Amplified Mass Immunosorbent Assay with a Quartz Crystal Microbalance

Amplified mass immunosorbent assays (AMISA) using a quartz-crystal microbalance (QCM) are described for the detection of adenosine 5'-phosphosulfate (APS) reductase and human chorionic gonadotropin (hCG). APS reductase detection is accomplished by binding APS reductase to an anti-APS reductase antibody immobilized on the surface of the QCM, followed by addition of an anti-APS-alkaline phosphatase reductase conjugate. Subsequent exposure of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to the bound sandwich complex results in enzymatically amplified deposition of the oxidized dimer of BCIP on the QCM surface, resulting in a frequency change that corresponds to the level of APS analyte. The enzymatic amplification leads to significant enhancement of the detection limits; levels of ca. 5 ng/mL (10^-14M) of APS reductase can be detected whereas the direct binding of APS reductase at even more elevated concentrations cannot be measured. Assay methodology providing for a reusable biosensor is also described. In this example, hCG is detected with a horseradish peroxidase conjugate in which a sandwich complex is immobilized on a separate nylon membrane positioned in close proximity to a polyvinylferrocene (PV-Fc) film on the QCM. This enzyme catalyzes the oxidation of I^-to I₂/I₃^-, which subsequently reacts with the PV-Fc film with concomitant insertion of I₃^-, resulting in an observable decrease of the QCM frequency. Since the formation of PV-Fc⁺I₃^-can be reversed electrochemically to regenerate the original PV-Fc film, this configuration provides for reuse of the detection crystal.