Dye/DNA conjugates as multiple labels for antibodies in sensitive fluorescence immunoassays

Fluorescence immunoassays are widely used in life science research, medical diagnostics, and environmental monitoring due to the intrinsically high specificity, simplicity, and versatility of immunoassays as well as the availability of a large variety of fluorescent labeling molecules. However, the sensitivity of immunoassays needs to be improved further to meet the ever-increasing demands of the new proteomics era. We have developed a novel and simple method to increase immunoassay sensitivity by attaching multiple fluorescent labels on an antibody with a dye/DNA conjugate. Our strategy is to use a DNA fragment as a molecular carrier to attach multiple fluorescent dyes to an antibody at a single site. The dye/DNA conjugate is not presynthesized, but rather formed in situ as part of the immunoassay. Our results demonstrate that by using a 219-bp DNA fragment in conjunction with SYBR Green I fluorescent DNA-binder, the sensitivity of both direct and competitive fluorescence immunoassays is improved by orders of magnitude, reaching a lower detection limit of 1.9 pg/mL for 17β-estradiol.