Superquenching as a detector for microsphere‐based flow cytometric assays

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Abstract
Background: Fluorescent conjugated polymers display high fluorescence quantum yields and enhanced sensitivity to quenching (superquenching) by oppositely charged quenchers through energy or electron transfer. Fluorescent polymers and their quenchers are used in bead‐based biosensor applications where the polymers are coated on particles. In this work, we investigate a detection method that utilizes superquenching on microspheres, which can be used for flow cytometric assays. Methods: Microspheres were coated with the fluorescent cationic polyelectrolyte poly(p‐phenylene‐ethynylene) (PPE), and its superquenching by 9,10‐anthraquinone‐2,6‐disulfonic acid (AQS) was examined by fluorometric methods in presence and in absence of a barrier to superquenching in the form of an anionic lipid bilayer. Results: Flow cytometry detected superquenching of PPE on microspheres (MS‐PPE) by AQS where high levels of reduction in fluorescence were observed. Adding different concentrations of AQS to MS‐PPE yielded a Stern–Volmer quenching constant of 0.8 × 106 M−1. While forming an anionic lipid bilayer around the MS‐PPE acted as a barrier to superquenching by AQS, disrupting the lipid bilayer allowed superquenching to take place. Conclusions: The sensitivity of flow cytometry in detecting fluorescence of microspheres and the amplified quenching sensitivity of fluorescent conjugated polymers both offer advantages over other fluorometric methods and conventional quenching detection. This study used superquenching of fluorescent polymers as a new tool in flow cytometry, thus combining the advantages offered by both method and detector. In addition, we employed the formation and the disruption of a supported lipid bilayer in mediating superquenching to offer new biosensing applications. © 2006 International Society for Analytical Cytology