On-Column Labeling of
Gram-Positive Bacteria with a Boronic Acid Functionalized Squarylium
Cyanine Dye for Analysis by Polymer-Enhanced Capillary Transient Isotachophoresis
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Abstract
A new asymmetric, squarylium cyanine dye functionalized
by boronic acid (“SQ-BA”) was designed and synthesized
for on-capillary labeling of gram-positive bacteria to provide for
high sensitivity detection by way of a modified form of capillary
electrophoresis with laser induced fluorescence detection (CE-LIF).
The CE-based separation employed a polymer-enhanced buffer with capillary
transient isotachophoresis in a new hybrid method dubbed “PectI.”
It was found that the addition of various monosaccharides to SQ-BA
in a batch aqueous solution greatly enhanced the emission of the boronic
acid functionalized dye by a factor of up to 18.3 at a long wavelength
(λ<sub>ex</sub> = 630 nm, λ<sub>em</sub> = 660 nm) with
a high affinity constant (<i>K</i> = ∼10<sup>2.80</sup> M<sup>–1</sup>) superior to other sugar probes. Semiempirical
quantum mechanics calculations suggest that the mechanism for this
high enhancement may involve the dissociation of initially nonemissive
dye associates (stabilized by an intramolecular hydrogen bond) upon
complex formation with sugars. The fluorescence emission of SQ-BA
was also significantly enhanced in the presence of a gram-positive
bacterial spore, <i>Bacillus globigii</i> (Bg), which serves
as a simulant of <i>B. anthracis</i> (or anthrax) and which
possesses a peptidoglycan (sugar)-rich spore coat to provide ample
sites for interaction with the dye. Several peaks were observed for
a pure Bg sample even with polyethyleneoxide (PEO) present in the
CE separation buffer, despite the polymer’s previously demonstrated
ability to focus microoorganisms to a single peak during migration.
Likewise, several peaks were observed for a Bg sample when capillary
transient isotachophoresis (ctITP) alone was employed. However, the
new combination of these techniques as “PectI” dramatically
and reproducibly focused the bacteria to a single peak with no staining
procedure. Using PectI, the trace detection of Bg spores (corresponding
to approximately three cells per injection) along with separation
efficiency enough to separate Bg from another gram-positive bacteria, <i>Saccharomyces cerevisiae</i> (resolution, <i>R</i><sub>s</sub> = 6.09, and apparent plate number, <i>N</i> = 2.7–3.3 × 10<sup>5</sup>), were successfully achieved