scholarly journals VEGF Detection via Simplified FLISA Using a 3D Microfluidic Disk Platform

Biosensors ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 270
Author(s):  
Dong Hee Kang ◽  
Na Kyong Kim ◽  
Sang-Woo Park ◽  
Hyun Wook Kang
Keyword(s):  
Three Dimensional ◽  
Rotating Disk ◽  
Microfluidic System ◽  
Antibody Binding ◽  
Vascular Endothelial ◽  
Binding Reaction ◽  
Incubation Chamber ◽  
Manual Intervention ◽  
Antigen Antibody ◽  
Endothelial Growth Factor

Fluorescence-linked immunosorbent assay (FLISA) is a commonly used, quantitative technique for detecting biochemical changes based on antigen–antibody binding reactions using a well-plate platform. As the manufacturing technology of microfluidic system evolves, FLISA can be implemented onto microfluidic disk platforms which allows the detection of trace biochemical reactions with high resolutions. Herein, we propose a novel microfluidic system comprising a disk with a three-dimensional incubation chamber, which can reduce the amount of the reagents to 1/10 and the required time for the entire process to less than an hour. The incubation process achieves an antigen–antibody binding reaction as well as the binding of fluorogenic substrates to target proteins. The FLISA protocol in the 3D incubation chamber necessitates performing the antibody-conjugated microbeads’ movement during each step in order to ensure sufficient binding reactions. Vascular endothelial growth factor as concentration with ng mL−1 is detected sequentially using a benchtop process employing this 3D microfluidic disk. The 3D microfluidic disk works without requiring manual intervention or additional procedures for liquid control. During the incubation process, microbead movement is controlled by centrifugal force from the rotating disk and the sedimentation by gravitational force at the tilted floor of the chamber.

VEGF Detection via Simplified FLISA Using a 3D Microfluidic Disk Platform

2021 ◽  
Author(s):  
Dong Hee Kang ◽  
Na Kyong Kim ◽  
Sang-Woo Park ◽  
Hyun Wook Kang
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Gravitational Force ◽  
Lower Proportion ◽  
Antibody Binding ◽  
Vascular Endothelial ◽  
Fluorogenic Substrates ◽  
Disk Rotation ◽  
Manual Intervention ◽  
Time Required ◽  
Endothelial Growth Factor

Fluorescence-linked immunosorbent assay (FLISA) is a commonly used, quantitative technique for detecting biochemical based on antigen–antibody binding reactions using a well-plate platform. With the developments in the manufacturing technology of microfluidic systems, FLISA can be implemented onto microfluidic disk platforms, which allows the detection of trace biochemical with high resolutions. Apart from requiring a lower proportion of reagent (1/10), this method also reduces the time required for the entire process to less than an hour. The incubation process involves antigen–antibody binding reactions as well as the binding of fluorogenic substrates to target proteins. The protocol for FLISA on a microfluidic platform necessitates the appropriate execution of liquid reagent movements during each step in order to ensure sufficient binding reactions. Herein, we propose a novel microfluidic disk comprising a 3D incubation chamber. Vascular endothelial growth factor as concentration with ng mL-1 is detected sequentially using a benchtop process employing this 3D microfluidic disk. The 3D microfluidic disk is implemented without requiring manual intervention or additional procedures for liquid control. During the incubation process, microbead movement is controlled through centrifugal force, generated due to disk rotation, and gravitational force via bead sedimentation on the sloped floor of the chamber.

Early changes in coronary artery wall structure detected by microcomputed tomography in experimental hypercholesterolemia

2007 ◽  
Vol 293 (3) ◽  
pp. H1997-H2003 ◽  
Author(s):  
Xiang-Yang Zhu ◽  
Michael D. Bentley ◽  
Alejandro R. Chade ◽  
Erik L. Ritman ◽  
Amir Lerman ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Coronary Artery ◽  
Growth Factor ◽  
Three Dimensional ◽  
Wall Structure ◽  
Vascular Endothelial ◽  
Micro Ct ◽  
Artery Wall ◽  
Coronary Artery Wall ◽  
Endothelial Growth Factor

Changes in the structure of the artery wall commence shortly after exposure to cardiovascular risk factors, such as hypercholesterolemia (HC), but may be difficult to detect. The ability to study vascular wall structure could be helpful in evaluation of the factors that instigate atherosclerosis and its pathomechanisms. The present study tested the hypothesis that early morphological changes in coronary arteries of hypercholesterolemic (HC) pigs can be detected using the novel X-ray contrast agent OsO4 and three-dimensional micro-computed tomography (CT). Two groups of pigs were studied after they were fed a normal or an HC (2% cholesterol) diet for 12 wk. Hearts were harvested, coronary arteries were injected with 1% OsO4 solution, and cardiac samples (6-μm-thick) were scanned by micro-CT. Layers of the epicardial coronary artery wall, early lesions, and perivascular OsO4 accumulation were determined. Leakage of OsO4 from myocardial microvessels was used to assess vascular permeability, which was correlated with immunoreactivity of vascular endothelial growth factor in corresponding histological cross sections. OsO4 enhanced the visualization of coronary artery wall layers and facilitated detection of early lesions in HC in longitudinal tomographic sections of vascular segments. Increased density of perivascular OsO4 in HC was correlated with increased vascular endothelial growth factor expression and suggested increased microvascular permeability. The use of OsO4 as a contrast agent in micro-CT allows three-dimensional visualization of coronary artery wall structure, early lesion formation, and changes in vascular permeability. Therefore, this technique can be a useful tool in atherosclerosis research.

390 Characterisation of a Platelet Rich Fibrin Membrane and Formation of an Autologous Fibrin Mesh

2021 ◽  
Vol 108 (Supplement_6) ◽  
Author(s):  
Joshua Burke ◽  
Jack Helliwell ◽  
Mikolaj Kowal ◽  
David Jayne
Keyword(s):  
Three Dimensional ◽  
Clinical Results ◽  
Vascular Endothelial ◽  
Blood Draw ◽  
Platelet Rich Fibrin ◽  
Fibrin Scaffold ◽  
Surgical Sutures ◽  
Endothelial Growth Factor ◽  
Range Of Values ◽  
Fibrin Membrane

Abstract Aim Platelet-rich fibrin (PRF) is a three-dimensional fibrin scaffold with associated platelets and leukocytes which releases high quantities of growth factors over a sustained period of time. PRF has shown promising clinical results in promoting wound healing and tissue regeneration. The aims of this feasibility study were to establish optimal spinning methods for production of PRF, to quantify the production of vascular endothelial growth factor (VEGF) by PRF and to explore new vehicles of clinical PRF delivery. Method Assessment of optimal production involved comparisons between Protocol 1 (EDTA bottle) and Protocol 2 (no additive) at three different centrifugation forces: 400g, 1000g and 1700g. VEGF production was analysed using ELISA with varied incubation periods and PRF plug segments. Novel methods for PRF delivery were explored using surgical sutures and a Zimmer® Skin Graft Mesher. Results Protocol 2 demonstrated shorter average time to blood draw (9.8s compared to 13.6s) and to centrifuge (25.5s compared to 33.1s) with a decreased range of values. All PRF segments exhibited a positive correlation between incubation time and amount of VEGF produced with the bottom segments producing on average more VEGF. A segment of the fibrin plug was successfully secured on a suture and meshed in a 1:1.5 ratio. Conclusions PRF production can be optimised using blood bottles with no additive and high centrifugation forces. VEGF production by PRF peaks at 120 hours with the bottom PRF segment exhibiting the highest rate of production. The first description of a PRF mesh enables new clinical applications.

Interaction between vascular endothelial cells and vascular intimal spindle-shaped cells in vitro

1983 ◽  
Vol 60 (1) ◽  
pp. 89-102
Author(s):  
D de Bono ◽  
C. Green
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Three Dimensional ◽  
Vascular Endothelial ◽  
Pure Cultures ◽  
Species Specific ◽  
Endothelial Growth Factor

The interactions between human or bovine vascular endothelial cells and fibroblast-like vascular intimal spindle-shaped cells have been studied in vitro, using species-specific antibodies to identify the different components in mixed cultures. Pure cultures of endothelial cells grow as uniform, nonoverlapping monolayers, but this growth pattern is lost after the addition of spindle cells, probably because the extracellular matrix secreted by the latter causes the endothelial cells to modify the way they are attached to the substrate. The result is a network of tubular aggregates of endothelial cells in a three-dimensional ‘polylayer’ of spindle-shaped cells. On the other hand, endothelial cells added to growth-inhibited cultures of spindle-shaped cells will grow in sheets over the surface of the culture. Human endothelial cells grown in contact with spindle-shaped cells have a reduced requirement for a brain-derived endothelial growth factor. The interactions of endothelial cells and other connective tissue cells in vitro may be relevant to the mechanisms of endothelial growth and blood vessel formation in vivo, and emphasize the potential importance of extracellular matrix in controlling endothelial cell behaviour.

Three-Dimensional Simulation of In Vitro Angiogenesis: Effects of Extracellular Matrix Structure and Density

2010 ◽  
Author(s):  
Lowell Taylor Edgar ◽  
James E. Guilkey ◽  
Clayton J. Underwood ◽  
Brenda Baggett ◽  
Urs Utzinger ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Three Dimensional ◽  
Vascular Endothelial ◽  
Chemical Factors ◽  
Dimensional Simulation ◽  
Endothelial Growth Factor ◽  
In Vitro Angiogenesis

The process of angiogenesis is regulated by both chemical and mechanical signaling. While the role of chemical factors such as vascular endothelial growth factor (VEGF) during angiogenesis has been extensively studied, the influence of the mechanostructural environment on new vessel generation has received significantly less attention. During angiogenesis, endothelial cells in the existing vasculature detach and migrate out into the surrounding extracellular matrix (ECM), forming tubular structures that eventually mature into new blood vessels. This process is modulated by the structure and composition of the ECM [1]. The ECM is then remodeled by endothelial cells in the elongating neovessel tip, resulting in matrix condensation and changes in fiber orientation [2]. The mechanism as to how angiogenic vasculature and the ECM influence each other is poorly understood.

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