. As shown in table 4.6, the transition energy, nature of excitation as well as the corresponding oscillator strength of both these structures is similar to each other. Naturally, it is expected for them to show similar spectroscopic behavior, particularly in non-interacting solvents. However, as these conformers differ considerably in their mode and extent of hydrogen bonding, it is possible to form different hydrogen bonded clusters in protic solvents with little difference in energy. The broad nature of 83

17 the emission spectra of LH 2 in protic solvents, as discussed before, may be due to the ensemble averaged spectral properties of all these microstructures. Figure 4.8 Ground state potential energy surface for water-mediated conversion of I to IIb obtained from IRC calculation using B3LYP/ G(d,p) methodology. The energy of the transition state (TS) structure (given in the right hand panel, bond lengths and angles are in Å and degrees, respectively) and six points on both the sides are shown Luminol Fluorescence in Aqueous Buffer and Mixed Solvents LH2 fluorescence in aqueous buffer: Presence of more than one species In homogeneous buffer medium LH 2 shows two absorption bands, one at 295 nm and the other at 350 nm. The emission obtained by exciting at both these absorptions show strongly intense, unstructured and broad spectra ranging from nm with the maximum at 420 nm. The excitation spectra corresponding to this emission resembles very closely with the absorption spectrum [119]. Picosecond time-resolved fluorescence decay measurement of LH 2 emission in homogeneous buffer solution indicates a bi-exponential decay function as confirmed by visual inspection of the fitting data as well by the statistical parameters shown in figure 4.9. It is observed that about 24% of the total excited fluorophore decays at about 2.4 ns, whereas, the remaining 76% population has an average fluorescence decay time of 9.8 ns. The measured 84

18 TCSPC data is in good agreement with the decay times of LH 2 reported previously in aqueous medium [77]. As discussed in the previous section, out of several possible isomers, only structures I and IIb (figure 4.1) are responsible for the fluorescence behavior of LH 2 in aqueous solution [119]. Density functional theory calculation using B3LYP/ G(d,p) formalism predicts that although the calculated spectroscopic properties like vertical transition energy and associated oscillator strength etc. are similar for both the isomers, structure IIb is more stable by 14.1 kj mol -1 energy compared to structure I in the ground state; whereas, in the excited state the energy difference is only 3.4 kj mol -1. From this excited state energy difference, a simple Boltzmann distribution calculation predicts about 25% of the fluorescence is contributed from hydrated I; whereas, the rest is due to the contribution from structure IIb. In analogy with this observation and the pre-exponential factors associated with LH 2 fluorescence decay time discussed above, the short and long decay time component of LH 2 fluorescence can be assigned to structures I and IIb, respectively. These two conformers differ considerably in their mode and extent of hydrogen bonding in aqueous medium. Very small energy difference among different hydrogen bonded clusters and a possible dynamic interconversion among them in the excited state may contribute towards the broad nature of LH 2 fluorescence spectra, particularly in protic solvent like water. Although, time-resolved data and theoretical calculation results give a definite indication of the presence of more than one fluorescing species, these conformers could not be resolved spectrally. Figure 4.9 Time-resolved fluorescence decay profile (open squares) and simulated data (solid line) with one and two exponential decay functions (represented by 1-exp and 2-exp, respectively) for LH2 in aqueous buffer (ph = 7.4). exc = 375 nm and mon = 430 nm. IRF indicates the instrument response function. Upper panel shows the distribution of weighted residuals for 2-exp fitting. Value of reduced chi-square ( 2 ) in different fitting model is also indicated. 85

19 Steady state spectral properties in mixed solvents Before going into the detail of the fluorescence properties in hydrophobic environment, it is worth discussing the behavior of the probe in the homogeneous mixture of water and 1,4 dioxane, as these mixtures are known to mimic the micellar environment very closely [122]. With increasing the volume fraction of water in 1,4-dioxane, both the fluorescence spectral position and intensity shift regularly and finally shows about 30 nm red shift along with almost three times increase in fluorescence quantum yield ( f ) in pure aqueous medium. Representative fluorescence emission spectral profiles along with the corresponding data are shown in figure 4.10a. However, the fluorescence excitation spectra (figure 4.10b) do not show any major change while going from 1.4-dioxane to water; only exception being the loss of vibrational structure in the high energy band accompanying by little broadening of the 350 nm peak. This difference in fluorescence properties in aqueous media is due to the stabilization of the excited state through formation of specific hydrogen bond of LH 2 with water [123]. The hydrogen bond formation occurs through the solvent hydrogen bond donation property towards the electron rich charge localized centers like imine nitrogen and carbonyl oxygen of the 2- and 4-positions of the phthalhydrazide ring system of LH 2. Figure 4.10 Steady state fluorescence emission (a) and excitation (b) spectra of LH 2 in varying water/1,4-dioxane content. (a) Volume percent of water = 0 (1), 10 (2), 50 (3), 75 (4) and 100 (5). Inset shows the fluorescence emission maxima ( max em ) and quantum yield ( f ) in different systems. (b) 1,4-dioxane (1) and water (2). 86

20 4.5. Interaction of Luminol with Surfactants & - Cyclodextrin Steady state spectral properties in micellar media The absorption maximum for the aqueous solution of LH 2 is practically unaffected by the presence of added surfactant, indicating very low absorbance sensitivity to the changes in surfactant concentration. However, the intensity of the fluorescence spectrum changes with the amount of surfactant in solution. Gradual addition of all the surfactants (CTAB, TX-100 and SDS) is associated with an initial increase within very small surfactant concentration followed by steady decrease in LH 2 fluorescence intensity. Figure 4.11 shows some representative spectral profile along with the pattern of intensity variation in each case. Figure 4.11 Change in LH 2 fluorescence emission profile ( exc = 360 nm) with increasing concentration of CTAB (a), TX-100 (b) and SDS (c). The concentration of the surfactants are (a) [CTAB]/ mm = 0.0 (i), 0.5 (ii), 1.4 (iii), 2.7 (iv), 4.7 (v), 7.8 (vi), 12.5 (vii), 18.7 (viii); (b) [TX-100]/ mm = 0.0 (i), 0.25 (ii), 0.30 (iii), 0.5 (iv) and (c) [SDS]/ mm = 0.0 (i), 2.7 (ii), 8.0 (iii), 11.9 (iv), 12.5 (v), 21.9 (vi). Inset shows the variation in fluorescence intensity at 425 nm in each case. 87

21 The increase in fluorescence intensity at very low surfactant concentration is due to the increased solubility of the organic fluorophore; however, it is clear that the fluorescence intensity decreases continuously after a certain concentration of surfactant in the solution, which is quite close to the critical micelle concentration (cmc) of the individual surfactant system. Once the micellar structure is formed, the fluorophore is partitioned in the hydrophobic micellar pseudo-phase from the bulk aqueous medium. As a result, the average steady state fluorescence intensity of the solution would decrease, which is in consistent with the observations made above for homogeneous solvents like water buffer and 1,4-dioxane. Interestingly, it is evident from the inset of figures 4.11(a) and 4.11(c) that the rate at which intensity decreases after the micelle formation is relatively higher in case of cationic micellar system CTAB in comparison with anionic SDS. However, it is rather difficult to quantitatively compare the TX-100 data given in inset of figure 4.11(b) as the concentration range used in this case are at least an order of magnitude lower than the other two. Apparently, the fluorescence peak position is not too sensitive towards the micellar medium. However, careful analyses of the data presented in table 4.7 as well as figure 4.11 reveal that fluorescence spectral blue shift is somewhat greater in case of interaction with CTAB ( 3 nm) in comparison with the other surfactant systems. In analogy with the results discussed above for water/1,4-dioxane solvent mixture, a blue shift of the fluorescence maximum suggests that the polarity of the micellar environments are less than the polarity of the bulk water. However, the fluorescence maxima of LH 2 in fully micellized condition (table 4.7) indicate that the probe is not incorporated into the core, rather, it stays more or less in the interfacial region of the micelle. Nevertheless, the data presented in table 4.7 indicates stronger interaction of LH 2 with CTAB. The decrease in LH 2 fluorescence intensity in micellar medium can be rationalized on the basis of the passage of the relatively non-polar fluorophore (structure IIb, figure 4.1) towards more hydrophobic interfacial region. However, due to extensive charge localization in the excited state [123 (a)], LH 2 interacts more strongly with cationic micelle like CTAB. The presence of this dipole-dipole type of interaction in addition to the hydrophobic force causes much stronger interaction with CTAB in comparison with other surfactant systems as evidenced by (a) rapid fall in fluorescence intensity, and also (b) detectable blue shift in fluorescence spectral position. This point is further confirmed from the time resolved studies as well as thermodynamics of ligand binding in BSA discussed below. Similar results were reported recently for the fluorescence behavior of 8- hydroxypyrene-1,3,6-trisulfonate, trisodium salt (HPTS) [124]. It was shown that the extent of 88

22 excited state deprotonation and quenching of neutral and/or anionic fluorescence of HPTS is drastically different and depends strongly on the nature (cationic, anionic or neutral) of the surfactant. It is to be noted here that while discussing the principle driving force and the stability of LH 2 -micelle complex in different surfactant systems, neither the dimension of the micelle nor the aggregation number was taken into consideration. However, a rigorous description of the forces responsible for binding the chromophore in the micellar sub-domain should involve the size of the respective micelles and its change with increasing surfactant concentration as well as the spatial distribution of the fluorophore in the interfacial region. Nevertheless, a qualitative description based on the averaged parameters of the probe as well as the micellar system is reasonable, given the tendency of the hydrophobic environments to quench LH 2 fluorescence and also the time-dependent fluorescence decay behavior in different media discussed in the following section [123 (b)]. Table 4.7 Fluorescence spectral behavior of LH 2 ( 5 M) in homogeneous buffer solution and in different heterogeneous medium. a Medium max em (nm) f Decay parameters 2 1 /ps ( 1 ) 2 /ns ( 3 ) 3 /ns ( 3 ) Buffer (ph=7.4) (0.24) 9.8 (0.76) 1.19 CTAB 0.5 mm (0.24) 9.9 (0.76) mm (0.61) 9.5 (0.39) 1.14 TX mM (0.25) 9.7 (0.75) mm (0.36) 9.6 (0.64) 1.15 SDS 2.4 mm (0.24) 9.9 (0.76) mm (0.34) 9.5 (0.66) 1.20 BSA 6 M <100 (0.65) 1.8 (0.12) 8.9 (0.23) M <100 (0.60) 1.0 (0.27) 8.8 (0.13) 1.2 HSA 6 M <100 (0.71) 2.0 (0.08) 9.0 (0.21) M <100 (0.73) 1.6 (0.09) 9.0 (0.18) 1.12 a The pre-exponential factor and the decay time is represented by and, respectively; the relative error in measuring values is within 0.1 ns. 89

23 Time-resolved fluorescence properties The fluorescence decay behavior was also monitored in presence of added surfactants. All the experimental decay curves could be well reproduced again with two-exponential decay functions as evidenced by the statistical parameters like reduced chi-square (χ 2 ) and distribution of weighted residuals. Figure 4.12 shows some representative decay profile in varying concentration of CTAB along with the fitting data and table 4.7 collects all the relevant data for different surfactants. From the table, it is clear that the fluorescence decay time of the two components remains practically unaffected in presence of all the surfactants. The relative contribution of the first decay component increases in presence of all the surfactant concentration above cmc. However, in case of CTAB, this increase is almost two and half times in comparison with that of the bulk aqueous medium. This observation further supports the idea of relatively stronger interaction in CTAB micelle as discussed before. Figure 4.12 Time resolved fluorescence decay profile (open circles) of aqueous LH 2 in presence of varying concentration of CTAB along with the simulated data (solid lines). [CTAB] /mm = 0.5 (1) and 12.5 (2), respectively. The upper panels show the distribution of weighted residuals for 2-exponential fitting in each case. IRF indicates the instrument response function. 90

24 Fluorescence behavior in β-cyclodextrin (CD) and mixed CDsurfactant system The unique property of cyclodextrin (CD) to encapsulate organic compounds inside its hydrophobic central cavity make them potential candidate as extremely efficient molecular vehicles for drug delivery [125]. Furthermore, the reduced polarity and restricted geometry of the interior cyclodextrin cavities gives an opportunity to study different photophysical properties in tailored environmental conditions. The inclusion of the organic probe is primarily controlled by the size fitting of the guest toward the host cavity [126]. The LH 2 fluorescence intensity seems to increase moderately on addition of CD till it reaches a plateau along with a spectral blue shift of about 5 nm (figure 4.13a). As seen in the previous section about the LH 2 fluorescence quenching in micellar medium, it is rather surprising to observe an enhancement in fluorescence intensity on binding with the hydrophobic CD cavity. In fact, little increase in molecular fluorescence of LH 2 derivatives in presence of CD was reported as early as in mideighties; however, no possible explanation was given for that [127]. Recently, Maeztu et. al. also reported the enhancement of chemiluminescence intensity of LH 2 and its derivatives at alkaline ph in presence of natural cyclodextrins [128,129]. In addition to the size requirement for the entire or partial inclusion of the guest molecule inside the CD cavity, additional hydrophobic forces are also important in determining the geometry of the complex [130,131]. Nevertheless, an increase in LH 2 fluorescence intensity in presence of CD can stem from the fact that considers the restricted non-radiative motion, and thereby reducing the vibrational deactivation of the caged analyte. The apparent binding constant and stoichiometric ratio of the inclusion complex can be determined from the modified Benesi-Hilderbrand (BH) equations described in the previous chapter [equations (3.19) and (3.20)] using the fluorescence data. Figure 4.13b shows the double reciprocal plot for LH 2 -CD system. The linearity for 1:2 case indicates that LH 2 is bound with two CD molecules and the binding constant is calculated to be M -2 from the slope and intercept of the simulated data. Interestingly, the fluorescence intensity is also found to increase upon gradual addition of CD in LH 2 solution containing a fixed amount (12.5 mm) of CTAB (figure 4.14). However, BH plot in this case indicates a complex with 1:1 stoichiometry with an apparent binding constant of M -1 (inset, figure 4.14). This difference in stoichiometry may be due to the fact that the nature and intensity of the feeble forces 91

25 responsible for the formation of the complex changes in presence of an ionic micellar medium like CTAB. Figure 4.13 (a) Variation of LH 2 fluorescence spectral profile on addition of -cyclodextrin (CD). [CD] /mm = 0 (i), 0.3 (ii), 0.7 (iii) and 3.8 (iv). Inset shows the intensity variation at 425 nm. (b) Double reciprocal plot for 1:1 (solid line) and 2:1 (dotted line) of the CD-LH 2 complex obtained from equations (3.19) and (3.20). Figure 4.14 Variation in intensity of fluorescence for LH 2 (12 µm) in fixed concentration of CTAB (17 mm) with increasing concentration of -cyclodextrin. [CD] /mm = 0 (i), 0.7 (ii), 1.2 (iii), 1.8 (iv), 3.2 (v) and 4.0 (vi). Inset shows the double reciprocal plots. 92

26 4.6. Binding of Luminol with Serum Albumins Interaction of luminol with human serum albumin (HSA) (i) Fluorescence quenching in presence of HSA Figure 4.15a shows the emission spectra of LH 2 in the presence of various concentration of HSA. It is observed that the fluorescence intensity of LH 2 decreases regularly with the increasing concentration of HSA without any significant shift in the emission maximum. The rapid quenching of LH 2 fluorescence indicates a strong interaction of LH 2 with HSA. The Stern- Volmer (SV) plot for the LH 2 fluorescence quenching (shown in the inset of figure 4.15a) shows a downward curvature. This kind of deviation from linear SV plot is a typical characteristic feature of fluorescence quenching involving two types of fluorophore populations; one of which is not accessible to the quencher [4(a)]. A modified form of SV equation, given in equation (4.1), can be used to analyze this type of fluorescence quenching data assuming the total fluorescence to be the contribution from both the accessible and inaccessible fluorophores. = [ ] + 4.1) where, F (= F 0 F) is the difference of fluorescence intensity in absence and presence of quencher (Q) concentration; K a is SV quenching constant of the accessible fraction (f a ). Figure 4.15 (a) Variation of LH 2 fluorescence with increasing concentration of HSA. [HSA] / M = 0 (1), 3 (2), 6 (3), 15 (4), 21 (5), 30 (6). The excitation wavelength is 360 nm. Inset shows the downward deviation of SV plot. (b) Modified Stern-Volmer plot for LH 2 fluorescence quenching by HSA at 308 K. Correlation coefficient (R) and standard deviation (SD) are given for the linear fitting. The numbers in parenthesis indicate the relative error in slope and intercept. 93

27 Apparent values of K a and the fraction of fluorophore accessible to the quencher (f a ) can be obtained from the slope and intercept of the plot of F 0 / F vs [Q] -1. Linear variation of the modified SV plot was obtained at all the temperatures with acceptable statistical parameters. A representative plot at 308 K is shown in figure 4.15b in case of LH 2 /HSA system. Apparently, the magnitude of the SV quenching constant (2.0±0.2 x 10 5 M -1 ) and fraction (40 47 %) of the accessible fluorophore for quenching does not vary significantly within the temperature range. Figure 4.16 (a) Fluorescence spectra of aqueous LH 2 in presence of 0.6 mm TX-100 (2) and 12.5 mm CTAB (3) relative to the normalized spectrum (1) at cmc (0.3 and 0.9 mm, respectively). (b) Increase in fluorescence intensity of the LH2/HSA solution in presence of bilirubin. [HSA] = 30 M and [BIL] / M = 0 (1), 0.5 (2) and 1.0 (3). The idea of fractional accessibility of LH 2 towards binding with protein and consequent fluorescence quenching is further supported from the steady state and time resolved fluorescence study in presence of different micellar medium discussed in the previous sections. The fluorescence intensity of LH 2 is found to decrease (figure 4.16a) in presence of different surfactants like SDS, CTAB and also TX-100, when the concentration of surfactants in solution is above critical micelle concentration (cmc). Moreover, time-resolved fluorescence decay analysis reveals that although the decay time on the first component (I) remains almost same ( 2.4 ns), the long component (IIb) decays relatively faster in presence of micellar environment when compared with that in aqueous buffer solution (table 4.7). Organic fluorophore are known to penetrate into the micellar core or can reside in the micelle-water interface when the surfactant concentration in solution is above cmc and therefore, experiences a relatively non-polar environment surrounding it [132]. In case of LH 2, the decrease in fluorescence intensity can also be ascribed due to the passage of component IIb in the micellar environment, thereby reducing 94

28 both the intensity as well as fluorescence decay time of this species. However, component I remains in the homogeneous (aqueous) phase of the micellar solution and the decay time remains unaltered. The calculated dipole moment of species I and IIb (2.12 and 1.92 D, respectively) is not too different to conclude about the preferential encapsulation of species IIb in the relatively hydrophobic micellar core with certainty; however, this definitely gives an indication towards this prediction necessary to explain the observed experimental results. It is known that HSA contains a hydrophobic portion on its surface (sub-domain IIA), which is normally interior to the protein and forms the primary binding region for a large number of hydrophobic ligands like fatty acids, bilirubin as well as several indole derivatives like tryptophan [22(b), 133]. It seems that out of two species of LH 2 (I and IIb) present in solution, the more hydrophobic IIb is accessible to bind to the albumin and consequently, the overall fluorescence intensity is quenched. The importance of sub-domain IIA in binding LH 2 is further reinforced by ligand replacement process in presence of bilirubin (BIL). On subsequent addition of BIL to an LH 2 /HSA solution, the fluorescence intensity of LH 2 increases continuously (figure 4.16b). BIL is known to have very strong affinity towards HSA sub-domain IIA with apparent binding constant varying within the range of approximately dm 3 mol -1 [134]. This value is higher by almost four order in magnitude compared to that in case of LH 2 (see below). With the preferential binding of BIL into the ligand binding site, the bound fraction of LH 2 is expelled in the more aqueous phase; consequently the fluorescence intensity increases. Figure 4.17 (a) Fluorescence decay profile (open circles) and simulated data (solid line) with three exponential decay function of LH 2 in presence of 30 M HSA. IRF indicates the instrument response function. Inset shows the same data in shorter time scale. 95

29 The fluorescence decay behavior of LH 2 in presence of albumin is markedly different from what is observed in homogeneous buffer and also the micellar medium (table 4.7). Although the decay time corresponding to species I (inaccessible for quenching) and IIb (accessible for quenching and therefore, bound to ligand binding domain of protein) still found to be 2 ns and 9 ns respectively, a major portion (ca. 70%) of the excited fluorophore decay with very short instrument limited life time (<100 ps). The presence of the first decay component in presence of HSA is obvious from figure 4.17 and also observed in presence of BSA (see below). A comparison of the statistical parameters reveals that two exponential fitting of the experimental data is insufficient to produce acceptable statistics and a minimum of threeexponential decay function is necessary for good fitting. However, no such fast decay was observed for LH 2 in presence of other heterogeneous micellar medium (table 4.7). As a first guess, a ground state complex formation may be approximated for the rapid fluorescence quenching along with a very fast LH 2 decay component in presence of albumins. However, relatively weak binding constant values (see below) and also the absence of any additional absorption peak for the LH 2 -HSA complex suggests that they do not really form a ground state complex. Instead, there may be a closely spaced fluorophore-quencher pair; where, the fluorescence is quenched rapidly in presence of the quencher and appears to be dark under this experimental condition. This rapid transient quenching is associated with the additional very fast decay time which is followed by the normal (slower) diffusion of the fluorophore and the quencher. (ii) Analysis of the nature and thermodynamics of binding equilibrium The equilibrium between the free and bound ligands for the binding of small molecules to a set of equivalent sites in a macromolecule is given by the following equation [135]: log = logk + n. log [Q] (4.2) where, K is apparent binding constant to a site and n is the number of binding site per macromolecule. F (=F 0 -F) is the difference in fluorescence intensity in absence and presence of quencher Q, respectively. The dependence of log( F/F) on log[q] is linear at all the temperatures studied here. Some representative plots are shown in figure The correlation coefficient (R) is 0.98±0.01 and the standard deviation (SD) values obtained in each case are within 0.05 indicates that the 96

30 assumptions made in deriving equation (4.2) is valid under the present experimental condition. The binding constant values obtained from the intercepts of figure 4.18 are further used to calculate the thermodynamic parameters of LH 2 -HSA interaction. Figure 4.18 Calculation of binding constant in LH 2 /HSA system using equation (4.2) at different temperatures. If the enthalpy change ( H) does not vary significantly in the temperature range studied, van t Hoff relation [equation (4.3)] can be used to evaluate the enthalpy and entropy change for LH 2 binding to HSA [136]; whereas the corresponding Gibb s free energy parameter is calculated from the relationship given in equation (4.4). logk = +.. (4.3) G = H T S (4.4) All the parameters are listed in table 4.8 and negative values of G assert that the binding process is spontaneous in the whole temperature range. Both the enthalpy and entropy change for LH 2 binding to HSA are also found to be negative. The interaction forces between drug and biomolecules may include electrostatic as well as van der Waals interaction, multiple hydrogen bond formation, hydrophobic and steric contact within the cavity etc. Ross and Subramanian [137] studied a series of protein association reaction with variety of ligands and proposed three possible reasons for the negative values of H and S. Those include non-bonded (van der Waals) interactions, hydrogen bond formation in low dielectric media and protonation due to the association process. The possibility of protonation during the LH 2 -HSA association at the experimental ph seems unlikely. Moreover, our previous report suggests that LH 2 possesses 97

31 several potential hydrogen bonding sites and even the hydrogen bonded conformers are thermodynamically far more stable ( 80 kj mol -1 for species IIb) than the corresponding nonhydrogen bonded structures [123]. So, the possibility of hydrogen bond formation even inside the protein binding domain cannot be ruled out. Overall, it is not possible to account for the thermodynamic parameters of LH 2 -HSA binding based exclusively on a single intermolecular force model. A combination of van der Waals interaction as well as possible hydrogen formation may contribute in the whole process. Meanwhile, it is clear from table 4.8, that major contribution for the G term comes from H rather than S; so the binding process is believed to be enthalpy driven. Table 4.8 Thermodynamic parameters corresponding to binding of LH 2 with HSA. a H /kj mol -1 Temperature /K Binding constant S /J K -1 mol -1 G /kj mol -1 /dm 3 mol a The binding constant values were calculated from the slope of equation (4.2). The other thermodynamic parameters were obtained using equations (4.3) and (4.4) with an error limit of 10%. Figure 4.19 Variation of LH 2 fluorescence spectral profile on addition of bovine serum albumin (BSA). [BSA] / M = 0 (i), 1.7 (ii), 3.5 (iii), 5.1 (iv), 6.9 (v), 8.6 (vi), 10.3 (vii) and 17.2 (viii). Inset shows the intensity variation at 425 nm. 98

32 Interaction of luminol with bovine serum albumin (BSA) (i) Fluorescence quenching in presence of BSA The fluorescence intensity of LH 2 decreases regularly with the increasing concentration of BSA accompanied with 10 nm blue shift in the emission maximum. Figure 4.19 shows the representative emission spectral profile in presence of various concentrations of BSA. The rapid quenching of LH 2 fluorescence indicates a strong interaction of LH 2 with BSA. In inset of figure 4.19, the fluorescence intensity was plotted against the quencher concentration. Interestingly, the decrease in intensity shows a clear sigmoidal shape. It is to be noted here that the LH 2 fluorescence intensity was also found to decrease with HSA concentration [138]; however, the nature of fluorescence intensity decrease is non-sigmoidal (inset of figure 4.20). The decrease in LH 2 fluorescence intensity can be assumed again as the passage of the probe molecule from the aqueous bulk phase and binding towards a more hydrophobic region in the ligand binding domain of the proteins. Figure 4.20 Variation of LH 2 fluorescence with increasing concentration of HSA. [HSA] / M = 0 (1), 3 (2), 6 (3), 15 (4), 21 (5), 30 (6). The excitation wavelength is 360 nm. Inset shows the intensity variation at 425 nm. Binding of LH 2 in albumin is further reinforced by ligand replacement process in presence of bilirubin (BIL). On subsequent addition of BIL to an LH 2 /BSA solution, the fluorescence intensity of LH 2 is recovered (figure 4.21). The binding constant value ( M -1 ) of BIL to BSA is higher by almost three order in magnitude compared to that in case of LH 2 in 99

33 BSA (see below). With the preferential incorporation of BIL into the ligand binding site, the bound LH 2 is expelled in the more aqueous phase; consequently the fluorescence intensity increases. Interestingly, this recovery of fluorescence in presence of BIL can be explored in further detail and used as an assay for efficient application of LH 2 for analytical and/or forensic purposes. Figure 4.21 Increase in fluorescence intensity of the LH 2 /BSA solution in presence of bilirubin (BIL). [HSA] = 26 M and [BIL] / M = 0 (i), 0.3 (ii), 0.5 (iii) and 1.0 (iv). The excitation wavelength is 360 nm in all the cases. Like HSA, the fluorescence decay behavior of LH 2 in presence of BSA is also markedly different from what is observed in homogeneous buffer and also the micellar medium (table 4.7). The presence of the first decay component in presence of BSA is also obvious from figure Furthermore, a comparison of the statistical parameters like reduced chi-square values as well as visual inspection of the weighted residuals confirm that two exponential fitting of the experimental data is insufficient and a minimum of three-exponential decay function is necessary. (ii) Stern-Volmer analysis of the quenching data The fluorescence quenching data was analyzed by Stern-Volmer (SV) relation [equation (1.20)]. As can be seen in figure 4.23, a plot of F 0 /F of LH 2 versus [BSA] exhibit a good linearity (R = 0.994) and affords K SV value 2.42± M -1 at 298 K. The amplitude weighted average lifetime in absence of quencher, <τ> 0, can be calculated from the bi-exponential decay data of 100

34 LH 2 in homogeneous buffer given in table 4.7 and found to be about 8.0 ns. From this data, the bi-molecular quenching rate constant, κ q, can be calculated as M -1 s -1. This value is almost three orders of magnitude greater than the maximum diffusion limited quenching rate constant, which is known to be of the order of M -1 s -1 [139]. Therefore, the quenching process is assumed mainly to be controlled by static quenching mechanism rather than dynamically controlled process. Figure 4.22 Fluorescence decay profile (open circles) and simulated data (solid line) with three exponential decay function of LH 2 in presence of 30 M BSA. exc = 375 nm and mon = 430 nm. IRF indicates the instrument response function. Inset shows the same data in shorter time scale. Upper panels show the distribution of weighted residuals and 2 values obtained from the fitting of the experimental data using three exponential (3-exp) and two exponential (2-exp) decay models, respectively. For a static quenching process, K SV can be regarded as the association constant (K a ) for the formation of the fluorophore-quencher complex in the ground state [4(a)]. The idea of static quenching can further be supported by doing temperature variation experiment. The calculated K a values from the linear SV plots given in figure 4.23 are collected in table 4.9. It is seen that the magnitude of K a decreases with increase in temperature, which further supports the formation 101