C hapter 2
6.4 Anion sensing aptitude of L 4
It has been reported that the AIE nature of a chromophore can be tuned by guest molecules.8-10 In the present study, the chemosensor L4 has potential anion binding sites (NH and OH). Hence, it is pertinent to scrutinize its anion sensing aptitude in water. To this end, changes in the UV- visible spectra of a 10 µM aqueous solution of L4 are recorded upon addition of 10 equivalents of sodium or tetrabutyl salts of various anions such as F-, Cl-, Br-, I-, NO3-, OAc- (CH3CO2-), HSO4-, SO42-, ClO4- and PPi. Interestingly, a distinct change in the UV-visible spectra is observed only after addition of pyrophosphate anion (PPi), which lead to the emergence of new absorption peaks at 270 nm and 439 nm (Figure 6.2). These spectral changes are accompanied by a distinct visual change in the color of the solution of the probe L4 from colorless to faint yellowish in case of the interaction of L4 with PPi (Figure 6.2b, inset). This visual color change TH-1462_10612241
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is encouraging as it renders facile naked eye detection of PPi in water. Further inspection of the UV-Vis spectra after the addition of PPi anion reveals the formation of isosbestic points at 266 nm, 321 nm and 391 nm, which indicates the formation of a new species. It may also be mentioned that addition of PPi induced further aggregation, which is evident from the trailing UV-visible trace of the probe solution in presence of PPi (Figure 6.2b). Our next endeavour is to determine the selectivity and sensitivity of L4 towards PPi for which fluorescence-based experiments are pursued.
Upon addition of increasing concentrations of PPi, the fluorescence intensity of L4 increases systematically to attain a magnitude, which is nearly 17 times more than the probe alone at 530 nm (Figure 6.3). The L4-PPi solution also exhibits intense yellowish-green like fluorescence when observed under the 365 nm UV lamp (Figure 6.3a).
Figure 6.2 UV-Visible changes of L4 with a) various anions (10 equivalents) and b) magnified view with PPi in water. Inset: Change in the color of L4 after PPi addition.
The steep enhancement in the fluorescence emission intensity of the aforesaid solution suggests the possibility that PPi promoted further aggregation of the probe L4. This tenet is validated by AFM and FESEM analysis of L4-PPi complex, which indicate the formation of aggregates (Figure 6.3b,6.4.c), whose average particle sizes (130-170 nm) appeared to be bigger than that of L4 alone in water (Figure 6.2c, 6.2d). Job’s plot analysis suggested that L4 and PPi form a 1:1 host guest complex (Figure A6.2). The apparent binding constant as calculated by B-H plot is 4.2×105 M-1 (Figure A6.2b). The limit of detection (LOD) of L4 for PPi is 1.67 nM, with a signal: noise ratio of 3:1 (Figure A6.3). This detection limit of PPi compares well with those reported in previous studies (Table A6.1).
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Figure 6.3 a) The systematic increase in fluorescence emission intensity change of L4 with increasing concentrations of PPi (λex = 430 nm, slit 2/2 nm). b) AFM and c) FESEM image of L4 after addition of PPi. d) Fluorescence based anion selectivity profile of L4.
The specificity of L4 towards PPi is verified in solution based experiments wherein other tested anions such as F-, Cl-, Br-, I-, NO3-, OAc-, HSO4-, SO42-, ClO4-, H2PO4-, PO43- HPO42-, ADP and AMP etc.cause no change to the emission spectra of L4. However, a prominent enhancement in the emission intensity of L4 is also recorded in presence of ATP (Figure 6.3d). Competitive binding studies are also carried out with these anions by mixing 10 equivalents of the aforesaid anions to a solution of L4 containing 1.0 equivalent of PPi (Figure A6.4). It is significant to mention that the PPi-induced manifold enhancement of the emission intensity of L4 remains almost unaffected by the presence of these anions, which indicates the strong selectivity of the developed sensor for PPi. We have also tested some biologically relevant anions such as citrate, tartrate, succinate and oxalate for possible interference. Interestingly, none of the aforesaid anions cause any significant change in the fluorescence emission intensity of L4 (Figure A6.5).
The chemosensor can also detect PPi in a mixed aqueous medium and cause similar changes.
Prior to PPi addition, L4 in a 1:1 (v/v) MeCN-5.0 mM HEPES buffer (pH~7.4) mixture exhibited weak fluorescence. However, addition of 10 equivalents of PPi resulted in strong fluorescence at 530 nm (Figure A6.6).
It can be construed that the higher acetonitrile (MeCN) content in this experimental solution prevent the L4 molecules to aggregate, which results in comparatively weak fluorescence emission of the solution. However, presence of PPi perhaps trigger the aggregation of L4
molecules, which results in enhanced fluorescence emission. Other anions fail to produce similar optical signal with L4 under alike experimental conditions. It may be mentioned here that even in pure dry acetonitrile medium (fw = 0), PPi-induced aggregation of the probe is visible (Figure A6.7).
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Ca2+ has a high affinity for PPi and is known to form a strong complex with the same. It is thus envisaged that addition of Ca2+ is likely to disrupt the L4-PPi ensemble owing to strong Ca2+-PPi interactions and this would also provide strong evidence for the involvement of PPi in inducing aggregation and thereby enhancing the fluorescence emission intensity of L4 manifold. As anticipated, upon addition of Ca2+, the solution turned colorless and the fluorescence diminished considerably, which implies the involvement of PPi in the enhancement of fluorescence emission (Figure A6.8). UV-Vis and fluorescence-based studies indicate that the initial aggregation induced emission of L4 in water is further intensified with the addition of PPi. This suggests that addition of PPi would lead to the generation of comparatively larger aggregates in aqueous medium. This is corroborated by FESEM and AFM analysis, as discussed earlier.
Further, dynamic light scattering (DLS) measurement shows an average particle size of 258 nm (PdI = 0.554, Figure A6.9) in a 10 µM solution of L4 in water interacted with 10 equiv. of pyrophosphate anion. Collectively, all these evidence support the PPi-induced aggregation of L4
in aqueous medium probably through the involvement of OH and NH groups (Scheme 6.2).