Detection of extracellular vesicles in non-Newtonian fluids using vibrating microcantilevers

Session

Stream 4 (AFM): Quantitative SPM for Biology, Biomedicine, and Bioinspired Technologies

Authors

Clodomiro Cafolla (1), Kislon Voitchovsky (1)

Affiliations

1. Durham University

Abstract text

Extracellular nanovesicles (EVs) are small (30-150 nm) phospholipid-based vesicles present in most, if not all bodily fluids. They are naturally released by cells into the surrounding medium and are used as vehicles to cargo small molecules, proteins and nucleic acids throughout the body. EVs mediate active communication between cells and can help regulate the growth and the fate of adjacent and distant cells [1]. Recently, EVs have drawn considerable attention for their potential in nanomedicine due to the fact that they carry distinct markers (proteins and nucleotides) which concentration may be correlated with diseases such as cancers, diabetes, and neurodegenerative diseases [1, 2]. In the case of cancer, for example, it has been shown that both cancer cells and Tumour MicroEnvironment stromal cells release EVs that promote tumour-induced immune suppression, angiogenesis and metastasis [3]. Aside from promoting tumour proliferation, multiple studies conducted on EVs extracted from cancer patients suggest the existence of distinct markers specific to most types of cancerous tumours [1], suggesting that EVs could be used as an early diagnostic tool, when cancer is most treatable. This would be further facilitated by the fact that EVs collection from liquid biopsies (mainly blood, saliva and urine) is relatively straightforward and minimally invasive compared to surgery.

However, characterising EVs from biopsies remains currently a significant challenge. Quantifying the proteomic and genetic content of the EVs is lengthy (> hours), costly, and typically requires significant quantities of biopsy liquid. This is because the EV sample are obtained by purification and concentration from the biopsies. There are no accepted standards for these crucial steps, and it is not clear whether the process alters the EVs, let alone the subsequent characterisation. This often results in contradictory or confusing findings, leading to difficulties in comparing studies and building a reliable picture. This confusion is best illustrated by the existence of a review paper for every three original publications in the field, with reviews attempting to bridge studies and build a global picture.

There is hence a strong need to develop techniques able to characterise EVs in-situ, directly in raw bodily fluid samples. Ideally such techniques should also be rapid from sample collection to quantitative results, relatively cheap, have inbuilt references, and be able to function on small volumes of fluid. 

Here we test the suitability of using atomic force microscopy (AFM) microcantilevers to quantify a set model EV sub-population exhibiting a specific marker directly inside saliva. Aside from bypassing extraction and purification-related issues, a microcantilever-based approach offers multiple advantages over existing approaches: (i) it uses using small amount of biopsy (<0.1 mL), (ii) it can be multiplexed easily, (iii) it is relatively flexible for functionalisation, and (iv) it can be integrated into microfluidics chips, potentially using self-actuated cantilever to bypass the expensive AFM detection. 

Microcantilevers have long been used for bio-detection with excellent sensitivity [4,5], but usually with the cantilever operated in air or in vapour, but not directly immersed into the fluid of interest. Here we use as a model system AFM microcantilevers functionalised with streptavidin to detect small quantities of synthetic phospholipid vesicles dissolved in raw saliva. 0.5% of the vesicles’ lipids are biotinylated, allowing for strong, irreversible binding to the cantilever’s streptavidin. The main challenge to overcome is the need for the cantilever to achieve reliable dynamic sensing inside a ‘dirty’ non-Newtonian fluid containing biopolymers in suspension while operating for extended periods of time. We show that it is possible to achieve a detection sensitivity better than 1 microgram/ml of vesicles in raw saliva within minutes, limited mainly by the vesicle’s diffusion timescale within the sample. The detection sensitivity can track model EVs at concentrations two orders of magnitude lower than the typical total EV concentration naturally occurring in blood [6]. This suggests microcantilever-based approaches to targeted EVs as a promising route for in-situ detection. 

 

References

[1] S. Fais, et al. ACS Nano 10 (2016) 3886–3899.

[2] L. J. Vella, et al. Int. J. Mol. Sci. 17, 173 (2016)

[3] J. Armstrong, et al. ACS Nano 11, 69 (2017).

[4] H. P. Lang, M. Hegner, & C. Gerber Materials Today 8 (2005) 30–36. 

[5] T. Braun, et al. Nat Nanotechnol 4 (2009) 179–185.

[6] I. Helwa, et al. PLoS ONE 12 (2017) e0170628.