Spend even a brief amount of time in a laboratory and you will quickly understand how important biological pH is to normal and aberrant cell functions. Yet, despite the growing recognition for the importance of pH directly surrounding a cell as an indicator of cell health, techniques to measure it thus far remain limited in terms of their sensitivity, the spatial resolution they can offer, and the speed of response to pH changes. However now, investigators at Imperial College London describe a new nanopipette pH biosensor that is sensitive to changes in pH of less than 0.01 units with a response time of 2 ms and 50 nm spatial resolution.

Findings from the new study were published recently in Nature Communications through an article titled “High-resolution label-free 3D mapping of extracellular pH of single living cells.”

“It is becoming clear that an acidic extracellular pH plays an essential role in cancer cell progression, invasiveness, and resistance to therapy,” explained senior study investigator Yuri Korchev, PhD, professor of biophysics at Imperial College London.

The researchers originally designed the new sensor as a nanopipette ionic field-effect transistor—where gates control the flow of ions in the nanopipette instead of electrons. However, while this tackled issues around pH sensitivity and spatial resolution, the device readings still took a few seconds to respond to pH changes due to ionic Coulomb blockade effects hampering the diffusion rate of ions.

The operation of the double-barrel nanoprobe for simultaneous SICM imaging and pH measurement. [Kanazawa University]
Interestingly, the solution that the research team proposes is to incorporate a zwitterionic membrane to enable faster responses. By using a twin-barrel nanopipette with the membrane in just one of the barrels the researchers were able to use the other barrel as a scanning ionic conductance microscope (SICM) for simultaneous topological measurements.

“We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity > 0.01 units, 2 ms response time, and 50 nm spatial resolution,” the authors wrote. “The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependence via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and extracellular pH (pHe) monitoring of living cancer cells.”

The team tested the device on live cancer cells and showed how the device could pick up on increases in extracellular pH from invasive phenotypes of breast cancer cells that had been deprived of estrogen. They could also detect pH changes from algae exposed to sunlight, caused by the uptake of inorganic carbon in photosynthesis, as well as identifying heterogeneities in aggressive melanoma cells from high-resolution pH maps.

Highlighting the real-time feedback-controlled dynamic 3D mapping of extracellular pH that their tool allows, and the heterogeneities of cancer cells that it can detect “label-free and at subcellular resolution,” the researchers noted. “This method could help with cancer diagnosis, prognosis, and in evaluating acidic targeted therapies.”

The most commonly used pH probes at present are based on microelectrodes that are quite large in comparison to the scale of the pH fluctuations of interest in studies of extracellular pH. Alternatives have been based on changes in the fluorescence of molecules, nuclear magnetic resonance imaging, and positron emission computed tomography. However, monitoring fluorescence is subject to background noise and photobleaching, and the other techniques have poor spatial resolution and raise difficulties in quantification because they are based on the distribution of probes within tissue.

By using a nanopipette as an ionic field effect transistor, the researchers were able to overcome most of the issues limiting previous techniques. However mutual same charge repulsion leads to the Coulomb blockade effect, which starts to inhibit the diffusion of positively charged protonated water molecules in the nanopipette and this slows down the response time.

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