Homogeneous immunoassays based on fluorescence emission intensity variations of zinc selenide quantum dot sensors Jun Wang, T.J. Mountziaris n Department of Chemical Engineering, University of Massachusetts, Amherst, MA 01003, USA article info Article history: Received 8 May 2012 Received in revised form 1 August 2012 Accepted 1 August 2012 Available online 14 August 2012 Keywords: Zinc selenide quantum dots Homogeneous separation-free immunoassay Fluorescence spectroscopy Optical biosensor Fluorescence emission intensity variations Rapid quantitative protein detection Point of care diagnostics Emergency care diagnostics abstract 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 mM 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. & 2012 Elsevier B.V. All rights reserved. 1. Introduction Semiconductor nanocrystals or quantum dots (QDs) are nano- meter-sized inorganic crystals that have unique optical properties due to confinement of electron-hole pairs (excitons) by the grain boundary of the nanocrystals (Alivisatos, 1996; Brus, 1991). These include size-tunable photoluminescence, high quantum yields and molar extinction coefficients, and high photostability (Murray et al., 2000). QDs have attracted significant attention as fluorescent labels of proteins and cells for in vitro and in vivo imaging and for biological sensing applications (Michalet et al., 2005; Smith et al., 2006). The excellent photochemical stability and high brightness of QDs can increase detection sensitivity in immunoassays and their narrow and tunable emission spectra enable multiplexing (Goldman et al., 2005a, 2006; Sapsford et al., 2006). A variety of targets have been detected using QDs as fluorescent labels, including small molecules, protein disease markers, bacteria, and viruses (Gill et al., 2008; Sapsford et al., 2006). For example, QDs have been employed in ELISA-type immunoassays (Wang et al., 2002; Woodbury et al., 2002), in Western blot analysis of proteins (Bakalova et al., 2005; Chen et al., 2009), in sandwich immunoassays that can detect prostate-specific antigen (PSA) (Kerman et al., 2007), in multiplexed sandwich immunoassays that can simultaneously detect four toxins (Goldman et al., 2004), and as donors for detection of small molecules and biological targets based on F ¨ orster Reso- nance Energy Transfer (FRET) between the QD and another fluor- ophore (Algar and Krull, 2008; Clapp et al., 2005; Goldman et al., 2005b; Medintz et al., 2003). However, the substitution of fluor- escent proteins by QDs in biological sensing applications is not always practical due to the high cost of QDs and the high toxicity of typical CdSe-based QDs. In this paper we demonstrate the development of novel homo- geneous (separation-free) assays that enable direct detection of target analytes by monitoring the variations of the peak emission intensity of QD-labeled biomolecular sensors upon binding to specific target analytes. The variations in the peak emission intensity of the QDs are caused by surface-induced electronic perturbations (Cadars et al., 2009). The detection of a target analyte by these assays is accomplished without employing a second fluorophore and without immobilizing the probe, target, or probe-target complex Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics 0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.08.003 n Corresponding author. Tel.: þ1 4135456145; fax: þ1 4135453540. E-mail address: tjm@ecs.umass.edu (T.J. Mountziaris). Biosensors and Bioelectronics 41 (2013) 143–149