Rational design of biotinylated probes: Fluorescent turn-on detection of (strept)avidin and bioimaging in cancer cells

Article abstract of DOI:10.1039/c4cc03315a

Two fluorescent probes SPS1 and SPS2 were designed by connecting biotin to an environment-sensitive coumarin fluorophore. Streptavidin and avidin induced dramatical fluorescence changes in both probes. SPS2 has potential in fluorescent imaging of biotin r

Full text of DOI:10.1039/c4cc03315a

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Rational design of biotinylated probes: fluorescent
turn-on detection of (strept)avidin and bioimaging
in cancer cells†
Cite this: Chem. Commun., 2014,
50, 8518
Received 4th May 2014,
Accepted 10th June 2014
Qian Sun,a Junhong Qian,*a Haiyu Tian,a Liping Duanb and Weibing Zhang*a
DOI: 10.1039/c4cc03315a
www.rsc.org/chemcomm
Two fluorescent probes SPS1 and SPS2 were designed by connecting 7-aminocoumarin showed solvent polarity-dependent spectral
biotin to an environment-sensitive coumarin fluorophore. Streptavidin properties, and its quantum yield decreased with increasing
and avidin induced dramatical fluorescence changes in both probes. solvent polarity.15 In view of the aforementioned results, we
SPS2 has potential in fluorescent imaging of biotin receptor-enriched present here two rationally designed biotinylated coumarin
tumor cells.
probes SPS1-2 for (strept)avidin (Scheme 1). The probes consist
of the following three components. (1) Biotin, a selective
The strong affinity between biotin and streptavidin (SA) and (strept)avidin receptor that was also chosen as the cancer
avidin (AV) (Ka B 1013 – 1015 À1)1 has found wide applications targeting unit. (2) The polarity-sensitive fluorophore coumarin
M
in bioanalytical areas such as biomolecule recognition,2 tumor to monitor the change in the microenvironmental polarity. An
cell diagnosis3 and immunoassay.4 Therefore, the development of enhanced fluorescence could be anticipated when the probes
sensitive and selective probes for (strept)avidin has attracted enter into the hydrophobic region of the protein or permeate
much attention.5 Using biotinylated fluorescence probes to moni- into tumor cells. (3) A phenyl group that quenches fluorescence
tor relative biomolecules is a simple and common approach.6 by internal rotation in fluid media that gives rise to an
However, most biotin–fluorophore conjugates were found to lose increased fluorescence intensity after the restriction of the
their fluorescence upon binding to (strept)avidin probably rotation. This design is expected to counteract the RET between
because of self-association in complexes with proteins.7,8 The the fluorophore and the proteins and results in fluorescence
reported fluorescence off–on probes for (strept)avidin were mainly enhancement. In addition, a 4-atom spacer was used as the
based on the FRET mechanism,9 luminescent biotin–transition linker between the fluorophore and the receptor to avoid the
metal complex conjugates with reduced RET,10 competition bind- self-quenching in complexes with (strept)avidin.
ing11 or hydrophobic ligand-binding domains of (strept)avidin.12
Probes SPS1-2 were fully characterized by 1H-NMR, 13C-NMR
The relatively low sensitivity, small Stokes shift and short emis- and ESI-MS data and were employed in the detection of
sion wavelengths, however, may limit their applications.9e,12b It is (strept)avidin. Compounds RC1-2 were used as the references
our interest to construct ‘‘off–on’’ fluorescent probes for (strept)- to verify the importance of the biotin unit in the detection of
avidin with high sensitivity. In addition, it is reported that the (strept)avidin, and RC3 was employed to investigate the role of
biotin receptors like (strept)avidin occur in tumor cells much the rotation group.
more frequently than in normal cells.13 Finally, we aim to apply
our rationally designed probes in living cell imaging.
Recently, many environment-sensitive fluorophores have
been exploited to detect certain proteins.14 Among those,
a Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry
and Molecular Engineering, East China University of Science and Technology,
Shanghai, 200237, China. E-mail: junhongqian@ecust.edu.cn,
weibingzhang@ecust.edu.cn
b National Institute of Parasitic Diseases, Chinese Center for Disease Control and
Prevention, Shanghai, 200032, China
† Electronic supplementary information (ESI) available: Experimental details of
Scheme 1 The chemical structures of probes SPS1-2 and their precursors
RC1-2 and the reference compound RC3.
synthetic procedures, characterization and cell cultures; fluorescence titrations,
confocal image analysis. See DOI: 10.1039/c4cc03315a
8518 | Chem. Commun., 2014, 50, 8518--8521
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First, the absorption and emission spectra of SPS1-2 in several
common solvents were measured to study their potential in
protein determination. The biotinylation of RC1-2 does not sub-
stantially affect the spectral properties. Similar to their precursors
RC1-2, both probes SPS1-2 exhibit clearly solvatochromic absorp-
tion and emission spectra (for SPS2, the absorption shifts from
454 nm in toluene to 475 nm in PBS, while the emission shifts
from 495 nm to 605 nm in the same series of solvents, Fig. S3 and
S4, ESI†). Generally, the absorption and emission maxima as well
as the Stokes shifts of both probes increase with the increasing
solvent polarity. Their quantum yields are much lower in water
than in other solvents (Table S1, ESI†), perhaps due to a quenching
mechanism related to hydrogen bonding.
Fig. 2 The relative fluorescence intensity of SPS2 (a) in the presence of
0.1 mg mLÀ1 of various proteins and (b) the competition of SPS2 toward SA/AV
over BSA and OVA. [SA] = [AV] = 0.1 mg mLÀ1, [BSA] = [OVA] = 1.0 mg mLÀ1
.
10 mM PBS, pH 7.0; [SPS2] = 3.0 mM; lex = 465 nm, lem = 576 nm.
upon binding of the biotinylated dyes to the proteins. As a
The lower polarity inside the binding pockets and the high result, the photophysical properties of the dyes are altered
affinity of biotin towards SA/AV inspired and promoted us to greatly.
apply these probes in the detection of SA and AV. We envi-
In the detection of SA/AV, SPS2 is preferred over SPS1
sioned that the addition of SA/AV would induce dramatic because of its higher sensitivity. Since the detection of SA/AV
enhancements in the fluorescence intensities of the probes with SPS2 is based on the polarity sensitivity of the coumarin,
with obvious blue shifts in the emission spectra. The effects of other biomolecules with non-polar cavities may interfere in the
SA and AV on the absorption and emission spectra of the detection of SA or AV through non-specific binding. Therefore,
probes turned out to agree with our expectations. SPS1 and the selectivity of SPS2 toward SA/AV over some other proteins
SPS2 are weakly fluorescent in phosphate buffer solution (PBS), including BSA, OVA, mucin, pepsin, casein, cytochrome C,
but they display clear spectral responses towards SA/AV (Fig. 1). myoglobin and biothiols (including cysteine, glutathione and
SPS1 has maximum fluorescence at 625 nm, the addition of SA homocysteine) was investigated (Fig. 2). The concentrations of
shifted the emission band to 587 nm without clear intensity the proteins were 0.1 mg mLÀ1, and the concentrations of thiols
change, while about 50 nm blue shift and 3-fold fluorescence were 0.3 mM. It can be seen in Fig. 2a that SA or AV induces
enhancement were induced by AV. The fluorescent responses of more than 5 times fluorescence enhancement of SPS2, but
SPS2 toward SA/AV are similar to those of SPS1 but more other proteins, except for casein and BSA, do not induce
pronounced: both SA and AV triggered evident fluorescence obvious spectral changes. BSA and casein lead to B2-fold
enhancement (46 folds) and a clear blue shift in the emission enhancement in the emission intensity.
spectrum. In order to understand the role of biotin in the
Considering the much higher concentrations of OVA and BSA
detection of SA and AV, the spectral responses of the reference in some biosamples, we also monitored the fluorescent response
compounds RC1-2 towards SA and AV were measured as well. It of SPS2 to OVA and BSA with higher concentrations. The results
can be seen in Fig. S5 (ESI†) that neither SA nor AV exerts much reveal that in the presence of 1.0 mg mLÀ1 BSA, the emission
influence on the emission spectra of RC1-2, which implies that maximum of SPS2 shifts from 607 nm to 548 nm and about
there are no strong interactions between RC1-2 and SA/AV. 12-fold enhancement of the fluorescence intensity is observed
Furthermore, a third reference compound RC3 (Fig. S6, ESI†) (Fig. 2b). Similar results were obtained for the reference com-
without a phenyl moiety was employed to investigate the role of pound RC2 (Fig. S7, ESI†), indicating that biotin did not exert
the rotation group in the detection of SA/AV. The results much influence on the interaction between BSA and the probes.
indicate that the rotation group is essential in the determina- Upon addition of 0.1 mg mLÀ1 AV (or SA) (one tenth of the
tion of SA. The above results suggest that the fluorescence BSA/OVA concentration) to SPS2-BSA (or OVA) solutions, the
enhancements and blue shifts in the emission spectra of SPS1-2 emission maximum shifted from 548 nm to 576 nm with slight
induced by SA/AV can be ascribed to the specific binding of fluorescence decrement, which was almost the same as that in
biotin in the probes to SA/AV. The fluorophores in the probes the absence of BSA/OVA (Fig. 2b). The above results indicate that
are supposed to reside in the hydrophobic pocket inside SA/AV the presence of 10 times excess of BSA or OVA does not affect the
detection of SA/AV. The blue shift and fluorescence enhance-
ment caused by BSA are attributed to the non-specific interaction
between the probes and BSA,15c which is much weaker than the
specific affinity between SPS2 and SA/AV.
In order to gain more insight into the sensing mechanism,
the time-resolved fluorescence data of SPS2 in different systems
were measured (Fig. S8, ESI†), and the results are summarized
in Table S2 (ESI†). Fig. S8 (ESI†) demonstrates that the fluores-
cence decay time of SPS2 is relatively short in PBS (0.9 ns) and
much longer in organic solvents (2.1–7.7 ns). In the presence of
1.0 mg mLÀ1 BSA in PBS, the fluorescence decay of SPS2 has a
Fig. 1 The emission spectra of SPS1 (a) and SPS2 (b) with or without SA/
AV. 10 mM PBS, pH 7.0; [dye] = 3.0 mM, [SA] = [AV] = 1.5 mM; lex = 465 nm.
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ratio of SPS2 to AV is about 2. When the ratio of SPS2/AV o 2,
most SPS2 molecules are located at the binding sites of AV
(Fig. S10a, ESI†), which results in the steep enhancement of
the fluorescence. When SPS2/AV 4 2, the excess probe mole-
cules are supposed to be dissolved in the bulk solution, and the
increasing trend of fluorescence is similar to that of the SPS2
solution without proteins.
Compared to AV, the loops in SA are shorter. When the
biotin moiety of the probe binds to the pocket in SA, the other
units including the fluorophore and the benzene ring are
relatively flexible, which benefits the entrance of a second
probe molecule into the neighbouring pocket. Therefore, the
Fig. 3 The emission spectra of SPS2 with different concentrations of AV
(a). The fluorescence intensities of SPS2 (b) in the absence (squares) and
presence of SA (triangles) or AV (circles). SA/AV was titrated with SPS2.
10 mM PBS, pH 7.0; lex = 465 nm, lem = 576 nm, both slits were 5 nm.
similar type of feature to that in toluene (7.7 ns) and the maximum binding ratio of SPS2 to SA is about 4 (Fig. S10b,
lifetime becomes longer (3.06 ns in the presence of BSA). The ESI†). Accordingly, the linear range is 0–4 PSP2/SA. When SPS2/
lifetime of SPS2 was a bit longer (1.07/1.79 ns) in the presence SA 4 4 the addition of SPS2 leads to an increment of unbound
of SA/AV (0.1 mg mLÀ1). Moreover, 10 times excess of BSA SPS2 concentration, and the increase of fluorescence is parallel
hardly changed the lifetime of SPS2 in the presence of SA to that of the control series (squares in Fig. 3b).
(1.71 ns, Fig. S8, ESI†). These results reveal that SPS2 can be
used for selective identification of SA/AV even under competi- imaging of biotin receptor-positive Hela cells (Fig. 4 and
tion from BSA or OVA. Fig. S11, ESI†). After Hela cells were incubated with 10 mM of
Finally, we evaluated the possibility of SPS2 for fluorescent
A linear relationship between the concentration of the analyte SPS2 for 1 h, strong intracellular green and red fluorescence
and the fluorescence signal is very important in the quantitative could be observed (Fig. 4a and b). Pretreated Hela cells incu-
detection. So, the effects of AV and SA concentrations on the bated with 10 mM biotin for 1 h to block the receptors in tumor
emission spectrum of SPS2 were further studied. It is observed in cells followed by incubation with SPS2 for another 1 h led to
Fig. 3a (see also Fig. S9, ESI†) that with increasing SA or AV dramatic decreases in both green and red emissions (Fig. 4e
concentration the fluorescence intensity of SPS2 increases and f). The above results indicate that SPS2 could specifically
progressively accompanied with a clear blue shift. A linear bind to the biotin-receptors in the cells after permeation into
relationship between the fluorescence intensity at 576 nm and the cell, which verifies the potential application of SPS2 for
AV concentration was found in the range of 0 to 0.84 mM. In the fluorescent imaging of living cells with biotin receptor-
case of SA, the linear range was 0 to 0.54 mM. The detection limits overexpression. The cell viability of HeLa cells pretreated with
of SA and AV were B3 nM (S/N = 3). The above results demon- various concentrations of SPS2 was assessed using a standard
strate the potential of SPS2 for quantitative detection of SA/AV.
MTT assay.13d After exposure of cells to SPS2 for 24 h, the
The tetrametric SA and AV have four biotin-binding sites, viability decreases by 33% at the concentration used for con-
which permits each protein molecule to bind at most four focal imaging (10 mM, Fig. S12, ESI†), suggesting that SPS2
molecules of the probe. In order to obtain the binding ratio of shows some toxicity to HeLa cells.
the probe to SA/AV, SA/AV samples with known concentrations
In conclusion, two ‘‘off–on’’ fluorescent probes SPS1-2 for
were titrated with SPS2. Upon titration of SPS2 to an AV SA/AV were designed and synthesized taking advantage of the
solution, a steep linear rise of the fluorescence signal was environment-sensitive solvatochromic coumarin fluorophore
observed between 0 and 2 equivalents of SPS2 per AV. Further and the mobility-sensitive phenyl rotor. SPS2 is highly sensitive
addition of SPS2 caused only a small rise in fluorescence.
Similar results were obtained when SPS2 was titrated to the
SA solution, but the break point was at SPS2/SA E 4 (Fig. 3b).
The weak linear rise at 42 SPS2/AV or at 44 SPS2/SA was
parallel to the control series (squares in Fig. 3b) and clearly
from the fluorescence of unbound probe molecules which were
in excess, the above results may indicate that the 1 : 2 complex
of AV-SPS2 and the 1 : 4 complex of SA-SPS2 were formed.
There are several differences in the structures of SA and AV,
which may be the cause of the different binding behaviours of
SPS2 to SA and AV. Compared to SA, there are some additional
hydrophobic and aromatic groups in the binding sites of AV,
and the length of the loop in AV is longer.1b We assume that
when the biotin moiety in SPS2 is located at a binding site, the
Fig. 4 Confocal laser fluorescence (a and b, e and f), bright-field (c, g) and
the merged (d, h) images of living HeLa cells incubated with SPS2 (10 mM)
for 60 min (a–d) and pretreated with 10 mM biotin for 1 h (e–h) followed by
benzene ring in SPS2 may act as a cover of the adjacent binding
pocket in AV to hamper a second probe molecule entering into
this site (Fig. S10a, ESI†). Therefore, the maximum binding
incubation with SPS2 for another 1 h. Excited at 488 nm; (a, e) green
channel; (b, f) red channel.
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This work was financially supported by National 973 Pro-
gram (No. 2011CB910403), NSFC (No. 21235005), Shanghai
Municipal Natural Science Foundation (12ZR1434900) and the
Opening Project of Shanghai Key Laboratory of New Drug
Design (Grant No. 11DZ2260600). We appreciate Prof. A. M.
Brouwer (University of Amsterdam) for improving our paper.
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