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.
| Original language | English |
|---|---|
| Pages (from-to) | 7161-7167 |
| Number of pages | 7 |
| Journal | Nano Letters |
| Volume | 15 |
| Issue number | 10 |
| DOIs | |
| Publication status | Published - Oct 14 2015 |
Fingerprint
Keywords
- botulinum neurotoxin
- FRET
- microarray
- protein sensor
- quantum dot
- scFv
ASJC Scopus subject areas
- Condensed Matter Physics
- Bioengineering
- Chemistry(all)
- Materials Science(all)
- Mechanical Engineering
Cite this
Fast, Ratiometric FRET from Quantum Dot Conjugated Stabilized Single Chain Variable Fragments for Quantitative Botulinum Neurotoxin Sensing. / Lee, Joonseok; Brennan, Melissa B.; Wilton, Rosemarie; Rowland, Clare E.; Rozhkova, Elena A.; Forrester, Sara; Hannah, Daniel C.; Carlson, Julia; Shevchenko, Elena V.; Schabacker, Daniel S.; Schaller, Richard D.
In: Nano Letters, Vol. 15, No. 10, 14.10.2015, p. 7161-7167.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Fast, Ratiometric FRET from Quantum Dot Conjugated Stabilized Single Chain Variable Fragments for Quantitative Botulinum Neurotoxin Sensing
AU - Lee, Joonseok
AU - Brennan, Melissa B.
AU - Wilton, Rosemarie
AU - Rowland, Clare E.
AU - Rozhkova, Elena A.
AU - Forrester, Sara
AU - Hannah, Daniel C.
AU - Carlson, Julia
AU - Shevchenko, Elena V.
AU - Schabacker, Daniel S.
AU - Schaller, Richard D
PY - 2015/10/14
Y1 - 2015/10/14
N2 - 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.
AB - 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.
KW - botulinum neurotoxin
KW - FRET
KW - microarray
KW - protein sensor
KW - quantum dot
KW - scFv
UR - http://www.scopus.com/inward/record.url?scp=84944348368&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84944348368&partnerID=8YFLogxK
U2 - 10.1021/acs.nanolett.5b03442
DO - 10.1021/acs.nanolett.5b03442
M3 - Article
C2 - 26397120
AN - SCOPUS:84944348368
VL - 15
SP - 7161
EP - 7167
JO - Nano Letters
JF - Nano Letters
SN - 1530-6984
IS - 10
ER -