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

Abstract

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 ion-selective 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.

Micro- and nano-devices for electrochemical sensing

“…The electrode with the smallest tip dimension showed a detection limit (LOD) of (3.96 ± 2.09) × 10 –7 M, whereas the larger one of (2.83 ± 0.47) × 10 –7 M. For this experiment, a standard macro-sized RE was used. A very recent example where both the indicator and the reference electrode were miniaturized reports the fabrication of a pH nanosensor for single intracellular measurements, consisting of two solid-contact carbon nanopipette electrodes whose tip dimension was 800 nm diameter, tailored to produce both the indicator (pH nanosensor) and RE [ 130 ]. The pH nanosensor was composed of a two-layer structure: a carbon film, to provide large conductivity to the electrode substrate and a solid contact to ensure a proper ion-to-electron transduction, and the proton-selective membrane.…”

Acidic pH‐Triggered Live‐Cell Lysosome Specific Tracking, Ratiometric pH Sensing, and Multicolor Imaging by Visible to NIR Switchable Cy‐7 Dyes

“…[1][2][3] Various subcellular parts of live cells have different pH allocation, for instance cytoplasm is nearly neutral (pH 7.2), mitochondria is weakly basic (pH 8), whereas the endosomes (pH 5-6) and lysosomes (pH 4-5) are acidic. [4,5] It is irrefutable that the upholding of precise pH in the discrete intracellular organelles is of supreme concern for their regular functions. All the mammalian cells possess lysosomes which are membraneenclosed acidic organelles, encompass a series of enzymes responsible for the breaking down of sugars, proteins, fats, and nucleic acids as well as involve in endocytosis.…”

Tuning the pH of Activation of Fluorinated Hydrazone-Based Switches─A Pathway to Versatile 19F Magnetic Resonance Imaging Contrast Agents

“…Measurements of pH are important in many areas of chemical research as well as in agriculture, food industry, microbiology, environmental sciences, and in medicine . The pH value is typically measured using colorimetric indicators such as methyl orange or phenolphthalein or with more accurate potentiometric methods . In medical diagnostics, besides classical chemical methods, imaging techniques such as positron emission tomography (PET) and magnetic resonance imaging (MRI) are also applied for pH measurements. , …”