Article

Cite This: ACS Sens. 2019, 4, 2952−2957 pubs.acs.org/acssensors

Microfluidic Immunoassay System for Rapid Detection and Semi-

Quantitative Determination of a Potential Serum Biomarker
Mesothelin
Xiaoxiao Duan,†,§ Linlin Zhao,‡,§ Heng Dong,†,§ Wang Zhao,† Sixiu Liu,*,† and Guodong Sui*,†
†
Shanghai Key Laboratory of Atmospheric Particle Pollution Prevention (LAP3), Department of Environmental Science &
Engineering, Fudan University, 220 Handan Road, Shanghai 200433, P. R. China
‡
Department of Gastroenterology, Changhai Hospital, Second Military Medical University, 168 Changhai Road, Shanghai 200433,
China
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Downloaded via INST FED EDU CIENCIA E TECH CEARA on July 12, 2020 at 21:25:59 (UTC).

*
S Supporting Information

ABSTRACT: Mesothelin (MSLN) is considered as a

potential serological tumor biomarker for early diagnosis of
pancreatic cancer. Nevertheless, low sensibility, high reagent
consumption, and time costs of traditional detection methods
limit their utility in clinical disease diagnoses. Here, we
combined the immunoassay technique with microfluidic chips
to develop a microfluidic immunoassay system (MIAS) that
can be used for rapid semi-quantitative detection of serum
MSLN levels. The MIAS was composed of 12 uniform
structures, including 12 inlets, 12 reaction chambers, and one
outlet allowing measurement of four samples with three
repeats, simultaneously. A unique microarray cylinder was
located at the end of each reaction chamber where immunoassay was performed to trap microspheres. A feasible interception
efficiency (∼80%) was attained, with microspheres filling the reaction column. It has been demonstrated that fluorescence
intensity is proportional to the MSLN concentration on the MIAS (R2 = 0.95). Subsequently, 16 clinical serum samples
collected from Changhai Hospital, Shanghai were selected from eight patients with pancreatic cancers, four with pancreatitis,
and four with other digestive system diseases (2 gastric cancer, 1 bile duct stricture, and 1 bile duct stones). MSLN levels for
these samples were detected via MIAS. The results showed a significant correlation between MIAS and traditional enzyme-
linked immunosorbent assay (ELISA), with the correlation coefficient, 0.93. The detection limit of MSLN fluorescence intensity
and concentration was ∼6 a.u. and ∼20 pg/mL, respectively. The entire duration of analysis by MIAS decreased to ∼40 min
compared to 2 h by ELISA. Statistical analysis of MIAS data revealed that MSLN was overexpressed in pancreatic cancer than in
the others (P value = 0.0014). Moreover, the diagnostic accuracies of MSLN detected by MIAS and CA19-9 detected by ELISA
in hospitals were 87.5 and 81.3%, respectively. MSLN is helpful for the early diagnosis of pancreatic cancer and other diseases,
and it had a significant ability to discriminate between pancreatic and nonpancreatic cancers (P value = 0.0159). The results
from this study show that MIAS has the potential to become a new serological tumor marker detection platform for rapid
detection and semi-quantitative determination of MSLN and would have broad applications in early clinical diagnosis.
KEYWORDS: MIAS, pancreatic cancer, MSLN, rapid detection, clinical serum samples

P ancreatic cancer is the deadliest carcinoma worldwide

with a 99% mortality rate and 5 year survival rate of 7%.1,2
The lethality associated with this cancer is attributed to the
unavailability of reliable tests for early diagnosis and ineffective
therapies for the metastatic form.3 Early diagnosis of pancreatic
cancer is typically poor due to the lack of symptoms during
early stages of the disease.4,5 Although the prognosis of
pancreatic cancer remains dismal, the 5 year survival rate
would rise to about 20%, provided that cancers could be
diagnosed early. Pancreatic cancer is often diagnosed by a
combination of medical imaging techniques and the detection
of serological tumor markers. CA19-9 is a tumor marker
frequently elevated in pancreatic cancer. Likewise, it also can
be used for other cancers, such as gastric, colorectal, and
gallbladder cancers and cholangiocarcinoma. Nevertheless, the
positive predictive value of CA19-9 as a screening marker in
pancreatic cancer is low (0.5−0.9%)6 and is based on the
detection of the Lewis (a) antigen, which depicts the ability to
produce CA19-9. This marker is 80% sensitive and 73%
specific in detecting pancreatic cancer and is often used for
certain known cases rather than diagnosis.7 Clearly, there is a
Received: July 29, 2019
Accepted: October 11, 2019
Published: October 11, 2019

© 2019 American Chemical Society 2952 DOI: 10.1021/acssensors.9b01430

ACS Sens. 2019, 4, 2952−2957
ACS Sensors
Figure 1. (A) MIAS with integrated immunoassay and microfluidic components. The MIAS image: (a) fluid layer and (b) pneumatic control layer.
(B) Image and illustration of the detection units on MIAS. (C) Image of microarray cylinders for the interception of microspheres in the reaction
column.
need to seek for a new tumor marker for the early diagnosis of
pancreatic cancer.
Mesothelin (MSLN) is a 40 kD glycosyl phosphatidylino-
sitol-linked cell surface glycoprotein expressed by a variety of
solid tumors, including pancreatic cancer, while only a limited
number of normal tissues, such as mesothelial cells lining the
pleura, peritoneum, and pericardium, stain positive for this
molecule.8,9 Current research indicates that MSLN has been
used as a new biomarker for the early diagnosis of pancreatic
cancer.10 In addition, it is overexpressed in pancreatic cancer
than pancreatitis11,12 and can help differentiate pancreatic
cancer from chronic pancreatitis. Due to its differential
overexpression, MSLN has shown significantly higher specific-
ity and sensitivity than CA19-9 in discriminating between
pancreatic and nonpancreatic cancers. Hence, it is emerging as
a new marker for differentiating pancreatic ductal adenocarci-
noma from chronic pancreatitis.13
Microfluidics is an emerging technique widely used in the
field of clinical diagnosis and for rapid serologic detection of
tumor markers. For instance, the microfluidic technique has
already been used to detect the levels of alpha-fetoprotein,14
serum lipoproteins,15 and human chorionic gonadotropin.16 Its
advantages include controllable liquid flow, low reagent
consumption, high throughput, and fast analysis.17,18 The
clinical serologic diagnosis often adopted is enzyme-linked
immunosorbent assay (ELISA), which is highly sensitive but
requires time-consuming antibody immobilization and long
incubation periods for the antigen−antibody reaction. With the
development of associated instruments, it becomes more and
more advanced for immunoassay. The associated semi-
automated microfluidic immunoassay instrument19 and the
mobile ELISA system20 provide new methods for automated
fluidic delivery and detection, allowing execution of all steps of
the immunoassay. Nevertheless, the capture antibody immobi-
lization and using antibodies with higher affinity are still a
concern, and the reaction efficiency needs to be enhanced by
using novel techniques. To determine the high sensitivity of
microfluidic analysis, a microfluidic immunoassay technique
based on microspheres was presented to detect response to
antigen and antibody. The technique integrated the superi-
orities over microfluidic chip and immunoassay and offered a
new approach to rapid detection of tumor markers.21
2953
Article
Herein, a microfluidic immunoassay system (MIAS) was
first presented for rapid semi-quantitative detection of serum
MSLN levels. Meanwhile, the semi-quantitative standard curve
of fluorescence intensity−MSLN concentration was estab-
lished to evaluate MIAS. Furthermore, the MSLN levels of 16
clinical serum samples collected from Changhai Hospital,
Shanghai were detected by MIAS, and the diagnosis accuracies
of serum MSLN and CA19-9 were calculated to further
estimate the clinical application potential of MIAS.
■
EXPERIMENTAL SECTION
Reagents and Materials. Photoresists SU-8 2025 and AZ-50XT
were purchased from Microchem and AZ Electronic Materials, USA,
respectively. Silicon wafers were obtained from Avago Technologies
(USA) and polydimethylsiloxane (PDMS, RTV-615-044) from
Momentive Performance Materials (USA). Carboxyl-modified micro-
spheres (PS-COOH) were purchased from Bang’s Laboratory (USA).
Anti-MSLN polyclonal (ab96869) and monoclonal antibodies
(ab168740) were purchased from Abcam (UK). Fluorescein
isothiocyanate (FITC)-labeled anti-mouse secondary antibodies
were purchased from Bio-Rad (USA). All other reagents were
purchased from Sigma (USA).
Design and Fabrication of the MIAS. The MIAS is shown in
Figure 1A, which is composed of 12 uniform structures. It was
integrated into a chip with multiple immunoassay units and one
outlet. The multiple immunoassay units can detect three repeated
analyses of four sample components. It could be integrated with more
individual immune-reaction columns for high throughput. One outlet
was shared by these columns through a microchannel while each
column had its own inlets and microvalves. These columns could be
used independently by controlling regular valves for different
component analyses, simultaneously or separately. The detailed
sizes of a detection unit (width 500 μm, depth 50 μm) are
demonstrated in Figure 1B. Microarray cylinders were specifically
designed in a 3 × 2 × 3 × 2 × 3 combination arrangement to
intercept microspheres which were used in the reaction column
(Figure 1C). The system was assembled from the fluid layer and the
pneumatic control layer, which served as a valve to control the flow of
the liquid. The fluid layer was used to add reagents and serum
samples.
The microfluidic immunoassay chip was fabricated by the standard
soft etch technology.22−24 The two (upper and lower) layers were also
made of PDMS and aligned under the microscope, with A/B ratios,
5:1 and 10:1, respectively. The mold of the two layers was made from
a positive photoresist (AZ-50XT) for the fluid layer and a negative
DOI: 10.1021/acssensors.9b01430
ACS Sens. 2019, 4, 2952−2957
ACS Sensors
Figure 2. (A) Schematic illustration of immunoassay on microspheres. (B) Procedures of MSLN on MIAS.
photoresist (SU-8 2025) for the pneumatic control layer. The reaction
column was heated to 115 °C for 30 min. The channel surface of the
chip was respectively rinsed with HCl (1.0% v/v), H2O2 (1.0% v/v),
and ultrapure water and then dried for use with clean nitrogen.
Detection Mechanism on MIAS. The schematic of the detection
mechanism is shown in Figure 2A, which is composed of several steps
(Figure 2B). Immunoassay was used to rapidly detect the MSLN
levels in different serum samples. First of all, carboxyl-modified
microspheres (PS-COOH), 20.38 μm in diameter and coated by
polyclonal antibody (ab96869), were loaded onto the channel
(reaction column) to establish the immune reaction column.
Subsequently, serum samples were added and incubated for 15 min,
and PBST washing buffer (1 mL Tween 20 dissolved in 1000 mL
phosphate buffered saline) was injected for continuous washing of the
microspheres. Blocking buffer (5% bovine serum albumin) was used
to rinse and seal. The mouse monoclonal antibody (ab168740) was
injected into the reaction column for 10 min, and the washing and
blocking steps were repeated. Mouse secondary antibody solution
with FITC was introduced into the reaction column for 10 min.
Finally, the microspheres were rinsed with PBST, and fluorescence
images of the microspheres were acquired with a blue light by using
an inverted fluorescence microscope (Nikon Eclipse Ti, Japan).
Serum Samples. Serum samples (16) were collected from
Changhai Hospital, Shanghai, and the institutional review board of
the hospital approved the research. To study MSLN expression in the
human serum of different patients, the samples were divided into
three groups (Table 1), including eight patients with pancreatic
cancer, four with pancreatitis, and four with other digestive diseases (2
gastric cancer, 1 bile duct stricture, and 1 bile duct stones). Patients in
the three groups were numbered 1−8, 9−12, and 13−16, respectively.
Meanwhile, these patients had complications, including hypertension,
diabetes, and other diseases. The average age of these patients was 55,
with ages ranging from 32 to 71 years old. There were 11 male
Table 1. Source of Serum Samples
cases
group (number) complicating diseases
pancreatic cancer 8 (1−8) hypertension, diabetes,
pancreatitis 4 (9−12) pancreatolithiasis, biliary
stricture, etc.
other digestive diseases (gastric 4
cancer, colon cancer,bile duct (13−16)
stricture, etc.)
2954
Article
patients, 37.5% more than female patients. These serum samples had
the data for CA19-9 cases diagnosed by ELISA in the hospital.
Rapid separation was used to collect the serum samples. Orange-
yellow coagulant-containing tubes were used to coagulate the blood
within 5 min, and the whole blood was collected. The blood
collection tubes were gently inverted 4−5 times to mix the coagulant
and blood and then were kept erect at 4 °C until the blood completely
coagulated. The tubes were centrifuged at 1000g for 10 min to
separate the serum. The collected serum samples were transferred to a
separate centrifuge tube, dispensed at 250 μL per tube, and stored at
−80 °C after liquid nitrogen flash-freezing.
Data Analysis. NIS-elements software was used to analyze the
brightness or gray level of the images. In order to guarantee the
uniformity of the analysis, the average intensity was calculated in the
same region (total intensity value of the area of analysis divided by the
area). Additionally, Graphpad Prism 7 (GraphPad software) was used
for graph plotting and data processing. SPSS version 22.0 software
(SPSS Inc., USA) was used for statistical analysis. The relationship
between MSLN concentration and fluorescence intensity was assessed
by linear regression and correlation analysis. The model was stratified
by pancreatic cancers, pancreatitis, and other digestive system diseases
with a correlation in univariate analysis. Baseline differences between
pancreatic and nonpancreatic cancer biomarkers were assessed by the
Student’s t-test, and P < 0.05 was considered to indicate statistical
significance.
■
RESULTS AND DISCUSSION
Design of the MIAS. Microarray cylinders, instead of sieve
valves, were employed to screen microspheres. Immunoassay
columns were formed in reaction columns, and their
subsequent performance depended on effective microsphere
interception. Microspheres were successfully trapped by the
multiple barriers of the microarray cylinders (Figure 4A) and
interception efficiency was up to 80%. Microarray cylinders
showed high detection sensitivity compared with traditional
sieve valves for microsphere interception.
Validation of the MIAS Function. The semi-quantitative
standard curve of fluorescence intensity−MSLN concentration
(standard products) was established to evaluate the accuracy of
the system. Six MSLN samples at 0, 20, 40, 60, 80, and 100 pg/
mL were selected to detect the fluorescence intensity. A fitting
DOI: 10.1021/acssensors.9b01430
ACS Sens. 2019, 4, 2952−2957
ACS Sensors
formula with a good R-squared (R2 = 0.95) was established
from the standard curve, which would be used for quantitative
analysis. The results, shown in Figure 3, demonstrate that
fluorescence intensity is proportional to MSLN concentration,
and our system accurately detected the MSLN levels.
Figure 3. Semi-quantitative standard curve of fluorescence intensity−
MSLN concentration (standard products) obtained via MIAS.
Detection of Serum Samples by the MIAS. To
ultimately investigate the function of the MIAS, semi-
quantitative fluorescence detection was performed on ultrapure
water and clinical serum samples. As shown in Figure 4, it is
clear that the MIAS performed well. When MSLN was present,
it became excited under blue light and the reaction column
emitted bright green fluorescence (Figure 4C−E). On the
contrary, the negative control showed no fluorescence (Figure
4B). These results have demonstrated that MSLN is
successfully captured by microspheres. It was apparent that
this MIAS was feasible in rapid detection of serum MSLN, and
MSLN levels of the 16 clinical samples were detected by
MIAS. The fluorescence intensities of clinical serum samples
were 6−15 a.u.
Figure 4. (A) Image of the reaction column filled with microspheres under visible light. (B) Image of the reaction column using serum samples
from a healthy person as the negative control. Panels (C−E) respectively represent fluorescence images of serum samples from pancreatic cancer,
pancreatitis, and other digestive diseases. Panels (a−f) are the representative cases from three groups and represent the patient numbers 7, 6, 9,10,
14, and 16, respectively. The reaction column, excited by blue light and MSLN, was used as an antigen. The region in the wireframe represents the
detection area, with the size, 500 μm × 600 μm.
2955
Article
To analyze the detection accuracy of MIAS, ELISA was also
performed on the clinical serum samples. The concentrations
of serum MSLN in samples detected by ELISA were 20−110
pg/mL, and Figure 5A shows the MSLN concentration
between ELISA and MIAS immunofluorescence detection;
the values measured by the MIAS were consistent with those
obtained by conventional ELISA conducted in 48-well plates
(correlation coefficient r = 0.93).
Using the MIAS, the consumption of samples and reagents
could decrease to 2 μL, whereas traditional immune-reactions
require 100 μL. The detection limit of MSLN fluorescence
intensity and concentration was ∼6 a.u. and ∼20 pg/mL,
respectively. Additionally, diffusion and binding of antibody
molecules can be completed fleetingly due to the nanoeffect
and microhydrodynamics of the microfluidic chip. Immuno-
reaction was accelerated by the microstructure of the
microfluidic chip. The duration of the immunoassay using
the MIAS is greatly shortened, compared with 2 h on ELISA,
enabling the rapid detection of serum MSLN within 40 min
after each sampling.
In addition, clinical serum samples detected by immuno-
fluorescence on MIAS were employed to authenticate the
specificity of the biomarker, serum MSLN. The average
fluorescence intensities of pancreatic cancer, pancreatitis, and
other digestive diseases were 10.22, 7.36, and 7.57 a.u,
respectively. Figure 6A shows that MSLN expression was
significantly different in pancreatic cancer, pancreatitis, and
other digestive disease samples. The serum MSLN levels in
pancreatic cancer patients was higher than those in patients
with pancreatitis (P = 0.0159 < 0.05) and other digestive
diseases (P = 0.0219 < 0.05). Moreover, serum MSLN levels in
pancreatic cancer patients were about 39% higher than those in
pancreatitis patients (Figure 6B). In distinguishing between
pancreatic cancer, pancreatitis, and other digestive diseases,
CA19-9 showed low specificity (Figure 5B), while MSLN
showed higher specificity.
DOI: 10.1021/acssensors.9b01430
ACS Sens. 2019, 4, 2952−2957
ACS Sensors Article

Figure 5. (A) MSLN concentration−fluorescence intensity for different patients, with the correlation coefficient, 0.93 (MSLN concentration was
detected by ELISA, and fluorescence intensity was detected by MIAS). (B) MSLN−CA19-9 concentration for different patients by ELISA, with the
correlation coefficient, 0.73.

Figure 6. (A) Fluorescence intensity of serum MSLN in pancreatic cancer, pancreatitis, and other digestive diseases (* represents significant
difference, 0.01 < P < 0.05). (B) Comparison of serum MSLN levels between pancreatic cancer with pancreatitis (the difference between the high
and low values divided by the low value). Panels (A,B) were both detected by immunofluorescence on MIAS.

CA19-9 is considered as a tumor marker frequently elevated
in pancreatic cancer. The difference between MSLN and
CA19-9 could reflect the diagnostic veracity of both serological
tumor markers. Because MSLN levels, using ELISA and MIAS
immunofluorescence detection methods, were consistent
(Figure 5A), the MSLN levels measured by ELISA were
selected to compare with CA19-9 measured by ELISA. There
Above all, the MIAS provides an effective method for the
rapid detection and semi-quantitative determination of a
potential serum biomarker, MSLN. It can detect MSLN
concentration accurately, with an identical value measured by
conventional ELISA. Interestingly, our data show that MSLN
is helpful in the field of differentiating pancreatic cancer from
nonpancreatic cancer.

was a certain difference between serum MSLN and CA19-9
levels, with a correlation coefficient of 0.73 (Figure 5B).
Subsequently, the diagnostic accuracies were calculated to
determine the clinical diagnostic value of MSLN (Table S1).
The results showed that the diagnostic accuracies were 87.5%
for MSLN detected by both MIAS and ELISA, while the
diagnostic accuracy for CA19-9 detected by ELISA was
relatively lower, 81.3%, most likely due to an accumulated
effect of serum total bilirubin.25−27 Thus, the discrimination
between benign and malignant disease should not be made on
the basis of an individual elevated CA19-9 measurement; joint
detection is required. Nevertheless, the combined detection of
the two tumor markers can increase the accuracy to 93.8% as
shown in Table S2. Hence, MSLN and CA19-9 can be used for
joint detection in diagnostic and prognostic assessments of
patients with pancreatic cancer.
P values showing the differences between the two serological
tumor markers in the detection of pancreatic cancer and other
diseases were calculated (Table S2). Results showed that
MSLN (0.0014) had a higher overall significant difference in
the diagnosis of pancreatic cancer than CA19-9 (0.0139).
Given this statistic, MSLN has a higher specificity than CA19-9
for the diagnosis of pancreatic cancer. Generally, MSLN might
have the potential to be a new biomarker for differentiating
pancreatic cancer from pancreatitis and other digestive
diseases.
2956
■
CONCLUSIONS
In this study, we preliminarily established a rapid, sensitive,
and high-throughput MIAS to detect MSLN levels in
pancreatic cancer serum samples. Compared with ELISA,
samples and reagent consumption decreased to 2 μL on the
MIAS, and the detection time was shortened from 2 h to ∼40
min. The detection limit for fluorescence intensity was ∼6 a.u.
The detection limit for concentration was ∼20 pg/mL.
Meanwhile, the MIAS offers an effective method for micro-
sphere interception by the multiple barriers of the microarray
cylinders (Figure 4A). Microarray cylinders showed high
detection sensitivity (∼80%) compared with conventional
sieve valves for microsphere interception.
The MIAS used in this study is high throughput, which not
only improves the credibility of the data but also shortens the
duration of analysis. Collectively, these facts suggest that
MSLN is concentration-dependent in pancreatic cancer,
confirmed by the clinical application values for serum MSLN
in differentiating pancreatic cancer, pancreatitis, and other
digestive system diseases. MIAS provides a rapid detection
method for the early diagnosis of pancreatic cancer.
The entire analysis system can be optimized by integrating
the whole laboratory on a chip. Blood samples could be
analyzed, following dilution and purification of the sample, on
one integrated chip. Meanwhile, parallel channels could be
DOI: 10.1021/acssensors.9b01430
ACS Sens. 2019, 4, 2952−2957
ACS Sensors
established on a chip to detect multiparameter samples.
Additionally, the detection of multiple tumor markers can be
completed based on an optimized immunoassay chip for early
cancer screening and diagnosis.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acssen-
sors.9b01430.
The diagnostic accuracies of MSLN and CA19-9, P
values, and supporting tables (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail: liusixiu@fudan.edu.cn (S.L.).
*E-mail: gsui@fudan.edu.cn (G.S.).
ORCID
Sixiu Liu: 0000-0001-7094-5892
Guodong Sui: 0000-0002-6751-0852
Author Contributions
§ nanofluidic preconcentrator. Microfluid. Nanofluid. 2010, 9, 973−979.
X.D., L.Z., and H.D. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
This work was funded by National Natural Science Foundation
of China (NSFC, grant numbers: 21577020, 21577019,
21377027, and 21876031) and the Shanghai Science and
Technology Committee (STCSM, grant numbers:
16441902100).
■
REFERENCES
(1) Siegel, R.; Ma, J.; Zou, Z.; Jemal, A. Cancer statistics, 2014. Ca-
Cancer J. Clin. 2014, 64, 9−29.
(2) Hassan, R.; Thomas, A.; Alewine, C.; Le, D. T.; Jaffee, E. M.;
Pastan, I. Mesothelin Immunotherapy for Cancer: Ready for Prime
Time? J. Clin. Oncol. 2016, 34, 4171−4179.
(3) Dovzhanskiy, D. I.; Arnold, S. M.; Hackert, T.; Oehme, I.; Witt,
O.; Felix, K.; Giese, N.; Werner, J. Experimental in vivo and in vitro
treatment with a new histone deacetylase inhibitor belinostat inhibits
the growth of pancreatic cancer. BMC Cancer 2012, 12, 226.
(4) Adamska, A.; Domenichini, A.; Falasca, M. Pancreatic Ductal
Adenocarcinoma: Current and Evolving Therapies. Int. J. Mol. Sci.
2017, 18, 1338.
(5) Tjensvoll, K.; Oddmund, N.; Rune, S. Circulating tumor cells in
pancreatic cancer patients: methods of detection and clinical
implications. Int. J. Cancer 2014, 134, 1−8.
(6) Ballehaninna, U. K.; Chamberlain, R. S. The clinical utility of
serum CA 19-9 in the diagnosis, prognosis and management of
pancreatic adenocarcinoma: An evidence based appraisal. J. Gastro-
intest. Oncol. 2012, 3, 105−119.
(7) Ryan, D. P.; Hong, T. S.; Bardeesy, N. Pancreatic
adenocarcinoma. N. Engl. J. Med. 2014, 371, 1039.
(8) Einama, T.; Kawamata, F.; Kamachi, H.; Nishihara, H.; Homma,
S.; Matsuzawa, F.; Mizukami, T.; Konishi, Y.; Tahara, M.; Kamiyama,
T.; Hino, O.; Taketomi, A.; Todo, S. Clinical impacts of mesothelin
expression in gastrointestinal carcinomas. World J. Gastrointest.
Pathophysiol. 2016, 7, 218−222.
(9) Chang, K.; Pastan, I. Molecular cloning of mesothelin, a
differentiation antigen present on mesothelium, mesotheliomas, and
ovarian cancers. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 136−140.
2957
Article
(10) Zhu, L.; Liu, Y.; Chen, G. Diagnostic value of mesothelinin
pancreatic cancer: a meta-analysis. Int. J. Clin. Exp. Med. 2014, 7,
4000−4007.
(11) Hassan, R.; Laszik, Z. G.; Lerner, M.; Raffeld, M.; Postier, R.;
Brackett, D. Mesothelin Is Overexpressed in Pancreaticobiliary
Adenocarcinomas but Not in Normal Pancreas and Chronic
Pancreatitis. Am. J. Clin. Pathol. 2005, 124, 838−845.
(12) Argani, P.; Iacobuzio-Donahue, C.; Ryu, B.; Rosty, C.; Goggins,
M.; Wilentz, R. E.; Murugesan, S. R.; Leach, S. D.; Jaffee, E.; Yeo, C.
J.; Cameron, J. L.; Kern, S. E.; Hruban, R. H. Mesothelin is
overexpressed in the vast majority of ductal adenocarcinomas of the
pancreas: Identification of a new pancreatic cancer marker by serial
analysis of gene expression (SAGE). Clin. Cancer Res. 2001, 7, 3862−
3868.
(13) Ibrahim, D. A.; Abouhashem, N. S. Diagnostic value of IMP3
and mesothelin in differentiating pancreatic ductal adenocarcinoma
from chronic pancreatitis. Pathol., Res. Pract. 2016, 212, 288−293.
(14) Lee, M.; Lee, K.; Kim, K. H.; Oh, K. W.; Choo, J. SERS-based
immunoassay using a gold array-embedded gradient microfluidic chip.
Lab Chip 2012, 12, 3720−3727.
(15) Zhuang, G.; Jin, Q.; Liu, J.; Cong, H.; Liu, K.; Zhao, J.; Yang,
M.; Wang, H. A low temperature bonding of quartz microfluidic chip
for serum lipoproteins analysis. Biomed. Microdevices 2006, 8, 255−
261.
(16) Lee, J. H.; Han, J. Concentration-enhanced rapid detection of
human chorionic gonadotropin (hCG) on a Au surface using a
(17) Sackmann, E. K.; Fulton, A. L.; Beebe, D. J. The present and
future role of microfluidics in biomedical research. Nature 2014, 507,
181−189.
(18) Jayamohan, H.; Sant, H. J.; Gale, B. K. Applications of
microfluidics for molecular diagnostics. Methods in Molecular Biology;
Humana Press, 2013; Vol. 949, pp 305−334.
(19) Dai, B.; Chen, S.; Li, W.; Zheng, L.; Han, X.; Fu, Y.; Wu, J.; Lin,
F.; Zhang, D.; Zhuang, S. Fully-functional semi-automated micro-
fluidic immunoassay platform for quantitation of multiple samples.
Sens. Actuators, B 2019, 300, 127017.
(20) Zhdanov, A.; Keefe, J.; Franco-Waite, L.; Konnaiyan, K. R.;
Pyayt, A. Mobile phone based ELISA (MELISA). Biosens. Bioelectron.
2018, 103, 138−142.
(21) Leng, C.; Zhang, X. Q.; Jue, H. X. Microfluidic Chip-Based
Immunoassay. Prog. Chem. 2009, 21, 687−695.
(22) Sia, S. K.; Whitesides, G. M. Microfluidic devices fabricated in
poly(dimethylsiloxane) for biological studies. Electrophoresis 2003, 24,
3563−3576.
(23) Thorsen, T.; Maerkl, S. J.; Quake, S. R. Microfluidic large-scale
integration. Science 2002, 298, 580−584.
(24) Bjørnsen, G.; Roots, J.; Henriksen, L. Patterning of soft
polydimethylsiloxane elastomers using plasma etching. J. Appl. Polym.
Sci. 2011, 119, 888−895.
(25) Jie, Y. L.; Liang, W. J. The value of serum CA19-9 in diagnosis
of pancreatic carcinoma and biliary diseases. China Trop. Med. 2009,
9, 871−872.
(26) Mann, D.; Edwards, R.; Ho, S.; Lau, W.; Glazer, G. Elevated
tumour marker CA19-9: clinical interpretation and influence of
obstructive jaundice. Eur. J. Surg. Oncol. 2000, 26, 474−479.
(27) Dumitra, S.; Jamal, M. H.; Aboukhalil, J.; Doi, S. A.;
Chaudhury, P.; Hassanain, M.; Metrakos, P. P.; Barkun, J. S.
Pancreatic cancer and predictors of survival: comparing the CA 19-
9/bilirubin ratio with the McGill Brisbane Symptom Score. HPB
2013, 15, 1002−1009.
DOI: 10.1021/acssensors.9b01430
ACS Sens. 2019, 4, 2952−2957