Harnessing Glycofluoroforms for Electrochemical Detection of Galectin-3

Despite underpinning biological communication, sugars have limited applications as biorecognition elements in biosensors, caused by promiscuity between sugar probes and protein binding partners. Here, site-specific fluorination was harnessed to introduce protein-sugar selectivity to specifically target galectin-3, a valuable diagnostic biomarker. Electrochemical detection methods were employed due to their speed, sensitivity, and affordability.

For the first time, electrochemical biosensing of galectin-3 was achieved by exploiting its enhanced binding affinity to glycofluoroforms (fluorinated analogues of native binding sugars). Polymer tethers were used to provide a passivated surface and the resulting biosensing platforms selectively bound galectin-3 compared to control glycans and proteins. Initially, the thiol functionality of the polymer, linked to the glycofluoroform via strain-promoted azide-alkyne cycloaddition, was used to immobilise the conjugate onto a gold electrode. Electrochemical impedance spectroscopy analysis quantified the increase in charge transfer resistance between the gold electrode surface and solution-phase potassium ferricyanide observed on galectin-3 binding. Titration experiments revealed an apparent Kd of 240 nM. However, a high level of irreproducibility was observed between sensors, and the electrochemical instability of the gold-thiol bond prevented the use of further electrochemical measurement techniques.

Subsequently, triazabutadienes were used as masked diazonium ions for covalent immobilisation of polymer-sugar conjugates onto carbon electrodes. Compared to gold-thiol self-assembly, electrografting yielded a higher coverage of glycofluoroform (5.201 pmol compared to 0.350 pmol) and conferred an apparent Kd of 13 nM. The higher affinity of this sensing platform and the wide range of detectable galectin-3 concentrations (1 ng mL 1 to 10 µg mL-1) is more suited to the expected biological galectin-3 levels. Furthermore, the increased stability of the modified surface due to the covalent carbon-electrode bond allowed for more complex voltammetry methods to be explored. Extraction of faradaic current from Fourier transformed alternating current voltammetry experiments demonstrated a simpler analysis of galectin-3 binding.