Biosensors and Bioelectronics 80 (2016) 18

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

journal homepage: www.elsevier.com/locate/bios

Evolution of DNA aptamers for malignant brain tumor gliosarcoma cell

recognition and clinical tissue imaging
Qiaoyi Wu a, Liang Wu a, Yuzhe Wang a, Zhi Zhu b,c, Yanling Song a,b, Yuyu Tan b,c,
Xing-Fu Wang a, Jiuxing Li b,c, Dezhi Kang a,n, Chaoyong James Yang b,c,nn
a
The First Clinical Medical College of Fujian Medical University, Department of Neurosurgery, Department of Pathology, the First Afliated Hospital of Fujian
Medical University, Fuzhou 350004, PR China
b
The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, State Key Laboratory of Physical Chemistry of Solid Surfaces, Key Laboratory for
Chemical Biology of Fujian Province, Xiamen University, Xiamen 361005, PR China
c
Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, PR China

art ic l e i nf o

a b s t r a c t

Article history:
Received 7 December 2015
Received in revised form
10 January 2016
Accepted 11 January 2016
Available online 14 January 2016

Gliosarcoma, a variant of glioblastoma multiforme (GBM), is a highly invasive malignant tumor. Unfortunately, this disease still marked by poor prognosis regardless of modern treatments. It is of great
signicance to discover specic molecular probes targeting gliosarcoma for early cancer diagnosis and
therapy. Herein, we have selected a group of DNA aptamers with high afnity and selectivity against
gliosarcoma cells K308 using cell-SELEX. All the dissociation constants of these aptamers against gliosarcoma cells were in the nanomolar range and aptamer WQY-9 has the highest afnity and good selectivity among them. Furthermore, truncated aptamer sequence, WQY-9-B, shows similar recognition
ability to aptamer WQY-9. In addition, WQY-9-B was found to be able to bind selectively and internalize
into cytoplasm of target cancer cell at 37 C. More importantly, compared to a random sequence, aptamer
WQY-9-B showed excellent recognition rate (73.3%) for tissue sections of clinical gliosarcoma samples.
These data suggests that aptamer WQY-9-B has excellent potential as an effective molecular probe for
gliosarcoma diagnosis.
& 2016 Elsevier B.V. All rights reserved.

Keywords:
Aptamer
Cell-SELEX
Gliosarcoma
Biomarker

1. Introduction
Gliosarcoma, a variant of glioblastoma multiforme (GBM), is a
malignant form of brain tumor (Biernat et al., 1995; Boerman et al.,
1996; Han et al., 2010) characterized by highly aggressive ability to
inltrate to critical brain areas, and to metastasize to extracranial
regions (Beaumont et al., 2007; Meis et al., 1991). Despite advances
in treatment over the past few decades, the prognosis of this
disease remains poor, and the median survival is generally less
than one year after diagnosis (Damodaran et al., 2014; Scheithauer,
2009). Conventional methods used to distinguish gliosarcoma
from other GBMs, such as computed tomography scan, magnetic
resonance imaging, and histopathology biopsy, mainly rely on
morphologic criteria. In most cases, however, these methods are
nonspecic or insensitive (Hussain and Nguyen, 2014; Kircher
et al., 2012). On the contrary, molecular imaging can detect
n

Correspondence to: 422 Siming South Road, Xiamen, Fujian 361005, PR China.
Correspondence to: 88 Jiaotong Road, Fuzhou, Fujian 350004, PR China.
E-mail addresses: kdz99988@vip.sina.com (D. Kang),
cyyang@xmu.edu.cn (C.J. Yang).
nn

http://dx.doi.org/10.1016/j.bios.2016.01.031
0956-5663/& 2016 Elsevier B.V. All rights reserved.

aberrant molecular changes in early stages of cancer (Blasberg,

2003; Weissleder, 2006). With the development of proteomics and
genomics, some molecular abnormalities in proteins, genes and
epigenetics have been found to correlate with cancer (Actor et al.,
2002; Reis et al., 2000; Singh et al., 2012). The specic molecular
characterization can not only accurately identify neoplasm, but
also provide the foundation for precise therapies. Unfortunately,
there are still no clinically effective biomarkers which can be used
to classify gliosarcoma from other GBMs. Consequently, it is very
necessary to develop novel and specic molecular probes capable
of identifying gliosarcoma to facilitate early diagnosis and therapy,
and thereby improve the survival rate and therapeutic efcacy of
gliosarcoma patients.
Aptamers, are short, single-stranded oligonucleotide molecules
that are generated from a large random oligonucleotide pool via
Systematic Evolution of Ligands by EXponential enrichment (SELEX) (Ellington and Szostak, 1990; Tuerk and Gold, 1990). As novel
biomedical probes, aptamers have attracted increasing attention in
recent years. Compared with antibodies, aptamers have signicant
advantages, including excellent afnity and seletivity for molecular recognition, economical and reproducible synthesis, stability

Q. Wu et al. / Biosensors and Bioelectronics 80 (2016) 18

Fig. 1. Scheme of systematic evolution of DNA aptamers target K308. SVGp12 cells were applied for counter selection to remove non-specic binding DNA. After eluted and
PCR amplied, sequences bound to K308 cells were separated from dsDNA and desalted for next round of selection. After enrichment, the PCR amplication product was
cloned and sequenced.

Fig. 2. Flow cytometry experiment of binding of aptamer candidates with K308 (A), SVGp12 (B), U251(C), U87 (D), T98G (E) and HuH-7 (F). The concentration of the
aptamers was 250 nM.

at room temperature, lack of immunogenicity, low molecular

weight, and rapid tissue penetration (de-los-Santos-Alvarez et al.,
2009; Iliuk et al., 2011; Raston and Gu, 2015; Tan et al., 2013; Zhu
et al., 2012). For example, DNA aptamer against alpha-methylacylCoA racemase, is not only a specic biomarker, but also a base of
the aptamer-based biosensors for prostate cancer diagnosis (Yang
et al., 2014). 99mTc radiolabeled aptamers bound to tenascin-C and
MUC1 protein, a type of membrane protein, have been used for
tumor nuclear imaging (Pieve et al., 2009). By conjugating nanoparticles with targeting aptamers, magnetic resonance imaging

has advanced from traditional anatomical imaging to molecular

level imaging (Das et al., 2015; Tian et al., 2014; Zhang et al., 2015;
Zhu et al., 2013a). Owing to their unique targeting property, aptamers have played very important roles in the monitoring of
environment and food safety (Wang et al., 2011; Yang et al., 2015),
the capture of circulating tumor cells (Shen et al., 2013; Zhao et al.,
2012), and in targeted therapy (Liang et al., 2015; Zhu et al.,
2013b). Mucagen, the rst aptamer-based drug approved by the US
Food and Drug Administration in 2004, is available towards agerelated macular degeneration (Ng and Adamis, 2006). In addition,

Q. Wu et al. / Biosensors and Bioelectronics 80 (2016) 18

Fig. 3. Confocal images of K308, SVGp12, U251, U87 and T98G cells stained with WQY-9 (250 nM) and initial library (250 nM).

some other aptamers are being evaluated in clinical trials for

treating diseases, including coagulation, oncology, and inammation (Cibiel et al., 2012; Lao et al., 2015; Sun et al., 2014).
Cell-SELEX (Daniels et al., 2003; Sefah et al., 2010; Shangguan
et al., 2006), a derivative of SELEX, is an efcient selection strategy
in which intact living cells are used to generate aptamers for most
cancer studies. In this way, the extracellular molecules are present
in their natural environment, and specically binding target molecules can be detected without prior knowledge of the biomarker
of target cells. Thereby, new, previously unknown biomarkers can
be discovered. Up to now, a number of aptamers obtained by cellSELEX against various types of cancer, including pancreatic ductal
adenocarcinoma (Wu et al., 2015), colon cancer (Li et al., 2015),
breast cancer (Li et al., 2014), leukemia (Sefah et al., 2009) and
liver cancer (Shangguan et al., 2008b; Xu et al., 2015), have been
identied and are being investigated for molecular imaging of the
above-mentioned tumors. In addition, several novel biomarkers
have been detected via this method, such as immunoglobulin
heavy mu chain (Mallikaratchy et al., 2007) and protein tyrosine
kinase-7 (Shangguan et al., 2008a).
In this study, for the rst time, we applied cell-SELEX to generate
a group of DNA aptamers that can selectively recognize gliosarcoma
cells. To identify aptamers with high binding afnity and good selectivity against human gliosarcoma cell line K308 (Yao et al., 2015),
normal human brain astroglia cell line SVGp12 was used for counter
selection. Flow cytometry was applied to monitor the process of the

DNA aptamer selection. The aptamer, WQY-9, with the best recognition capability was identied and further study has revealed
its target type and essential truncated sequence. Moreover, uorescence imaging was utilized to assess the binding ability of truncated aptamer WQY-9-B to clinical gliosarcoma tissue.

2. Materials and methods

2.1. Cell lines and cell culture
Gliosarcoma cells K308 were provided by the First Afliated
Hospital of Fujian Medical University (Yao et al., 2015). HeLa cells
(Cervical cancer) were obtained from the Cell Bank of the Chinese
Academy of Science (Shanghai). Glioblastoma cells T98G, U87,
U251, liver cancer cell HuH-7, lung cancer cell A549, colon carcinoma cells SW620, SW480, breast cancer cell MDA-MB-231, normal astroglia cells SVGp12 and breast cancer cell MCF-7 were
purchased from the American Type Culture Collection. All cells
were incubated at 37 C in a humidied atmosphere of 5% CO2.
K308, T98G, U87, U251, HuH-7, SW480 and Hela were cultured in
Dulbecco's minimal essential medium (Hyclone) with 10% fetal
bovine serum (FBS, Invitrogen). The growth medium for MDA-MB231, A549, MCF-7 and SW620 was composed of RPMI-1640 (Hyclone) with 10% FBS. SVGp12 was cultured and propagated in Eagle's minimal essential medium (ATCC) with 10% FBS.

Q. Wu et al. / Biosensors and Bioelectronics 80 (2016) 18

Fig. 4. (A) Secondary structure of WQY-9 predicted by NUPACK. (B) Binding assay of ve truncated sequences to K308 cells. (C) The equilibrium dissociation constant (Kd) of
WQY-9-B to target cell K308. (D) Binding abilities of aptamers (250 nM) to target K308 cells. Error bar represents standard deviation of three repeated experiments (n3).

2.2. SELEX procedures

The DNA library and primers used in this work were obtained
from the laboratory of reference (Sefah et al., 2009). Gliosarcoma cells
K308 were used as target cells and astroglia cells SVGp12 as negative
cells for the counter selection. Cells and ssDNA library process
methods were similar to our previous work (Li et al., 2015). Signicant
changes were as following. 1
106 negative cells SVGp12 was rst
added and incubated with the prepared ssDNA starting from the third
round of selection. For the purpose of obtaining sequences with high
afnity and specicity, the incubation time for negative control was
increased from 30 min to 1 h and the number of negative cells was
enhanced gradually from 1
106 to 1
107. In addition, FBS was
added starting at the fourth round. After 16 rounds of selection, the
enrichment pool were cloned, sequenced and characterized.
2.3. Flow cytometry analysis
Pool enrichment was monitored using FACS (Becton Dickinson
Immunocytometry Systems). 3
105 cells were incubated with
ssDNA pools (250 nM) labeled with FAM at 4 C for 30 min in the
dark. Cells were washed with 0.5 mL of washing buffer twice to
remove the unbound sequences and resuspended in 0.25 mL of
washing buffer for analysis. The mean uorescence intensity was

analyzed by counting 10,000 events. The unselected DNA library

was used as the negative control. To assess the seletivity of aptamers, positive cells K308, negative cells SVGp12 and other related
glioma cells T98G, U87, U251, were treated with FAM-labeled aptamers and subjected to FACS analysis. In addition, human liver
cancer cells HuH-7, breast cancer cells MDA-MB-231 and MCF-7,
cervical cancer cell line HeLa, and colon carcinoma cells SW620,
SW480 were also used as negative control.
2.4. Determination of aptamers binding afnity
3
105 K308 cells were incubated with a series of concentrations of aptamers at 4 C in the dark for 0.5 h. Cells were washed as
described above and investigated via ow cytometry analysis. The
unselected DNA library was adopted as control. By tting the dependence of mean uorescence intensity of aptamer-cell complex
on the concentration of aptamers, the dissociation constants (Kd)
were calculated with the equation Y Bmax X/(Kd X), using SigmaPlot (Jandel Scientic).
2.5. Proteinase treatment
K308 cells (3
105) were washed three times with 1 mL of
washing buffer and then incubated with 0.5 mL of 0.05% trypsin/

Q. Wu et al. / Biosensors and Bioelectronics 80 (2016) 18

Fig. 5. (A) Flow cytometry experiment of aptamer WQY-9-B binding to trypsin-treated K308 cells. (B) Binding of aptamer WQY-9-B to K308 cells at 4 C and 37 C.
(C) Confocal images of aptamer WQY-9-B to K308 cells at 4 C and 37 C, respectively. The nal concentration of WQY-9-B was 250 nM.

0.53 nM EDTA in PBS for 30 s and 60 s, respectively. Before

washing with 2 mL of washing buffer, an equal amount of FBS was
added to terminate the reaction. Cells were then incubated with
aptamers on ice in the dark for 30 min and the afnity was determined by ow cytometry.

antigens. Afterwards, the array slides were incubated with normal

goat serum at room temperature for 60 min. Finally, the prepared
array slides were stained with FAM-labeled aptamer (250 nM,
0.2 mL) at 4 C for 30 min.

2.6. Effect of temperature on binding

3. Results and discussion

After washing with 1 mL of washing buffer, 3
105 K308 cells

were incubated with aptamer (250 nM) labeled with FAM at 4 C
and 37 C for 30 min in the dark. The FAM-labeled random sequence was used as control. Then prepared samples were analyzed
via ow cytometry.

3.1. Selection of aptamers against gliosarcoma cells K308

2.7. Imaging of cells or tissue bound with aptamers

Cells cultured in a confocal dish were washed with washing
buffer and stained with aptamers (250 nM) labeled with FAM on
ice in the dark for 30 min. After washing, a LeicaTCS SP5 confocal
uorescence microscope was applied for imaging. Gliosarcoma
tissue was obtained from the First Afliated Hospital of Fujian
Medical University. The other tissue array slides were purchased
from US Biomax (Alenabio Co., Ltd, Xian, China). According to the
approach described previously (Chen et al., 2008; Zhao et al.,
2009), briey, array slides were deparafnized in xylene, rinsed in
PBS twice for 5 min each, and then rehydrated through series of
ethanol (100%, 95%, 80% and 70%) for 5 min each, PBS (pH 8.0) for
5 min, and 95 C heated buffer (1 mM EDTA) for 15 min to retrieve

To generate aptamers against gliosarcoma, K308 cell line derived from a primary gliosarcoma of the temporal lobe was
adopted as positive cells, and human normal astroglia cell line
SVGp12 was used for counter selection. The schematic of cell-SELEX is illustrated in Fig. 1. The initial pool that contained 45-mer
random bases was incubated with K308 cells. After washing several times, cells were scraped from the dish, and the bound DNA
was eluted from the cell-DNA complex and amplied by PCR. From
the third round of selection, ssDNA library was rst incubated with
SVGp12 cells for counter selection. The unbound sequences were
collected and then incubated with K308 cells for positive selection.
The process of selection was monitored by ow cytometry. As
shown in Supplementary Fig. S1A, with the number of selection
rounds increased, a signicant increase in uorescence intensity
was observed for K308 cells. In the contrary, no evident shift in
uorescence intensity was detected with the SVGp12 cells (Supplementary Fig. S1B), suggesting that DNA sequences were enriched and selectively bound to K308 cells. After 16 cycles of

Q. Wu et al. / Biosensors and Bioelectronics 80 (2016) 18

Fig. 6. Confocal orescence images of normal human brain tissues and gliosarcoma tissues stained with WQY-9-B (250 nM) and random sequence (250 nM).

selection, the evolved library was amplied, cloned and sequenced

for identication of aptamer candidates.
3.2. Afnity of aptamer candidates to gliosarcoma cells
To identify the DNA aptamer candidates, 50 clones of enrichment
of DNA pool were sequenced. The sequences acquired from cloning
and sequencing were analyzed by software Clustal X 2.0.3 (Thompson et al., 1997). According to the homology of the sequences, the
sequences were classied as ve families. Sequences with over 5%
enrichment were selected and sent to synthesize for further characterization. The selected four aptamers WQY-4, WQY-5, WQY-9, and
WQY-11 accounted for 13%, 9%, 50%, and 6% of all identied sequences, respectively. Flow cytometry analysis was carried out to
validate the binding afnity of the selected aptamers. The Kd of four
aptamers were in the nanomolar range, as shown in Supplementary
Fig. S2 and Supplementary Table 1. The results indicated that these
sequences possess good binding afnity to K308 cells.
3.3. Selectivity of DNA aptamer WQY-9
Among the four aptamers, WQY-9 was selected for specicity
tests because of its highest degree of enrichment and lowest Kd
value. The binding of the FAM-labeled aptamers WQY-9 showed
the highest uorescence signal, and signicant uorescence shift
over that of unselected library against K308 cells (Fig. 2A), while
not bound to SVGp12 (Fig. 2B). Although aptamers WQY-9 were
selected against gliosarcoma cells, the possibility of binding to
GBM cells could not be excluded. To verify this, aptamers were
assessed with glioma cell lines U87, U251 and T98G. Compared
with random sequence (RS), WQY-9 hardly changed in uorescence intensity, as shown in Fig. 2CE, suggesting that WQY-9 can
be used to differentiate gliosarcoma cell line from other GBM cells.
Next, the specicity of WQY-9 was further evaluated with cell
lines derived from other organs, including liver cancer cells HuH-7
(Fig. 2F), colon cancer cells SW480 and SW620, lung cancer cells

A549, cervical cancer cells HeLa, breast cancer cells MCF-7 and
MDA-MB-231 (Supplementary Fig. S3). There was no obvious
signal with these tested cells. The results imply that the selected
aptamers are probably exclusive to gliosarcoma. Then we further
assessed the specic cell recognition of the selected FAM-labeled
aptamers by confocal uorescence imaging. As shown in Fig. 3, the
results clearly proved that aptamer WQY-9 displayed high binding
ability towards K308 but no binding to SVGp12. More remarkably,
compared to U251, U87 and T98G, K308 cells gave signicantly
brighter uorescence signal after incubating with WQY-9.
3.4. Sequence optimization of WQY-9
To some extent, short sequences may be superior because of
their high permeability, easy modication and low cost of synthesis, all factors signicantly promoting the application of aptamers. Therefore, we optimized the sequence of WQY-9 with
minimal loss in binding capacity, according to its secondary
structure predicted by NUPACK (Zadeh et al., 2011) (Fig. 4A). Five
kinds of truncated versions of aptamer WQY-9 were obtained by
gradually removing its marginal sequences. The sequences WQY9-A (63nt, 1981), WQY-9-B (63nt, 163), WQY-9-C (45nt, 1963),
WQY-9-D (50nt, 3281) and WQY-9-E (27nt, 3157) are shown in
Supplementary Table 2. Subsequently, the binding experiment of
aptamer by ow cytometry demonstrated that only WQY-9-B
maintained similar binding afnity to K308 cells compared to the
primitive WQY-9 (Fig. 4B). WQY-9-A, WQY-9-C, WQY-9-D and
WQY-9-E hardly bound to target cells K308, suggesting that the
forward primer is indispensable to form a stem-loop structure for
binding with K308 cells. However, further truncation of aptamer
WQY-9-B resulted in loss of binding ability towards K308 cells.
3.5. Binding afnity of truncated sequence WQY-9-B
Next, we investigated the binding ability of truncated sequence
WQY-9-B to target cells K308, and found that the Kd value of WQY-

Q. Wu et al. / Biosensors and Bioelectronics 80 (2016) 18

9-B was 237 5 nM (Fig. 4C) with binding afnity to K308 cells as
strong as WQY-9 (Fig. 4D). The results suggested that we generated an aptamer with shorter sequence and similar recognition
capacity as WQY-9.

potential as a valid molecular diagnostic and therapeutic agent for

gliosarcoma.

4. Conclusions
3.6. Analysis of target type and the impact of temperature on WQY9-B
Because of its remarkable binding afnity and specicity, the
target of aptamer WQY-9-B was further characterized. To investigate whether the target molecule of the aptamer is an extracellular membrane protein, K308 cells were pretreated with
trypsin before incubation with FAM-labeled aptamer. In Fig. 5A,
after incubated with trypsin for 30 s and 60 s, aptamer WQY-9-B
lost its binding abilities to K308. The signicant decrease in
binding ability suggested that WQY-9-B targets are probably extracellular membrane proteins. Since future studies need to occur
in physiological conditions, the binding of aptamer WQY-9-B were
tested at 37 C. As shown in Fig. 5B, there was little inuence on
uorescence intensity of target cells K308 at the higher temperature. In addition, the selectivity of WQY-9-B were also tested at
37 C, as shown in Supplementary Fig. S4, and the result suggested
that there was no impact of temperature on the selectivity. Although aptamers were mainly localized on the cytomembrane of
K308 cells at 4 C, at 37 C, there were parts of intracellular
uorescence signals observed by confocal uorescence imaging
(Fig. 5C), suggesting the internalization of the aptamer under this
condition. The ability for WQY-9-B to bind selectively to target
cancer cells and internalize into cytoplasm under physiological
condition offers exciting new opportunity to develop new targeting molecular therapeutic agents.

In summary, we have successfully selected a group of DNA

aptamers selectively targeting gliosarcoma cells by cell-SELEX
after 16 cycles of evolved enrichment. The Kd of these DNA aptamers are all in the nanomolar range. These aptamers can specically distinguish gliosarcoma cells from human normal astroglia
cells. Among them, WQY-9 has found to have highest afnity and
good specicity against gliosarcoma cells with a Kd of 21 74 nM.
The target of aptamer WQY-9 has been preliminarily veried as an
extracellular membrane protein. An optimized sequence WQY-9-B
holds the same recognition ability to bind gliosarcoma cells as that
of WQY-9. In addition, WQY-9-B was found to be able to bind
selectively and internalize into cytoplasm of target cancer cell at
37 C. More importantly, tissue imaging results demonstrated that
aptamer WQY-9-B has high binding afnity to target tumor tissue
and exhibited as high as 73.3% positive detection rate against
gliosarcoma tissue. The DNA aptamer WQY-9-B has great potential
for further application to image gliosarcoma, deliver chemotherapy drugs and discover tumor biomarkers.

Conict of interest
All authors have declared that no competing interest exists.

Acknowledgments
3.7. Imaging of gliosarcoma tissues with aptamer WQY-9-B
To test the recognition ability of aptamer WQY-9-B in clinical
gliosarcoma samples, laser scanning confocal microscopy was
applied to detect gliosarcoma tissues with FAM-labeled aptamer
WQY-9-B and the results were analyzed statistically. Therefore,
formalin-xed parafn-embedded slides of normal human brain
tissues and gliosarcoma tissues were stained with WQY-9-B. As a
result, it was observed that 11 of the 15 gliosarcoma cases exhibited strong green uorescence, and the binding ratio of WQY-9B to gliosarcoma tissues was 73.3%. Because cancer is characterized
by its genetic heterogeneity, even the same kind of tumor would
encompass different phenotypes with distinct biologic features.
WQY-9-B was selected targeting gliosarcoma cells K308, which
could not represent all kinds of clinical gliosarcoma. As a result,
some of clinic tumor tissues cannot be recognized by aptamer
WQY-9-B. In comparison, binding rate for random sequence to
gliosarcoma tissues from the same patients was only 13.3%. On the
contrary, a 16.7% positive rate was observed among 12 normal
brain tissue sections stained with WQY-9-B, while positive rate of
sections stained with random sequence was 20%, as shown in
Supplementary Table 3 and Fig. 6. We further tested some other
tissue arrays from 54 kinds of human cancer tissues and 18 normal
tissues, including bladder transitional cell carcinoma, breast cancer, colon adenocarcinoma, esophageal cancer, hepatocellular
carcinoma, kidney clear-cell carcinoma, lung cancer, lymphoma,
malignant meningioma, nose squamous cell carcinoma, ovarian
cancer, pancreatic ductal adenocarcinoma, prostate cancer, stomach adenocarcinoma and uterine endometriioid adenocarcinoma. Almost all of them exhibited extremely weak uorescence
signals after incubation with FAM-labeled WQY-9-B (Supplementary Figs. S5S8). These results suggest that aptamer WQY-9-B
selected by cell-SELEX is highly specic to the corresponding
gliosarcoma tissues. Hence, the aptamer WQY-9-B has great

This study is supported by the National Natural Science Foundation of China (No. 81372345, 21325522, 21275122, and
21521004); Joint Research Program of Health and Planning Committee and Education Department of Fujian, P.R.C (WKJ-FJ-07);
Type JK Program of Education Department of Fujian, P.R.C
(JK2012018); Key Clinical Speciality Discipline Construction Program of Fujian province and Fundamental Research Funds for the
Central Universities (20720150141, and 20720150159).

Appendix A. Supplementary material

Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.bios.2016.01.031.

References
Actor, B., Cobbers, J.M., Buschges, R., Wolter, M., Knobbe, C.B., Lichter, P., Reifenberger, G., Weber, R.G., 2002. Gene Chromosom. Cancer 34, 416427.
Beaumont, T.L., Kupsky, W.J., Barger, G.R., Sloan, A.E., 2007. J. Neuro-Oncol. 83,
3946.
Biernat, W., Aguzzi, A., Sure, U., Grant, J.W., Kleihues, P., Hegi, M.E., 1995. J. Neuropathol. Exp. Neurol. 54, 651656.
Blasberg, R.G., 2003. Mol. Cancer Ther. 2, 335343.
Boerman, R.H., Anderl, K., Herath, J., Borell, T., Johnson, N., SchaefferKlein, J.,
Kirchhof, A., Raap, A.K., Scheithauer, B.W., Jenkins, R.B., 1996. J. Neuropathol.
Exp. Neurol. 55, 973981.
Chen, H.W., Medley, C.D., Sefah, K., Shangguan, D., Tang, Z., Meng, L., Smith, J.E., Tan,
W., 2008. ChemMedChem. 3, 9911001.
Cibiel, A., Pestourie, C., Duconge, F., 2012. Biochimie 94, 15951606.
Damodaran, O., van Heerden, J., Nowak, A.K., Bynevelt, M., McDonald, K., Marsh, J.,
Lee, G., 2014. J. Clin. Neurosci. 21, 478481.
Daniels, D.A., Chen, H., Hicke, B.J., Swiderek, K.M., Gold, L., 2003. Proc. Natl. Acad.
Sci. USA 100, 1541615421.
Das, M., Duan, W., Sahoo, S.K., 2015. Nanomed. Nanotechnol. 11, 379389.
de-los-Santos-Alvarez, N., Lobo-Castanon, M.J., Miranda-Ordieres, A.J., Tunon-

Q. Wu et al. / Biosensors and Bioelectronics 80 (2016) 18

Blanco, C.J., 2009. Biosens. Bioelectron. 24, 25472553.

Ellington, A.D., Szostak, J.W., 1990. Nature 346, 818822.
Han, S.J., Yang, I., Tihan, T., Prados, M.D., Parsa, A.T., 2010. J. Neuro-Oncol. 96,
313320.
Hussain, T., Nguyen, Q.T., 2014. Adv. Drug. Deliv. Rev. 66, 90100.
Iliuk, A.B., Hu, L., Tao, W.A., 2011. Anal. Chem. 83, 44404452.
Kircher, M.F., Hricak, H., Larson, S.M., 2012. Mol. Oncol. 6, 182195.
Lao, Y.H., Phua, K.K., Leong, K.W., 2015. ACS Nano 9, 22352254.
Li, X.L., Zhang, W.Y., Liu, L., Zhu, Z., Ouyang, G.L., An, Y., Zhao, C.Y., Yang, C.J., 2014.
Anal. Chem. 86, 65966603.
Li, X.L., An, Y., Jin, J., Zhu, Z., Hao, L.L., Liu, L., Shi, Y.Q., Fan, D.M., Ji, T.H., Yang, C.Y.J.,
2015. Anal. Chem. 87, 49414948.
Liang, C., Guo, B.S., Wu, H., Shao, N.S., Li, D.F., Liu, J., Dang, L., Wang, C., Li, H., Li, S.H.,
Lau, W.K., Cao, Y., Yang, Z.J., Lu, C., He, X.J., Au, D.W.T., Pan, X.H., Zhang, B.T., Lu,
C.W., Zhang, H.Q., Yue, K.M., Qian, A.R., Shang, P., Xu, J.K., Xiao, L.B., Bian, Z.X.,
Tan, W.H., Liang, Z.C., He, F.C., Zhang, L.Q., Lu, A.P., Zhang, G., 2015. Nat. Med. 21,
288.
Mallikaratchy, P., Tang, Z.W., Kwame, S., Meng, L., Shangguan, D.H., Tan, W.H., 2007.
Mol. Cell. Proteom. 6, 22302238.
Meis, J.M., Martz, K.L., Nelson, J.S., 1991. Cancer 67, 23422349.
Ng, E.W., Adamis, A.P., 2006. Ann. N. Y. Acad. Sci. 1082, 151171.
Pieve, C.D., Perkins, A.C., Missailidis, S., 2009. Nucl. Med. Biol. 36, 703710.
Raston, N.H.A., Gu, M.B., 2015. Biosens. Bioelectron. 70, 261267.
Reis, R.M., Konu-Lebleblicioglu, D., Lopes, J.M., Kleihues, P., Ohgaki, H., 2000. Am. J.
Pathol. 156, 425432.
Scheithauer, B.W., 2009. Brain Pathol. 19, 551564.
Sefah, K., Shangguan, D., Xiong, X., ODonoghue, M.B., Tan, W., 2010. Nat. Protoc. 5,
11691185.
Sefah, K., Tang, Z.W., Shangguan, D.H., Chen, H., Lopez-Colon, D., Li, Y., Parekh, P.,
Martin, J., Meng, L., Phillips, J.A., Kim, Y.M., Tan, W.H., 2009. Leukemia 23,
235244.
Shangguan, D., Cao, Z.H., Meng, L., Mallikaratchy, P., Sefah, K., Wang, H., Li, Y., Tan,
W.H., 2008a. J. Proteome Res. 7, 21332139.
Shangguan, D., Li, Y., Tang, Z., Cao, Z.C., Chen, H.W., Mallikaratchy, P., Sefah, K., Yang,
C.J., Tan, W., 2006. Proc. Natl. Acad. Sci. USA 103, 1183811843.
Shangguan, D.H., Meng, L., Cao, Z.H.C., Xiao, Z.Y., Fang, X.H., Li, Y., Cardona, D.,
Witek, R.P., Liu, C., Tan, W.H., 2008b. Anal. Chem. 80, 721728.
Shen, Q.L., Xu, L., Zhao, L.B., Wu, D.X., Fan, Y.S., Zhou, Y.L., OuYang, W.H., Xu, X.C.,
Zhang, Z., Song, M., Lee, T., Garcia, M.A., Xiong, B., Hou, S., Tseng, H.R., Fang, X.H.,
2013. Adv. Mater. 25, 23682373.

Singh, G., Mallick, S., Sharma, V., Joshi, N., Purkait, S., Jha, P., Sharma, M.C., Suri, V.,
Julka, P.K., Mahapatra, A.K., Singh, M., Kale, S.S., Sarkar, C., 2012. Neuropathology 32, 534542.
Sun, H., Zhu, X., Lu, P.Y., Rosato, R.R., Tan, W., Zu, Y., 2014. Mol. Ther. Nucleic Acids 3,
e182.
Tan, W., Donovan, M.J., Jiang, J., 2013. Chem. Rev. 113, 28422862.
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. Nucl.
Acids Res. 25, 48764882.
Tian, J.W., Ding, L., Ju, H.X., Yang, Y.C., Li, X.L., Shen, Z., Zhu, Z., Yu, J.S., Yang, C.J.,
2014. Angew. Chem. Int. Ed. 53, 95449549.
Tuerk, C., Gold, L., 1990. Science 249, 505510.
Wang, L., Ma, W., Chen, W., Liu, L., Ma, W., Zhu, Y., Xu, L., Kuang, H., Xu, C., 2011.
Biosens. Bioelectron. 26, 30593062.
Weissleder, R., 2006. Science 312, 11681171.
Wu, X., Zhao, Z., Bai, H., Fu, T., Yang, C., Hu, X., Liu, Q., Champanhac, C., Teng, I.T., Ye,
M., Tan, W., 2015. Theranostics 5, 985994.
Xu, J.H., Teng, I.T., Zhang, L.Q., Delgado, S., Champanhac, C., Cansiz, S., Wu, C.C., Shan,
H., Tan, W.H., 2015. Plos One 10, e0125863.
Yang, D.K., Chen, L.C., Lee, M.Y., Hsu, C.H., Chen, C.S., 2014. Biosens. Bioelectron. 62,
106112.
Yang, L.L., Zhang, Y., Li, R.B., Lin, C.Y., Guo, L.H., Qiu, B., Lin, Z.Y., Chen, G.N., 2015.
Biosens. Bioelectron. 70, 268274.
Yao, P.S., Kang, D.Z., Wang, X.F., Lin, R.Y., Ye, Z.C., 2015. In Vitro Cell. Dev. Biol.
Anim. 51, 345352.
Zadeh, J.N., Steenberg, C.D., Bois, J.S., Wolfe, B.R., Pierce, M.B., Khan, A.R., Dirks, R.M.,
Pierce, N.A., 2011. J. Comput. Chem. 32, 170173.
Zhang, H.M., Ma, Y.L., Xie, Y., An, Y., Huang, Y.S., Zhu, Z., Yang, C.Y.J., 2015. Sci. Rep.
UK 5, 10099.
Zhao, W.A., Cui, C.H., Bose, S., Guo, D.G., Shen, C., Wong, W.P., Halvorsen, K., Farokhzad, O.C., Teo, G.S.L., Phillips, J.A., Dorfman, D.M., Karnik, R., Karp, J.M., 2012.
Proc. Natl. Acad. Sci. USA 109, 1962619631.
Zhao, Z., Xu, L., Shi, X., Tan, W., Fang, X., Shangguan, D., 2009. Analyst 134,
18081814.
Zhu, G.Z., Zhang, S.F., Song, E.Q., Zheng, J., Hu, R., Fang, X.H., Tan, W.H., 2013a.
Angew. Chem. Int. Ed. 52, 54905496.
Zhu, G.Z., Zheng, J., Song, E.Q., Donovan, M., Zhang, K.J., Liu, C., Tan, W.H., 2013b.
Proc. Natl. Acad. Sci. USA 110, 79988003.
Zhu, L., Li, C., Zhu, Z., Liu, D., Zou, Y., Wang, C., Fu, H., Yang, C.J., 2012. Anal. Chem. 84,
83838390.