Label-Free and Substrate-Free Potentiometric Aptasensing Using Polycation-Sensitive Membrane Electrodes

Abstract

A potentiometric label-free and substrate-free (LFSF) aptasensing strategy which eliminates the labeling, separation, and immobilization steps is described in this paper. An aptamer binds specifically to a target molecule via reaction incubation, which could induce a change in the aptamer conformation from a random coil-like configuration to a rigid folded structure. Such a target binding-induced aptamer conformational change effectively prevents the aptamer from electrostatically interacting with the protamine binding domain. This could either shift the response curve for the potentiometric titration of the aptamer with protamine as monitored by a conventional polycation-sensitive membrane electrode or change the current-dependent potential detected by a protamine-conditioned polycation-sensitive electrode with the pulsed current-driven ion fluxes of protamine across the polymeric membrane. Using adenosine triphosphate (ATP) as a model analyte, the proposed concept offers potentiometric detection of ATP down to the submicromolar concentration range and has been applied to the determination of ATP in HeLa cells. In contrast to the current LFSF aptasensors based on optical detection, the proposed strategy allows the LFSF biosensing of aptamer/target binding events in a homogeneous solution via electrochemical transduction. It is anticipated that the proposed strategy will lay a foundation for development of potentiometric sensors for LFSF aptasensing of a variety of analytes where target binding-induced conformational changes such as the formation of folded structures and the opening of DNA hairpin loops are involved.

Potentiometric flow injection sensing system for determination of heparin based on current-controlled release of protamine

“…This effect may be due to the fact that the protamine molecules with high molecular weights need a rather long time to cross the membrane [13]. Recently, it has been found that the ion fluxes across the membrane can be modulated and controlled precisely by applying an external current [23,[30][31][32]. Previous reports have shown that ions across the polymeric membrane ion-selective electrode can be selectively and precisely delivered to the sample solution for calibration-free titrations [33].…”

Potentiometric determination of trypsin using a polymeric membrane polycation-sensitive electrode based on current-controlled reagent delivery

“…1A). The procedures for switching between the galvanostatic and potentiostatic steps were designed as described before [22,23]. The opencircuit potential of the electrode was first recorded for 1 s (pulse 1).…”

“…1A shows that the current-dependent potential decreases with increasing the concentration of trypsin in the sample solution, which is due to the decrease of the concentration of free protamine released at the sample-membrane interface via the degradation by trypsin. As described before [18,22], the consumption of protamine would facilitate the stripping of protamine out of the membrane surface via the ion-exchange process with sodium ions from the sample solution, which can dramatically decrease the potential values. Fig.…”

“…Tris-HCl buffer (pH 7.4) was used as the inner filling solution of the protamine-conditioned polycation-sensitive electrode [22]. Before measurements, the electrode was conditioned for 12 h in a solution identical to the inner filling solution at 25 ± 1°C.…”

Pulsed galvanostatic control of a solid-contact ion-selective electrode for potentiometric biosensing of microcystin-LR

“…In recent years, potentiometry based on ion-selective electrodes (ISEs) have evolved to provide a promising transducer for biosensing [22][23][24][25][26]. Typically, these electrodes have been extensively investigated for biosensing via zero-current potentiometry.…”