Antibody microarrays have become a powerful tool in multiplexed immunoassay technologies. The advantage of microarray technology is the possibility of rapid analysis of multiple targets in a single sample with a high sensitivity, which makes them ideal for high throughput screening. Usually these microarrays contain biological recognition molecules, such as full-size antibodies, antigen-binding fragments, and single-domain antibodies, and a label for detection. Organic fluorophores are the most popular labels, but they suffer from low sensitivity and instability due to their photodegradation. Here, we describe a protocol for fabricating an antibody microarray with highly fluorescent semiconductor nanocrystals or quantum dots (QDs) as the source of fluorescent signals, which may significantly improve the properties of microarrays, including their sensitivity and specificity. Our approach to analyte detection is based on the use of sandwich approach with streptavidin-biotin to assess and monitor the fluorescence signal instead of direct labeling of samples, which helps improve the reproducibility of results and sensitivity of the microarrays. The antibody microarray developed has been tested for its capacity of detecting DNA-PKcs in glial cell lines and measuring cell protein phosphorylation changes caused by camptothecin-induced DNA damage with different protein kinase inhibitors in HeLa cells.
Keywords: DNA damage; DNA-PKcs kinase activity; Diagnostic tool; Protein arrays; Semiconductor quantum dot (QD).