Abstract
Protein glycosylation is one of the key processes that play essential roles in biological functions and dysfunctions. However, progress in glycomics has considerably lagged behind genomics and proteomics, due in part to the enormous challenges in analysis of glycans. Here we present a new integrated and automated microfluidic lectin barcode platform to substantially improve the performance of lectin array for focused glycomic profiling. The chip design and flow control were optimized to promote the lectin-glycan binding kinetics and speed of lectin microarray. Moreover, we established an on-chip lectin assay which employs a very simple blocking method to effectively suppress the undesired background due to lectin binding of antibodies. Using this technology, we demonstrated focused differential profiling of tissue-specific glycosylation changes of a biomarker, CA125 protein purified from ovarian cancer cell line and different tissues from ovarian cancer patients in a fast, reproducible, and high-throughput fashion. Highly sensitive CA125 detection was also demonstrated with a detection limit much lower than the clinical cutoff value for cancer diagnosis. This microfluidic platform holds the potential to integrate with sample preparation functions to construct a fully integrated “sample-to-answer” microsystem for focused differential glycomic analysis. Thus, our technology should present a powerful tool in support of rapid advance in glycobiology and glyco-biomarker development.
Highlights
Method, sandwich types of lectin array assisted by antibodies have been developed for the study of glycosylation of specific proteins[11,12]
A lectin barcode array was patterned on the substrate surface as the sensing elements for the antibody-lectin assay involving lectin capture of glycoproteins and fluorescence detection using biotinylated antibodies and dye-labeled streptavidin (Fig. 1A)
Compared to many biological interactions, glycan-lectin affinity is intrinsically weak, underscoring the importance of controlling reaction kinetics to improve the performance of lectin-based glycomic assays
Summary
Method, sandwich types of lectin array assisted by antibodies have been developed for the study of glycosylation of specific proteins[11,12]. Kuno et al reported the antibody-overlay lectin array (abbreviated as “antibody-lectin array” hereafter) in which specific antibodies were used to detect the target glycoproteins captured by the lectins on the surface[12] This method can use the same antibodies for enrichment and expression analysis of targeted glycoproteins, which enables profiling of glycosylation changes on disease-specific or tissue-specific biomarkers, and greatly increases the sensitivity, specificity and reproducibility than the direct detection method that requires pre-labeling of samples. We developed an on-chip sandwich antibody-lectin barcode assay and a very simple blocking method to effectively suppress the background noise Using this system, specific glyco-profiling and sensitive detection of two standard N-linked glycoproteins were achieved with a substantially improved sensitivity and speed than conventional lectin arrays. Our technology should provide a useful tool that complements the existing lectin array technologies to facilitate the elucidation of complex human glycome and the development of glyco-biomarkers
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