title: Fast, Ratiometric FRET from Quantum Dot Conjugated Stabilized Single Chain Variable Fragments for Quantitative Botulinum Neurotoxin Sensing.
abstract: Botulinum neurotoxin (BoNT) presents a significant hazard under numerous realistic scenarios. The standard detection scheme for this fast-acting toxin is a lab-based mouse lethality assay that is sensitive and specific, but slow (∼2 days) and requires expert administration. As such, numerous efforts have aimed to decrease analysis time and reduce complexity. Here, we describe a sensitive ratiometric fluorescence resonance energy transfer scheme that utilizes highly photostable semiconductor quantum dot (QD) energy donors and chromophore conjugation to compact, single chain variable antibody fragments (scFvs) to yield a fast, fieldable sensor for BoNT with a 20-40 pM detection limit, toxin quantification, adjustable dynamic range, sensitivity in the presence of interferents, and sensing times as fast as 5 min. Through a combination of mutations, we achieve stabilized scFv denaturation temperatures of more than 60 °C, which bolsters fieldability. We also describe adaptation of the assay into a microarray format that offers persistent monitoring, reuse, and multiplexing.
authors.affiliation: Center for Nanoscale Materials, Argonne National Laboratory , Argonne, Illinois 60439, United States.
authors.forename: Joonseok
authors.initials: J
authors.lastname: Lee
authors.affiliation: Biosciences, Argonne National Laboratory , Argonne, Illinois 60439, United States.
authors.forename: Melissa B
authors.initials: MB
authors.lastname: Brennan
authors.affiliation: Biosciences, Argonne National Laboratory , Argonne, Illinois 60439, United States.
authors.forename: Rosemarie
authors.initials: R
authors.lastname: Wilton
authors.affiliation: Center for Nanoscale Materials, Argonne National Laboratory , Argonne, Illinois 60439, United States.|Department of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Clare E
authors.initials: CE
authors.lastname: Rowland
authors.affiliation: Center for Nanoscale Materials, Argonne National Laboratory , Argonne, Illinois 60439, United States.
authors.forename: Elena A
authors.initials: EA
authors.lastname: Rozhkova
authors.affiliation: Global Security Sciences, Argonne National Laboratory , Argonne, Illinois 60439, United States.
authors.forename: Sara
authors.initials: S
authors.lastname: Forrester
authors.affiliation: Department of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Hannah
authors.affiliation: College of Agricultural & Life Sciences, University of Wisconsin , Madison, Wisconsin 53706, United States.
authors.forename: Julia
authors.initials: J
authors.lastname: Carlson
authors.affiliation: Center for Nanoscale Materials, Argonne National Laboratory , Argonne, Illinois 60439, United States.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Shevchenko
authors.affiliation: Global Security Sciences, Argonne National Laboratory , Argonne, Illinois 60439, United States.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Schabacker
authors.affiliation: Center for Nanoscale Materials, Argonne National Laboratory , Argonne, Illinois 60439, United States.|Department of Chemistry, Northwestern University , Evanston, Illinois 60208, United States.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Schaller
mesh_terms: D001905:Botulinum Toxins / Q000032:analysis*; D031541:Fluorescence Resonance Energy Transfer; D057230:Limit of Detection; D045663:Quantum Dots; D011874:Radiometry / Q000379:methods*; D057127:Single-Chain Antibodies / Q000737:chemistry*
chemical_list: D057127:Single-Chain Antibodies; D001905:Botulinum Toxins
keywords: FRET; botulinum neurotoxin; microarray; protein sensor; quantum dot; scFv