Improved ELISA for tumor marker detection using electro-readout-mode based on label triggered degradation of...

Enzyme-linked immunosorbent assay (ELISA), a gold-standard method for protein detection, has been widely utilized in disease diagnosis. Nevertheless, the method is constrained by its dependence on a colorimetric readout that, due to its relatively low sensitivity, prevents its use in tumor marker detection. Here we demonstrate an ELISA that incorporates an electro-readout mode in place of a colorimetric readout to achieve ultrasensitive and convenient tumor marker detection. Briefly, because hemin molecules supported by carbon sphere (CS) can catalyze dye degradation upon H₂O₂ addition, CS incorporating hemin was employed as a label. A hydrogel of sodium alginate-graphene oxide (SA-GO) affixed to an electrode was utilized as a support matrix to immobilize methylene blue (MB) for electrochemical signal generation. Using an ELISA like approach, a solution containing H₂O₂ was added to wells of 96-well plates containing preformed sandwiched immunocomplexes comprised of analyte, capture monoclonal antibody and signal antibody labeled with CS-supported hemin. When the hydrogel-modified electrode was immersed into a well containing a solution containing analyte immunocomplexes, the degradation of MB on the electrode was immediately triggered by label present within the immunocomplexes, resulting in a signal decrease dependent upon analyte concentration. Cancer antigen 125 was used as a model analyte to evaluate the improved ELISA. The calculated limit of detection was 0.048 mU mL^-1, which was over six-fold more sensitive than traditional ELISA, indicating that this strategy greatly improves ELISA sensitivity and holds great promise for the quantitative determination of biomarkers for use in clinical diagnosis.