Q. Xiao, X. Zhao and H. Xiong
Chinese Chemical Letters 32 (2021) 1687–1690
cells with a compromised plasma membrane and cluster in the
nucleus by endocytosis. Firstly, the concentration of bio-probes
was been inquired to screen, the AIE efficiency is very obvious with
10 mmol/L of TPE-dC or TPE-dU incubated for 12 h at 37 ℃ (Fig. S3A
in Supporting information). Further, the effect of incubation time
has also been explored by time course (Fig. S3B in Supporting
information).
After analyzing the effect of concentration and incubated time,
fibroblasts L929 cells were treated with 10
m
mol/L of TPE-dC for
ꢁ
4
h at 37 C as optimized condition, strong fluorescence has
exhibited an emission at 460 nm by CLSM imaging, and TPE-dU
was used as a comparison (Figs. 3B and E). The AIE efficiency of
TPE-dC is obviously higher than that of the same concentration for
TPE-dU (Figs. 3C and F).
The contour plot shows the intermolecular AIE phenomenon.
The bio-probes were used to exhibit the fluorescent quality scores
of the stained nucleus in L929 cells, and the four cells were located
and calculate by Image J (Fig. 4). The density of sharp peaks on the
plot represented fluorescence intensity of TPE-dC is much higher
than that of TPE-dU, which reveals the AIE efficiency is stronger for
the L929 cells incubated in TPE-dC (Figs. 4A and C). Two distance
dispersion diagrams show that the effective numbers of TPE-dC
binding with the DNA/RNA of the nucleus in living cells is more
than that of TPE-dU, counting the integrated area of peaks (Figs. 4B
and D).
Fig. 4. The 3D surface plot was analyzed by Image J software. In vitro confocal laser
scanning microscopy (CLSM) images of L929 cells treated with 10
m
mol/L of TPE-dC
ꢁ
(A, B) or TPE-Du (C, D) for 4 h at 37 C.
To further explore the possibility of whether the probes can be
performed in gel electrophoresis, the gel was stained in TPE-dC or
TPE-dU buffer (10 mmol/L). It shows the detection limit of DNA for
TPE-dC is low as 250 ng per lane, and TPE-dU is around 500 ng per
lane (Figs. 5A and B). The DNA gel imaging of TPE-dC shows a
higher resolution due to its stronger binding affinity with the
nucleic acid. The result demonstrates this methodology can be
applied for the detection and quantification of both DNA and RNA.
In aqueous solutions, TPE-dC or TPE-dU form complexes with
nucleic acid are mainly driven by molecule affinity forces. In-gel
electrophoresis, the intramolecular motions between TPE-dC or
TPE-dU and nucleic acid are restricted, and the fluorescence
emission of the binding complex is enhanced with increasing DNA
or RNA concentration in the gel state. It directly reveals that the AIE
process is caused by their gelation aggregate and agrees with
previous literature [30,31]. Relative survival rates of cells are
almost identical both in TPE-dC and TPE-dU, and the survival rate
of fibroblast cells L929 keep above 90% even at a high concentra-
tion (Fig. 5B). The MTT assay for cell viability revealed that these
AIEgens bio-probes are noncytotoxic in living cells.
Fig. 5. (A) Staining of DNA (human Histone H3 DNA chain) on PAGE by TPE-dC (up)
and TPE-dU (down). The amount of DNA in lanes 1–5: 0, 100, 250, 500, 1000 ng;
concentrations of TPE-dC or TPE-dU: 10
of yeast tRNA (Invitrogen, 15401029) on PAGE by TPE-dC (up) and TPE-dU (down).
The amount of RNA in lanes 1–5: 0, 1, 2.5, 5, 10 g concentration of TPE-dC or TPE-
dU: 10 mol/L. Staining time: 45 min. (C) Cell viability assay was treated with the
mmol/L. Staining time: 45 min. (B) Staining
m
m
different concentrations of TPE-dC or TPE-dU for 12 h.
tetraphenylethene as AIEgens bio-probes and their biological
applications were also investigated. When TPE-dC or TPE-dU
binds to the negatively charged DNA or RNA with the affinity
interaction, the emission of the binding complexes is turned on
due to the restriction of their intramolecular motions. Such an AIE
process enables quantitative analysis or visualization of nucleic
acids in solution or gels state. Unlike conventional nucleic acid
stains, aggregates of TPE-dC and TPE-dU show a high affinity for
the nucleus in living cells, and they exhibit intense fluorescence
and superior stability. The AIEgens probes can easily penetrate
cells with compromised plasma membranes and enter the
nucleus of live cells, which indicates that TPE-dC and TPE-dU
have a strong nuclear targeting ability. That may because the
probes’ affinity for DNA is much more similar to the natural
structure of DNA biosynthesis, which would minimize the
damage to DNA. It is remarkable that TPE-dC has better AIE
performance than TPE-dU in the most of above cases. It is possible
that cytidine has a better affinity with DNA/RNA sequences or
chromosome compared with uridine due to their different
chemical structures or pKa values. Furthermore, the biocompati-
bility and safety of chiral TPE-dC and TPE-dU analogues are being
investigated by cell culture assay with potential applications in
the delivery of nuclear-targeted drugs or the development of
time-lapse microscopy.
To our knowledge, this work is the first example of the AIEgens
0
0
sensor based on 2 -deoxycytosine or 2 -deoxyuridine conjugating
Fig. 3. In vitro confocal laser scanning microscopy (CLSM) images of L929 cells
treated with TPE-dC (A) bright, (B) at Ex = 370 nm, (C) merge; and TPE-dU (D) bright,
(
E) at Ex = 370 nm, (F) merge; The incubation concentration of 10
mmol/L using TPE-
dC or TPE-dU in the PBS buffer at pH 7.4 for 4 h, and scale bar = 10
m
m.
1689
Xiao, Qiuyun
