Analytical Chemistry Chemical Cytometry Quantitates Superoxide

Figure 2. (A) Binding isotherm of HSA to BSPOTPE in the artificial urine and PBS buffer. (B) Dependence of the FL intensity of BSPOTPE at
470 nm on different proteins in artificial urine and PBS buffer. [BSPOTPE] ) 5 µM; [protein] ) 100 µg/mL; λex ) 350 nm.
nm keeps rising with an increase in the HSA concentration. The
rate of the FL enhancement is fast at lower HSA concentrations
and becomes almost constant at [HSA] > 1 µM (Figure 1B). At
an HSA concentration of 10 µM, the FL intensity is increased by
as high as ∼300-fold. The detection limit can be squeezed to as
low as 1 nM. In the protein concentration range of 0-100 nM,
the plot of FL enhancement (I/I0 - 1) as a function of HSA
concentration is a linear line with a high correlation coefficient
(0.995).
To examine the feasibility of protein assay in body fluids, we
performed HSA quantitation in artificial urine. 31 As shown in
Figure 2A, the binding isotherm is practically identical to that in
PBS. The assay sensitivity is not affected by the presence of
miscellaneous bioelectrolytes and physiological level of urea.
Thanks to the intriguing AIE characteristic of BSPOTPE, the
detection limit and linear range can be tuned by adjusting the
luminogen concentration. 21b Besides the superior sensitivity,
BSPOTPE shows excellent selectivity to albumin proteins, such
as HSA and bovine serum albumin (BSA; Figure 2B). A variety
of human proteins [pepsin, human immunoglobulin G (IgG),
trypsin, etc.] with isoelectric points ranging from 1 to 10 and DNA
[e.g., calf thymus DNA] were chosen for the binding tests. None
of these biomolecules, however, can turn on the BSPOTPE
emission. The selectivity of BSPOTPE toward the albumin proteins
remains unperturbed in the artificial urine. These exciting results
encourage us to further explore its clinical utility.
Protein Visualization in PAGE. The “lighting-up” of BSPOTPE
luminogen by albumin in the PBS buffer inspired us to check the
possibility of the use of the AIE luminogen as a protein staining
reagent in the PAGE assay. Figure 3A shows the gel images of
electrophoresed HSA after staining with a BSPOTPE solution for
∼5 min. The protein bands become visible under the illumination
of a hand-held UV lamp. The detection limit can be down to 50
(31) Brooks, T.; Keevil, C. W. Lett. Appl. Microbiol. 1997, 24, 203.
7038 Analytical Chemistry, Vol. 82, No. 16, August 15, 2010
Figure 3. PAGE analyses of HSA using (A) BSPOTPE and (B)
Coomassie Blue R-250 as the staining reagents. Lanes correspond
to the protein bands with amounts of HSA of (1) 1000, (2) 500, (3)
100, (4) 50, and (5) 2.5 ng. [BSPOTPE] ) 1 mg/100 mL; staining
time ≈ 5 min.
ng per lane (Lane 4). CBB is one of the most commonly used
protein stains in the bioassay. 32 The gel stained by CBB is shown
in Figure 3B for comparison. Although we have made efforts to
enhance the contrast of the image, only a few bands are
discernible in the gel. Those fine bands containing trace amounts
of HSA are ignored by CBB. It must be pointed out that a high
CBB concentration is generally needed for staining. Evidently,
BSPOTPE shows a much higher sensitivity than CBB.
Another advantage of the use of BSPOTPE as gel stain is its
simplicity and promptness, in contrast to the conventional methods.
Commercially available protein staining reagents such as
colorimetric CBB and fluorimetric SYPRO Ruby require a long
staining time (normally overnight). A destaining step is often
required to remove the excess dye in the gel. 33 Silver staining is
expensive and entails several labor-intensive and time-sensitive
steps. 13 Unlike the above probes, neither careful timing nor a
destaining step is necessary for BSPOTPE staining. The protein
bands can be visualized after 5 min of staining. Soaking the gel
(32) (a) Blakesley, R. W.; Boezi, J. A. Anal. Biochem. 1977, 82, 580. (b)
Candiano, G.; Bruschi, M.; Musante, L.; Santucci, L.; Ghiggeri, G. M.;
Carnemolla, B.; Orecchia, P.; Zardi, L.; Righetti, P. G. Electrophoresis 2004,
25, 1327.
(33) Lopez, M. F.; Berggren, K.; Chernokalskaya, E.; Lazarev, A.; Robinson, M.;
Patton, W. F. Electrophoresis 2000, 21, 3673.