A highly sensitive switch-on fluorescent probe for protein quantification and visualization based on aggregation-induced emission

Article abstract of DOI:10.1039/c2cc33172a

A highly sensitive and water-soluble switch-on fluorescent probe with aggregation-induced emission characteristics was developed for protein quantification and visualization. It offers a rapid, economic and effective way for the assay of complete serum proteins and disease-marker proteins.

Full text of DOI:10.1039/c2cc33172a

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COMMUNICATION
A highly sensitive ‘‘switch-on’’ fluorescent probe for protein
quantification and visualization based on aggregation-induced emissionw
Fangfang Wang,a Jiying Wen,a Lingyun Huang,b Jinjiu Huanga and Jin Ouyang*a
Received 3rd May 2012, Accepted 30th May 2012
DOI: 10.1039/c2cc33172a
A highly sensitive and water-soluble ‘‘switch-on’’ fluorescent
probe with aggregation-induced emission characteristics was
developed for protein quantification and visualization. It offers
a rapid, economic and effective way for the assay of complete
serum proteins and disease-marker proteins.
A thorny problem encountered by conventional FL reagents
in aqueous medium or physiological buffers is the ubiquitous
aggregation-caused quenching (ACQ) effect,8 which is attributed
to the non-radiative relaxation of excited states of aggregates
forcing researchers to study and employ them as single molecules
in very dilute solutions. However, the use of dilute solution causes
many problems such as weak emissions and poor sensitivity in
fluorescent imaging bioassays. Even in dilute solutions, the ACQ
effect can still not be avoided, because, in biological analysis, the
fluorophore molecules may accumulate on the surfaces of the
biomacromolecules and aggregate in the hydrophobic cavities.9
Recently we have synthesized 9,10-bis[4-(3-sulfonatopropoxyl)-
styryl]anthracene sodium salt (BSPSA, Fig. 1A) and discovered
that BSPSA is not only soluble in aqueous medium but also
behaves in the absolute opposite way: aggregation plays a
constructive, instead of a destructive, role in the light-emitting
process (aggregation-induced emission,10 AIE) and BSPSA is
found to be non-luminescent in the solution but emissive in the
aggregate sate. Hence, the strong points of BSPSA prompted
us to further explore its potential in bioanalysis. In this work,
we investigated the performance of BSPSA as a fluorescent
Protein analysis is of prime importance to proteomics research
that aims at interpreting biological and physiological processes
at the protein level. A quantity of molecular probes for protein
assays have been developed by utilizing their photophysical
changes, which is caused by physical interaction or chemical
reaction with proteins.1 Colorimetry, such as Lowry and
Brandford assays, is a traditional analytical method for protein
quantitation in solutions.2 When the chromophores bind with
neutral organic or inorganic molecules or ions, they can exhibit a
color change, which enables researchers to investigate the
changes in the free guest ions or in the concentration of molecules
by utilizing absorption spectroscopy.3 However, these methods
lack sensitivity and accuracy and require lengthy staining and
destaining procedures. Biosensors based on fluorescent (FL)
materials have been proven useful for the assay of proteins in
solution and attracted wide-spread attention owing to their
rapidity, low background signals and selectivity. Several FL
probes that target proteins, including Nile Red, SYPRO Ruby,
NanoOrange and fluorescamine, have been developed and
widely used for proteomics research.4,5 Although FL techniques
offer high sensitivity, low background noise and a broad dynamic
range, some FL bioprobes are insoluble in aqueous solutions,
require lengthy procedures with time-sensitive steps,6 some are
unstable under ambient conditions (e.g., fluorescamine and
Nile Red),7 and some are extremely expensive and sold only as
a formulated solution.4,5 These shortcomings greatly limit the
scope of their real-life applications. Therefore, it is necessary
for us to develop environmentally stable and synthetically readily
accessible bioprobes with high sensitivity, low background signals
and high selectivity.
a College of Chemistry, Beijing Normal University, Beijing, 100875,
P. R. China. E-mail: jinoyang@bnu.edu.cn; Fax: 86-10-6279 9838;
Tel: 86-10-5880 5373
b Key laboratory for Cell Proliferation and Regulation Biology,
Ministry of Education, Beijing Normal University, Beijing,
100875, P. R. China.
w Electronic supplementary information (ESI) available. See DOI:
10.1039/c2cc33172a
Fig. 1 (A) Chemical structure of water-soluble AIE fluorophore
molecules BSPSA; (B) Illustration of the mechanism of protein
visualization after PAGE using BSPSA as a fluorescent probe with
AIE characteristics.
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 7395–7397 7395

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probe for the detection and quantitation of human serum
proteins. The FL ‘‘turn-on’’ properties of BSPSA after
aggregation in the hydrophobic cavities of proteins facilitated
the rapid fluorescent imaging and quantitative of complete
proteins in human serum after polyacrylamide gel electrophoresis
(PAGE).
In this context, BSPSA has received special attention for the
following reasons: (1) BSPSA can realize fluorescent imaging
of complete proteins in human serum in ten minutes, fixation
and destaining procedures are not required; (2) BSPSA has
high sensitivity (0.78 ng mLÀ1 of ferritin in gel), which is
comparable to that of silver staining; (3) BSPSA is soluble
and stable in aqueous medium, and organic solvents are not
required, for this reason BSPSA is a ‘‘safe’’ stain. Hence,
BSPSA is a good protein indicator for its rapid and sensitive
detection.
Fig. 2 Comparison of the sensitivities of (A) BSPSA staining,
(B) CBB-R250 staining and (C) SYPRO-Ruby staining. Serial dilutions
of marker proteins loaded in the wells (for ferritin from left to right)
The mechanism of protein visualization after PAGE using
BSPSA as a fluorescent probe is schematically illustrated in
Fig. 1B. The solutions of BSPSA are weakly luminescent, that
is because the intramolecular rotation of these probes in
solution is active and will form a relaxation channel for
non-radiative decay of the excited state. When a certain amount
of protein is introduced, the BSPSA may accumulate in the
hydrophobic cavities or pockets of the folding structures,11 and
the intramolecular rotation of BSPSA is restricted in the
aggregates due to physical constraint, which will prevent the
energy from losing by non-radiative jump and activate
the emission transition. As a result, the fluorescence of BSPSA
would increase significantly in the presence of proteins.12 Thus,
the purpose to use BSPSA as a fluorescent probe for detecting
proteins after PAGE would be achieved. The effect of increasing
the concentration of HSA on the fluorescence spectra of BSPSA
and the dependence of the FL intensity of BSPSA on different
proteins are shown in Fig. S1 and S2, respectively (ESIw).
To demonstrate the application of BSPSA as a fluorescent
probe, protein markers in the gel obtained after PAGE were
stained by BSPSA. Fig. 2A shows an emissive image of a gel
containing electrophoresized protein markers (for ferritin
from left to right: 0.20–100 ng mLÀ1) after staining with
BSPSA (0.3 mg/100 mL) for 10 min. The lowest concentration
of ferritin detected after staining with BSPSA is 0.78 ng mLÀ1
(lane 8 in Fig. 2A). Conventional protein detection methods
include CBB-R250 staining, SYPRO-Ruby staining, silver
staining, double-metal-chelate (DMC) staining and so on.13,14
However, many of these methods involve careful timing and
time-consuming procedures. The most frequently used and
commercially available protein dyes, such as colorimetric
CBB-R250 and fluorimetric SYPRO-Ruby, require lengthy
staining and destaining steps. Unlike the conventional dyes,
neither lengthy destaining nor careful timing procedures were
required for BSPSA staining, and there was no overstaining and
decrease in signal intensity after staining or destaining for 24 h.
For comparison, conventional protein staining methods,
including CBB-R250 staining and SYPRO-Ruby staining,
are shown in Fig. 2B and C, respectively. When compared
the three detection methods, the BSPSA staining shows clearer
fluorescent protein bands with relatively higher resolution and
signal intensity. Taking the band of ferritin for example,
although we made great efforts to enhance the contrast and
were (1) 100.00 ng mLÀ1, (2) 50.00 ng mLÀ1, (3) 25.00 ng mLÀ1
,
(4) 12.50 ng mLÀ1, (5) 6.25 ng mLÀ1, (6) 3.13 ng mLÀ1, (7) 1.56 ng mLÀ1
,
(8) 0.78 ng mLÀ1, (9) 0.39 ng mLÀ1, and (10) 0.20 ng mLÀ1, respectively.
brightness of the images that were stained by conventional
methods, it can only be recorded in lane 6 for SYPRO-Ruby
staining and CBB-R250 staining. As indicated, the sensitivity
of BSPSA-based fluorescent imaging is higher than the two
conventional methods.
In order to evaluate the performance of BSPSA in clinical
applications, we applied BSPSA in the detection of human
serum proteins. Because of the high resolution and high
quantity of proteome information, native 2D-PAGE was
introduced to examine the applicability of BSPSA for detecting
human serum samples. Three gels were electrophoresed in
parallel and the proteins after 2D-PAGE were stained with
BSPSA, CBB-R250, and silver respectively. When comparing
the results of the three different staining methods, the proteins
marked in the rectangle regions could be hardly detected with
the conventional methods. However, because of the high FL
intensity of proteins and low background in BSPSA-based
fluorescent imaging, several new protein spots marked in
Fig. 3A have been observed. In addition, we also detected
the human serum proteins after native 1D-PAGE with
BSPSA. Compared with conventional methods, BSPSA-based
fluorescent imaging offers clearer protein bands, stronger
signals, lower backgrounds (Fig. S6w), and the detection limit
for tranferrin is comparable to that of the most sensitive silver
staining (Fig. S7w). As shown in Fig. S8,w a linear relationship is
obtained between the relative fluorescent intensity of the scanned
bands in the gel (transferrin) and the protein concentration, and
the linear dynamic range for BSPSA-based fluorescent imaging is
5.5–55 ng mLÀ1 (R2 = 0.998), which is the same as silver staining.
Thus, BSPSA shows great potential as an excellent dye for the
detection of complete proteins.
To identify the proteins only detected by BSPSA-based
fluorescent imaging, three protein spots indicated by the
dotted arrows on the gel image in Fig. 3A were identified
using MS/MS techniques. The results are shown in Fig. 3D.
From the results of the peptide mass data, spots a, b, and c
were zinc-a2-glycoprotein (ZAG), thyroxine-binding globulin
c
7396 Chem. Commun., 2012, 48, 7395–7397
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The authors gratefully acknowledge the support from the
National Nature Science Foundation of China (20975016,
21175014, 91027034), National Grant of Basic Research Program
of China (No. 2011CB915504), and the Fundamental Research
Funds for the Central Universities (2009SC-1).
Notes and references
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Fig. 3 The detection of human serum after 2D-PAGE. (A) BSPSA-
based fluorescent imaging, (B) CBB-R250 staining, (C) silver staining,
and (D) proteins identified by MS/MS.
(TBG) and a2-HS-glycoprotein (AHSG), respectively. Zinc-
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In this work, a novel, synthetically readily accessible and
environmentally stable AIE bioprobe has been developed for
the detection and quantitation of human serum proteins.
BSPSA is successfully used for protein staining after PAGE.
The detection limit of BSPSA-based fluorescent imaging for
ferritin in gel is 0.78 ng mLÀ1, which was much lower than that
of CBB-R250 staining and SYPRO-Ruby staining. In comparison
with conventional protein dyes, BSPSA-based fluorescent imaging
showed great potential in protein assays because of its simple and
rapid staining procedure, high sensitivity, low detection limits and
low background. In addition, we have demonstrated that several
relatively low-abundance proteins in serum can be easily detected
with the proposed method.
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c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 7395–7397 7397