Application of ion-sensitive field effect transistors for ion channel screening

Abstract

Cell-based screening assays are now widely used for identifying compounds that serve as ion channel modulators. However, instrumentation for the automated, real-time analysis of ion flux from clonal and primary cells is lacking. This study describes the initial development of an ion-sensitive field effect transistor (ISFET)-based screening assay for the acquisition of K+ efflux data from cells cultured in multi-well plates. Silicon-based K+-sensitive ISFETs were tested for their electrical response to varying concentrations of KCl and found to display a linear response relationship to KCl in the range of 10 µM to 1 mM. The ISFETs, along with reference electrodes, were inserted into fast-flow chambers containing either human colonic T84 epithelial cells or U251-MG glioma cells. Application of the Ca2+ ionophore A23187 (1 µM), to activate Ca2+-activated non-selective cation (NSC) channels (T84 cells) and large conductance Ca2+-activated K+ (BK) channels (U251 cells), resulted in time-dependent increases in the extracellular K+ concentration ([K+]o) as measured with the ISFETs. Treatment of the cells with blockers of either the NSC or BK channels, caused a strong inhibition of the A23187-induced increase in [K+]o. These results were consistent with ion current measurements obtained using the whole-cell arrangement of the patch clamp procedure. In addition, K+ efflux data could be acquired in parallel from multiple cell chambers using the ISFET sensors. Given the non-invasive properties of the probes, the ISFET-based assay should be adaptable for screening ion channels in various cell types.

Direct measurement of K+ ion efflux from neuronal cells using a graphene-based ion sensitive field effect transistor

“…The observed transient response is very similar to that obtained with Si-ISFET test on live-cells [ Fig. 5(b)] conducted in exactly the same way, as well as result reported in an earlier article by Walsh et al 21 However, we note that the K + concentration change (the difference between the responses corresponding to cellular efflux and control) measured by the GISFET is almost 2 times lower than that estimated by the Si ISFET. We attribute this discrepancy to inaccuracies in the conversion factor (from a voltage or current change to K + concentration) for the sensors.…”

“…[18][19][20] In addition to the investigation in DI water medium, the sensor's ion sensing performance was also examined in a NaCl buffer solution consisting of 140 mM NaCl, 1 mM CaCl 2, and 1 mM MgCl 2 , which are at the levels present in the buffer solution used for cell culture. 21 The measurements were carried out using the same set up shown in Fig. 1(b) to mimic the conditions of the live-cell assay conducted following these measurements.…”

Graphene field effect transistors for highly sensitive and selective detection of K+ ions

“…commercial Si ISFETs) is to look at the shift in threshold voltage (equivalent to the shift in Dirac point of the ISFET) as a function of the change in K + ion concentration [35]. The change in Dirac point as the K + ion concentration was varied from 1 µM to 20 mM (this includes the range of interest for cellular efflux of 10 µM -1 mM [36] ) can be determined from Fig. 3(b) to be ~280 mV, and hence the sensitivity can be calculated as 64 mV/decade (= 280 mV/4.3 decade), which is very comparable to that obtained from the Si-based ISFET devices [37].…”

Porous Graphene Oxide Decorated Ion Selective Electrode for Observing Across-Cytomembrane Ion Transport

“…From the perspective of the non-invasive and selective detection, the electrochemical sensors are also good candidates [ 17 ], both the ion-selective field effect transistors (ISFETs) [ 18 , 19 ] and ion selective electrodes (ISEs) [ 20 , 21 ] have been developed to observe the transmembrane ions’ movement. In these researches, an interfacial micro-environment (IME) between cells and sensors was fabricated by the micromachining techniques.…”

“…In these researches, an interfacial micro-environment (IME) between cells and sensors was fabricated by the micromachining techniques. Not only the weak ionic variations in IME, which were caused by the ion transport across the epithelial cell layer, like the fluxes of Cl − [ 18 ] and K + [ 19 ], could be detected, but also the multi-ions’ detection of Na + , K + , Cl − and pH could be realized [ 20 , 21 ]. However, for I − , whose uptake and outflow mediated by NIS are of great significance for cancer treatment [ 4 ], it is still the minority in the detection of transmembrane ion transport.…”