||Detection of Trace Vascular Endothelial Growth Factor (VEGF) Using a 3D-Dielectrophoresis-based Microfluidic Chip
||Department of BioMedical Engineering
Vascular Endothelial Growth Factor
Vascular Endothelial Growth Factor (VEGF) is a signal protein produced by cells to stimulate the formation of new blood vessels. Although VEGF is essential for physiologic vascular homeostasis, it has been demonstrated to be important in pathogenesis of tumor growth and metastasis, and retinopathy associated with several blinding eye diseases. Studies also suggest the upregulation of VEGF due to various viruses. VEGF detection is difficult to achieve because it is present at extremely low levels (pg/mL), and is currently achieved in the clinical setting through Enzyme-Linked Immunosorbent Assay (ELISA). However, this method has disadvantages such as being a tedious process and takes a long time to perform. Here, we propose a method for trace VEGF detection by using a microfluidic chip integrated with a dielectrophoretic function. This chip can either capture or repel particles by utilizing a dielectrophoresis (DEP) force. Silica beads immobilized with an aptamer specific to VEGF is injected into the chip, which is then captured by the microelectrodes in the chip. A solution of VEGF conjugated to a labeled antibody is subsequently injected into the chip. When the VEGF is captured by the beads, a fluorescence signal is produced, which can then be observed with a fluorescence microscope. The amount of VEGF can then be correlated with the area of fluorescence produced from the beads. This method offers the advantage of being able to detect trace amounts of VEGF (<100 pg/mL) while requiring only a small sample volume (15 μL) and a short assay time (15 min).
1. INTRODUCTION 1
1.1 Vascular Endothelial Growth Factor 1
1.1.1 Functions of VEGF 1
1.1.2 VEGF isoforms 2
1.1.3 VEGF production in the body 2
1.2 Significance of VEGF for diagnosis of disease 3
1.2.1 VEGF in cancer 3
1.2.2 VEGF in intraocular neovascular syndromes 3
1.2.3 VEGF in Viral Diseases 4
1.2.4 Human Papillomavirus 4
1.2.5 Dengue Virus 4
1.3 Current Methods for VEGF detection 5
1.3.1 Enzyme-linked immunosorbent assay 5
1.3.2 Surface Plasmon Resonance 5
1.3.3 Surface-enhanced Raman Spectroscopy 5
1.3.4 Field-effect Transistor 6
1.3.5 Electrochemical Techniques 6
1.4 Microfluidics 7
1.4.1 Electrokinetics 7
1.4.2 Theory of Dielectrophoresis (DEP)7
1.4.3 Limitations of DEP 9
1.4.4 Advantages to using a 3D format for DEP 9
1.4.5 Previous studies using 3D DEP 10
1.5 Motivation and framework of this Study 11
2. MATERIALS AND METHODS 12
2.1 Chip fabrication 12
2.1.1 Microelectrode fabrication 12
2.1.2 Microchannel fabrication 13
2.1.3 Chip alignment and bonding 13
2.2 Immobilization of capture molecule on microparticles 14
2.3 Sample preparation 15
2.4 Evaluation of binding affinity using flow cytometry 16
2.5 Experiment design 17
2.6 Quantification of the fluorescence signal 18
3. RESULTS AND DISCUSSION 19
3.1 Binding affinity evaluation of the aptamer with VEGF 19
3.2 Evaluation of the bead capturing performance of the chip 21
3.3 Detection of VEGF based on the fluorescence signal 23
3.4 VEGF calibration curve 26
3.5 Detection of VEGF in a matrix similar to human plasma 28
4. CONCLUSION 29
5. PROSPECTS AND FUTURE WORK 30
Personal Information 33
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