Zeineldin Reema, Piyasena Menake E, Bergstedt Troy S, Sklar Larry A, Whitten David, Lopez Gabriel P
Department of Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico 87131, USA.
Cytometry A. 2006 May;69(5):335-41. doi: 10.1002/cyto.a.20250.
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.
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.
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 0.8x10(6) 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.
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.
荧光共轭聚合物具有高荧光量子产率，并且通过能量或电子转移，对带相反电荷的猝灭剂的猝灭作用（超猝灭）具有增强的敏感性。荧光聚合物及其猝灭剂用于基于微珠的生物传感器应用，其中聚合物被涂覆在颗粒上。在这项工作中，我们研究了一种利用微球上的超猝灭的检测方法，该方法可用于流式细胞术分析。
用荧光阳离子聚电解质聚对苯撑乙炔（PPE）包被微球，并通过荧光法在存在和不存在以阴离子脂质双层形式的超猝灭屏障的情况下，检测其被9,10-蒽醌-2,6-二磺酸（AQS）的超猝灭情况。
流式细胞术检测到AQS对微球上PPE（MS-PPE）的超猝灭，观察到荧光有高水平的降低。向MS-PPE中加入不同浓度的AQS，得到的斯特恩-沃尔默猝灭常数为0.8×10⁶ M⁻¹。虽然在MS-PPE周围形成阴离子脂质双层可作为AQS超猝灭的屏障，但破坏脂质双层会使超猝灭发生。
流式细胞术检测微球荧光的灵敏度以及荧光共轭聚合物增强的猝灭灵敏度，都比其他荧光法和传统猝灭检测具有优势。本研究将荧光聚合物的超猝灭用作流式细胞术的一种新工具，从而结合了方法和检测器两者的优势。此外，我们利用支撑脂质双层的形成和破坏来介导超猝灭，以提供新的生物传感应用。
