A heterogeneous ion transfer NH4+ detector core amperometric genosensor method with sandwich nanohybridization coupled enzymatic signal-on amplification

This work extends the application of heterogeneous ion transfer (HIT) voltammetry at the polarizable oil/ water (O|W) interface by presenting an amperometric genosensor developed on HIT based NH4+ detector core coupled with a urease/urea/NH4+ nano-enzymatic amplification of signal. It is demonstrated for the detection of a 20 base (b) oligo-RNA target (tORN) related to the genomic insertion sequence (IS6110) of Mycobacterium tuberculosis (MTB). A semi-permeable cellulose membrane (CM)-supported and internally Pt-wire-contacted O-phase (1,2-dichloroethane solution of ethyl violet tetraphenylborate (10 mM) and dibenzo-18-crown-6 (50 mM)) in a hydrophobic plastic tube (PTFE, tip ID 5 mm) served as an NH4+-selective amperometric core sensor (Pt|O|CM). 3′-end alkylamine-modified 10b long oligo-DNA capture-probe (cODN) was covalently attached to the CM to fabricate the genosensor (Pt|O|CM-cODN). Silver nanoparticles (Agn, 10–40 nm) co-conjugated with urease and a 5′-end-alkylthiol-modified, 21b oligo-DNA signal-probe (sODN) served as the signal-amplification reagent (sODN-Agn-Urs). After sequential incubation in aq. tORN (5–50 nM, 15 min) and aq. sODN-Agn-Urs (54 mg/mL, 10 min), cyclic voltammograms (CV) were acquired in aq. 50 mM MgSO4 solution (W-phase) with or without urea (2 mM). Sensitivity of 55.8 µA cm−2 decade−1 and limit of detection (LoD) of about 44 pM were achieved based on background subtracted CV peak currents. The peaks were much less sensitive to a double-mismatch oligo-RNA than the tORN. Impedance spectra were also consistent with the CV results. The analytical merits compare well with some genosensors in the literature, thus the Pt|O|CM-cODN combined with Urs/urea/NH4+ nano-enzymatic signal amplification is promising. Further work to optimize fabrication and measurement factors and improve ruggedness is underway.