Fabrication and characterization of 3D hydrogel microarrays to measure antigenicity and antibody functionality for biosensor applications
| Autor: | Chris R. Taitt, Paul T. Charles, Ellen R. Goldman, Jermain G. Rangasammy, Caroline L. Schauer, Mu-San Chen |
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| Rok vydání: | 2004 |
| Předmět: |
Polyacrylamide Hydrogel
Antigenicity Polyacrylamide Protein Array Analysis Biomedical Engineering Biophysics Analytical chemistry Antigen-Antibody Complex Biosensing Techniques Sensitivity and Specificity law.invention chemistry.chemical_compound Confocal microscopy law Fluorescence Polarization Immunoassay Electrochemistry Chromatography technology industry and agriculture Reproducibility of Results Hydrogels Equipment Design General Medicine Fluorescence Equipment Failure Analysis chemistry Covalent bond Self-healing hydrogels Biosensor Biotechnology |
| Zdroj: | Biosensors and Bioelectronics. 20:753-764 |
| ISSN: | 0956-5663 |
| Popis: | We report the fabrication, characterization and evaluation of three-dimensional (3D) hydrogel thin films used to measure protein binding (antigenicity) and antibody functionality in a microarray format. Protein antigenicity was evaluated using the protein toxin, staphylococcal enterotoxin B (SEB), as a model on highly crosslinked hydrogel thin films of polyacrylamide and on two-dimensional (2D) glass surfaces. Covalent crosslinking conditions were optimized and quantified. Interrogation of the modified 3D hydrogel was measured both by direct coupling of a Cy5-labeled SEB molecule and Cy5-anti-SEB antibody binding to immobilized unlabeled SEB. Antibody functionality experiments were conducted using three chemically modified surfaces (highly crosslinked polyacrylamide hydrogels, commercially available hydrogels and 2D glass surfaces). Cy3-labeled anti-mouse IgG (capture antibody) was microarrayed onto the hydrogel surfaces and interrogated with the corresponding Cy5-labeled mouse IgG (antigen). Five different concentrations of Cy5-labeled mouse IgG were applied to each microarrayed surface and the fluorescence quantified by scanning laser confocal microscopy. Experimental results showed fluorescence intensities 3-10-fold higher for the 3D films compared to analogous 2D surfaces with attomole level sensitivity measured in direct capture immunoassays. However, 2D surfaces reported equal or greater sensitivity on a per-molecule basis. Reported also are the immobilization efficiencies, inter-and intra-slide variability and detection limits. |
| Databáze: | OpenAIRE |
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