Abstract
In addition to disease diagnostics, there is a need for biomarkers to predict severity of cancer therapy side effects such as oral mucositis. Oral mucositis is an inflammatory lesion of oral mucosa caused by high-dose chemotherapy and/or radiation that is especially prevalent during oral cancer treatment. We describe here a semi-automated, modular microfluidic immunoarray optimized for ultrasensitive detection of pro-inflammatory cytokines involved in pathobiology of oral mucositis. Our goal is to methodologically identify risk of mucositis early in oral cancer treatment, before the onset of lesions. Biomarkers include tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and C-reactive protein (CRP). Protein analytes were captured from serum in a capture chamber by 1-μm magnetic beads coated with antibodies and enzyme labels. Beads are then transported downstream to a detection chamber containing an eight-sensor array coated with glutathione-coated gold nanoparticles (GSH-AuNP) and a second set of antibodies to capture the beads with analyte proteins. In this first application of the immunoarray to four-protein multiplexed measurements, ultralow detection limits of 10–40 fg mL−1 in 5 μL serum were achieved for simultaneous detection in 30 min. Mass detection limits were 2.5–10 zmol, as few as 1500 molecules. Accuracy and diagnostic utility of the arrays were demonstrated by correlation of levels of the four biomarker proteins in serum from head and neck cancer patients with results from standard ELISA. This approach may lead to rapid, low-cost estimates of projected risk for severity of oral mucositis in cancer patients to enable improved therapeutic management.
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Acknowledgments
This work was supported financially by grant no. EB014586 from the National Institute of Biomedical Imaging and Bioengineering (NIBIB), NIH. Serum samples were obtained in a study supported by grant no. K23DE016946 from the National Institute of Dental and Craniofacial Research and grant no. M01RR06192 from the National Center for Research Resources. We thank Colleen Holewa for initial assay development of TNF-α.
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Colleen E. Krause and Brunah A. Otieno contributed equally to this work.
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Krause, C.E., Otieno, B.A., Bishop, G.W. et al. Ultrasensitive microfluidic array for serum pro-inflammatory cytokines and C-reactive protein to assess oral mucositis risk in cancer patients. Anal Bioanal Chem 407, 7239–7243 (2015). https://doi.org/10.1007/s00216-015-8873-1
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DOI: https://doi.org/10.1007/s00216-015-8873-1