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Dynamic single-cell intracellular pH sensing using a SERS-active nanopipette.

ABSTRACT: Glass nanopipettes have shown promise for applications in single-cell manipulation, analysis, and imaging. In recent years, plasmonic nanopipettes have been developed to enable surface-enhanced Raman spectroscopy (SERS) measurements for single-cell analysis. In this work, we developed a SERS-active nanopipette that can be used to perform long-term and reliable intracellular analysis of single living cells with minimal damage, which is achieved by optimizing the nanopipette geometry and the surface density of the gold nanoparticle (AuNP) layer at the nanopipette tip. To demonstrate its ability in single-cell analysis, we used the nanopipette for intracellular pH sensing. Intracellular pH (pH_i) is vital to cells as it influences cell function and behavior and pathological conditions. The pH sensitivity was realized by simply modifying the AuNP layer with the pH reporter molecule 4-mercaptobenzoic acid. With a response time of less than 5 seconds, the pH sensing range is from 6.0 to 8.0 and the maximum sensitivity is 0.2 pH units. We monitored the pH_i change of individual HeLa and fibroblast cells, triggered by the extracellular pH (pH_e) change. The HeLa cancer cells can better resist pH_e change and adapt to the weak acidic environment. Plasmonic nanopipettes can be further developed to monitor other intracellular biomarkers.

SUBMITTER: Guo J

PROVIDER: S-EPMC7425357 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The merging of the molecular specificity of Raman spectroscopy with the extraordinary optical properties of metallic nanoarchitectures is at the heart of Surface Enhanced Raman Spectroscopy (SERS), which in the last few decades proved its worth as powerful analytical tool with detection limits pushed to the single molecule recognition. Within this frame, SERS-based nanosensors for localized pH measurements have been developed and employed for a wide range of applications. Nevertheless, to improve the performances of such nanosensors, many key issues concerning their assembling, calibration and stability, that could significantly impact on the outcome of the pH measurements, need to be clarified. Here, we report on the detailed characterization of a case study SERS-active pH nanosensor, based on the conjugation of gold nanoparticles with the pH-sensitive molecular probe 4-mercaptobenzoic acid (4MBA). We analyzed and optimized all the aspects of the synthesis procedure and of the operating conditions to preserve the sensor stability and provide the highest responsiveness to pH. Exploiting the dependence of the SERS spectrum on the protonation degree of the carboxylic group at the edge of the 4MBA molecules, we derived a calibration curve for the nanosensor. The extrapolated working point, i.e., the pH value corresponding to the highest sensitivity, falls at pH 5.6, which corresponds to the pKa value of the molecule confined at the nanoparticle surface. A shift of the pKa of 4MBA, observed on the molecules confined at the nanostructured interface respect to the bulk counterpart, unveils the opportunity to assembly a SERS-based pH nanosensor with the ability to select its working point in the sensitivity region of interest, by acting on the nanostructured surface on which the molecular probe is confined. As a proof-of-concept, the nanosensor was successfully employed to measure the extracellular pH of normal and cancer cells, demonstrating the capability to discriminate between them.

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Dynamic single-cell intracellular pH sensing using a SERS-active nanopipette.

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