Author interview: Debashish Paul is a Ph.D. scholar in the Department of Chemistry, Shiv Nadar Institution of Eminence, working under the supervision of Dr. Tatini Rakshit, trained in Atomic Force Microscopy of biological samples. He received his MSc. degree in physical chemistry from Ramakrishna Mission Vivekananda Centenary College, Rahara in 2019 and CSIR-Direct SRF fellowship in 2024 for his research on hyaluronan-coated cancer EV sensing.
Lab: Dr. Tatini Rakshit, University: Shiv Nadar Institution of Eminence, Delhi NCR, India.
Electrical sensing of cancer cell-derived hyaluronan-coated extracellular vesicles
This study investigates the electrical properties of hyaluronan-coated cancer extracellular vesicles using advanced scanning probe microscopy approaches, offering a label-free sensing of cancer EVs from normal EVs.
What was the core problem you aimed to solve with this research?
There is a lack of effective methods for distinguishing cancer extracellular vesicles from normal EVs based on their electrical properties. The study utilizes advanced scanning probe microscopy (SPM) techniques to analyze cancer EVs’ surface potential, charge distribution, and piezoelectric electro-mechanical response at the single-vesicle resolution.
The correlative, label-free multiplexed scanning probe microscopy approach can be utilized for sensing cancer HA-EVs from normal counterparts. — Prof. T. Rakshit
How did you go about solving this problem?
Our previous findings suggested HA molecules are highly abundant on cancer EV surfaces where FT-IR, Raman, CD spectroscopy and AFM unbinding force spectroscopy were used to distinguish HA-coated cancer EVs from normal EV counterparts. These HA molecules are short (MW < 200 kDa with contour length <450 nm), and HA-EVs are intrinsically more flexible than normal EVs. Due to its attractive properties, HA-EVs offer a versatile and promising platform for biosensing applications. Since HA is an anionic polymer densely populated on cancer EV surfaces, investigating HA-EVs’ electrostatic properties could be an exciting gateway in biosensing. The electrical characteristics of EVs have been only briefly evaluated in a few recent reports until date. Very few techniques are available for characterizing the surface electrical properties of EVs, viz. Zeta potential measurements and nanopore technology which have some limitations regarding electro-osmotic effect, resolution, etc. In this work, our approach introduces collective scanning probe microscopy (SPM) based nanoelectrical modes, which offer potential advantages in biosensing cancer HA-EVs from normal counterparts. Atomic force microscopy (AFM), a subset of SPM, is a powerful technique for visualizing EVs in single vesicle resolution. The key concept involves approaching a sharp AFM probe to a specific nanoscale region of the sample surface, where the interaction between the probe and the sample is measured through various electrical responses.
Considering the nanoscale dimensions of individual HA-EVs and the zeta potential-derived evidence of distinct surface charges (−ve), we aimed to investigate the surface charge distribution at the single-vesicle level using advanced electrical modes of AFM, EFM, and KPFM. In a broader context, we explored whether these differences are confined to the surface level or extend to the bulk properties of the entire EV. Notably, cancer extracellular vesicles (CEVs) exhibit greater vesicle flexibility compared to normal extracellular vesicles (NEVs). Under mechanical deformation, this flexibility may lead to alterations in the orientation of polar groups, potentially resulting in macroscopic polarization. To investigate this, piezoresponse force microscopy (PFM) could be utilized to examine how piezoelectricity in these vesicles is influenced by their structural integrity and mechanical deformability. Furthermore, piezoelectricity, although primarily a bulk property, has significant contributions from surface charges in biomolecules. It originates from their intrinsic asymmetry, polar functional groups, and ordered structural arrangements. Such piezoelectric properties are crucial for various EV-related biological processes and hold substantial potential for applications. Vesicles are known to facilitate ion transport through embedded channels, and biomolecules are not purely insulating but exhibit a degree of electrical conductivity. Significant structural differences in vesicles, along with variations in their ionic affinities, can influence the transport properties of different types of EVs. C-AFM provides a valuable tool for characterizing the electrical transport properties of EVs, enabling the distinction of whole-vesicle properties beyond surface-specific characteristics. Collectively, EFM/KPFM techniques effectively could differentiate cancer-EVs from normal EVs based on their distinct surface electrical characteristics simply by imaging techniques. Additionally, for a more comprehensive analysis, PFM and C-AFM techniques could be employed to investigate the electro-mechanical properties and electrical-transport mechanism of EVs. These insights significantly contribute to the effective discrimination of HA-EVs from normal counterparts through advanced sensing approaches.
How would you explain your research outcomes (Key findings) to the non-scientific community?
Cancer cells release nano-sized sugar-coated pouches. This study addresses electrical sensing this cancer nanopouches from normal counterparts using advanced single molecule imaging and current-voltage measurements, which could contribute to non-invasive cancer detection.
What are the potential implications of your findings for the field and society?
Imaging and current-voltage measurements-based sensing of cancer HA-EVs from normal EVs using advanced electrical AFM modes opens up new possibilities for non-invasive cancer detection and monitoring. EVs are abundant in body fluids and could be used for liquid biopsy applications.
What was the exciting moment during your research?
It was fascinating to visualize these tiny cancer EVs with EFM and KPFM. We discovered that the anionic HA coating on the cancer EVs are contributing in image contrast formation and we could successfully distinguish cancer EVs from normal counterparts by AFM imaging in EFM and KPFM modes.
Reference: Paul, D.; Bera, S.; Agrawal, T.; Karmodak, N.; Rakshit, T. Unveiling the Electrical Properties of Hyaluronan Coated Cancer Extracellular Vesicles Using Correlative Scanning Probe Microscopy- Based Nano-Electrical Modes. ACS Appl. Mater. Interfaces, 2025. https://pubs.acs.org/doi/10.1021/acsami.4c17247
