Signal-On Fluorescent Imprinted Nanoparticles for Sensing of Phenols in Aqueous Olive Leaves Extracts

Abstract

The activation of signals in fluorescent nanosensors upon interaction with their targets is highly desirable. To this aim, several molecularly imprinted nanogels have been synthetized for the recognition of tyrosol, hydroxytyrosol and oleuropein in aqueous extracts using the non-covalent approach. Two of them contain fluorescein derivatives as co-monomers, and their fluorescence emission is switched on upon binding of the target phenols. The selection of functional monomers was previously done by analyzing the interactions by nuclear magnetic resonance (NMR) in deuterated dimethylsulfoxide (DMSO-d₆) of the monomers with tyrosol and hydroxytyrosol. Polymers were synthetized under high dilution conditions to obtain micro- and nano-particles, as verified by transmission electron microscopy (TEM). 1,4-Divinylbenzene (DVB) was used in the fluorescent polymers in order to enhance the interactions with the aromatic ring of the templates tyrosol and hydroxytyrosol by π-π stacking. The results were fully satisfactory as to rebinding: DVB-crosslinked molecularly imprinted polymers (MIPs) gave over 50 nmol/mg rebinding. The sensitivity of the fluorescent MIPs was excellent, with LODs in the pM range. The sensing polymers were tested on real olive leaves extracts, with very good performance and negligible matrix effects.

Keywords: fluorescence; imprinted nanogel; olive leaf extracts; phenols.

Figure 1

Chemical structures of tyrosol (TY) 1, hydroxytyrosol (HT) 2, ligstroside (LG) 3, oleuropein (OL, Glc: glucose) 4 and of monomers 5–9, co-monomers 10, 13, 14 and cross-linkers 11, 12.

Figure 2

(a) Equilibria of fluorescein in water solution [23]; (b) optimized geometry of the complex of tyrosol and the H₂Fquin form of fluorescein.

Figure 3

TEM images; (a) MIP 4VP-TY (200,000×, bar 20 nm); (b) MIP 4VP-HT (200,000×, bar 20 nm); (c) MIP 13-TY (200,000×, bar 20 nm); (d) MIP 13-HT (100,000×, bar 50 nm).

Figure 3

TEM images; (a) MIP 4VP-TY (200,000×, bar 20 nm); (b) MIP 4VP-HT (200,000×, bar 20 nm); (c) MIP 13-TY (200,000×, bar 20 nm); (d) MIP 13-HT (100,000×, bar 50 nm).

Figure 4

(a) Decay of TY concentration in 100 μM solutions upon incubation with the MIPs; (b) decay of TY concentration in 100 μM solutions upon incubation with the NIPs; (c) decay of HT concentration in 100 μM solutions upon incubation with the MIPs; (d) Decay of HT concentration in 100 μM solutions upon incubation with the NIPs.

Figure 5

Competition tests for MIPs 13-TY, 14-TY, 13-HT and 14-HT towards the templates TY and HT.

Figure 6

(a) fluorescence emission of MIPs 13-TY and 13-HT upon increasing concentrations of TY, HT and OL. (b) emission spectra of MIP 13-TY along the titration with TY.

Figure 7

(a) HPLC analysis of olive leaves extracts, (b) capture of OL from olive leaf extracts after addition of MIPs, (c) calibration curve of fluorescence enhancement after addition of OL to MIP 13-TY (r² = 0.991, intercept 1.796 ± 0.067, slope 0.1461 ± 0.0069).