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

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.