Specific detection of cancer cells through aggregation-induced emission of a light-up bioprobe

Article abstract of DOI:10.1039/c7cc00122c

A cancer cell specific aptamer was labeled with an aggregation-induced emission (AIE) probe for the first time. Using it as a light-up bioprobe, a specific cancer cell detection method is developed.

Full text of DOI:10.1039/c7cc00122c

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Specific Detection of Cancer Cells through Aggregation-Induced
Emission of a Light-Up Bioprobe
Jian Chen,‡ab Hong Jiang,‡ab Huipeng Zhou,*ab Zhenzhen Hu,ab Niu Niu,ab Sohail Anjum Shahzad,ac
and Cong Yu*ab
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A cancer cell specific aptamer was labeled with an aggregation-
Aptamers are singleꢀstranded DNA or RNA oligonucleotides
induced emission (AIE) probe for the first time. Using it as a light- which can specifically and tightly bind to a variety of targets,
up bioprobe,
developed.
a
specific cancer cells detection method is such as metal ions, small molecules, proteins, and even entire
viruses and cells.14 Compared with other recognition molecules
such as antibodies, aptamers are more stable, inexpensive and
can be synthesized and labeled chemically with ease.15 As a
result, a variety of aptamerꢀbased biosensing methods have
been developed.14,16 In recent years, specific aptamers against
entire cancer cells have been reported.17 Using those aptamers
as novel and powerful affinity recognition ligands, several
aptamerꢀbased cancer cell detection methods have been
developed.3b–3g
Cancer is a group of diseases involving abnormal cell growth.
Cancer cells grow and divide at a very rapid and unregulated
pace, and could invade or spread to other parts of the body.1
Highly sensitive and selective detection of cancer cells is of
great importance for both early diagnosis of cancer and
investigation of cancer metastasis.2 In recent years, a number of
cancer cell detection methods have been developed, which
include the fluorescent,3 chemiluminescent,4 electrochemical,5
surfaceꢀenhanced Raman scattering,6 surface plasmon
resonance,7 colorimetric8, and electrochemiluminescent9
methods. Compared with other methods, fluorescenceꢀbased
method has drawn growing attentions due to its high sensitivity,
rapid response, and simple manipulation.
Aggregationꢀinduced emission (AIE) is
a
novel
photophysical phenomenon which has attracted considerable
interest in recent years.10 The molecules with AIE
characteristics are nonꢀemissive in the monomeric form but
become highly emissive in the aggregated form as a result of
the restriction of intramolecular rotations, which could
significantly reduce the energy dissipation through nonradiative
means.11 Based on this observation, a number of AIE probes
have been developed for the applications in bioanalysis and
biosensing.12 The AIE probes are usually propellerꢀshape
molecules. Tetraphenylethene (TPE) is one of the most
important AIE probes, which has been widely used for the
construction of novel biosensing techniques.13
Scheme 1. Schematic illustration of the Ramos cells detection
strategy.
In this work, a cancer cell specific aptamer was labeled with
an AIE probe for the first time. Using it as a lightꢀup bioprobe,
a specific cancer cell detection method is developed. Ramos
cells (human Burkitt’s lymphoma cells) were selected as the
model cancer cells. The overall sensing strategy is
schematically illustrated in Scheme 1. A Ramos cell specific
aptamer with both ends labeled with TPE probe (TPE-aptamer
was designed and successfully synthesized. The TPE-aptamer
showed only very weak emission in an aqueous buffer solution.
Upon the addition of Ramos cells, specific binding between the
a State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of
Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China
E-mail: hpzhou@ciac.ac.cn; congyu@ciac.ac.cn; Fax: (+86)-431-8526-2710
b University of the Chinese Academy of Sciences, Beijing 100049, P. R. China
c Department of Chemistry, COMSATS Institute of Information Technology,
Abbottabad 22060, Pakistan
Electronic Supplementary Information (ESI) available: Experimental details,
supporting figures. See DOI: 10.1039/x0xx00000x
‡These authors contributed equally to this work.
)
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aptamer and the Ramos cells resulted in restrictions of the digested into small fragments by nuclease S1. Figure S10
intramolecular rotations of the TPE molecules. Turnꢀon shows that, in an aqueous solution, after the digestion of the
emission of the TPE-aptamer was observed, and a specific TPE-aptamer with nuclease S1, a significant increase of TPE
fluorescence cancer cell detection method is therefore AIE emission was observed. The results confirm that the
established.
aptamer conjugated TPE molecules mainly existed in the
The synthetic route for the TPEꢀlabeled Ramos cell specific monomeric form, and the negatively charged oligonucleotide
aptamer (TPE-aptamer) is shown in Scheme 2. A succinimidyl effectively diminished the tendency of the TPE probe selfꢀ
esterꢀmodified TPE derivative (compound
1) was prepared aggregation. After the digestion with nuclease S1, the TPE
(Figures S1–S6, Supporting Information).18 The aptamer with probe was released, and strong hydrophobic interactions among
amino functional groups labeled on both ends was coupled the probe molecules caused strong AIE emission.
directly with two molecules of compound
1. The resulting
crude product was purified by reverseꢀphase HPLC. Figure S7
shows the HPLC chromatograms of the crude product. The
peaks 1–4 are the unreacted aptamer (with absorption at 260 nm
only), singleꢀTPEꢀlabeled aptamer (with absorption at both 260
and 350 nm), dualꢀTPEꢀlabeled aptamer (with absorption at
both 260 and 350 nm), and unreacted TPE (with absorption at
350 nm mainly), respectively. Peak
3 was collected, and pure
TPE-aptamer was obtained.
Scheme 2. Synthetic route for the TPE-aptamer
.
Figure S8 shows the UVꢀvis absorption spectra of the
aptamer, compound and the TPE-aptamer, respectively. It
1
was observed that the TPE-aptamer contained the
characteristic absorption of both the aptamer (maximum
absorption at 260 nm) and compound
at 350 nm). The results clearly indicate that the aptamer has
been successfully labeled with the TPE probe.
1 (maximum absorption
Figure 1. (a) Changes in emission spectrum of the TPE-
aptamer with the amount of Ramos cells added (0, 100, 200,
500, 1000, 2000, 3000, 4000, 5000, 10000, 15000 and 20000,
respectively). (b) Plot of the changes in emission intensity of
the TPE-aptamer at 470 nm against the amount of Ramos cells
added. Inset: expanded linear region of the curve.
Figure S9 shows the emission spectra of compound
TPE-aptamer in aqueous and organic solvents. In DMSO,
compound (a hydrophobic molecule) mainly existed in the
monomeric form and showed no AIE emission. While in a
DMSOꢀwater solvent mixture (v/v = 1/99), compound had a
1 and the
1
1
The Ramos cell specific aptamer can specifically recognize
the immunoglobulin heavy mu chain on the surface of the
Ramos cells.3f Upon the addition of Ramos cells to the solution
of TPE-aptamer, specific binding between the aptamer and the
Ramos cells resulted in restrictions of the intramolecular
rotations of the TPE molecules. And strong AIE emission of the
TPE-aptamer was thus observed. Figures 1 and S11 show that,
strong tendency to selfꢀaggregate due to the strong hydrophobic
interactions among the probe molecules, thus strong compound
1
AIE emission was observed. In sharp contrast, the TPE-
aptamer showed only very weak emission in water but strong
emission in the DMSOꢀwater solvent mixture (v/v = 99/1). The
results clearly suggest that the TPE-aptamer had better
solubility in an aqueous solution. Since the TPE molecules
were covalently labeled to the aptamer (an anionic polymer),
the negatively charged oligonucleotide phosphate backbone
diminished the tendency of TPE selfꢀaggregation.
in the presence of 2.5
µM of TPE-aptamer, the emission
intensity of the aptamer increased gradually with the addition of
increasing amounts of the Ramos cells (0 – 20,000). A linear
relationship was obtained at a Ramos cell range of 0 – 5,000.
The linear regression equation is E = 0.067A + 84.55
To investigate further the property of the TPE-aptamer
control experiments were conducted. The TPE-aptamer was
,
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(correlation coefficient R2 = 0.999), in which E is the emission
The assay could also be used for the specific fluorescence
intensity of the TPE-aptamer at 470 nm and A is the amount imaging of cancer cells. Ramos, CCRFꢀCEM and HeLa cells
of Ramos cells. Our assay is quite sensitive compared with the were tested. Figure 3 shows that, in the presence of 2.5
µM
previously reported fluorometric methods.3b,3e,3g Without the TPE-aptamer and 10,000 of Ramos cells, the AIE emission of
use of any signal amplification, 100 Ramos cells could be the TPE-aptamer could be easily observed. Under the same
easily detected.
experimental conditions, CCRFꢀCEM and HeLa cells could not
Selectivity of the assay was studied. Five cancer cell lines give noticeable AIE fluorescence. The results clearly suggest
(HeLa, CCRFꢀCEM, MCFꢀ7, A549 and HepG2) and one that the assay could also be used for the specific fluorescence
normal cell line (L929) were selected. Figure 2 shows that, imaging of cancer cells. In addition, cytotoxicity study of the
under the same experimental conditions, 20,000 Ramos cells TPE-aptamer was conducted (Figure S13). The results clearly
could induce obvious AIE emission increase of the TPE- indicate that our probe is nontoxic to the cells.
aptamer. In contrast, the other cells of the same amount could
In summary, a cancer cell specific aptamer is labeled with an
not induce noticeable emission increase. The results clearly AIE probe for the first time, and a selective detection method
indicate that the assay is highly selective for Ramos cells.
for cancer cells based on aggregation induced emission is
developed. A TPE labeled Ramos cell specific aptamer (TPE-
aptamer) was synthesized. The TPE-aptamer shows very
weak emission in an aqueous buffer solution. Upon the addition
of Ramos cells, specific binding between the aptamer and
Ramos cells resulted in restrictions of intramolecular rotations
of the TPE probe molecules. Strong AIE emission of the TPE-
aptamer was observed, and a selective Ramos cell detection
method is established.
Our assay has several important features. First, it offers a
convenient “mixꢀandꢀdetect” approach, which is simple and
fast. Second, it is based on the fluorescence “turnꢀon” mode,
which could considerably reduce the likelihood of falseꢀ
positive signals associated with the “turnꢀoff” assay. Third,
without the use of any signal amplification, highly sensitive
cancer cell detection could be easily accomplished. Fourth, our
assay could be used in complex sample mixtures, and also be
used for the specific fluorescence imaging of cancer cells. Fifth,
the AIE probe has certain unique characteristics, the
fluorescence turn on does not rely on energy transfer or
nanomaterials. We envision that the principle of our assay
could be employed for the development of novel AIE and
nucleic acid aptamer based sensing techniques for the detection
of various targets.
Figure 2. Selectivity of the assay. Columns a–g: Ramos, HeLa,
CCRFꢀCEM, MCFꢀ7, A549, HepG2 and L929, respectively.
Assay conditions: 2.5
µM TPE-aptamer; 20 mM phosphate
buffer, pH 7.4; the amount of cell added, 20,000 each.
The assay was also tested in complex sample mixtures. TPE-
aptamer (2.5 µM) and Ramos cells of different amounts were
added to human serum (80%), and the sample mixtures were
tested. Figure S12 shows that the more Ramos cells were
spiked, the larger emission intensity increase of the TPE-
aptamer was obtained. The results clearly indicate that the
assay could also be used in complex sample mixtures.
This work was supported by the National Natural Science
Foundation of China (21561162004, 21275139), the Science
and Technology Development Project (International
Collaboration Program) of Jilin Province (20160414040GH),
and the Natural Science Foundation of Jilin Province
(20150101183JC).
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