Homogeneous immunoassays based on fluorescence emission intensity variations of zinc selenide quantum dot sensors

The fluorescence emission intensity of ZnSe quantum dots (QDs) conjugated to proteins to form QD-based biomolecular sensors increases significantly upon binding of the sensors to target proteins in solution. This phenomenon enables the development of homogeneous, separation-free immunoassays for rapid quantitative detection of proteins in solution. Proof-of-principle assays were developed by dosing a solution containing a biomolecular target with a solution containing the corresponding QD-based sensor and monitoring the changes in the peak fluorescence emission intensity of the QDs. Direct immunoassays for detecting basic fibroblast growth factor (bFGF) and prostate-specific antigen (PSA) in solution were demonstrated using QD-anti-bFGF and QD-anti-PSA sensors. A competitive immunoassay for detecting human serum albumin (HSA) was also demonstrated by dosing samples containing HSA with QD-HSA sensors and free anti-HSA antibodies. The QD-HSA sensors were tested in 1000× diluted human serum and found to be unaffected by interference from other proteins. The lower limit of detection of the assays was equal to the lowest sensor concentration in the solution that can be unambiguously detected, typically less than 1 nM. The dynamic range of the assays was determined by identifying the sensor concentration above which optical interference between QDs affected adversely the observed fluorescence emission intensity. The upper limit of this concentration was 2.5 μM for 4 nm QDs. The ZnSe QD-based sensors were stable and preserved ~80% of their initial peak emission intensity after two months in refrigerated storage. These biosensors have potential applications in rapid sensing of target proteins for emergency and point-of-care diagnostic applications.