title: A mass-tagging approach for enhanced sensitivity of dynamic cytokine detection using a label-free biosensor.
abstract: Monitoring cytokine release by cells allows the investigation of cellular response to specific external stimuli, such as pathogens or candidate drugs. Unlike conventional colorimetric techniques, label-free detection of cytokines enables studying cellular secretions in real time by eliminating additional wash and labeling steps after the binding step. However, label-free techniques that are based on measuring mass accumulation on a sensor surface are challenging for measuring small cytokines binding to much larger capture agents (usually antibodies) because the relative signal change is small. This problem is exacerbated when the capturing antibodies desorb from the surface, a phenomenon that almost inevitably occurs in immunoassays but is rarely accounted for. Here, we demonstrate a quantitative dynamic detection of interleukine-6 (IL-6), a pro-inflammatory cytokine, using an interferometric reflectance imaging sensor (IRIS). We improved the accuracy of the quantitative analysis of this relatively small protein (21 kDa) by characterizing the antibody desorption rate and compensating for the antibody loss during the binding experiment. By correcting for protein desorption, we achieved an analytical limit of detection at 19 ng/mL IL-6 concentration. We enhanced the sensitivity by 7-fold by using detection antibodies that recognize a different epitope of the cytokine. We demonstrate that these detection antibodies, which we call "mass tags", can be used concurrently with the target analyte to eliminate an additional wash and binding step. Finally, we report successful label-free detection of IL-6 in cell culture medium (with 10% serum) with comparable signal to that obtained in PBS. This work is the first to report quantitative dynamic label-free detection of small protein in a complex biological fluid using IRIS.
authors.affiliation: Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215, USA.
authors.forename: Sunmin
authors.initials: S
authors.lastname: Ahn
authors.forename: David S
authors.initials: DS
authors.lastname: Freedman
authors.forename: Paola
authors.initials: P
authors.lastname: Massari
authors.forename: Mario
authors.initials: M
authors.lastname: Cabodi
authors.forename: M Selim
authors.initials: MS
authors.lastname: Ünlü
mesh_terms: D015374:Biosensing Techniques; D002478:Cells, Cultured; D006801:Humans; D015850:Interleukin-6 / Q000032:analysis*; D011994:Recombinant Proteins / Q000032:analysis
chemical_list: D015850:Interleukin-6; D011994:Recombinant Proteins