2 research outputs found
Development of Escherichia coli Asparaginase II for Immunosensing: A Trade-Off between Receptor Density and Sensing Efficiency
The clinical success
of Escherichia coli l-asparaginase
II (EcAII) as a front line chemotherapeutic
agent for acute lymphoblastic leukemia (ALL) is often compromised
because of its silent inactivation by neutralizing antibodies. Timely
detection of silent immune response can rely on immobilizing EcAII,
to capture and detect anti-EcAII antibodies. Having recently reported
the use of a portable surface plasmon resonance (SPR) sensing device
to detect anti-EcAII antibodies in undiluted serum from children undergoing
therapy for ALL (AubeĢ et al., <i>ACS Sensors</i> <b>2016</b>, <i>1</i> (11), 1358ā1365), here we
investigate the impact of the quaternary structure and the mode of
immobilization of EcAII onto low-fouling SPR sensor chips on the sensitivity
and reproducibility of immunosensing. We show that the native tetrameric
structure of EcAII, while being essential for activity, is not required
for antibody recognition because monomeric EcAII is equally antigenic.
By modulating the mode of immobilization, we observed that low-density
surface coverage obtained upon covalent immobilization allowed each
tetrameric EcAII to bind up to two antibody molecules, whereas high-density
surface coverage arising from metal chelation by N- or C-terminal
histidine-tag reduced the sensing efficiency to less than one antibody
molecule per tetramer. Nonetheless, immobilization of EcAII by metal
chelation procured up to 10-fold greater surface coverage, thus resulting
in increased SPR sensitivity and allowing reliable detection of lower
analyte concentrations. Importantly, only metal chelation achieved
highly reproducible immobilization of EcAII, providing the sensing
reproducibility that is required for plasmonic sensing in clinical
samples. This report sheds light on the impact of multiple factors
that need to be considered to optimize the practical applications
of plasmonic sensors
General CāH Arylation Strategy for the Synthesis of Tunable Visible Light-Emitting Benzo[<i>a</i>]imidazo[2,1,5ā<i>c</i>,<i>d</i>]indolizine Fluorophores
Herein
we report the discovery of the benzoĀ[<i>a</i>]ĀimidazoĀ[2,1,5-<i>c</i>,<i>d</i>]Āindolizine motif displaying tunable
emission covering most of the visible spectrum. The polycyclic core
is obtained from readily available amides via a chemoselective process
involving Tf<sub>2</sub>O-mediated amide cyclodehydration, followed
by intramolecular CāH arylation. Additionally, these fluorescent
heterocycles are easily functionalized using electrophilic reagents,
enabling divergent access to varied substitution. The effects of said
substitution on the compoundsā photophysical properties were
rationalized by density functional theory calculations. For some compounds,
emission wavelengths are directly correlated to the substituentās
Hammett constants. Easily introduced nonconjugated reactive functional
groups allow the labeling of biomolecules without modification of
emissive properties. This work provides a straightforward platform
for the synthesis of new moderately bright fluorescent dyes remarkable
for their chemical stability, predictability, and unusually high excitationāemission
differential