Flow cytometry-based biosensor for detection of multivalent proteins.
Microsphere-based flow cytometric detection of cholera toxin (CT) through distance-dependent fluorescence resonant energy transfer (FRET) has been developed. Simultaneous double-fluorescence changes induced by multivalent interactions between CT and fluorophore (both fluorescence donor and acceptor)-labeled ganglioside GM1 on a biomimetic membrane surface (supported bilayers of phospholipids) can be measured by a commercial flow cytometer, providing a convenient and sensitive detection method for CT. The flow cytometry-based biosensor is capable of detecting less than 10 pM CT within 30 min. The signal generation strategy coupled with flow cytometry also provides a convenient method for kinetic studies of multivalent interactions. The surface density and the ratio of donor/acceptor-labeled GM1 on the surfaces of phospholipid bilayers are optimized to achieve high sensitivity.
Song
X
X
Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87544, USA. xsong@lanl.gov
Shi
J
J
Swanson
B
B
D015374:Biosensing Techniques / Q000379:methods*; D001896:Boron Compounds / Q000494:pharmacology; D002772:Cholera Toxin / Q000032:analysis; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D005677:G(M1) Ganglioside / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism; D010713:Phosphatidylcholines / Q000494:pharmacology; D011506:Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000032:analysis; D013997:Time Factors
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D011506:Proteins; D012709:Serum Albumin; D005677:G(M1) Ganglioside; D002772:Cholera Toxin; C028694:1-palmitoyl-2-oleoylphosphatidylcholine