A single-step enzyme immunoassay capillary sensor composed of functional multilayer coatings for the diagnosis of marker proteins

Abstract

A single-step, easy-to-use enzyme immunoassay capillary sensor, composed of functional multilayer coatings, was developed in this study. The coatings were composed of substrate-immobilized hydrophobic coating, hydrogel coating, and soluble coating containing an enzyme-labeled antibody. The response mechanism involved a spontaneous immunoreaction triggered by capillary action-mediated introduction of a sample antigen solution and subsequent separation of unreacted enzyme-labeled antibodies and antigen-enzyme-labeled antibody complexes by the molecular sieving effect of the hydrogel. An enzyme reaction at the substrate-immobilized hydrophobic coating/hydrogel coating interface resulted in a protein-selective fluorescence response. An antigen concentration-dependent response was obtained for diagnostic marker protein samples (hemoglobin A1c (HbA1c), 7.14–16.7 mg mL⁻¹; alpha-fetoprotein (AFP), 1.4–140 ng mL⁻¹; C-reactive protein (CRP), 0.5–10 μg mL⁻¹) that cover a clinically important concentration range. The successful measurement of CRP in diluted serum samples demonstrated the application of this capillary sensor.