Immunoassay for Cardiac Troponin I with Fluorescent Signal Amplification by Hydrolyzed Coumarin Released from a Metal-Organic Framework

Despite its simplicity and specificity, enzyme-linked immunosorbent assay (ELISA) requires conjugation of the enzyme to an antibody and preservation of enzymatic activity during storage and assay. In light of the high porosity and enormous surface area possessed by metal-organic frameworks (MOFs), we constructed an immunosensor by integrating antibody and fluorescent probe (coumarin or COU) with zeolitic imidazolate framework-8 (ZIF-8), in which about 34 COU molecules can be encapsulated per ZIF-8. After coating of ZIF-8 with the secondary antibody specific to cardiac troponin I (cTnI), the final MOF@COU/Ab(2) composite renders an enzyme-free assay that amplifies the detection signal via release of alkaline-hydrolyzed COU from the MOF interior. Owing to the isolation of the fluorescent probe before the readout step, our immunosensors are highly stable and reproducible, with a relative standard deviation of less than 1.5% for repetitive assays in a 30-day period. Our MOF-based immunoassay is also highly sensitive and possesses a wide dynamic range (11.1 fM to 35.6 pM). The detection limit was estimated to be 4.4 fM (0.099 pg.mL(-1)), which is similar to 180-fold lower than that of ELISA. We quantified cTnI in clinical serum samples, yielding results highly comparable to those measured by ELISA. Particularly remarkable is that the low detection limit enabled us to quantify cTnI in sera of healthy persons, including levels that are close to the high-risk threshold but not detectable by ELISA. The generality of this cost-effective and stable fluorescent immunosensor renders versatilities for sensitive detections of many other protein biomarkers.