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.
Highlights
We present a pH nanosensor conceived for single intracellular measurements
The potentiometric responses of pH nanosensors prepared with CNPEs of different tip dimensions were investigated in the pH range of 6.0 to 8.5, which includes the expected physiological range and pH levels related to other conditions, such as the pH expected in cancerous cells.[3]
The tailoring of a nano-sized potentiometric ion-selective electrode for single-cell pHi measurements has been demonstrated in this work
Summary
We present a pH nanosensor conceived for single intracellular measurements. The sensing architecture consisted of a two-electrode system evaluated in the potentiometric mode. The portfolio of analytical techniques available for the determination measurements, of pHi including primarily relies on spectroscopic fluorescence imaging,[7−10] and surface-enhanced Raman scattering.[11,12] The main disadvantages of these approaches are that they often require extensive cell manipulation[13] and, in particular for fluorescence studies, signal intensity is difficult to quantify with direct assays and is influenced by some experimental conditions and factors, such as dye localization, photobleaching and quenching.[3] In contrast, electrochemical sensing is a label-free option for pH detection, and the electrode tip needed for measurements can be miniaturized down to nano-dimensions (nanotips) This approach provides real-time and continuous signals with high spatial resolution.[3−5] Another advantage is that the electrode tip can be tailored for the determination of different ions (e.g., sodium, potassium, chloride, and metals) as well as biomolecules (such as glucose) inside and outside of a single cell.[14−17]. To the best of our knowledge, the very first attempts of pHi measurements date back to the seventies and were based on glass microelectrodes containing an internal liquid contact, with relatively large tip dimensions and rather slow response time, which were limited by the configuration of the electrode (e.g., tip geometry, the working principle of the glass membrane, and the use of inner-filling solutions as an internal reference).[21−23] Seemingly, the use of an internal liquid contact has been the only approach used, until the time of this writing, in pHi potentiometric measurements, limiting the reported applications to relatively big-sized cells, as following described
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