1-Acryloly-3-hydroxyaminopropanol (AAP) Synthesis. AAP
has been synthesized using a modified procedure.8 Briefly, a 10%
solution of acryloyl chloride in dichloromethane was slowly added
to 2-fold excess of 10% 3-aminopropanol in dichloromethane at
-35 °C. The AAP solution in dichloromethane was separated from
a viscous layer and purified on a silica gel column with acetone
as the eluent. AAP was stabilized by 0.004% (w/w) of p-hydroxy-
anisol, ∼10-fold excess of D2O was added (D2O was used instead
of H2O to simplify further monitoring of quality), and the acetone
was evaporated at 0 °C. The product was analyzed by NMR and
LC-MS and stored as ∼10% solution in D2O at -20 °C.
Capillary Modification. To decrease interactions between the
analyte particles and the capillary walls, the walls were modified
by AAP. The sol-gel procedure was used for introduction of allyl
anchors, and copolymerization of AAP and the anchors was
performed as described.8 A 5 m long piece of fused-silica capillary
(50 µm i.d.) was flushed with 0.1 M NaOH for 3 h at 35 psi,
followed by water for 10 min, 2% HCl for 1 h, and methanol for 1
h at the same pressure. Next, the capillary was dried with Ar (40
psi) at 140 °C for 1 day. Then a 5% solution of allyltriethoxysilane
in toluene was flushed through the capillary for 10 min at 35 psi.
The capillary ends were capped, and the capillary was incubated
in an oven at 140 °C for 2 days, followed by a toluene flush.
Electroosmotic flow (EOF) was measured as previously described9
and was found to have decreased from 1.28 × 10-4 cm2 V-1 s-1 in
the bare capillary to 0.70 × 10-4 cm2 V-1 s-1 in the treated one,
indicating a decrease in the amount of free siloxane groups. Two
1 m long capillary pieces were flushed with 3% AAP, 0.04%
ammonium persulfate, and 0.1% TEMED in water for 5 min at 35
psi. This treatment was followed by 1 h of incubation without
pressure. Next, the capillary contents were flushed out with water
at 35 psi. The capillary was cut into ∼30-35 cm long segments.
After measuring the final EOF (ex. 2.87 × 10-6 cm2 V-1 s-1), the
capillaries were flushed with water and Ar at 35 psi for 15 min
each, capped, and stored at room temperature.
Mitochondria Sample Preparation, Characterization, and
Labeling. The mitochondria samples were prepared from liver
of freshly sacrificed rat (kindly provided by Dr. LaDora Thomp-
son). The liver was cut into ∼1 mm3 pieces, suspended in isolation
buffer, and homogenized mechanically (clearance 0.0035-0.0055′′
× 90 strokes; clearance 0.001-0.001′′ × 90 strokes) at 0 °C. The
cell debris was removed by 2 × 10 m centrifugation at 2kG, and
mitochondria were sedimented at 12 kG during 20 m, resuspended
in isolation buffer, and immediately used for labeling or frozen in
liquid nitrogen until needed (typically 1-2 months).
Figure 1. Scheme of the LS-LIF detector. A: The detector for
simultaneous analysis of scattering and fluorescence signals, 1s
PMTs, 2sbeam splitter, 3sscattering filter set (narrow band-pass for
488 nm and neutral density filters), 4sfluorescence filter set (long-
pass above 510 and 535 nm band-pass), 5spinhole, 6ssignal
collection objective, 7slaser-focusing objective, 8slaser beam, 9s
sheath-flow cuvette, 10scollected light. B: The side view of the
detection volume defined by the overlapping region of the capillary
outflow and the laser beam, 11scapillary walls, 12scapillary outflow,
13slaser beam, 14svolume from which light is collected.
Briefly, the instrument uses a sheath flow cuvette to house the
end of the CE capillary and employs postcolumn laser excitation
and collection of the optical signal. A 488 nm line from an argon-
ion laser (Melles Griot, Irvine, CA) was used for excitation. Both
fluorescence and scattering signals were collected 90° from the
laser beam with a 60× objective with NA 0.7 (Mitutoyo, Japan).
A 1 mm diameter pinhole was used to spatially select the
fluorescence and scattering light from the detection volume. The
selected light was then separated into two channels by a glass
beam splitter positioned at 45°. This splitter was made from a
microscope glass slide, which has an expected reflection efficiency
of 6%. The less intense reflected light was used for scattering
detection. In this detector, the light passed through a neutral
density filter (OD ) 2), narrow band-pass filter (488 ( 1.5 nm,
NB3, Omega Optical, Bratteboro, VT), and was detected by a
photomultiplier tube (PMT) (R1477, Hamamatsu Corp., Bridge-
water, NJ). The light transmitted by the beam splitter (∼94%) was
directed toward the fluorescence detector. In this detector, the
light passed through a 505 nm long-pass filter and a 530 nm band-
pass filter (Omega Optical, Bratteboro, VT) and was detected by
a second PMT (Figure 1A). The PMT outputs were electronically
filtered (RC ) 0.01 s), digitized using a PCI-MIO-16E-50 I/O card
at 100 Hz data collection frequency, and stored as a binary file.
No significant cross-talking was observed for signals from the
analyzed particles.
The identity of the mitochondria in the samples used (fresh
or frozen) was confirmed by TEM (Figure 4C-F). The samples
for TEM were prepared by fixing the mitochondria using gluter-
aldehyde, followed by embedding them in a resin.
Fluorescent labeling was performed by resuspension of ∼1
mm3 of the mitochondrial pellet in 1 mL of sucrose-HEPES
buffer, followed by addition of 10 µL of 100 µM NAO in the same
buffer and incubation for 20 min at 4 °C. The labeled mitochondria
were immediately used in CE experiments.
This detector uses a sheath flow cuvette to house the end of
the CE capillary and employs postcolumn laser excitation and
collection of fluorescence and scattering signals, thereby avoiding
scattering from capillary walls (Figure 1B). The sheath flow was
driven by application of 300 Pa pressure, which was applied by
elevating a reservoir containing the same buffer used for the CE
separation. This pressure results in a 1.25 mm/s sheath flow linear
Capillary Electrophoresis. A custom-built CE-LIF instrument,
used as the basis for this work, has been previously described.5
(8) Gelfi, C.; Curcio, M.; Righetti, P. G.; Sebastiano, R.; Citterio, A.; Ahmadza-
deh, H.; Dovichi, N. J. Electrophoresis 1998, 19, 1677-1682.
(9) Fuller, K. M.; Duffy, C. F.; Arriaga, E. A. Electrophoresis 2002, 23, 1571-
1576.
Analytical Chemistry, Vol. 79, No. 14, July 15, 2007 5475