SCIENCE AND TECHNOLOGY OF ADVANCED MATERIALS, 2016
VOL .17, NO . 1, 618625
http://dx.doi.org/10.1080/14686996.2016.1227222

OPEN ACCESS

FOCUS ISSUE ARTICLE

An immuno-wall microdevice exhibits rapid and sensitive detection of
IDH1-R132H mutation specific to grade II and III gliomas
Akane Yamamichia,d, Toshihiro Kasamab, Fumiharu Ohkaa, Hiromichi Suzukia, Akira Katoa, Kazuya Motomuraa,
Masaki Hiranoa, Melissa Ranjita, Lushun Chalisea, Michihiro Kurimotoa, Goro Kondoa, Kosuke Aokia, Noritada
Kajib, Manabu Tokeshic, Toshio Matsubarad, Takeshi Sengae, Mika K. Kanekof, Hidenori Suzukid, Masahito Haraa,
Toshihiko Wakabayashia, Yoshinobu Babab, Yukinari Katof and Atsushi NatsumeaaDepartment of Neurosurgery, Nagoya University Graduate School of Medicine, Nagoya, Japan
Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Nagoya, Japanc
Division of Applied Chemistry, Hokkaido University, Sapporo, Japand
Department of Neurosurgery, Mie University Graduate School of Medicine, Tsu, Japane
Division of Cancer Biology, Nagoya University Graduate School of Medicine, Nagoya, Japanf
Department of Regional Innovation, Tohoku University Graduate School of Medicine, Sendai , Japanb

ABSTRACTWorld Health Organization grade II and III gliomas most frequently occur in the central
nervous system (CNS) in adults. Gliomas are not circumscribed; tumor edges are irregular andconsist of tumor cells, normal brain tissue, and hyperplastic reactive glial cells. Therefore, thetumors are not fully resectable, resulting in recurrence, malignant progression, and eventual
death. Approximately 69–80% of grade II and III gliomas harbor mutations in the isocitrate
dehydrogenase 1 gene (IDH1), of which 83–90% are found to be the IDH1-R132H mutation.
Detection of the IDH1-R132H mutation should help in the differential diagnosis of grade II and III
gliomas from other types of CNS tumors and help determine the boundary between the tumor
and normal brain tissue. In this study, we established a highly sensitive antibody-based device,referred to as the immuno-wall, to detect the IDH1-R132H mutation in gliomas. The immuno-wallcauses an immunoreaction in microchannels fabricated using a photo-polymerizing polymer.
This microdevice enables the analysis of the IDH1 status with a small sample within 15 minwith substantially high sensitivity. Our results suggested that 10% content of the IDH1-R132H
mutation in a sample of 0.33 μl volume, with 500 ng protein, or from 500 cells is theoreticallysufficient for the analysis. The immuno-wall device will enable the rapid and highly sensitive
detection of the IDH1-R132H mutation in routine clinical practice.

1. Introduction
Glioma accounts for approximately 30% of all of central
nervous system (CNS) tumors in adults.[1] Gliomas are
divided into World Health Organization (WHO) grades
I–IV[2]; grade IV glioma, namely glioblastoma (GBM),

ARTICLE HISTORY

Received 8 March 2016
Revised 11 August 2016
Accepted 18 August 2016
KEYWORDS

Glioma; isocitrate
dehydrogenase 1 mutation;
immuno-wall microdevice;rapid diagnosis; precision
medicine
CLASSIFICATION

30 Bio-inspired andbiomedical materials; 404
Materials informatics /Genomics

is the most malignant subtype with a median survival
time of approximately 15 months despite extensive surgical resection and chemo-radiotherapy.[3,4] Although
grade II and III gliomas (lower grade gliomas, LGGs) areless aggressive than GBM, because of their infiltrative

CONTACT Atsushi Natsume
anatsume@med.nagoya-u.ac.jp
Department of Bioengineering, School of Engineering, The University of Tokyo,
Tokyo, Japan.
Supplemental data for this article can be accessed here. [http://dx.doi.org/10.1080/14686996.2016.1227222]© 2016 The Author(s). Published by National Institute for Materials Science in partnership with Taylor & Francis.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.

Sci. Technol. Adv. Mater. 17 (2016) 619

nature, complete surgical removal cannot be achieved.
Pathologically, the surrounding marginal tissue is acomplex mixture of normal white and gray matters,
infiltrative tumor cells, and hyperplastic reactive glial
cells. Moreover, in most cases, LGGs appear similar to
the normal tissue without clear boundaries between
the two. Such non-circumscribed tumors are not fully
resectable, resulting in recurrence, malignant progression, and eventual fatality.[5]Recent comprehensive genetic studies in large
cohorts of LGGs revealed that the isocitrate dehydrogenase 1 (IDH1) mutation was found in 69–80% of these
gliomas.[6–8] The most significant of the IDH1 mutations in LGGs substitutes the amino acid residue 132
from arginine to histidine (R132H), accounting for
83–90% of IDH1 mutations.[9–11] While the wild-type
IDH1 catalyzes the oxidative decarboxylation of isocitrate and produces alpha-ketoglutarate (alpha-KG) inthe tricarboxylic acid cycle,[12] the mutant IDH1 converts alpha-KG further into 2-hydroxyglutarate, which,as an oncogenic metabolite, plays several crucial roles in
the initiation of glioma.[13] More importantly, the IDH1
mutation is rarely found in other CNS tumors,[14] andregardless of their locations (i.e. in the tumor core or in
the margin), and every tumor cell in an LGG harboring
the mutation of IDH1 expresses the mutated IDH1.[6]These facts suggest that detection of the IDH1 mutation would enable clinicians to distinguish LGGs fromother CNS tumors and to better delineate the ambiguous
tumor margin from the normal brain. The IDH1 mutation is a potential biomarker; however, the only means ofdetecting this mutation in routine clinical practice thus
far are direct sequencing [15] and immunohistochemistry with anti-IDHR132H antibody,[16] both of which
are time-consuming and labor-intensive.
We previously constructed immuno-wall devices toenable rapid molecular analysis (manuscript in preparation). These immuno-wall structures were fabricatedwith a photo-polymerizing polymer placed inside of
microchannels on a plastic chip. This device enables theanalysis of molecular characteristics in under 15 minusing only a small sample.
In this study, we developed a novel immuno-wall
device to detect the IDH1-R132H mutation in glioma.
We found high sensitivity for IDH1-R132H even in small
amounts of tumor tissue.

2. Methodology
2.1. Ethics statement
This study was approved by the institutional review
board at Nagoya University Hospital and complied withall provisions of the Declaration of Helsinki. Informed
consent was obtained before the operation from all thepatients.

A. YAMAMICHI et al.

2.2. Cell lines
U87 and immortalized normal human astrocytoma(NHA), expressing either mutated IDH1 (U87-IDH1R132H, NHA-IDH1-R132H, respectively) or wild-type
IDH1 (U87-wtIDH1, NHA-wtIDH1, respectively) werekindly donated by Dr Russell O. Pieper of the Universityof California, San Francisco, CA, USA. These cell lines
were maintained in Dulbecco’s modified Eagle’s medium(DMEM; Sigma-Aldrich, St Louis, MO, USA), containing 10% heat-inactivated fetal bovine serum (FBS;
Thermo Fisher Scientific Inc., Waltham, MA, USA), 100
units ml–1 of penicillin and 100 μg ml–1 of streptomycin(Thermo Fisher Scientific Inc.) at 37°C in a humidified
atmosphere of 5% CO2.
2.3. Intra-operative collection of tumor tissues
Fresh tumor samples, 5–10 mm in diameter, were collected intraoperatively from 10 patients whose tumors
were resected at Nagoya University Hospital in 2015. The
location of each sample was recorded stereotactically in
an intraoperative navigation system (Brainlab, Munich,Germany). Each tumor tissue was dissected into threepieces for the immuno-wall assay, immunohistochemistry, and DNA sequencing.
2.4. Preparation of protein lysate
Cell pellets were mechanically broken down in RIPA
buffer (Wako, Osaka, Japan), which contained proteaseinhibitor (Wako), and centrifuged at 15,000 rpm for
5 min at 4 °C. Supernatants were collected and analyzed
with the immuno-wall assay. In order to lyse the tumor
tissues, the tissues were placed in 1.5 ml tubes containing
200 μl RIPA buffer, a protease inhibitor, and resin beads,
which were then collectively ground using pestles froma sample-grinding kit (GE Healthcare, Little Chalfont,
UK). The lysate was then centrifuged at 15,000 rpm for
5 min at 4 °C and the supernatants were collected andanalyzed. Approximately 100 μg of protein was extracted
from 105 cells.
2.5. Western blot analysis
Cell lysates were boiled at 100 °C for 5 min in SDS
sample buffer (New England Biolabs, Ipswich, MA,
USA) containing 42 mM dithiothreitol (Cell Signaling
Technologies, Danvers, MA, USA). Next, the samples(50 μg) were applied to each well and resolved on a
10% polyacrylamide gel (Bio-rad, Hercules, CA, USA).
Proteins were transferred to a polyvinylidene difluoride membrane (GE Healthcare), blocked with 5%
skim milk in phosphate-buffered saline (PBS) containing 0.1% Tween-20 for 1 h at room temperature, andincubated with HMab-2, RcMab-1, and anti-β-actin(Sigma-Aldrich) for 60 min at room temperature. The

Sci. Technol. Adv. Mater. 17 (2016) 620

A. YAMAMICHI et al.

Figure 1. Schematic representation of the immuno-wall device. (A) Immuno-wall chips with 40 microchannels (each 1 mm
in width, 40 μm in height and 8.5 mm in length) in a cyclic olefin polymer substrate were constructed using photolithography.
(B) The channels were filled with 6% azide-unit pendant water-soluble photopolymer (AWP) and 10 mg ml–1 streptavidin. UV light(313 nm, 20 mW cm–2) through slits in a photomask was used to immobilize the photoreactive polymer in the center of the channels,
before the uncured polymer was washed with PBS. (C, D) A biotinylated anti-R132H-IDH1 antibody (HMab-2), an anti-wild-type IDH1
antibody (RcMab-1), and a fluorescent DyLight650-conjugated goat anti-rat IgG antibody were used to label IDH1-R132H. Note that
RcMab-1 is interacted with multiple numbers of DyLight 650-conjugated anti-rat IgGs.

membrane was then washed, incubated with horseradish
peroxidase-labeled secondary antibodies for 30 min atroom temperature, and visualized using an enhanced
chemiluminescence method.
2.6. Immuno-wall assay
The structure of the immuno-wall device is shown in
Figure 1. Immuno-wall chips with 40 microchannels(1 mm width, 40 μm height and 8.5 mm length each)in a cyclic-olefin-polymer substrate were constructedusing photolithography. The channel was filled with 6%
azide-unit pendant water-soluble photopolymer (AWP;
Toyo Gosei, Tokyo, Japan) and 10 mg ml–1 streptavidin(Prospec, East Brunswick, NJ, USA). Through slits in aphotomask, UV light (313 nm, 20 mW cm–2) immobilized the photoreactive polymer in the center of channels, before uncured polymer was removed by washingwith PBS.
A biotinylated anti-R132H mutated IDH1 antibody (HMab-2),[17] an anti-wild-type IDH1 antibody (RcMab-1),[18] and a fluorescent DyLight650-conjugated goat anti-rat IgG antibody (Abcam,
Cambridge, UK) were used to label R132H mutated
IDH1. PBS, containing 1% bovine serum albumin(BSA; Roche, Basel, Switzerland), was used to dilute
the antibodies. PBS, containing 0.5% Tween-20 and
0.5% BSA, was used as a washing buffer.

Biotinylated HMab-2 (1 μl, 50 μg ml–1) was injected into inlets,
and incubated for 1 h. Microchannels were washed five times
with washing buffer. The sample lysate (1.5 μl) was then injected
into the inlet and incubated for 5 min followed by incubationof RcMab-1 (1 μl, 50 μg ml–1) for 30 s. DyLight650-conjugatedgoat anti-rat IgG was applied. Finally, the fluorescence wasmeasured and quantified using a fluorescence microscope witha CCD camera and ImageJ software (National Institute of Health,Bethesda, MD, USA).
2.7. Direct sequencing and pyrosequencing for IDH1
mutation
DNA was prepared using the QIAmp DNA Mini kit (Qiagen,Hilden, Germany) according to the manufacturer’s instructions.
The amount of DNA obtained from the tumor was sufficientfor the subsequent genomic analyses. For IDH1 sequencing, a
129-bp fragment, spanning the sequence encoding the catalytic domain of IDH1, including codon 132, was amplified. We
applied conventional polymerase chain reaction (PCR) for 35
cycles with denaturation at 95 °C for 30 s, annealing at 56 °C for
40 s, and extension at 72 °C for 50 s, followed by
72 °C for 7 min to complete the extension. Primer sequences wereas follows: forward primer: CGGTCTTCAGAGAAGCCATT:
reverse primer: GCAAAATCACATTATTGCCAAC. Direct
sequencing was performed using the BigDye Terminator v1.1
Cycle Sequencing Kit (Applied Biosystems, Foster City, CA,
USA). The reactions were carried out using an ABI 3100 Genetic

Sci. Technol. Adv. Mater. 17 (2016) 621

Analyzer (Applied Biosystems). For pyrosequencing,
we applied PCR at 50 cycles with denaturation at 95 °C
for 30 s, annealing at 58 °C for 30 s, and extension at
72 °C for 45 s, followed by 72 °C for 2 min to complete the extension. Primer sequences were as follows:forward primer: GGCTTGTGAGTGGATGGGTA:
reverse primer:
G G G AC AC C G C T G AT C G T T TAT G T G T T G A
GATGGACGCCTA and universal primer (5′-biotin)
GGGACACCGCTGATCGTTTA. Detection and calculation of the frequency of the mutant allele was performed
using pyrosequencing technology (Pyrosequencing
AB, Uppsala, Sweden) with the sequencing primer:
TGGATGGGTAAAACCTATCATCA, according to the
manufacturer’s instructions.[19]2.8. ImmunohistochemistryHMab-2 antibody was used for immunohistochemistry.
The tumor samples were fixed with 10% formalin andembedded with paraffin. Sections (5-μm thick) were prepared using a microtome (RM2125RT, Leica, Wetzlar,
Germany). After deparaffinization and hydration, thesections were incubated in retrieval solution, Tris-EDTA
buffer pH 9.0, for 30 min at 100 °C in an electric pot
and then blocked with 1.5% normal goat serum (Vector
Laboratories, Burlingame, CA, USA) in PBS containing
0.05% Tween-20, at room temperature for 1 h, and wereincubated with HMab-2, diluted to 1 μg ml–1, overnightat 4 °C. The second labeled polymer from the EnVision
HRP kit (Dako; Agilent Technologies, Inc., Santa Clara,
CA, USA) was applied and the sections were incubatedfor 30 min. The substrate-chromogen solution from theDAB Substrate Kit (Vector Laboratories) was applied
for 10 min. After washing, the sections were counterstained with hematoxylin and mounted in multi-Mount(Matsunami Glass Ind., Kishiwada, Japan).
2.9. Statistical analysis
The statistical significance of the differences between twocell line groups was determined using Student’s t-test forthe mean fluorescence intensity. P-values < 0.05 wereconsidered significant.

3. Results
3.1. Sensitivity and specificity of immuno-wallassaysOn the basis of our previous study which detected amutation in lung cancer (manuscript in preparation),
we found that 1.5 μl of cell lysate at a concentration of
1.0 mg ml–1 was sufficient for conducting an immuno-wallassay. The cell lysate extracted from U87-IDH1-R132H,
at a protein concentration of 0.5–2.5 mg ml–1, yielded apositive fluorescence intensity along the immuno-wall

A. YAMAMICHI et al.

(Figure S1). Thus, our device detected IDH1-R132H inas little as 0.5 mg ml–1 of protein lysates.
Western blotting confirmed that the anti-IDH1R132H antibody, HMab-2, was specific to the lysate
from NHA-IDH1-R132H and U87-IDH1-R132H cells,
whereas the anti-IDH1 antibody, RcMab-1, recognizedboth wild-type and mutant IDH1 in NHA and U87 cells(Figure 2(A)). In the immuno-wall assay, at the highest
protein concentration (3.0 mg ml–1), U87 and NHA cells
expressing wild-type IDH1 did not show fluorescence
while both types of cells expressing IDH1-R132H displayed strong fluorescence. The mean fluorescence intensity was significantly different between cells expressing
wild-type IDH1 and IDH1-R132H (Figure 2(B)).
Next, in order to determine the detection limit of
the ratio of mutated IDH1/wild-type IDH1, we mixed
3.0 mg ml–1 cell lysate from mutant cells with the same
concentration of wild-type cell lysate in various ratios.
When mutated IDH1 comprised more than 10% of thecell lysate, strong fluorescence was observed. However,when the mutated cells accounted for less than 5% of thesolution, fluorescence was difficult to distinguish frombackground intensity (Figure 3). The results indicatethat the device can detect the IDH1-R132H mutation if
the sample contains more than 10% of mutated IDH1.
However, because the data shown here were determinedusing artificial cell lines overexpressing mutated or wildtype IDH1, further studies are needed to explore thedetection threshold in clinical samples.
3.2. Patient characteristics and protein extraction
from tumor samples
Next, we analyzed brain tumor samples to investigate theutility of our method in the clinical setting. We analyzed
tumors from 10 patients (mean age, 47 ± 20, three males
and seven females); three GBMs, four LGGs, one gliomatosis cerebri, and two non-gliomas (Table 1). The DNA
sequencing showed that all four LGGs harbored the IDH1R132H mutation. We lysed 5–14 mg tumor samples (meanweight: 9.6 mg) in 200 μl RIPA buffer. The lysates contained
1.8–3.5 (mean, 2.9 ± 0.5) mg ml–1 of protein.
3.3. Immuno-wall assay of tumor samples
All four LGGs containing the IDH1-R132H mutation
as determined by DNA sequencing displayed positive
intensity in the immuno-wall assay. Next, we evaluatedthe mutant allele frequencies (MAFs) by pyrosequencing
the LGG samples exhibiting the IDH1-R132H mutation(Table 1). A tumor sample with an MAF of 40% wasselected, and its lysate was mixed with another lysate
from a tumor with wild-type IDH1 such that the MAF
was decreased stepwise from 40% to 20, 10, 5, 2, and
1%. Our immuno-wall assay for IDH1-R132H detected
as low as 10% of MAF (Figure 4). The detection limit inclinical samples was consistent with that in cell lines.

Sci. Technol. Adv. Mater. 17 (2016) 622

A. YAMAMICHI et al.

Figure 2. Sensitivity and specificity of the immuno-wall assays. (A) Western blotting confirmed that the anti-IDH1-R132H antibody,HMab-2, was specific to the lysate from NHA-IDH1-R132H and U87-IDH1-R132H cells, whereas the anti-IDH1 antibody, RcMab-1,
recognized both wild-type IDH1 and IDH1-R132H in NHA and U87 cells. (B) U87 and NHA cells expressing wild-type IDH1 did notshow fluorescence, while both types of cells expressing IDH1-R132H displayed strong fluorescence.

3.4. Detection of tumor border with immuno-wallassay
As the immuno-wall was able to detect mutated cellswith an MAF as low as 10%, we sought to utilize thedevice to estimate the gross tumor margin. A representative case of diffuse astrocytoma (Patient #8 in Table 1)
is shown in Figure 5. During tumor removal surgery forthis patient, two specimens were collected. A specimen
from the center of the tumor showed positive intensity inthe immuno-wall (Figure 5(A)), while, a specimen fromthe marginal region that appeared normal was negative(Figure 5(B)). The IDH1 status of these samples agreed
with the immunohistochemistry and DNA sequencing
results. These data demonstrate that our immuno-wallassay can guide surgeons to the boundary between thetumor and surrounding normal brain. The immuno-wallassay requires only 15 min, indicating that the assay canbe used as an intraoperative modality to image the border between the tumor and normal brain.

4. Discussion
Figure 3. Eﬀect of mutated IDH1/wild-type IDH1 ratio on thedetection of mutated IDH1. When mutated IDH1 comprised
more than 10% of the cell lysate, strong fluorescence wasobserved. However, when the proportion of mutated cells was
below 5%, fluorescence was diﬃcult to distinguish from thebackground.

In this study, we established a highly sensitive
antibody-based device to detect the IDH1-R132H
mutation in glioma. Several techniques for detecting
the IDH1 mutation were recently developed, such as
modified PCR protocols.[20–23] Even the most rapid

Sci. Technol. Adv. Mater. 17 (2016) 623

A. YAMAMICHI et al.

Table 1. Patient characteristics and summarized results.
Case
no.
1

Age
30

Sex
F

2

53

M

3

50

F

4
5

78
19

F
F

6

58

F

7
8

59
25

M
F

9
10

47
48

M
M

Pathological diagnosis
Anaplastic astrocytoma (grade
III glioma)
Gliomatosis cerebri (grade III
glioma)Diﬀuse astrocytoma (grade II
glioma)Glioblastoma (grade IV glioma)
Dysembryoplastic neuroepithelial tumor(DNT, non-glioma)
Pineal parenchyma tumor
of intermediate diﬀerentiation(PPTID, non-glioma)
Glioblastoma (grade IV glioma)
Anaplastic astrocytoma (grade
III glioma)
Glioblastoma (grade IV glioma)
Diﬀuse astrocytoma (grade II
glioma)Sampleweight(mg)
11

Protein lysate
concentration(mg ml–1)
2.71

Amount of
total protein
(μg)
542.4

DNA
sequencing
Mut

Immuno-wall
Mut

Mutant allele
frequencies
(%)
31.5

7

2.89

578.4

WT

WT

0

10

3.47

694

Mut

Mut

45.8

11
9

2.69
3.21

538
642.2

WT
WT

WT
WT

0
0

5

1.86

371.6

WT

WT

0

10
12

2.56
2.89

511.4
578.6

WT
Mut

WT
Mut

0
34.7

14
7

3.56
2.35

712.8
470.2

WT
Mut

WT
Mut

0
27.1

Figure 4. Eﬀect of the mutant allele frequency in clinicalsamples on the detection of mutated IDH1. The lysate from atumor sample with a mutant allele frequency of 40% was mixed
with the lysate from a tumor sample with wild-type IDH1 suchthat the mutant allele frequency decreased stepwise from 40%
to 20, 10, 5, 2, and 1%. The immuno-wall assay IDH1-R132H
detected the mutant allele frequency in samples containing as
low as 10% mutant IDH1.

method requires more than 60 min to obtain results. Our
immuno-wall device detected the IDH1 mutation within
15 min with substantially high sensitivity. Our immuno-wall generates an immunoreaction in microchannels
fabricated using a photo-polymerizing polymer. This
device enables the analysis of the IDH1 status using asmall sample, which shows potential for intra-operative
rapid diagnosis.

Our immuno-wall device was constructed using ahigh concentration of streptavidin (10 mg ml–1) mixed
with photoreactive polymer. Then primary antibody was
immobilized to both sides of the walls via biotin–avidin
binding. We estimated that the final density per area ofprimary antibody at the surface of the immuno-wall wasapproximately 0.155 μg mm–2 because the primary antibody was concentrated in the reaction with a high concentration of streptavidin during the 1-h incubation. We
estimated the density of primary antibody at the bottomof the plate by using sandwich enzyme linked immunosorbent assay (ELISA; this method has high specificity)by using two antibodies and found a value of approximately 0.0014–0.0057 μg mm–2. Furthermore, fluorescence of the immuno-wall was integrated because thefluorescence signal extended laterally and all extended
signals were observed from above. In contrast, theWestern blotting signals were not integrated spatiallybecause the membrane transfer signal was observed
through a monolayer. A high density of primary antibody and integrated fluorescence increase the sensitivityof the immuno-assay compared to other immunoassays
such as ELISA and Western blotting. Additionally, molecule movement is limited in the microchannels, enabling rapid detection because the primary antibody cancapture molecules very quickly.
The rapid detection of IDH1 mutation may also assist
in determining the extent of tumor removal. During asurgical resection of glioma, it is difficult to visuallydifferentiate the tumor from the surrounding normal
tissue.[24] Intra-operative IDH1 profiling may be usefulfor detecting the border between the tumor and normal brain. In this study, we evaluated a representative
LGG case in which IDH1-R132H was detected at thecenter of the tumor concurrent with wild-type IDH1
at the margin.
The emergence of molecular-targeting anti-cancer
agents requires an individual molecular profiling of

Sci. Technol. Adv. Mater. 17 (2016) 624

A. YAMAMICHI et al.

Figure 5. Tumor boundary detected using the immuno-wall assay. During tumor removal surgery in a patient, two specimens werecollected. (A1, A2) A specimen from the center of the tumor tested positive in the immuno-wall assay. (B1, B2) In contrast, a specimen
from the margins of the tumor, which appeared normal, tested negative. (A3, A4, B3, B4) The IDH1 status of these samples was
confirmed using immunohistochemistry and DNA sequencing.

tumors performed in a non-invasive manner. ‘Precision
medicine’ for cancer patients trends toward sensitive
analysis of circulating tumor cells or cell-free DNA in
liquids such as blood and cerebrospinal fluid.[25,26]
In this regard, the rapid molecular diagnostic method
developed in this study may be an important milestone
in precision medicine. In this study, we demonstrated
that our immuno-wall assay could detect mutated IDH1
in a tissue lysate containing 10% IDH1 mutant allele at
a protein concentration of 1.0–1.5 mg ml–1 (Figure S2).
Because the minimal sample volume for a microchannel
is 0.33 μl, 500 ng protein or 500 cells is sufficient for
determining IDH1 status. Total protein concentration in
cerebrospinal fluid is more than 1.0 mg ml–1 in patientswith glioma.[27] Recent studies reported that the number of circulating tumor cells in the blood of glioma
patients is approximately 10 cells ml–1.[28,29]. Thesesuggest that there is a possibility that the IDH1 status
can be determined with our device using cerebrospinal
fluid and blood.
Some challenges remain before this diagnostic tool
can be applied clinically, but our immuno-wall assay may
lead to rapid and highly sensitive detection of the IDH1R132H mutation preoperatively and intra-operatively.
Clinical application, using a newly developed portable
CCD camera, is currently under development; this will

facilitate rapid on-site diagnosis. Our immuno-wall
device will enable non-invasive rapid liquid biopsy inprecision medicine.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by a JSPS KAKENHI [grant number 25462242]; Grant-in Aid for Scientific Research on
Innovative Areas from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT) of Japan [grant
number 23107010]; Practical Research for Innovation
Cancer Control from Japan Agency for Medical Research anddevelopment, AMED; by ‘Knowledge Hub Aichi’, Priority
Research Project from Aichi Prefectural Government; by the
Platform for Drug Discovery, Informatics, and Structural Life
Science (PDIS) from AMED; and by the Regional Innovation
Strategy Support Program from MEXT of Japan.

References[1] Ostrom QT, Gittleman H, Liao P, et al. CBTRUS
statistical report: primary brain and central nervous
system tumors diagnosed in the United States in 20072011. Neuro-oncology. 2014;16 Suppl 4, iv1-iv63.

Sci. Technol. Adv. Mater. 17 (2016) 625[2] Louis DN, Ohgaki H, Wiestler OD, et al. The 2007
WHO classification of tumours of the central nervous
system. Acta Neuropathol. 2007;114:97–109.
[3] Stupp R, Mason WP, van den Bent MJ, et al. Radiotherapy
plus concomitant and adjuvant temozolomide forglioblastoma. N Engl J Med. 2005;352:987–996.
[4] Stupp R, Hegi ME, Mason WP, et al. Effects of radiotherapy with concomitant and adjuvant temozolomide
versus radiotherapy alone on survival in glioblastoma
in a randomised phase III study: 5-year analysis of the
EORTC-NCIC trial. Lancet Oncol. 2009;10:459–466.
[5] Claus EB, Walsh KM, Wiencke JK, et al. Survivaland low-grade glioma: the emergence of genetic
information. Neurosurg Focus. 2015;38:E6.
[6] Suzuki H, Aoki K, Chiba K, et al. Mutational landscapeand clonal architecture in grade II and III gliomas.
Nature Genet. 2015;47:458–468.
[7] Gorovets D, Kannan K, Shen R, et al. IDH mutationand neuroglial developmental features define clinicallydistinct subclasses of lower grade diffuse astrocytic
glioma. Clin Cancer Res: J Am Assoc Cancer Res.
2012;18:2490–2501.
[8] Hartmann C, Meyer J, Balss J, et al. Type and frequency
of IDH1 and IDH2 mutations are related to astrocytic andoligodendroglial differentiation and age: a study of 1,010
diffuse gliomas. Acta Neuropathol. 2009;118:469–474.
[9] Parsons DW, Jones S, Zhang X, et al. An integrated
genomic analysis of human glioblastoma multiforme.
Sci (New York, N.Y.). 2008;321:1807–1812.
[10] Arita H, Narita Y, Yoshida A, et al. IDH1/2 mutation
detection in gliomas. Brain Tumor Pathol. 2015;32:79–89.
[11] Horbinski C. What do we know about IDH1/2 mutations
so far, and how do we use it? Acta Neuropathol.
2013;125:621–636.
[12] Reitman ZJ, Yan H. Isocitrate dehydrogenase 1 and 2
mutations in cancer: alterations at a crossroads of cellular
metabolism. J Natl Cancer Inst. 2010;102:932–941.
[13] Dang L, White DW, Gross S, et al. Cancer-associated
IDH1 mutations produce 2-hydroxyglutarate. Nature.
2009;462:739–744.
[14] Yan H, Parsons DW, Jin G, et al. IDH1 and IDH2
mutations in gliomas. N Engl J Med. 2009;360:765–773.
[15] Balss J, Meyer J, Mueller W, et al. Analysis of the IDH1
codon 132 mutation in brain tumors. Acta Neuropathol.
2008;116:597–602.
[16] Kato Y, Jin G, Kuan CT, et al. A monoclonal antibody
IMab-1 specifically recognizes IDH1R132H, the mostcommon glioma-derived mutation. Biochem Biophys
Res Commun. 2009;390:547–551.

A. YAMAMICHI et al.

[17] Fujii Y, Ogasawara S, Oki H, et al. A high-sensitive
HMab-2 specifically detects IDH1-R132H, the mostcommon IDH mutation in gliomas. Biochem Biophys
Res Commun. 2015;466:733–739.
[18] Kato Y. Specific monoclonal antibodies against IDH1/2
mutations as diagnostic tools for gliomas. Brain Tumor
Pathol. 2015;32:3–11.
[19] Shinjo K, Okamoto Y, An B, et al. Integrated analysis
of genetic and epigenetic alterations reveals CpG island
methylator phenotype associated with distinct clinicalcharacters of lung adenocarcinoma. Carcinogenesis.
2012;33:1277–1285.
[20] Kurimoto M, Suzuki H, Aoki K, et al. Rapid sensitive
analysis of IDH1 mutation in lower-grade gliomas byautomated genetic typing involving a quenching probe.
Cancer Invest. 2016;34:12–15.
[21] Pang B, Durso MB, Hamilton RL, et al. A novel COLDPCR/FMCA assay enhances the detection of lowabundance IDH1 mutations in gliomas. Diagn Mol
Pathol: Am J Surg Pathol Part B. 2013;22:28–34.
[22] Horbinski C, Kelly L, Nikiforov YE, et al. Detection of
IDH1 and IDH2 mutations by fluorescence melting
curve analysis as a diagnostic tool for brain biopsies.
J Mol Diagn JMD. 2010;12:487–492.
[23] Felsberg J, Wolter M, Seul H, et al. Rapid and sensitive
assessment of the IDH1 and IDH2 mutation status in
cerebral gliomas based on DNA pyrosequencing. Acta
Neuropathol. 2010;119:501–507.
[24] Silbergeld DL, Chicoine MR. Isolation and characterization of human malignant glioma cells from histologicallynormal brain. J Neurosurg. 1997;86:525–531.
[25] Nagrath S, Sequist LV, Maheswaran S, et al. Isolationof rare circulating tumour cells in cancer patients by
microchip technology. Nature. 2007;450:1235–1239.
[26] Nayak, L, Fleisher, M, Gonzalez-Espinoza, R, et al.
Rare cell capture technology for the diagnosis of
leptomeningeal metastasis in solid tumors. Neurology.
2013;80:1598–1605. discussion 1603.
[27] Khwaja FW, Reed MS, Olson JJ, et al. Proteomic
identification of biomarkers in the cerebrospinal
fluid (CSF) of astrocytoma patients. J Proteome Res.
2007;6:559–570.
[28] MacArthur KM, Kao GD, Chandrasekaran S, et al.
Detection of brain tumor cells in the peripheral bloodby a telomerase promoter-based assay. Cancer Res.
2014;74:2152–2159.
[29] Sullivan JP, Nahed BV, Madden MW, et al. Brain tumor
cells in circulation are enriched for mesenchymal gene
expression. Cancer Discov. 2014;4:1299–1309.