Stereoselective Voltammetric Biosensor for Myo-Inositol and D-Chiro-Inositol Recognition

Abstract

This paper describes the development of a simple voltammetric biosensor for the stereoselective discrimination of myo-inositol (myo-Ins) and D-chiro-inositol (D-chiro-Ins) by means of bovine serum albumin (BSA) adsorption onto a multi-walled carbon nanotube (MWCNT) graphite screen-printed electrode (MWCNT-GSPE), previously functionalized by the electropolymerization of methylene blue (MB). After a morphological characterization, the enantioselective biosensor platform was electrochemically characterized after each modification step by differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). The results show that the binding affinity between myo-Ins and BSA was higher than that between D-chiro-Ins and BSA, confirming the different interactions exhibited by the novel BSA/MB/MWCNT/GSPE platform towards the two diastereoisomers. The biosensor showed a linear response towards both stereoisomers in the range of 2-100 μM, with LODs of 0.5 and 1 μM for myo-Ins and D-chiro-Ins, respectively. Moreover, a stereoselectivity coefficient α of 1.6 was found, with association constants of 0.90 and 0.79, for the two stereoisomers, respectively. Lastly, the proposed biosensor allowed for the determination of the stereoisomeric composition of myo-/D-chiro-Ins mixtures in commercial pharmaceutical preparations, and thus, it is expected to be successfully applied in the chiral analysis of pharmaceuticals and illicit drugs of forensic interest.

Keywords: D-chiro-inositol; MWCNTs; biosensor; myo-inositol; stereoselective recognition; voltammetric detection.

Scheme 1

Schematic representation of myo-Ins/D-chiro-Ins enantioselective biosensor fabrication steps and sensing mechanism.

Figure 1

SEM images and EDX spectra for: MWCNT/GSPE (a,b); MB/MWCNT/GSPE (c,d) and BSA/MB/MWCNT/GSPE (e,f). Experimental conditions: magnification: 25 K X; voltage: 1.50 kV.

Figure 2

CVs (a) and EIS spectra (b) of bare/SPGE (black line) and MWCNT/GSPE (red line). Redox probe: 5 mM [Fe(CN)₆]^3−/4− containing 0.1 M KCl solution. Scan rate: 25 mV s⁻¹. Inset: (1) [R(Q[RW])] and (2) [R(C[RW])] circuits used for fitting the experimental data.

Figure 3

DPV in PBS 50 mM, pH 7.0 of MB/MWCNT/GSPE (blue line), BSA/MB/MWCNT/GSPE (red line), HSA/MB/MWCNT/GSPE (green line) in absence (dotted line) and in presence (continuous line) of 50 μM myo-Ins. Incubation time: 15 min.

Figure 4

Nyquist’s plots of MWCNT/GSPE (black curve), MB/MWCNT/GSPE (red curve), and BSA/MB/MWCNT/GSPE (green curve). Redox probe: 5 mM [Fe(CN)₆]^3−/4− containing a 0.1 M KCl solution.

Figure 5

DPV of BSA/MB/MWCNT/GSPE in PBS 50 mM, pH 7.0, in absence (dotted red curve) and in presence (continuous line) of 50 μM myo-Ins (red curve), 50 μM D-chiro-Ins (violet curve), and a mixed solution of 25 mM myo-Ins and 25 mM D-chiro-Ins (green curve). Incubation time: 15 min.

Figure 6

Calibration curves of BSA/MB/MWCNT/SPGE at different myo-inositol (●) and D-chiro-inositol (Δ) concentrations.

Figure 7

Relationship between log(ΔI/(ΔI_max − ΔI)) and log[conc] of myo-inositol (●) and D-chiro-inositol (Δ) using a BSA/MB/MWCNT/SPGE based biosensor.

Figure 8

(a) Best conformation of whole BSA in carton ribbons with the three ligands: myo-Ins (orange color), D-chiro-Ins (blue color), folic acid (red color); (b) zoom of the predicted binding site with myo-Ins and D-chiro-Ins; (c) zoom of the predicted binding site with folic acid; (d,e) zoom of the H-bonds of the predicted binding sites for myo-Ins and D-chiro-Ins, respectively.

Figure 9

Current response of BSA/MB/MWCNT/SPGE with stereoisomer mixture of myo- and D-chiro-inositols at different total concentrations: (□)10 μM; (■) 50 μM.