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:Analysis of protein phosphorylation in extracellular vesicles (EVs) offers an unprecedented potential for understanding cancer signaling and early stage disease diagnosis. However, prior to the phosphoproteome analysis step, the isolation of EVs from biofluids remains a challenging issue to overcome due to the low yield and impurity from current isolation methods. Here, we carry out an extensive assessment of several EV isolation methods including a novel rapid isolation method EVTRAP for highly efficient capture of extracellular vesicles from human urine sample. We demonstrate that over 95% recovery yield can be consistently achieved by EVTRAP, a significant improvement over current standard techniques. We then applied EVTRAP to identify over 16 000 unique peptides representing 2000 unique EV proteins from 200 μL urine sample, including all known EV markers with substantially increased recovery levels over ultracentrifugation. Most importantly, close to 2000 unique phosphopeptides were identified from more than 860 unique phosphoproteins using 10 mL of urine. The data demonstrated that EVTRAP is a highly effective and potentially widely implementable clinical isolation method for analysis of EV protein phosphorylation.
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Project description:Endothelialization by materials provides a promising approach for the rapid re-endothelialization of a cardiovascular implantation. Although previous studies have focused on improving endothelialization through the immobilization of bioactive molecules onto the surface of biodegradable implants, comparative studies of effective surface modification have not yet been reported. Here, we conducted a comparative study on the surface modification of poly(lactide-co-glycolide) (PLGA)-based composites to graft mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) using three different materials, fibronectin (FN), polyethylenimine (PEI), and polydopamine (PDA), which have different bond strengths of ligand-receptor interaction, ionic bond, and covalent bond, respectively. Further in vitro analysis exhibited that MSC-EVs released from all modified films sustainably, but the MSC-EVs grafted onto the surface coated with PEI are more effective than other groups in increasing angiogenesis and reducing the inflammatory responses in endothelial cells. Therefore, the overall results demonstrated that PEI is a desirable coating reagent for the immobilization of MSC-EVs on the surface of biodegradable implants.
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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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Project description:Detection of analytes using streaming current has previously been explored using both experimental approaches and theoretical analyses of such data. However, further developments are needed for establishing a viable microchip that can be exploited to deliver a sensitive, robust, and scalable biosensor device. In this study, we demonstrated the fabrication of such a device on silicon wafer using a scalable silicon microfabrication technology followed by characterization and optimization of this sensor for detection of small extracellular vesicles (sEVs) with sizes in the range of 30 to 200 nm, as determined by nanoparticle tracking analyses. We showed that the sensitivity of the devices, assessed by a common protein-ligand pair and sEVs, significantly outperforms previous approaches using the same principle. Two versions of the microchips, denoted as enclosed and removable-top microchips, were developed and compared, aiming to discern the importance of high-pressure measurement versus easier and better surface preparation capacity. A custom-built chip manifold allowing easy interfacing with standard microfluidic connections was also constructed. By investigating different electrical, fluidic, morphological, and fluorescence measurements, we show that while the enclosed microchip with its robust glass-silicon bonding can withstand higher pressure and thus generate higher streaming current, the removable-top configuration offers several practical benefits, including easy surface preparation, uniform probe conjugation, and improvement in the limit of detection (LoD). We further compared two common surface functionalization strategies and showed that the developed microchip can achieve both high sensitivity for membrane protein profiling and low LoD for detection of sEV detection. At the optimum working condition, we demonstrated that the microchip could detect sEVs reaching an LoD of 104 sEVs/mL (when captured by membrane-sensing peptide (MSP) probes), which is among the lowest in the so far reported microchip-based methods.
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Project description:Urinary extracellular vesicles (uEVs) are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.
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Project description:We performed RNA-Seq analysis of plasma and urinary EVs collected before and after radical prostatectomy, and matched tumor and normal prostate tissues of 10 patients with prostate cancer. To identify putative cancer-derived RNA biomarkers, we searched for RNAs that were overexpressed in tumor as compared to normal tissues, present in the pre-operation EVs and decreased in the post-operation EVs in each RNA biotype.
2022-11-16 | E-MTAB-11910 | biostudies-arrayexpress
Project description:Background: Renal cell carcinoma (RCC) accounts for about 2% of all cancers. Renal biopsy is the gold standard among the diagnostic tools, but it is invasive and not suitable for all patients. Therefore, new reliable and non-invasive biomarkers for ccRCC detection are required. Secretion of extracellular vesicles (EVs), containing RNA molecules that can be transferred between cells, seems to be a general characteristic of malignant transformation. Consistently, cancer-derived EVs are enriched in the blood, urine and various malignant effusions of cancer patients. Therefore, urinary samples can be a non-invasive approach for discovering diagnostic biomarkers. Methods: We enrolled 33 clear-cell RCC (ccRCC) patients and 22 healthy subjects (HS), age and sex-matched, for urine collection and extracellular vesicles isolation by differential centrifugation. Transcriptional profiles of urinary EVs from 12 patients with ccRCC and 11 HS were generated using the Illumina HumanHT-12 v4 BeadChip oligonucleotide arrays. Microarray analysis led to the identification of RNA that were then validated using RT-qPCR. Results: We showed for the first time that urinary exosomal shuttle RNA (esRNA) was significantly different in ccRCC patients compared to HS and we identified three EVs esRNA involved in the tumor biology that are potentially suitable as non-invasive biomarkers. GSTA1, CEBPA and PCBD1 RNA levels decreased in urinary EVs of patients compared to HS. After 1 month post-operation, the levels of RNA increased to reach the normal level. Conclusions: This study suggests, for the first time, the potential use of the RNA content of urinary EVs to provide a non-invasive first step to diagnose the ccRCC. Total RNA obtained from urinary extracellular vesicles isolated from ccRCC patients and healthy subjects.
2016-09-01 | E-GEOD-72922 | biostudies-arrayexpress