An immunosensor for the near real-time and site of inflammation detections of multiple proinflammatory cytokines.

Diseases mediated by cytokine storms are often characterized by an overexuberant pace of pathogenesis accompanied by significant morbidity and mortality. Thus, near real-time (NRT) detections via a site-of-inflammation (SOI) sampling of proinflammatory cytokines are essential to ensure a timely and effective treatment of acute inflammations, which up to now, has not been fully possible. In this work, we proposed a novel NRT and SOI immunosensor using ZIF-8 signal amplification together with an off-on strategy. To achieve NRT detections via a SOI sampling, the body fluid of choice is the dermal interstitial fluid (ISF). The significant merits of ISF over blood are the quality, quantity and diversity of ISF-based biomarkers; the fluid is non-coagulating, making it feasible to perform multiple or continuous samplings and the sampling is minimally invasive. Our immunosensor requires only 5 µL of ISF to achieve a simultaneous detection of five highly potent proinflammatory cytokines: IL-6, IFN-γ, IL-1ß, TNF-α, IP-10. We employed a microneedle array patch (MAP) together with a trifurcated nozzle pump to extract a mean volume of between 30 and 60 µL of ISF in 20 min. Under optimal conditions, the biosensor is capable of high-quality performance that exhibits a lower limit of detection (LOD) of 5.761 pg/mL over a wide linear range of 5.761-3 ‒ 20.00 ng/mL. We believe our immunosensor for NRT detections via a SOI sampling of ISF-biomarkers offers new theranostic opportunities that may not be possible with blood-based biomarkers.

Using Functionalized Liposomes to Harvest Extracellular Vesicles of Similar Characteristics in Dermal Interstitial Fluid.

Extracellular vesicles (EVs) are used by living cells for the purpose of biological information trafficking from parental-to-recipient cells and vice versa. This back-and-forth communication is enabled by two distinct kinds of biomolecules that constitute the cargo of an EV: proteins and nucleic acids. The proteomic-cum-genetic information is mediated by the physiological state of a cell (healthy or otherwise) as much as modulated by the biogenesis pathway of the EV. Therefore, in mirroring the huge diversities of human communications, the proteins and nucleic acids involved in cell communications possess seemingly near limitless diversities, and it is this characteristic that makes EVs so highly heterogeneous. Currently, there is no simple and reliable tool for the selective capture of heterogeneous EVs and the delivery of their undamaged cargo for research in extracellular protein mapping and spatial proteomics studies. Our work is a preliminary attempt to address this issue. We demonstrated our approach by using antibody functionalized liposomes to capture EVs from tumor and healthy cell-lines. To characterize their performance, we presented fluorescence and nanoparticle tracking analysis (NTA) results, TEM images, and Western blotting analysis for EV proteins. We also extracted dermal interstitial fluid (ISF) from healthy individuals and used our functionalized synthetic vesicle (FSV) method to capture EVs from their proteins. We constructed three proteomic sets [EV vs ISF, (FSV+EV) vs ISF, and (FSV+EV) vs EV] from the EV proteins and the free proteins harvested from ISF and compared their differentially expressed proteins (DEPs). The performance of our proposed method is assessed via an analysis of 1095 proteins, together with volcano plots, heatmap, GO annotation, and enriched KEGG pathways and organelle localization results of 213 DEPs.