Carter, E. P. | bioRxiv Science

bioRxiv2020

Configurable Digital Virus Counter on Robust Universal DNA Chips

Here, we demonstrate real-time multiplexed virus detection by applying DNA-directed antibody immobilization technique to a single-particle interferometric reflectance imaging sensor (SP-IRIS). In this technique, the biosensor chip surface spotted with different DNA sequences is converted to a multiplexed antibody array by flowing antibody-DNA conjugates and allowing specific DNA-DNA hybridization. The resulting antibody array is shown to detect three different recombinant Vesicular Stomatitis Viruses (rVSVs) genetically engineered to express surface glycoproteins of Ebola, Marburg, and Lassa viruses in real-time in a disposable microfluidic cartridge. We also show that this method can be modified to produce a single-step, homogeneous assay format by mixing the antibody-DNA conjugates with the virus sample in solution phase prior to flowing in the microfluidic cartridge, eliminating the antibody immobilization step. This homogenous approach achieved detection of the model Ebola virus, rVSV-EBOV, at a concentration of 100 PFU/ml in 1 hour. Finally, we demonstrate the feasibility of this homogeneous technique as a rapid test using a passive microfluidic cartridge. A concentration of 104 PFU/ml was detectable under 10 minutes for the rVSV-Ebola virus. Utilizing DNA microarrays for antibody-based diagnostics is an alternative approach to antibody microarrays and offers advantages such as configurable sensor surface, long-term storage ability, and decreased antibody use. We believe these properties will make SP-IRIS a versatile and robust platform for point-of-care diagnostics applications.

bioengineering↗

bioRxiv2020

Centrosome amplification mediates small extracellular vesicles secretion via lysosome disruption

Bidirectional communication between cells and their surrounding environment is critical in both normal and pathological settings. Extracellular vesicles (EVs), which facilitate the horizontal transfer of molecules between cells, are recognized as an important constituent of cell-cell communication. In cancer, alterations in EV secretion contribute to the growth and metastasis of tumor cells. However, the mechanisms underlying these changes remain largely unknown. Here, we show that centrosome amplification is associated with and sufficient to promote small extracellular vesicle (SEV) secretion in pancreatic cancer cells. This is a direct result due of lysosomal dysfunction, caused by increased reactive oxygen species (ROS) downstream of extra centrosomes. Defects in lysosome function promotes multivesicular body fusion with the plasma membrane, thereby enhancing SEV secretion. Furthermore, we find that SEVs secreted in response to amplified centrosomes are functionally distinct and activate pancreatic stellate cells (PSCs). These activated PSCs promote the invasion of pancreatic cancer cells in heterotypic 3-D cultures. We propose that SEVs secreted by cancer cells with amplified centrosomes influence the bidirectional communication between the tumor cells and the surrounding stroma to promote malignancy.

cell biology↗