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Immunoassays are analytical methods that use the highly specific binding of antibodies in order to detect and quantify an analyte. The technique has become a staple in modern biopharmaceutical research and diagnostics, however the measurement of biomarkers like dysregulated cytokines require ultra-sensitive immunoassays that can detect molecules at sub pg/mL concentrations. One such method is the Exazym^® signal amplification. Based on a method called Binding Oligo Ladder Detection (BOLD), it is a set of add-on reagents where a primer is conjugated to a detection antibody which is then combined with a template, polymerase and modified DNA nucleotides to generate a oligonucleotide ladder that is detected with a secondary detection antibody; this amplifies the signal by a factor of 10-100 in an existing immunoassay. 

By applying this method to the Gyrolab^® microfluidic immunoassay system, a sensitivity increase of 880x-1800x was achieved between a pre-synthesised BOLD product and the polymerised BOLD product. Several key factors for successful polymerisation in the microfluidic system were identified: adding the template separately before the polymerase and using a buffer with low ionic strength for the secondary detection antibody. Applying the BOLD amplification to an existing Gyrolab TNF-α assay only resulted in similar sensitivity as previous methods however. This report demonstrates that BOLD amplification can be successfully performed in a flow-through format on miniaturized affinity columns in the Gyrolab system to increase the sensitivity by orders of magnitude, where both the immunoassay and the amplification steps are automated in the system. However, further optimisation is needed for application in biomarker assays.