Superquenching as a detector for microsphere-based flow cytometric assays.
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 0.8x10(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.


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.
Zeineldin
Reema
R
Department of Chemical and Nuclear Engineering, University of New Mexico, Albuquerque, New Mexico 87131, USA.
Piyasena
Menake E
ME
Bergstedt
Troy S
TS
Sklar
Larry A
LA
Whitten
David
D
Lopez
Gabriel P
GP
D000880:Anthraquinones / Q000737:chemistry; D015374:Biosensing Techniques; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D005470:Fluorometry; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008863:Microspheres; D008954:Models, Biological; D011108:Polymers / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry
D000880:Anthraquinones; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D011108:Polymers; C473966:poly(phenylene ethynylene); C023554:anthraquinone sulfonate; D012822:Silicon Dioxide