Design and Optimization of a Biosensor Surface Functionalization to Effectively Capture Urinary Extracellular Vesicles.
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ABSTRACT:
SUBMITTER: Kaminska A
PROVIDER: S-EPMC8399133 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Metallic material functionalization with Extracellular Vesicles (EVs) is a desirable therapeutic approach to improve regenerative procedures. Among the different functionalization strategies available, here we have compared drop casting on machined Ti surfaces, drop casting on nanostructured TiO2 surfaces and polymeric entrapment with polydopamine. EVs are a heterogeneous population of communication nanovesicles released by cells that are being intensively investigated for their use in therapeutics. We have selected platelet derived EVs for Ti surface coating due to their demonstrated osteoinductive properties. Our results show that each functionalization strategy leads to differences in the size of EV populations attached to and released from the metallic implants, which, in turn, leads to variations in their osteogenic capability measured through alkaline phosphatase activity and calcium deposition. In conclusion, the functionalization strategy used has an important effect on the resulting implant functionality, probably due to the heterogeneous EVs nature. Thus, the methodological approach to metallic material functionalization should be carefully chosen when working with extracellular vesicles in order to obtain the desired therapeutic application.
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Project description:We highlight microgel/enzyme thin films that were deposited onto solid interfaces via two sequential steps, the adsorption of temperature- and pH-sensitive microgels, followed by their complexation with the enzyme choline oxidase, ChO. Two kinds of functional (ionic) microgels were compared in this work in regard to their adsorptive behavior and interaction with ChO, that is, poly(N-isopropylacrylamide-co-N-(3-aminopropyl)methacrylamide), P(NIPAM-co-APMA), bearing primary amino groups, and poly(N-isopropylacrylamide-co-N-[3-(dimethylamino) propyl]methacrylamide), P(NIPAM-co-DMAPMA), bearing tertiary amino groups. The stimuli-sensitive properties of the microgels in the solution were characterized by potentiometric titration, dynamic light scattering (DLS), and laser microelectrophoresis. The peculiarities of the adsorptive behavior of both the microgels and the specific character of their interaction with ChO were revealed by a combination of surface characterization techniques. The surface charge was characterized by electrokinetic analysis (EKA) for the initial graphite surface and the same one after the subsequent deposition of the microgels and the enzyme under different adsorption regimes. The masses of wet microgel and microgel/enzyme films were determined by quartz crystal microbalance with dissipation monitoring (QCM-D) upon the subsequent deposition of the components under the same adsorption conditions, on a surface of gold-coated quartz crystals. Finally, the enzymatic responses of the microgel/enzyme films deposited on graphite electrodes to choline were tested amperometrically. The presence of functional primary amino groups in the P(NIPAM-co-APMA) microgel enables a covalent enzyme-to-microgel coupling via glutar aldehyde cross-linking, thereby resulting in a considerable improvement of the biosensor operational stability.
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Project description:BackgroundUrinary extracellular vesicles (UEVs) represent an ideal platform for biomarker discovery. They carry different types of RNA species, and reported profile discrepancies related to the presence/absence of 18s and 28s rRNA remain controversial. Moreover, sufficient urinary RNA yields and respective quality RNA profiles are still to be fully established.MethodsUEVs were enriched by hydrostatic filtration dialysis, and RNA content was extracted using 7 different commercially available techniques. RNA quantity was assessed using spectrophotometry and fluorometry, whilst RNA quality was determined by capillary electrophoresis.ResultsThe presence of prokaryotic transcriptome was stressed when cellular RNA, as a control, was spiked into the UEVs samples before RNA extraction. The presence of bacteria in hydrostatic filtration dialysis above 1,000 kDa molecular weight cut-off and in crude urine was confirmed with growth media plates. The efficiency in removing urinary bacteria was evaluated by differential centrifugation, filtration (0.22 µm filters) and chemical pretreatment (water purification tablet). For volumes of urine >200 ml, the chemical treatment provides ease of handling without affecting vesicle integrity, protein and RNA profiles. This protocol was selected to enrich RNA with 7 methods, and its respective quality and quantity were assessed. The results were given as follows: (a) Fluorometry gave more repeatability and reproducibility than spectrophotometry to assess the RNA yields, (b) UEVs were enriched with small RNA, (c) Ribosomal RNA peaks were not observed for any RNA extraction method used and (d) RNA yield was higher for column-based method designed for urinary exosome, whilst the highest relative microRNA presence was obtained using TRIzol method.ConclusionOur results show that the presence of bacteria can lead to misidentification in the electrophoresis peaks. Fluorometry is more reliable than spectrophotometry. RNA isolation method must be selected in conjunction with appropriate UEV collection procedure. We also suggested that a minimum 250 ml of urine should be processed to gather enough RNA for robust quantification, qualification and downstream analysis.
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2022-11-16 | E-MTAB-11910 | biostudies-arrayexpress