Potentiometric pH Nanosensor for Intracellular Measurements: Real- Time and Continuous Assessment of Local Gradients

Abstract

Se presenta un nanosensor de pH de dos electrodos para mediciones intracelulares precisas. Este sistema utiliza nanopipetas de carbono de contacto sólido para crear tanto el electrodo indicador, sensible a iones de hidrógeno, como el de referencia. El nanosensor mostró una respuesta nernstiana ideal (pendiente de −59.5 mV/pH), rápido tiempo de respuesta (<5 s) y excelente reproducibilidad. El rango de medición lineal abarca los niveles de pH celulares normales y patológicos (6.0−8.5).

Se evaluaron diversas configuraciones de los electrodos para asegurar mediciones imparciales dentro y entre células. El dispositivo se aplicó con éxito para medir gradientes de pH al insertar el sensor en una célula PC12 y para monitorear cambios de pH intracelular inducidos por fármacos. Se prevé que esta herramienta analítica sin etiquetas sea muy útil en la investigación de enfermedades relacionadas con la desregulación del pH celular, sin importar el tipo de célula.

We present a pH nanosensor conceived for single intracellular measurements. The sensing architecture consisted of a two-electrode system evaluated in the potentiometric mode. We used solid-contact carbon nanopipette electrodes tailored to produce both the indicator (pH nanosensor) and reference electrodes. The indicator electrode was a membrane-based ionselective electrode containing a receptor for hydrogen ions that provided a favorable selectivity for intracellular measurements. The analytical features of the pH nanosensor revealed a Nernstian response (slope of −59.5 mV/pH unit) with appropriate repeatability and reproducibility (variation coefficients of <2% for the calibration parameters), a fast response time (<5 s), adequate medium-term drift (0.7 mV h−1 ), and a linear range of response including physiological and abnormal cell pH levels (6.0−8.5). In addition, the position and configuration of the reference electrode were investigated in cell-based experiments to provide unbiased pH measurements, in which both the indicator and reference electrodes were located inside the same cell, each of them inside two neighboring cells, or the indicator electrode inside the cell and the reference electrode outside of (but nearby) the studied cell. Finally, the pH nanosensor was applied to two cases: (i) the tracing of the pH gradient from extra-to intracellular media over insertion into a single PC12 cell and (ii) the monitoring of variations in intracellular pH in response to exogenous administration of pharmaceuticals. It is anticipated that the developed pH nanosensor, which is a label-free analytical tool, has high potential to aid in the investigation of pathological states that manifest in cell pH misregulation, with no restriction in the type of targeted cells